Novel antibacterial silver-silica surface coatings prepared by chemical vapour deposition for infection control.

AIMS: Environmental contamination plays an important role in the transmission of infections, especially healthcare-associated infections. Disinfection transiently reduces contamination, but surfaces can rapidly become re-contaminated. Antimicrobial surfaces may partially overcome that limitation. The antimicrobial activity of novel surface coatings containing silver and silica prepared using a flame-assisted chemical vapour deposition method on both glass and ceramic tiles was investigated. METHODS AND RESULTS: Antimicrobial activity against a variety of bacteria including recent clinical isolates was investigated based on the BS ISO 22196:2007 Plastics--Measurement of antibacterial activity on plastics surfaces, British Standards Institute, London, method. Activity on natural contamination in an in use test in a toilet facility was also determined. Activity on standard test strains gave a log10 reduction of five after 1-4 h. The hospital isolates were more resistant, but MRSA was reduced by a log10 reduction factor of >5 after 24 h. Activity was maintained after simulated ageing and washing cycles. Contamination in situ was reduced by >99.9% after 4 months. Activity was inhibited by protein, but, although this could be overcome by increasing the amount of silver in the films, this reduced the hardness of the coating. CONCLUSIONS: The coatings had a good activity against standard test strains. Clinical isolates were killed more slowly but were still sensitive. The optimum composition for use therefore needs to be a balance between activity and durability. SIGNIFICANCE AND IMPACT OF THE STUDY: The coatings may have applications in health care by maintaining a background antimicrobial activity between standard cleaning and disinfection regimes. They may also have applications in other areas where reduction in microbial contamination is important, for example, in the food industry.

Microbiological aspects of public health planning and preparedness for the 2012 Olympic Games.

Although communicable diseases have hitherto played a small part in illness associated with Olympic Games, an outbreak of infection in a national team, Games venue or visiting spectators has the potential to disrupt a global sporting event and distract from the international celebration of athletic excellence. Preparation for hosting the Olympic Games includes implementation of early warning systems for detecting emerging infection problems. Ensuring capability for rapid microbiological diagnoses to inform situational risk assessments underpins the ability to dispel rumours. These are a prelude to control measures to minimize impact of any outbreak of infectious disease at a time of intense public scrutiny. Complex multidisciplinary teamwork combined with laboratory technical innovation and efficient information flows underlie the Health Protection Agency's preparation for the London 2012 Olympic and Paralympic Games. These will deliver durable legacies for clinical and public health microbiology, outbreak investigation and control in the coming years.

Survey of Salmonella contamination of raw shell eggs used in food service premises in the United Kingdom, 2005 through 2006.

This survey was launched after an unusual number of Salmonella Enteritidis outbreaks associated with the use of eggs in food service premises in England and Wales. Between November 2005 and December 2006, 9,528 eggs (1,588 pooled samples of 6 eggs) were collected from 1,567 food service premises in the United Kingdom, most of which (89%) were produced in the United Kingdom. Salmonella was isolated from 6 (0.38%) pools of eggs. Of these, 5 (0.31%) were Salmonella Enteritidis, which were further characterized to phage types (PTs): PT 4 (0.19%), PT 8 (0.06%), and PT 12 (0.06%). Salmonella Mbandaka was also isolated (0.06%). Salmonella was detected from five and one of pooled eggs samples that were produced in the United Kingdom and Germany, respectively; these were from different producers. The study showed evidence of poor egg storage and handling practices in food service premises, in that 55% did not store eggs under refrigerated conditions; 20.7% of eggs had expired "best before" dates or were in use after 3 weeks of lay, indicating poor stock rotation; and 37.1% pooled eggs not intended for immediate service. Eggs are a commonly consumed food that may occasionally be contaminated with Salmonella at different rates, according to their country of origin. The food service sector needs to be aware of this continuing hazard, receive appropriate food safety and hygiene training on storage and usage of raw shell eggs, adopt appropriate control measures, and follow advice provided by national food agencies in order to reduce the risk of infection.

Comparison of virulence-associated in vitro properties of typed strains of Campylobacter jejuni from different sources.

Campylobacter jejuni is a major cause of human diarrhoeal disease, but specific virulence mechanisms have not been well defined. This blinded study was undertaken with 40 C. jejuni isolates from different sources to determine their haemolytic, cytotoxic and adhesion and invasion activities towards mammalian cells. The results were correlated with source of isolation and genetic makeup by amplified fragment length polymorphism (AFLP) typing. The isolates had variable degrees of haemolytic activity against rabbit erythrocytes and cytotoxicity towards CaCo-2, HeLa and Vero cells. The data indicated that the haemolytic and cytotoxic activities were due to separate factors. A range of cytotoxicity was exhibited, whereby some strains had no activity against the target cells and others had activity against all three cell lines. Certain strains had activity against CaCo-2 cells but little or no activity against the other cells, while others exhibited the opposite phenotype. The data suggested that the cytotoxicity assay with the different cell lines may have detected more than one cytotoxin. A wide variation between isolates was observed for both adherence and invasion with all three cell lines, yet, overall, the strains showed a significantly greater invasion capacity for CaCo-2. There was no clear relationship between source of isolation or disease manifestation and possession of statistically significantly higher levels of particular virulence-associated factors although, in some cases, a correlation between cytotoxicity and cell invasion was evident. Five AFLP clusters, each representing two to eleven isolates with similar profiles, were observed at the 90 % similarity level. Some AFLP groups contained isolates with a common serotype, but each group had C. jejuni isolates from more than one source with the exception of group IV, which contained only human isolates. Isolates with high cytotoxic activity against CaCo-2 cells were confined to groups I, III and IV and a group of unrelated strains (U). Group II isolates had uniformly low cytotoxicity. Isolates in groups I, V and U were more invasive for CaCo-2 cells than isolates in groups II, III and IV. The strain differences in cytotoxicity or invasion did not correlate with source of isolation.

Real-time single-nucleotide polymorphism profiling using Taqman technology for rapid recognition of Campylobacter jejuni clonal complexes.

The rapid identification of Campylobacter jejuni isolates to strain level would significantly inform the public health investigation of C. jejuni infection. Conceptual advances provided by multilocus sequence typing (MLST) have established the clonal complex as an important epidemiological group at the strain level, enabling accurate and phylogenetically valid strain identification for C. jejuni. The development of real-time PCR assays for allelic discrimination of strain-associated single-nucleotide polymorphisms (SNPs) based upon MLST locus alleles offers one possible approach for rapid strain detection. SNPs defining key alleles diagnostic for the most prevalent clonal complexes were identified following a detailed analysis of the available MLST data. Real-time Taqman allelic discrimination assays designed to detect the SNPs specific for six major clonal complexes, ST-21, ST-45, ST-48, ST-61, ST-206 and ST-257, were developed, allowing the rapid detection of C. jejuni isolates and preliminary strain identification. This will provide an important complementary technique to sequence typing for rapid detection and strain characterization to inform in real-time the public health management and investigation of C. jejuni infections.

A case of infant botulism with a possible link to infant formula milk powder: evidence for the presence of more than one strain of Clostridium botulinum in clinical specimens and food.

Infant botulism was confirmed in a 5-month-old female by both isolation of Clostridium botulinum type B and by detection of type B botulinum neurotoxin in rectal washout and faeces. DNA fingerprinting of nine isolates from faeces yielded two different amplified-fragment length polymorphism (AFLP) patterns. C. botulinum was isolated from two of 14 food and drink items from the patient's home: C. botulinum type A was recovered from an opened container of dried rice pudding and C. botulinum type B from opened infant formula milk powder. Ten C. botulinum type B isolates from the opened infant formula yielded four AFLP patterns, two of which were indistinguishable from the clinical isolates. Fifteen unopened foods were tested and C. botulinum type B of a unique AFLP pattern was recovered from one unopened infant formula of the same batch as the opened container. It is suggested that multiple C. botulinum were present in both food and the intestine during infant botulism.

A study of the oxygen and carbon dioxide requirements of thermophilic campylobacters.

The oxygen and carbon dioxide requirements of different biotypes of thermophilic campylobacters were investigated by means of (a) quantitative studies, and (b) total growth studies. Oxygen tolerance of the five test organisms differed markedly and varied with the carbon dioxide concentration. At most carbon dioxide concentrations tested, Campylobacter jejuni strains NCTC 11168 and NCTC 11392 tolerated 21% oxygen (growth reduced), C coli NCTC 11353 tolerated 15% oxygen (growth reduced), and C jejuni ATCC 3036 and (nalidixic acid resistant thermophilic campylobacter) NCTC 11352 tolerated 10% oxygen (growth not reduced). Total growth studies indicated that 10% oxygen was the optimal concentration for growth of the five test organisms. All exhibited a requirement for carbon dioxide, and only C jejuni strains NCTC 11168 and NCTC 11392 tolerated its absence (growth reduced), when the oxygen concentration was low. The studies indicated that atmospheres containing 5% to 10% oxygen and 1.0% to 10% carbon dioxide are suitable for growth of the various biotypes of thermophilic campylobacters. The oxygen and carbon dioxide concentrations produced in anaerobic jars by variations of the evacuation-replacement technique were determined and suitable practices identified.

A selective medium for isolating Campylobacter jejuni/coli.

Skirrow's medium is effective for isolating Campylobacters from human faeces but is less suitable for animal and environmental specimens owing to the presence of contaminating species. After determining the sensitivity of 104 strains of Campylobacters to several antimicrobial agents, used singly and in various combinations, a selective medium incorporating polymixin, rifampicin, trimethoprim and actidione, was developed. The medium, called Preston medium, was shown to be more selective than Skirrow's medium and suitable for any kind of specimen.

Evaluation of three Campylobacter pylori antigen preparations for screening sera from patients undergoing endoscopy.

A surface antigen (SA), acid glycine extract (AGE), and urease preparation (UP) were evaluated using sera from patients undergoing endoscopy and from subjects with gastric or duodenal ulcers. Sera were tested for the presence of IgG and IgA antibodies by a conventional indirect enzyme linked immunosorbent assay (ELISA). In patients with confirmed Campylobacter pylori associated gastritis, raised IgG antibody titres were indicated by absorbance values of greater than or equal to 500, greater than or equal to 500, and greater than or equal to 1500 for the SA, AGE, and UP, respectively. Corresponding values for the IgA assay were greater than or equal to 500, greater than or equal to 500, and greater than or equal to 1000. The specificity of the IgG assays were 94%, 92%, and 90% for the AGE, SA, and UP, respectively. In contrast, the UP was the most sensitive (97%); the other two antigen preparations gave values of 82%. In the IgA assay the UP showed the greatest specificity (90%) and sensitivity (90%). The predictive value for a true positive for the IgG assay was the same for all antigens (93%), whereas the UP gave a predictive value for a true negative of 96% compared with 79% for the other two antigen preparations. Of the patients with gastric or duodenal ulcers, raised antibody titres to SA were found in 72% (IgG) and 73% (IgA), to AGE in 75% (IgG) and 63% (IgA), and to UP in 77% (IgG) and 75% (IgA). The use of a urease antigen preparation to determine IgG antibody is recommended for screening patients undergoing endoscopy.

Is enrichment culture necessary for the isolation of Campylobacter jejuni from faeces?

The role of enrichment culture for the isolation of Campylobacter jejuni from faeces is discussed. It is concluded that enrichment culture is only necessary for those specimens where it is anticipated that the number of organisms is likely to be low. In a trial of a blood free enrichment broth (CCD broth) and the modified Preston enrichment broth the latter gave significantly superior results.

Comparison of selective media for isolation of Campylobacter jejuni/coli.

A comparison of Skirrow's, Butzler's, Blaser's, Campy-BAP and Preston media for Campylobacter spp was made using human, animal and environmental specimens. Butzler's medium gave the lowest isolation rate and Preston medium, which was the most selective, the highest isolation rate. Enrichment culture using Preston enrichment broth gave a higher isolation rate than direct plating onto Preston medium.

Campylobacter biotyping scheme of epidemiological value.

A biotyping scheme has been developed which utilises 12 tests, including growth at 28 degrees C, hippurate hydrolysis, and 10 resistotyping tests. These tests are arranged in groups of three, and by assigning a numerical value to each positive test a four figure code is produced for each strain. The order of the tests is such that campylobacters are both speciated and biotyped . This scheme recognises Campylobacter jejuni, C coli, "C laridis ," C fetus fetus, and C fetus subspecies venerealis. The reproducibility of the biotyping technique and the stability of the biotype code have been determined by testing campylobacter reference strains. The routine application of the scheme has also been evaluated by biotyping 1000 recent campylobacter isolates, and the epidemiological value has been confirmed by testing serotyped isolates from several milk borne outbreaks.

Campylobacter pylori: clinical, histological, and serological studies.

The presence of Campylobacter pylori, histologically diagnosed gastritis, and antibodies to C pylori were determined in a series of 113 patients undergoing endoscopy. Paired biopsy specimens from the fundus, body, and antrum were collected from 59 patients and from the antrum of 54 patients. The presence of C pylori was confirmed by either culture or silver stain in 30 of 59, 31 of 59, and 54 of 103 biopsy specimens from the fundus, body, and antrum, respectively. Of the specimens which contained C pylori 20 of 30 (66%) from the fundus, 25 of 31 (80%) from the body, and 54 (100%) from the antrum showed gastritis. C pylori and gastritis were shown in seven of nine (78.1%) of patients with gastric ulcers and in nine of 11 (82%) of patients with duodenal ulcers. Using an enzyme linked immunosorbent assay (ELISA) technique to detect IgG antibody to C pylori, all patients with histologically diagnosed gastritis and organisms present had titres of greater than or equal to 640; eight of 39 (21%) of patients without gastritis and without organisms gave similar titres. Hence the presence of C pylori was associated with gastritis and with raised titres of IgG antibody.

Characterisation of 16 Campylobacter jejuni and C. coli typing bacteriophages.

Taxonomic classification of bacteriophages specific for Campylobacter jejuni and C. coli has not been reported previously. A set of 16 virulent phages, distinguishable by their lytic spectra, has been used extensively for epidemiological typing of C. jejuni and C. coli at Preston Public Health Laboratory. These phages were investigated by electron microscopy, pulsed-field gel electrophoresis and restriction endonuclease analysis. All phages had icosahedral heads and long contractile tails. Accordingly, they were classified as members of the Myoviridae family. These phages could be subdivided into three groups according to genome size and head diameter: group I, two phages with head diameters of 140.6 and 143.8 nm and genome sizes of 320 kb; group II, five phages with average head diameters of 99 nm and average genome sizes of 184 kb; and group III, nine phages with average head sizes of 100 nm and average genome sizes of 138 kb. Phages NCTC12676 and NCTC12677 of group I had unusually large genomes of c. 320 kb which are two of the largest phage genomes to be described. Restriction endonuclease analysis demonstrated that DNA from the 16 phages was refractory to digestion by a number of restriction enzymes.

An investigation into the microflora of heroin.

In 2000, an unusual increase of morbidity and mortality among illegal injecting drug users in the UK and Ireland was reported and Clostridium novyi was identified as the likely source of the serious infection, although infections due to C. botulinum and Bacillus cereus were also reported. Because heroin was a possibile source of infection, this study investigated the microflora of heroin samples seized in England during 2000 and 2002. Two methods were developed for the examination of the microflora of heroin. The first consisted of suspension of the drug in maximum recovery diluent (MRD) which was inoculated directly into Clostridium Botulinum Isolation Cooked Meat Broth (CBI). The second method rendered the heroin soluble in citric acid, concentrated particulate material (and bacterial cells) by filtration and removed heroin residues by washing with citric acid and phosphate-buffered saline before placing the filter in CBI broth. Duplicate CBI broths from both methods were incubated without heating and after heating at 60 degrees C for 30 min. Subcultures were made after incubation for 7 and 14 days on to eight different solid media. The methods were evaluated with heroin samples spiked with either C. botulinum or C. novyi spore suspensions; recovery of 10 spores in the original sample was demonstrated. Fifty-eight heroin samples were tested by citric acid solubilisation and 34 by the MRD suspension technique. Fifteen different gram-positive species of four genera were recognised. No fungi were isolated. Aerobic endospore-forming bacteria (Bacillus spp. and Paenibacillus macerans) were the predominant microflora isolated and at least one species was isolated from each sample. B. cereus was the most common species and was isolated from 95% of all samples, with B. licheniformis isolated from 40%. Between one and five samples yielded cultures of B. coagulans, B. laterosporus, B. pumilus, B. subtilis and P. macerans. Staphylococcus spp. were isolated from 23 (40%) samples; S. warneri and S. epidermidis were the most common and were cultured from 13 (22%) and 6 (10%) samples respectively. One or two samples yielded cultures of S. aureus, S. capitis and S. haemolyticus. The remainder of the flora detected comprised two samples contaminated with C. perfringens and two samples with either C. sordellii or C. tertium. Multiple bacterial species were isolated from 43 (74%) samples, a single species from the remaining 15. In 13 samples B. cereus alone was isolated, in one B. subtilis alone and in one sample B. pumilus alone. C. botulinum and C. novyi were not isolated from any of the heroin samples. Recommendations for the optimal examination of the microflora of heroin are given.

A study of photobactericidal activity in the phenothiazinium series.

The photodynamic antibacterial properties of a closely related series of commercially available phenothiazinium dyes were tested against a range of pathogenic strains of Gram-positive (Staphylococcus aureus, Enterococcus faecalis, Bacillus cereus) and Gram-negative organisms (Escherichia coli, Pseudomonas aeruginosa). The photosensitisers were illuminated using a non-laser light source at a fluence of 1.75 mW cm-2 and this resulted in the enhancement of antibacterial activity in liquid culture. In several cases, illumination resulted in considerable decreases in the minimum lethal concentrations required, giving up to 100-fold increases in bactericidal activity.

Quality assurance in food microbiology--a novel approach.

The introduction of quality systems as a requirement of laboratory accreditation is causing microbiologists to review current practices. The need for Quality Assurance (QA) in food microbiology is of growing importance and this paper presents a novel approach to implementing QA based on a system which is analogous to the Hazard Analysis Critical Control Point approach adopted by the food industry. The basis of the QA system is the recognition of Quality Assessment Points (QAPs). Several Quality Control and monitoring practices are suggested for each of the QAPs with the overall aim of developing a Total Quality Assurance system for food microbiology laboratories.

Strategies in the development of media for the detection of food-borne pathogens.

Successful medium development is dependent on using a systematic approach and also by giving due consideration to the factors which can influence the performance of the medium at the various stages of assessment. The most important factors to be considered are: (i) the properties of the target organisms, (ii) the selection of test strains, (iii) the methods of evaluation, (iv) the basal medium and growth supplements, (v) the properties of the medium, and (vi) the intended use of the medium. If these are investigated fully then culture media can be optimised to fulfil the demands of modem microbiology techniques used for detection and confirmation of bacteria.

Prevalence and numbers of Salmonella and Campylobacter spp. on raw, whole chickens in relation to sampling methods.

Salmonella and Campylobacter continue to be major foodborne pathogens and raw poultry is considered to be an important source of these bacteria. In this study, the prevalence and numbers of Salmonella and Campylobacter spp. in relation to isolation/sampling methods were determined in 241 whole raw chickens purchased from retail outlets in England during the winters of 1998/1999 (101 chickens) and 1999/2000 (140 chickens). The packaging of the 140 chickens was also examined for the presence of the above pathogens. The prevalence and numbers of enterococci were examined in 21 of the 101 chickens. In total, Salmonella and Campylobacter spp. were present in 25% and 83% of the chickens, respectively. Salmonella were isolated from a sample representing both the inside and outside of the packaging in 19% of the chickens, while the corresponding figure for Campylobacter spp. was 56%. Both of these pathogens were isolated from the outside of the packaging in 6% of the chickens. Salmonella was more frequently isolated from samples containing chicken skin in comparison with those containing carcass-rinse fluid only. Two chickens (0.8%) were positive for Salmonella by direct enumeration methods with contamination levels of log10 3.8 and 4.5 colony forming units (cfu) per carcass, respectively. The most prevalent serotypes were S. Hadar, S. Enteritidis and S. Indiana and two different serotypes were identified in 5/20 salmonella-positive chickens. Resistance to at least one antibiotic was found in 70% of the strains, 46% were multiresistant (resistant to > or = four drugs) and 52% showed a lowered susceptibility to ciprofloxacin. The likelihood of isolating Campylobacter spp. from neck-skin, carcass-rinse or carcass-rinse plus whole skin samples was similar, Campylobacter spp. were found in higher levels in carcass-rinse or carcass-rinse plus whole skin samples than in neck-skin. The log10 cfu of Campylobacter spp. were 2.70-4.99 in 18% of the chickens and 5.00-6.99 in 20%. Campylobacter isolates (425) comprised Campylobacter jejuni (98%) and C. coli (2%) and 98 different sero/phagetypes of these two species were identified. Resistance to at least one antibiotic was found in 73% of the strains and 13% were multiresistant. Thirteen percent of the strains showed lowered susceptibility to ciprofloxacin, while 4.9% were resistant to erythromycin. Vancomycin-resistant enterococci (VRE), able to grow on agar containing 15 mg l(-1) vancomycin (VRE15), were present in 19 chickens. The log10 cfu of VRE15 was 2.90-3.99 in 10 chickens and between 4.00 and 4.99 in two chickens. The data presented here contribute to risk assessment and highlight the need to continue to emphasise the safe handling of raw retail poultry.

Campylobacter contamination of raw meat and poultry at retail sale: identification of multiple types and comparison with isolates from human infection.

Campylobacter species are the major cause of acute bacterial enteritis reported in the United Kingdom, nonetheless many aspects of campylobacteriosis epidemiology remain poorly understood. The aim of this study was to determine the prevalence of Campylobacter jejuni and Campylobacter coli in fresh bovine, ovine, and porcine liver and chicken portions from retail outlets and compare strain subtype distributions with those associated with cases of human campylobacteriosis occurring within the same period and study area. Meat samples were examined by both enrichment culture and direct plating, and Campylobacter isolates were subjected to the same test procedures (identification, serotyping, phagetyping, resistotyping) applied to the clinical strains. Campylobacter species were isolated from 73.2% of 489 samples examined. Chicken exhibited the highest contamination rate (83.3%), followed by lamb (72.9%), pig (71.7%), and ox (54.2%) liver. C. jejuni predominated in chicken (77.3%), lamb (75.0%), and ox (49.0%) liver, and C. coli predominated in pigs' liver (42.4%). Campylobacter fetus was identified in 12.5% of ox liver samples and also in pig and lamb. Of the human isolates, 89.3% were C. jejuni and 10.7% C. coli. The greatest variation in C. jeuni subtypes was observed among the chicken isolates (57 sero/phage-types), followed by human (48 types) and lamb (30 types). A significant proportion of the chicken and lamb isolates shared identical subtypes with the human strains, indicative of their role as potential sources of infection. Almost 30% of samples yielded multiple strains of Campylobacter, a finding that reinforces the epidemiological importance of selecting and testing more than one presumptive isolate per sample.