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Abstracts of Posters Presented at 9th Tripartite Meeting of the Celtic Microbiology Associations (Hosted by the Welsh Microbiological Association) Cardiff, 5-7th June 2009 www.wma-wales.org.uk 001 Identification of clinical isolates of alpha-haemolytic streptococci by 16S rRNA gene amplification, Matrix-Assisted Laser Desorption Ionization coupled with Time Of Flight analysis Mass Spectrometry (MALDI-ToF/MS) and conventional phenotypic methods – a comparison. S Hadfield1, M Reid1, WY Wan1, S Johnston1, N Berry1, K El-Bouri1, A Lewis1, D Mack1,2, AP Davies1,2 1Molecular Diagnostics Unit, NPHS Microbiology Swansea, Singleton Hospital, Abertawe-Bro Morgannwg University NHS Trust, Swansea. 2Medical Microbiology and Infection, Institute of Life Science, School of Medicine, Swansea University, Swansea. Background The alpha-haemolytic streptococci (other than Streptococcus pneumoniae) are notoriously difficult to identify reliably in the clinical laboratory. They have important clinical significance, causing serious infections such as endocarditis, brain and liver abscess and other deep-seated pyogenic infections. Correct identification of these isolates is important clinically. For example, Abiotrophia sp (previously referred to as nutritionally variant streptococci) require the use of gentamicin as well as penicillin for therapy, and the milleri-group streptococci have a particular propensity for causing pyogenic infection. Identification is currently attempted using phenotypic methods based on biochemical reactions, such as API 20S or the automated BD Phoenix system. Other methods such as MALDI-ToF/MS are becoming available but there is very little experience with this as yet. Broad range 16S rRNA gene sequence analysis offers a genotypic method capable of definitive identification. Objectives To identify clinically significant isolates of alpha-haemolytic streptococci using MALDI-ToF/MS, API 20S, BD Phoenix, and 16S rRNA gene sequence analysis to compare their reliability, and consider the clinical implications. Methods Forty clinically significant isolates of alpha-haemolytic streptococci were tested by MALDI-ToF/MS, API 20S, BD Phoenix, and 16S rRNA gene sequencing. Using 16S rRNA gene sequencing as the gold standard, the relative reliability of each method was assessed. Conclusions Alpha-haemolytic streptococci pose well-known difficulties in laboratory identification. Currently, universal 16S rRNA gene PCR and sequencing remains the best method and its application greatly facilitates laboratory identification in a time-frame which aids clinical decision-making. 2 002 Dissociated Clindamycin Resistance in Staphylococci. Katherine McBride1, Elaine McCulloch2, Leigh Williams1, Elizabeth Kilgour1, Craig Williams2, Alistair Leanord1 1Microbiology 2Microbiology Department, Monklands Hospital, Airdrie Department, Yorkhill Hospital, Glasgow Objective Macrolide-lincosamide-streptogramin B group (MLSB) resistance can be caused by two mechanisms. The msrA gene encodes for an efflux pump causing constitutive resistance, whilst the erm genes confer resistance to the macrolide-lincosamide-streptogramin B group of antibiotics (MLSB) by alteration of the ribosomal target. MLSB resistance may be inducible or constitutive. Inducible MLSB resistance is not recognised using standard susceptibility testing methods, including automated systems. Failure to identify this resistance mechanism may lead to clinical failure of clindamycin therapy. Previous work showed that 96% of our local S aureus population carried inducible MLSB resistance. We undertook this study in order to establish the prevalence of genes coding for MLS B resistance in a group of 55 MRSA, and 56 MSSA isolates and to discover which, if any standard phenotypic laboratory techniques are efficient in detecting inducible resistance. Methods A resistance profile was built up using disk diffusion, Vitek automation and E tests. Real-time PCR was used as the ‘gold standard’ in order to probe for resistance genes erm A, erm B, erm C and msrA. Results All 74 strains (MSSA and MRSA) which phenotypically showed macrolide resistance carried a resistance gene. Of the 35 clindamycin-susceptible, erythromycin-resistant MSSA strains 14 (40%) carried erm A, 17 (49%) erm C, and 4 (11%) msrA. Of the 19 clindamycin-susceptible, erythromycin-resistant MRSA strains 2 (11%) carried erm A and 17 (89%) erm C. All isolates that contained an erm gene were D test positive. Of the 4 isolates that harboured msrA, all were D test negative. Erythromycin MIC of >256ug/l was associated with the erm C gene in 90% of MRSA strains but only 11% of MSSA. Conclusions In S aureus the D test is an effective method of identifying MLSBi resistance. In this strain collection, erm A and erm C where equally distributed within MSSA, whilst MRSA had a predominance of erm C. Although no phenotypic tests could reliably differentiate between the erm genes, within MRSA, an erythromycin MIC of >256ug/l was associated with the erm C gene 3 003 Asymptomatic Carriage of Protozoan Parasites in Children in Day Care Centers in the United Kingdom. AP Davies1,2, B Campbell1, M R Evans3,4, A Bone5, A Roche5, RM Chalmers2 1Institute of Life Sciences, School of Medicine, Swansea University Cryptosporidium Reference Unit, National Public Health Service Microbiology Swansea 3National Public Health Service Communicable Disease Surveillance Centre, Cardiff 4Department of Primary Care and Public Health, Cardiff University 5South West London Health Protection Unit, Tooting, London 2UK Background Cryptosporidium and Giardia are protozoan parasites, endemic worldwide, which cause diarrhea in healthy children. Most Cryptosporidium infections in the UK are caused by two species, C. hominis and C. parvum, although occasionally other species or genotypes are detected. Previous studies have found a significant association between infection with both Cryptosporidium hominis and Giardia duodenalis and changing children’s nappies, suggesting that very young children may be an important reservoir for these organisms in the community. Objective To investigate the prevalence of asymptomatic carriage of these parasites amongst infants and preschool children attending day-care nurseries in two UK cities, using highly sensitive immunomagnetic separation of Cryptosporidium oocysts and a commercially available ELISA kit to detect Giardia antigens. The species/genotype of Cryptosporidium isolates was also defined. Methods Carers of healthy children attending daycare nurseries in Swansea and London were asked to send faeces from soiled nappies for testing using immunomagnetic separation, an extremely sensitive technique recently validated for use in human faecal samples at the UK Cryptosporidium Reference Unit. Isolates were typed by PCR-sequencing ssu rDNA. The presence of Giardia was also sought, using ELISA (Giardia II, Techlab®) and questionnaire data was collected on possible risk factors. Results In total, 230 samples were collected. The point prevalence of both Cryptosporidium and Giardia was 1.3% (95%CI 0.3 to 3.8%), with no dual infections. Only one of the three Cryptosporidium isolates was C. hominis, the other two being Cryptosporidium skunk and cervine genotypes which are rarely found in samples from symptomatic human cases. Conclusions The results do not lend support to the suggestion that young children are a significant reservoir of Cryptosporidium and Giardia in the UK. The finding of genotypes of Cryptosporidium rarely identified in samples from symptomatic patients raises the possibility that acquisition of nonhominis/parvum species may be relatively more likely to result in asymptomatic carriage than in clinical disease, which may reflect lower pathogenicity 4 004 Comparison and evaluation of commercially available identification methods for Candida albicans. Catherine Price, Lorna Vale, Alan Paull NPHS Microbiology Cardiff, University Hospital of Wales, Cardiff Candida species are known to cause superficial and systemic infections. Superficially they cause irritation of the mucosal membranes or dermal rashes, but systemic candida infections may result in death. Systemic infections are rated as the forth causative agent of mortality in the UK and third in the US. Candida albicans is the most commonly isolated organism, accounting for over 75% of all candidiasis. Treating infections caused by Candida albicans is relatively straight forward as there is generally very little acquired resistance to the commonly available antimycotics. Treatment failure is likely to be a result of an infection by a non-albicans strain of Candida; some of which are intrinsically resistant to the commonly used antimycotics. Candida albicans is a commensal organism of the skin, oral and gastrointestinal flora, therefore obtaining mixed cultures is very likely. The performance of a germ tube test on a mixed culture of Candida species containing Candida albicans would yield a positive result, which would confirm the presence of Candida albicans but would mask the presence of the non-albicans species. The gold standard method of identifying Candida albicans, thus differentiating it from non-albicans species is the germ tube method using human plasma. However, due to risks of blood borne viruses with human plasma, animal sera or solid media, such as Mueller-Hinton agar can be used as an alternative. In this study four different methods of identifying Candida albicans were performed, evaluated and compared to the Gold standard method, in order to determine which method would be a better alternative to using human plasma. These methods were: germ tube test using horse serum and Mueller-Hinton agar, “spiking” on heated horse blood agar and the chlamydospore test using cornmeal/Tween 80 agar. It was discovered that horse serum was superior for sensitivity and specificity; results were 100%. However, use of Mueller-Hinton germ tube method, allowed for easier determination of true hyphae and pseudohyphae. The usefulness of using chromogenic agar as a primary plate, identification plate and for the determination of mixed cultures in comparison to Sabouraud agar with chloramphenicol (SABC) was also investigated in this study. Four commercially available chromogenic agars were used: As an identification plate, the best agar was: ColorexTM Candida agar As a primary plate the best agars were: SABC agar and ChromIDTM Candida agar (CANID2) by Biomérieux The best agar for determining mixed cultures was ChromIDTM Candida agar (CANID2) by Biomérieux 5 005 Evaluation of Single and Multiplex PCR Assays for the Rapid Identification of Dermatophytes from Nail and Skin Samples within Twenty Four Hours. C.L.Alexander, L.Brown, R.Dunsmuir, G.S.Shankland. Clinical Mycology, Microbiology Department, Yorkhill Hospital, Glasgow Clinical Mycology, Glasgow receives over 10,000 specimens each year for the diagnosis and identification of fungal infections from superficial sites. The causative fungi are known as dermatophytes. Audits during 2007 and 2008 indicate Trichophyton rubrum is the most common dermatophyte (84% of all positive superficial samples). The second is Trichophyton interdigitale (14%). Objectives: To evaluate and validate rapid PCR-based methods with modifications based on studies by Arabatzis et al* involving single and multiplex assays. To demonstrate prompt, accurate diagnosis and identification of dermatophytes from nail and skin samples. The aim is to improve turnaround times for dermatophyte identification which is currently 14 days by culture for Trichophyton rubrum and Trichophyton interdigitale. Method: A total of 398 samples which included 362 nail samples and 36 skins were subjected to; 1) Microscopical analysis after 3 hours incubation in 20% KOH for the presence of fungal spores and/or hyphae; 2) Culture on Sabourauds and Mycosel agar for 3 weeks. Identification is based on colony morphology and micromorphology. 3) DNA extraction by BioRobot EZ1 then real-time PCR using two separate assays to detect T. rubrum or T. interdigitale. Multiplex assays have also been evaluated to detect Microsporum canis, Trichophyton tonsurans, Trichophyton violaceum, Microsporum audouinii. Results: In 254 samples, 155 were microscopy, culture and PCR negative. The other 99 samples were microscopy, culture and PCR positive (n=96 for T.rubrum, n=3 for T.interdigitale). T.rubrum was detected in a further 55 samples by PCR which were microscopy positive but failed to grow any organism on culture. Microscopy failed to detect fungi in 12 samples which grew T.rubrum and which were PCR positive. 2 samples previously reported as T.interdigitale were PCR positive for T.rubrum. PCR detected T.rubrum in a further 2 samples which yielded Scopulariopsis brevicaulis or Candida albicans from culturing. 8 samples grew organisms which were not detected using these assays. Conclusions: PCR successfully identified dermatophytes within 24 hours compared to 14 days by culturing. It was more sensitive than culturing, detecting a greater number of infections and produced accurate identifications. Key Findings: Identification of a range of dermatophytes from nail and skin samples is achievable within 24 hours using this PCR methodology and samples negative for fungi can be reported within 24-48 hours compared to 3 weeks. References *Arabatzis et al, Br.J Dermatol Vol 157, pg 681-689. 6 006 Contamination of Laryngoscope Handles David Williams1, Nidhika Berry2, John Dingley1, Ceri Jones2 1Deptartment 2NPHS of Anaesthetics, Abertawe Bro Morganwwg NHS Trust, Swansea Microbiology Swansea, Singleton Hospital, Swansea Objectives To assess the nature and extent of microbial contamination on the handles of laryngoscopes that were considered to be clean and ready for use in the anaesthetic rooms within the operating department of our hospital. Background Despite use of sterile or disposable laryngoscope blades for each patient, disinfection of laryngoscope handles does not routinely occur. Handles may be contaminated by splashes and contact with surfaces or the hands of the anaesthetist. Laryngoscope handles are typically knurled to provide a good grip, however the fissures in this surface may harbour pathogens. The tip of the blade usually makes contact with an area on the lower third of the handle when folded after use, creating a potential route for transmission of pathogens between patients. Method Specimens collected from 64 laryngoscope handles which were deemed to be “ready for patient use” were assessed semi-quantitatively for bacterial contamination. Further identification of all isolates by routine methods and a MicroflexTM LT MALDI-TOF mass spectrometer was performed. Samples were taken from three sites on each laryngoscope handle: A. Hook mount (smooth) B. Upper third (knurled) C. Contact point (knurled) Results One or more species of bacteria were isolated from 61 (86%) of the handles. These included the potential pathogens Enterococci, meticillin-sensitive Staphylococcus aureus, Klebsiella and Acinetobacter. The cultures did not yield any anaerobes, fungi, meticillin-resistant Staphylococcus aureus, vancomycin resistant enterococci or multiresistant gram negative bacilli. 192 samples yielded 130 positive cultures, of which Site A: 33 (25%); Site B: 44 (34%); Site C: 53 (41%). Site C was the only site to demonstrate heavy contamination, and the only site from which Streptococcus viridans were isolated. Conclusions & Key Findings Contamination with aerobic bacteria, some of which were potentially pathogenic, was demonstrated on the majority of laryngoscope handles studied. A greater range of species and heavier growth were found on knurled surfaces (B,C) compared with smooth surfaces (A). A greater range of species (including oral flora) and heavier growth were found at the contact point where the tip of the laryngoscope blade made contact with the handle (C) compared with other knurled surfaces (B), highlighting a potential route for transmission of pathogens between patients. Laryngoscope handles present a potential route for transmission of infection. Strategies to address this include: revision of procedures for disinfection and storage prior to use, introduction of disposable handles or sheaths, and re-design of handles to eliminate knurled surfaces and contact points. 7 007 Improved laboratory diagnosis of herpes virus infections Jenna Dawson, Jenny Bayliss, Michael Isaac, Nidhika Berry Virology Department, NPHS Microbiology Swansea, Singleton Hospital, Swansea Background The traditional method of tissue culture confirmed by immunofluorescence (TC/ DIF) for diagnosis of herpes and varicella infections has been replaced by molecular methods which have proved to be more sensitive in other centres within the UK and elsewhere. Therefore, we compared the sensitivity of our routine tissue culture method for diagnosis of herpes (HSV-1, HSV-2) and varicella zoster (VZV) with a real time polymerase chain reaction. Methods Included in the 230 samples were 222 from genital sites, skin lesions (5) and CSF (3). The swabs were transported in skimmed milk virus transport medium (SVTM), inoculated into MRC-5 human fibroblast cells and incubated for up to 10 days. Cultures with a CPE were tested by DIF using Chemicon HSV and VZV typing reagents. A simultaneous detection system for HSV-1, HSV-2, and VZV DNA via multiplex real-time PCR using different fluorophores was used. Viral DNA was extracted on a Corbett Xtractorgene using Corbett VX reagent pack. A mastermix of Invitrogen Platinum Quantitative PCR SuperMix-UDG and primer/ probes manufactured by Metabion and Applied Biosystems was added using the Corbett CAS1200. Amplification was performed on the Rotorgene 6000 and results were read and analysed. Discrepant specimens (TC/ DIF negative and PCR positive for HSV- 1 or HSV-2) were referred to Bristol HPA laboratory for confirmation. Results Comparison of TC/DIF with RT-PCR (n=227) in swab specimens PCR +ve PCR-ve HSV-1 +ve 36 0 TC/ DIF HSV-1 –ve 7 184 HSV-2 +ve 49 0 HSV-2 -ve 11 167 For discrepant specimens referred for confirmation, complete agreement was seen with our in house real time PCR for HSV-1 (4/4 of these specimens sent). 2 of the 11 HSV-2 discrepant specimens did not confirm on repeat testing in our laboratory and were negative at Bristol. VZV DNA was detected in 4/ 230 specimens, all of which were negative by TC/ DIF. Conclusions 1. The detection rate of HSV-1, HSV-2 increased by 7.9%. 2. We were able to demonstrate VZV by real time PCR in clinically diagnosed chickenpox patients and from other suspect lesions. 3. The turnaround time for the results was reduced to 24 hours using PCR. 4. We propose using this method as a timely, sensitive and reliable test for the diagnosis of HSV1, HSV-2 and VZV in genital and skin swabs. 5. Further comparison of CSF specimens will be carried out to enable us to use this test locally for diagnosis of meningitis/ meningo-encephalitis. 8 008 The development of an in-house real-time PCR assay for Bordetella pertussis and Bordetella parapertussis and its use in a tertiary referral paediatric hospital Elaine McCulloch, Kathleen Harvey-Wood, Alison Balfour, Craig Williams Department if Microbiology, Royal Hospital for Sick Children , Glasgow B. pertussis, the cause of whooping cough, and B. parapertussis which causes a milder form of disease, are responsible for significant disease burden, especially in those aged <1 year. Primary vaccination against B. pertussis is in infancy, but immunity wanes and does not last lifelong. Infected adults with chronic cough not only suffer illness themselves, but also act as a source of infection to partially or non-immunised infants, in whom considerable morbidity and mortality can occur. Early diagnosis is crucial as it allows effective treatment to be commenced, limits transmission and reduces complications. Culture has poor sensitivity and it can take up to 5 days for growth to appear on charcoal blood agar plates. In November 2006 we developed an in-house real-time PCR (QPCR) assay for B. pertussis and B. parapertussis which initially was accessed only by paediatric inpatients and those presenting at the Accident and Emergency Department of our tertiary referral paediatric hospital. In July 2007 the service was extended to other hospitals, local GPs and community based paediatricians. Our specific objectives were: o To determine specimen suitability (ie recoverability from standard swabs in charcoal transport medium, dry pernasal swabs with no transport medium, and naso-pharyngeal aspirates) o To determine specimen stability (ie length of time specimens could be left prior to successful nucleic acid recovery) o To compare turnaround times of standard culture versus QPCR DNA was extracted from all specimens using an EZ1 Biorobot (QIAGEN) and a tissue extraction kit. All specimens were then tested using specific B. pertussis and B. parapertussis primers and probes. Amplification and detection was performed using ABI 7000 QPCR system. Culture was on charcoal blood agar plates incubated in a moist atmosphere at 350C for 7 days. Analysis of stability of specimens showed that all specimens regardless of time to processing were positive by QPCR. There was some decrease in DNA recovered after 6 days, particularly when testing for B. parapertussis. This was not deemed to be of significance provided samples were processed within 7 days. Over 200 clinical specimens have been analysed to date. The sensitivity of PCR versus culture has improved by 50% and the turn-around time is just under 2 days for QPCR versus an average of 7 days for culture. In conclusion, improved turn-around time using QPCR and a decrease in the reliance of culture has allowed this service to be accessed by a wide range of clinicians. 9 009 Q-PCR screening of BK viraemia in paediatric renal transplant patients Kathleen-Harvey-Wood, Alison Balfour, Craig Williams, Jim Beattie Department if Microbiology, Royal Hospital for Sick Children, Glasgow Polyomavirus BK virus (BKV) is part of the Polyomaviridae family, which includes JC virus (JCV) and the simian virus SV40. Primary infection with BKV occurs in early childhood and leads to viral latency. In immunosuppressed post renal transplant patients BKV may reactivate. BKV nephropathy has increasingly been recognised as a significant cause of renal dysfunction affecting 1 –10% of renal transplant patients and graft loss after renal transplantation of between 10 –80 %. Routine screening for the virus is recommended so that early diagnosis and appropriate treatment strategies can be instituted. Specific objectives To validate a commercial Q-PCR kit which can analyse urine and blood specimens for the presence of BKV To develop an algorithm for screening patients post-transplant for BKV which would complement our current screening strategy for EBV, CMV and Adenovirus Methods EDTA blood and urine samples were extracted using EZ1 BioRobot (Qiagen). Blood was extracted as per our established protocol for CMV and EBV using the EZ1 DNA blood kit with an elution volume of 200 ul. Urines were pre-treated and extracted using the EZ1 DNA Tissue kit with 50 ul elution volume. The Q-PCR assay was performed using the BKV Q-PCR Alert Kit (Nanogen) supplied by Inverness Medical. Samples were run in duplicate and amplification was performed on an ABI 7500 (standard mode). Water samples were extracted and analysed. QCMD BK/JC 2008 panel was examined. The assay was validated to determine the optimal number of amplification cycles. Results In order to validate the BKV Q-PCR kit, the results were compared with Micropathology Ltd where samples are currently sent for testing. 100 specimens were analysed to date. All 43 EDTA Blood samples tested were negative and the internal positive control was positive. 49 Urine specimens were tested : 27 positive, 18 negative and 4 equivcocal. 8 extracted water samples showed no contamination. EBV, CMV and Adenovirus positive samples were negative showing specificity of the PCR reaction. QCMD BKV 2008 panel samples examined : the lowest value ( 245 copies) was not detected. Conclusions Recent studies indicate detectable virus in the blood is more predictive of BKV nephropathy than viruria alone. We have developed and implemented a cost-effective algorithm for screening paediatric patients post-renal transplant for BKV which complements our already established regimen for screening for other viruses. 10 010 Rapid identification of microorganisms using Matrix-Assisted Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) Laser- Eugene Rees, Stuart Johnston, K El Bouri NPHS Microbiology Swansea, Singleton Hospital, Swansea A MALDI-TOF-MS system is available commercially that offers analysis and identification of bacterial cultures within 10 minutes of isolation. This offers major benefits to the clinical laboratory: More rapid isolate identification will support earlier clinical management decisions regarding appropriate treatment; Specific sensitivity testing could be undertaken in the context of identification data; Control of infection alerts could be generated at an earlier stage. Isolates Over 2,000 sequential, clinically isolated organisms identified by conventional methods, were investigated using MALDI-TOF. Gram negative bacteria were identified by either API20E,API20NE or BD Phoenix. Staphylococcus aureus was identified by Staph latex, coagulase reaction, Dnase reaction or BD Phoenix. Coagulase negative Staphylococci were speciated by BD Phoenix. ß haemolytic streptococci were identified by Lancefield grouping . Other streptococci, either non haemolytic or α haemolytic were identified by Optochin sensitivity testing and BD Phoenix. Miscellaneous bacteria (Neisseria sp, Campylobacter sp, Haemophilus sp, Clostridium sp etc.) were identified by conventional biochemical methods) MALDI Biotyper Part of a colony from each overnight culture was smeared in duplicate onto the MALDI-TOF steel target plate, overlaid with 1цl of matrix solution and allowed to air dry. The plate was placed into the MALDI-TOF-MS target receptor and an emission spectrum produced. Spectral analysis and identification was undertaken using the system’s ‘MALDI Biotyper’ software. The MALDI Biotyper typically collects 240 spectra from each well and compares the spectra against a library of known reference bacterial spectra. Results The identification software reports findings as one of the following: i. Excellent genus identification, good species identification ii. Good genus identification iii. No reliable identification iv. No peaks (spectra) found Data comparison (conventional versus MALDI-TOF) When an identification level of (i) or (ii) were obtained, the MALDI TOF results were compared with the conventional results follows A. Excellent match of species B. Match of genus, different species C. No match Results Coliforms - There was an 85% ‘Excellent Match’ (A) from at least one of the two duplicate wells. Of the rest 12% gave a B match. No match (C) was found in only 3% of the coliforms (all of these were confirmed as either non-fermenters or ‘unusual’ isolates e.g. Achromobacter. Mucoid strains of Klebsiella sp. Consistently yielded no emission spectrum using the smear method. These were identified by the MALDI-TOF later using an extraction method. Staphylococci – over 98% ‘Excellent Match’ (A) with S.aureus and the coagulase negative species. Streptococci – 95% ‘Excellent Match’ (A) of groupable streptococci, and 60% match(A or B) with greening or non haemolytic streptococci Conclusion MALDI-TOF MS is a reliable method for identification of clinically isolated microrgansims, is extremely fast compared to conventional methods, and requires significantly less staff time and lower consumable costs. 11 011 Disruption of Staphylococcus epidermidis biofilms by medicinal maggot Lucilia sericata excretions/secretions Llinos G Harris1, Alyson Bexfield2, Yamni Nigam3, Holger Rohde4, Norman Ratcliffe2, Dietrich Mack1 1Medical Microbiology and Infectious Diseases, Institute of Life Science, School of Medicine, Swansea University, Swansea 2Department of Biological Sciences, School of the Environment & Society, Swansea University, Swansea 3School of Health Sciences, Swansea University, Swansea, SA2 8PP, UK 4Institut für Medizinische Mikrobiologie, Virologie und Hygiene, Universitätsklinikum HamburgEppendorf, Universität Hamburg, Germany Objectives Chronic infections frequently cause substantial morbidity and mortality, and are commonly associated with biofilms formed by bacteria such as Staphylococcus epidermidis. Biofilms have been shown to protect bacteria from phagocytosis and antibiotic treatments. Thus with the increase in antibiotic resistant bacteria, maggot debridement therapy has been reintroduce for the treatment of chronic wounds. Studies have suggested that the beneficial effect of Lucilia sericata larvae (maggots) are present within their excretion/secretions (ES), which contain several different proteases, a glycosidase and an antibacterial compound. This present study evaluated the effect of L. sericata ES on formation and disruption of biofilms formed by two S. epidermidis strains employing different mechanisms of intercellular adhesion namely polysaccharide intercellular adhesin (PIA) or accumulation associated protein (AAP), respectively. Methods Native excretions/secretions (ES) were collected from third instar L. sericata larvae (ZooBiotic Ltd., Bridgend, UK ). A semiquantitative biofilm assay was used and the OD 550 of stained adherent bacterial biofilms was quantitated with a FLUOstar OPTIMA microplate reader. The presence of a biofilm was defined to have a mean OD550 greater than 0.2. 10µl ES was either added to test wells immediately after inoculation with bacteria (nascent biofilm formation) or after 24h of culturing (preformed biofilm). The effect of culturing for different times and temperatures, stability of ES by boiling the extracts, and 3 different ES fractions were analysed applied to nascent biofilms and pre-formed biofilms. The results were substantiated by immunolabelling using specific antibodies against PIA and AAP. Results S. epidermidis biofilm formation was inhibited in the presence of ES. Whilst ES also disrupted preformed biofilms, the extent of disruption differed depending on the mechanisms of biofilm accumulation employed by the respective S. epidermidis strain. The inhibitory activities were temperature and time dependent and were inactivated by heat treatment. The active compounds in ES against S. epidermidis biofilms were >10kDa in size, and appeared to have protease or glucosaminidase activity, respectively. ES disintegrated cell aggregates by degrading PIA or AAP as substantiated by the immunolabelling results. Conclusions In summary, ES interferes with S. epidermidis biofilm formation by specifically degrading factors employed in biofilm accumulation, which are similarly employed by Staphylococcus aureus. In a purified form ES-factors may have general applicability for the treatment or prevention of chronic biofilm infections caused by staphylococci. 12 012 Molecular epidemiology of extended-spectrum beta-lactamase (ESBL) carrying Enterobacteriaceae in Swansea Caron Jones1, Llinos G. Harris1, Eugene Rees2, Khalid El-Bouri2, Angharad P. Davies1, Dietrich Mack1 1Medical Microbiology and Infectious Diseases, Institute of Life Science, School of Medicine, Swansea University, Swansea 2NPHS Microbiology Swansea, Singleton Hospital, Swansea Extended-spectrum beta-lactamases (ESBL) mediate resistance to 3rd generation cephalosporins and aztreonam in Enterobacteriaceae and pose major clinical problems. Enterobacteria were collected from Swansea NPHS Microbiology laboratory and ESBL status confirmed by BSAC phenotypic methods. Of 167 isolates 118 were Escherichia coli, 38 Klebsiella spp., 5 Enterobacter spp., 2 Citrobacter spp., 2 Pseudomonas spp., 1 Morganella morganii and 1 Acinetobacter baumannii. Primer sets were used to specifically amplify TEM, SHV, CTX-M, and plasmid-encoded ampC genes. The most predominant ESBLs were CTX-M (n=139). 128, 5, 4, and 1 ESBLs belonged to CTX-M groups-1, -9, -25/26, and -2, respectively. 66/128 CTX-M group 1-positive isolates (exclusively E. coli) generated a 400 bp fragment of insertion sequence IS26-CTX-M-15 link region characteristic for epidemic E. coli strain A, which were all clonally related by PFGE. Nucleotide sequencing revealed 7 TEM-116 and 1 TEM-52 ESBLs, 1 TEM-33 (IRT), and 45 nonESBL TEM-1. 5 SHV-2 ESBLs were found, while 17 SHV-89, 6 SHV-11 and 10 SHV-1 genes, all non-ESBL, were present. 6 isolates carried ampC genes (2 CIT, 1 DHA, 1 EBC, 2 Fox). 83% of isolates carried CTX-M ESBLs, of which 92% belonged to CTX-M group 1. ESBL carrying E. coli outnumber the other Enterobacteriaceae species studied. 13 013 Evaluation of glycopeptide discs for Staphlococcus aureus with reduced susceptibility to glycopeptides (hGISA/GISA) Leanne Davies1, Mandy Wootton1, Robin Howe2 1SACU, 2NPHS NPHS Microbiology Cardiff, University Hospital of Wale, Cardiff Microbiology Cardiff, University Hospital of Wale, Cardiff OBJECTIVES GISA/hGISA strains are not detected by current laboratory disc diffusion testing. The GISA/hGISA detection method using Population Area Profile-Area Under Curve is proven to be accurate however it is time consuming. Disc testing with a range of vancomycin (V) and teicoplanin (T) concentrations was evaluated with Glycopeptide susceptible S aureus (GSSA), hGISA and GISA strains. METHOD 83 strains from a worldwide collection were tested in triplicate including; 14 meticillin resistant GSSA, 49 hGISA and 20 GISA. Vancomycin discs containing 5µg and 30µg, and teicoplanin discs containing 5µg, 10µg, 20µg and 30µg were tested on IsoSensitest (ISO) and Mueller Hinton Agar (MHA). Variation in zone diameter (ZD) and reproducibility according to media and phenotype were assessed. RESULTS ISO MHA GISA hGISA GSSA GISA hGISA GSSA V5 Mean 13.9 17.1 18.6 14.1 17.2 19.1 SE 0.34 0.09 0.19 0.32 0.08 0.16 V30 Mean SE 21.1 0.15 22.1 0.09 23 0.24 21.1 0.15 22.2 0.09 22.9 0.25 T5 Mean 11.3 14.1 17.1 10.8 13.7 16.6 SE 0.32 0.19 0.13 0.32 0.19 0.15 T10 Mean SE 14.2 0.26 16.5 0.17 18.4 0.15 13.8 0.26 15.9 0.18 18.4 0.15 T20 Mean SE 16.9 0.22 18.9 0.15 20.5 0.18 16.2 0.24 18.1 0.15 20 0.15 T30 Mean SE 18.7 0.17 20 0.14 21.2 0.15 17.9 0.21 19.3 0.15 20.7 0.17 Mean ZDs (mm) and standard error (SE) are given in the table. ZD was related to the level of susceptibility for all discs. Differentiation was greatest for low content discs. T5 on ISO/MHA and V5 on MHA distinguished between the GSSA and GISA strains. No disc could differentiate hGISA from GISA and GSSA. CONCLUSIONS Low concentrations of vancomycin and Teicoplanin can differentiate between GISA and hGISA strains. None of the glycopeptides concentrations tested were adequate for the accurate identification of hGISA. 14 014 Blood Culture Results in Welsh Special Care Baby Units SJA Froude1, RA Howe1, M Heiginbothom1, S Kotecha2 1NPHS Microbiology Cardiff, University Hospital of Wale, Cardiff of Child Health, School of Medicine, UHW Cardiff 2Department Introduction: Neonates are especially vulnerable to infection which can lead to significant morbidity and mortality. The All Wales Perinatal Survey (2006) reported that 11% of neonatal mortality was as a consequence of infection. Serious infections often manifest as bacteraemia, and the analysis of blood culture (BC) isolates presented here allows a better understanding of the patterns of serious infections presenting in Special Care Baby Units (SCBUs) across Wales. Methods: Data from all blood cultures submitted from SCBUs across Wales for the period 2005-07 were downloaded from the microbiology DataStore repository. The numbers of live births (LB) for each unit were obtained from the All Wales Perinatal Survey and used as a denominator of clinical activity. Results: For 2005-7, 10,314 blood cultures were taken. Overall sampling rates in 2007 were 85 BC/1000LB (being highest in level 3 units) with a positivity rate of 8%. The most frequent isolates were Coagulase Negative Staphylococci (CNS) followed by Group B Streptococci (GBS), Staphylococcus aureus, Escherichia coli, and Enterococci. CNS and coryneforms accounted for 71% of positive cultures. GBS incidence increased from 0.37 – 0.60/1000LB for 2005-07, remaining lower than published UK rates (0.72/1000LB). Conclusions: Traditional neonatal pathogens (eg GBS) were cultured at rates in keeping with UK experience. However the commonest isolates were CNS and coryneforms that, although sometimes implicated in line-associated infections, frequently may be “contaminants”. Strategies to improve the taking of blood cultures and assessment of potential contaminants should be developed to avoid unnecessary antimicrobial therapy. 15 015 Antibiotic Usage in Welsh Special Care Baby Units SJA Froude1, RA Howe1, M Heiginbothom1, S Kotecha2 1NPHS Microbiology Cardiff, University Hospital of Wale, Cardiff of Child Health, School of Medicine, UHW Cardiff 2Department Introduction: Neonates, particularly those admitted to Special Care Baby Units (SCBUs), are especially vulnerable to infection. Since early effective antibiotic therapy is critical in reducing morbidity and mortality, and signs of infection are often non-specific, SCBUs usually have clear empiric therapy guidance. However there is little data available on antibiotic use on SCBUs and inter-unit variability. This study provides such data. Methods: Ward stock data for antibiotics was obtained from the MEDUSA data repository for 11 of 13 SCBUs in Wales. Data was transformed into WHO Defined Daily Doses (DDDs) to facilitate inter-unit comparison. Occupied bed days (BD) data obtained from Health Solutions Wales was used as a denominator of clinical activity. Results: Antibiotic use varied from 4 DDDs/100BD (level 1 unit) to 18 DDDs/100BD (level 3 unit), with more different agents used (23) in level 3 than in level 1 (14). Of 9 units with complete data, 4 clearly used benzylpenicillin as the primary beta-lactam, while 4 used amoxicillin. Two of the 3 highest users of glycopeptides (up to 3 DDDs/100BD) were level 2 units that used predominantly teicoplanin; other units used vancomycin. Conclusions: There is predictable variability in antibiotic use with greater use in level 2 & 3 units. Differences in beta-lactam selection are presumably due to local preference rather than specific infection-related requirements. The high use of glycopeptides (teicoplanin) in level 2 units may warrant further investigation. Given the variance in antibiotic use, consideration could be given to ‘All Wales’ guidance for antibiotic prescribing in SCBUs. 16 016 C-Reactive Protein Response Associated with Candidaemia K.Marden1, S.Schelenz2, R.A.Barnes1 1NPHS Microbiology Cardiff, University Hospital of Wales, Cardiff and Norwich University Hospital 2Norfolk Background The literature relating C-reactive protein (CRP) levels in response to candidaemia is small. In clinical practice one might suspect candidaemia in a high risk patient with an elevated CRP when no other cause for an acute phase response is evident. Objectives To determine the relationship between CRP levels and candidaemia in a hospital case series. Method Cases included were a consecutive series of positive Candida species blood cultures identified in Cardiff and Vale NHS Trust between 2000 and 2006. Source hospital unit and contemporaneous CRP, white blood cells (WBC) and liver function tests (LFT) were recorded. The CRP taken at the shortest interval to the positive blood culture, up to a maximum of +/- 5 days, was recorded. All cases with concurrent bacteraemia or without a contemporaneous CRP were excluded. Results There were a total of 290 candidaemia episodes. After exclusion criteria were applied 118 episodes were eligible for study. The majority (51%) of the isolates were Candida albicans with C.glabrata and C.parapsilosis contributing 17% and 15% respectively. CRP in all the candidaemia episodes ranged from <6 (normal) to 476 mg/l. 7% of CRP values were normal. CRP was elevated in 93% of episodes, 41% were between 6 and 99 and 52% were 100. 44% of all candidaemia episodes occurred during a stay on an intensive care unit (n=52), be it adult general (GITU), cardiothoracic, paediatric or neonatal. For episodes occurring on an adult ITU, 2% of CRP values were normal, 36% were between 6 and 99 and 62% were 100. In adult haematology (n=22) all CRP values were elevated, 27% were between 6 and 99 and 73% were 100. There were 25 candidaemia episodes in children, including 11 in paediatric oncology, 6 in neonates and 8 from general paediatrics. CRP was normal in 17% of neonatal episodes and 45% of paediatric oncology episodes. CRP values did not rise above 89 in all paediatric oncology episodes. There was no relationship with CRP and LFT. The correlation with WBC will be described. Conclusion In most cases candidaemia is associated with a markedly elevated CRP. However in paediatric oncology and neonates CRP was normal in a substantial minority. This may suggest these Candida isolates were contaminants or that CRP response is absent in some cases. Further studies are warranted. 17 017 Evaluation of IQ200 Sprint urine analyser as a primary screen for bacteriuria Julie Varga, Teresa Peach, Peter James NPHS Microbiology Cardiff, University Hospital of Wales, Cardiff Introduction: The IQ200 Sprint analyser employs real-time video flow imaging and patented Automated Intelligent Microscopy (AIM) technology for automating urine microscopy. It is validated here as a primary screen for bacteriuria. Objectives: To evaluate the utility of selected parameters measured by the IQ200 Sprint analyser to screen urine samples for bacteriuria. To compare the negative predictive value (NPV) of the analyser when compared to two culture methods (CLED and UTI clear, Oxoid), both independently and collectively. A comparison of RBC and WBC counts from the analyser and from inverted manual microscopy is also presented. Methods: 1020 urine samples were processed using the IQ200 Sprint Urine analyser. The samples were inoculated onto CLED (Oxoid) and inverted light microscopy performed as part of the routine screening procedure according to the laboratory’s Standing Operating Procedure. Samples were also inoculated onto UTI clear (Oxoid) using 1uL sterile disposable loops. Comparisons were made between WBC and RBC counts obtained by the two methods. A linearity check was performed by selecting two urine samples with large numbers of RBCs and WBCs respectively and processing doubling dilutions of each sample individually and mixed together to reveal any quenching effects on either cell type. Results: 246 organisms (24% positivity), were reported using CLED agar compared with 294 organisms (29% positivity) using chromogenic agar. There was complete agreement for 186 organisms, 58 organisms were grown on CLED agar only, and 106 organisms were grown on chromogenic agar only. The NPV of the IQ200 Sprint was 94.9% when compared with chromogenic agar and 94.0% when compared with CLED agar. This compares favourably with the NPV of chromogenic agar when compared with CLED agar (94.7%) and CLED when compared with chromogenic agar (93.3%). There was at least a four-fold increase in average RBC counts determined by the automated analyser in comparison with manual counts. In samples where the WBC count was <100, more than 50% of samples had RBC counts <1 per uL by manual microscopy, whereas only 6% had RBC counts <1 per uL. WBC counts, though variable between methods were broadly comparable. There was no distortion of RBC counts by large numbers of WBCs and visa versa obtained from the urine analyser and counts were linear over a wide dilution range. Conclusions: The IQ200 Sprint urine analyser performs (using the parameters described here) at least as well as culture when used as a primary negative screen for bacteriuria. The heightened sensitivity of the IQ200 Sprint analyser for RBCs requires careful introduction of this technology to the clinical setting. We recommend continuation of culture on all samples from critical patient groups such as paediatrics and ICU patients. 18 018 A review of Staphylococcus aureus bacteraemia in a Welsh district general hospital A Towers1, A Omrani2 1Department 2Department of Medicine, Royal Glamorgan Hospital, Llantrisant of Microbiology & Infectious Diseases, Royal Glamorgan Hospital, Llantrisant Aim: To review diagnosis, management and follow up of patients with Staphylococcus aureus bacteraemia with a view to creating a management pathway Method: Data was collected from Royal Glamorgan Hospital in South Wales. Patients were identified using MSSA and MRSA positive blood cultures. A proforma was designed to collect relevant data. The case notes and medication charts were reviewed and data collected. There are no evidence-based UK or European guidelines on the management of S aureus bacteraemia. For the purpose of this study, a minimum of 14 days of effective antibiotic therapy in addition to adequate assessment for a source of sepsis and, where applicable, source control were considered minimal standards of care. Furthermore, follow up blood cultures and regular clinical monitoring were assessed. Results: There were 65 MSSA/MRSA positive blood cultures in 2008. Notes were available for 32 patients. The majority of the study population were over 50 years of age and had a medical underlying diagnosis. Furthermore, 13% were on haemodialysis, 3% were intravenous drug users, 22% immunocompromised, 13% were known to have a heart murmur, 16% had diabetes mellitus and 13% had an underlying malignancy. The overall in-hospital mortality was 44% with all deaths occurring in the over 50 years age groups. MRSA-associated in-hospital mortality was 64%, compared with 28% in patients with MSSA bacteraemia. The majority of cases were healthcareassociated (59%). The relapse rate was low at 6%. In the majority of cases the source of bacteraemia was respiratory, intravascular catheter or uncertain. Of those cases with an intravascular catheter source, approximately 70% were MRSA-associated. Two thirds of the patients received the minimum standard of care outlined above. In those who were considered to have received suboptimal therapy, half received less than 14 days of effective antibiotic therapy. Seventy eight per cent were considered to have had adequate monitoring of inflammatory markers and regular clinical reviews by the team in charge of their care. However, only 31% of cases had repeat blood cultures within 72 hours of the first episode of S aureus bacteraemia. Advice from Microbiology & Infectious Diseases was documented on the blood culture reports, but documentation of such advice in the clinical notes was poor. Conclusion: In-hospital mortality associated with S aureus bacteraemia was high despite adequate treatment in most cases. This is probably because most patients had significant co-morbidities. A considerable proportion of S aureus bacteraemia was nosocomial. Perhaps focussing on prevention and rapid identification of cases would be a more effective management strategy. One possible strategy to improve documentation in clinical notes could be insertion of alert stickers in the patients’ notes once a diagnosis of S aureus bacteraemia is made, with advice on further assessment, monitoring and therapy. 19 019 Audit of Compliance with Antimicrobial Therapy Guidelines for Patients at Increased Risk of C difficile Infection at Cwm Taf NHS Trust (South) Taha Akhtar1, Natalie Collings2, Huma Changez1, Ali Omrani3 1Department of Medicine, Royal Glamorgan Hospital, Llantrisant University School of Medicine, Cardiff 3Department of Microbiology & Infectious Diseases, Royal Glamorgan Hospital, Llantrisant 2Cardiff Introduction: Similar to many hospitals in UK, an increase in Clostridium difficile infection (CDI) rate was noted in our institution, in association with a general excessive use of antibiotics. The increase was particularly notable in medical wards in the acute and rehabilitation hospitals. Along with increased infection control precautions and a review of cleaning procedures, new antimicrobial therapy guidelines were introduced. The guidelines were only applicable to patients at increased risk of CDI, defined as those aged over 65 years, were on long-term haemodialysis or had history of previous CDI. The guidelines discouraged the use of cephalosporins, fluoroquinolones and Clindamycin for the target patient group. Tetracyclines, sulphonamides (co-trimoxazole) and betalactam/beta-lactamase inhibitors (co-amoxiclav & piperacillin/tazocin) were utilised, were indicated. The first aim of this study was to audit antimicrobial prescribing and its compliance with those guidelines. The second was to review CDI rates before and after the introduction of the guidelines and assess the relationship between any change in rates and antimicrobial therapy guidelines. Methods: The first part of the study was a point-prevalence study whereby all patients in a particular ward had their medication charts reviewed. Notes of patients who were an increased risk of CDI and were on antimicrobial agents were reviewed to assess the indication for prescribing and whether that was consistent with the recommendations set out in the guidelines. The second part was a retrospective review of computer records of CDI and a calculation of the CDI rates for individual wards per 1,000 admissions. The rate was calculated for the months of October and November 2008, the period of the study, as well as the same two months in the two preceding years. Results: One hundred and forty two patients were receiving care on the acute medical wards at the point of study. Fifty four patients (38%) were on antibiotics, of which thirty eight were considered to be at higher risk of CDI (70%). Over all, two thirds of the antimicrobial therapy was consistent with the guidelines. Twenty two out of one hundred and twenty nine patients (17%) in the non-acute medical wards were on antibiotics, all of whom were at an increased risk of CDI. Compliance with the recommendations was generally excellent (83%) in those wards. The situation was different in the surgical wards, where eighteen (19%) of the ninety four patients were at higher risk of CDI and on antibiotics. However, only half of the prescribing was compliant with antimicrobial therapy guidelines (range 33-67%).Rates/1000 admissions in wards Wards Acute medical wards Non acute medical wards Surgical wards October 06 5.6 101.7 4.6 October 07 17.5 89.3 0 October 08 3.6 52.6 7.27 Rates of CDI per 1000 admissions fell considerable following the introduction of the new guidelines on medical wards. However in surgical wards, the rates increased. In medical wards, although other interventions were introduced at the same time, the guidelines appear to have contributed to the decline in CDI rates. Conclusions: Compliance with newly introduced antimicrobial therapy guidelines for patients at increased risk of CDI is general good in medical wards where there historically higher rates of CDI. The guidelines appear to have contributed to the fall in CDI rates. Further interventions are required to address antimicrobial prescribing in the surgical wards in our hospital. 20 020 An Audit of Rubella Susceptibility Rates in the Cwm Taf (South) NHS Trust Over a Four Year Period LA Matthews1, L Lawrance2, S Gray2, D Gray2 1Virology Laboratory, Department of Microbiology & Infectious Diseases, Royal Glamorgan Hospital, Llantrisant 2University of the West of England, Bristol Background Rubella is an insignificant childhood disease that has devastating results in pregnancy. Immunisation using Rubella single vaccine was replaced by Measles, Mumps and Rubella vaccine (MMR) in 1988. Adverse publicity (1998) caused a fall in uptake of MMR. Anecdotal evidence suggests that rubella susceptibility levels in pregnancy have subsequently increased. Aims Using data collected in one NHS Trust area in Wales: To determine the rate of rubella susceptibility in antenatal women by age group, in first and subsequent pregnancies and to compare with National figures. Methods A retrospective analysis (2005 – 2007) of the results of rubella immunity in ante-natal screening tests was carried out. Information given on the request forms was also examined. Analysis was also planned prospectively for another 3 years from 2008 to 2010. To date 3 years retrospective and one year prospective have been analysed. Results The number and percentage of all women who are susceptible to rubella (as defined by an antibody level of <10 IU/ml) has risen over the period 2005-2008. The susceptibility rate in the most vulnerable group, namely women in their first pregnancy have increased from 6.6% to 9.5% in this four year period. Data will presented by age group, first pregnancy and ethnicity. Conclusions Poor uptake in the MMR Immunisation programme has been associated with an increase in rubella susceptibility rates particularly in the under twenties. The reduction in uptake of MMR may result in the re-emergence of rubella and cases of congenital rubella. 21 021 Evaluation of the Early (2005) HAIN GenotypeR Staphylococcus to Identify MRSA from Cultured Specimens in a District General Hospital Kelly Ward Department of Microbiology & Infectious Diseases, Royal Glamorgan Hospital, Llantrisant The major reservoir of MRSA in the hospital setting is the infected or colonised patient (Wernitz et al., 2005). The screening of MRSA at hospital admission offers an early opportunity to detect MRSA and so aiding in the reduction of the noscomial spread of MRSA. Molecular methods to detect MRSA are now increasingly common and a range of primers have been designed to amplify species-specific targets. The aim of this project was to evaluate the use of the HAIN GenoTypeR Staphylococcus to identify MRSA from cultured specimens in a District General Hospital The HAIN GenoTypeR Staphylococcus is a molecular genetic assay. The test is based on Line Probe Assay DNA STRIP technology and permits the identification of a range of Staphylococcal species. The strip also detects the methicillin resistance mediating mecA gene and the cytotoxic virulence factor, Panton-Valentine Leukocidin (PVL). The method consists of 3 steps. Step 1 is the isolation of the DNA from the bacterial cells. Step 2 is an End Point PCR Amplification with Biotinylated primers to exponentially amplify the DNA using Taq DNA Polymerase. The final step is a hybridisation process to allow the development of coloured bands on nitrocellulose strips. Seventeen isolates of S.aureus were confirmed as MRSA by the BD Phoenix analyser. The GenoTypeR Staphylococcus also confirmed they were MRSA. The banding pattern on the nitrocelluolose strips consisted of the two control bands which demonstrated the steps in the PCR and Hybridisation worked correctly. They also all had the band for mecA which confirmed methicillin resistance. They all contained bands 5 and 9 which are specific oligonucleotide bands for S.aureus. None of the isolates were carrying the gene for PVL toxin however we were able to demonstrate it using a laboratory control. Due to the high cost of the molecular kit, only 1 kit (25 tests) was purchased. However this system was excellent for the confirmation of MRSA and its ability to detect PVL was very useful. However the cost and expertise required to perform the test makes it unlikely that it would be introduced into a district general hospital for routine MRSA screening. A disadvantage this test has is that it is based on confirmation using an already cultured organism. The culture and incubation process still takes 24hrs so there would be no gain in the time it would take to achieve a result compared to the current culture alone method. 22 022 Case Study: Toxic Shock Syndrome Associated with a Prior Sports Injury S. Iyer1, B. Tan2, A. Gandhe3, T. Andarde3 1Department of Microbiology, Royal Berkshire NHS Foundation Trust, Reading of Pharmacy, Royal Berkshire NHS Foundation Trust, Reading 3Department of Orthopaedics, Royal Berkshire NHS Foundation Trust, Reading 2Department Background: The case study presented highlights the importance of obtaining a detailed clinical history and the need for a multi-disciplinary approach in the management of unusual emergency admissions. Case study: A previously healthy 33 year-old female presented in Accident and Emergency with severe back pain. One week prior to onset of pain she had fallen on her back whilst snowboarding but did not report any obvious injury. On admission her temperature was 36.4 oC, RR 16/min, PR 88/min CRP 270 and WBC 6.33X109/L. There was no sensory loss or weakness but she had difficulty in flexion of both hips. Nothing abnormal was visible on lumber X-ray. MRI showed a haemorrhagic cyst on the right ovary. Ultrasound showed no obvious adenexal mass or cysts. 24 hours post admission temperature spiked to >39oC; CRP increased to 360 and WBC decreased to 3.77. Blood culture drawn at admission was positive for Staphylococcus aureus and, due to history of ‘penicillin allergy’, IV teicoplanin 400 mg 3 loading doses followed by 400 mg once daily was initiated. The patient continued to deteriorate and she became clinically septic. On CT scan an abscess was found in front of the right iliac crest adjacent to the sacral joint. Frank pus was drained and sent for culture that grew S. aureus. Clindamycin 1.2g 6h was added.The blood culture and pus isolates subsequently on typing were confirmed to be toxic shock syndrome toxin 1 (TSST-1) positive. The patient continued to deteriorate and developed right sided consolidation with pleural effusion. She was transferred to high dependency care. IV linezolid 600mg 12h was added. 48h blood culture was S. aureus positive, indicating continued bacteraemia. Teicoplanin was replaced with IV daptomycin 8 mg/kg once daily. Blood cultures 48 and 96h post initiation of daptomycin were culture negative and the patient improved clinically. Discussion: Patients with TSS exhibit a number of clinical symptoms, including fever, diarrhoea, inability to maintain homeostasis and multiple-organ involvement, with the most severe cases being fatal. In the presence of fever of unknown origin the early involvement of microbiology and the need for molecular typing are highlighted by this case study. In vitro studies have shown that daptomycin has high activity against toxin producing strains of S. aureus, and the successful outcome in this case confirms its activity in clinical practice. 23 023 Highlighting a Clinical Conundrum – The Need for Microbiological Clinical Expertise S. Iyer1, G. Boden2, T. Pollard3, R. Dodds3 , B. Tan4, A. Andrade3 1Department of Microbiology, Royal Berkshire NHS Foundation Trust, Reading of Paediatrics, Royal Berkshire NHS Foundation Trust, Reading 3Department of Orthopaedics, Royal Berkshire NHS Foundation Trust, Reading 4Department of Pharmacy, Royal Berkshire NHS Foundation Trust, Reading 2Department Background: This case study highlights the need for a team approach in usual presentations of infection of unknown origin. Case study: A 16 year-old boy presented to the Paediatric Assessment Unit with fever, chills, diarrhoea and vomiting of 3 days duration. He had right knee pain, progressively increasing in intensity. Two days prior to the onset of symptoms he had been playing football, but could not remember being injured. On examination his temperature was 39.5oC, RR 20/min, PR 120/min CRP 183 and WBC 10.34X109/L. His right knee was slightly swollen, with pain on rotation and standing. Initial diagnosis was viral illness with reactive arthritis. Analgesics and anti-inflammatory drugs were initiated. Over the next 48h the pain continued to worsen, his temperature was spiking WBC fell to 6X109/L and CRP increased to 300. Knee aspirate obtained at admission was culture-ve at 24h. The knee was re-aspirated. Results from the blood sample obtained at admission were positive for Gram-positive cocci in clusters (2/2). IV flucloxacillin 1g 6h and IV clindamycin 1.2g 6h were commenced. He underwent arthroscopic washout of his knee that did not reveal any evidence of synovitis. On day-3 he became afebrile but complained of chest pain on coughing and swollen left ankle. The chest X-ray showed right midzone shadowing. The patient was admitted to the paediatric HDU with clinical sepsis and respiratory distress. Antibiotics were continued and PVLtyping of S. aureus was requested by microbiology. At MRI, right proximal tibial and left distal tibial osteomyelitis was confirmed. Surgical debridement of right tibia was performed. On day-8, he developed multifocal lung infiltrates and lesions on chest X-ray and CT scan. The patient was transferred to ITU. IV linezolid replaced flucloxacillin. On day-9 the patient had stabilised and was transferred back to the HDU. IV Daptomycin 8mg/kg was added on day-10. On day-12 the isolate was confirmed as PVL-+ve. By day-12 the patient was progressively improving and was blood culture-ve by day-15. One month after admission the patient was discharged on oral clindamycin. Discussion: This case study of a child presenting with fever, chills, diarrhoea and vomiting highlights the need for a team approach and the early involvement of microbiological services. In the presence of the increasing incidence of PVL-producing strains clinicians need to maintain a high level of suspicion for these organisms, especially in the presence of sports associated injuries. 24 024 Chromogenic Agar – a change of culture on the Urine Bench Linda Sim, Glen Widdows, Dr Andrew Hay Bacteriology Laboratory, Microbiology Department, Raigmore Hospital Inverness Objectives Traditionally, our laboratory identified all significant urinary isolates either by an automated system (Vitek, Biomérieux UK Ltd) or other methods such as Lancefield grouping for Streptococci/Enterococci. Although reliable, these methods are relatively time consuming and expensive. The development of chromogenic agars for simultaneous primary isolation and initial identification of specific urinary pathogens gave the opportunity to review our methods our ways. The aims of this study are (i) to examine the primary isolation on chromogenic UTI medium (Oxoid UK Ltd) as a means of reliably identifying E.coli and Enterococci, which account for two thirds of our urinary isolates, (ii) produce an algorithm for identification of urinary isolates and (iii) to identify potential cost savings. Methods and Results 100 presumptive E.coli (pink colonies), from chromogenic agar, were identified through Vitek and results compared. Similarly 100 presumptive Enterococci (small dark blue colonies) on chromogenic agar were identified by Lancefield’s grouping (Pro Lab) and results compared. Results show ≥ 98% agreement between standard methods of identification and morphological appearance on chromogenic agar. This is considered acceptable for both E.coli and Enterococcus species. From these results an algorithm for identification of urinary isolates was developed which has realised significant savings in the laboratory. Conclusions This study showed that, in our laboratory, Oxoid chromogenic UTI medium can be used as a cost effective identification method for E.coli isolates and Enterococcus species. It also realised significant savings without compromising clinical care or epidemiological analysis. 25 025 Physiology of Clostridium difficile Spores: Lovleen Joshi, Prof. Les Baillie Welsh School of Pharmacy, Cardiff University, Cardiff Clostridium difficile, an anaerobic, spore-forming bacterium, is commonly associated with Clostridium difficile-associated diarrhoea and pseudomembranous colitis – both serious hospital– acquired infections. In Wales there were over 3000 cases of C. difficile in patients over 65 between July 2007 and June 2008. Literature and high infection rates have highlighted some of the spores’ complexities, such as their ability to remain viable on contaminated surfaces for months and their resistance to biocides. Objectives: To characterise the physiological properties of Clostridium difficile spores to assist combat of infection in the hospital environment, and to understand if these properties play a role in disease. Methods: Sporulation: 20 strains of C. difficile from the National Anaerobic Reference Unit, Cardiff, including hyper-virulent ribotypes 001, 027 and 106, were compared for sporulation efficiency. Spores were grown using methods described by Perez et al., 2005. Spore yield was determined by assessing germination via Miles-Misra method on Brain-Heart Infusion agar. BHI agar was then supplemented with 1% Sodium taurocholate to further deduce if there were differences in sporulation between strains. Spore yield was determined on Columbia agar, with and without 1% Sodium taurocholate, and germination assessed. Hydrophobicity: Spores persist on surfaces for months, which is thought to be linked to their surface charge. The relative hydrophobicity of 20 C. difficile strains was ascertained using the microbial adhesion to hydrocarbon test, using Hexadecane as the organic solvent. Transmission Electron Microscopy: Strains with the highest and lowest relative hydrophobicity were further examined using TEM at the Electron Microscopy Unit, Cardiff University School of Biosciences. Results & Key Findings: Germination & Sporulation: There was variation in germination between strains on BHI and Columbia agar. Addition of 1% Sodium taurocholate to both media resulted in consistent yield with little variation. Thus there is no difference in ability of Clostridium difficile strains to produce spores. However there is a difference in germination ability according to media. Sodium taurocholate appears to stabilise germination conditions for C. difficile spores. Hydrophobicity: Downward trend in percentage hydrophobicity according to ribotype exhibited. Strains with hyper-virulent ribotypes seem to be more hydrophobic than other strains. TEM: TEM revealed significant physiological differences between highly hydrophobic DS1813 strain and least hydrophobic DS1748 strain. Some hydrophobic DS1813 spores exhibit what may be an exosporium. This suggests that Clostridium difficile spores vary physiologically across strains. Conclusion: This research is designed to underpin efforts to combat and eradicate C. difficile infection in hospital environments. 26 026 MRSA in Companion Animals Tracey Jolliffe Ninewells Hospital, Dundee Bacteria belonging to the genus Staphylococcus are catalase positive, Gram-positive cocci, which form part of the normal flora of humans and animals, and in general have an innocuous association with the host. They are, however, capable of causing opportunistic infection in the host species. Staphylococcus aureus is the predominant coagulase positive Staphylococci found in humans. In Methicillin* Resistant Staphylococcus aureus (MRSA), the presence of the mecA gene allows the bacteria to produce the normal penicillin binding protein as well as an alternative penicillin binding protein (PBP2a). Unlike the normal penicillin binding protein, PBP2a is not inhibited by antibiotics such as the β-lactams, so can bypass the effect of these antibiotics. It is known that animals can be infected and colonised by MRSA, but prevalence studies tend to focus on clinical infections and animals presented at veterinary surgeries. In this study, I sampled 262 dogs and cats, both healthy and unwell, to attempt to discover the prevalence of Staphylococcus aureus and MRSA carriage. Questionnaires where given to the owners to establish possible risk factors such as recent antibiotic use, recent in-patient stays at the veterinary surgery, owners who worked in the healthcare professions, and households that had an MRSA positive person as a member. Nasal swabs were taken and processed using an enrichment broth, and both chromogenic MRSA media and Staphylococcal selective media. Identification used phenotypical and biochemical methods, and MRSA positive isolates were sent to the Scottish MRSA reference laboratory for confirmation and further tests. In total, twenty Staphylococcus aureus isolates were found, two of which were MRSA. The two MRSA isolates, from one dog and one cat, had only one risk factor in common: the owners of both animals were currently, or had been, MRSA positive. 27 027 The Effect of Health-Care Associated Infection Surveillance and the Introduction of Care Bundles in a Regional Intensive Care Unit in Northern Ireland. Hilda Crookshanks1, Colin Lavelle2, Gerard McIlvenny3, Irene Thompson2, Joanna McCormick2, Gavin Lavery2, Anthony Stevens2, Edward T. M. Smyth1,2 1Northern 2Belfast Ireland Healthcare-associated Infection Surveillance Centre, Belfast HSC Trust, Belfast Background: Our Trust participated in the Safer Patient Initiative (SPI) project coordinated by the Institute of Healthcare Improvement (IHI, USA) and the Health Foundation (UK). Objective: To reduce ventilator associated pneumonia (VAP) and central line associated bloodstream infection (CLABSI) rates in a 17-bedded, level 3 Regional Intensive Care Unit (RICU) by the introduction of care bundles. Methods: In March 2007 care bundles recommended by the IHI were applied to patients requiring mechanical ventilation and central line insertions. No care bundle was used for urinary catheters. Ventilator care and central line insertion bundle compliance was recorded daily. To ascertain the effect of the bundles on patient care, a dedicated surveillance nurse was employed to conduct active, prospective surveillance of device-associated infections (including catheter associated urinary tract infection [CAUTI]). Device associated infections were recorded using Centers for Disease Control (CDC) definitions of infection. Denominator data was collected daily to record device utilisation rates and the surveillance nurse actively participated in the microbiology round. Data was recorded on a scannable form and data fed back monthly as per the SPI protocol. Results: When surveillance was implemented in the RICU in February 2007, care bundle compliance was 72% and the VAP rate was 8.99 per 1000 ventilator days. The CLABSI rate was 10.75 per 1000 catheter days. Bundle compliance has shown an upward trend reaching 95% compliance in October 2007 and has remained consistent. In October 2008, the VAP rate was been 0.00 per 1000 ventilator days and the CLABSI rate was 6.5 per 1000 patient days. CAUTI rates were 4.17 per 1000 catheter days in March 2007 and have demonstrated a gradual upward trend to 10.02 per 1000 days by October 2008. Ventilator-Figure 1 - Ventilator-associated pneumonia rate 100 90 14.00 RICU 12.00 NHSN 10.00 Bundle Compliance 80 70 60 8.00 50 6.00 40 4.00 30 2.00 20 0.00 Percentage compliance VAP rate /1000 ventilator days 16.00 10 0 M ar 0 Ap 7 r0 M 7 ay 0 Ju 7 n 07 Ju l0 Au 7 g 0 Se 7 p 0 O 7 ct 0 N 7 ov 0 D 7 ec 0 Ja 7 n 0 Fe 8 b 0 M 8 ar 0 Ap 8 r0 M 8 ay 0 Ju 8 n 08 Ju l0 Au 8 g 0 Se 8 p 0 O 8 ct 08 -2.00 Conclusions: Where care bundles were implemented in the management of the prevention of VAPs and CLABSIs, a steady improvement was recorded in the infection rate particularly in the occurrence of VAPs (Figure 1). This improvement was associated with increasing care bundle compliance. No improvement was observed in CAUTI rates where a care bundle had not been employed. A CAUTI care bundle devised by the Department of Health (England) Saving Lives Campaign 2007 is currently being reviewed for implementation in the unit. The care bundle concept and surveillance system is currently being developed to enable regional participation. 28 028 Prevalence Survey of Healthcare Associated Infections (HCAI) In Argentina Ricardo Durlach1, Gerard McIlvenny2, Geraldine Reid3, Lorraine Doherty4, Edward TM Smyth2,3 1Instituto Técnico de Acreditación de Establecimientos de Salud, Buenos Aires, Argentina HSC Trust, Belfast 3Northern Ireland Healthcare-associated Infection Surveillance Centre, Belfast 4Department of Health, Social Services and Public Safety, Northern Ireland 2Belfast Background: The use of scannable forms has now been used in many countries with minimal cost implications. In collaboration with the Northern Ireland Healthcare Associated Infection Surveillance Centre an healthcare-associated infection (HCAI) prevalence survey was carried out in hospitals in Argentina. Objectives: Determine prevalence rates of HCAIs and provide data, compatible for international comparisons, which will assist in the prioritization of measures to reduce the occurance of HCAIs. Methods: The NHSN definitions of infections were used. Participation in the survey was organized by the Instituto Técnico de Acreditación de Establecimientos de Salud. Between March and and June 2008, infection prevention & control (IP&C) staff with assistance from clinical colleagues across Argentina, completed the survey. Data was collected for all types of HCAI and included specific questions regarding MRSA. The dataset was identical to the recent Hospital Infection Society’s HCAI prevalence survey of England, Wales, Northern Ireland and the Republic of Ireland. Standardised methods and training were used and the CDC definitions of infection employed. Questionnaires were returned to HISC in Northern Ireland for processing utilizing optical mark reader technology (OMR). Feedback of data to individual hospitals will be by means of individual reports. Results: The prevalence survey included 4 249 patients from 43 hospitals. There were 480 patients with one or more healthcare associated infections giving a prevalence of 11.30% (95%CI 10.38 to 12.28). Results for types of HCAI are given in the table below: Table: Types and prevalence of healthcare-associated infection in Argentinian hospitals HCAI Primary bloodstream infection Pneumonia Surgical site infection Urinary tract infection Skin and soft tissue infection MRSA infections Number of patients Number of infections Prevalence 95% Confidence Interval 4 249 62 1.46 1.14 – 1.87 4 249 1 207 (Surgical patients only) 4 249 141 3.32 2.82 – 3.90 123 10.19 8.61 – 12.03 133 2.76 2.22 – 3.42 4 249 51 1.20 0.91 – 1.57 4 249 48 1.13 0.85 – 1.49 Conclusions: The initial results of the survey are encouraging. The methodology, definitions and use of the scannable surveillance questionnaires proved highly acceptable and economical to those involved. This survey has established a baseline for HCAI prevalence in Argentina. 29 029 Two Into One Won’t Go? Reporting Surgical Site Infections in Hip Arthroplasty and Hemiarthroplasty of the Hip as Opposed to Hip Prosthesis Geraldine Reid1, Hilda Crookshanks1, Gavan McAlinden2, Chris Andrews3, Gerard McIlvenny1, Lorraine Doherty4, Edward T. M. Smyth1,5 1Northern Ireland Healthcare-associated Infection Surveillance Centre, Belfast Hospital Dundonald, Belfast & Musgrave Park Hospital, Belfast 3The Royal Hospitals, Belfast, & Musgrave Park Hospital, Belfast 4Department of Health Social Services & Public Safety, Northern Ireland 5Belfast HSC Trust, Belfast 2Ulster Background/Objective: In Northern Ireland SSI surveillance is performed on all orthopaedic procedures. We currently report aggregate and country specific rates for arthroplasty and hemiarthroplasty of the hip as separate categories. The National Healthcare Safety Network (NHSN) Atlanta, Georgia report rates of SSI for the operative procedure hip prosthesis which includes both arthroplasty and hemiarthroplasty of the hip. The objective was to compare the validity of reporting results of arthroplasty and hemiarthroplasty against hip prosthesis. Methods: We compared the published rates for Northern Ireland with the NHSN published rates. Comparisons were also made with the proportions of patients that fall into the different risk categories Results: The SSI rates for arthroplasty of hip and hemiarthroplasty of hip in Northern Ireland (2003 – 2007) are compared with the NHSN SSI rates (2007 – 2008) [Table]. The SSI rate for hemiarthroplasty of hip with RI 0 of 2.05 was significantly higher than the rate of 0.48 for arthroplasty hip RI0 [OR 4.37, p<0.001] and similarly, the SSI rate for hemiarthroplasty of hip with RI1 of 3.32 was significantly higher than the rate of 1.47 for arthroplasty hip RI1 [OR 2.38, p<0.001]. Patients undergoing arthroplasty of hip were predominately in risk index category 0 (74%) and the majority of hemiarthroplasty of hip were in risk index category 1 (74%). However, patients in both operative categories covered the range of risk index categories, i.e. 0 – 3. Operative procedure category Reporting Number Risk index SSI rate Northern Ireland 5676 0 0.48 Arthroplasty of hip 1764 1 1.47 Arthroplasty of hip 245 2,3 1.63 635 0 2.05 Hip hemiarthroplasty 1900 1 3.32 Hip hemiarthroplasty 26 2,3 - 17521 0 0.75 Hip prosthesis 22681 1 1.68 Hip prosthesis 5492 2,3 2.79 Arthroplasty of hip Hemiarthroplasty of hip Hip prosthesis Northern Ireland NHSN Conclusions: The NHSN operative procedure categories are groups of clinically similar operative procedures. The purpose of the categories is to make it possible to compare SSI rates in groups of patients that have similar procedures. Until 2004 NNIS reported the operative procedure category ’vascular surgery’. In 2008 the NHSN subdivided the vascular surgery operative procedure category into three separate operative procedure categories, i.e. abdominal aortic aneurysm repair, carotid endartectomy and peripheral vascular bypass surgery. It may be appropriate to adopt this approach for hip prosthesis and report the rates for arthroplasty and hemiarthroplasty separately, due to the fact that the SSI rates for hemiarthroplasty of hip are significantly higher than the SSI rates for arthroplasty of hip. 30