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The Control of Microorganisms LC D R B R I A N B E A R D E N , M S , P E U S P U B L I C H E A LT H S E R V I C E / U S E PA R 9 M A R I A N A I S L A N D S WAT E R O P E R ATO R S A S S O C I AT I O N FEBRUARY 5, 2015 Topics • Review of pathogens • Disinfection terminology • Physical methods of disinfection and sterilization • Heat, radiation, filtration • Chemical disinfection • Oxidizers • Metals • Other chemicals Source: Madigan, Martinko, Dunlap, & Clark (2008 ) Microorganisms of interest HARDEST TO KILL (most resistant) • Protozoa: • Cryptosporidium cysts • Giardia cysts • Bacterial endospores • Viruses EASIEST TO KILL (least resistant) • Bacteria • Cryptoporidium: • Giardia: Source: Madigan, Martinko, Dunlap, & Clark (2008 ) • Bacterial endospores • Diseases: • Anthrax • Tetanus • botulism Source: Madigan, Martinko, Dunlap, & Clark (2008 ) • Viruses • Bacteria Cholera: (Vibrio chlolerae) Tuberculosis: (Mycobacterium tuberculosis) http://www.microbiologyinpictures.com/bacteria%20photo s/mycobacterium%20tuberculosis%20photos/mycobacteriu m%20tuberculosis%20030.jpg http://remf.dartmouth.edu/Cholera_SEM/images/03_Cholera %200395%20area1%2020kX.jpg Terminology • Sterilization • The killing or removal of all microorganisms • Inhibition • The limitation of the growth of microorganisms • Decontamination: The treatment of an object or surface to make it safe to use. Example: wiping a table or cleaning dishes after a meal. • Disinfection: Can involve killing or just inhibiting the growth of microorganisms. Not all are eliminated. Example: bleach. Terminology: Chemical Methods • Sterilants • Destroy all forms of microbial life, including bacterial endospores • Disinfectants • Chemicals that kill most, but not all microorganisms. • Used on inanimate objects – not people! • Sanitizers • Reduce (but may not eliminate) microorganisms to a level that is considered safe • Used to sanitize food equipment, household objects, and laundry • Antiseptics & Germicides • Kill or inhibit growth of microorganisms, but non-toxic enough to be applied to living tissues (people can use these on themselves) Physical Methods of Control • Heat • Ionizing Radiation • Filtration • Ultraviolet Radiation Heat (Thermal Destruction) • Kills by: “Denaturation” – macromolecules lose structure & function. In other words, the basic components of the cell (proteins, DNA, outer membrane) and damaged and no longer function. http://kimwootae.com.ne.kr/apbiology/Protein%20Denaturation.jpg http://www.btci.org/k12/bft/pcr/PCR_st udentscenario_files/denaturation.JPG Heat (Thermal Destruction) • AUTOCLAVE most common form of heat sterilization • 121°C for 4 to 5 minutes will kill endospores • Entire object must remain in autoclave long enough to achieve this – typically 10-15 minutes, more for larger, more moist materials • Pasteurization another form (milk) • BOIL water to sterilize: • 1 minute at sea level (100°C); • 3 minutes at altitudes above 1 mile (95°C) Heat (Thermal Destruction) • Autoclave (lab) http://www.medsupplier.com/productimages/Autoclaves/Midmark/midmark-ritter-m11autoclave-sterilizer-hi-res-3.jpg • Autoclave (medical waste) http://www.packworld.com/sites/default/files/styles/lightbox/p ublic/images/issues/10_10/Images%20Features/Autoclave.jpg? itok=qISshWMT http://features.cgsociety.org/gallerycri ts/86785/86785_1194093509_large.jpg Ionizing radiation • Kills by: damage to macromolecules, esp. DNA • Gamma rays, X-rays, electrons • Used mostly in food processing; some experiments with sludge http://people.chem.duke.edu/~jds/cruise_chem/nuclear/pics/fruit.gif Filtration • Kills by: Physically removing pathogens • Must be sized for target organisms Pore size 5 µm: Algae and aquatic bacteria Pore size 0.2 µm: Source: Madigan, Martinko, Dunlap, & Clark (2008 ) Leptospira interrogans Ultraviolet (UV) Radiation • Kills by: Damaging the DNA/RNA, which prevents replication. Some wavelengths can do broader damage to other cell structures • Commonly used for water disinfection http://www.oemcollect.com/uv2.jpg Ultraviolet (UV) Radiation Organism Ultraviolet Dose (pW-s/cm2) Required for 90% Reduction Bacterial endospores 45,000 to 56,000 Adenovirus 23,600 to 56,000 Coxsackievirus 11,900 to 15,600 Hepatitis A virus 3,700 to 7,300 Cryptosporidium cysts 3,000 Giardia cysts 2,000 E. coli Vibrio cholerae (Cholera) 1,300 to 3,000 650 to 3,400 • Note sensitivity of Crypto & Giardia to UV – cysts are transparent and allow UV radiation to penetrate easily. Ultraviolet (UV) Radiation • Strange phenomenon: photoreactivation • Some UV-damaged bacteria can repair DNA when exposed to sunlight • Total and fecal coliform are capable • Fecal streptococci are not • To prevent: • Requires sufficient UV dose • Prevent direct exposure of disinfected water to sunlight • Use medium pressure or pulsed UV lamp (damages more parts of the cell than just DNA) http://www.nature.com/nature/journal/v421/n6921/images/nature014 08-f1.2.jpg Chemical Methods of Control • Strong oxidants: • Chlorine, chloramines, chlorine dioxide, ozone • Bromine and Iodine • Metal ions • Spray & wipe disinfectants • Alcohol Strong Oxidants • Kill by: reacting with any organic molecule. • Bacterial inactivation: damage to cell membrane, impairment of cellular functions, destruction/damage of DNA • Virus inactivation: reaction with outer coating of virus (capsid), reaction with RNA/DNA Source: Pepper, Gerba, & Gentry, (2014) Strong oxidants MOST POWERFUL • Ozone • Chlorine Dioxide • Chlorine LEAST POWERFUL • Chloramines Strong Oxidants • Conditions that can interfere: • pH • Particulate matter & turbidity • Organic matter Controls strength of some oxidants Reacts with , blocks, or “uses up” oxidants • Disinfection byproducts (“DBPs”): • All oxidants react with many substances in water to create byproducts that are harmful to humans and regulated by EPA: • • • • THMs (trihalomethanes) HAAs (haloacetic acids) Chlorite Bromate Comparison of effects of agents Source: Pepper, Gerba, & Gentry, (2014) Chlorine • “Free Chlorine” = HOCl + OCl• HOCl (hypochlorous acid) more effective than OCl(hypochlorite ion) • Effectiveness depends on pH: less effective at high pH • “Combined Chlorine” results from reaction with ammonia (chloramines) Source: Pepper, Gerba, & Gentry, (2014) Chloramines • Chlorine reacts with Ammonia to form chloramines • Less powerful but longer lasting than free chlorine • Created intentionally to provide longer-lasting residual in some water distribution systems • “secondary” disinfection, following “primary” disinfection, such as by ozone • Occurs naturally when ammonia present in water (especially in wastewater) – results in need to achieve “breakpoint chlorination” Chloramines • Breakpoint chlorination – satisfaction of organic and ammonia demand required before free chlorine can be achieved Source: Pepper, Gerba, & Gentry, (2014) Chlorine Dioxide • Very strong oxidizer • pH does not affect disinfection strength • does not create THMs (important when there is organic matter in water), but does create chlorite as a DBP • Dangerous – potentially explosive. Must be generated on-site by combining chlorine gas and sodium hypochlorite Ozone • Made by passing electric discharge (arc) through a stream of air or oxygen • Does not produce THMs, but can make other byproducts that have health concerns (aldehydes and bromates) • Effectiveness not influenced by pH or ammonia • Much more powerful oxidant than Chlorine – lower C·t values • Can even kill Cryptosporidium with C·t of 1 to 3 Ozone – kills Cryptosporidium Source: Pepper, Gerba, & Gentry, (2014) Strong Oxidants – Comparison of strength (by C·t values) Organism C·t Values for 99% inactivation Chlorine Chloramines Chlorine dioxide Ozone Cryptosporidium cysts 9,740 11,300 11,400 64,600 1,000 3.3 - 40 Giardia cysts 54 - 192 430 - 1,400 2.7 to 10.7 053 - 1.94 -- -- 25 -- 0.15 360 - 990 0.28 0.02 0.15 - 2.16 -- -- 0.64 - 2.6 592 1.7 -- Bacterial endospores Adenovirus Coxsackie virus Hepatitis A Polio virus 1.7 1420 0.2 to 6.7 0.2 E. coli 0.6 113 0.48 0.006 to 0.02 Source: Pepper, Gerba, & Gentry, (2014) Bromine & Iodine • Bromine: • used in hot tubs and spas • Not as fast acting as Chlorine (larger C·t values) • Effective against bacteria, viruses, and protozoa • Iodine • Used in small applications such as for camping and survival • Not effective against all protozoa: cryptosporidium cysts are very resistant to iodine • Physiologically active; not recommended for very long periods Metal Ions • Metals that exhibit antimicrobial activity: • Copper, Silver, Zinc, Lead, Cadmium, Nickel, Cobalt • Only Silver (Ag) and Copper (Cu) used for disinfection, due to toxicity of other metals • Cu & Ag used as swimming pool and hot tub disinfectants • Cu used in hospital distribution systems to control legionella growth • Ag used in home faucet filters to prevent growth in activated charcoal • Action is slow, but effective for long periods of time in water Summary of water disinfectant attributes Disinfection method Attribute Killing Power Crypto & Giardia Residual Toxicity/ Lack of DBPs Filtration (membrane) Boiling UV Radiation Chlorine Chloramines Chlorine dioxide Ozone Bromine Iodine Silver & Copper Spray & Wipe Disinfectants • Quats (quaternary ammonium compounds) • Antibacterial handsoaps, antiseptic wipes, mouthwashes, household & workplace cleaners • Common compounds: benzalkonium chloride; cetylpyridinium chloride • Kills common bacteria, but not endospores • Effective against enveloped viruses (influenza, Ebola?) • Some specific formulations effective against nonenveloped viruses • Some microorganisms may develop increased tolerance to quats over time • Tolerance does not mean the same thing as reistance! Quats remain effective at higher doses Spray & Wipe Disinfectants • Triclosan • Anti-bacterial and antifungal agent • Hand soaps, mouthwashes, shampoos, toothpastes, and also incorporated into materials such as cutting boards • Mild to skin • Some cases of increased tolerance, but still useful • Some bacteria have built-in resistance to triclosan Alcohol • Kills by: dissolving/damaging cell membrane, damaging cellular proteins • Used in: medical and laboratory sterilization, hand sanitizers http://www.bizpacreview.com/wp-content/uploads/2015/01/sanitizer-2.jpg Summary • Methods of disinfection: • Physical • Heat • Radiation • Filtration • Chemical • No method of disinfection is “perfect” • Physical methods can removal all pathogens, but do not prevent re-growth • Chemical methods vary in effectiveness and create byproducts or side effects Summary HARDEST TO KILL (most resistant) • Protozoa: • Cryptosporidium cysts • Giardia cysts • Bacterial endospores • Viruses EASIEST TO KILL (least resistant) • Bacteria Summary • Only sterilization results in 100% removal of microorganisms • Heat • Filtration • Radiation • But only chemical disinfection can prevent regrowth of microorganisms Final word • Microbial resistance: does the widespread use of disinfectants risk the development of “superbugs”, as we often hear about in reference to antibiotic drugs? NO - the methods of disinfection and sterilization in use today act very aggressively and non-specifically on all of the organic molecules which make up a microorganism, not like antibiotics which act with very specific mechanisms, against which microorganisms can evolve defenses. Chlorine has been in use for over 100 years now, with no change in microbial resistance to it. References Madigan, M. T., Martinko, J. M., Dunlap, P. V., & Clark, D. P. (2008). Brock Biology of Microorganisms (12th ed.). Benjamin Cummings. Pepper, I. L., Gerba, C. P., & Gentry, T. J. (2014). Environmental Microbiology (Third.). Academic Press.