Anthony C. Hilton

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.

Penetration of Chlorhexidine into Human Skin

ABSTRACT 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 μm 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 μg/mg tissue within the top 100 μm), and levels of chlorhexidine were minimal at deeper skin depths (less than 0.002 μg/mg tissue below 300 μm). After 24 h of exposure, there was more chlorhexidine within the upper 100-μm sections (7.88 ± 1.37 μg/mg tissue); however, the levels remained low (less than 1 μg/mg tissue) at depths below 300 μm. 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.

Isolation of Salmonella from urban wild brown rats (Rattus norvegicus) in the West Midlands, UK

A 6-month study was undertaken to investigate the prevalence of Salmonella enterica in wild urban brown rats (Rattus norvegicus) in the West Midlands. Samples were obtained of faecal droppings (n = 100) and from rectal swabs (n = 50) of rat carcases collected from active infestation sites. A subset of the rats (n = 25) had additional swab samples taken of the fur, paws and tail. Five (10%) of the rectal swabs were positive for Salmonella by direct plating onto XLD media. No further samples were positive following pre-enrichment and selective culture. A total of eight (8%) faecal samples were positive for Salmonella; two by direct plating and a further six following enrichment. All positive faecal samples were fresh or moist upon collection. None of the samples obtained from the outer surfaces of the rat were positive. Additionally, rat faeces were spiked with Salmonella and sampled periodically to determine survival in drying faeces exposed to a typical indoor environment. Salmonella could be recovered by direct culture up to 86 days. These results demonstrate a regional variability in the carriage of Salmonella in urban rats compared to other studies and that Salmonella longevity in faecal pellets is sufficient to present a potential contamination risk in the absence of an active infestation.

Genotypic and antimicrobial characterisation of Propionibacterium acnes isolates from surgically excised lumbar disc herniations

The anaerobic skin commensal Propionibacterium acnes is an underestimated cause of human infections and clinical conditions. Previous studies have suggested a role for the bacterium in lumbar disc herniation and infection. To further investigate this, five biopsy samples were surgically excised from each of 64 patients with lumbar disc herniation. P. acnes and other bacteria were detected by anaerobic culture, followed by biochemical and PCR-based identification. In total, 24/64 (38%) patients had evidence of P. acnes in their excised herniated disc tissue. Using recA and mAb typing methods, 52% of the isolates were type II (50% of culture-positive patients), while type IA strains accounted for 28% of isolates (42% patients). Type III (11% isolates; 21% patients) and type IB strains (9% isolates; 17% patients) were detected less frequently. The MIC values for all isolates were lowest for amoxicillin, ciprofloxacin, erythromycin, rifampicin, tetracycline, and vancomycin (≤1mg/L). The MIC for fusidic acid was 1-2 mg/L. The MIC for trimethoprim and gentamicin was 2 to ≥4 mg/L. The demonstration that type II and III strains, which are not frequently recovered from skin, predominated within our isolate collection (63%) suggests that the role of P. acnes in lumbar disc herniation should not be readily dismissed.

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.

RT-PCR for the pseudogene-free amplification of the glyceraldehyde-3-phosphate dehydrogenase gene (gapd)

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme which catalyses the conversion of glyceraldehyde-3-phosphate to 1,3 diphosphoglycerate. It is considered to be constitutively expressed in all cells, and as such the gene for GAPDH (gapd) is commonly used as a benchmark reference in expression studies. However, previous investigations have demonstrated that gapd may show altered gene expression in a number of disease states and under certain experimental conditions, suggesting that results of experiments using gapd as a control should be interpreted with caution. Furthermore, consideration must be given to the potential co-amplification of pseudogenes of gapd during RT-PCR. Here, we describe a method to avoid the amplification of contaminating pseudogenes through the design of primers that bind only to genuine gapd mRNA transcript.

Analysis of clinical isolates of Propionibacterium acnes by optimised RAPD

Random amplification of polymorphic DNA (RAPD) was evaluated as a genotypic method for typing clinical strains of Propionibacterium acnes. RAPD can suffer from problems of reproducibility if parameters are not standardised. In this study the reaction conditions were optimised by adjusting template DNA concentration and buffer constituents. All isolates were typeable using the optimised RAPD protocol which was found to be highly discriminatory (Simpsons diversity index, 0.98) and reproducible. Typing of P. acnes by optimised RAPD is an invaluable tool for the epidemiological investigation of P. acnes for which no other widely accepted method currently exists.

Enhanced chlorhexidine skin penetration with eucalyptus oil

BackgroundChlorhexidine digluconate (CHG) is a widely used skin antiseptic, however it poorly penetrates the skin, limiting its efficacy against microorganisms residing beneath the surface layers of skin. The aim of the current study was to improve the delivery of chlorhexidine digluconate (CHG) when used as a skin antiseptic.MethodChlorhexidine was applied to the surface of donor skin and its penetration and retention under different conditions was evaluated. Skin penetration studies were performed on full-thickness donor human skin using a Franz diffusion cell system. Skin was exposed to 2% (w/v) CHG in various concentrations of eucalyptus oil (EO) and 70% (v/v) isopropyl alcohol (IPA). The concentration of CHG (μg/mg of skin) was determined to a skin depth of 1500 μm by high performance liquid chromatography (HPLC).ResultsThe 2% (w/v) CHG penetration into the lower layers of skin was significantly enhanced in the presence of EO. Ten percent (v/v) EO in combination with 2% (w/v) CHG in 70% (v/v) IPA significantly increased the amount of CHG which penetrated into the skin within 2 min.ConclusionThe delivery of CHG into the epidermis and dermis can be enhanced by combination with EO, which in turn may improve biocide contact with additional microorganisms present in the skin, thereby enhancing antisepsis.

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.

Permeation of chlorhexidine from alcoholic and aqueous solutions within excised human skin

Chlorhexidine digluconate (CHG) is widely used in the clinical setting for skin antisepsis prior to incision or insertion of medical devices, e.g., central venous catheters (11-13); however, its permeation into skin is limited (6, 7, 9, 10, 16). In a recent study, we demonstrated the limited penetration of CHG in a skin model comprising full-thickness excised human skin following the application of 2% (wt/vol) aqueous CHG (9). The aim of this current study was to compare the penetration of chlorhexidine into skin following the topical application of 2% (wt/vol) CHG in 70% (vol/vol) isopropyl alcohol (IPA) with that of aqueous CHG.