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1 TRANSFER OF MICROORGANISMS FROM FOMITES TO HANDS AND RISK ASSESSMENT OF CONTAMINATED AND DISINFECTED SURFACES by Gerardo Urquijo Lopez _______________________ A Dissertation Submitted to the Faculty of the DEPARTMENT OF SOIL, WATER AND ENVIRONMENTAL SCIENCE In Partial Fulfillments of the Requirements for the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 2013 2 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Gerardo Urquijo Lopez entitled TRANSFER OF MICROORGANISMS FROM FOMITES TO HANDS AND RISK ASSESSMENT OF CONTAMINATED AND DISINFECTED SURFACES and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Doctor in Philosophy Charles P. Gerba, PhD Date: December 14, 2012 Kelly A. Reynolds, PhD Date: December 14, 2012 Ian L. Pepper, PhD Date: December 14, 2012 Kelly R. Bright, PhD Date: December 14, 2012 Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. Dissertation Director: Charles P. Gerba, PhD Date: December 14, 2012 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of the requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Request for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in interests of scholarship. In all other instances, however, permission must be obtained by the author. SIGNED: GERARDO URQUIJO LOPEZ_______ 4 ACKNOWLEDGMENTS I first would like to say thank you Dr. Charles Gerba for the opportunity you gave me to study in your laboratory. It was a challenging and rewarding experience and I am forever grateful to you. I would also like to express gratitude to Dr. Kelly Reynolds for your support, advice, and for giving me the opportunity to collaborate with you on your projects. Thank you Dr. Ian Pepper for taking the time to mentor me in preparation for my life after graduate school. I am also grateful to Dr. Kelly Bright for the support and opportunity to work on your projects. I would also like to express gratitude to Dr. Masaaki Kitajima for all the support, advice, and guidance he gave me. Your time and energy is greatly appreciated. Dr. Akrum Tamimi, thank you for advising me on the transfer data analysis. Sheri Carlino thank you for the technical assistance you provided. I also would like to recognize everyone in the Gerba lab. You made my time in the lab enjoyable and I will cherish these memories. I also would like to show gratitude to Dr. Joan Rose and the Center for Advancing Microbial Risk Assessment (CAMRA) for funding. I am also grateful to the Alfred P. SLOAN Foundation for their support in while pursuing my Doctorate. I also greatly appreciate the support Dr. Maria Teresa Velez and her office staff provided throughout my graduate studies. I also would like to thank Dr. Silvertooth and his office staff for the support and guidance. 5 DEDICATION This dissertation is dedicated to the people in my life who provided me with support and encouragement through out this endeavor. I could not have accomplished this goal without you’re backing and unwavering encouragement. I am forever grateful to all of you for helping me obtain this Ph.D. degree. To my wife Stephanie and daughter Lucia, my parents Leonardo Gonzalez Lopez and Guadalupe Urquijo Lopez (†), my in laws Edward and Lucy Chavez, and my family and friends. 6 TABLE OF CONTENTS ABSTRACT...................................................................................................................... 15 INTRODUCTION ............................................................................................................ 17 Problem Definition .................................................................................................. 17 Literature Review .................................................................................................... 18 Role of Fomites in Disease Transmission ..................................................... 18 Viruses on fomites ............................................................................... 19 Bacteria on fomites .............................................................................. 20 Survival of Disease on Fomites and Fingers ................................................. 23 Survival of viruses on fomites and fingers ........................................... 23 Survival of bacteria on fomites and fingers ......................................... 25 Role of Hands in Disease Transmission ........................................................ 25 Hands ................................................................................................... 25 Viral transmission ................................................................................ 26 Bacterial transmission .......................................................................... 28 DISSERTATION FORMAT ............................................................................................ 31 PRESENT STUDY ........................................................................................................... 33 REFERENCES ................................................................................................................. 39 APPENDIX A: MINIREVIEW: TRANSFER OF BACTERIA AND VIRUSES TO HANDS ............................................................................................................................. 59 ABSTRACT ............................................................................................................ 60 INTRODUCTION ................................................................................................... 61 7 TABLE OF CONTENTS - Continued FACTORS INFLUENCING TRANSFER .................................................... 62 Relative Humidity ................................................................................ 62 Damp versus dry fomites or hands ....................................................... 62 Duration of drying time........................................................................ 63 Type of fomite material ........................................................................ 64 Type of Organism ................................................................................ 65 Level of fomite contamination ............................................................. 67 Hand washing ....................................................................................... 67 Direction of transfer ............................................................................. 68 Friction and pressure ............................................................................ 70 DISCUSSION ......................................................................................................... 71 CONCLUSIONS ..................................................................................................... 73 ACKNOWLEDGEMENTS .................................................................................... 74 REFERENCES ........................................................................................................ 75 FIGURES ................................................................................................................ 83 TABLE .................................................................................................................... 88 APPENDIX B: COMPARISON OF TWO APPROACHES IN DETERMINING TRANSFER EFFICIENCY OF ESCHERICHIA COLI FROM NONPOROUS FOMITES TO FINGERS.................................................................................................................... 89 ABSTRACT ............................................................................................................ 90 TEXT ....................................................................................................................... 90 8 TABLE OF CONTENTS - Continued ACKNOWLEDGEMENTS .................................................................................... 94 REFERENCES ........................................................................................................ 95 TABLE .................................................................................................................. 102 APPENDIX C: TRANSFER EFFICIENCY OF BACTERIA AND VIRUSES FROM POROUS AND NONPOROUS FOMITES TO FINGERS UNDER DIFFERENT RELATIVE HUMIDITY ................................................................................................ 103 ABSTRACT .......................................................................................................... 104 INTRODUCTION ................................................................................................. 105 MATERIALS AND METHODS .......................................................................... 106 Subjects ........................................................................................................ 106 Bacteria, Virus and preparation of inocula .................................................. 106 Control wash and disinfection ..................................................................... 108 Relative Humidity Conditions and Temperature ......................................... 108 Fomites tested .............................................................................................. 109 Inoculation of fomites .................................................................................. 110 Fomite-to-finger transfer, sampling, and assays .......................................... 111 Transfer efficiency calculation and statistical analyses ............................... 112 RESULTS.............................................................................................................. 113 Influence of relative humidity on microbial transfer ................................... 113 Influence of fomite type on microbial transfer ............................................ 114 DISCUSSION ....................................................................................................... 115 9 TABLE OF CONTENTS - Continued CONCLUSIONS ................................................................................................... 120 ACKNOWLEDGEMENTS .................................................................................. 120 REFERENCES ...................................................................................................... 122 TABLES ................................................................................................................ 131 FIGURES .............................................................................................................. 135 APPENDIX D: COMPARATIVE PERSISTENCE OF BACTERIA AND VIRUSES UNDER DIFFERENT RELATIVE HUMIDITY ON POROUS AND NONPOROUS FOMITES ....................................................................................................................... 138 ABSTRACT .......................................................................................................... 139 INTRODUCTION ................................................................................................. 140 MATERIALS AND METHODS .......................................................................... 141 Bacteria, Virus and preparation of inocula .................................................. 141 Relative Humidity Conditions and Temperature ......................................... 143 Fomites tested .............................................................................................. 144 Inoculation of fomites .................................................................................. 144 Surface sampling and assays ....................................................................... 145 Reduction and Statistical analysis ............................................................... 146 RESULTS.............................................................................................................. 147 Influence of relative humidity on microbial survival .................................. 147 Influence of fomite type on microbial survival ........................................... 147 DISCUSSION ....................................................................................................... 149 10 TABLE OF CONTENTS - Continued ACKNOWLEDGEMENTS .................................................................................. 154 REFERENCES ...................................................................................................... 155 TABLES ................................................................................................................ 163 FIGURES .............................................................................................................. 167 APPENDIX E: THE EFFECT OF A DISINFECTANT WIPE ON MICROBIAL TRANSFER .................................................................................................................... 170 ABSTRACT .......................................................................................................... 171 INTRODUCTION ................................................................................................. 172 MATERIALS AND METHODS .......................................................................... 175 Subjects ........................................................................................................ 175 Bacteria, endospores, and preparation of inocula ........................................ 175 Study organisms ................................................................................. 175 Gram negative and Gram positive inoculum preparation .................. 175 Endospore-forming bacteria inoculum preparation ........................... 176 Virus inoculum preparation................................................................ 176 Control wash and disinfection ..................................................................... 176 Fomites tested .............................................................................................. 177 Disinfectant wipe and Neutralizing Solution............................................... 178 Relative Humidity and Temperature ........................................................... 178 Inoculation of fomites .................................................................................. 179 11 TABLE OF CONTENTS - Continued Layout of fomites ............................................................................... 179 Organism concentration ..................................................................... 179 Fomite sampling .......................................................................................... 179 Intervention application ............................................................................... 180 Transfer experiments ................................................................................... 180 Sampling the fingers .................................................................................... 181 Organism assays .......................................................................................... 181 Transfer efficiency, log10 reduction, and statistical analysis ....................... 182 Calculation of transfer efficiency ....................................................... 182 RESULTS.............................................................................................................. 183 Removal of microbial contamination from nonporous surfaces ................. 183 Influence of disinfectant wipes on Microbial Transfer................................ 184 DISCUSSION ....................................................................................................... 184 CONCLUSIONS ................................................................................................... 188 ACKNOWLEDGEMENTS .................................................................................. 188 REFERENCES ...................................................................................................... 189 TABLES ................................................................................................................ 198 APPENDIX F: RISK OF CAMPYLOBACTER JEJUNI INFECTION FROM PREPARING RAW CHICKEN IN DOMESTIC KITCHENS AND CROSSCONTAMINATION REDUCTION FROM DISINFECTANT WIPES ....................... 201 ABSTRACT .......................................................................................................... 202 12 TABLE OF CONTENTS - Continued 1. INTRODUCTION ............................................................................................ 203 2. METHODS....................................................................................................... 206 2.1 The Exposure Model ............................................................................. 206 2.1.1 Input Distribution ...................................................................... 207 2.1.2 Fomite-to-finger Dose ............................................................... 209 2.1.3 Risk of Infection......................................................................... 209 2.1.4 Risk of Illness ............................................................................ 210 2.1.5 Risk of Death ............................................................................. 210 2.1.6 Annual Risk ............................................................................... 211 2.1.7 Percent Reduction ..................................................................... 211 2.1.8 Model Implementation .............................................................. 212 3. RESULTS.......................................................................................................... 213 3.1 Risk Comparison of Scenario 1 and 2 under Condition A .................... 213 3.1.1 Risks per person per event ........................................................ 213 3.1.2 Annual Risks per person per year ............................................. 213 3.2 Risk Comparison of Scenario 1 and 2 under Condition B .................... 213 3.2.1 Risks per person per preparation event .................................... 213 3.2.2 Annual Risks per person per year ............................................. 214 4. DISCUSSION ................................................................................................... 214 ACKNOWLEDGEMENTS .................................................................................. 219 REFERENCES ...................................................................................................... 220 13 TABLE OF CONTENTS - Continued FIGURES .............................................................................................................. 226 TABLES ................................................................................................................ 227 APPENDIX G: SUPPLEMENTAL MATERIALS ........................................................ 232 TABLES ................................................................................................................ 233 REFERENCES ...................................................................................................... 252 APPENDIX H: SUPPLEMENTAL MATERIALS ........................................................ 254 ADDTIONAL DETAILS TO METHODS AND MATERIALS ......................... 255 ADDITIONAL DETAILS TO RESULTS ............................................................ 255 TABLE .................................................................................................................. 256 FIGURES .............................................................................................................. 257 REFERENCES ...................................................................................................... 259 APPENDIX I: SUPPLEMENTAL MATERIALS ......................................................... 260 TABLES ................................................................................................................ 261 FIGURES .............................................................................................................. 264 APPENDIX J: SUPPLEMENTAL MATERIALS ......................................................... 267 ADDITIONAL DETAILS TO METHODS AND MATERIALS ........................ 268 Calculation of percent reduction.................................................................. 268 ADDITIONAL DETAILS TO RESULTS ............................................................ 268 Removal of microbial contamination from nonporous surfaces ................. 268 TABLES ................................................................................................................ 270 FIGURES .............................................................................................................. 272 14 TABLE OF CONTENTS - Continued APPENDIX K: SUPPLEMENTAL MATERIALS ........................................................ 275 TABLES ................................................................................................................ 276 15 ABSTRACT It is now widely accepted that surface contamination plays an important role in the transmission of both respiratory and gastrointestinal infections in the domestic environment and community setting. The efficiency of transfer of a pathogen to the hand from a fomite is important in modeling transmission in microbial risk assessment models. The objective of this study was to use published literature to assess the role of fomites and hands in disease transmission, and to conduct fomite-to-finger transfer studies from various porous and nonporous fomites under different relative humidity condition using non-pathogenic strains of Escherichia coli, Staphylococcus aureus, MS2 coliphage, Bacillus thuringiensis spores, and poliovirus 1; to evaluate the persistence of bacteria and viruses on surfaces; to examine bacteria and virus transfer from treated surfaces; and to conduct a foodborne quantitative microbial risk assessment using Campylobacter jejuni from the data obtained in these studies. It was found that numerous factors influence the transfer efficiency of microorganisms, with moisture being the most important, with greater transfer under humid conditions. Other factors influencing transfer include drying time, contact time, pressure, friction, type of material, and porosity of the fomite. Percent transfer was greater under high relative humidity for both porous and nonporous surfaces. Most organisms on average had greater transfer under high relative humidity (40 – 65%) compared to low relative humidity (15 – 32%). Relative humidity and fomite type influenced the survival of all studied organisms; survival was greater on nonporous surfaces than those for porous surfaces. Test organisms were reduced up to 99.997% on 16 the fomites after the surfaces were wiped with a disinfectant wipe. Microbial fomite-tofinger transfer from disinfectant wipe-treated surfaces were, lower than from non-treated surfaces. The disinfectant-wipe intervention reduced the risk of Campylobacter infection, illness, and death by 2 to 3 orders on all fomites. The disinfectant-wipe intervention reduced the annual risk of illness below the reported national average of diagnosed Campylobacteriosis cases 1.3E-04. This risk assessment demonstrates that the use of disinfectant wipes to decontaminate surface areas after chicken preparation reduces the risk of C. jejuni infections up to 99.2%. 17 INTRODUCTION Problem Definition The role of fomites in the transmission of infectious diseases has been a controversial subject (1). However, this controversy is dimensioning through evidence from studies showing contamination of inanimate surfaces occurs in homes and community settings through cross-contamination from colonized or infected people, raw animal meat, or by contaminated sponges and cleaning cloths who serve as reservoirs and vehicles in microbial transmission (2-6). Indoor environments play a key role in the dissemination of microbial pathogens (7, 8). The home environment, specifically the kitchen and bathroom, serves as reservoir of large numbers of microorganisms, and infectious disease transmission has been shown to occur in 6 – 60% of households in which one member is ill (9). Scott (10) suggested that during outbreaks of infection in the home, the transfer between animate and inanimate surfaces promotes a cycle of re-infection. The community setting in hospitals, nursing homes, schools, and day-care centers are known to harbor viral and bacterial pathogens. Health-care associated infections (HAI) remain a major cause of patient morbidity and mortality (11). From these studies we have learned the importance of environmental contamination cannot be sufficiently emphasized. There is strong evidence that person-to-person transmission via the hands and contaminated fomites plays a key role in the spread of viral infections (12). Contact with contaminated surfaces varies for each person depending on age, personal habits, and type of activities (13-15). Once contact with the host is achieved, microbial pathogens can 18 gain entry into the host through portals of entry, including the mouth, nasopharynx or eyes (16-18). The objective of this study was to use existing published literature to assess the role of fomites and hands in disease transmission; to conduct fomite-to-finger transfer studies from various porous and nonporous fomites under different relative humidity conditions; to evaluate the persistence of bacteria and viruses on surfaces; to examine bacteria and virus transfer from treated surfaces; and to conduct a quantitative microbial risk assessment using the findings of these transfer studies as input parameters in a Monte Carlo 20,000 iteration simulation and determine the risk of Campylobacter jejuni infection in household kitchens. Literature Review Role of Fomites in Disease Transmission Fomites consist of animate or inanimate porous and nonporous surfaces that can become contaminated with pathogenic microorganisms and serve as reservoirs of microbial pathogens and vectors for cross-transmission in the domestic environment and in community settings (14, 18, 19). Potential pathogens from sources such as raw foods, infected persons and animals can be transferred between inanimate and animate surfaces through either direct or indirect contact (10, 20-25). Environmental surfaces become contaminated with virus, bacteria or parasites shed by infected or colonized individuals by direct contact with body secretions such as blood, feces, urine, saliva, and nasal fluid (17, 18, 26-29), contact with soiled hands, contact with aerosolized virus or bacteria generated by talking, sneezing, coughing, or vomiting (12, 17, 18, 28, 30). Boyce and 19 Pittet (31) described that surfaces near or in close contact with individuals can even become contaminated from colonized normal skin with S. aureus (4, 32-38), Enterococcus facium and faecalis (2, 39-43), Klebsiella spp. (44-47) and with Gram – negative bacteria (35, 44, 47-50). Patients who have gastrointestinal colonization with Clostridium difficile may also serve as reservoirs and thus contaminate the surrounding environment (51-56). Layton et al. (6) found that the major reservoirs of MRSA within institutions are patients with either S. aureus infection or colonization. Zervos et al. (43) reported that the potential hospital reservoirs for gentamicin-resistant enterococci could include colonized as well as infected patients, hospital personnel, and the inanimate hospital environment. After a fomite becomes contaminated, the transfer of infectious virus or bacteria easily occur between hand to fomite, fomite to hand, hand to hand, hand to fomite to hand, or fomite to hand to fomite (1, 13, 30, 57-68). Viruses on fomites Bellamy et al. (17) surveyed the home environment for the presence of viruses and body fluids and found haemoglobin on 2% of surfaces (taps, washbasins, toilets bowls and seats) and amylase, an indicator or saliva, sweat and urine. Reynolds et al. (18) found fecal and total coliform bacteria, protein, and biochemical markers to be widespread in public places. These studies indicate that surfaces may remain soiled for long periods of times and may not be thoroughly cleaned. Studies conducted on home and community settings have provided a better understanding of how infectious disease is spread in these environments (16). Day care centers and elementary schools have been investigated for 20 the presence of influenza A virus and norovirus and it has been found that influenza A virus was detected on over 50% of the fomites in homes and day care centers during influenza season (14, 69, 70). Bacteria on fomites Several studies have identified bacteria prevalent on environmental surfaces both in domestic environments and in community settings. In a hospital study, Bonten et al. (2) reported that vancomycin-resistant enterococci (VRE) were recovered more frequently from rooms with contaminated surfaces 60-70% in which there was a high proportion of VRE-colonized body sites than from rooms with only a few colonized body sites. In another study, patients with VRE who had diarrhea had 46% of environmental cultures contaminated, compared to 15% of surfaces cultured in the rooms of VRE patients without diarrhea (3). In a similar study, MRSA was recovered from 58.8% of surfaces in rooms with patients with diarrhea, compared to 23.3% of surfaces in rooms of patients without diarrhea (51). Boyce et al. (4) found that when patients had methicillin-resistant Staphylococcus aureus (MRSA) in a wound or urine, 36% of surfaces were contaminated, while when MRSA was isolated from sputum, blood, or conjunctivae, only 6% of surfaces were contaminated. In the same study, Boyce et al. (4) reported environmental contamination of MRSA occurred in rooms of 73% of infected patients and 69% of colonized patients. McFarland et al. (53) described that patients with symptomatic C. difficile resulted in 49% of their rooms were contaminated, compared to 29% of room occupied by asymptomatic patients were contaminated. The frequency of positive 21 personnel hand contamination with C. difficile has been strongly correlated with the intensity of environmental contamination in rooms (5, 54, 56, 71). One study reported room site contamination ranging from 1% to >50%, with the most heavily contaminated rooms having >90% of sites positive for C. difficile (54). Environmental surveillance studies have investingated the prevalence of nosocomial pathogens in intensive care units (ICUs) and the most commonly reported surfaces to be contaminated with MRSA, VRE, Enterococcus spp., Enterobacter spp., C. difficile, and Gram negative rods are the floor next to the bed, bathroom floor, bathroom hand rails, mops, bed linens, patient gowns, healthcare worker’s (HCWs) gowns or uniforms, gloves, drawsheet, overbed tables, bedside tables/stands, bedside rails, bedsheets, door handle, bedside commodes, toilet seat, toilet rail, keyboard, computer table, faucet, tubs, washbasins, chairs, dresser, nurse call button, TV remote, shelves, cabinet, and window sill (2-4, 42, 51, 53, 54, 72-81). Medical equipment reported to have been contaminated with MRSA, VRE, mupirocin-resistant S. aureus are infusion pump buttons, electrocardiogram-EKG wires and monitor, intravenous-IV poles and fluid pump, pulse oximeters, stethoscopes, monitors, oxygen flow meters, rectal and ear probe electronic thermometers, blood-pressure cuff, blood glucose monitor, suction canisters, enteral feed, ventilator tubing, automated medication dispenser, and urine container (2-4, 6, 42, 51, 72, 74-77, 82-85). Even paper-towel dispensers near wash basins have been known to be contaminated with Staphylococci bacteria (86). Patients’ charts have also been shown to be potential reservoirs and vectors for cross-contamination of pathogenic bacteria MRSA, 22 VRE and several other to HCW hands and to patients (87-90). One study reported 81.1% of patients’ charts in a surgical ICU and surgical ward were contaminated by infectious agents (90). Inanimate surfaces near or in close contact with affected patients as well as those that are frequently touched by hospital personnel commonly become contaminated with MRSA, VRE, and other pathogens and that the frequency of contamination is affected by the body site at which patients are colonized or infected (4, 91). The evidence that personnel may contaminate their gloves or possibly their hands, by touching surfaces or medical equipment suggests that contaminated environmental surfaces serve as microbial reservoirs and vehicles of transmission in hospitals (2-6). Paper currency used in several countries has been reported to harbor bacteria and parasites including E. coli, B. cereus, S. aureus, and Salmonella ranging from (0.1 CFU/cm2 to 128 CFU/cm2) (92) and has been identified as a reservoir and vehicle of microbial transmission (92-94). Several studies have demonstrated the potential for cross-contamination in the kitchen after preparation of raw chicken, contaminated naturally with Campylobacter and Salmonella (24, 95) or seeded with marker organisms to contaminate hands, counter tops, sink basins, cutting boards, utensils, dishcloths, and sponges (20, 22, 68, 96-105). A number of studies have investigated the domestic environment and have determined the prevalence and quantity of bacterial contamination on surfaces (76, 106-111). The results indicate bacteria to be found on kitchen surfaces (0.2 log10 CFU/ swab area to 8.4 CFU/ swab area), cleaning cloths and sponges (0.90 log10 CFU/ swab area or ml to 10.12 CFU/ 23 swab area or ml) and in the bathroom (0.02 log10 CFU/ swab area to 8.5 CFU/ swab area). Surface contamination has been shown to spread by using cleaning cloths that are harboring viruses and bacteria (112). Public areas and homes have been investigated for the occurrence of bacteria and biochemical markers (17, 18) and the results indicate that they are prevalent. Reynolds et al. (18) found biochemical markers, i.e., hemoglobin (blood marker), amylase (mucus, saliva, sweat, and urine marker), and urea (urine and sweat marker) were detected on 3% (26/801); 15% (120/801), and 6% (48/801) of the surfaces, respectively. Medrano-Felix et al. (107) found up to 3.27 log10 per 900 cm2 of counter surface in kitchens. Survival of Disease on Fomites and Fingers Survival of viruses on fomites and fingers Viruses outside a host may be considered inert particles, without an intrinsic metabolism, they do not require any nutrients to persist (113). Nonetheless, they possess a degree of robustness which allows them to remain infectious during the various conditions that they may encounter between one host and another (113). The longer a virus can survive outside a host, the greater are its chances for transmission (113). Virus and bacterial survival on fomites is influenced by intrinsic factors which include fomite properties such as hydrophobicity of the surface, virus and bacteria characteristics such as isoelectric points and extrinsic factors including environmental temperature, humidity and other factors (14).Virus are infectious at very low doses and have been shown to survive for minutes to days on nonporous surfaces (17, 18). Both respiratory and enteric viruses 24 have been shown to survive on nonporous and porous fomites from a few minutes to several days, with non-enveloped viruses such as rotavirus, adenovirus, poliovirus, herpes simplex virus, and hepatitis A virus (HAV) having greater resistance to drying and thus surviving longer and spreading more easily than enveloped viruses, such as influenza A virus, coronaviruses, and respiratory syncytial virus (RSV) which are less stable in the environment (12). Enveloped viruses have been shown to survive on fomites. Respiratory viruses shown to persist on fomites for 24 – 48 h are influenza A and B (nonporous stainless steel and plastic surfaces) while surviving for < 8 – 12 h on cloth, paper, and tissues (114). Mahl and Sadler (115) showed herpes simplex virus to survive on glass slides for 56 days, while vaccinia virus for at least 14 days. Both of these viruses were also found to survive in hospital floor sweepings for at least 14 days (115). Non-enveloped viruses in general have been shown to survive longer (days to several weeks) than enveloped viruses on fomites. Enteric viruses can persist on fomites for long periods of time. Mahl and Sadler (115) showed adenovirus and poliovirus to survive on glass slides for at least 56 days, while coxsackie virus for at least 14 days. Abad et al. (116) found that hepatitis A virus (HAV) and human rotavirus were more resistant to inactivation than enteric adenovirus (ADV) and poliovirus (PV), yet they all survived up to 60 d. In a later study, Abad et al. (117) found that astroviruses can survive on nonporous china (up to 60 d) and porous paper surfaces (up to 90 d) under different 25 environmental conditions. Both respiratory and enteric viruses have been shown to survive on fingers or gloves from a few minutes to several hours and again nonenveloped viruses surviving longer than enveloped viruses. Influenza A and B has been shown to survive on hands for up to 5 min (114). Survival of bacteria on fomites and fingers Both Gram negative and Gram positive bacteria have been shown to survive on nonporous and porous fomites from a few minutes to several days. Noskin et al. (118) showed vancomycin-resistant enterococci (VRE) to survive on several fomites. They found E. faecalis and E. faecium surviving 5 days and 7 days on countertops, respectively, 24 h on bedrails, 60 min on telephone handpiece, and 30 min on the surface of stethoscopes (118). Neely and Maley (119) reported the survival of MRSA and VRE on five common hospital materials, clothing, towels, scrub suits and lab coats, privacy drapes, and splash aprons to be from 1 day to as long as 90 days. VRE, E. faecium, and E. faecalis have been shown to survive for at least 60 min on gloved and ungloved fingertips (118). Role of Hands in Disease Transmission Hands Multiple studies prove that environmental contamination in the domestic environment and community setting plays an important role in the transmission of microbial pathogens. Epidemiologic data have suggested for more than a century and a half that healthcare 26 workers (HCWs) spread microbes from patient to patient via contaminated hands causing health care-associated infections (HAI) (11, 120). It is estimated that in the United States there are 1.7 million HAI each year, with approximately 99,000 deaths (11). An estimated 20% to 40% of these HAI have been attributed to cross infection via the hands of health care personnel, that have become contaminated from direct contact with the patient or indirectly by touching contaminated environmental surfaces (11). Touching fomites animate (hands) or inanimate, such as counter tops, toys, eating utensils, towels or doorknobs, inadvertently contaminated with secretions, vomit, or nasal mucosa from infected person and then transferring the virus from the hands to the eyes, nose or mouth, are further routes of spread (12). Transfer of gastrointestinal viruses to food and food contact surfaces during handling and preparation is an important route of spread of viral gastroenteritis (12). Most documented foodborne viral outbreaks can be traced to food that has been manually handled by an infected foodhandler (28). Viral transmission Viruses are undoubtedly the most common cause of infectious disease acquired within indoor environments and cause considerable morbidity (12). Close personal contact within the home and community settings, such as daycare centers, schools, hospitals, nursing homes, and cruise chips, make them ideal places for the rapid spread of viral infections. A study by Jiang et al. (121) investigated virus transmission in childcare facilities using modified cauliflower virus DNA as an environmental marker. The viral DNA introduced through treated toy balls spread within a few hours of children handling 27 the toys, even after the treated balls were removed after one day, the viral DNA continued to circulate in the day-care center for up to 2 weeks. The markers could also be detected in the children’s homes, on the hands of family members, and on surfaces within those homes (121). Studies have shown that the home is conducive to the transmission of respiratory and enteric viruses. A respiratory infected person will deposit virus onto their hands from their nasal mucous and spread it to their surrounding environment where family members can transfer the virus onto their hands and self-inoculate. This has been demonstrated with natural and laboratory rhinovirus infections (7, 8, 65, 122, 123), and influenza virus (114, 124). Bean et al. (114) showed influenza A and B viruses to transfer from stainless steel surfaces to hands for 24 h and from tissues to hands for up to 15 min. The spread of respiratory syncytial virus (RSV) from contaminated surfaces in the hospital demonstrated that the infectious virus could be transferred to the hands of hospital personnel touching the contaminated surfaces (26). The link between school and day-care centers with homes of children has been identified as critical in the chain of dissemination of rhinovirus (8), and influenza virus (69). It has been estimated that 30-40% of infectious gastroenteritis cases in developed countries are attributable to viruses (125). The presence of microorganisms associated with diarrhea has been isolated from the household environment (25, 126). Enteric viral 28 infections such as rotavirus are shed from children and adults in large numbers with feces containing greater than 1012 particles per gram and can excreted up to 34 d (12, 127, 128). Common foodborne viruses for which we become reservoirs of while ill are norovirus and HAV and some less common viruses are enteric adenovirus (types 40/41), rotavirus sapovirus, astrovirus, coronavirus, and aichivirus (28, 113). Human norovirus (HuNoV) transmission via contaminated fomites has been well documented and several outbreaks have been reported in cruise ships (129), U.S. Navy ships (130), house boats (131), and river rafters (132), long-term care facility (133) nursing homes (16). Norwalk-like viruses are the number one cause of foodborne illness, accounting for much higher incidence than that of the two highest bacterial etiologies combined (63). It is estimated that vomiting associated with Norwalk-like viruses (NLVs) can have up to 3.0 x107 particles are distributed as an aerosol into the environment (12) and shedding can persist up to 10 d. Noroviruses account for greater than 90% of nonbacterial and approximately 50% of all epidemic gastroenteritis (11). They are responsible for an estimated 267 million infections annually worldwide and 23 million infections annually in the United States (11). Bacterial transmission There is substantial evidence to show that bacterial transmission occurs in both homes and community settings. Studies have shown that the same strain of gentamicin-resistant E. faecalis and MRSA has been isolated in two separate hospitals, suggesting that interhospital spread of gentamicin-resistant enterococci may have been caused by 29 transient carriage on the hands of personnel who rotate between the two hospitals or by the transfer of patients from one facility to the other (91, 120). Muto et al. (120) reported that there has even been evidence to suggest transmission of MRSA clones from one city to city, from country to country, and even from continent to continent traced to the transfer of patients infected or colonized with MRSA. The hands of HCWs are a major source of transmission of nosocomial pathogens and frequently acquire transient flora, which colonize the superficial layers of the skin (31, 134). Multiple studies have shown that HCWs hands or gloves frequently acquire multidrug-resistant MRSA, VRE, Klebsiella spp., C. difficile and other pathogenic flora during direct contact with patients or contact with contaminated surfaces found in close proximity of infected or colonized patients (31, 40, 44, 54, 56, 81, 83, 118, 135-141). MRSA and VRE have even contaminated gloves of personnel who had no direct contact with patients, but had touched contaminated surfaces (4, 140). Tenorio et al. (140) reported that HCWs who tended a patient with diarrhea resulted in 100% gloves positive for VRE, as compared to 44% (12/27) HCWs gloves who tended to a patient without diarrhea. Studies in hospitals have shown that transmission via the contaminated hands of HCWs to be an important mechanism and risk factor in previously uncolonized patients acquiring the same VRE strain as colonized patients in other rooms (3, 142). Another study identified HCWs hands were becoming contaminated with MRSA while tending to patients in their rooms and then possibly cross-contaminating hospital computer keyboards used only by clinicians (143). Yet in another hospital outbreak of 30 gentamicin-resistant E. faecalis the HCWs hands were suggested as the cause of spread due to frequent contact with a contaminated door handle leading out of the surgical ICU (43). Duckro et al. (83) conducted a study to determine the frequency of VRE transmission from VRE-colonized patients’s skin or contaminated surface to the HCWs hands or gloves and then to a VRE-negative site, VRE was transferred 10.6% (16/151) to negative sites. C. difficile has been recovered from the hands of infected patients and HCWs, with one study reporting 59% (20.5/35) HCWs hands being positive after tending a culture positive patient (56). In a similar study, Samore et al. (54) found 14% (10/73) personnel hands were positive for C. difficile. 31 DISSERTATION FORMAT This dissertation contains eleven appendices: six manuscripts and five supplemental materials. Appendix A is a Minireview entitled “Minireview: Transfer Bacteria and Viruses to Hands” that will be submitted for publication. This review assesses the importance of factors affecting the transfer of bacteria and viruses, and the degree of transfer, which might be expected under different environmental conditions. Appendix B is a short form manuscript entitled “Comparison of Two Approaches in Determining Transfer of Escherichia coli from Nonporous Fomites to Fingers” that will be submitted for publication. This manuscript evaluates the fomite-to-finger percent transfer of E. coli from fomites using two approaches; one determines the percent transfer based on control fomite, while the other is based on the sum of bacteria recovered from a finger and donor fomite. Appendix C is a long form manuscript entitled “Percent Transfer of Bacteria and Viruses from Nonporous and Porous Fomites to Fingers Under Different Relative Humidity” that will be submitted for publication. This research examines the effect of low and high relative humidity on fomite-to-finger percent transfer of four model organisms: Escherichia coli, Staphylococcus aureus, Bacillus thuringiensis spores, MS2 coliphage, and poliovirus 1 from nine common inanimate surfaces (fomites). Appendix D is a manuscript entitled “Comparative Persistence of Bacteria and Viruses under different Relative Humidity on Porous and Nonporous Fomites” that will be submitted for publication. This study evaluates the survival of several model organisms under low and high relative humidity on various types of fomites to better understand 32 microorganism survival in modeling microbial exposure and risk assessment. Appendix E is a manuscript entitled “The Effect of a Disinfectant Wipe on Microbial Transfer” that will be submitted for publication. This research investigated the effects of a disinfectant wipe on removing microorganisms from fomites and reducing microbial fomite-to-finger transfer. Appendix F is a manuscript entitled “Risk of Campylobacter jejuni infection from Preparing Raw Chicken in Domestic Kitchens and Cross-contamination Reduction from Disinfectant Wipes” that will be submitted for publication. This manuscript describes the Quantitative Microbial Risk Assessment conducted on the risk of infection after the preparation of raw chicken fillet in a domestic kitchen and cross-contamination to fomite-to-hand exposure and the reduction in risk by disinfectant wipes on the contaminated surfaces. Appendices G through K provide additional materials for the corresponding manuscripts. 33 PRESENT STUDY The manuscript in Appendix A is a Minireview of the transfer of microorganisms to hands from fomites and vice versa. This review assesses the importance of factors affecting the transfer of bacteria and viruses, and the degree of transfer, which might be expected under different environmental conditions. Such information is important to reduce the uncertainty in quantitative microbial risk assess models, which can be used to estimate the risk of infection from contact with contaminated surfaces and the impact of interventions (disinfectants, hand sanitizers, self-sanitizing surfaces). It was found that numerous factors influence the transfer efficiency of microorganisms, with moisture being the most important, with greater transfer occurring in humid environments. Other factors influencing transfer include drying time, contact time, pressure, friction type of material, and nature of the fomite. The greater the pressure applied to a surface and longer the contact time the greater the amount of transfer that can be expected. Much greater transfer occurs with hard surfaces (stainless steel) than porous surfaces (paper, cloth). Overall trends can be observed but transfer is organism dependent. Both Gram negative and positive bacteria have a greater transfer from hand-to-fomite than virus. Fomite-to-hand transfer is similar for both virus and bacteria. Hand-to-hand, hand-tofomite-to-hand and the fomite-to-hand-to-fomite transfer direction had an overall lower transfer than hand-to-fomite and fomite-to-hand transfer. Hand-to-fomite-to-hand showed the least transfer. Uncertainty in risk models can best be reduced by studies on specific data on transfer from hard nonporous surfaces, since the greatest transfer for most 34 organisms occurs under these conditions. It is important that future studies on microbial transfer include data on the important environmental factors that influence transfer for better comparison among groups of organisms. The manuscript in Appendix B evaluates the fomite-to-finger percent transfer of Escherichia coli from fomites using two approaches: approach 1 defines transfer efficiency (TE) as colony forming units (CFU) recovered from finger relative to CFU recovered from control fomite, while approach 2 defines TE as the CFU recovered from the finger (recipient) relative to the sum of the CFU recovered from the finger (recipient) and the donor fomite. Fomite-to-finger transfer efficiency of microbial pathogens is important in modeling the potential for transmission. Consistent approaches for determining microbial transfer efficiencies are essential in producing accurate input values for risk assessment modeling. Five fomites of different compositions were studied representing nonporous surfaces. E. coli were placed on fomites in 10 µl drops and allowed to dry for 15 to 32 min under low (20% to 34%) relative humidity. Fomite-tofinger transfers were performed using a 1.0 kg/cm2 of pressure for 10 ± 2 s. Index, middle, and ring finger pads were sampled and E. coli was enumerated by the spread plate technique and transfer efficiencies were calculated using the two approaches. There were no statistical differences between transfer efficiencies determined by the two approaches (P = 0.05), demonstrating that the transfer efficiencies determined by each approach are comparable. 35 The manuscript in Appendix C examines the effect of low and high relative humidity on fomite-to-finger transfer efficiency of four model organisms from several common inanimate surfaces (fomites). The efficiency of transfer of a pathogen to the hand from the fomite is important in modeling the potential for transmission. Nine fomites of different compositions were studied representing porous and nonporous surfaces. Escherichia coli, Staphylococcus aureus, Bacillus thuringiensis, MS2 coliphage, and poliovirus 1 were placed on fomites in 10 µl drops and allowed to dry for 30 min under low (15% to 32%) or high (40% to 65%) relative humidity. Fomite-to-finger transfers were performed using a 1.0 kg/cm2 of pressure for 10 s. Index, middle, and ring finger pads were sampled and organisms were enumerated and transfer efficiencies were calculated. Transfer efficiencies were greater under high relative humidity for both porous and nonporous surfaces. Most organisms on average had greater transfer efficiencies under high relative humidity compared to low relative humidity. Nonporous surfaces had a greater transfer efficiency (up to 57%) than porous surfaces (< 6.8%) under low relative humidity, as well as under high relative humidity; nonporous (up to 79.5%) and porous (< 13.4%). Transfer efficiency also varied by fomite material and organism type. These results better qualify the transfer efficiencies of several different types of organisms under control conditions to provide data that could be used in quantitative microbial risk assessment (QMRA) models. The manuscript in Appendix D evaluates the survival of several surrogate pathogens under low and high relative humidity on various types of fomites to better understand 36 microorganism survival in modeling microbial exposure and risk assessment. Nine fomites of different compositions were studied representing porous and nonporous surfaces. Escherichia coli, Staphylococcus aureus, Bacillus thuringiensis, MS2 coliphage, and poliovirus 1 were placed on various fomites in 10 µl drops and allowed to dry for 30 min under low (15% to 32%) or high (40% to 65%) relative humidity. Relative humidity influenced organism survival on fomites; greater survival was observed under high relative humidity for E. coli, S. aureus, MS2, and poliovirus 1 . B. thuringiensis spores survived longer at low relative humidity. Fomite type did influence the survival of all model organisms; survival was greater on nonporous surfaces than those for porous surfaces. Organism survival was greatest with paper currency and least with cotton for most pathogens under both low and high relative humidity. Generally most organisms had greater survival under high relative humidity compared to low relative humidity with B. thuringiensis spores showing the opposite effect. Survival was less on porous surfaces compared to nonporous surfaces. The data obtained in our study can be used to better model survival parameters in microbial risk assessment models. The manuscript in Appendix E quantitatively assesses the percent microbial transfer to and from various surfaces and the influence of a disinfectant intervention to inhibit such transfers. Such data is needed for the development of quantitative microbial risk assessment models to assess the impact of interventions on reduction in the risk of infection. Escherichia coli, Staphylococcus aureus, Bacillus thuringiensis spores and poliovirus 1 were inoculated onto ceramic tile, laminate and granite in 10 µl drops and 37 allowed to dry for 30 min under relative humidity of 15% - 32%. Non-treated control fomites were sampled and remaining surfaces were wiped with a disinfectant wipe and allowed to dry for 10 min. Disinfectant wipe-treated control fomites were sampled and fomite-to-finger transfers were performed on the remaining surfaces using a 1.0 kg/cm2 pressure for 10 sec. The test organisms were reduced by 98.1% to 99.997% on the fomites after the surfaces were wiped. Microbial fomite-to-finger transfer from disinfectant wipe-treated surfaces were, lower (less than 0.5%) than from non-treated surfaces (up to 36.3%). This study demonstrated a disinfectant wipe intervention to be very effective in reducing bacterial contamination on several different types of fomites found in homes, office building, and healthcare facilities. These findings also showed that less fomite-to-finger transfer occurs when surfaces have been treated with disinfectant wipes compared to non-treated surfaces (fomites). The manuscript in Appendix F describes the QMRA modeling the probability of a C. jejuni infection from a cross-contamination to the surface and a subsequent fomite-tofinger transfer then a hand-to-mouth transfer occurring resulting in a dose. The risk of infection C. jejuni without and with the use of a disinfectant wipe is compared. A Monte Carlo simulation was used to compare the risk of transferring C. jejuni strain, from various surfaces to hands and subsequent transfer it to the mouth. Several assumptions were used as input parameters along with α and N50 values in the classical Beta-Poisson model to determine the risk of infection. The disinfectant-wipe intervention reduced the risk of Campylobacter infection, illness, and death by 2 to 3 orders on all fomites. 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Wells CL, Juni BA, Cameron SB, Mason KR, Dunn DL, Ferrieri P, Rhame FS. 1995. Stool Carriage, Clinical Isolation, and Mortality During an Outbreak of Vancomycin-Resistant Enterococci in Hospitalized Medical and/or Surgical Patients. Clin. Infect. Dis. 21:45-50. 58 142. Byers KE, Anglim AM, Anneski CJ, Germanson TP, Gold HS, Durbin LJ, Simonton BM, Farr BM. 2001. A Hospital Epidemic of Vancomycin-Resistant Enterococcus: Risk Factors and Control. Infect. Control Hosp. Epidemiol. 22:140-147. 143. Devine J, Cooke RPD, Wright EP. 2001. Is methicillin-resistant Staphylococcus aureus (MRSA) contamination of ward-based computer terminals a surrogate marker for nosocomial MRSA transmission and handwashing compliance? J. Hosp. Infect. 48:72-75. 59 APPENDIX A: MINIREVIEW: TRANSFER OF BACTERIA AND VIRUSES TO HANDS Running title: Transfer of Microorganisms to Hands Gerardo U. Lopez and Charles P. Gerba# Department of Soil, Water and Environmental Science, University of Arizona, Tucson, Arizona 85721 # Corresponding author. Tel: (520) 621-6910, Fax (520) 621-6366, E-mail: [email protected] A manuscript for Applied and Environmental Microbiology 60 ABSTRACT Fomites are known to play a role in the transmission of bacterial and viral pathogens. Advances in quantitative microbial risk assessment have allowed for the development of models, which can be used to determine the probability of infection via the hand and the likely success of interventions. Critical to these models is information on the effectiveness of organism transfer from fomites to hands and vice versa. Numerous factors influence the transfer efficiency of microorganisms, with environmental humidity being the most important factor with increased transfer. Other factors influencing transfer include drying time, contact time, pressure, friction type of material, and nature of the fomite. The greater the pressure applied to a surface and longer the contact time the greater the amount of transfer that can be expected. Much greater transfer occurs with hard surfaces (stainless steel) than porous surfaces (paper, cloth). Overall trends can be observed but transfer is organism dependent. Both Gram negative and positive bacteria have a greater transfer from hand-to-fomite than virus. Fomite-to-hand transfer is similar for both virus and bacteria. Hand-to-hand, hand-to-fomite-to-hand and the fomite-tohand-to-fomite transfer direction showed an overall lower transfer than hand-to-fomite and fomite-to-hand transfer. Hand-to-fomite-to-hand showed the least transfer. 61 INTRODUCTION It is widely accepted that environmental contamination plays an important role in the transmission of both respiratory and gastrointestinal infections in homes and in public settings (1). Fomites found in indoor environments are known reservoirs of microbial pathogens and a number of studies have shown human bodily fluids commonly contaminate fomites (2-5) becoming vectors for direct or indirect transfer of pathogens (6, 7). Fomites found in day care centers (4, 8-12), schools (13), office building (14), homes (15-22), public areas (23) and hospitals (24-30) have been identified as reservoirs and vectors for secondary modes of transmission. The presence and survival of pathogens on fomites found in indoor environments is an important factor in evaluating exposure potential and in the development of quantitative microbial risk assessment models to assess risk and the likely success of interventions (23, 31, 32). A number of studies have shown that nosocomial and foodborne pathogens can persist on inanimate surfaces for weeks or even months (26, 30, 33, 34). They can also remain viable up to several hours on fingers (35-37). Bacterial and viral persistence on fomites has been shown to be dependent on the numbers deposited, type of organism, and environmental conditions (26, 38, 39). Routes of transmission studies using different transfer scenarios; hand-to-fomite, fomiteto-hand, hand-to-hand, hand-to-fomite-to-hand, and fomite-to-hand-to-fomite have been conducted (Figure 1). The purpose of this review was to assess the importance of factors affecting the transfer of bacteria and viruses, and the degree of transfer, under different 62 environmental conditions. Such information is important to reduce the uncertainty in quantitative microbial risk assess models, which can be used to estimate the risk of infection from contact with contaminated surfaces and the impact of interventions (disinfectants, hand sanitizers, self-sanitizing surfaces) (40-42). FACTORS INFLUENCING TRANSFER Relative Humidity Few studies have reported the influence of relative humidity on microbial transfer. The recommended indoor relative humidity is 40 to 60% (43) although it may be as low as 10% during indoor heating and in arid regions (44, 45). Lopez et al., (submitted for publication) investigated the transfer efficiency of Escherichia coli, Staphylococcus aureus, Bacillus thuringiensis spores, coliphage MS2, and poliovirus type 1 from porous and nonporous fomites under high (40% to 65%) and low (15% to 32%) relative humidity. It was found that greater transfer typically occurs under high relative humidity (up to 79.5%) compared to low relative humidity (up to 57%) with a few exceptions. Poliovirus transfer however was not affected by relative humidity. Damp versus dry fomites or hands A number of studies have shown that moist, damp fomites or hands result in greater microbial transfer than dry surfaces. Reed (46) reported under dry conditions rhinovirus transfer rates were less efficient than under damp conditions (Figures 2, 3, and 5). Damp 63 mucus was more efficient in transfer than mucous dried on fingers. Marples and Tower (47) found 85% transfer of Staphylococcus saprophyticus from wet hands contaminated after firmly grasping a bottle with a rectangle kitchen cloth taped to it (Figure 2). The greater transfer efficiency was attributed to wet hands (47). Sattar et al. (48) found that moist poly cotton fabric and all-cotton fabric had greater S. aureus transfer than transfer from dry fabrics. Duration of drying time A number of studies have shown that drying time fomites or hands decreases microbial transfer. This most likely do to inactivation of the organisms during drying of the surface (14). Ansari et al. (35) found that longer drying times of rotavirus inoculum from 20 to 60 min on fingers or fomites resulted in lower transfer. Transfer of rotavirus from 20 and 60 min observed were (16.1% and 1.8% hand-to-fomite), (16.8% and 1.6% fomite-to-hand), and (6.6% and 2.8% hand-to-hand), respectively (Figures 2, 3, and 5) (35). Similar observations were observed by Marples and Towers (47) when fomite-to-hand transfers were conducted using 200 cm2 rectangle kitchen cloths taped to glass bottles and contaminated with S. saprophyticus and immediately grasped firmly by hands resulting in a 9.8% and 6.2% transfer efficiencies using the palm-index finger and thumb wash elution method, respectively (Figure 4). Compared to transfer efficiencies drying for 1.0 h showed 0.05% and 0.4% (Figure 4) (47). They also observed a decrease of S. saprophyticus transfer from contaminated fabric to hands and then to a second donor 64 fabric when hands were not allowed to dry compared to when they were air dried for 15 min. Scott and Bloomfield (49) found Staphylococcus aureus, Escherichia coli, Klebsiella aerogenes, and Salmonella spp. to be consistent with Ansari et al. (35) observations with a decrease in transfer from laminate to hands with a longer drying time from zero to 24 h (Figure 4). Similar results were observed with cloth to hands however at the 24 and 48 h the to numerous to count bacterial data suggested there was regrowth in the cleaning cloth (Figure 4). Mbithi et al. (36) performed a series of hepatitis A virus (HAV) transfer studies examining the effects of drying time from 20 to 240 min. Mbithi et al. (36) reported a decrease in HAV transfer after the longer drying time (Figures 2, 3, and 5). Paulson (50) reported on a series of transfer studies using feline calicivirus strain F9 as surrogate for noroviruses (Figure 2). Generally there was no difference in transfer between the 5 and 15 min drying with the spatula, fork, and the cutting board except for the doorknob where there was a greater transfer at 15 min than at 5 min (50). Type of fomite material Several studies have shown that fomite type influences microbial transfer. Scott and Bloomfield (49) found Staphylococcus aureus and Escherichia coli had a greater transfer from nonporous fomite laminate to hands than porous cloth to hands (Figure 4). Similar 65 results were reported by Rusin et al. (51) for PRD 1, Micrococcus luteus, Serratia rubidea observing transfer rates from hard, nonporous surfaces; faucet handles and phone receivers to hands, to be greater than from porous surfaces; dishcloth, sponge, and laundry (Figures 3 and 4). Despite the low percent transfer for the dishcloth and sponge they had the highest contamination of hands compared to the other surfaces because of the large numbers of bacteria on these fomites (51). Lopez et al., (submitted for publication) also observed that E. coli, S. aureus, B. thuringiensis, and MS2 had greater transfer efficiencies from ceramic tile, glass, granite and other nonporous fomites than from nonporous fomites; cotton, polyester, and paper currency (Figure 3 and 4). Transfer rates under high relative humidity for nonporous fomites were greater (30% to 79.5%) compared to porous fomites (0.3% to 2.3%) and under low relative humidity nonporous fomites were also greater (5.4% to 57.0%) compared to porous (0.03% to 0.4%) (Figures 3 and 4). Sattar et al. (48) found that 50% polyester 50% cotton had greater transfer of S. aureus to fingers than 100% cotton. These findings are similar to that observed with cotton, polyester, and paper currency (Figure 4) (Lopez et al., submitted for publication). Type of Organism A number of studies have shown that microorganism type does influence transfer. Ansari et al. (2) compared the transfer of rhinovirus 14, a non enveloped virus, and human 66 parainfluenza virus 3 (HPIV), an enveloped virus, and found HPIV 3 to transfer less than rhinovirus 14 who transferred (less than 1.0%) in hand-to-fomite and hand-to-hand transfers (Figures 2 and 5). However, in fomite-to-hand transfer direction HPIV 3 had a slightly greater transfer than rhinovirus 14 with 1.5% (Figure 3) (2). Rusin et al. (51) reported the transfer of Micrococcus luteus a Gram positive bacterium, Serratia rubidea a Gram negative bacterium, and PRD-1 coliphage from faucet handles and phone receivers. They found PRD-1 to have a transfer of (33.47% and 65.8%) (Figure 3), M. luteus to have a slightly greater transfer (40.03% and 41.81%) than S. rubidea (27.59% and 38.47%), respectively (Figure 4). All three organisms had (< 1.0%) transfer from porous objects to hands. In contrast M. luteus had the greatest transfer while PRD-1 and S. rubidea were similar (Figure 2) (51). These transfer differences are attributed to the ability of the virus to remain viable and tolerant of the environmental conditions and the interaction with hand characteristics. Mackintosh and Hoffman (52) observed a transfer difference between Staphylococcus saprophyticus, a Gram positive bacteria, that had a greater fomite-to-hand transfer (1.67%) than the Gram negative bacteria E. coli, Klebsiella aerogenes, Serratia marcescens, and Pseudomonas aeruginosa with (0.29% to 0.5%), however Streptococcus pyogens a Gram positive bacteria did not follow this observation (Figure 4). 67 Level of fomite contamination Pancic et al. (53) found that increasing the amount of mucous containing rhinovirus increased transfer from hand to fomite from 13.6% to 44.6% (Figure 2). However the opposite was seen from fomite-to-hand transfer with the lower inoculum showing a greater transfer (61.5%) (Figure 3) (53). Hand washing Marples & Tower (47) also tested the effects of various hand washing procedures on transfer of organisms from fabrics. They found 70% ethanol to reduce transfer the most compared to detergent and bar soap. Marples and Tower (47) also compared the effect of rubbing hands with alcohol-impregnated wipes and with 80% ethanol and found less transfer with hands rubbed with ethanol (21%). Mackintosh and Hoffman (52) also investigated the influence of hand washing methods and found both the use of 70% or 80% alcohol and soap and water were effective in reducing transfer. Julian et al. (54) found that hand washing prior to reduced transfer of the coliphages ΦX174, fr, and MS2. The reduction in coliphage transfer was greater for finger-to-glass than glass-to-finger transfer (Figure 2 and 3) (54). Rheinbaben et al. (37) reported ΦX174 fomite-to-hand transfer efficiencies of 0.000005% from washed hands and an average of 0.0004% for three individuals with unwashed hands after 6 h (Figure 3). The low transfer was due to the rapid inactivation of the virus. 68 In the finger-to-finger hand washing transfer a 16.7% was determined (Figure 5) (37). Virus re-isolated from the soap could have contributed to the transmission to recipient fingers (37). Direction of transfer Direction of transfer may influence transfer but appears species dependent. Mackintosh and Hoffman (52) observed a distinct difference in transfer of S. saprophyticus, S. pyogens E. coli, K. aerogenes, S. marcescens, and P. aeruginosa from fabric to fabric via the hands. Transfer stage fomite-to-hand (0.021% to 1.67%) (Figure 4), was 100 times less than hand-to-fomite (17% to 88%) (Figure 2), overall fomite-to-hand-to-fomite transfer was only (0.01% to 0.37%). The greater transfer rate in the hand to fomite is consistent with Marples and Tower (47) observations of 85% transfer efficiency of Staphylococcus saprophyticus contaminated hands to fabric taped to bottles (Figure 2). Both studies also observed very low fomite-to-hand-to-fomite transfer of less than 0.5%. Hubner et al. (55) demonstrated the transfer of E. coli by inoculating the index finger of a volunteer, air-drying it and then touched a sterilized paper, then a second volunteer’s index finger was moistened with saline to simulate licking the finger before turning pages and then pressed on the contaminated swatch to simulate cross-contamination. They found a 0.009% transfer rate (Figure 6) that is consistent with (Lopez et al., submitted for publication) found with paper currency transfers to fingers (Table 3). 69 A transfer rate of 0.03% for M. luteus and 0.04% for S. marcescens was observed from contaminated hands to paper-towel dispenser, while greater transfer rates were observed from contaminated dispenser to hands for two separate towels for M. luteus (13.1% and 6.0%, respectively) and for S. marcescens (12.4% and 6.7%, respectively) (Figure 2 and 4) (56). In contrast Pancic et al. (53) observed similar rhinovirus transfer from both hand-tofomite and fomite-to-hand transfer directions. The hand-to-fomite-to-hand transfer directions for rhinovirus did show greater transfer than observed in the fomite-to-hand-tofomite by Mackintosh and Hoffman (52) and Marples and Tower (47). The degree of virus transfer is dependent on virus type. Rhinovirus 14 was found to have similar transfer regardless of direction (0.67% to 0.92%) while human parainfluenza virus 3 (HPIV) showed very different transfer rates depending on the direction of transfer, hand-to-fomite and hand-to-hand (2) (Figure 2, 3, and 5). Different transfer rates for ΦX174, fr, and MS2 coliphage are influenced by direction of transfer and is specific to viral species (54). All three types of coliphage had similar transfer rates from finger-tofomite (15% to 28%) and ΦX174 and MS2 coliphage had comparable transfers from fomite-to-finger as observed from hand-to-fomite (Figure 2 and 3). However fr coliphage had greater transfer (37% to 39%) from fomite-to-finger. Coliphage ΦX174 could be horizontally transferred to 14 consecutive individuals with an average percent transfer of 0.04% after touching a contaminated door handle allowed to 70 initially dry for 15 min and with 15 s intervals between each consecutive contact (37). In the finger-to-finger transfer experiments they reported an average of 0.004% and 0.03% for the horizontal (14 consecutive fingers) and vertical (finger-to-finger-to-finger) transfers, respectively (Figure 5) (37). Even though the transfer efficiencies for the horizontal and vertical transfers were < 1.0% and generally decreased after each contact, a 2 to 3 log transfer of ΦX174 resulted (37). In contrast Pancic et al. (53) observed greater rhinovirus hand-to-hand transfer (5.9%) (Figure 5) than observed by Rheinbaben et al. (37) for ΦX174. Friction and pressure Contact time, friction, and pressure have been shown to influence transfer efficiency. Transfer of S. aureus contaminated fabric to fingers is greater with friction than without friction independent if the fabric was moist or dry (48). Friction increased the level of transfer by two- to five fold from both types of fabric with a transfer range for the dry fingerpad without friction from 0.01% to < 0.2%, and with friction 0.01% to 1%, while the moistened fingerpad without friction 0.01% to < 2.%, and with friction 0.01% to < 3%. Friction also increases the transfer of viruses (36). The mean HAV transfer was almost 3 times greater when friction was applied (36). The amount transferred to fingers increased almost 5 times when the finger pressure was increased from 0.2 kg/cm2 to 1.0 kg/cm2 (36). 71 DISCUSSION Factors that influence transfer are not independent of each other, but rather interact with each other to either promote or reduce overall transfer. Microbial transfer is influenced by environmental factors such as relative humidity, moisture, drying time, fomite material, direction of microbial transfer as well as biological factors such as bacterial or virus species, and the characteristics of the fingers washed or unwashed hands (Table 1). Usually higher relative humidity conditions reduce the desiccation of the microbial suspension and as a result greater bacterial and viral transfer occurs. Lower relative humidity conditions allow the microbial suspension to dry faster and promote bacterial and viral inactivation resulting in lower transfer of viable microorganisms. Longer drying times have been shown to reduce transfer rates by promoting the desiccation of the suspension medium resulting in microbial inactivation. Moisture found on fomites or hands has been shown to increase transfer rates. Greater microbial contamination levels found on donor or recipient fomites increases transfer. Friction and pressure have also been shown to increase transfer rates of microbial pathogens. Washing hands has been shown to reduce transfer compared to unwashed hands. Relative humidity, moisture, level of contamination, friction, pressure, and drying time interact with each other and in combination along with hand characteristics pH and washed or unwashed hands influence microbial transfer rates. 72 Type of fomite material has been shown to have a significant impact on transfer rates. Transfer rates between nonporous fomites have been shown to be much greater than from porous fomites. Moisture in the form of microbial suspension is able to persist longer on nonporous fomites compared to porous fomites that can absorb the suspension and provide crevices for bacteria and viruses to attach to and reduce transfer. Direction of transfer has also been indicated to influence transfer but is species dependent. Bacterial and viral species have been shown to have different transfer rates due to the unique interaction that a species has with the suspension medium, fomite, and environmental conditions. In general Gram negative bacteria seem to be transferred less readily than Gram positive bacteria. Coliphage are generally transferred at similar rates, however they seem to be dependent on the direction of the transfer, fomite-to-hand or hand-to-fomite. Enteric viruses usually are more readily transferred than respiratory viruses with direction playing a major role. There is plenty of evidence to suggest that the environment could contribute significantly to contamination of healthcare workers hands, child care givers, and foodhandlers (57). The long-term survival of viruses and bacteria on common surfaces found in many environments, in addition to the laboratory, emphasizes the possible role of nonporous and porous fomites in the transmission of viruses and bacteria (1, 34, 58). These studies suggest that the transmission of virus from donors who are shedding large amounts of 73 virus through coughing, sneezing, or nasal mucosa can cross-contaminate surrounding surfaces (58). CONCLUSIONS Transfer of pathogens in the indoor environment is complex being dependent on a number of factors. Transfer is very organism dependent, with relative humidity and type of fomite having the greatest influence on transfer. Obviously hard surfaces will present a greater risk than porous surfaces. While general assumptions can be made on the degree of transfer for bacteria and viruses, it is organism dependent. Uncertainty in risk models can best be reduced by studies on specific data on transfer from hard nonporous surfaces, since the greatest transfer for most organisms occur under these conditions. It is important that future studies on microbial transfer include data on the important environmental factors that influence transfer for better comparison among groups of organisms. The lack of a standard transfer method along with the different factors influencing transfer makes evaluating transfer rates difficult. Future transfer studies should standardize key factors such as pressure, moisture, contact time, and drying time. Bacterial hand-to-hand transfer studies are still lacking to better understand the transmission between individuals. There is also a need for hand-to-fomite-to-hand bacterial and viral studies as well as fomite-to-hand-to-fomite viral studies. 74 ACKNOWLEDGEMENTS This research was supported by the Center for Advancing Microbial Risk Assessment, funded by the U.S. Environmental Protection Agency Science to Achieve Results (STAR) program. And the U.S. Department of Homeland Security University Program Grant R83236301. 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Dis. 146:4751. 83 FIGURES Hand Fomite Hand Fomite Portal Entry FIG 1 Transfer direction; hand-to-fomite, fomite-to-hand, hand-to-fomite-to-hand, fomite-to-hand-to-fomite. 84 100 90 Transfer efficiency (%) 80 70 60 50 40 30 20 10 0 Nonporous Nonporous Nonporous Rhinovirus Human Parainfluenza virus-3 Rotavirus Respiratory Viruses Nonporous Hepa s A Virus Enteric Viruses Lips - skin Nonporous Nonporous Nonporous Porous PRD-1 Φ-X174 fr MS2 Staphylococcus saprophy cus Coliphage Virus FIG 2 Hand-to-fomite bacterial and viral transfer efficiency Lips - skin Nonporous Micrococcus luteus Porous Porous Porous Nonporous Lips - skin Porous Escherichia coli Klebsiella aerogenes Psuedomonas aeruginosa Enterobacter aerogenes Serra a rubidea Serra a marcescens Gram (+) bacteria Gram (-) bacteria Bacteria 85 100 90 Transfer efficiency (%) 80 70 60 50 40 30 20 10 0 Nonporous Nonporous Nonporous Rhinovirus Human Para Influenza Rotavirus Respiratory Viruses Nonporous Hepa s A Virus Nonporous Nonporous Feline Calicivirus Poliovirus type 1 Nonporous Enteric Viruses Nonporous Nonporous ΦX174 fr Coliphage Virus FIG 3 Fomite-to-hand viral transfer efficiency Porous PRD-1 Nonporous Porous MS2 86 90 80 Transfer efficiency 70 60 50 40 30 20 10 0 Porous Porous Staphylococcus saprophy cus Streptococcus pyogenes Nonporous Porous Staphylococcus aureus Nonporous Porous Micrococcus luteus Nonporous Porous Escherichia coli Porous Porous Klebsiella aerogenes Serra a marcescens Gram (+) bacteria Porous Serra a rubidea Gram (-) bacteria Bacteria FIG 4 Fomite-to-hand bacterial transfer efficiency Nonporous Porous Nonporous Nonporous Psuedomonas aeruginosa Enterobacter aerogenes Salmonella spp. Nonporous Porous Bacillus thuringiensis Endospores 87 Transfer efficiency (%) 40 35 30 25 20 15 10 5 0 Finger Finger Finger Rhinovirus Human Parainfluenza virus-3 Rotavirus Respiratory Viruses Hepa Enteric Viruses Virus FIG 5 Hand-to-hand viral transfer efficiency Finger s A Virus Finger ΦX174 Coliphage 88 TABLE TABLE 1 Factors influencing transfer Factor Remarks Relative Humidity Usually greater transfer at high relative humidity Drying time Greater transfer with short drying time Contact time Greater transfer with longer contact time Pressure Greater transfer with greater contact pressure Friction Greater transfer with friction Moisture Greater transfer with moister than under dry conditions Contamination level Greater contamination level the greater the transfer Transfer direction Usually greater transfer from fomite-tohand than hand-to-fomite but is species dependent Type of fomite material Greater transfer with nonporous than with porous surfaces Washed vs unwashed hands Usually greater transfer with unwashed hand than with washed hands Finger vs hand Similar transfer in relation to surface area Bacteria, virues, and ensospores Generally, Gram positive bacteria transfer more readily, then viruses, Gram negative bacteria and then endospores Gram negative, Gram positive, and endosprores Generally, Gram positive bacteria transfer more readily than Gram negative bacteria and endospores Enteric and respiratory viruses Enteric viruses usually transfer more readily than respiratory viruses Coliphage transfer is similar among different types but is dependent on direction Coliphage 89 APPENDIX B: COMPARISON OF TWO APPROACHES IN DETERMINING TRANSFER EFFICIENCY OF ESCHERICHIA COLI FROM NONPOROUS FOMITES TO FINGERS Running title: Determining Bacterial Percent Transfer Gerardo U. Lopez1, Masaaki Kitajima1, Kelly A. Reynolds2, and Charles P. Gerba1 # 1 Department of Soil, Water and Environmental Science, The University of Arizona, Tucson, Arizona 85721 USA; 2The University of Arizona, Mel and Enid Zuckerman College of Public Health, 1295 N. Martin Ave., Tucson, Arizona 85724, USA # Corresponding author. Tel: (520) 621-6910, Fax (520) 621-6366, E-mail: [email protected] A manuscript for Applied and Environmental Microbiology 90 ABSTRACT Fomites were inoculated with Escherichia coli and fomite-to-finger transfer efficiency was evaluated using two approaches: one determines the efficiency based on control fomite; the other is based on the sum of bacteria recovered from a finger and donor fomite. No statistical difference was observed between the two approaches. TEXT Inanimate surfaces (fomites) are reservoirs of microbial pathogens and play a critical role in fomite-hand routes of transmission (1-4). Several studies have demonstrated that fomites found in indoor environments such as day care centers and schools (5-11), homes (12-19), public areas (20, 21) and hospitals (22-27) have been contaminated with microbes and human bodily fluids (6-8, 13, 20, 21, 28-30) that persist on fomites for hours to months (23, 31, 32) and contribute to secondary modes of transmission. Fomite-to-finger transfer efficiency of microbial pathogens is important in modeling the potential for transmission (1, 33-35). Consistent approaches for determining microbial transfer efficiencies are essential in producing accurate exposure input values for risk assessment modeling. In the present study, we compared the following two approaches in determining fomite-to-finger transfer efficiency: approach 1 defines transfer efficiency (TE) as colony forming units (CFU) recovered from finger relative to CFU recovered from control fomite, while approach 2 defines TE as the CFU recovered from the finger 91 (recipient) relative to the sum of the CFU recovered from the finger (recipient) and the donor fomite. One subject conducted the fomite-to-finger transfer experiments. Project approval obtained from the University of Arizona’s Office for Human Subjects Research. Subject’s hands were washed, rinsed and sprayed with 70% ethanol. Study organism, Escherichia coli C-3000 (ATCC 15597) obtained from the American Type Culture Collection (ATCC Washington DC), was grown as previously described (36). Temperature (19°C to 25°C) and relative humidity (20% to 34%) were monitored with a High Accuracy Thermo – Hygro (VWR, Mississauga, ON). Five nonporous fomite materials were tested with surface areas of 16 to 25 cm2; acrylic (poly-methyl methacrylate) matte non-glare finish, ceramic tile porcelain (Home Depot, Atlanta, GA), glass slides (VWR, Mississauga, ON), laminate various colors (Wilsonart International, Temple, TX), and stainless steel 304 (AK Steel Corporation, West Chester, OH). Acrylic was radiated under UV light for 30 min on each side, while the other fomites were sprayed with 70% ethanol, washed, and autoclaved. Baseline control, transfer fomite, and negative control swatch/coupons were placed on a laboratory bench. A protocol adapted from previous publications (37, 38) was used. Briefly, approximately 107 to 109 CFU/cm2 of E. coli in 10 µl droplets were spread over approximately a 1.0 cm2 area on the center of each fomite using the tip of the pipet and allowed to air-dry (15 to 32 min) on the laboratory bench. Fomites were sampled using a cotton-tipped swab 92 applicator (Puritan Medical Products Company, Guilford, ME). Swabs were wet in 1.0 ml of phosphate buffer saline (PBS) (PBS, SIGMA, St. Louis, MO) and a 6.0 cm2 area was swabbed with applicator pressed firmly against the surface before being placed back into the remaining PBS and vortexed. To assess fomite-to-finger transfer, the fomite was placed at the center of a scale with a digital readout and a finger transfer was performed by placing the right hand index finger on the center covering the inoculated area of the fomite for 10 ± 2 s with a 1.0 kg/cm2 (98.0665 kPa) of average pressure (range of 700 g – 1500 g) (FIG S1) (37, 38). Using a cotton-tipped swab applicator (Puritan Medical Products Company, Guilford, ME), moistened in 1.0 ml of PBS, the index, middle, and ring finger pads were sampled and placed in the PBS vial and vortexed directly after each transfer event. E. coli was enumerated by the spread plate technique using MacConkey agar (EMD Chemicals Inc., Gibbstown, NJ). Transfer efficiency was calculated using two approaches; equations to calculate TE (%) by approaches 1 and 2 can be expressed with the following equations (1) (39, 40) and (2) (36, 41, 42), respectively. % 100 (1) % 100 (2) 93 To assess the statistical significance of untransformed percent transfer efficiencies between the two approaches, a Student’s t-test (two-tailed) was conducted using Microsoft Excel 2011 and the software package StatPlus: mac, 2009, (AnalystSoft). Transfer efficiency for E. coli was determined for a total of 72 fomite-to-finger transfer events. Transfer efficiencies were slightly greater with approach 1 (6.3% to 27.3%) than with approach 2 (3.8% to 20.7%) (Table 1). E. coli recovered on fingers and fomites are in (Table 1S) and (FIG S2) provides transfer efficiency in log10 scale and geometric mean in order to give the range and variability of the data. The greatest transfer efficiency was obtained with glass (27.3%) with approach 1, while glass and acrylic (20.7%) with approach 2. Laminate showed the least transfer efficiency for approach 1 and 2 (6.3% and 3.8%, respectively). There were no statistical differences between transfer efficiencies determined by the two approaches (P = 0.05), demonstrating that the transfer efficiencies determined by each approach are comparable (Table 1). Approach 1 has been previously used to investigate the transfer of Staphylococcus saprophyticus from fabric to hands (39) and to examine the transfer of feline calicivirus from various fomites to foodhandler gloves (40). Variations of approach 1 have been previously used to evaluate the transmission of microbial pathogens immediately following inoculation of fomites (42-46). A limitation to approach 1 is that transfer efficiencies greater than 100% can result and would need to be truncated to 100% as done 94 with glass and ceramic tile (Table 1) in order to prevent skewing the data. Approach 2 has also been used by several studies for determining transfer efficiency of both bacteria and viruses from fomites to fingers (36-38, 41, 47). To the best of our knowledge, this is the first study directly comparing the two approaches that are widely used to calculate microbial transfer efficiencies and demonstrating that the efficiencies determined by the two approaches are comparable showing no statistical difference. The data obtained in the present study can be used as input values for E. coli transfer efficiency parameters in quantitative microbial risk assessment (QMRA) to assess risk of infection associated with fomite-to-finger transmissions and relative exposure from different types of nonporous fomites. ACKNOWLEDGEMENTS This research was supported by the Center for Advancing Microbial Risk Assessment, funded by the U.S. Environmental Protection Agency Science to Achieve Results (STAR) program. And the U.S. Department of Homeland Security University Program Grant R83236301. 95 REFERENCES 1. Haas CN, Rose JB, Gerba CP. 1999. Quantitative microbial risk assessment. John Wiley, New York. 2. Hall CB, Douglas JRG. 1981. Modes of transmission of respiratory syncytial virus. J. Pediatr. 99:100-103. 3. Hendley JO, Wenzel RP, Gwaltney JM. 1973. Transmission of Rhinovirus Colds by Self-Inoculation. N. Engl. J. Med. 288:1361-1364. 4. Nicas M, Sun G. 2006. An Integrated Model of Infection Risk in a Health-Care Environment. Risk Anal. 26:1085-1096. 5. Barker J, Stevens D, Bloomfield SF. 2001. Spread and prevention of some common viral infections in community facilities and domestic homes. J. Appl. Microbiol. 91:7-21. 6. Boone SA, Gerba CP. 2005. The occurrence of influenza A virus on household and day care center fomites. J. Infect. 51:103-109. 7. Bright KR, Boone SA, Gerba CP. 2010. Ocurrence of Bacteria and Viruses on Elementary Classroom Surfaces and the Potential Role of Classroom Hygiene in the Spread of Infectious Diseases. J. Sch. Nurs. 26:33-41. 8. Butz AM, Fosarelli P, Dick J, Cusack T, Yolken R. 1993. Prevalence of Rotavirus on High-Risk Fomites in Day-Care Facilities. Pediatrics 92:202-205. 9. Keswick BH, Pickering LK, DuPont HL, Woodward WE. 1983. Prevalence of rotavirus in children in day care centers. J. Pediatr. 103:85-86. 96 10. Keswick BH, Pickering LK, DuPont HL, Woodward WE. 1983. Survival and detection of rotaviruses on environmental surfaces in day care centers. Appl. Environ. Microbiol. 46:813-816. 11. Laborde DJ, Weigle KA, Weber DJ, Kotch JB. 1993. Effect of Fecal Contamination on Diarrheal Illness Rates in Day-Care Centers. Am. J. Epidemiol. 138:243-255. 12. 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Microbiol. 29:2115-2119. 102 TABLE TABLE 1: E. coli fomite-to-finger transfer efficiency Approach 1a Approach 2b Transfer efficiency (%) Transfer efficiency (%) (n) Average ± SD (Range) (n) Average ± SD (Range) Acrylic (15) 22.2 ± 21.1 (1.8 - 74.8) (15) 20.7 ± 24.4 (1.4 - 93.9) 0.856 Glass (15) 27.3 ± 32.3 (3.6 - 100)d (15) 20.7 ± 22.6 (< 70.6)c 0.526 Ceramic Tile (21) 13.3 ± 28.3 (1.3 - 100)d (21) 7.8 ± 8.4 (0.5 - 29.8) 0.405 Laminate (15) 6.3 ± 4.8 (1.5 - 18.0) (15) 3.8 ± 3.0 (0.8 - 9.6) 0.096 Stainless Steel (6) 17.1 ± 15.2 (0.2 - 37.4) (6) 16.3 ± 16.0 (0.5 - 44.9) 0.928 Surface Type a Transfer efficiency (%) = (CFU finger/CFU baseline control fomite) X 100 using untransformed data b Transfer efficiency (%) = (CFU finger/CFU finger + CFU donor fomite) X 100 using untransformed data c E. coli recovery from fomite or finger were below the detectible limit of 10 CFU/2-cm2 indicated by < d Transfer event was > 100% and was truncated to 100% Student t-test P-value 103 APPENDIX C: TRANSFER EFFICIENCY OF BACTERIA AND VIRUSES FROM POROUS AND NONPOROUS FOMITES TO FINGERS UNDER DIFFERENT RELATIVE HUMIDITY Running title: Fomite to Finger Microbial Transfer Gerardo U. Lopez1, 3, Charles P. Gerba1, 3#, Akrum Tamimi1, Masaaki Kitajima1, Sheri Maxwell1, and Joan B. Rose2, 3 1 Department of Soil, Water and Environmental Science, The University of Arizona, Tucson, Arizona 85721 USA; 2Department of Fisheries and Wildlife, and 3Center for Advancing Microbial Risk Assessment, Michigan State University, East Lansing, Michigan # Corresponding author. Tel: (520) 621-6906, Fax (520) 621-6366, E-mail: [email protected] Manuscript for Applied and Environmental Microbiology 104 ABSTRACT Fomites can serve as routes of transmission for both enteric and respiratory pathogens. The present study examined the effect of low and high relative humidity on fomite-tofinger transfer efficiency of four model organisms from several common inanimate surfaces (fomites). Nine fomites of different compositions were studied representing porous and nonporous surfaces. Escherichia coli, Staphylococcus aureus, Bacillus thuringiensis, MS2 coliphage, and poliovirus 1 were placed on fomites in 10 µl drops allowed to dry for 30 min under low (15% to 32%) or high (40% to 65%) relative humidity. Fomite-to-finger transfers were performed using 1.0 kg/cm2 of pressure for 10 s. Transfer efficiencies were greater under high relative humidity for both porous and nonporous surfaces. Most organisms on average had greater transfer efficiencies under high relative humidity compared to low relative humidity. Nonporous surfaces had a greater transfer efficiency (up to 57%) than porous surfaces (< 6.8%) under low relative humidity, as well as under high relative humidity; nonporous (up to 79.5%) and porous (< 13.4%). Transfer efficiency also varied with fomite material and organism type. The data generated can be used in quantitative microbial risk assessment models to assess risk of infection from fomite transmitted pathogens to humans and relative exposure from different types of fomites and microorganisms. 105 INTRODUCTION Inanimate objects, or fomites, are a potential reservoir in the transmission of pathogens either directly, by surface-to-mouth contact, or indirectly, by contamination of fingers and subsequent hand-to-mouth, hand-to-eye, or hand-to-nose contact (1-5). Bodily fluids such as saliva, mucus, nasal secretions, blood, urine, and feces may all potentially contain pathogens that can be transmitted via fomites (6-9). A number of studies have shown that enteric and respiratory pathogens are capable of surviving from hours to months on fomites, depending on the numbers deposited, the type of microorganism, and the variable environmental conditions (10-12). Several studies have shown that inanimate surfaces found in day care centers (8, 13-17), schools (18), office buildings (19), homes (20-27), public areas (28), or hospitals (12, 29-33) can be reservoirs for secondary modes of transmission with contaminated hands playing a critical role as a route of exposure. The efficiency of transfer of a pathogen to the hand from the fomite is important in modeling the potential for its transmission (11, 34-36). This information can be used to understand the spread of disease in indoor environments, and the potential for designing surfaces that reduce transfer efficiency and /or are antimicrobial (5). The purpose of this work was to better elucidate the transfer efficiencies of several different types of organisms under control conditions to provide data that may be used in quantitative microbial risk assessment (QMRA) models. 106 MATERIALS AND METHODS Subjects A single subject conducted the fomite-to-finger transfer experiments. Permission was obtained from the University of Arizona’s Office for Human Subjects Research prior to the study. Bacteria, Virus and preparation of inocula Study organisms. Escherichia coli (ATCC 15597), Staphylococcus aureus (ATCC 25923), Bacillus thuringiensis (ATCC 10792), and coliphage MS2 (ATCC 15597-B1) were obtained from the American Type Culture Collection (ATCC Manassas, VA). Poliovirus 1 (PV-1; strain LSc-2ab) was obtained from the Department of Virology and Epidemiology at the Baylor College of Medicine (Houston, TX). These organisms were selected as model organisms for pathogenic Gram negative and Gram positive bacteria, spore-forming bacteria, and viruses. Gram negative and Gram positive bacteria inoculum preparation. Frozen aliquots of E. coli and S. aureus were transferred into separate flasks containing100 to 150 ml of Tryptic Soy Broth, (TSB, EMD, Gibbstown, NJ) incubated for 18 ± 2 h at 37°C on an orbital shaker (150 to 180 rpm), and streaked for isolation onto Tryptic Soy Agar plates (TSA, EMD Gibbstown, NJ). The bacteria were then subcultured in a flask of TSB and incubated for 18 ± 2 h at 37°C on an orbital shaker (150 to 180 rpm) (37). 107 Endospore-forming bacteria inoculum preparation. B. thuringiensis spores were prepared as previously described with minor modifications (38). Briefly, spores were suspended in Difco sporulation media with supplements (DSM + S; Becton, Dickinson and Company, Sparks, MD), cultivated for 24 h at 37°C on an orbital shaker (150 to 180 rpm), and resuspended in fresh DSM + S to obtain a final OD600 of 0.1 (Spectronic Genesys 5, Milton Roy, Ontario Canada). Virus inoculum preparation. MS2 coliphage were prepared as previously described with minor modifications (37). Briefly, 0.1 ml of phage suspension and 0.5 ml of a log phase E. coli 15597 (host bacterium) culture 0.5 ml were added to top agar, melted, and maintained at 48°C. The inoculated top agar was mixed and poured over the TSA. The solidified agar overlay plates were then inverted and incubated at 37°C for 24 h. TSB was then added to each plate and maintained at room temperature for 2 h. The TSB eluent was aspirated and centrifuged at (1,989 X g for 10 min), after which, the supernatant was filtered through 0.22 µm pore size Acrodisc® Syringe Filters (Pall Corporation, Ann Arbor, MI) pre-moistened with 3% beef extract (Bacto™, Becton, Dickinson and Company, Sparks, MD), the virus tittered, and stored at 4°C. PV-1 propagation and plaque-forming assays were conducted as described previously (39, 40). Briefly, PV-1 was propagated on buffalo green monkey kidney (BGM; ATCC CCL81; American Type Culture Collection, Manassas, VA) cell line monolayers with minimal essential media (MEM) containing 5% calf serum (HyClone Laboratories, 108 Logan, UT) at an incubation temperature of 37°C with 5% CO2. Plaque-forming assays were performed using six-well plates with confluent monolayers of the BGM cells. Control wash and disinfection Prior to all experiments, the subject’s hands were washed with warm water and nonantibacterial soap (Liquid Joy; Procter and Gamble, Cincinnati, OH) for 45 s, rinsed with water, and dried with paper towels. Each hand was then sprayed twice with 70% ethanol, rubbing the alcohol thoroughly over the hands and wrists for 15 s, and subsequently airdried. After conducting fomite-to-finger transfer experiments with the prepared inocula, fingers were disinfected twice with 70% ethanol, wrapped with a 70% ethanol saturated paper towel for 30 s, then washed and rinsed using warm water and Softsoap® antibacterial liquid hand soap (Colgate-Palmolive, Morristown, NJ) for 45 s, and then dried with paper towels. After sampling fingers for B. thuringiensis spores and PV-1, fingers were placed in 10% bleach (The Clorox Company, Oakland, CA) for 15 s and then neutralized with 10% sodium thiosulfate (EMD, Gibbstown, NJ). The hands were then washed as described above to prepare for subsequent trials. Relative Humidity Conditions and Temperature The study consisted of two relative humidity conditions - high (40% to 65%) and low (15% to 32%) relative humidity. To achieve both humidity conditions, two separate incubators were turned off and used. Incubator temperatures thus reflected room temperature ranges of 19°C to 25°C. The temperature and relative humidity were 109 monitored with a High Accuracy Thermo – Hygro (VWR, Mississauga, ON). During days with higher ambient relative humidity in the laboratory, t.h.e.® Desiccant (EMD, Gibbstown, NJ) and Drierite desiccant (Drierite, Xenia, OH) were utilized in the incubator to decrease the relative humidity to the low relative humidity range (15% to 32%). During laboratory ambient lower relative humidity conditions a BIONAIRE® humidifier (Milford, MA) was used in the specific incubator to increase the relative humidity to the high relative humidity range (40% to 65%). Fomites tested Nine different types of fomite materials were tested ranging in surface areas of 16 to 25 cm2 six nonporous and three porous (Table 1). With the exception of acrylic, all fomites were sterilized by autoclave whereas acrylic was radiated under UV light (254 nm) for 30 min on each side. After fomite-to-finger transfers with E. coli, S. aureus, and MS2, nonporous fomites were sprayed three times with 70% ethanol and allowed to dry for 10 min. Nonporous fomites used in the finger transfers with B. thuringiensis spores and PV1 were disinfected with 10% bleach (The Clorox Company, Oakland, CA), then allowed to sit for 10 min, and subsequently neutralized in 10% sodium thiosulfate (EMD, Gibbstown, NJ). Fomites were then washed under warm running water with nonantibacterial soap (Liquid Joy, Procter and Gamble, Cincinnati, OH), rubbed with a wet paper towel on the surface area of inoculation, rinsed thoroughly with RO treated water, air-dried, and autoclaved. Cotton and polyester fomites were discarded after use. Paper currency was autoclaved and reused. 110 Inoculation of fomites Layout of fomites. For each of the six nonporous and three porous fomites, seven swatches/coupons were evenly spaced on the middle shelf of an incubator. Each trial consisted of three control swatches/coupons, three fomite-to-finger transfer swatches/coupons, and one negative control swatch/coupon to ensure that the fomites were not previously contaminated. Organism concentration. The concentration of organisms added to the fomites was approximately 107 to 108 colony forming units (CFU)/cm2 of E. coli in (TSB, EMD, Gibbstown, NJ), 108 to 109 CFU/cm2 of S. aureus in TSB, 106 to 107 CFU/cm2 of B. thuringiensis spores in (DSM + S; Becton, Dickinson and Company, Sparks, MD), 109 to 1011 plaque forming units (PFU)/cm2 of MS2 in TSB, and 108 PFU/cm2 of PV-1 in (PBS, Sigma-Aldrich, St. Louis, MO) in 10 µl droplets. Using a pipet tip, the 10 µl inoculum droplets were spread over approximately a 1.0 cm2 area on the center of each fomite. The paper currency were divided into four 24 cm2 sections; two one dollar bills were used to make the seven swatches. With paper money for each set of transfer experiments, a new 1.0 cm2 area on each of the 24 cm2 sections were inoculated. Using an ink marker, an identifying spot was placed near the inoculated area on cotton, polyester, and paper money due to the absorbance. The fomites were allowed to dry for 30 min. 111 Fomite-to-finger transfer, sampling, and assays Fomite sampling. Fomites were sampled using a cotton-tipped swab applicator (Puritan Medical Products Company, Guilford, ME), when inoculated with E. coli, S. aureus, B. thuringiensis, and MS2. In the case of PV-1, a polyester fiber-tipped applicator swab (Falcon, Becton Dickinson and Company, Cockeysville, MD) was used. Swabs were wet in 1.0 ml of phosphate buffer saline (PBS, Sigma-Aldrich, St. Louis, MO), and then a 6.0 cm2 area on the fomite was swabbed using firm sweeping and rotating motions. The swab was then placed back into the remaining PBS and vortexed for 5 s. Transfer experiment. One transfer trial consisted of three separate fomite-to-finger transfer events using the index, middle, and ring fingers of the right hand for each surface type. Two transfer trials were conducted, resulting in six transfers total for each fomite under both low and high relative humidity. A protocol adapted from Ansari et al. (41) and Mbithi et al. (42) was used to perform the fomite-to-finger transfer of test organisms from the previously mentioned nonporous and porous fomites to hands after a 30 min drying time. To assess transfer, the fomite was placed at the center of a scale with a digital readout, and a finger transfer was performed by placing the right hand finger on the center, covering the inoculated area of the fomite, for 10 s with 1.0 kg/cm2 (98.0665 kPa) of average pressure (range of 700 g – 1500 g) (41, 42). Sampling the fingers. Using a cotton-tipped swab applicator (Puritan Medical Products Company, Guilford, ME) moistened in 1.0 ml of PBS (PBS, SIGMA, St. Louis, MO), the 112 index, middle, and ring finger pads were sampled using a sweeping, rotating motion and placed in the PBS vial and vortexed directly after each transfer event. A polyester-tipped swab (Puritan Medical Products Company, Guilford, ME) was used to sample PV-1. Organism assays. E. coli, S. aureus, and B. thuringiensis spores were enumerated using the spread plate technique on MacConkey (EMD Chemicals Inc, Gibbstown, NJ), Mannitol salt agar (MSA, EMD Chemicals Inc, Gibbstown, NJ), and TSA (EMD Chemicals Inc., Gibbstown, NJ) agar plates, respectively. The plates were incubated at 37°C for 18 ± 2 h. B. thuringiensis spore samples were heat shocked at 81 ± 2°C for 10 min prior to spread plating, to stimulate germination. The MS2 plaque assay was conducted using the double agar overlay method using TSA (EMD Chemicals Inc., Gibbstown, NJ) (39, 43). PV-1 titrations were performed using 10-fold serial dilution plaque-forming assays as described previously (39, 40). All dilutions were assayed in duplicate. Transfer efficiency calculation and statistical analyses Calculation of transfer efficiency. Bacterial colonies and viral plaques were enumerated and the transfer efficiencies were calculated using Equation (1) below (44, 45). The transfer efficiency (TE) is defined as the number of CFU or PFU recovered from finger relative to the CFU or PFU recovered from the control fomite. If bacteria or phage were not recovered from the finger pad, the lower detection limit of 10 CFU or PFU was used as an estimate for the microorganism recovered as previously described (46, 47). A less 113 than value (<) is used to indicate that the transfer efficiency was lower than the least detectable limit. Finger transfers greater than 100% were truncated to 100%. % 100 (1) Statistical analyses. Data were entered in Microsoft Excel 2010 and the software package StatPlus:mac, 2009, (AnalystSoft) to compute the descriptive statistic measures of mean percent transfer efficiency, the standard deviation, and statistical significance. To assess the statistical significance at the (P = 0.05) level, the percent transfer efficiencies between low and high relative humidity and nonporous and porous fomites were compared in a Student’s t-test. RESULTS Influence of relative humidity on microbial transfer The percent transfer efficiency was determined for 468 finger transfers; 234 transfer events were performed for both low and high relative humidity conditions. Tables 2, 3, and 4 summarize the fomite to finger percent transfer efficiency results. Relative humidity influenced the transfer rate of pathogens from fomites to fingers except for PV1. Most organisms had greater transfer efficiencies under high relative humidity (< 0.1% to 79.5%) compared to low relative humidity (0.03% to 57%) with a few exceptions (Table 2 and 3). E. coli and MS2 had greater transfer efficiencies under high relative humidity for all the fomites. S. aureus had greater transfer rates under high relative 114 humidity except for cotton and paper currency. B. thuringiensis spores had greater transfer rates under high relative humidity except for paper currency. PV-1 seemed not to be influenced by relative humidity with transfer efficiencies for ceramic tile, laminate, and granite under low relative humidity of 23.1%, 36.3%, 33.8%, respectively compared to 29.2%, 25.5%, 25.9% under high relative humidity (Table 4). Figures 1, 2, and 3 show the distribution of each of the six transfer efficiencies in logarithmic scale along with the geometric mean to show the variability in the transfer efficiencies. The statistical significance (P = 0.05) between low and high transfer efficiencies for each microorganism are indicated on (Tables 2 and 3). No statistical significance (P = 0.05) between low and high transfer efficiencies for PV-1 were found (Table 4). Influence of fomite type on microbial transfer Fomite type did influence the transfer efficiency of all model organisms except PV-1. In general, the transfer efficiencies were greater from nonporous surfaces (0.04% to 57% under low relative humidity; 12.8% to 79.5% under high relative humidity) than those for porous surfaces (< 6.8% under low relative humidity and < 13.4% under high relative humidity) (Tables 2 and 3). Under low relative humidity, acrylic provided the highest transfer rate for E. coli, B. thuringiensis, and MS2 coliphage (40.7%, 57%, and 21.7%, respectively), while S. aureus had the highest transfer efficiency from glass (20.3%) (Table 2). Under high relative humidity, glass provided the highest transfer rate for E. coli (78.6%), laminate for S. aureus (61.9%), and acrylic for B. thuringiensis and MS2 115 coliphage (65.6% and 79.5%, respectively) (Table 3). PV-1 transfer did not seem to be influenced by the type of nonporous surfaces (Table 4). The transfer efficiency was least with paper currency under both low and high relative humidity for E. coli, S. aureus, and B. thuringiensis (< 0.05%, 0.2%, < 0.1% under low relative humidity; 0.1%, 0.2%, < 0.1% under high relative humidity, respectively). Cotton produced the lowest transfer efficiencies for MS2 under both low and high relative humidity (0.03% and 0.3%, respectively). DISCUSSION The goal of this study was to obtain detailed quantitative information on fomite-to-finger transfer that could be used to model the probability of infection from exposure to various types of pathogens, a parameter needed for quantitative microbial risk assessments (1, 5, 48, 49). Unfortunately, there are no standard methods for quantifying transfer rates, making it difficult to compare the results from various studies. Our results indicate that while transfer is influenced by the relative humidity and fomite type, different organisms vary greatly in the relative influence of these environmental factors. The relative humidity greatly influenced the transfer for most of the microorganisms except for PV-1; however, on average, most had greater transfer rates at a high relative humidity. Under low relative humidity, E. coli, MS2, and PV-1 had the greatest transfer efficiencies. No substantial difference of transfer efficiencies between different organisms was observed at a high relative humidity, although E. coli and MS2 exhibited slightly greater transfer rates than the other organisms. B. thurigiensis spores were poorly transferred under low 116 relative humidity conditions from all surfaces except acrylic. Desiccation did not seem to account for these differences since E. coli is more influenced by desiccation than S. aureus and endospores (50). These differences may also reflect differences in the composition of the cell or endospore outer surface, and the hydrophobicity or other chemical/structural characteristics of the organisms. Generally, it would appear that the smoother surfaces provide greater transfer efficiencies. As might be expected, porous surface had much lower transfer rates than the nonporous surfaces. Porous surfaces may entrap organisms within their matrix and provide a much greater surface area for attachment. Overall, acrylic tended to exhibit greater transfer, especially under low relative humidity. Acrylic (poly-methyl methacrylate) is a transparent thermoplastic, often used as a lightweight or shatter-resistant alternative to glass and paints. S. aureus and MS2 tended to have greater transfer from the porous surfaces than the other organisms. PV-1 transfer rates were similar to the ranges (16% to 32%) reported by Mbithi et al. (42) for hepatitis A virus; Ansari et al. (41) for rotavirus; and Paulson (45) for feline calicivirus. However it is important to note that different methods were used to assess these transfer efficiencies. Rheinbaben et al. (51) found lower transfer of ΦX174 coliphage from door handles to volunteers hands (0.001 to 0.4%) than that of MS2 from stainless steel (37.4%) under 117 high relative humidity observed in the present study. Rusin et al. (37) found PRD 1 phage to have a transfer efficiency of 33.5% and 65.8% from faucet handle and phone receiver, respectively. Julian et al. (47) investigated the effects of washed and unwashed hands on the transfer rates of MS2, ΦX174, and fr coliphages from glass slides to fingers and fingers to glass slides and found similar transfer rates of 25%, 21%, 37% for unwashed hands and 26%, 11%, 39% for washed hands, respectively. The fomite-tofinger transfer efficiency of MS2 observed in our study agrees with the findings of these previous reports. Transfer rates for MS2 under high relative humidity for glass was greater (67.3%) than under low relative humidity (19.3%) (Tables 2 and 3). Transfer rates for porous fomites, cotton, polyester, and paper currency under low and high relative humidity were also comparable to the PRD 1 transfer rates found by Rusin et al. (37). Previous studies have used the 1.0 kg/cm2 pressure which has been estimated to be equivalent to the pressure applied in a handshake without friction, or to opening a door with a door handle with friction (6, 41, 42). Other studies have used lower levels of contact pressure to simulate children handling and grasping objects (47), or to represent ordinary touching of environmental surfaces (42). Mbithi et al. (42) found a significant difference in the amount of pressure and friction applied in determining the amount of hepatitis A virus transferred from stainless steel disks to fingers. Another factor that might influence the results is drying time. We selected a 30 min drying time based on our preliminary studies. The seeded suspension medium on various 118 surfaces became visibly dry between 15 min to 32 min under a relative humidity range of 20% to 34%, which allowed us to compare the effects of low and high relative humidity on fomite-to-finger transfer efficiencies. Other investigators have used drying times varying from 5 min to 48 h and contact time varying from 5 s to 30 s (6, 37, 41, 42, 45-47, 50-55). Based on these previous studies, we can expect the transfer rates to be lower over longer drying periods, allowing the seeded suspension medium to become drier, resulting in lower transfer rates. Different contact pressures and contact times could have been examined, providing a more broad view of the transfer rates during variable contact times and pressures. Incorporating more volunteer hands could also better characterize the effects of individual hand characteristics such as pH (56). The transfer efficiencies observed in our study for S. aureus from nonporous surfaces under high relative humidity (39.6% to 61.9%) were greater compared with (9% to 43%) reported by Scott and Bloomfield (50). The difference in contact time and drying time could have influenced the different transfer rates. Our low relative humidity transfer efficiencies seem to agree with Scott and Bloomfield (50) transfer efficiency at the 24 h drying time of 9.05%. Transfer efficiencies for porous fomites, under low and high relative humidity were lower than those reported by Scott and Bloomfield (50) and Sattar et al. (57) with the exception of polyester at a high relative humidity (5% transfer). Sattar et al. (57) observed the same greater transfer efficiency of S. aureus from poly-cotton fabric than from 100% cotton and explained that this might be due to the higher hydrophobic nature of polyester material than cotton. The hydrophobic nature reduced 119 the ability of the bacterial cells to penetrate deeper into individual fibers. Data reported by Hubner et al. (58) study on the hand-to-paper-to-hand transfer cycle of E. coli (0.009%) agree with our fomite-to-finger observations of E. coli with paper currency under low relative humidity. A possible reason of higher transfer efficiency under high relative humidity observed in our study was that the high humidity prevented the inoculum from drying, resulting in greater transfer efficiencies. Previous studies have shown that virus transfer efficiencies between contaminated surfaces were greater when the seeded suspension medium was not completely dry (4, 7, 41, 42, 55, 59). This was seen with coliphage MS2, which had the most significant difference between low and high relative humidity with the nonporous inanimate surfaces, and to a lesser degree with the porous surfaces (Fig. 1, 2, and 3). It was also seen with Gram negative E. coli, Gram positive S. aureus, and spores of B. thuringiensis. In most cases, there was a significant difference (P ≤ 0.05) between nonporous and porous surfaces between organism transfer efficiencies. The transfer efficiency for each organism on a specific surface can be influenced by the seeded suspension medium along with the physicochemical properties of the species and how it interacts with the physical properties of the environmental surfaces (60-63). Both the isolectric point and the hydrophobicity of the surface can influence the interactions between the fomite and the organism. MS2 has a relatively low isoelectric point of pH 3.9 compared to many non-enveloped enteric viruses and is often less attracted to 120 common fomites (62, 64). In the case of the low transfer efficiencies seen with B. thuringiensis spores and S. aureus from nonporous surfaces under low relative humidity, a possible explanation could be that the spores and the Gram positive bacteria had stronger electrostatic interactions through van der Waals forces, resulting in a stronger attachment to the fomites (61). CONCLUSIONS In the present study, a number of different fomites were tested, providing a broad data set to estimate the distributions in the transfer efficiency that depend on the type of fomite and the microorganism. Our results highlight the importance of relative humidity in the organism fomite-to-finger transfer efficiency rates. Most species on average had greater transfer efficiencies under high relative humidity in comparison to low relative humidity. 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Microbiol. 64:405-410. 131 TABLES TABLE 1 Fomites tested Fomite Nonporous Notes Manufactures Acrylic Poly-methyl methacrylate, matte nonglare finish Home Depot, Atlanta, GA Glass Slides VWR, Mississauga, ON Ceramic Tile Porcelain Home Depot, Atlanta, GA Laminate Various colors Wilsonart International, Temple, TX Stainless Steel Gage 304 AK Steel Corporation, West Chester, OH Granite One from India and two from Brazil GRANITE Kitchen & Bath Countertops, Tucson, AZ Cotton 100% fabric Polyester 100% fabric Paper Currency Cotton-based one dollar bills Home Trends Brand, Walmart, Bentonville, AR Home Trends Brand, Walmart, Bentonville, AR United States Porous 132 TABLE 2 Fomite-to-finger transfer efficiencya of microorganisms under low relative humidity 15% - 32% E. coli S. aureus B. thuringiensis MS-2 (n) Average (%) ± SD (Range) (n) Average (%) ± SD (Range) (n) Average (%) ± SD (Range) (n) Average (%) ± SD (Range) Acrylic (6) 40.7 ± 37.7 (6.4 - 93.5) (6) 3.4 ± 2.5 (0.9 - 8.0)c (6) 57.0 ± 12.0 (45.8 - 74.8) (6) 21.7 ± 15.0 (3.0 - 40.6)c Glass (6) 5.1 ± 5.4 (0.7 - 15.1)c (6) 20.3 ± 33.4 (0.6 - 85.4) (6) < 0.5 ± 0.2 (< 0.3 - 0.9)b, c (6) 19.3 ± 13.2 (2.9 - 40.5)c Ceramic Tile (6) 11.6 ± 11.8 (0.1 - 33.3)c (6) 2.7 ± 2.3 (0.8 - 6.7)c (6) < 0.2 ± 0.1 (< 0.1 - 0.4)b (6) 7.1 ± 4.0 (3.8 - 15.0)c Laminate (6) 21.7 ± 23.9 (5.2 - 66.5) (6) 4.3 ± 2.4 (1.3 - 7.4)c (6) 0.2 ± 0.1 (< 0.1 - 0.3)b, c (6) 5.4 ± 3.6 (1.0 - 10.0)c Stainless Steel (6) 3.8 ± 2.5 (1.5 - 7.1)c (6) 4.0 ± 4.0 (1.1 - 11.9)c (6) < 0.5 ± 0.2 (< 1.0)b, c (6) 6.9 ± 8.9 (1.4 - 24.2)c Granite (6) 7.3 ± 10.6 (< 0.1 - 28.0)b (6) 3.9 ± 5.0 (0.7 - 13.9) (6) 0.04 ± 0.03 (< 0.02 - 0.1)b (6) 10.2 ± 5.0 (4.8 - 16.9) Cotton (6) < 6.8 ± 7.0 (< 15.4)b (6) < 1.0 ± 0.6 (< 1.9)b (6) < 0.6 ± 0.1 (< 0.8)b (6) 0.03 ± 0.02 (0.01 - 0.1) Polyester (6) < 0.37 ± 0.28 (< 0.9)b (6) 0.37 ± 0.48 (0.04 - 1.3)b (6) < 0.6 ± 0.6 (< 1.7)b (6) 0.3 ± 0.2 (0.1 - 0.7)c Paper Currency (6) < 0.05 ± 0.04 (< 0.02 - 0.1)b (6) 0.2 ± 0.1 (0.1 - 0.4) (6) < 0.1 ± 0.1 (< 0.02 - 0.2)b (6) 0.4 ± 0.4 (0.1 - 0.9) Surface Type Nonporous Porous a Transfer efficiency (%) = (CFU or PFU finger/CFU or PFU control fomite) X 100 b Transfer of organisms from fomite to fingers for one or more transfer events, were below the detectible limit of 10 CFU/2-cm2 indicated by < c There was a statistical difference (Student t-test P = 0.05) of the transfer efficiency between low and high relative humidity 133 TABLE 3 Fomite-to-finger transfer efficiencya of organisms under high relative humidity 40% - 65% E. coli S. aureus B. thuringiensis MS-2 (n) Average (%) ± SD (Range) (n) Average (%) ± SD (Range) (n) Average (%) ± SD (Range) (n) Average (%) ± SD (Range) Acrylic (6) 53.3 ± 27.5 (30.4 - 98.0) (6) 47.2 ± 17.9 (24.4 - 67.3)d (6) 65.6 ± 15.9 (48.8 - 94.9) (6) 79.5 ± 21.2 (54.1 - 100)c, d Glass (6) 78.6 ± 27.1 (38.0 - 100)c, d (6) 45.5 ± 15.5 (25.7 - 65.5) (6) 33.8 ± 24.0 (< 4.3 - 65.9)b, d (6) 67.3 ± 25.0 (37.4 - 96.9)d Ceramic Tile (6) 60.7 ± 45.4 (3.7 - 100)c, d (6) 54.7 ± 18.8 (27.7 - 77.6)d (6) 21.2 ± 28.2 (< 1.3 - 76.4)b (6) 41.2 ± 18.8 (18.7 - 74.7)d Laminate (6) 27.4 ± 30.2 (1.9 - 77.0) (6) 61.9 ± 24.7 (30.9 - 89.8)d (6) 53.5 ± 19.6 (33.8 - 79.0)d (6) 63.5 ± 24.0 (36.2 - 100)c, d Stainless Steel (6) 54.1 ± 23.5 (29.4 - 99.0)d (6) 48.3 ± 25.4 (16.6 - 85.5)d (6) 57.0 ± 9.7 (47.5 - 71.4)d (6) 37.4 ± 16.0 (19.5 - 62.4)d Granite (6) 36.5 ± 39.3 (0.3 - 100)c (6) 39.6 ± 41.5 (1.3 - 100)c (6) 12.8 ± 19.8 (0.1 - 42.7) (6) 30.0 ± 24.3 (4.9 - 59.3) Cotton (6) < 13.4 ± 11.7 (< 33.3)b (6) 0.5 ± 0.5 (0.1 - 1.3)b (6) < 3.5 ± 3.5 (< 10.0)b (6) 0.3 ± 0.3 (0.04 - 0.6) Polyester (6) < 0.7 ± 0.8 (< 2.2)b (6) 5.0 ± 6.9 (0.1 - 15.5) (6) < 4.6 ± 6.1 (< 16.3)b (6) 2.3 ± 0.8 (1.2 - 3.2)d Paper Currency (6) 0.1 ± 0.3 (< 0.01 - 0.7)b (6) 0.2 ± 0.1 (0.1 - 0.3) (6) < 0.1 ± 0.1 (< 0.2)b (6) 0.7 ± 0.5 (0.1 - 1.5) Surface Type Nonporous Porous a b Transfer efficiency (%) = (CFU or PFU finger/CFU or PFU control fomite) X 100 Transfer of organisms from fomite to fingers for one or more transfer events, were below the detectible limit of 10 CFU/2-cm2 indicated by < c Transfer of organism from fomite-to-fingers for one or more transfer events were > 100% and were truncated to 100% d There was a statistical difference (Student t-test P = 0.05) of the transfer efficiency between low and high relative humidity 134 TABLE 4 Fomite-to-finger transfer efficiencya of poliovirus 1 Surface Type (n) Average (%) ± SD (Range) Low RH (15% - 32%) Ceramic Tile (6) 23.1 ± 24.0 (0.4 - 52.7) Laminate (6) 36.3 ± 8.7 (24.1 - 50.0) Granite (6) 33.8 ± 40.4 (0.4 - 100)b High RH (40% - 65%) a b Ceramic Tile (6) 29.2 ± 6.4 (19.4 - 35.4) Laminate (6) 25.5 ± 15.5 (3.4 - 50.0) Granite (6) 25.9 ± 4.1 (19.7 - 32.1) Transfer efficiency (%) = (PFU finger/PFU control fomite) X 100 Transfer event was > 100% and was truncated to 100% 135 FIGURES A B Geometric Mean Geometric Mean 100 % Transfer Efficiency % Transfer Efficiency 100 10 1 0.1 0.01 10 1 0.1 0.01 Acrylic Glass Ceramic Laminate Stainless Granite Tile Steel Co on Polyester Nonporous Paper Currency Acrylic Glass Porous Ceramic Laminate Stainless Granite Tile Steel Co on Polyester Nonporous C Porous D Geometric Mean Geometric Mean 100 100 % Transfer Efficiency % Transfer Efficiency Paper Currency 10 1 0.1 0.01 10 1 0.1 0.01 Acrylic Glass Ceramic Laminate Stainless Tile Steel Granite Nonporous Co on Polyester Porous Paper Currency Acrylic Glass Ceramic Laminate Stainless Tile Steel Nonporous Granite Co on Polyester Paper Currency Porous FIG 1 Transfer efficiency (%) of (A) E. coli 15597, (B) S. aureus 25923, (C) B. thuringiensis spores, and (D) MS2 under low relative humidity (15% - 32%). 136 A B Geometric Mean Geometric Mean 100 % Transfer Efficiency % Transfer Efficiency 100 10 1 0.1 0.01 10 1 0.1 0.01 Acrylic Glass Ceramic Laminate Stainless Tile Steel Granite Co on Nonporous Polyester Paper Currency Acrylic Glass Porous Ceramic Laminate Stainless Tile Steel Granite Co on Nonporous C Polyester Paper Currency Porous D Geometric Mean Geometric Mean 100 % Transfer Efficiency % Transfer Efficiency 100 10 1 0.1 0.01 10 1 0.1 0.01 Acrylic Glass Ceramic Laminate Stainless Tile Steel Granite Nonporous Co on Polyester Porous Paper Currency Acrylic Glass Ceramic Laminate Stainless Tile Steel Nonporous Granite Co on Polyester Paper Currency Porous FIG 2 Transfer efficiency (%) of (A) E. coli 15597, (B) S. aureus 25923, (C) B. thuringiensis spores, and (D) MS2 under high relative humidity (40% - 65%). 137 A B Geometric Mean 100 10 10 % Transfer Efficiency % Transfer Efficiency Geometric Mean 100 1 0.1 1 0.1 0.01 0.01 Ceramic Tile Laminate Nonporous Granite Ceramic Tile Laminate Granite Nonporous FIG 3 Transfer efficiency (%) of Poliovirus 1 under (A) low relative humidity (15% - 32%) and (B) high relative humidity (40% - 65%). 138 APPENDIX D: COMPARATIVE PERSISTENCE OF BACTERIA AND VIRUSES UNDER DIFFERENT RELATIVE HUMIDITY ON POROUS AND NONPOROUS FOMITES Abbreviated running headline: Persistence of Microbes on Fomites Gerardo U. Lopez, Masaaki Kitajima, Akrum Tamimi, Charles P. Gerba# Department of Soil, Water and Environmental Science, University of Arizona, Tucson, Arizona 85721 USA # Corresponding author. Tel: (520) 621-6910, Fax (520) 621-6366, E-mail: [email protected] A manuscript for Journal of Applied Microbiology 139 ABSTRACT Aims: Fomites can play a role in the transmission of many types of human pathogens. Information on the persistence of bacteria and viruses on fomites is needed for the development of quantitative microbial risk assessment models to reduce uncertainty in predicting risk from contact with contaminated fomites. Survival of bacteria and virus on several common fomites under different relative humidity conditions was determined. Methods and Results: Nine fomites of different compositions were studied representing porous and nonporous surfaces. Escherichia coli, Staphylococcus aureus, Bacillus thuringiensis, MS2 coliphage, and poliovirus 1 were placed on various fomites in 10 µl drops allowed to dry for 30 min under low (15% to 32%) or high (40% to 65%) relative humidity. Relative humidity influenced organism survival on fomites; greater survival was observed under high relative humidity for E. coli, S. aureus, MS2, and PV-1. B. thuringiensis spores survived longer at low relative humidity. Fomite type did influence the survival of all model organisms; survival was greater on nonporous surfaces than those for porous surfaces. Organism survival was greatest with paper currency and least with cotton for most pathogens under both low and high relative humidity. Conclusions: Generally most organisms had greater survival under high relative humidity compared to low relative humidity with B. thuringiensis spores showing the opposite effect. Survival was less on porous surfaces compared to nonporous surfaces. Significance and Impact of Study: This data can be used to predict microorganism log10 reduction parameters in microbial risk assessment models, in order to be able to develop 140 more sophisticated and realistic models taking into account bacterial and virus survival on various fomites under different relative humidity. Keywords: Bacilus thuringiensis, Staphylococcus aureus, relative humidity, nonporous, porous, fomites, surfaces, survival INTRODUCTION Fomites can serve as reservoirs of microbial pathogens and contribute to direct or indirect transmission of infectious agents (Haas et al., 1999, Nicas and Sun, 2006). Several studies have shown that environmental surfaces found in indoor environments are contaminated with human bodily fluids indicating the potential presence of infectious agents (Ansari et al., 1991, Barker et al., 2001, Barker et al., 2004, Hall et al., 1980). A number of indoor fomites in day care centers (Barker et al., 2001, Boone and Gerba, 2005, Butz et al., 1993, Keswick et al., 1983a, Keswick et al., 1983b, Laborde et al., 1993), schools (Bright et al., 2010), office building (Boone and Gerba, 2010), homes (Bellamy et al., 1998, Fekety et al., 1981, Josephson et al., 1997, Medrano-Félix et al., 2010, Rusin et al., 1998, Scott et al., 1982, Sinclair and Gerba, 2011, Speirs et al., 1995), public areas (Reynolds et al., 2005) and hospitals (Huslage et al., 2010, Kramer et al., 2006, Otter et al., 2011, Pittet et al., 2006, Pittet et al., 1999, Weber et al., 2010) have been identified as reservoirs and vectors for secondary modes of transmission. In order for a microbial pathogen to be spread via contaminated fomites the bacteria or virus needs to maintain infectivity long enough to come into contact with a susceptible host. A number of studies 141 have shown that microbial pathogens can persist from hours to months on fomites depending on the numbers deposited, type of organism and environmental conditions (Abad et al., 2001, Boone and Gerba, 2007, Kramer et al., 2006). However, few studies have examined the effects of relative humidity on microbial survival. Data on the survival of bacteria and viruses on fomites is important in modeling the efficiency of transfer of a pathogens from fomite to hands in risk assessment models (Atkinson and Wein, 2008, Boone and Gerba, 2007, Brankston et al., 2007, Nicas and Best, 2008). The purpose of the present study was to evaluate the survival of five model organisms under low and high relative humidity on various types of fomites to better understand microorganism survival in modeling microbial exposure and risk assessment. MATERIALS AND METHODS Bacteria, Virus and preparation of inocula Escherichia coli (ATCC 15597), Staphylococcus aureus (ATCC 25923), Bacillus thuringiensis (ATCC 10792), and coliphage MS2 (ATCC 15597-B1) were obtained from the American Type Culture Collection (ATCC Manassas, VA). Poliovirus 1 (PV-1; strain LSc-2ab) was obtained from the Department of Virology and Epidemiology at the Baylor College of Medicine (Houston, TX). These organisms were selected as model organisms for pathogenic Gram negative, Gram positive bacteria, spore-forming bacteria, and viruses. 142 Frozen aliquots of E. coli and S. aureus were transferred into separate flasks containing 100 to 150 ml of Tryptic Soy Broth, (TSB, EMD, Gibbstown, NJ) incubated for 18 ± 2 h at 37°C on an orbital shaker (150 to 180 rpm), and streaked for isolation onto Tryptic Soy Agar plates (TSA, EMD Gibbstown, NJ), the bacteria were then subcultured in a flask of TSB, and incubated for 18 ± 2 h at 37°C on a orbital shaker (150 to 180 rpm) (Rusin et al., 2002). B. thuringiensis spores were prepared as previously described with minor modification (Nicholson and Setlow, 1990). Briefly, spores were suspended in Difco sporulation media with supplements (DSM + S; Becton, Dickinson and Company, Sparks, MD), cultivated for 24 h at 37°C on an orbital shaker (150 to 180 rpm), and resuspended in fresh DSM + S to obtain a final OD600 of 0.1 (Spectronic Genesys 5, Milton Roy, Ontario Canada). MS2 coliphage were prepared as previously described with minor modifications (Rusin et al., 2002). Briefly, 0.1 ml of phage suspension and 0.5 ml of a log phase E. coli 15597 (host bacterium) culture 0.5 ml were added to top agar, melted, and maintained at 48°C. The inoculated top agar was mixed and poured over the TSA. The solidified agar overlay plates were then inverted and incubated at 37°C for 24 h. TSB was then added to each plate and maintained at room temperature for 2 h. The TSB eluent was aspirated and centrifuged at (1,989 X g for 10 min), after which, the supernatant was filtered through 0.22 µm pore size Acrodisc® Syringe Filters (Pall Corporation, Ann Arbor, MI) pre- 143 moistened with 3% beef extract (Bacto™, Becton, Dickinson and Company, Sparks, MD), the virus tittered, and stored at 4°C. PV-1 propagation and plaque-forming assays were conducted as previously described (Bidawid et al., 2003, Ikner et al., 2011). Briefly, poliovirus 1 was propagated on buffalo green monkey kidney (BGM; ATCC CCL-81); obtained from the American Type Culture Collection (ATCC), Manassas, VA) cell line monolayers with minimal essential media (MEM) containing 5% calf serum (HyClone Laboratories, Logan, UT) at an incubation temperature of 37°C with 5% CO2. Plaque-forming assays were performed using sixwell plates with confluent monolayers of the BGM cells. Relative Humidity Conditions and Temperature Two relative humidity ranges were studied, high (40% to 65%) and low (15% to 32%) relative humidity. To achieve both humidity conditions, two separate incubators were turned off and used. Incubator temperatures thus reflected room temperature ranges of 19°C to 25°C. The temperature and relative humidity were monitored with a High Accuracy Thermo – Hygro (VWR, Mississauga, ON). During days with higher ambient relative humidity in the laboratory, t.h.e.® Desiccant (EMD, Gibbstown, NJ) and Drierite desiccant (Drierite, Xenia, OH) were utilized in the incubator to decrease the relative humidity to the low relative humidity range (15% to 32%). During laboratory ambient lower relative humidity conditions a BIONAIRE® humidifier (Milford, MA) was used in 144 the specific incubator to increase the relative humidity to the high relative humidity range (40% to 65%). Fomites tested Nine different types of fomite materials were tested ranging in surface areas of 16 to 25 cm2 six nonporous and three porous (Table 1). With the exception of acrylic, all fomites were sterilized by autoclave whereas acrylic was radiated under UV light (254 nm) for 30 min on each side. After fomite-to-finger transfers with E. coli, S. aureus, and MS2, nonporous fomites were sprayed three times with 70% ethanol and allowed to dry for 10 min. Nonporous fomites used in the finger transfers with B. thuringiensis spores and PV1 were disinfected with 10% bleach (The Clorox Company, Oakland, CA), then allowed to sit for 10 min, and subsequently neutralized in 10% sodium thiosulfate (EMD, Gibbstown, NJ). Fomites were then washed under warm running water with nonantibacterial soap (Liquid Joy, Procter and Gamble, Cincinnati, OH), rubbed with a wet paper towel on the surface area of inoculation, rinsed thoroughly with RO treated water, air-dried, and autoclaved. Cotton and polyester fomites were discarded after use. Paper currency was autoclaved and reused. Inoculation of fomites For each of the six nonporous and three porous fomites, seven swatches/coupons were evenly spaced on the middle shelf of an incubator. Each trial consisted of three control 145 swatches/coupons, three fomite-to-finger transfer swatches/coupons, and one negative control swatch/coupon to ensure that the fomites were not previously contaminated. The concentration of organisms added to the fomites was approximately 107 to 108 colony forming units (CFU)/cm2 of E. coli in (TSB, EMD, Gibbstown, NJ), 108 to 109 CFU/cm2 of S. aureus in TSB, 106 to 107 CFU/cm2 of B. thuringiensis spores in (DSM + S; Becton, Dickinson and Company, Sparks, MD), 109 to 1011 plaque forming units (PFU)/cm2 of MS2 in TSB, and 108 PFU/cm2 of PV-1 in (PBS, Sigma-Aldrich, St. Louis, MO) in 10 µl droplets. Using a pipet tip, the 10 µl inoculum droplets were spread over approximately a 1.0 cm2 area on the center of each fomite. The paper currency were divided into four 24 cm2 sections; two one dollar bills were used to make the seven swatches. With paper money for each set of transfer experiments, a new 1.0 cm2 area on each of the 24 cm2 sections were inoculated. Using an ink marker, an identifying spot was placed near the inoculated area on cotton, polyester, and paper money due to the absorbance. The fomites were allowed to dry for 30 min. Surface sampling and assays Fomites were sampled using a cotton-tipped swab applicator (Puritan Medical Products Company, Guilford, ME), when inoculated with E. coli, S. aureus, B. thuringiensis, and MS2. In the case of PV-1, a polyester fiber-tipped applicator swab (Falcon, Becton Dickinson and Company, Cockeysville, MD) was used. Swabs were wet in 1.0 ml of phosphate buffer saline (PBS, Sigma-Aldrich, St. Louis, MO), and then a 6.0 cm2 area on 146 the fomite was swabbed using firm sweeping and rotating motions. The swab was then placed back into the remaining PBS and vortexed for 5 s. E. coli, S. aureus, and B. thuringiensis spores were enumerated using the spread plate technique on MacConkey (EMD Chemicals Inc, Gibbstown, NJ), Mannitol salt agar (MSA, EMD Chemicals Inc, Gibbstown, NJ), and TSA (EMD Chemicals Inc., Gibbstown, NJ) agar plates, respectively. The plates were incubated at 37°C for 18 ± 2 h. B. thuringiensis spore samples were heat shocked at 81 ± 2°C for 10 min prior to spread plating, to stimulate germination. The MS2 plaque assay was conducted using the double agar overlay method using TSA (EMD Chemicals Inc., Gibbstown, NJ) (Adams, 1959, Ikner et al., 2011). PV-1 titrations were performed using 10-fold serial dilution plaqueforming assays as described previously (Bidawid et al., 2003, Ikner et al., 2011). All dilutions were assayed in duplicate. Reduction and Statistical analysis Log10 reduction was calculated using Eqn (1) and is described as the negative Log10 of the value of CFU or PFU concentration recovered from fomite divided by CFU or PFU of the stock titer. (1) 147 Data were entered in Microsoft Excel 2010 and the software package StatPlus:mac, 2009, (AnalystSoft) to compute the descriptive statistic measures of mean percent transfer efficiency, the standard deviation, and statistical significance. To assess the statistical significance at the (P = 0.05) level, the percent transfer efficiencies between low and high relative humidity and nonporous and porous fomites were compared in a Student’s t-test. RESULTS Influence of relative humidity on microbial survival In general relative humidity influenced organism survival. S. aureus had greater survival, less log10 reduction (Table 2 and 3) (Fig. 1 and 2), under high relative humidity on all fomites except granite. PV-1 had slightly greater survival under high relative humidity for the three fomites tested (Table 4) and (Fig. 3). E. coli survived better under high relative humidity for most fomites except for granite, cotton, and polyester (Table 2 and 3) (Fig. 1 and 2). MS2 was less influenced by relative humidity with the exception of stainless steel, polyester, and paper currency where MS2 survived better under high relative humidity (Table 2 and 3) (Fig.1and 2). B. thuringiensis spores on the other hand seemed to be more stable under low relative humidity with most fomites except for glass, stainless steel, and paper currency (Table 2 and 3) (Fig. 1 and 2). Influence of fomite type on microbial survival Fomite type also influenced survival. In general, survival was greater for nonporous surfaces (less than 2.6 reduction log10 CFU or PFU/2-cm2 under both low and high 148 relative humidity) than those for porous surfaces (up to 5.0 reduction log10 CFU or PFU/2-cm2 under low and high relative humidity) (Fig. 1, 2, and 3). Granite showed the least reduction between low and high relative humidity for all microorganisms (less than 0.05 reduction log10 CFU or PFU/2-cm2 except with PV-1 (Fig. 3). Cotton showed the greatest reduction for all organisms under low and high relative humidity, while paper currency showed the least reduction. MS2 also showed a greater PFU recovery than the initial titer on ceramic tile, stainless steel, polyester, and paper currency resulting in a negative reduction (Table 2 and 3) (Fig. 2 and 3). The statistical significance (Student t-test P = 0.05) between low and high transfer efficiencies for each microorganism are indicated on (Tables 2, 3, and 4). No statistical significance (P = 0.05) between low and high transfer efficiencies for PV-1 were found (Table 4). A statistical difference (Student t-test P = 0.05) between high and low relative humidity log10 reduction at 30 min (Fig. 1, 2, and 3) occurred with E. coli between acrylic, glass, ceramic tile, and laminate; S. aureus for acrylic, glass, ceramic tile, laminate, stainless steel, cotton, and polyester; B. thuringiensis for glass, ceramic tile, laminate, stainless steel, and cotton; MS2 coliphage for all fomites except laminate and granite; poliovirus for the three fomites tested. No statistical difference between high and low relative humidity log10 reduction at 30 min was seen with PV-1 (Fig. 3). 149 DISCUSSION The objective of the present study was to quantitate the survival of several surrogate pathogens in order to determine log10 reduction under low and high relative humidity on various types of fomites to better quantify microorganism survival in modeling microbial exposure and risk assessment. Our results indicate that relative humidity as well as fomite type influenced survival on various surfaces. The different organisms studied varied greatly among the influence of these environmental factors. Over all microorganisms were more stable resulting in a lower reduction under high relative humidity however, did seem to be fomite dependent. Greater organism reduction was observed with porous fomites than with nonporous fomites. The observed reduction difference between the relative humidity ranges was up to 2.5 reduction log10 CFU or PFU/2-cm2. Figures 1, 2, and 3 compares the distribution of the microbial reduction log10 CFU or PFU/2-cm2 and the geometric mean between low and high relative humidity for each organism. Reduction plotted below the zero axis indicated that there was a greater recovery of the organism than the initial titer. Such results were seen more often under high relative humidity and could be due to aggregated bacteria cells and virus particles that were not as easily able to break apart under low relative humidity, while at high relative humidity the drying process was inhibited by the relative humidity conditions which was conducive for the organisms to break apart. 150 Our observations with both bacteria and viruses were consistent with Abad et al. (1994) that found viruses to have a lower survival on porous fomites than nonporous surfaces with up to a four log difference in survival under different relative humidity and temperatures over 60 days. A recent study by Hubner et al. (2011) also showed that several strains of bacteria that have been found on medical records can survive on paper for several days and can be transferred to healthcare professions leading to crosscontamination to patients and themselves. Our study looked at both bacteria and virus survival on mostly different nonporous and porous fomites than used in these previous studies under different relative humidity at the same temperature range. Even at a short time period overall organisms survived least on cotton, even compared to polyester than on nonporous fomites. Organisms survived the greatest on paper currency which had similar results as nonporous fomites indicating that bacteria and viruses can persist on paper currency. Studies have found the presence of organisms on paper currency (Khin Nwe and al., 1989, Uneke and Ogbu, 2007, Vriesekoop et al., 2010) suggesting that dollar bills can be reservoirs for potential pathogens. The overall lower survival observed in porous fomites over time? could be due to entrapment of organisms within their matrix over time or they provide a much greater surface for attachment. Most organisms showed a statistical significance (P = 0.05) between low and high relative humidity survival measured in reduction (Fig. 1, 2, and 3). Even though the reduction for most organisms was on average less than 1.0 to 2.0 log10 CFU or PFU/2- 151 cm2. This statistical difference might have reflected the small standard deviation within each group under the different relative humidity conditions. Poliovirus has been reported to survive 4 h to < 8 days (Kramer et al., 2006) on stainless steel disks. In the present study poliovirus seemed to be less influenced by relative humidity, however still showing a lower reduction at 40% to 65% relative humidity (Fig. 3). These results agree with (Mbithi et al., 1991) and (Abad et al., 1994) observations of PV-1 surviving better on nonporous fomites at 95% and 85% relative humidity, respectively. Greater stability under higher relative humidity was also reported by (Sattar et al., 1988) for enterovirus 70 (EV-70) and Abad et al. (1994) reported no effects of relative humidity on enteric adenovirus (ADV). These findings support the general idea that nonenveloped viruses are more stable at relative humidity levels higher than 80% (Buckland and Tyrrell, 1962, Sattar et al., 1988). On the other hand other studies reported contradictory results for other nonenveloped viruses; Mbithi et al. (1991) indicated hepatitis A virus (HAV) to be more stable at lower relative humidity (25% ± 5%), while Abad et al. (1994) demonstrated HAV to have greater stability at higher relative humidity. Similar contrasting observations were reported for human rotavirus (HRV) where Moe and Shirley (1982) showed greater HRV survival under both low and high relative humidity, while Sattar et al. (1986) reported more stability at low or medium level, and Abad et al. (1994) observed greater stability at high relative humidity. Explanation for this variableness in virus survival among different studies is unknown. Never the less the present study shows that the lower relative humidity results in increased poliovirus 152 survival while higher relative humidity enhances poliovirus stability on nonporous surfaces. Such results indicate that the influence of relative humidity is very virus specific. Even though the relative humidity tested in this study did not have a great effect on MS2 coliphage survival (Tables 2 and 3) (Fig. 1 and 2) a slightly lower reduction was observed under high relative humidity. Under both low and high relative humidity conditions MS2 was very stable, however, there was a greater reduction under low relative humidity particularly with porous fomites (Figure 1 and 2). Using feline calicivirus as a surrogate for norovirus foodborne outbreaks (Paulson, 2005) found that feline calicivirus can persist on various fomites after a 15 min drying time with little inactivation. B. thuringiensis spores were more stable under low relative humidity. Bacterial spores such as Clostridium difficile are known to be very stable in the environment and have been reported to persist on fomites for several months (Kim et al., 1981). Persons infected with acute diarrhea can excrete 107 to 109 micro-organisms per gram of feces resulting in wide spread contamination of the surrounding surfaces with spores (Mutters et al., 2008). Up to 3.5 log10 CFU of C. difficile spores on surfaces in rooms of patients positive with C. difficile infections have been reported (Mutters et al., 2008). B. anthracis and B. thuringiensis strains have been shown to survive better (> 6.5 log10 CFU) at lower temperatures (60°C) and lower relative humidity (60%) than at higher temperatures and relative humidity (Buhr et al., 2012) and (Van Cuyk et al., 2011) found B. thuringiesis subsp. Kurstaki to persist in urban environments for at least 4 years. Even though we did 153 not test the effects of temperature we also observed similar results showing that B. thuringiensis is less stable at higher relative humidity. E. coli has been described to persist on inanimate surfaces from 1.5 hours to 16 months (Kramer et al., 2006). A study by (Abrishami et al., 1994) found a log10 reduction 0.06 after 24 h of drying on plastic cutting boards in a laminar flow hood. Whereas S. aureus has been reported to persist for 7 days to 7 months on dry inanimate surfaces (Kramer et al., 2006). In the present study we found S. aureus to be more stable with a 0.4 reduction log10 CFU/2-cm2 on nonporous surfaces under high relative humidity than E. coli (2.4 reduction log10 CFU/2-cm2). Our results are consistent with (Scott and Bloomfield, 1990) observations where S. aureus a Gram positive bacteria survives better than E. coli a Gram negative bacteria on laminate. In conclusion, most organisms in this study generally had greater survival under high relative humidity compared to low relative humidity with B. thuringiesis showing the opposite. The present study also demonstrated that fomite type influenced survival with microorganisms having the least reduction from nonporous surfaces. 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Am J Infect Control 38, S25-S33. 163 TABLES TABLE 1 Fomites tested Fomite Nonporous Notes Manufactures Acrylic Poly-methyl methacrylate, matte nonglare finish Home Depot, Atlanta, GA Glass Slides VWR, Mississauga, ON Ceramic Tile Porcelain Home Depot, Atlanta, GA Laminate Various colors Wilsonart International, Temple, TX Stainless Steel Gage 304 AK Steel Corporation, West Chester, OH Granite One from India and two from Brazil GRANITE Kitchen & Bath Countertops, Tucson, AZ Cotton 100% fabric Polyester 100% fabric Paper Currency Cotton-based one dollar bills Home Trends Brand, Walmart, Bentonville, AR Home Trends Brand, Walmart, Bentonville, AR United States Porous 164 TABLE 2 Survival of organisms on fomites under low relative humidity 15% - 32% E. coli Surface Type S. aureus 2 B. thuringiensis 2 MS-2 2 (n) Mean CFU/2-cm log10 ± SD (Range) (n) Mean CFU/2-cm log10 ± SD (Range) (n) Mean CFU/2-cm log10 ± SD (Range) (n) Mean PFU/2-cm2 log10 ± SD (Range) Acrylic (6) 4.8 ± 0.4 (4.5 - 5.4)a (6) 6.2 ± 0.1 (6.1 - 6.4)a (6) 4.8 ± 0.1 (4.6 - 4.9) (6) 8.5 ± 0.2 (8.3 - 8.7)a Glass (6) 5.0 ± 0.4 (4.4 - 5.4) (6) 6.2 ± 0.2 (5.9 - 6.4)a (6) 4.4 ± 0.2 (4.1 - 4.5)a a a Nonporous a (6) 6.2 ± 0.2 (6.0 - 6.4) (6) 4.9 ± 0.1 (4.9 - 5.0) (6) 8.5 ± 0.1 (8.3 - 8.6) (6) 8.8 ± 0.1 (8.6 - 9.0)a Ceramic Tile (6) 4.9 ± 0.2 (4.5 - 5.2) Laminate (6) 4.3 ± 0.3 (4.1 - 4.9)a (6) 6.2 ± 0.1 (6.1 - 6.3)a (6) 4.9 ± 0.05 (4.8 - 5.0) (6) 8.8 ± 0.1 (8.7 - 9.0)a Stainless Steel (6) 4.5 ± 0.6 (3.8 - 5.2)a (6) 6.1 ± 0.2 (5.8 - 6.4)a (6) 4.3 ± 0.1 (4.2 - 4.4)a (6) 7.1 ± 0.9 (6.2 - 8.6)a Granite (6) 4.5 ± 0.3 (4.3 - 5.0) (6) 6.1 ± 0.2 (5.9 - 6.3) (6) 4.7 ± 0.1 (4.7 - 4.8) (6) 8.1 ± 0.2 (7.7 - 8.3) Cotton (6) 2.5 ± 0.7 (1.8 - 3.5) (6) 3.1 ± 0.2 (2.7 - 3.4)a (6) 3.2 ± 0.1 (3.1 - 3.3)a (6) 7.0 ± 0.3 (6.6 - 7.4)a Polyester (6) 3.6 ± 0.4 (3.1 - 4.1) (6) 4.1 ± 0.3 (3.7 - 4.4)a (6) 3.3 ± 0.4 (2.8 - 3.8)a (6) 7.2 ± 0.4 (6.9 - 7.9) (6) 6.0 ± 0.4 (5.6 - 6.6) (6) 4.6 ± 0.1 (4.5 - 4.8) (6) 7.3 ± 0.2 (7.0 - 7.4)a Porous Paper Currency a (6) 4.6 ± 0.2 (4.4 - 4.8) a There was a statistical difference (Student t-test P = 0.05) of the organism survival between low and high relative humidity 165 TABLE 3 Survival of organisms on fomites under high relative humidity 40% - 65% E. coli Surface Type S. aureus 2 B. thuringiensis 2 MS-2 2 (n) Mean CFU/2-cm log10 ± SD (Range) (n) Mean CFU/2-cm log10 ± SD (Range) (n) Mean CFU/2-cm log10 ± SD (Range) (n) Mean PFU/2-cm2 log10 ± SD (Range) Acrylic (6) 6.7 ± 0.2 (6.3 - 6.9)a (6) 6.8 ± 0.2 (6.6 - 7.1)a (6) 4.8 ± 0.1 (4.6 - 4.9) (6) 7.0 ± 0.2 (6.8 - 7.2)a Glass (6) 5.0 ± 0.3 (4.7 - 5.5) (6) 6.9 ± 0.3 (6.6 - 7.1)a (6) 4.9 ± 0.3 (4.4 - 5.3)a a (6) 4.7 ± 0.2 (4.4 - 5.0) a Nonporous a (6) 6.5 ± 0.2 (6.2 - 6.8) (6) 8.4 ± 0.2 (8.2 - 8.7) (6) 8.6 ± 0.2 (8.3 - 8.7)a Ceramic Tile (6) 5.7 ± 0.3 (5.5 - 6.2) Laminate (6) 6.7 ± 0.2 (6.4 - 7.0)a (6) 6.7 ± 0.2 (6.4 - 6.9)a (6) 4.8 ± 0.2 (4.6 - 4.9) (6) 8.6 ± 0.2 (8.3 - 8.8)a Stainless Steel (6) 5.9 ± 0.3 (5.6 - 6.4)a (6) 6.8 ± 0.2 (6.6 - 7.1)a (6) 5.0 ± 0.2 (4.7 - 5.2)a (6) 8.8 ± 0.1 (8.7 - 9.1)a Granite (6) 4.4 ± 1.6 (3.8 - 5.2) (6) 6.4 ± 0.3 (6.1 - 6.7) (6) 4.8 ± 0.1 (4.7 - 4.8) (6) 8.3 ± 0.1 (8.1 - 8.5) Cotton (6) 2.5 ± 0.4 (1.5 - 2.6) (6) 3.7 ± 0.4 (3.2 - 4.3)a (6) 2.6 ± 0.4 (2.0 - 3.1)a (6) 6.3 ± 0.1 (6.1 - 6.5)a Polyester (6) 3.4 ± 0.5 (2.7 - 4.2) (6) 5.0 ± 0.2 (4.8 - 5.4)a (6) 2.9 ± 0.1 (2.8 - 3.2)a (6) 7.1 ± 0.2 (6.9 - 7.3) (6) 6.3 ± 0.1 (6.1 - 6.4) (6) 4.6 ± 0.2 (4.3 - 4.9) (6) 6.9 ± 0.2 (6.7 - 7.2)a Porous Paper Currency a (6) 5.0 ± 0.2 (4.8 - 5.3) a There was a statistical difference (Student t-test P = 0.05) of the organism survival between low and high relative humidity 166 TABLE 4 Survival of PV-1on fomites Surface Type (n) Mean PFU/2-cm2 log10 ± SD (Range) Low RH (15% - 32%) Ceramic Tile (6) 5.5 ± 0.3 (5.1 - 5.9)a Laminate (6) 5.8 ± 0.2 (5.4 - 6.0)a Granite (6) 5.1 ± 0.6 (4.4 - 5.8)a High RH (40% - 65%) a Ceramic Tile (6) 6.2 ± 0.1 (6.0 - 6.4) Laminate (6) 6.5 ± 0.4 (6.3 - 7.3) Granite (6) 6.2 ± 0.1 (6.0 - 6.3) There was a statistical difference (Student t-test P = 0.05) of the organism survival between low and high relative humidity 167 FIGURES B 3.0 1.4 2.5 1.2 Reduc on log10 CFU/2-cm2 Reduc on log10 CFU/2-cm2 A 2.0 1.5 1.0 0.5 1.0 0.8 0.6 0.4 0.2 0.0 0.0 -0.5 -0.2 LRH HRH Acrylic LRH HRH Glass LRH HRH Ceramic Tile LRH HRH Laminate LRH HRH Stainless Steel LRH LRH HRH Average C LRH HRH Glass LRH HRH Ceramic Tile LRH HRH Laminate LRH HRH Stainless Steel LRH HRH Granite D 1.2 2.0 1.0 Reduc on log10 PFU/2-cm2 Reduc on log10 CFU/2-cm2 HRH Acrylic Average Granite 0.8 0.6 0.4 0.2 0.0 1.5 1.0 0.5 0.0 -0.5 -1.0 -0.2 LRH HRH Acrylic LRH HRH Glass LRH HRH Ceramic Tile LRH HRH Laminate LRH HRH Stainless Steel LRH HRH Granite Average LRH HRH Acrylic LRH HRH Glass LRH HRH Ceramic Tile LRH HRH Laminate LRH HRH Stainless Steel LRH HRH Granite Average Figure 1 Reduction of organisms from nonporous fomites after 30 min drying time (A) E. coli 15597, (B) S. aureus 25923, (C) B. thuringiensis spores, and (D) MS2 under low (15% - 32%) and high (40% - 65%) relative humidity. Reduction = -log10 (CFU or PFU fomite/(CFU or PFU titer/100)). 168 A B 4.5 4.0 5.0 Reduc on log10 CFU/2-cm2 Reduc on log10 CFU/2-cm2 6.0 4.0 3.0 2.0 1.0 2.5 2.0 1.5 1.0 0.5 0.0 0.0 LRH HRH LRH Co on HRH Polyester LRH LRH HRH C HRH Co on Paper Currency LRH HRH Polyester LRH HRH Paper Currency Average Average D 3.5 3.5 3.0 3.0 Reduc on log10 PFU/2-cm2 Reduc on log10 CFU/2-cm2 3.5 3.0 2.5 2.0 1.5 1.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 0.5 -1.0 0.0 LRH HRH Co on LRH HRH Polyester LRH LRH HRH Average HRH Co on Paper Currency LRH HRH Polyester LRH HRH Paper Currency Average Figure 2 Reduction of organisms from porous fomites after 30 min drying time (A) E. coli 15597, (B) S. aureus 25923, (C) B. thuringiensis spores, and (D) MS2 under low (15% - 32%) and high (40% - 65%) relative humidity. Reduction = -log10 (CFU or PFU fomite/(CFU or PFU titer/100)). 169 Reduc on log10 PFU/2-cm2 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 -1.0 LRH HRH Ceramic Tile LRH HRH Laminate LRH HRH Granite Average Figure 3 Reduction of Poliovirus 1 from nonporous fomites after 30 min drying time under low (15% - 32%) and high (40% 65%) relative humidity. Reduction = -log10 (CFU or PFU fomite/(CFU or PFU titer/100)). 170 APPENDIX E: THE EFFECT OF A DISINFECTANT WIPE ON MICROBIAL TRANSFER Gerardo U. Lopez1, Masaaki Kitajima1, Aaron Havas1, and Kelly A. Reynolds2 1 Department of Soil, Water and Environmental Science, The University of Arizona, Tucson, Arizona 85721, USA 2 Mel and Enid Zuckerman College of Public Health, The University of Arizona, 1295 N. Martin Ave., Tucson, Arizona 85724, USA Corresponding author. Tel: (520) 626-8230, Fax (520) 626-8009, E-mail: [email protected] A manuscript for International Journal of Hygiene and Environmental Health 171 ABSTRACT Inanimate environmental surfaces, or fomites, can serve as routes of transmission for microbial pathogens. This study investigated the effects of a disinfectant wipe on removing microorganisms from fomites and reducing microbial fomite-to-finger transfer rates. Escherichia coli, Staphylococcus aureus, Bacillus thuringiensis spores and poliovirus 1 were inoculated onto ceramic tile, laminate and granite in 10 µl drops and allowed to dry for 30 min under relative humidity of 15% - 32%. Non-treated control fomites were sampled and remaining surfaces were wiped with a disinfectant wipe and allowed to dry for 10 min. Fomite-to-finger transfers were then performed on the remaining surfaces using a 1.0 kg/cm2 pressure for 10 sec. Test organisms were reduced by 98.1% to 99.997% on the fomites after the surfaces were wiped. Microbial fomite-tofinger transfer from disinfectant wipe-treated surfaces were, lower (less than 0.5%) than from non-treated surfaces (up to 36.3%). This is the first study providing data on the reduction of fomite-to-finger microbial transfer with a disinfectant wipe. Keywords: Disinfectant wipes, Fomites, Fomite-to-finger transfer, Escherichia coli, Staphylococcus aureus, B. thuringiensis spores 172 INTRODUCTION Inanimate objects and surfaces (fomites) are known to play a role as a reservoir for the transmission of pathogens in the environment either directly, by surface-to-mouth contact, or indirectly, by contamination of fingers and subsequent hand-to-mouth, hand-to-eye, hand-to-nose contact and even through cuts in the skin (Haas et al., 1999, Nicas and Best, 2008). Studies have been conducted in laboratory settings to model food preparation in domestic kitchens (De Jong et al., 2008, Van Asselt et al., 2008) to better understand crosscontamination of foodborne pathogens. In addition the occurrence and spread of pathogens throughout the home have been conducted to better understand the role of fomites in exposure (Fekety et al., 1981, Josephson et al., 1997, Medrano-Félix et al., 2010, Rusin et al., 1998, Scott et al., 1982, Sinclair and Gerba, 2011, Speirs et al., 1995). It is estimated that up to 87% of reported foodborne outbreaks are associated with food prepared or consumed in the home (Van Asselt et al., 2008). In the United States, foodborne pathogens cause an estimated 48 million cases of illnesses, 128,000 hospitalizations and 3,000 deaths per year (CDC, 2011). Microbial contaminated surfaces (fomites) in hospitals and nursing homes contribute to healthcare-acquired infections (HAI) (Bhalla et al., 2004, Bonten et al., 1996, Boyce et al., 1997, Hayden et al., 2006, Ray et al., 2002, Weber and Rutala, 2011, Weber et al., 2010). Fomites have been implicated in the transmission of hospital-acquired infections (HAIs) 173 because they are in close proximity to the patient and are frequently touched by hands (Bhalla et al., 2004, Dancer, 2009, Dancer et al., 2008, Kramer et al., 2006, Murphy et al., 2012, Sexton et al., Siani et al., 2011, Weber et al., 2010, White et al., 2008). In addition, HAIs caused by methicillin-resistant Staphylococcus aureus (MRSA), methicillinsusceptible S. aureus (MSSA), and Clostridium difficile are associated with high morbidity and mortality (Cosgrove et al., 2005, Humphreys, 2009, Murphy et al., 2012, Schweickert et al., 2011, Siani et al., 2011, Weber et al., 2010, Williams et al., 2007, Williams et al., 2009). Pathogen presence and survival on fomites found in domestic homes, public places, hospitals, and other healthcare facilities are important factors in evaluating human exposure potentials (Reynolds et al., 2005). Most nosocomial and foodborne pathogens can persist on fomites for weeks or even months (Kramer et al., 2006, Masago et al., 2008, Neely and Maley, 2000, Weber et al., 2010) and up to several hours on fingers (Ansari et al., 1988, Mbithi et al., 1992, Rheinbaben et al., 2000). Hand and environmental hygiene are crucial in preventing the spread of infectious disease in homes, healthcare facilities, and public places. Numerous studies have focused specifically on hand hygiene interventions (Bloomfield et al., 2008, Boyce and Pittet, 2002, Muto et al., 2003, Sattar et al., 2000, Scott et al., 2010, Weber et al., 2010). Including specific studies of healthcare workers’ hand known as vehicles of nosocomial pathogen transmission (Boyce and Pittet, 2002, Farrington et al., 1990, Laborde et al., 174 1993, Mermel et al., 1997, Muto et al., 2003). Publications by the Centers for Disease Control and Prevention (CDC), such as The Guidelines for Hand Hygiene in Health-Care Settings and other organizations recommend that healthcare workers routinely sanitize or wash their hands after contact with inanimate objects in the immediate vicinity of all patients (Bhalla et al., 2004, Bloomfield et al., 2008, Boyce and Pittet, 2002). Improved cleaning and disinfection of the environment has been shown to reduce transmission of pathogens (Bhalla et al., 2004, Bloomfield et al., 2008, Boyce et al., 2010, Hayden et al., 2006, Kramer et al., 2006, Mayfield et al., 2000, Muto et al., 2003). Several studies have examined the efficacy of surface cleaning and hygiene to reduce pathogen exposure in households (Bloomfield et al., 2008, Medrano-Félix et al., 2010, Scott et al., 2010), hospitals (Bhalla et al., 2004, Bloomfield et al., 2008, Boyce et al., 2010, Dancer, 2009, Dancer et al., 2008, Hayden et al., 2006, Kramer et al., 2006, Mayfield et al., 2000, Muto et al., 2003, Weber et al., 2010, White et al., 2008, Williams et al., 2007, Williams et al., 2009), and nursing homes (Murphy et al., 2012). However, few studies have quantitatively assessed the efficiency of microbial transfer to and from various surfaces or the efficacy of a disinfectant intervention to inhibit such transfers. Such data is needed for the development of quantitative microbial risk assessment models to assess the impact of interventions on reduction in the risk of infection (Atkinson and Wein, 2008, Boone and Gerba, 2007, Brankston et al., 2007, Gerba et al., 1996, Nicas and Best, 2008). This study provides information to reduce the 175 uncertainty in risk models and aids to better quantitatively predict the public health benefit of targeted hygiene interventions. MATERIALS AND METHODS Subjects One subject conducted the fomite-to-finger transfer experiments. Permission was obtained from the University of Arizona Office for Human Subjects Research prior to the study. Bacteria, endospores, and preparation of inocula Study organisms Escherichia coli C-3000 (ATCC 15597), Staphylococcus aureus (ATCC 25923), and Bacillus thuringiensis (ATCC 10792) were obtained from the American Type Culture Collection (ATCC Manassas, VA). Poliovirus 1 (PV-1; strain LSc-2ab) was obtained from the Department of Virology and Epidemiology at the Baylor College of Medicine (Houston, TX). These organisms were selected as model organisms Gram negative and Gram positive bacteria, spore-forming bacteria, and viruses. Gram negative and Gram positive inoculum preparation Frozen aliquots of E. coli and S. aureus were transferred into separate 150 ml volumes of tryptic soy broth, (TSB) (EMD, Gibbstown, NJ) incubated for 18 ± 2 h at 35 ± 2°C on an orbital shaker (180 rpm), and streaked for isolation onto tryptic soy agar (TSA) (EMD 176 Gibbstown, NJ). The bacteria were then subcultured to a new flask of TSB, and incubated as before (Rusin et al., 2002). Endospore-forming bacteria inoculum preparation B. thuringiensis spores were prepared as previously described with slight modification (Nicholson and Setlow, 1990). Briefly, spores were suspended in Difco sporulation media with supplements (DSM + S; Becton, Dickinson and Company, Sparks, MD), cultivated for 24 h at 37°C on an orbital shaker (150 to 180 rpm), and resuspended in fresh DSM + S to obtain a final OD600 of 0.1 (Spectronic Genesys 5, Milton Roy, Ontario Canada). Virus inoculum preparation PV-1 propagation and plaque-forming assays were conducted as described previously (Bidawid et al., 2003, Ikner et al., 2011). Briefly, poliovirus 1 was propagated on buffalo green monkey kidney (BGM; ATCC CCL-81; American Type Culture Collection, Manassas, VA) cell line monolayers with minimal essential media (MEM) containing 5% calf serum (HyClone Laboratories, Logan, UT) at an incubation temperature of 37°C with 5% CO2. Plaque-forming assays were performed using six-well plates with confluent monolayers of the BGM cells. Control wash and disinfection Prior to all experiments volunteer’s hands were washed with warm water and nonantibacterial soap (Liquid Joy, Procter and Gamble, Cincinnati, OH) for 45 s, rinsed with 177 water, and dried with paper towels. Each hand was then sprayed twice with 70% ethanol, rubbing the alcohol thoroughly over hands and wrists for 15 s, and allowing them to airdry. After seeded experiments with E. coli and S. aureus, fingers were disinfected twice with 70% ethanol and wrapped with a 70% ethanol saturated paper towel for 30 s, then washed and rinsed using warm water and Softsoap® liquid hand soap (Colgate-Palmolive, Morristown, NJ) for 45 s, and then dried with paper towels. After seeded experiments with B. thuringiensis spores and PV-1, fingers were placed in 10% sodium hypochlorite solution (The Clorox Company, Oakland, CA) for 15 s and then neutralized in 10% sodium thiosulfate (EMD, Gibbstown, NJ). The hands were then washed as described above to prepare for subsequent trials. Fomites tested Three types of fomite materials were tested ranging in surface areas of 16 to 25 cm2: ceramic tile porcelain (Home Depot, Atlanta, GA), laminate (Wilsonart International. Temple, TX), and three different types of granite, one from India and two from Brazil (GRANITE Kitchen & Bath Countertops, Tucson AZ). After fomite to finger transfers with E. coli, and S. aureus, nonporous fomites were sprayed three times with 70% ethanol and allowed to sit for 10 min. After seeded experiments with B. thuringiensis spores and PV-1, were disinfected with 10% sodium hypochlorite solution (The Clorox Company, Oakland, CA) allowed to sit for 10 min and subsequently neutralized in 10% sodium thiosulfate (EMD, Gibbstown, NJ). All fomites were then washed under warm running water with non-antibacterial soap (Liquid Joy, Procter and Gamble, Cincinnati, 178 OH), rubbed with a wet paper towel on the surface area of inoculation, rinsed thoroughly with RO treated water, air-dried, autoclaved, and reused. Disinfectant wipe and Neutralizing Solution The disinfectant tested in this study was a ready-to-use disinfectant wipe CLOROX Disinfecting Wipes (The Clorox Company, Oakland, CA) with a square measure of 20.5 cm x 18 cm. The active ingredients were: n-Alkyl (C14, 60%; C16, 30%; C12, 5%; C18, 5%;) Dimethyl Benzl Ammonium Chloride 0.184%, n-Alkyl (C12, 68%; C14, 32%) Dimethyl Ethylbenzyl Ammonium Chloride 0.184%. Other ingredients 99.632%. D/E neutralizing medium was used to neutralize the bactericidal effects of the disinfectant wipe (D/E, EMD, Gibbstown, NJ). Relative Humidity and Temperature The study was conducted at a relative humidity of 15% - 32% (typical indoor relative humidity in Tucson, AZ) and temperature of 19°C to 25°C (i.e., room temperature). Temperature and relative humidity were monitored with a High Accuracy Thermo – Hygro (VWR, Mississauga, ON). During high relative humidity in the laboratory, t.h.e.® Desiccant (EMD, Gibbstown, NJ) and Drierite desiccant (Drierite, Xenia, OH) were used to decrease the relative humidity to the low relative humidity range (15% to 32%). 179 Inoculation of fomites Layout of fomites For each of the three nonporous fomites ten fomite test coupons were evenly spaced in three rows on the middle shelf of an incubator. Each trial consisted of three control coupons without the disinfectant wipe application, three control coupons with disinfectant wipe use, three fomite-to-finger transfer coupons with disinfectant wipe use, and one negative control coupon to ensure fomites were not previously contaminated. Organism concentration The concentration of organisms added to the fomites was approximately 108 to 109 colony forming units (CFU)/cm2 of E. coli in (TSB, EMD, Gibbstown, NJ), 109 CFU/cm2 of S. aureus in TSB, 107 to 108 CFU/cm2 of B. thuringiensis spores in (DSM + S; Becton, Dickinson and Company, Sparks, MD), and 108 plaque forming units (PFU)/cm2 of PV-1 in (PBS, Sigma-Aldrich, St. Louis, MO) in 10 µl droplets. Using a pipet tip, the 10 µl inoculum droplets were spread over approximately 1.0 cm2 area on the center of each fomite. Fomites were seeded in time intervals to provide a consistent 30 min drying time to each coupon. Fomite sampling Fomites inoculated with E. coli, S. aureus, and B. thuringiensis were sampled using a cotton-tipped swab applicator (Puritan Medical Products Company, Guilford, ME). In the case of poliovirus a polyester fiber-tipped applicator swab (Falcon, Becton Dickinson 180 and Company, Cockeysville, MD) was used. Swabs were moistened with 1.0 ml of D/E neutralizing medium (D/E, EMD, Gibbstown, NJ), a 6.0 cm2 area was swabbed with the applicator firmly against the surface and in a rotating motion before placed back into the remaining D/E and vortexed for 5 s. Control fomites without the disinfectant wipe application were sampled as described after a 30 min drying time. Intervention application A protocol adapted from William et al. (2007) and Siani et al. (2011) were used for the use of the disinfectant wipe to assess bacterial and viral removal from contaminated surfaces. Three control coupons and three fomite-to-finger transfer coupons were removed one at a time from the incubator placed on the counter and held stationary with the left hand. Coupons were treated with disinfectant wipes using a surface pressure of approximately 300 – 500 g ± 50 applied in one sweeping motion from the far corner of the coupon towards the proximal corner as previously described (Williams et al., 2009). The disinfectant wipe was then discarded and the fomite was placed back into the controlled humidity and room temperature incubator for an additional 10 min of drying. Transfer experiments The three control fomites with disinfectant wipe application were sampled as described above at the end of 10 min. Fomite-to-finger transfer experiments were then conducted with the remaining three swatches/coupons using the index, middle, and ring fingers of the right hand. The transfer was done using a method adapted from Ansari et al. (1988) 181 and Mbithi et al. (1992) by placing the fomite at the center of a scale with a digital readout and performing a finger transfer by placing the right hand index finger on the center covering the inoculated area of the fomite, for ten seconds with a 1.0 kg/cm2 (98.0665 kPa) of average pressure (range of 700 g to 1500 g). Finger transfers with the middle and ring fingers were conducted in the same fashion to complete one trial. Sampling the fingers Using a cotton-tipped swab applicator (Puritan Medical Products Company, Guilford, ME), moistened in 1.0 ml of D/E neutralizing medium (D/E, EMD, Gibbstown, NJ), the index finger pad was sampled by a sweeping, rotating motion and placed into the remaining D/E vial and vortexed. Middle and ring fingers were sampled in the same way. To sample PV-1 to finger transfers, a polyester fiber-tipped applicator swab (Falcon, Becton Dickinson and Company, Cockeysville, MD) was used. Organism assays Organisms were eluted from the swab with 100 µl of D/E neutralizing medium (D/E, EMD, Gibbstown, NJ), diluted, and quantitated for E. coli, S. aureus, and B. thuringiensis spores using the spread plate technique on MacConkey, mannitol salt agar (MSA), and tryptic soy agar (TSA) (EMD Chemicals Inc, Gibbstown, NJ), respectively. Agar plates were incubated at 35 ± 2°C for 18 ± 2 h. B. thuringiensis spore samples were heat shocked at 81 ± 2°C for 10 min prior to spread plating, to stimulate germination. PV-1 titrations were performed using 10-fold serial dilution plaque-forming assay previously 182 described (Bidawid et al., 2003, Ikner et al., 2011). Dilutions and plating were done in duplicate. Transfer efficiency, log10 reduction, and statistical analysis Calculation of transfer efficiency Bacterial colonies and plaques were enumerated and transfer efficiencies were calculated using Eqn (1) (Marples and Towers, 1979, Paulson, 2005). Transfer efficiency (TE) is defined as CFU or PFU recovered from finger relative to CFU or PFU recovered from control fomite without intervention. If bacteria or phage were not recovered from the finger pad, the lower detection limit of 10 CFU or PFU was used as an estimate for the microorganism recovered as previously described (Chen et al., 2001, Julian et al., 2010). A less than value (<) was used to indicate the transfer efficiency was less than the least detectable limit. Finger transfers greater than 100% were truncated to 100%. % / / . 100 (1) Log10 reduction was calculated using Eqn (2) and is described as the negative Log10 of the value of CFU or PFU concentration recovered from the control fomite with intervention divided by CFU or PFU recovered from fomite without intervention. / . / . (2) 183 Data were entered into the software package StatPlus:mac, 2009, (AnalystSoft) to compute descriptive statistical measures of mean survival, standard deviation, minimum, maximum and statistical significance. Student’s t-test was used to assess the statistical significance (P = 0.05) of the CFU/2-cm2, CFU/finger, and percent transfer efficiency between the treated and non-treated surfaces. RESULTS Removal of microbial contamination from nonporous surfaces E. coli, S. aureus, B. thuringiensis spores and poliovirus recovered from disinfectant wipe-treated fomites was compared to concentrations recovered from non-treated fomites. Greater numbers of test organisms were recovered from non-treated surfaces (4.5 – 6.9 log10 CFU or PFU/2-cm2) than from disinfectant wipe-treated surfaces (< 1.2 – 2.7 log 10 CFU or PFU/2-cm2) (Table 1). There were significantly fewer (Student’s t-test, P < 0.05) E. coli, S. aureus, B. thuringiensis spores, and poliovirus recovered from ceramic tile, laminate, and granite following the application of a disinfectant wipe. Reduction of B. thuringiensis spores was always lower than the other microorganisms (Table 1), indicating that B. thuringiensis spores are less efficiently removed/killed by the disinfectant wipe. 184 Influence of disinfectant wipes on Microbial Transfer Transfer of E. coli, S. aureus, B. thuringiensis and PV-1 from disinfectant wipe-treated fomites to fingers were compared to microbial transfer from non-treated fomites to fingers reported by Lopez et al., (submitted for publication) (Table 2 and 3). All model microorganisms had lower transfer and recovery to fingers from disinfectant wipe-treated fomites (< 0.0002% to 0.1%) and (< 1.0 – 1.7 log10 CFU or PFU/2-cm2), respectively than from non-treated fomites (< 0.04% to 36.3%) and (1.2 – 5.3 log10 CFU or PFU/2cm2), respectively (Tables 2 and 3). Transfer from disinfectant wipe-treated fomites to fingers was significantly lower than that from non-treated fomites for E. coli for ceramic tile, S. aureus, B. thuringiensis, and PV-1 for ceramic tile and laminate (Student t-test, P ≤ 0.05). There was also a statistical difference (Stuent’s t-test, P ≤ 0.05) for E. coli, S. aureus and PV-1 recovered from fingers in contact with all disinfectant wipe-treated surfaces and B. thuringiensis for ceramic tile and laminate than those recovered from fingers in contact with non-treated surfaces. DISCUSSION The purpose of this study was to determine the impact on fomite-to-finger transfer of various types of microorganisms from surfaces contaminated treated with a disinfectant wipe. Such information can be used to assess this type of intervention in reducing exposure (Gibson et al., 1999, Haas et al., 1999, Nicas and Sun, 2006, Wein and 185 Atkinson, 2009). As expected the present study showed that the use of disinfectant wipes greatly reduced the number of bacteria and virus on contaminated surfaces that are commonly found in indoor environments (Table 1). Our results demonstrate that the use of disinfectant wipes on surfaces greatly reduced the potential transfer of E. coli, S. aureus, B. thuringiensis spores, and PV-1 to fingers (Tables 2 and 3). S. aureus was reduced by four orders, while E. coli and PV-1 was reduced by three. B. thuringiensis spores are the most resistant to wipe disinfectants and were therefore the most resistant to removal. Williams et al. (2007) using a three-stage efficacy protocol tested grapefruit extractcontaining surface wipes for the removal of several different strains of methicillinresistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) on stainless steel discs (step 1). Williams et al. (2007) inoculated stainless steel discs with 20 uL of test suspension 6-7 log10 cfu/disk and dried at 37°C for 25 min. Test suspension consisted of bovine serum albumin (BSA) concentration to mimic clean (0.3 g/L) and dirty (3 g/L) conditions. Cell removal was assessed by the attachment of a disinfectant-containing wipe to a steel rod to allow mechanical rotation with a drill using an exerting weight between the disc and the wipe. They used these parameters to follow the observation of staff using the wipes in Intensive Therapy Units (Williams et al., 2007) and found cell removal of 3 – 6.73 log10 CFU/disc and 2.55 – 4.66 log10 CFU/disc under clean and dirty conditions, respectively. Our study observed similar result with a bacterial removal for S. aureus (4.2 – 5.7 log10, 4.4 – > 6.3 log10, and 3.4 – > 6.2 log10) for ceramic tile, laminate, 186 and granite, respectively, (data not shown) using a wipe containing quaternary ammonium compounds (QAC), even though we used different methodologies and parameters than (Williams et al., 2007). Our study used a recommendation by Williams et al. (2009) of a “one wipe – one application – one direction” approach to wipe the surface, with a surface pressure similar to Siani et al. (2011) of 300 – 500 g. This wiping method is possibly a more reasonable application of the disinfectant wipe. A 10 min drying time followed the application of the intervention wipe. The additional 10 min drying time after the intervention was applied allowed any residual quaternary ammonium compounds (QAC) remaining on the fomite, to interact with the bacteria and spores resulting in greater bactericidal and sporicidal affects. As expected the present study showed an effective cell removal with S. aureus showing a % reduction (up to 99.997%) and a viability loss of a log10 reduction (up to 5.0 log10 CFU/2-cm2) for the nonporous fomites (Table 1). In a similar study Siani et al. (2011) examined the efficacy of nine sporicidal wipes on two strains of Clostridium difficile spores. Using a modified three-stage protocol described by Williams et al. (2007) stainless steel discs were inoculated with C. difficile suspension medium and dried for 1.0 h at 37°C. Siani et al. (2011) reported bacterial removal for both C. difficile strains to be < 2.5 log10 CFU/disk for most wipes tested. Our model bacteria for C. difficile was B. thuringiensis spores. B. thuringiensis was the most resistant organism to the intervention with a % reduction up to 99.4%, a log10 reduction 187 of up to 2.5 log10 CFU/2-cm2, and spore removal of > 6 log10 CFU/2-cm2 on nonporous surfaces. The influence of disinfectant wipes on E. coli and poliovirus from ceramic tile, laminate, and granite was similarly observed with the other microorganisms resulting in a percent reduction, log10 reduction, and a total cell removal for E. coli of > 99.97%, > 4.0 log10 CFU/2-cm2, > 6.2 log10 CFU/2-cm2 and for PV-1 of up to 99.98%, 4.0 log10 PFU/2-cm2, 5.0 log10 PFU/2 cm2, respectively (Table 1). This present study demonstrated that there were significantly fewer (Student’s t-test, P ≤ 0.05) E. coli, S. aureus, B. thuringiensis spores, and poliovirus recovered from treated surfaces than non-treated and while this is an expected outcome, quantitative reduction levels are necessary to detail future intervention efficacy can be mathematically modeled. To our knowledge, this is the first study that quantitates microbial transfer rates after the use of disinfectant wipes as an intervention (Table 2). Fomite-to-finger transfer efficiencies from non-treated surfaces compared to treated surfaces were: E. coli (7.3 to 21.7% and < 0.004), S. aureus (2.7 to 4.3% and < 0.002), B. thuringiensis spores (0.04 to 0.2% and < 0.03), and poliovirus type 1 (23.1 to 36.3% and < 0.004), respectively. S. aureus, B. thuringiensis, and poliovirus had a significant difference (Student t-test, P ≤ 0.05) with ceramic tile and laminate. All tested organisms did not show any significant difference in pre/post intervention reduction with granite. This could be attributed to the more porous nature of granite surface, it was sealed by the manufacturer, or specific 188 differences, in microbial attachment mechanisms to various surfaces. Unlike the transfer efficiencies observed by the other model organisms from non-treated fomites B. thuringiensis was transferred less than the other organisms (Table 2 and 3). These results showed the importance of disinfectant wipes in reducing fomite-to-finger transfer of microorganisms. The fomite-to-finger transfer will improve model accuracy where this information could not previously be modeled to predict disinfectant efficacy and exposure following intervention use and public health benefit of the intervention (Boone and Gerba, 2007, Nicas and Best, 2008). With this data, we can mathematically predict these outcomes, which will help to make cost benefit decisions based on the wipe methods efficacy and the interventions role in reducing human exposures and risk. CONCLUSIONS The present study showed disinfectant wipes to be very effective in reducing fomite-tofinger bacterial transfer in indoor environments. Our findings also showed that less fomite-to-finger transfer occurs when surfaces have been treated with disinfectant wipes compared to non-treated surfaces (fomites). 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Epidemiol. 30, 570-573. 198 TABLES TABLE 1 Recovered microorganisms from fomites pre and post disinfectant wipe interventionb Surface Type E. coli Mean ± SD (Range) S. aureus c Mean ± SD (Range) B. thuringiensis c Mean ± SD (Range) Poliovirus Type 1 c Mean ± SD (Range)c Ceramic Tile Non-treated 5.1 ± 0.4 (4.8 - 5.5) Treated < 1.6 ± 0.9 (< 1.0 - 3.2) Reduction 3.6 ± 0.9 (2.3 - > 4.5) a 6.8 ± 0.1 (6.7 - 6.8) 4.6 ± 0.2 (4.4 - 4.8) 5.6 ± 0.4 (5.1 - 6.2) 2.3 ± 0.6 (1.5 - 2.9) 2.7 ± 0.4 (2.2 - 3.4) 1.6 ± 0.4 (1.0 - 2.1) 4.5 ± 0.6 (3.7 - 5.3) 1.9 ± 0.5 (1.1 - 2.5) 3.9 ± 0.8 (3.0 - > 5.2) Laminate Non-treated 5.1 ± 0.9 (4.2 - 6.6) Treated < 1.2 ± 0.4 (< 1.0 - 1.9) Reduction 4.0 ± 0.9 (3.2 - > 5.6) 6.9 ± 0.1 (6.8 - 7.1) a < 1.9 ± 0.8 (< 1.0 - 2.9) 4.6 ± 0.1 (4.5 - 4.7) a 5.0 ± 0.8 (4.0 - > 5.8) < 2.1 ± 0.6 (< 1.0 - 2.6) 5.6 ± 0.1 (5.5 - 5.7) a 2.5 ± 0.7 (1.9 - > 3.6) < 1.8 ± 0.5 (< 1.0 - 2.5)a 3.8 ± 0.5 (3.2 - > 4.7) Granite Non-treated 5.4 ± 0.1 (5.3 - 5.6) Treated < 1.9 ± 1.0 (< 1.0 - 3.2) Reduction 3.5 ± 0.9 (2.4 - > 4.3) 6.8 ± 0.3 (6.4 - 7.1) a < 2.5 ± 1.2 (< 1.0 - 3.8) 4.4 ± 1.1 (3.1 - > 5.7) 4.5 ± 0.3 (4.1 - 4.9) a < 2.4 ± 0.8 (< 1.0 - 3.2) 2.1 ± 0.9 (1.3 - > 3.8) 5.5 ± 0.2 (5.0 - 5.7) a < 1.8 ± 0.8 (< 1.0 - 2.8)a 3.7 ± 0.9 (2.8 - > 4.5) a Transfer of organisms from fomite to fingers for one or more transfer events, were below the detectible limit of 10 CFU/2-cm2 b There were six replicates for each organism c Mean ± SD (Range) are expressed as CFU or PFU/2-cm2 log10 199 TABLE 2 Fomite-to-finger percent transfer of microorganismsd E. coli S. aureus B. thuringiensis Poliovirus Type 1 Transfer (%) ± SD (Range) Transfer (%) ± SD (Range) Transfer (%) ± SD (Range) Transfer (%) ± SD (Range) Without Interventionb 11.6 ± 11.8 (0.1 - 33.3) 2.7 ± 2.3 (0.8 - 6.7) < 0.2 ± 0.1 (< 0.1 - 0.4)a 23.1 ± 24.0 (0.4 - 52.7) With Intervention < 0.01 ± 0.01 (< 0.003 - < 0.02)a < 0.003 ± 0.01 (< 0.0001 - < 0.02)a < 0.04 ± 0.03 (< 0.02 - < 0.1)a < 0.01 ± 0.02 (< 0.003 - 0.05) 21.7 ± 23.9 (5.2 - 66.5) 4.3 ± 2.4 (1.3 - 7.4) < 0.2 ± 0.1 (< 0.1 - 0.3)a Surface Type Ceramic Tile Laminate Without Interventionb a 36.3 ± 8.7 (24.1 - 50.0) < 0.02 ± 0.03 (< 0.0002 - < 0.1) < 0.0002 ± 0.0001 (< 0.0001 - < 0.0004) < 0.03 ± 0.01 (< 0.02 - < 0.03) < 0.004 ± 0.004 (< 0.002 - 0.01)a Without Interventionb < 7.3 ± 10.6 (< 0.1 - 28.0)a 3.9 ± 5.0 (0.7 - 13.9) < 0.04 ± 0.03 (< 0.02 - 0.1)a 33.8 ± 40.4 (0.4 - 100)c With Intervention < 0.004 ± 0.001 (< 0.002 - < 0.01)a < 0.001 ± 0.001 (< 0.0002 - < 0.004)a < 0.1 ± 0.1 (< 0.01 - < 0.2)a < 0.01 ± 0.01 (< 0.002 - 0.02)a With Intervention a a Granite a Transfer of organisms from fomite to fingers for one or more transfer events, were below the detectible limit of 10 CFU/2-cm2 b Percent transfer data from (Lopez et al., submitted for publication) c Transfer event was > 100% and was truncated to 100% d There were six replicates for each organism 200 TABLE 3 Recovered microorganims from fingersc Surface Type E. coli Mean ± SD (Range) S. aureus d Mean ± SD (Range) B. thuringiensis d Mean ± SD (Range) Poliovirus Type 1 d Mean ± SD (Range)d Ceramic Tile Without Interventionb 3.6 ± 0.7 (2.4 - 4.1) 4.5 ± 0.3 (4.1 - 4.9) < 2.1 ± 0.2 (< 2.0 - 2.5)a 4.4 ± 0.6 (3.4 - 5.0) With Intervention < 1.0 ± 0.0 (< 1.0 - < 1.0)a < 1.7 ± 0.7 (< 1.0 - 2.9)a < 1.1 ± 0.2 (< 1.0 - < 1.4)a < 1.6 ± 0.5 (< 1.0 - 2.3)a 3.5 ± 0.4 (2.8 - 3.9) 4.7 ± 0.3 (4.2 - 5.0) < 2.1 ± 0.2 (< 2.0 - 2.4)a 5.3 ± 0.2 (5.0 - 5.6) Laminate Without Interventionb With Intervention < 1.0 ± 0.0 (< 1.0 - < 1.0) a < 1.2 ± 0.3 (< 1.0 - 1.5) a < 1.0 ± 0.0 (< 1.0 - < 1.0) a < 1.1 ± 0.2 (< 1.0 - 1.5)a Granite Without Interventionb With Intervention < 2.9 ± 0.7 (< 2.2 - 3.7)a < 1.0 ± 0.0 (< 1.0 - < 1.0) < 1.2 ± 0.3 (< 1.0 - 1.6)a 4.5 ± 0.3 (4.1 - 5.0) a < 1.5 ± 0.6 (< 1.0 - 2.5) a < 1.2 ± 0.2 (< 1.0 - 1.5) a a Transfer of organisms from fomite to fingers for one or more transfer events, were below the detectible limit of 10 CFU/2-cm2 b Recovery of organism without intervention reported in (Lopez et al., submitted for publication) c There were six replicates for each organism d Mean ± SD (Range) are expressed as CFU or PFU/2-cm2 log10 4.1 ± 1.3 (2.5 - 5.5) < 1.3 ± 0.2 (< 1.0 - 1.4)a 201 APPENDIX F: RISK OF CAMPYLOBACTER JEJUNI INFECTION FROM PREPARING RAW CHICKEN IN DOMESTIC KITCHENS AND CROSSCONTAMINATION REDUCTION FROM DISINFECTANT WIPES Gerardo U. Lopez1, 2, Masaaki Kitajima1, Jonathan Sexton1, 2, Charles P. Gerba1, and Kelly A. Reynolds2* 1 Department of Soil, Water and Environmental Science, University of Arizona, Tucson, Arizona 85721, USA 2 The University of Arizona, Mel and Enid Zuckerman College of Public Health, 1295 N. Martin Ave., Tucson, Arizona 85724, USA * Corresponding author. Tel: (520) 626-8230, Fax (520) 626-8009, E-mail: [email protected] A manuscript for Risk Analysis 202 ABSTRACT Chicken bought at a retail store has a 71% chance of being contaminated with Campylobacter jejuni. Using a new Consumer Phase Model (CPM) we conducted a quantitative microbial risk assessment on the risk of infection following the preparation of raw chicken filet in a domestic Kitchen. Using a Monte Carlo simulation of the risk of transferring C. jejuni strain A3249, from various surfaces to hands and subsequently transferring it to the mouth was forecasted. The use of a disinfectant wipe intervention to disinfect contaminated surface area was also assessed. Several assumptions were used as input parameters in the classical Beta-Poisson model to determine the risk of infection. The disinfectant-wipe intervention reduced the risk of Campylobacter infection, illness, and death by 2 to 3 orders on all fomites. The disinfectant-wipe intervention reduced the annual risk of illness below the reported national average of diagnosed Campylobacteriosis cases 1.3E-04. This risk assessment demonstrates that the use of disinfectant wipes to decontaminate surface areas after chicken preparation reduces the risk of C. jejuni infections up to 99.2%. KEY WORDS: Campylobacter jejuni, Quantitative Microbial Risk Assessment, disinfectant wipes, nonporous surfaces, fomite-to-hand transfer 1. INTRODUCTION Foodborne infection remains a significant worldwide problem (1-3). Foodborne pathogens cause a broad range of diarrhoeal to flu like symptoms (4). It is estimated that 1.8 million childhood deaths are associated with disease-causing organisms acquired from food consumption with developing countries having the greatest number of cases (5). In addition more than 130 million people in European countries and over 17 million people in Australia suffer from foodborne diseases (6). In the United States, foodborne pathogens cause an estimated 48 million cases of illnesses, 128,000 hospitalizations and 3,000 deaths per year (7). The annual cost of foodborne illnesses caused by the four most common bacterial pathogens alone (Salmonella strains, Shigella, Campylobacter species, and Escherichia coli has been estimated at 6.9 billion (8). There is growing awareness of the foodborne illness transmission from household exposures (6, 8). Even though the most common cause of foodborne acquired infections has been attributed to the global food distribution and international travels, in European countries such as England, Wales and the Netherlands, 80% of Salmonella and Campylobacter infections are acquired in the home (3, 4). According to Van Asselt (9) up to 87% of reported foodborne outbreaks are associated with food prepared or consumed in the home. Several studies conducted in homes and in the laboratory show that Campylobacter jejuni, Clostridium perfringes, Escherichia coli 0157:H7, Listeria monocytogenes, non-typhoid Salmonella and Staphylococcus aureus are the primary pathogens of concern (1, 4, 10-14). 204 Raw food, whether from vegetable or uncooked/undercooked animal meat sources can be contaminated with human pathogens and transmitted to humans (4, 13-17). The food with the greatest potential risk is raw poultry meat. Handling of poultry in domestic kitchens has been shown to widely disseminate bacteria throughout the kitchen (18, 19). The potential for cross-contamination in the kitchen via counters, cutting boards, and other hand and food contact surfaces, after preparation of raw chicken contaminated either naturally or seeded with marker organisms, has been demonstrated by several studies. Dewit et al. (10) found that 60 families were each given a frozen chicken artificially contaminated with Escherichia coli K12, they were able to recover the indicator bacterium from many sites within the kitchen, often after routine cleaning. Another study isolated Campylobacter jejuni from 170 (61%) of 279 chicken products, while Salmonella was isolated from 44 (54%) of 81 chicken products(18). Several studies have demonstrated that the preparation and cooking of chicken resulted in the frequent transfer of Campylobacter and Salmonella to various surfaces throughout the kitchen (1, 20, 21). A study conducted by Humphrey et al. (11) found that Salmonella enteritidis PT4 was recovered kitchen utensils, from fingers, and work surfaces after breaking of artificially contaminated eggs. Potential pathogens from sources such as raw foods, humans, and animals can be transferred between inanimate and animate surfaces through either direct or indirect contact (9). During outbreaks of infection within the home, this transfer between animate and inanimate surfaces may even give rise to cyclic infection (3). According to Kagan et al. (2) , secondary infectious disease transmission occur between 6 to 60% of the time within 205 households in which one member is ill. Evidence from cross-contamination studies, both in the field and in simulated laboratory scenarios exists for several areas in the home, including toilets, kitchen counters, and cutting boards, together with other hand and food contact surfaces, kitchen sponges and cloths, and household laundry (12, 22). In a review of consumer phase models (CPM) on quantitative microbial risk assessment (QMRA), Nauta et al. (23) found that cross-contamination from fresh chicken meat to other foods and hands, is the dominant route of exposure in all risk assessments and not undercooking. This risk assessment study focuse specifically on forecasting the exposure to C. jejuni contaminated surfaces and the transfer of C. jejuni to hands and then finally to the mouth. Therefore, the risk of infection from C. jejuni was evaluated after a consumer prepared chicken filets at home and C. jejuni cross-contaminated the surface and is transferred to a household members fingers after touching the contaminated surface (fomite). We also investigate how using disinfectant wipe intervention to clean the possible contaminated work area decreases the risk. We used a CPM on C. jejuni strain A3249 for quantitative microbial risk assessment in order to compare the risk of infection under two different scenarios. Both scenarios involve the preparation of a chicken filet dinner resulting in cross-contamination to three different surface (fomite) materials (ceramic tile, laminate, and granite) where the chicken was prepared prior to cooking. In one scenario the food preparation surface is not wiped with a disinfectant wipe, where as in the second scenario the work area is wiped with a disinfectant wipe. In both scenarios the food preparer touches the disinfectant wipe, treated or non-treated surfaces and then 206 touches their mouth transferring. An assumption made in this study is that 100% of C. jejuni that transfers to the mouth is capable of causing infection. 2. METHODS 2.1 The Exposure Model Our CPM exposure scenario considers input parameters that predict the risk in transferring C. jejuni from treated and non-treated surfaces to fingers. Input parameters are shown in Table 1. The exposure assessment begins in the domestic kitchen where the consumer is preparing a chicken dinner using a package of raw chicken filets purchased at a retail store (Fig. 1). The probability that the consumer purchased C. jejuni contaminated chicken meat is considered (input 1a). C. jejuni concentration is variable and is also considered (input 1b). These two variables combined predict the concentration of C. jejuni on the chicken filet portions. The consumer begins to prepare the chicken filet by opening the package and placing the chicken portions on a cutting board. Portion size is not considered in the scenario for simplification. Raw chicken juice contaminated with C. jejuni spills (input 2) onto a certain surface area (input 3) of the kitchen counter (fomite); ceramic tile, laminate, and granite. The consumer or a household member touches the contaminated surface with their finger (input 4) and transfers C. jejuni to their fingers (input 5). At this point the consumer touches their mouth with their finger. The mouth is assumed to be an absorbing state as, described by Nicas and Sun (24). Thus we assume that the C. jejuni concentration in contact with the mouth is absorbed into the body as a total dose. 207 2.1.1 Input Distribution Concentrations of C. jejuni on raw chicken filet, Cchicken, are not static. Rather two variable parameters were used to forecast the concentration on the chicken meat. The forecast of the C. jejuni contamination parameter, Pcontamination, was based on the data reported from Zhao et al. (25) where 71% of chicken samples randomly collected from retail stores in the Washington, D.C. area were contaminated with C. jejuni. Based on these findings a Yes-No distribution was used to forecast the probability that a consumer purchasing a chicken filet package would have a 71% chance (Yes) of preparing chicken meat contaminated with C. jejuni. There would then remain a 29% chance that the consumer would purchase a chicken package that was not contaminated (Table I). C. jejuni concentrations on raw chicken meat are variable and are given a Lognormal distribution (mean 1.5, standard deviation 1.2), Cdistribution, provided by Nauta et al. (26), (Table I). We convert the output value produced after the 20,000 iterations from the Monte Carlo simulation back to actual CFU before multiplying by the probability of contamination. (1) The volume assumed to have spilled, Vspilled, while opening and preparing the chicken filet is 1 ml and the assumed spilled area, Afomite, on the fomite is 100 cm2. The surface area in contact between the fomite and the finger, Sfomite/finger, is assumed to be 2 208 cm2 based on Nicas and Best (27) the average surface area of a finger tip (Table I). We also assumed that every time the individual touches the surface they also then touch their mouth. Fomite-to-finger transfer efficiencies, TEFomite Finger, of C. jejuni were based on Gram negative Escherichia coli transfer rates found by Lopez et al., (submitted for publication) under both treated and non-treated transfer scenarios (Table I). Transfer rates from non-treated ceramic tile and laminate were assumed to have a lognormal distribution based on the best-fit distribution on transfer efficiencies of E. coli from Lopez et al. (submitted for publication). Lognormal distributions had the mean and standard deviation as input parameters. Transfer rates from non-treated granite as well as the transfer rates from treated surfaces were assumed to have a triangular distribution in order to take into account the least detectable limit of 10 CFU/cm2 seen in some transfers. The triangular distribution considers the minimum (0.0), likeliest (half of the maximum), and maximum as distribution parameters. The hand-to-fomite transfer efficiency, TEFinger Mouth, parameter of 34% is a point estimate based on Rusin et al. (22) using Gram negative Serratia rubidea hand-tomouth transfer data (Table I). Decay rates are not considered in this model because transfer rates are based on a 30 min survival time of E. coli. C. jejuni exposure is assumed to take place within or at the 30 min exposure window. The surface area in contact between the fomite and the finger Sfomite/finger, was assumed to be 2 cm2 based on Nicas and Best (27) previous assumptions of the average surface area of a finger tip. Condition A, used one contact between the fomite and the 209 finger Sfomite/finger equaling 2 cm2 surface area and condition B, used 4 contacts between the fomite and the finger Sfomite/finger equaling 8 cm2 surface area as an input parameter (Table I). The number of times an individual touches their mouth was based on the observations reported by Nicas and Best (27). who found a total of 24 contacts in 3 hours with an average contact of 8 h-1. Since our transfer data (Lopez et al., submitted for publication) is based on a 30 min drying time and decay rate is not considered we assumed 4 contacts per 30 min-1 equaling Sfomite/finger 8 cm2 with the surface for condition b (Table I). We also assume that each surface contact resulted in a hand-to-mouth transfer and, as Nicas and Sun (24) previously described, the mouth is assumed to be an absorbing state. Based on this assumption we consider the C. jejuni in contact with the mouth to be absorbed into the body as a dose. 2.1.2 Fomite-to-finger Dose This fomite-to-finger CPM is developed to show the probability distribution of the ingested dose of C. jejuni (CFU/finger). A single Dose (D) can be represented with: / (2) 2.1.3 Risk of Infection The C. jejuni risk of infection (Pinfection) was determined by modeling the doseresponse relationship. The dose-response relationship in risk assessment describes the 210 relationship between exposure, expressed as the number of Camplobacter bacteria ingested, and the probability of a response. The Beta-Poisson equation (3) suggested by the QMRA wiki (28) and the parameters provided were used in modeling this risk assessment. The QMRA wiki provides the N50 of 8.9E+02 and α 0.144 (28) for the BetaPoisson model. 1 1 (3) 2.1.4 Risk of Illness The C. jejuni risk of illness (Pillness) was calculated by using equation (4). Morbidity ratio was determined by dividing the 2.4 million people estimated by the CDC (29) to be affected by Campylobacteriosis by the United States population estimated to be 315,000,000 people (30). Therefore the morbidity ratio is estimated to be 7.62E-03. (4) 2.1.5 Risk of Death The C. jejuni risk of death (Pdeath) was calculated by using equation (5). Mortality ratio was determined by dividing the estimated 124 deaths per year from Campylobacter infections reported by the CDC (29) by the 2.4 million people estimated to be affected by Campylobacteriosis (29). Therefore the mortality ratio is estimated to be 5.17E-05. 211 (5) 2.1.6 Annual Risk Annual risk of infection (Pinfection), illness (Pillness), and death (Pdeath) were determined by using equation 6 and the results of a National Chicken Council frequency of eating chicken survey (31). The survey did not distinguish between cooked and raw chicken meat so the total biweekly average was used for each year. Consumers purchasing no chicken filets, cooked or raw, were included in the average. The 2010, 2011, and 2012 survey found that the average people purchased chicken from a retail grocery store was 3.6, 3.6, and 3.4, respectively within the previous two weeks from the time of the survey. The three year average was 3.53 multiplied by 26 biweekly periods resulted in 91.87 ≈ 92 times people purchased chicken filets and prepared a meal at home in a year (Table I). 1 1 (6) 2.1.7 Percent Reduction Percent reduction was calculated using equation (7) and is defined as the risk of touching a non-treated surface minus the risk touching a treated surface relative to the risk from the non-treated surface. 212 % – 100 (7) 2.1.8 Model Implementation The CPM simulations were conducted with Monte Carlo (Oracle Crystal Ball, Fusion Edition, Redwood Shores, CA) and Excel Mac version (Microsoft Excel 2011). Monte Carlo simulations consisted of 20,000 iterations to predict the risk of infection from non-treated and treated surfaces. The Monte Carlo sampling generates model parameter inputs from defined probability distributions, previously mentioned, allowing the incorporation of parameter uncertainty. Each simulation produced an input value based on the assumption of the distribution for the input 1a probability for contamination, input 1b concentration distribution of C. jejuni contamination on the chicken portions, and input 5 fomite-to-finger transfer efficiency. The output values produced for distribution assumptions 1a and 1b were used to calculate concentrations on chicken (Equation 1). This output value along with the previously mentioned point estimates and input value 5 were used in calculating the dose (Equation 2). In turn the dose output value was an input value in the Beta Poisson model (Equation 3), considered a forecast cell in the Monte Carlo simulation to determine the risk of infection (Pinfection). The risk of infection output value was then an input value in (Equation 4), considered then the forecast cell in the Monte Carlo simulation to determine the risk of illness (Pillness). In turn the risk of illness output value was an input value in (Equation 5), considered the new forecast cell in the Monte Carlo simulation to determine 213 the risk of death (Pdeath). The Monte Carlo simulation was performed the same way for determining the annual risk of infection, illness, and death. 3. RESULTS 3.1 Risk Comparison of Scenario 1 and 2 under Condition A 3.1.1 Risks per person per event Scenario 2, fomites treated with the disinfectant-wipe intervention resulted in a > 99% reduction for ceramic tile, laminate, and granite for risk of infection, illness, and death, compared to scenario 1, non-treated fomites. Condition A, depicted one fomite to hand contact and subsequently a hand to mouth transfer. Risk of infection, illness, and death was 2 to 3 orders greater under non-treated fomites, scenario 1, than treated fomites (Table II). 3.1.2 Annual Risks per person per year The percent reduction of annual risk of infection, illness, and death were all > than 99% except for a laminate for risk of infection. The annual risk of the infection was up to 1.67E-01 for scenario 1, non-treated fomites, compared to as low as 1.00E-03 for scenario 2, treated fomites. Annual risk of death was up to 2.9E-07 with non-treated fomites, while with treated fomites as low as 2.2E-10 (Table III). 3.2 Risk Comparison of Scenario 1 and 2 under Condition B 3.2.1 Risks per person per preparation event Condition B, describes 4 fomite to hand contacts with 4 subsequent hand to mouth transfers. Risk of infection under scenario 1, non-treated surfaces, was an up to 1.8E-02, 214 while treated fomites, scenario 2, showed 2 to 3 orders lower risk (Table IV). Most fomites showed a 99% reduction. Risk of illness and death were also 2 to 3 orders lower with the use of the disinfectant-wipe intervention. 3.2.2 Annual Risks per person per year The annual risks under Condition B were all greater than under Condition A. The percent reduction of the annual risk of infection, illness, and death were all > than 95%. The annual risk of infection was up to 2.71E-01 for scenario 1, non-treated fomites, compared to as low as 2.00E-03 for scenario 2, treated fomites. Annual risk of illness was up to 1.21E-02 with non-treated laminate, while with treated laminate 1.38E-04 (Table V). 4. DISCUSSION A number of QMRA have been conducted using Consumer Phase Models (CPM) showing the cross-contamination of C. jejuni and the risk associated with preparing raw poultry in domestic kitchens (23, 26). However, this is the first QMRA modeling the probability of a C. jejuni infection from a cross-contamination to the surface and a subsequent fomite-to-finger transfer and then a hand-to-mouth transfer occurring resulting in a dose. Further we provide a novel comparison of the risk of infection with and without the use of a disinfectant wipe. The probability of infection given the CPM presented in this QMRA is relatively high. With a 71% chance that chicken purchased at a retail store would be contaminated with C. jejuni (25), along with the other assumptions, resulted in a higher mean risk of 215 infection per person per preparation event for scenario 1, non-treated surfaces, compared to scenario 2, disinfectant-wipe treated surfaces, under both Condition A and B. Under Condition A, 1-fomite to hand to mouth transfers, the risk of infection with scenario 1 non-treated surfaces was (5.6E-03 to 8.00E-03), while with scenario 2 treated surfaces (1.5E-05 to 6.00E-06) (Table II). As expected the risk of infection per person per preparation event increased by 2 to 3 orders under Condition B, 4-fomite to hand to mouth contacts, scenario 1 non-treated surfaces (1.23E-02 to 1.80E-02), compared with scenario 2 treated fomites (2.17E-04 to 4.6E-05) (Table IV). The annual risk of infection per person per year (92 preparation events) under Condition A resulted in (1.26E-01 to 1.67E-01) under scenario 1, compared to (1.00E-03 to 4.00E-03) under scenario 2 (Table III). As expected the annual risk of infection per person per year under Condition B showed (2.21E-01 to 2.71E-01) for scenario 1 nontreated fomites, compared to (2.00E-03 to 1.2E-02) for scenario 2, disinfectant-wipe treated fomites (Table V). The annual risk of illness under Condition A for scenario 1 non-treated surfaces (3.80E-03 to 5.50E-03) is greater than scenario 2 treated surfaces (4.9E-05 to 7.9E-06) (Table III). The same trend is seen under Condition B with 4fomite to hand to mouth transfers, where risk of illness is greater without the use of the disinfectant wipe and lower with the use of the disinfectant-wipe intervention. The same pattern is observed with the risk of death for both Conditions and scenarios. The FoodNet surveillance data shows 13 cases are diagnosed each year for each 100,000 persons in the population (29), which means 1.3E-04, 1.3 in 10,000 persons become ill with Campylobacteriosis annually. The risk of illness with scenario 1 non- 216 treated surfaces under Condition A, resulted in 38.0E-04 to 55.0E-04, while under scenario 2 treated fomites was 0.49E-04 to 0.079E-04 (Table III). Compared to the risk of illness under Condition B scenario 1, 8.50E-03 to 1.05E-02 (85.0E-04 to 105.0E-04), while scenario 2 showed 1.38E-04 to 2.92E-05 (1.38E-04 to 29.2E-04) (Table V). Sensitivity analysis showed the probability of contamination having the greatest influence on the probability of infection. Most Monte Carlo simulations reported a greater than 70% influence on the outcome of infection with concentration most of the time having a 25% influence. Transfer efficiency showed the least influence compared to the other two assumption distributions. The probability of contamination had a Yes-No distribution, which meant that 29% of the time the output was zero, which gave a final dose of zero. This means one-third of the 20,000 iterations resulted in a zero risk. This input parameter seems to have caused the annual risk of infection, illness and death to be up to 30 times greater than the national average. A retail study by Gupta et al. (32) found 44% of the chicken products samples where contaminated with Campylobacter. This Yes-No distribution will be used on future QMRA studies to see if the risk is closer to the national average. It is important to compare surveillance, diagnosed cases from a region with the retail survey study to see if Campylobacter contamination on chicken products transfer over to reported cases. Our study clearly showed an over estimation of Campylobacteriosis in the Washington, DC area where the retail study was conducted (25). The surface area in contact between the fomite and the finger Sfomite/finger, was assumed to be 2 cm2 by Nicas and Best (27) the average surface area of a finger tip. Condition A, used one contact between the fomite and the finger Sfomite/finger equaling 2 217 cm2 surface area as an input parameter and condition B, used 4 contacts between the fomite and the finger Sfomite/finger equaling 8 cm2 surface area as an input parameter (Table I). The surface area between the fomite and finger and the number of times an individual touches their mouth was based on the observations reported by Nicas and Best (27), who found a total of 24 contacts in 3 hours with an average contact of 8 h-1. Since our transfer data (Lopez et al., submitted for publication) is based on a 30 min drying time and decay rate is not considered we assumed 4 contacts per 30 min-1 equaling Sfomite/finger 8 cm2 with the surface for condition b (Table I). Nicas and Best (27) described that the hand contact with environmental surfaces depends on the individual and the activities being performed, they assumed 1 min-1 in their influenza QMRA model. We also assume that each surface contact resulted in a hand-to-mouth transfer and, as Nicas and Sun (24) previously described, the mouth is assumed to be an absorbing state. Based on this assumption we consider the C. jejuni in contact with the mouth to be absorbed into the body as a dose. The classic Beta-Poisson model was used to describe the probability of infection based on the feeding study by Black et al. (33) fitted by Medema et al. (34) and Teunis and Havelaar (35). The maximum likelihood estimates for the Beta-Poisson model parameters based on these data are α = 0.145 and β = 7.59 (34). The Beta-Poisson equation (3) suggested by the QMRA wiki (28) and the parameters provided were used in modeling this risk assessment. Even though there are updated parameters for C. jejuni developed based on milk outbreaks by Teunis et al. (36) the parameters used in this risk assessment are well accepted and do not seem to over estimate the probability risk of infection (37). These 218 input parameters are more often used in C. jejuni risk assessments models, with some variation in the model equation (23, 26). The results of this QMRA study show that the use of a disinfectant wipe intervention lowers the risk of infection, illness, and death by 2 to 3 orders of magnitude, percent reduction 95% to >99%. The risk of infection and illness is over estimated compared to the national average of 1.3 in 10,000 persons with Campylobacteriosis each year. However, FoodNet surveillance has shown that risk of illness is greater in some surveillance locations up to 6.4E-04 (38). Since the probability of contamination showed to have the greatest influence on the probability of infection another retail study isolated Campylobacter from 44% (80/180) chicken products (32) probability of contamination will be used compared to the 71% (130/184) used in this study (25). The “farm-to-fork” approach to food safety places a responsibility on the consumer to protect both themselves and those for whom they prepare food. Proper precaution and effective home hygiene practices should be practiced in order to prevent cross-contamination of pathogenic microbes to our foods and hands. This risk assessment shows that the use of disinfectant wipes after the preparation of raw chicken meat could reduce the risk of C. jejuni infection. In conclusion, the QMRA study show that the use of disinfectant wipes reduces the risk of Campylopacter infection, illness, and death by 2 to 3 orders of magnitude. The disinfectant-wipe intervention reduced the annual risk of illness below the reported national average of diagnosed Campylobacteriosis cases. The present study also found that risk increase with the number of fomite to hand to mouth transfers. The sensitivity 219 analysis showed the Yes-No distribution had the greatest influence. Based on these results future studies will use retail surveillance of the probability of chicken product contamination with FoodNet surveillance states in order to reduce the disparity in the risk to the actual cases. This risk assessment shows that the use of disinfectant wipes to decontaminate surface areas after chicken preparation reduces the risk of C. jejuni infections up to 99.2%. ACKNOWLEDGEMENTS This research was supported by the Center for Advancing Microbial Risk Assessment, funded by the U.S. Environmental Protection Agency Science to Achieve Results (STAR) program and the U.S. Department of Homeland Security (Grant R83236301), and the Clorox Company. 220 REFERENCES 1. Cogan TA, Bloomfield SF, Humphrey TJ. The effectiveness of hygiene procedures for prevention of cross-contamination from chicken carcases in the domestic kitchen. Letters in Applied Microbiology, 1999; 29:354-358. 2. Kagan LJ, Aiello AE, Larson E. 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Clinical Infectious Diseases, 2004; 38(Suppl 3):S165-174. 226 FIGURES Chicken Fillet Cu ng Board Surface (fomite) Ceramic Tile, Laminate, Granite Interven on Disinfectant wipe Hands (fingers) Mouth (Lips) Fig. 1. Describes the Consumer Phase Model and direction of cross-contamination. 227 TABLES Table I. In put parameters used in CPM Variable Description C. jejuni chicken concentration Symbol Probability of contamination Pcontamination Distribution of concentration Cdistribution Chicken concentration Units Log 2 Input Parameters Source/Reference Yes (0.71), No (0.29) (Zhao et al., 2001) Lognormal (1.5, 1.2) (Nauta and Christensen, 2011) Cchicken CFU/cm Forecaset Monte Carlo simulation Vspilled ml 1 Assumption Afomite cm2 100 Assumption Sfomite/finger cm2 2 (Nicas and Best 2008) Sfomite/finger 2 8 (Nicas and Best 2008) (Lopez et al. submitted for publication) Chicken package juice Volume spilled Total contaminated Area of fomite Surface area Between fomite/finger Condition A Condition B cm Transfer rate from non-treated surfaces Ceramic Tile to finger TEceramic tile --> finger Lognormal (0.12, 0.12) Laminate to finger TElaminate --> finger Lognormal (0.22, 0.24) Granite to finger TEgranite --> finger Triangular (0.0, 0.14, 0.28) Ceramic Tile to finger TEceramic tile --> finger Triangular (0.0, 0.0001, 0.0002) Laminate to finger TElaminate --> finger Triangular (0.0, 0.0005, 0.001) Granite to finger TEgranite --> finger Triangular (0.0, 0.00005, 0.0001) Transfer rate from treated surfaces TEfinger --> mouth 0.34 (Rusin et al. 2002) Frequency of eating chicken survey Fpreparation 92 (National Chicken Council 2012) Beta Piosson Parameters α and N50 0.144 and 8.9E+02 (CAMRA QMRA Wiki, 2012) Dose D Forecast Monte Carlo simulation Transfer rate from treated surfaces CFU/finger (Lopez et al. submitted for publication) 228 Table II Risks associated with one preparation of chicken filet (/person/event): Condition A Intervention Ceramic tile Laminate Granite Fomite Non-treatment Risk of Infection 5.60E-03 Risk of Illness 4.30E-05 Risk of Death 2.20E-09 Treatment 1.50E-05 7.30E-08 5.20E-12 Percent Reduction 99.73 99.83 99.76 Non-treatment 8.00E-03 6.00E-05 3.10E-09 Treatment 6.60E-05 4.01E-07 2.10E-11 Percent Reduction 99.18 99.33 99.32 Non-treatment 6.00E-03 4.80E-05 2.40E-09 Treatment 6.00E-06 4.50E-08 2.20E-12 Percent Reduction 99.90 99.91 99.91 229 Table III Annual Risk (/ person/year): Condition A Intervention Ceramic tile Laminate Granite Fomite Non-treatment Risk of Infection 1.26E-01 Risk of Illness 3.80E-03 Risk of Death 1.90E-07 Treatment 1.00E-03 7.90E-06 3.90E-10 Percent Reduction 99.21 99.79 99.79 Non-treatment 1.67E-01 5.50E-03 2.90E-07 Treatment 4.00E-03 4.90E-05 2.30E-09 Percent Reduction 97.60 99.11 99.21 Non-treatment 1.51E-01 4.50E-03 2.40E-07 Treatment 1.00E-03 7.90E-06 2.20E-10 Percent Reduction 99.34 99.82 99.91 230 Table IV Risks associated with one preparation of chicken filet (/person/event): Condition B Intervention Ceramic tile Laminate Granite Fomite Non-treatment Risk of Infection 1.23E-02 Risk of Illness 1.03E-04 Risk of Death 5.20E-09 Treatment 4.60E-05 3.52E-07 1.61E-11 Percent Reduction 99.63 99.66 99.69 Non-treatment 1.80E-02 1.39E-04 6.80E-09 Treatment 2.17E-04 1.54E-06 8.20E-11 Percent Reduction 98.79 98.89 98.79 Non-treatment 1.50E-02 1.21E-04 6.10E-09 Treatment 2.70E-05 1.92E-07 9.60E-12 Percent Reduction 99.82 99.84 99.84 231 Table V Annual Risk (/ person/year): Condition B Intervention Ceramic tile Laminate Granite Fomite Non-treatment Risk of Infection 2.21E-01 Risk of Illness 8.50E-03 Risk of Death 4.90E-07 Treatment 4.00E-03 2.92E-05 1.89E-09 Percent Reduction 98.19 99.66 99.61 Non-treatment 2.71E-01 1.21E-02 6.90E-07 Treatment 1.20E-02 1.38E-04 7.80E-09 Percent Reduction 95.57 98.86 98.87 Non-treatment 2.57E-01 1.05E-02 5.70E-07 Treatment 2.00E-03 1.69E-05 9.10E-10 Percent Reduction 99.22 99.84 99.84 232 APPENDIX G: SUPPLEMENTAL MATERIALS Transfer of Bacteria and Viruses to Hands Running title: Transfer of Microorganisms to Hands Gerardo U. Lopez and Charles P. Gerba# Department of Soil, Water and Environmental Science, University of Arizona, Tucson, Arizona 85721 # Corresponding author. Tel: (520) 621-6910, Fax (520) 621-6366, E-mail: [email protected] A manuscript for Applied and Environmental Microbiology Supplemental Material Not Included in Publication 233 TABLES TABLE 1 Hand-to-Fomite Transfer Efficiency (%) Organism Type Respiratory Viruses Species (Reference) Rhinovirus (1) Pen, SSb Pen, SS Rhinovirus (2) Rhinovirus 14 (3) Human Parainfluenza virus-3 (3) Enteric Viruses Fomite Rotavirus (4) Hepatitis A Virus (5) Brass Door Knob Faucet handle Brass Door Knob Stainless Steel Stainless Steel Stainless Steel N/D Drying time (min) Dryd Natural Contact Time (s) Hand to Fomite (%) 3 min Undetected 3 min 14 N/D 0 e c S/B/F N/D 13.6 N/D 0e S/B/F N/D 35.7 N/D 0 f S/B/F N/D 44.6 50 ± 5 20g 1 5 0.92 50 ± 5 20 g 1 5 Undetected 20 g 1 10 16.1 g 1 10 1.8 50 Damp d Contact Pressure kg/cm2 Natural Stainless Steel 50 60 Stainless Steel 45 ± 5 20g 0.2 10 26.8 45 ± 5 g Stainless Steel Stainless Steel Stainless Steel Stainless Steel a Relative Humidity (%) N/Da 45 ± 5 45 ± 5 45 ± 5 60 0.2 10 21.2 120 g 0.2 10 14.1 180 g 0.2 10 3.5 240 g 0.2 10 1.6 N/D - Not determined; b SS - Stainless Steel spoons; c S/B/F - Sliding back and forth; d Inoculum (ml) ≤ 0.005; e Inoculum (ml) 0.05; f Inoculum (ml) 0.1; g Inoculum (ml) 0.01 234 TABLE 1 Continued Organism Type Coliphage Species (Reference) Fomite Drying time (min) N/Da 30 sb Contact Pressure kg/cm2 N/D Contact Time (s) Hand to Fomite (%) 10 33.9 PRD-1 (6) Lips - skin Φ-X174 (7) Glass cover slip 45 - 60 Visibly dryb 0.2 - 0.4 10 26c Glass cover slip 45 - 60 Visibly dryb 0.2 - 0.4 10 17d Glass cover slip 45 - 60 Visibly dryb 0.2 - 0.4 10 28c Glass cover slip 45 - 60 Visibly dryb 0.2 - 0.4 10 20d Glass cover slip 45 - 60 Visibly dryb 0.2 - 0.4 10 24c Glass cover slip 45 - 60 Visibly dryb 0.2 - 0.4 10 15d fr (7) MS2 (7) a Relative Humidity (%) N/D - Not Determined; b Inoculum (ml) 0.005; c Unwashed hands; d Washed hands 235 TABLE 1 Continued Organism Type Gram (+) bacteria Staphylococcus saprophyticus (8) Kitchen cloth N/Da 0d Contact Pressure kg/cm2 G/Fb Staphylococcus saprophyticus (9) Fabric N/D 0d G/F 10 17c Micrococcus luteus (6) Lips - skin N/D 30 se Species (Reference) Fomite Micrococcus luteus (10) Gram (-) bacteria Paper-towel Disp. Drying time (min) N/D Contact Time (s) Hand to Fomite (%) N/D 85 N/D 10 40.99 f N/D Natural 0.03 d G/F 10 88c 0 Escherichia coli (9) Fabric N/D 0 Klebsiella aerogenes (9) Fabric N/D 0d G/F 10 86c Psuedomonas aeruginosa (9) Fabric N/D 0d G/F 10 76c Enterobacter aerogenes (11) Spigot N/D 0g N/D N/D 1.02 N/D 10 33.97 N/D Natural 0.04 Serratia rubidea (6) Lips - skin Serratia marcescens (10) a Relative Humidity (%) N/D - Not Determined; b G/F - Grasped firmly; Contamination Paper-towel Disp. c N/D N/D e 30 s 0 f Considered second stage of transfer; d Inoculum (ml) 1.0; e Inoculum (ml) 0.005; f 1.0x108 CFU; g Cross- 236 TABLE 2 Fomite-to-Hand Transfer Efficiency (%) Organism Type Respiratory Viruses Species (Reference) Rhinovirus (1) Rhinovirus (2) Pen, Table, SSb Relative Humidity (%) N/Da Pen, Table, SS N/D Fomite Brass Door Knob Faucet Handle Brass Door Knob Rhinovirus 14 (3) Human Parainfluenza virus-3 (3) Enteric Viruses Rotavirus (4) Hepatitis A Virus (5) Stainless Steel Stainless Steel Stainless Steel Dampf F/R Fomite to Hand (%) 15e 15 15e 21 10 d S/B/F N/D 61.5 10 g S/B/F N/D 16.3 N/D 10 h S/B/F N/D 32.9 50 ± 5 20i 1 5 0.67 50 ± 5 20 i 1 5 1.5 20 i 1 10 16.8 i 1 10 1.6 0.2 10 22.1 N/D N/D 50 50 60 Stainless Steel 45 ± 5 20i 45 ± 5 i Stainless Steel Contact Time (s) g Stainless Steel Stainless Steel a Dryf Contact Pressure kg/cm2 F/Rc Drying time (min) 45 ± 5 60 0.2 10 14.6 120 i 0.2 10 9.0 i 0.2 10 4.6 0.2 10 0 Stainless Steel 45 ± 5 180 Stainless Steel 45 ± 5 240i N/D - Not determined; b SS - Stainless Steel spoons; c F/R - Firmly rubbing table top or fingers; d S/B/F - Sliding back and forth; e Contact with table top 15 s and 3 min with plastic pen and stainless steel spoon; f Inoculum (ml) ≤ 0.005; g Inoculum (ml) 0.05; h Inoculum (ml) 0.1; i Inoculum (ml) 0.01 237 TABLE 2 Continued Organism Type Enteric Viruses Relative Humidity (%) Drying time (min) Spatula N/Da 5b Contact Pressure kg/cm2 0.2 - 0.4 Spatula N/D 15b Species (Reference) Feline Calicivirus, Strain F9 (12) Fomite Fork Fork Cutting Board N/D 5 b Cutting Board N/D 15b N/D 5 b b N/D 15 Stainless Steel N/D 5 b Stainless Steel N/D 15b Ceramic Tile Ceramic Tile 15 - 32 40 - 65 Fomite to Hand (%) 5 - 10 31.62 0.2 - 0.4 5 - 10 31.62 0.2 - 0.4 5 - 10 25.12 0.2 - 0.4 5 - 10 25.12 0.2 - 0.4 5 - 10 25.12 0.2 - 0.4 5 - 10 25.12 0.2 - 0.4 5 - 10 6.31 0.2 - 0.4 5 - 10 12.59 0.2 - 0.4 5 - 10 19.95 0.2 - 0.4 5 - 10 12.59 30 b 1 10 23.1 30 b 1 10 29.2 b 1 10 36.3 Laminate 15 - 32 30 Laminate 40 - 65 30b 1 10 25.5 15 - 32 30 b 1 10 33.8 30 b 1 10 25.9 Granite Granite N/D - Not Determined; b Inoculum (ml) 0.01 b 15 Door Knob a 5 N/D Door Knob Poliovirus Type 1 (Lopez et al., submitted for publication) N/D b Contact Time (s) 40 - 65 238 TABLE 2 Continued Organism Type Enteric Viruses Species (Reference) Poliovirus Type 1 (Lopez et al., submitted for publication) Relative Humidity (%) 15 - 32 Fomite Ceramic Tile Laminate Granite Coliphage PRD-1 (6) Faucet handle a 10 < 0.004 15 - 32 N/D 30 + 10 Contact Time (s) Fomite to Hand (%) 10 0.01 1 10 0.01 1 f N/D 30 33.47 1 f N/D 30 65.8 1 g N/D 10 0.03 h N/D 10 0.02 N/D N/D < 0.01 Sponge N/D 1 Laundry 100%b N/D 1i N/D h Laundry 50:50% MS2 (7) 1 e N/D b fr (7) 30 + 10e N/D Dishcloth ΦX174 (7) 15 - 32 a Phone receiver ΦX174 (13) 30 + 10e Contact Pressure kg/cm2 1 Drying time (min) 1 N/D N/D < 0.01 j N/D Natural 0.04 Door handles N/D 15 Door handles N/D 15j N/D Natural 0.000005d Door handles N/D 15j N/D Natural 0.0004c Glass cover slip 45 - 60 Visibly dryk 0.2 - 0.4 10 21c Glass cover slip 45 - 60 Visibly dryk 0.2 - 0.4 10 11d Glass cover slip 45 - 60 Visibly dryk 0.2 - 0.4 10 37c Glass cover slip 45 - 60 Visibly dryk 0.2 - 0.4 10 39d Glass cover slip 45 - 60 Visibly dryk 0.2 - 0.4 10 25c Glass cover slip 45 - 60 Visibly dryk 0.2 - 0.4 10 26d N/D - Not Determined; b Laundry 100% - cotton, Laundry 50% polyester: 50% cotton; c Unwashed hands; d Washed hands; e Inoculum (ml) 0.01; f Inoculum N/D; g Inoculum (ml) 50; h Inoculum (ml) 100; i Inoculum (ml) 200; j Inoculum (ml) 0.4; k Inoculum (ml) 0.005 239 TABLE 2 Continued Organism Type Coliphage Relative Humidity (%) Drying time (min) 15 - 32 30a Acrylic 40 - 65 30 a Glass 15 - 32 40 - 65 Species (Reference) MS2 (Lopez et al., submitted for publication) Fomite Acrylic Glass Ceramic Tile Fomite to Hand (%) 10 21.7 1 10 79.5 30a 1 10 19.3 30 a 1 10 67.3 30 a 1 10 7.1 a 1 10 41.2 40 - 65 30 Laminate 15 - 32 30a 1 10 5.4 40 - 65 30 a 1 10 63.5 30 a 1 10 6.9 a 1 10 37.4 Stainless Steel 15 - 32 Stainless Steel 40 - 65 30 Granite 15 - 32 30a 1 10 10.2 40 - 65 30 a 1 10 30.0 30 a 1 10 0.03 a 1 10 0.3 Granite Cotton 15 - 32 Cotton 40 - 65 30 Polyester 15 - 32 30a 1 10 0.3 40 - 65 30 a 1 10 2.3 30 a 1 10 0.4 30 a 1 10 0.7 Polyester Paper Currency Paper Currency Inoculum (ml) 0.01 Contact Time (s) Ceramic Tile Laminate a 15 - 32 Contact Pressure kg/cm2 1 15 - 32 40 - 65 240 TABLE 2 Continued Organism Type Species (Reference) Gram (+) bacteria Staphylococcus saprophyticus (8) Relative Humidity (%) Drying time (min) N/Da 0e Fabric N/D 0 e Fabric 26 26 Fomite Fabric Fabric Fomite to Hand (%) N/D 9.8 G/F N/D 6.2 60e G/F N/D 0.05 e 60 G/F N/D 0.4 e G/F 10 1.67c G/F 10 0.021c Fabric N/D 0 Streptococcus pyogenes (9) Fabric N/D 0e Staphylococcus aureus (14) Laminate 40 - 45 0f 25.24 N/D 30 43.81 f 1h 2h N/D 30 30.48 Laminate 40 - 45 24 hf N/D 30 9.05 Cloth c 30 40 - 45 Cloth N/D - Not Determined; b G/F - Grasped firmly; 0.1; g Inoculum (ml) 3.0 40 - 45 N/D f Laminate Cloth a Contact Time (s) Staphylococcus saprophyticus (9) Laminate Contact Pressure kg/cm2 G/Fb 60 60 60 0 g N/D 30 3.6 g N/D 30 3.86 g 1h 2h N/D 30 2.49 g Cloth 60 24 h N/D 30 TNTCd Cloth 60 48 hg N/D 30 TNTC Considered first stage of transfer; d TNTC - To Numerous To Count; e Inoculum (ml) 1.0; f Inoculum (ml) 241 TABLE 2 Continued Organism Type Species (Reference) Gram (+) bacteria Staphylococcus aureus (Lopez et al., submitted for publication) Fomite Acrylic Acrylic Glass 15 - 32 30a 40 - 65 30 a 30 a a 15 - 32 Contact Pressure kg/cm2 1 Contact Time (s) Fomite to Hand (%) 10 3.4 1 10 47.2 1 10 20.3 1 10 45.5 40 - 65 30 Ceramic Tile 15 - 32 30a 1 10 2.7 40 - 65 30 a 1 10 54.7 a 1 10 4.3 Laminate 15 - 32 30 Laminate 40 - 65 30a 1 10 61.9 15 - 32 30 a 1 10 4 30 a 1 10 48.3 30 a 1 10 3.9 a 1 10 39.6 Stainless Steel Stainless Steel Granite 40 - 65 15 - 32 Granite 40 - 65 30 Cotton 15 - 32 30a 1 10 < 1.0 40 - 65 30 a 1 10 0.5 a 1 10 0.37 Cotton Polyester 15 - 32 30 Polyester 40 - 65 30a 1 10 5 15 - 32 30 a 1 10 0.2 30 a 1 10 0.2 Paper Currency Paper Currency Inoculum (ml) 0.01 Drying time (min) Glass Ceramic Tile a Relative Humidity (%) 40 - 65 242 TABLE 2 Continued Organism Type Species (Reference) Gram (+) bacteria Staphylococcus aureus (Lopez et al., submitted for publication) Micrococcus luteus (6) Relative Humidity (%) 15 - 32 Fomite Ceramic Tile 30 + 10c N/D 10 < 0.003 < 0.0002 15 - 32 N/D Fomite to Hand (%) 10 Granite Faucet handle Contact Time (s) 1 30 + 10 a Contact Pressure kg/cm2 1 c 15 - 32 1 10 0.001 1 d N/D 30 40.03 1 d N/D 30 41.81 e N/D 10 0.04 Dishcloth N/D 1 Sponge N/D 1f N/D 10 0.03 N/D g N/D ND 0.13 f N/D ND 0.06 h N/D Natural 13.1 N/D Natural 6.0 b Laundry 100% b Laundry 50:50% a 30 + 10c Laminate Phone receiver Micrococcus luteus (10) Drying time (min) N/D 1 1 Paper-towel Disp. N/D 0 Paper-towel Disp. N/D 0h N/D - Not Determined; b Laundry 100% - cotton, Laundry 50% polyester: 50% cotton; c Inoculum (ml) 0.01; d Inoculum N/D; e Inoculum (ml) 50.0; f Inoculum (ml) 100.0; g Inoculum (ml) 200.0; h Inoculum (ml) 1.0x108 CFU/hand 243 TABLE 2 Continued Organism Type Gram (-) bacteria Species (Reference) Fomite Escherichia coli (9) Fabric Escherichia coli (14) Laminate Laminate 0e 40 - 45 0f 40 - 45 60 f 60 30 22.69 30 23.79 N/D 30 1.4 N/D 30 0.67 g 120 N/D N/D g 60 0.47c 25.95 24 hf 60 10 30 40 - 45 0 Fomite to Hand (%) N/D Laminate 60 Contact Time (s) f 120 Cloth c N/Da Contact Pressure kg/cm2 G/Fb 40 - 45 Cloth N/D - Not Determined; b G/F - Grasped firmly; (ml) 0.1; g Inoculum (ml) 3.0 Drying time (min) Laminate Cloth a Relative Humidity (%) N/D 30 0.61 g N/D 30 0.36 g Cloth 60 24 h N/D 30 TNTCd Cloth 60 48 hg N/D 30 TNTC Considered first stage of transfer; d TNTC - To Numerous To Count; e Inoculum (ml) 1.0; f Inoculum 244 TABLE 2 Continued Organism Type Species (Reference) Gram (-) bacteria Escherichia coli (Lopez et al., submitted for publication) Relative Humidity (%) Drying time (min) Acrylic 15 - 32 30a Contact Pressure kg/cm2 1 Acrylic 40 - 65 30a 15 - 32 30 a 30 a 30 a a Fomite Glass Glass Ceramic Tile 10 40.7 1 10 53.3 1 10 5.1 1 10 78.6 1 10 11.6 1 10 60.7 40 - 65 30 Laminate 15 - 32 30a 1 10 21.7 40 - 65 30 a 1 10 27.4 a 1 10 3.8 Stainless Steel 15 - 32 30 Stainless Steel 40 - 65 30a 1 10 54.1 15 - 32 30 a 1 10 7.3 30 a 1 10 36.5 30 a 1 10 < 6.8 a 1 10 < 13.4 Granite Granite Cotton 40 - 65 15 - 32 Cotton 40 - 65 30 Polyester 15 - 32 30a 1 10 < 0.37 40 - 65 30 a 1 10 < 0.7 a 1 10 < 0.05 1 10 0.1 Polyester Inoculum (ml) 0.01 15 - 32 Fomite to Hand (%) Ceramic Tile Laminate a 40 - 65 Contact Time (s) Paper Currency 15 - 32 30 Paper Currency 40 - 65 30a 245 TABLE 2 Continued Organism Type Species (Reference) Gram (-) bacteria Escherichia coli (Lopez et al., submitted for publication) Relative Humidity (%) Drying time (min) 15 - 32 30 + 10e 15 - 32 30 + 10 e 15 - 32 30 + 10 e Acrylic 21 - 27 25 - 32 e Glas 23 - 34 29 - 30 Fomite Ceramic Tile Laminate Granite Escherichia coli (Lopez et al., submitted for publication) Ceramic Tile Fomite to Hand (%) 10 < 0.01 1 10 < 0.02 1 10 < 0.004 1 13 ± 2 22.17 20 - 26e 1 13 ± 2 27.26 15 - 29 e 1 13 ± 2 13.26 e 1 13 ± 2 6.28 20 - 24 27 - 30 Stainless Steel 21 - 32 30e f Klebsiella aerogenes (9) Fabric N/D 0 Klebsiella aerogenes (14) Cloth 60 0g Cloth a Contact Time (s) Laminate a N/D - Not Determined; b G/F - Grasped firmly; 1.0; g Inoculum (ml) 3.0 c 60 Contact Pressure kg/cm2 1 60 g 1 13 ± 2 26.67 b G/F 10 0.29c N/D 30 9.8 N/D 30 6.58 g N/D 30 4.55 Cloth 60 120 Cloth 60 24 hg N/D 30 TNTCd Cloth 60 48 hg N/D 30 TNTC Considered first stage of transfer; d TNTC - To Numerous To Count; e Inoculum (ml) 0.01; f Inoculum (ml) 246 TABLE 2 Continued Organism Type Gram (-) bacteria Species (Reference) Relative Humidity (%) N/Da Fomite Serratia marcescens (9) Fabric Serratia marcescens (10) Paper-towel Disp. Paper-towel Disp. Serratia rubidea (6) Faucet handle Phone receiver N/D 0f N/D Natural 12.4 N/D 0f N/D Natural 6.7 N/D 1 g N/D 30 27.59 1 g N/D 30 38.47 h N/D 10 < 0.01 N/D Fomite to Hand (%) 10 0.46c N/D 1 Sponge N/D 1i N/D 10 < 0.01 N/D 1 j N/D N/D < 0.01 1 i N/D N/D < 0.01 e G/F 10 0.36 N/D N/D 10.08 N/D 30 13.05 Laundry 100% d Laundry 50:50% N/D Psuedomonas aeruginosa (9) Fabric N/D 0 Enterobacter aerogenes (11) Spigot N/D 0k Salmonella spp.Wild type strains (14) Contact Time (s) Dishcloth d a 0e Contact Pressure kg/cm2 G/Fb Drying time (min) Laminate Laminate 40 - 45 40 - 45 0 l 60 l N/D 30 17.15 l N/D 30 1.17 N/D 30 0.54 Laminate 40 - 45 120 Laminate 40 - 45 24 hl N/D - Not Determined; b G/F - Grasped firmly; c Considered first stage of transfer; d Laundry 100% - cotton, Laundry 50% polyester: 50% cotton; e Inoculum (ml) 1.0; f Inoculum 1.0x108 CFU/hand; g Inoculum N/D; h Inoculum (ml) 50.0; i Inoculum (ml) 100.0; j Inoculum (ml) 200.0; k CrossContamination; l Inoculum (ml) 0.1 247 TABLE 2 Continued Organism Type Species (Reference) Endospores Bacillus thuringiensis (Lopez et al., submitted for publication) Relative Humidity (%) Drying time (min) 15 - 32 30a Acrylic 40 - 65 30 a Glass 15 - 32 40 - 65 Fomite Acrylic Glass Ceramic Tile Fomite to Hand (%) 10 57.0 1 10 65.6 30a 1 10 < 0.5 30 a 1 10 33.8 30 a 1 10 < 0.2 a 1 10 21.2 40 - 65 30 Laminate 15 - 32 30a 1 10 0.2 40 - 65 30 a 1 10 53.5 30 a 1 10 < 0.5 a 1 10 57.0 Stainless Steel 15 - 32 Stainless Steel 40 - 65 30 Granite 15 - 32 30a 1 10 0.04 40 - 65 30 a 1 10 12.8 30 a 1 10 < 0.6 a 1 10 < 3.5 Granite Cotton 15 - 32 Cotton 40 - 65 30 Polyester 15 - 32 30a 1 10 < 0.6 40 - 65 30 a 1 10 < 4.6 30 a 1 10 < 0.1 30 a 1 10 < 0.1 Polyester Paper Currency Paper Currency Inoculum (ml) 0.01 Contact Time (s) Ceramic Tile Laminate a 15 - 32 Contact Pressure kg/cm2 1 15 - 32 40 - 65 248 TABLE 2 Continued Organism Type Endospores Species (Reference) Bacillus thuringiensis (Lopez et al., submitted for publication) Fomite Ceramic Tile Laminate Granite a Inoculum (ml) 0.01 Relative Humidity (%) Drying time (min) 15 - 32 30 + 10a 15 - 32 30 + 10 a 30 + 10 a 15 - 32 Contact Pressure kg/cm2 1 Contact Time (s) Fomite to Hand (%) 10 < 0.04 1 10 < 0.03 1 10 0.1 249 TABLE 3 Hand-to-Hand Transfer Efficiency (%) Organism Type Respiratory Viruses Species (reference) Rhinovirus (1) Rhinovirus (2) Rhinovirus 14 (3) Enteric Viruses Fingerb Relative Humidity (%) N/Da Fingerb N/D Fomite Finger-skin Finger F/R N/D g d Hand to Hand (%) 15e 17 15e 34 G/R N/D 5.9 20 h 1 5 0.71 h 1 5 Undetected Finger 50 ± 5 20 Rotavirus (4) Finger 50 20h 1 10 6.6 50 60 h 1 10 2.8 20 h 0.2 10 23.5 h 0.2 10 23.5 Hepatitis A Virus (5) Fingerpad 45 ± 5 60 Fingerpad 45 ± 5 120h 0.2 10 16.9 45 ± 5 180 h 0.2 10 5.1 240 h Fingerpad ΦX174 (13) 45 ± 5 Fingerpad Fingerpad 45 ± 5 0.2 10 0.9 i, j N/D N/D 0.004 Finger tip skin N/D 15 Finger tip skin N/D 15i, k N/D N/D 0.03 N/D i, l N/D N/D 16.7 Finger tip skin a 50 ± 5 Dampf Contact Time (s) Human Parainfluenza virus-3 (3) Finger Coliphage N/D Dryf Contact Pressure kg/cm2 F/Rc Drying time (min) 15 N/D - Not determined; b Finger, back of hand skin; c F/R - Firmly rubbing table top or fingers; d Gently rubbing; e Contact with table top 15 s and 3 min with plastic pen and stainless steel spoon; f Inoculum (ml) ≤ 0.005; g Inoculum (ml) 0.05; h Inoculum (ml) 0.01; i Inoculum (ml) 0.1; j Horizontal transmission with 14 other fingers; k Vertical transmission finger-to-finger-to-finger; l Hand washing with bar soap then touching fingers 250 TABLE 4 Hand-to-Fomite-to-Hand Transfer Efficiency (%) Organism Type Respiratory Virus Gram (-) bacteria a Relative Humidity (%) Drying time (min) N/Da 10c, d Faucet handle N/D 10 c, d Brass Door Knob N/D 10c, e Species (reference) Rhinovirus (2) Escherichia coli (15) Fomite Brass Door Knob Paper N/D f Air-dry Contact Pressure kg/cm2 S/B/Fb N/D Hand to Fomite to Hand (%) 11.2 S/B/F N/D 5.63 S/B/F N/D 12.6 N/D 30 0.009 Contact Time (s) N/D - Not determined; b S/B/F - Sliding back and forth; e 10 min drying on the fomite before recipeint handles fomite; d Inoculum (ml) 0.05; e Inoculum (ml) 0.1; f Inoculum (ml) 0.025 251 TABLE 5 Fomite-to-Hand-to-Fomite Transfer Efficiency (%) G/F N/D 4.29d 15j G/F N/D 1.57d N/D 0j G/F N/D 1.62e Fabric N/D 15j G/F N/D 4.58e Fabric N/D 0j G/F N/D 0.01f Fabric N/D 15j G/F N/D 0.0f Fabric N/D 0j G/F N/D 31g Fabric N/D 0j G/F N/D 21h Staphylococcus saprophyticus (9) Fabric N/D 0j G/F 10 0.37i Streptococcus pyogenes (9) Fabric N/D 0j G/F 10 0.01i Escherichia coli (28) Fabric N/D 0j G/F 10 0.018i Klebsiella aerogenes (28) Fabric N/D 0j G/F 10 0.076i Serratia marcescens (28) Fabric N/D 0j G/F 10 0.11i Psuedomonas aeruginosa (28) Fabric N/D 0j G/F 10 0.14i Species (reference) Gram (+) bacteria Staphylococcus saprophyticus (8) Gran (-) bacteria a N/D Fomite to Hand to Fomite (%) 0.04c Organism Type Relative Humidity (%) Drying time (min) N/Da 0j Fabric N/D 0 j Fabric N/D Fabric Fomite Fabric Contact Pressure kg/cm2 G/Fb Contact Time (s) N/D - Not Determined; b G/F - Grasped firmly; c Average transfer efficiency from multiple handling methods of the recipient fabric; d Hand washing with detergent; e Hand washing with bar soap; f Hand washing with70% ethanol; g Rubbing procedure using alcohol-impregnated wipes; h Rubbing procedure using 0.2 ml 80% ethanol; i Considered a complete transfer; j Inoculum (ml) 1.0 252 REFERENCES 1. Reed SE. 1975. An Investigation of the Possible Transmission of Rhinovirus Colds through Indirect Contact. J. Hyg. 75:249-258. 2. Pancic F, Carpentier DC, Came PE. 1980. Role of Infectious Secretions in the Transmission of Rhinovirus. J. Clin. Microbiol. 12:567-571. 3. Ansari SA, Springthorpe VS, Sattar SA, Rivard S, Rahman M. 1991. Potential role of hands in the spread of respiratory viral infections: studies with human parainfluenza virus 3 and rhinovirus 14. J. Clin. Microbiol. 29:2115-2119. 4. Ansari SA, Sattar SA, Springthorpe VS, Wells GA, Tostowaryk W. 1988. Rotavirus survival on human hands and transfer of infectious virus to animate and nonporous inanimate surfaces. J. Clin. Microbiol. 26:1513-1518. 5. Mbithi JN, Springthorpe VS, Boulet JR, Sattar SA. 1992. Survival of hepatitis A virus on human hands and its transfer on contact with animate and inanimate surfaces. J. Clin. Microbiol. 30:757-763. 6. Rusin P, Maxwell S, Gerba C. 2002. Comparative surface-to-hand and fingertip-tomouth transfer efficiency of gram-positive bacteria, gram-negative bacteria, and phage. J. Appl. Microbiol. 93:585-592. 7. Julian TR, Leckie JO, Boehm AB. 2010. Virus transfer between fingerpads and fomites. J. Appl. Microbiol. 109:1868-1874. 8. Marples RR, Towers AG. 1979. A Laboratory Model for the Investigation of Contact Transfer of Micro-Organisms. J. Hyg. 82:237-248. 253 9. MacKintosh CA, Hoffman PN. 1984. An Extended Model for Transfer of MicroOrganisms via the Hands: Differences between Organisms and the Effect of Alcohol Disinfection. J. Hyg. 92:345-355. 10. Harrison WA, Griffith CJ, Ayers T, Michaels B. 2003. Bacterial transfer and crosscontamination potential associated with paper-towel dispensing. Am. J. Infect. Control 31:387-391. 11. Chen Y, Jackson KM, Chea FP, Schaffner DW. 2001. Quantification and variability analysis of bacterial cross-contamination rates in common food service tasks. J. Food Prot. 64:72-80. 12. Paulson DS. 2005. The Transmission of Surrogate Norwalk Virus - From Inanimate Surfaces to Gloved Hands: Is It a Threat? Food Prot. Trends 25:450-454. 13. Rheinbaben Fv, Schunemann S, Grob T, Wolff MH. 2000. Transmission of viruses via contact in ahousehold setting: experiments using bacteriophage 0X174 as a model virus. J. Hosp. Infect. 46:61-66. 14. Scott E, Bloomfield SF. 1990. The survival and transfer of microbial contamination via cloths, hands and utensils. J. Appl. Microbiol. 68:271-278. 15. Hubner N-O, Hubner C, Kramer A, Assadian O. 2011. Survival of Bacterial Pathogens on Paper and Bacterial Retrieval from Paper to hands: Preliminary Results. Am. J. Nurs. 111:30-34. 254 APPENDIX H: SUPPLEMENTAL MATERIALS Comparison of two approaches in determining transfer efficiency of Escherichia coli from nonporous fomites to fingers Running title: Determining Bacterial Percent Transfer Gerardo U. Lopez1, Masaaki Kitajima1, Kelly A. Reynolds2 and Charles P. Gerba1, # 1 Department of Soil, Water and Environmental Science, The University of Arizona, Tucson, Arizona 85721 USA; 2The University of Arizona, Mel and Enid Zuckerman College of Public Health, 1295 N. Martin Ave., Tucson, Arizona 85724, USA # Corresponding author. Tel: (520) 621-6910, Fax (520) 621-6366, E-mail: [email protected] A manuscript for Applied and Environmental Microbiology 255 ADDTIONAL DETAILS TO METHODS AND MATERIALS Figure S1 shows a diagram of the experimental design of the method used in assessing the fomite-to-finger transfer efficiency. The donor fomite was placed on the scale as previously described (1, 2). After the transfer event the donor fomite and the finger were swabbed and assayed. The baseline control fomite was also swabbed and assayed. Approach 1 used E. coli recovered on the finger relative to E. coli recovered from baseline control fomite, where as approach 2 used E. coli recovered on the finger relative to E. coli recovered on the donor fomite. ADDITIONAL DETAILS TO RESULTS Table S1 shows the log10 CFU/2-cm2 of E. coli recovered from fingers after the transfer event with the donor fomite (Fig. S1) and E. coli recovered from the baseline control fomite (Fig. S1). E. coli recovered from fomites are based on 2-cm2 area as used by Nicas and Best (3) in their modeling of quantifying hand-to-face contact rate. The inoculum was spread with the tip of a pipet over a 1.0 cm2 area and dried on laboratory bench for 30 min. The index, middle, and ring fingers each had an average 2.0 cm2 surface area that covered the entire seeded inoculum. Figure S2 provides the transfer efficiency in log10 scale and geometric mean. Transfer efficiencies calculated by both approaches show considerable variability that has been observed in previous studies (2, 4, 5). 256 TABLE TABLE S1: E. coli recovered from fomites and fingers Approach 1 Approach 2 Recovered from finger Recovered from baseline control fomite Recovered from donor fomite Mean CFU/2-cm2 log10 ± SD (Range) Mean CFU/2-cm2 log10 ± SD (Range) Mean CFU/2-cm2 log10 ± SD (Range) Acrylic 3.4 ± 0.5 (2.5 - 4.4) 4.3 ± 0.5 (3.5 - 4.7) 4.2 ± 0.6 (3.2 - 5.2) Glass 4.0 ± 0.4 (3.4 - 4.8) 4.8 ± 0.5 (4.2 - 5.3) 4.8 ± 0.6 (< 3.7 - 5.6) Ceramic Tile 3.9 ± 0.6 (3.0 - 4.9) 5.3 ± 0.7 (4.3 - 6.2) 5.2 ± 0.5 (4.4 - 5.9) Laminate 4.1 ± 0.3 (3.7 - 4.6) 5.5 ± 0.1 (5.3 - 5.6) 5.7 ± 0.2 (5.3 - 5.9) Stainless Steel 4.5 ± 0.7 (3.2 - 5.2) 5.6 ± 0.3 (5.1 - 5.9) 5.5 ± 0.2 (5.3 - 5.7) Surface Type a E. coli recovered from fomite were below the detectible limit of 10 CFU/2-cm2 indicated by < 257 FIGURES FIG S1 Diagram showing experimental design. 258 A B 100 % Transfer efficiency % Transfer efficiency 100 10 1 0.1 Acrylic Geometric Mean 10 1 0.1 Glass Ceramic Tile Laminate Stainless Steel Acrylic Geometric Mean Glass Ceramic Tile Laminate Stainless Steel FIG S2 Transfer efficiency log scale (%) of E. coli determined with (A) approach 1 and (B) approach 2. 259 REFERENCES 1. Ansari SA, Sattar SA, Springthorpe VS, Wells GA, Tostowaryk W. 1988. Rotavirus survival on human hands and transfer of infectious virus to animate and nonporous inanimate surfaces. J. Clin. Microbiol. 26:1513-1518. 2. Mbithi JN, Springthorpe VS, Boulet JR, Sattar SA. 1992. Survival of hepatitis A virus on human hands and its transfer on contact with animate and inanimate surfaces. J. Clin. Microbiol. 30:757-763. 3. Nicas M, Best D. 2008. A Study Quantifying the Hand-to-Face Contact Rate and Its Potential Application to Predicting Respiratory Tract Infection. J. Occup. Environ. Hyg. 5:347-352. 4. Chen Y, Jackson KM, Chea FP, Schaffner DW. 2001. Quantification and variability analysis of bacterial cross-contamination rates in common food service tasks. J. Food Prot. 64:72-80. 5. Paulson DS. 2005. The Transmission of Surrogate Norwalk Virus - From Inanimate Surfaces to Gloved Hands: Is It a Threat? Food Prot. Trends 25:450454. 260 APPENDIX I: SUPPLEMENTAL MATERIALS Transfer efficiency of bacteria and viruses from porous and nonporous fomites to fingers under different relative humidity Running title: Fomite to Finger Microbial Transfer Gerardo U. Lopez1, 3, Charles P. Gerba1, 3#, Akrum Tamimi1, Masaaki Kitajima1, Sheri Maxwell1, and Joan B. Rose2, 3 1 Department of Soil, Water and Environmental Science, The University of Arizona, Tucson, Arizona 85721 USA; 2Department of Fisheries and Wildlife, and 3Center for Advancing Microbial Risk Assessment, Michigan State University, East Lansing, Michigan # Corresponding author. Tel: (520) 621-6910, Fax (520) 621-6366, E-mail: [email protected] Manuscript for Applied and Environmental Microbiology Supplemental Material Not Included in Publication 261 TABLES TABLE S1 Recovered organisms from fingers under low relative humidity 15% - 32% E. coli Surface Type S. aureus 2 B. thuringiensis 2 MS-2 2 (n) Mean CFU/2-cm log10 ± SD (Range) (n) Mean CFU/2-cm log10 ± SD (Range) (n) Mean CFU/2-cm log10 ± SD (Range) (n) Mean CFU/2-cm2 log10 ± SD (Range) Acrylic (6) 4.2 ± 0.5 (3.7 - 5.1) (6) 4.6 ± 0.3 (4.3 - 5.0) (6) 4.5 ± 0.04 (4.5 - 4.6) (6) 7.7 ± 0.4 (7.1 - 8.1) Glass (6) 3.4 ± 0.3 (2.9 - 3.8) (6) 4.9 ± 0.7 (4.2 - 6.0) (6) < 2.1 ± 0.2 (< 2.0 - 2.4) (6) 7.7 ± 0.3 (7.0 - 7.9) Ceramic Tile (6) 3.6 ± 0.7 (2.4 - 4.1) (6) 4.5 ± 0.3 (4.1 - 4.9) (6) < 2.1 ± 0.2 (< 2.0 - 2.5) (6) 7.6 ± 0.2 (7.4 - 8.0) Laminate (6) 3.5 ± 0.4 (2.8 - 3.9) (6) 4.7 ± 0.3 (4.2 - 5.0) (6) 2.1 ± 0.2 (< 2.0 - 2.4) (6) 7.5 ± 0.3 (7.0 - 7.7) Stainless Steel (6) 3.0 ± 0.7 (2.0 - 4.0) (6) 4.5 ± 0.4 (3.9 - 5.2) (6) < 2.0 ± 0.1 (< 2.0 - 2.2) (6) 5.8 ± 1.2 (4.6 - 7.5) (6) 4.5 ± 0.3 (4.1 - 5.0) (6) 1.2 ± 0.3 (< 1.0 - 1.6) (6) 7.1 ± 0.2 (6.9 - 7.3) (6) < 1.1 ± 0.1 (< 1.3)a (6) < 1.0 ± (< 1.0)a (6) 3.3 ± 0.2 (3.1 - 3.6) Nonporous Granite (6) 2.9 ± 0.7 (< 2.2 - 3.7) a Porous Cotton Polyester Paper Currency a (6) < 1.0 ± (< 1.0)a (6) < 1.0 ± 0.1 (< 1.2) a (6) < 1.1 ± 0.3 (< 1.0 - 1.7) (6) 1.4 ± 0.3 (1.0 - 1.9) a (6) 3.3 ± 0.4 (2.8 - 4.0) a (6) < 1.0 ± (< 1.0) a (6) < 1.2 ± 0.3 (< 1.0 - 1.8) (6) 4.5 ± 0.5 (3.9 - 5.1) a (6) 4.7 ± 0.3 (4.5 - 5.3) Transfer of organisms from fomite to fingers for one or more transfer events, were below the detectible limit of 10 CFU/2-cm2 indicated by < 262 TABLE S2 Recovered organisms from fingers under high relative humidity 40% - 65% E. coli Surface Type S. aureus 2 B. thuringiensis 2 MS-2 2 (n) Mean CFU/2-cm log10 ± SD (Range) (n) Mean CFU/2-cm log10 ± SD (Range) (n) Mean CFU/2-cm log10 ± SD (Range) (n) Mean CFU/2-cm2 log10 ± SD (Range) Acrylic (6) 6.4 ± 0.2 (6.2 - 6.7) (6) 6.4 ± 0.2 (6.1 - 6.6) (6) 4.6 ± 0.1 (4.5 - 4.7) (6) 7.0 ± 0.3 (6.6 - 7.3) Glass (6) 5.0 ± 0.5 (4.2 - 5.6) (6) 6.5 ± 0.1 (6.3 - 6.7) (6) 4.3 ± 0.4 (< 3.7 - 4.8) (6) 8.2 ± 0.1 (8.0 - 8.3) Ceramic Tile (6) 5.4 ± 0.4 (4.8 - 5.8) (6) 6.3 ± 0.2 (6.0 - 6.5) (6) 3.7 ± 0.6 (< 3.0 - 4.4) (6) 8.1 ± 0.2 (7.8 - 8.3) Laminate (6) 5.8 ± 0.8 (5.0 - 6.6) (6) 6.5 ± 0.3 (6.1 - 6.8) (6) 4.5 ± 0.2 (4.1 - 4.7) (6) 8.4 ± 0.1 (8.2 - 8.5) Stainless Steel (6) 5.6 ± 0.3 (5.3 - 6.1) (6) 6.5 ± 0.3 (6.0 - 6.8) (6) 4.7 ± 0.2 (4.5 - 5.0) (6) 8.4 ± 0.2 (8.1 - 8.5) Granite (6) 3.6 ± 1.1 (2.3 - 5.0) (6) 5.6 ± 0.9 (4.2 - 6.6) (6) 2.7 ± 1.3 (1.7 - 4.4) (6) 7.6 ± 0.6 (6.8 - 8.3) Cotton (6) < 1.0 ± (< 1.0)a (6) 1.2 ± 0.3 (< 1.0 - 1.9)a (6) < 1.0 ± (< 1.0)a (6) 3.5 ± 0.5 (3.0 - 4.2) Polyester (6) < 1.1 ± 0.1 (< 1.2)a (6) 3.0 ± 1.0 (1.9 - 4.0) (6) < 1.3 ± 0.5 (< 2.0)a Nonporous Porous Paper Currency a (6) < 1.6 ± 0.8 (< 1.0 - 3.2) a (6) 3.4 ± 0.3 (2.9 - 3.8) (6) < 1.5 ± 0.5 (< 1.0 - 2.0) (6) 5.4 ± 0.2 (5.2 - 5.7) a (6) 4.6 ± 0.4 (4.2 - 5.2) Transfer of organisms from fomite to fingers for one or more transfer events, were below the detectible limit of 10 CFU/2-cm2 indicated by < 263 TABLE S3 Recovered poliovirus 1 from fingers Surface Type (n) Mean CFU/2-cm2 log10 ± SD (Range) Low RH (15% - 32%) Ceramic Tile (6) 4.4 ± 0.6 (3.4 - 5.0) Laminate (6) 5.3 ± 0.2 (5.0 - 5.6) Granite (6) 4.1 ± 1.3 (2.5 - 5.5) High RH (40% - 65%) Ceramic Tile (6) 5.7 ± 0.04 (5.6 - 5.7) Laminate (6) 5.8 ± 0.2 (5.6 - 6.2) Granite (6) 5.6 ± 0.1 (5.4 - 5.7) 264 FIGURES B 8.0 Recovered from fingers log10 CFU/2-cm2 Recovered from fingers log10 CFU/2-cm2 A 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 LRH HRH Acrylic Geometric Mean LRH HRH Glass LRH HRH Ceramic Tile LRH HRH Laminate LRH HRH Stainless Steel LRH 6.8 5.8 4.8 3.8 2.8 1.8 0.8 -0.2 HRH LRH Granite C HRH Acrylic Geometric Mean LRH HRH Glass LRH HRH Ceramic Tile LRH HRH Laminate LRH HRH Stainless Steel LRH HRH Granite D 6.0 Recovered from fingers log10 PFU/2-cm2 Recovered from fingers log10 CFU/2-cm2 7.8 5.0 4.0 3.0 2.0 1.0 0.0 LRH HRH Acrylic Geometric Mean LRH HRH Glass LRH HRH Ceramic Tile LRH HRH Laminate LRH HRH Stainless Steel LRH HRH Granite 11.0 9.0 7.0 5.0 3.0 1.0 -1.0 LRH HRH Acrylic Geometric Mean LRH HRH Glass LRH HRH Ceramic Tile LRH HRH Laminate LRH HRH Stainless Steel LRH HRH Granite FIG S1 Organisms recovered from fingers after contact with nonporous fomites (A) E. coli 15597, (B) S. aureus 25923, (C) B. thuringiensis spores, and (D) MS2 under low (15% - 32%) and high (40% - 65%) relative humidity. 265 B 3.5 Recovered from fingers log10 CFU/2-cm2 Recovered from fingers log10 CFU/2-cm2 A 3.0 2.5 2.0 1.5 1.0 0.5 0.0 LRH HRH LRH Co on Geometric Mean HRH Polyester LRH HRH 2.5 2.0 1.5 1.0 0.5 0.0 HRH LRH Co on Geometric Mean HRH Polyester LRH HRH Paper Currency D Recovered from fingers log10 PFU/2-cm2 2.5 Recovered from fingers log10 CFU/2-cm2 3.5 3.0 LRH Paper Currency C 4.5 4.0 2.0 1.5 1.0 0.5 0.0 LRH Co on Geometric Mean HRH LRH HRH Polyester LRH HRH Paper Currency 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 -1.0 LRH Co on Geometric Mean HRH LRH HRH Polyester LRH HRH Paper Currency FIG S2 Organisms recovered from fingers after contact with porous fomites (A) E. coli 15597, (B) S. aureus 25923, (C) B. thuringiensis spores, and (D) MS2 under low (15% - 32%) and high (40% - 65%) relative humidity. Recovered from fingers log10 PFU/2-cm2 266 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 LRH HRH Ceramic Tile Geometric Mean LRH HRH Laminate LRH HRH Granite FIG 3 Poliovirus 1 recovered from fingers after contact with nonporous fomites under low (15% - 32%) and high (40% - 65%) relative humidity. 267 APPENDIX J: SUPPLEMENTAL MATERIALS The Effect of a Disinfectant Wipe on Microbial Transfer Gerardo U. Lopez1, Masaaki Kitajima1, Aaron Havas1, and Kelly A. Reynolds2 1 Department of Soil, Water and Environmental Science, The University of Arizona, Tucson, Arizona 85721, USA 2 Mel and Enid Zuckerman College of Public Health, The University of Arizona, 1295 N. Martin Ave., Tucson, Arizona 85724, USA Corresponding author. Tel: (520) 626-8230, Fax (520) 626-8009, E-mail: [email protected] A manuscript for International Journal of Hygiene and Environmental Health Supplemental Material Not Included in Publication 268 ADDITIONAL DETAILS TO METHODS AND MATERIALS Calculation of percent reduction Percent reduction was calculated using Eqn (1) and is defined as the CFU or PFU recovered from control fomite without intervention minus CFU or PFU recovered from control fomite with intervention relative to CFU or PFU recovered from control fomite without intervention. % / . – / . / . 100 (1) Log10 organism removal from fomite surface was calculated using Eqn (2) by subtracting the mean Log10 CFU or PFU recovered from the control fomite with intervention from the Log10 CFU or PFU inoculated onto the fomite factoring the 10 ul inoculum volume . / . (2) ADDITIONAL DETAILS TO RESULTS Removal of microbial contamination from nonporous surfaces Percent reduction from ceramic tile, laminate, and granite for E. coli (> 99.8%, > 99.97%, 99.9%), S. aureus (99.9%, 99.997%, 99.98%), B. thuringiensis (99.4%, 99.4%, 98.1%), and Poliovirus (99.98%, 99.8%, 99.9%) respectively (Tables S1 and S2). Calculated using Eqn 1. 269 The removal from ceramic tile, laminate, and granite for E. coli was (> 5.8, > 6.2, 5.1 log10 CFU/2 cm2), S. aureus (4.9, 5.3, 4.7 log10 CFU/2 cm2), B. thuringiensis (2.5, 3.1, 3.8 log10 CFU/2 cm2), and for Poliovirus was (5.0, 4.9, 4.9 log10 PFU/2 cm2) respectively (Tables S1 and S2). Calculated using Eqn 2. Figure S1 shows recovery of E. coli, S. aureus, B. thuringiensis spores, and poliovirus 1 from control non-treated and intervention treated fomites. Figure S2 shows fomite-tofinger transfer efficiency % of E. coli, S. aureus, B. thuringiensis spores, and poliovirus 1 from control non-treated (Lopez et al., submitted for publication) and intervention treated fomites. Figure S3 shows recovery of E. coli, S. aureus, B. thuringiensis spores, and poliovirus 1 from fingers in contact with non-treated (Lopez et al., submitted for publication) and intervention treated fomites. 270 TABLES Table S1. Recovered microorganisms from fomitesa E. coli S. aureus Mean CFU/2-cm2 log10 ± SD (Range) Mean CFU/2-cm2 log10 ± SD (Range) % Reduction > 99.8 ± 0.3 (> 99.5 - > 99.997) 99.9 ± 0.01 (99.98 - 99.9995) Organism Removal > 5.8 ± 0.9 (> 4.4 - > 6.6) 4.9 ± 0.6 (4.2 - 5.7) % Reduction > 99.97 ± 0.02 (> 99.9 - > 99.9998) 99.997 ± 0.004 (99.99 - 99.9999) Organism Removal > 6.2 ± 0.4 (> 5.7 - > 6.6) 5.3 ± 0.8 (4.4 - > 6.3) % Reduction 99.9 ± 0.2 (99.6 - > 99.995) 99.98 ± 0.03 (99.9 - > 99.9998) Organism Removal 5.1 ± 0.9 (3.9 - > 5.8) 4.7 ± 1.2 (3.4 - > 6.2) Surface Type Ceramic Tile Laminate Granite a There were six replicates for each organism 271 Table S2. Recovered microorganisms from fomitesa B. thuringiensis Poliovirus Type 1 Mean CFU/2-cm2 log10 ± SD (Range) Mean CFU/2-cm2 log10 ± SD (Range) % Reduction 99.4 ± 0.4 (91.8 - 99.7) 99.98 ± 0.01 (99.9 - > 99.999) Organism Removal 2.5 ± 0.3 (2.0 - 2.8) 5.0 ± 0.4 (4.5 - 5.7) % Reduction 99.4 ± 0.5 (98.7 - > 99.98) 99.97 ± 0.02 (99.9 - > 99.998) Organism Removal 3.1 ± 0.5 (2.6 - > 4.0) 4.9 ± 0.5 (4.2 - 5.7) % Reduction 98.1 ± 1.7 (95.3 - > 99.98) 99.94 ± 0.1 (99.8 - > 99.997) Organism Removal 3.8 ± 1.3 (2.5 - > 6.0) 4.9 ± 0.8 (3.9 - 5.7) Surface Type Ceramic Tile Laminate Granite a There were six replicates for each organism 272 FIGURES A B log10 Reduction 3.6 3.9 log10 Reduction 3.5 Recovery of bacteria CFU /2-cm 2 Recovery of bacteria CFU/2-cm2 4.5 5.0 Ceramic Tile Laminate 4.4 8 8 7 6 5 4 3 2 1 7 6 5 4 3 2 1 0 0 Ceramic Tile Control non-treatment log10 CFU/2-cm2 Laminate Granite Surfaces Interven on treatment log10 CFU/2-cm2 C Control non-treatment log10 CFU/2-cm2 Granite Surfaces Interven on treatment log10 CFU/2-cm2 D 1.9 2.5 2.1 8 log10 Reduction Recovery of virus PFU/2cm 2 Recovery of spores CFU/2cm2 log10 Reduction 7 6 5 4 3 2 3.9 3.8 3.7 8 7 6 5 4 3 2 1 1 0 0 Ceramic Tile Laminate Granite Ceramic Tile Control non-treatment log10 CFU/2cm2 Interven on treatment log10 CFU/2cm2 Laminate Granite surfaces surfaces Control non-treatment log10 PFU/2-cm2 Interven on treatment log10 PFU/2-cm2 Fig. S1. Recovery of (A) E. coli, (B) S. aureus, (C) B. thuringiensis spores, and (D) poliovirus 1 from control non-treated and intervention treated fomites 273 A B Geometric Mean 100 100 10 10 % Transfer Efficiency % Transfer Efficiency Geometric Mean 1 0.1 0.01 1 0.1 0.01 0.001 0.001 0.0001 0.0001 Ceramic Tile Laminate Granite Ceramic Tile Without Interven on Laminate Granite Ceramic Tile With Interven on Laminate Granite Ceramic Tile Without Interven on C Laminate D Geometric Mean Geometric Mean 100 100 10 10 % Transfer Efficiency % Transfer Efficiency Granite With Interven on 1 0.1 0.01 0.001 1 0.1 0.01 0.001 0.0001 0.0001 Ceramic Tile Laminate Without Interven on Granite Ceramic Tile Laminate With Interven on Granite Ceramic Tile Laminate Granite Ceramic Tile Without Interven on Laminate Granite With Interven on Fig. S2. Transfer efficiency % of (A) E. coli, (B) S. aureus, (C) B. thuringiensis spores, and poliovirus 1 from control nontreated (Lopez et al., submitted for publication) and intervention treated fomites 274 A B Geometric Mean Recovery of S. aureus log10 CFU/2-cm 2 Recovery of E. coli log 10 CFU/2-cm 2 Geometric Mean 6 5 4 3 2 1 0 Ceramic Tile Laminate Granite Ceramic Tile Without Interven on Laminate 5 4 3 2 1 0 Ceramic Tile Granite Laminate Granite Ceramic Tile Without Interven on With Interven on C Laminate Granite With Interven on D Geometric Mean Geometric Mean 6 6 Recovery of virus log 10 PFU/2-cm 2 Recovery of spores log10 CFU/2-cm 2 6 5 4 3 2 1 0 Ceramic Tile Laminate Without Interven on Granite Ceramic Tile Laminate With Interven on Granite 5 4 3 2 1 0 Ceramic Tile Laminate Granite Ceramic Tile Without Interven on Laminate Granite With Interven on Fig. S3. Recovery of (A) E. coli, (B) S. aureus, (C) B. thuringiensis spores, and poliovirus 1 from fingers in contact with nontreated (Lopez et al., submitted for publication) and intervention treated fomites. 275 APPENDIX K: SUPPLEMENTAL MATERIALS Risk of Campylobacter jejuni Infection From in Preparing Raw Chicken in Domestic Kitchens and Cross-contamination Reduction From Disinfectant Wipes Gerardo U. Lopez1, 2, Masaaki Kitajima1, Jonathan Sexton1, 2, Charles P. Gerba1, and Kelly A. Reynolds2* 1 Department of Soil, Water and Environmental Science, University of Arizona, Tucson, Arizona 85721, USA 2 The University of Arizona, Mel and Enid Zuckerman College of Public Health, 1295 N. Martin Ave., Tucson, AZ 85724, USA * Corresponding author. Tel: (520) 626-8230, Fax (520) 626-8009, E-mail: [email protected] A manuscript for Risk Analysis Supplemental Material Not Included in Publication 276 TABLES Table SI Risk of Infection (/person/event): Condition A Fomite Ceramic tile Laminate Granite Intervention Non-treatment Condition A 5.60E-03 Standard Deviation 2.62E-02 Treatment 1.50E-05 5.76E-04 Non-treatment 8.00E-03 3.30E-02 Treatment 6.60E-05 1.03E-03 Non-treatment 6.00E-03 2.70E-02 Treatment 6.00E-06 7.40E-05 Table SII Risk of Illness (/person/event): Condition A Fomite Ceramic tile Laminate Granite Intervention Non-treatment Condition A 4.30E-05 Standard Deviation 2.03E-04 Treatment 7.30E-08 6.74E-07 Non-treatment 6.00E-05 2.42E-04 Treatment 4.01E-07 5.13E-06 Non-treatment 4.80E-05 1.99E-04 Treatment 4.50E-08 5.65E-07 277 Table SIII Risk of Death (/person/event): Condition A Fomite Ceramic tile Laminate Granite Intervention Non-treatment Condition A 2.20E-09 Standard Deviation 1.02E-08 Treatment 5.20E-12 1.22E-10 Non-treatment 3.10E-09 1.22E-08 Treatment 2.10E-11 3.49E-10 Non-treatment 2.40E-09 1.04E-08 Treatment 2.20E-12 2.49E-11 Table SIV Annual Risk of Infection (/person/year): Condition A Fomite Ceramic tile Laminate Granite Intervention Non-treatment Condition A 1.26E-01 Standard Deviation 2.55E-01 Treatment 1.00E-03 1.20E-02 Non-treatment 1.67E-01 2.96E-01 Treatment 4.00E-03 3.10E-02 Non-treatment 1.51E-01 2.78E-01 Treatment 1.00E-03 1.20E-02 278 Table SV Annual Risk of Illness (/person/year): Condition A Fomite Ceramic tile Laminate Granite Intervention Non-treatment Condition A 3.80E-03 Standard Deviation 1.68E-02 Treatment 7.90E-06 1.11E-04 Non-treatment 5.50E-03 2.09E-02 Treatment 4.90E-05 1.01E-03 Non-treatment 4.50E-03 1.84E-02 Treatment 7.90E-06 5.29E-04 Table SVI Annual Risk of Death (/person/year): Condition A Fomite Ceramic tile Laminate Granite Intervention Non-treatment Condition A 1.90E-07 Standard Deviation 9.00E-07 Treatment 3.90E-10 4.23E-09 Non-treatment 2.90E-07 1.17E-06 Treatment 2.30E-09 4.12E-08 Non-treatment 2.40E-07 9.90E-07 Treatment 2.20E-10 3.23E-09 279 Table SVII Risk of Infection (/person/event): Condition B Fomite Ceramic tile Laminate Granite Intervention Non-treatment Condition B 1.23E-02 Standard Deviation 4.11E-02 Treatment 4.60E-05 5.88E-04 Non-treatment 1.80E-02 5.20E-02 Treatment 2.17E-04 2.33E-03 Non-treatment 1.50E-02 4.60E-02 Treatment 2.70E-05 6.39E-04 Table SVIII Risk of Illness (/person/event): Condition B Fomite Ceramic tile Laminate Granite Intervention Non-treatment Condition B 1.03E-04 Standard Deviation 3.46E-04 Treatment 3.52E-07 4.92E-06 Non-treatment 1.39E-04 4.12E-04 Treatment 1.54E-06 1.44E-05 Non-treatment 1.21E-04 3.63E-04 Treatment 1.92E-07 2.96E-06 280 Table SIX Risk of Death (/person/event): Condition B Fomite Ceramic tile Laminate Granite Intervention Non-treatment Condition B 5.20E-09 Standard Deviation 1.70E-08 Treatment 1.61E-11 1.55E-10 Non-treatment 6.80E-09 2.01E-08 Treatment 8.20E-11 1.43E-09 Non-treatment 6.10E-09 1.87E-08 Treatment 9.60E-12 2.00E-10 Table SX Annual Risk of Infection (/person/year): Condition B Fomite Ceramic tile Laminate Granite Intervention Non-treatment Condition B 2.21E-01 Standard Deviation 3.38E-01 Treatment 4.00E-03 2.80E-02 Non-treatment 2.71E-01 3.68E-01 Treatment 1.20E-02 6.70E-02 Non-treatment 2.57E-01 3.60E-01 Treatment 2.00E-03 1.90E-02 281 Table SXI Annual Risk of Illness (/person/year): Condition B Fomite Ceramic tile Laminate Granite Intervention Non-treatment Condition B 8.50E-03 Standard Deviation 2.72E-02 Treatment 2.92E-05 3.12E-04 Non-treatment 1.21E-02 3.42E-02 Treatment 1.38E-04 1.19E-03 Non-treatment 1.05E-02 3.16E-02 Treatment 1.69E-05 2.37E-04 Table SXII Annual Risk of Death (/person/year): Condition B Fomite Ceramic tile Laminate Granite Intervention Non-treatment Condition B 4.90E-07 Standard Deviation 1.58E-06 Treatment 1.89E-09 3.78E-08 Non-treatment 6.90E-07 2.01E-06 Treatment 7.80E-09 8.29E-08 Non-treatment 5.70E-07 1.71E-06 Treatment 9.10E-10 1.99E-08
