The drugs don't work: evaluation of educational theatre to gauge and influence public opinion on antimicrobial resistance.

Increased public awareness of antimicrobial resistance (AMR) is a key component of effective antimicrobial stewardship strategies. Educational theatre combined with an expert panel was used to engage the public about AMR through delivery of a play entitled 'The drugs don't work'. Audience knowledge and understanding of AMR were measured by pre- and post-play questionnaires. Performance of the play and discussion with the expert panel significantly improved audience knowledge and understanding of AMR, including antibiotic misuse and prescribing. Educational theatre provides a positive learning experience and is an innovative method of public engagement to disseminate important public health messages.

Acquisition and retention of Clostridium difficile by Musca domestica larvae and pupae during metamorphosis.

BACKGROUND: Transfer of Clostridium difficile by Musca domestica has been demonstrated, revealing their potential for disseminating infection in the hospital environment. AIM: To determine the ability of M. domestica larvae to acquire and retain C. difficile throughout their metamorphosis into adult flies. METHODS: Larvae were exposed to spores of C. difficile in a faecal emulsion, and examined externally and internally to determine carriage and internalization of spores through their development to adults. FINDINGS: Larvae harboured C. difficile externally, with means of 21.56+/-5.76 colony-forming units (cfu) at Day 0, 22.44+/-9.90 cfu at Day 2, decreasing to 0.56+/-0.34 cfu at Day 4, with no C. difficile isolated thereafter. The same larvae harboured C. difficile internally, with means of 587.33+/-238.29 cfu at Day 0, decreasing to 297.44+/-155.21 cfu at Day 2, decreasing further to 73.67+/-46.74 cfu at Day 4, with no C. difficile isolated thereafter. The zero recovery of C. difficile coincided with the development of M. domestica larvae into pupae. From Day 6 onwards, all larvae had developed into the pupal stage and no C. difficile was recoverable from any pupae. No C. difficile was recovered from adult flies (emerged on Day 12) or empty puparia. CONCLUSION: Although C. difficile spores are readily acquired and internalized by larvae during feeding, they are not retained through development to adults. Adult flies therefore acquire C. difficile contamination as adults. The potential antimicrobial action of M. domestica larvae and their extracts against C. difficile spores warrants further investigation.

The housefly Musca domestica as a mechanical vector of Clostridium difficile.

BACKGROUND: Clostridium difficile is a bacterial healthcare-associated infection that may be transferred by houseflies (Musca domestica) due to their close ecological association with humans and cosmopolitan nature. AIM: To determine the ability of M. domestica to transfer C. difficile both mechanically and following ingestion. METHODS: M. domestica were exposed to independent suspensions of vegetative cells and spores of C. difficile, then sampled on to selective agar plates immediately postexposure and at 1-h intervals to assess the mechanical transfer of C. difficile. Fly excreta was cultured and alimentary canals were dissected to determine internalization of cells and spores. FINDINGS: M. domestica exposed to vegetative cell suspensions and spore suspensions of C. difficile were able to transfer the bacteria mechanically for up to 4h upon subsequent contact with surfaces. The greatest numbers of colony-forming units (CFUs) per fly were transferred immediately following exposure (mean CFUs 123.8 +/- 66.9 for vegetative cell suspension and 288.2 +/- 83.2 for spore suspension). After 1h, this had reduced (21.2 +/- 11.4 for vegetative cell suspension and 19.9 +/- 9 for spores). Mean C. difficile CFUs isolated from the M. domestica alimentary canal was 35 +/- 6.5, and mean C. difficile CFUs per faecal spot was 1.04 +/- 0.58. C. difficile could be recovered from fly excreta for up to 96h. CONCLUSION: This study describes the potential for M. domestica to contribute to environmental persistence and spread of C. difficile in hospitals, highlighting flies as realistic vectors of this micro-organism in clinical areas.

Epidemiology of community-acquired meticillin-resistant Staphylococcus aureus obtained from the UK West Midlands region.

SUMMARY: Between January 2005 and December 2005, 199 meticillin-resistant Staphylococcus aureus (MRSA) isolates were obtained from non-hospitalised patients presenting skin and soft tissue infections to local general practitioners. The study area incorporated 57 surgeries from three Primary Care Trusts in the Lichfield, Tamworth, Burntwood, North and East Birmingham regions of Central England, UK. Following antibiotic susceptibility testing, pulsed-field gel electrophoresis, Panton-Valentine leukocidin gene detection and SCCmec element assignment, 95% of the isolates were shown to be related to hospital epidemic strains EMRSA-15 and EMRSA-16. In total 87% of the isolate population harboured SCCmec IV, 9% had SCCmec II and 4% were identified as carrying novel SCCmec IIIa(-mecI). When mapped to patient home postcode, a diverse distribution of isolates harbouring SCCmec II and SCCmec IV was observed; however, the majority of isolates harbouring SCCmec IIIa(-mecI) were from patients residing in the north-west of the study region, highlighting a possible localised clonal group. Transmission of MRSA from the hospital setting into the surrounding community population, as demonstrated by this study, warrants the need for targeted patient screening and decolonisation in both the clinical and community environments.

Penetration of chlorhexidine into human skin.

This study evaluated a model of skin permeation to determine the depth of delivery of chlorhexidine into full-thickness excised human skin following topical application of 2% (wt/vol) aqueous chlorhexidine digluconate. Skin permeation studies were performed on full-thickness human skin using Franz diffusion cells with exposure to chlorhexidine for 2 min, 30 min, and 24 h. The concentration of chlorhexidine extracted from skin sections was determined to a depth of 1,500 microm following serial sectioning of the skin using a microtome and analysis by high-performance liquid chromatography. Poor penetration of chlorhexidine into skin following 2-min and 30-min exposures to chlorhexidine was observed (0.157 +/- 0.047 and 0.077 +/- 0.015 microg/mg tissue within the top 100 microm), and levels of chlorhexidine were minimal at deeper skin depths (less than 0.002 microg/mg tissue below 300 microm). After 24 h of exposure, there was more chlorhexidine within the upper 100-microm sections (7.88 +/- 1.37 microg/mg tissue); however, the levels remained low (less than 1 microg/mg tissue) at depths below 300 microm. There was no detectable penetration through the full-thickness skin. The model presented in this study can be used to assess the permeation of antiseptic agents through various layers of skin in vitro. Aqueous chlorhexidine demonstrated poor permeation into the deeper layers of the skin, which may restrict the efficacy of skin antisepsis with this agent. This study lays the foundation for further research in adopting alternative strategies for enhanced skin antisepsis in clinical practice.

Antimicrobial efficacy of copper surfaces against spores and vegetative cells of Clostridium difficile: the germination theory.

OBJECTIVES: Persistent contamination of surfaces by spores of Clostridium difficile is a major factor influencing the spread of C. difficile-associated diarrhoea (CDAD) in the clinical setting. In recent years, the antimicrobial efficacy of metal surfaces has been investigated against microorganisms including methicillin-resistant Staphylococcus aureus. This study compared the survival of C. difficile on stainless steel, a metal contact surface widely used in hospitals, and copper surfaces. METHODS: Antimicrobial efficacy was assessed using a carrier test method against dormant spores, germinating spores and vegetative cells of C. difficile (NCTC 11204 and ribotype 027) over a 3 h period in the presence and absence of organic matter. RESULTS: Copper metal eliminated all vegetative cells of C. difficile within 30 min, compared with stainless steel which demonstrated no antimicrobial activity (P < 0.05). Copper significantly reduced the viability of spores of C. difficile exposed to the germinant (sodium taurocholate) in aerobic conditions within 60 min (P < 0.05) while achieving a >or=2.5 log reduction (99.8% reduction) at 3 h. Organic material did not reduce the antimicrobial efficacy of the copper surface (P > 0.05). CONCLUSIONS: The use of copper surfaces within the clinical environment and application of a germination solution in infection control procedures may offer a novel way forward in eliminating C. difficile from contaminated surfaces and reducing CDAD.

Sporicidal activity of two disinfectants against Clostridium difficile spores.

The sporicidal activity of an odour-free peracetic acid-based disinfectant (Wofasteril) and a widely-used dichloroisocyanurate preparation (Chlor-clean) was assessed against spores of the hyper-virulent strain of Clostridium difficile (ribotype 027), in the presence and absence of organic matter. In environmentally clean conditions, dichloroisocyanurate achieved a >3 log10 reduction in 3 minutes, but a minimum contact time of 9 minutes was required to reduce the viable spore load to below detection levels. Peracetic acid achieved a >3 log10 reduction in 30 minutes and was overall significantly less effective (P<0.05). However, in the presence of organic matter - which reflects the true clinical environment - there was no significant difference between the sporicidal activity of dichloroisocyanurate and peracetic acid over a 60-minute period (P=0.188). Given the greater occupational health hazards generally associated with chlorine-releasing agents, odour-free peracetic acid-based disinfectants may offer a suitable alternative for environmental disinfection.

Physical and chemical factors influencing the germination of Clostridium difficile spores.

AIMS: To investigate the influence of chemical and physical factors on the rate and extent of germination of Clostridium difficile spores. METHODS AND RESULTS: Germination of C. difficile spores following exposure to chemical and physical germinants was measured by loss of either heat or ethanol resistance. Sodium taurocholate and chenodeoxycholate initiated germination together with thioglycollate medium at concentrations of 0.1-100 mmol l(-1) and 10-100 mmol l(-1) respectively. Glycine (0.2% w/v) was a co-factor required for germination with sodium taurocholate. There was no significant difference in the rate of germination of C. difficile spores in aerobic and anaerobic conditions (P > 0.05) however, the initial rate of germination was significantly increased at 37 degrees C compared to 20 degrees C (P < 0.05). The optimum pH range for germination was 6.5-7.5, with a decreased rate and extent of germination occurring at pH 5.5 and 8.5. CONCLUSIONS: This study demonstrates that sodium taurocholate and chenodeoxycholate initiate germination of C. difficile spores and is concentration dependant. Temperature and pH influence the rate and extent of germination. SIGNIFICANCE AND IMPACT OF THE STUDY: This manuscript enhances the knowledge of the factors influencing the germination of C. difficile spores. This may be applied to the development of potential novel strategies for the prevention of C. difficile infection.

RAPD for the typing of coagulase-negative staphylococci implicated in catheter-related bloodstream infection.

OBJECTIVES: A rapid random amplification of polymorphic DNA (RAPD) technique was developed to distinguish between strains of coagulase-negative staphylococci (CoNS) involved in central venous catheter (CVC)-related bloodstream infection. Its performance was compared with that of pulsed-field gel electrophoresis (PFGE). METHODS: Patients at the University Hospital Birmingham NHS Foundation Trust, U.K. who underwent stem cell transplantation and were diagnosed with CVC-related bloodstream infection due to CoNS whilst on the bone marrow transplant unit were studied. Isolates of CoNS were genotyped by PFGE and RAPD, the latter employing a single primer and a simple DNA extraction method. RESULTS: Both RAPD and PFGE were highly discriminatory (Simpson's diversity index, 0.96 and 0.99, respectively). Within the 49 isolates obtained from blood cultures of 33 patients, 20 distinct strains were identified by PFGE and 25 by RAPD. Of the 25 strains identified by RAPD, nine clusters of CoNS contained isolates from multiple patients, suggesting limited nosocomial spread. However, there was no significant association between time of inpatient stay and infection due to any particular strain. CONCLUSION: The RAPD technique presented allows CoNS strains to be genotyped with high discrimination within 4h, facilitating real-time epidemiological investigations. In this study, no single strain of CoNS was associated with a significant number of CVC-related bloodstream infections.

Adaptive resistance to biocides in Salmonella enterica and Escherichia coli O157 and cross-resistance to antimicrobial agents.

The mechanisms by which bacteria resist killing by antibiotics and biocides are still poorly defined, although repeated exposure to sublethal concentrations of antibacterial agents undoubtedly contributes to their development. This study aimed both to investigate the potential of Salmonella enterica and Escherichia coli O157 for adaptive resistance to commonly used biocides and to determine any cross-resistance to antibiotics. Strains were repeatedly passaged in media containing increasing concentrations of a biocide or antibiotic until adaptive resistance was obtained. A wide panel of antimicrobial agents was then screened by using the adapted strain to determine cross-resistance, if any. Adaptive resistance was readily achieved for both S. enterica and E. coli O157. Cross-resistance in adaptively resistant S. enterica varied with the serotype; Salmonella enterica serovar Enteritidis expressed cross-resistance to chloramphenicol, whereas Salmonella enterica serovar Typhimurium expressed cross-resistance to chlorhexidine. Benzalkonium chloride-resistant Salmonella enterica serovar Virchow showed elevated resistance to chlorhexidine; however, chlorhexidine-resistant Salmonella serovar Virchow did not demonstrate reciprocal cross-resistance to benzalkonium chloride, suggesting specific rather than generic resistance mechanisms. E. coli O157 strains acquired high levels of resistance to triclosan after only two sublethal exposures and, when adapted, repeatedly demonstrated decreased susceptibilities to various antimicrobial agents, including chloramphenicol, erythromycin, imipenem, tetracycline, and trimethoprim, as well as to a number of biocides. These observations raise concern over the indiscriminate and often inappropriate use of biocides, especially triclosan, in situations where they are unnecessary, whereby they may contribute to the development of microbial resistance mechanisms.

Use of multiple primers in RAPD analysis of clonal organisms provides limited improvement in discrimination.

Randomly amplified polymorphic DNA (RAPD) analysis using two or more primers has been reported to provide additional discriminatory ability over one primer used individually. This may be of particular application in epidemiological typing of clonal organisms, such as Shiga toxin-producing E. coli O157, where strain differentiation can be difficult. Using four arbitrary primers individually, and in all possible permutations, E. coli O157 isolates and other arbitrarily chosen E. coli strains were typed using RAPD analysis. For most nonclonal strains, the use of two primers resulted in increased differentiation between isolates; however, more than two primers did not increase further the discriminatory capacity. E. coli O157 isolates that produced virtually identical profiles using one primer did not show increased differentiation when using two or more primers, demonstrating that in some cases, where strains of an organism are highly related, there is limited advantage to using more than one primer in RAPD analysis.

Use of representational difference analysis to identify Escherichia coli O157-specific DNA sequences.

Whereas several important virulence factors in Escherichia coli O157 have been identified, studies suggest they are not always essential and are probably insufficient to account for the severe clinical manifestation of E. coli O157 infection. Identification of putative virulence determinants is crucial to the understanding of bacterial pathogenesis and genomic comparison analysis may aid the characterisation of unidentified virulence attributes. In this study, representational difference analysis (RDA) was used for genomic comparison of E. coli O157 with the proposed ancestral strain, E. coli O55. Unique E. coli O157 gene sequences were isolated and one, termed RDA-1, taken forward for further analysis. Southern blotting with labelled RDA-1 as a probe showed it to be present in 77% of E. coli O157 isolates and absent in all non-E. coli O157 screened. Sequence flanking RDA-1 was obtained from a genomic clone identified by hybridisation, and contained an open reading frame predicted to encode a novel iron-regulated outer membrane protein.

Optimization of random amplification of polymorphic DNA analysis for molecular subtyping of Escherichia coli O157.

Random amplification of polymorphic DNA (RAPD) analysis using the polymerase chain reaction has proved to be a useful technique in the epidemiological investigation of micro-organisms but may suffer from a lack of reproducibility in poorly optimized protocols. In this study a method of obtaining reproducible genomic fingerprints using RAPD analysis of Escherichia coli O157 is described. By systematic optimization of reaction conditions and selection of suitable primers, reproducible and discriminatory profiles could be obtained from all E. coli O157 strains tested. In addition, two other methods of obtaining reproducible profiles from E. coli O157 strains without the need to purify genomic DNA are described.

Simultaneous molecular subtyping and shiga toxin gene detection in Escherichia coli using multiplex polymerase chain reaction.

A robust random amplification of polymorphic DNA (RAPD)-polymerase chain reaction (PCR) protocol was developed for the combined epidemiological typing and shiga toxin detection of clinical shiga toxin-producing O157 and non-O157 Escherichia coli isolates. Using shiga toxin gene-specific primers, combined with two short 10-mer primers, in a multiplex shiga toxin/RAPD-PCR the fingerprints generated allowed differentiation between epidemiologically unrelated strains and allowed identification of a band amplified from the shiga toxin gene(s). Hybridization with a digoxigenin-labelled probe specific for stx1 and stx2 confirmed its identity. The combination of primers in this way allows valuable additional information to be gained from discriminatory RAPD profiles, with further benefits of time and cost savings over tests performed individually.

Plasma total homocysteine measurement by ion-paired reversed-phase HPLC with electrochemical detection.

Homocysteine is thought to be a risk factor for vascular disease and this has led to an increase in demand for its assay in clinical laboratories. An HPLC method, incorporating electrochemical detection, for measurement of plasma total homocysteine is presented. The method is simple to perform, precise and suitable for clinical applications.

Restriction enzyme analysis of randomly amplified polymorphic DNA amplicons of Salmonella enterica ser. Enteritidis PT4 and Typhimurium DT104.

Salmonella enterica serotype Enteritidis PT4 and Typhimurium DT104 isolates were characterized using a random amplification of polymorphic DNA (RAPD) protocol found previously to be highly discriminatory for isolates of Salmonella. Profiles generated with a single primer 1254, and independently 1283, successfully characterized an outbreak strain of Enteritidis PT4 but could not differentiate epidemiologically unrelated strains of Enteritidis PT4 from the outbreak strains. Primer 1254 differentiated one strain, and 1283 two strains of Typhimurium DT104 previously undifferentiated on the basis of biochemical and physical properties. Subsequent analysis using a combination of RAPD and restriction enzyme analysis could not provide additional differentiation of Enteritidis PT4 and Typhimurium DT104 isolates but did, however, exhibit the potential to be a useful combination of molecular techniques.

Comparison of ribotyping and arbitrarily primed PCR for molecular typing of Salmonella enterica and relationships between strains on the basis of these molecular markers.

Arbitrarily primed PCR (AP-PCR) using a discriminatory 10-mer primer and an automated EcoRI ribotyping technique (Riboprinter) were compared for their ability to discriminate between 100 serovars of Salmonella, including multiple isolates representing Salm. Enteritidis PT4 and Salm. Typhimurium DT104. Profiles generated by each method were subjected to numerical analysis using GelCompar software, resulting in the construction of phylogenetic trees and calculation of Simpson's numerical index of diversity (DI). Both methods were highly discriminatory for isolates of Salmonella (Ribotype DI = 0.990, AP-PCR DI = 0.997) with EcoRI ribotyping proving more discriminatory than AP-PCR for isolates of Typhimurium DT104. The population structure was found to be clonal by numerical analysis of markers generated by both methods with serovars being polyphyletic in some cases and grouped in a single cluster in others. No absolute correlation was observed in the relationships between strains formed on the basis of ribo- and AP-PCR markers and serological characteristics.