Norovirus attribution study: Detection of norovirus from the commercial food preparation environment in outbreak and non-outbreak premises.

AIMS: Norovirus remains the most significant virological risk that is transmitted via food and the environment to cause acute gastroenteritis. This study aimed to investigate the hypothesis that the contamination of the commercial food production environment with norovirus will be higher in premises that have recently reported a foodborne norovirus outbreak than those that have not. METHODS: Sampling of commercial food production environments was carried out across a 16-month period between January 2015 and April 2016 in the South East and the North West of England by local authority environmental health departments as part of routine surveillance visits to premises. A total of 2982 samples, 2038 virological and 944 bacteriological, were collected from 256 premises. Sixteen of these premises, six from South East and ten from North West England, were sampled as part of a public health outbreak investigation. RESULTS & CONCLUSIONS: Overall, 2038 swabs were submitted for norovirus testing, with an average of eight swabs per premises (range 4 to 23) and a median of seven. Of the premises sampled, 11.7% (30/256) yielded at least one norovirus-positive sample (environmental, and/or food handler hand swab), and 2.5% of the swabs were positive for norovirus. A peak in the positivity rate was seen in the South East in April 2016. No associations were found between norovirus positivity and bacteriology indicators, or between bacteriology indicators and hygiene ratings. SIGNIFICANCE AND IMPACT OF STUDY: This study demonstrates that food premises and food handlers remain a potential source of norovirus transmission and outbreaks.

Optimized methods for detecting Salmonella Typhi in the environment using validated field sampling, culture and confirmatory molecular approaches.

AIMS: This study evaluated detection methods for Salmonella Typhi (S. Typhi) in the environment, to establish a novel pathway from field sampling to isolation of viable organisms and molecular confirmation from complex environmental samples, thus enabling environmental surveillance of typhoid. METHODS AND RESULTS: Multiple media were assessed using clinical isolates from the Public Health England's (PHE) Culture collection. The culture pathway selected consisted of a primary 2% bile broth and secondary Selenite F broth, followed by modified Chromogenic Agar for Salmonella Esterase (mCASE). A qPCR assay was adapted from a validated S. Typhi PCR panel for confirmation of isolates, with comparison to biochemical and serological tests showing good specificity. Sampling locations in Blantyre, Malawi were used to compare sampling methods. Viable S. Typhi were isolated from a mixture of trap and grab river water samples on six occasions. CONCLUSIONS: Culture of viable S. Typhi from environmental samples was possible using effective capture and culture techniques. SIGNIFICANCE AND IMPACT OF STUDY: Whilst several studies have attempted to detect S. Typhi from the environment, this is the first successful attempt to isolate the organism from river water since the 1980s. Supplementing clinical data with environmental screening offers the potential for enhanced surveillance, which might inform interventions and assess vaccination programmes.

Amoxicillin therapy of poultry flocks: effect upon the selection of amoxicillin-resistant commensal Campylobacter spp.

BACKGROUND: The aim of this study was to investigate the effect of amoxicillin therapy of poultry flocks upon the persistence of commensal Campylobacter spp. and the incidence of antibiotic resistance. METHODS: Four poultry flocks naturally colonized with Campylobacter were treated with amoxicillin and monitored before, during and up to 4 weeks post-treatment. The numbers of Campylobacter were determined and the isolates speciated and typed by flaA short variable region (SVR) sequence analysis and PFGE. The susceptibility of the isolates to antibiotics, presence of the Cj0299 gene encoding a beta-lactamase and beta-lactamase production (nitrocefin hydrolysis) were also determined. RESULTS: Amoxicillin-resistant Campylobacter were isolated from Flock 1 before and during treatment, but Campylobacter were not detected afterwards. Flock 2 was colonized by amoxicillin-susceptible strains throughout sampling. No amoxicillin-resistant isolates arose during or after treatment. Flock 3 contained amoxicillin-susceptible and -resistant types pre-treatment. Resistant isolates were detected during treatment, while antibiotic-susceptible isolates re-emerged at 3 weeks post-treatment. All Campylobacter isolates from Flock 4 were amoxicillin resistant, irrespective of sampling time. All but one of the 82 amoxicillin-resistant (MICs 16 to >128 mg/L) Campylobacter jejuni and Campylobacter coli tested for the presence of Cj0299 carried the gene and all of these produced beta-lactamase. Co-amoxiclav remained active against amoxicillin-resistant isolates. CONCLUSIONS: Amoxicillin therapy had little effect on the numbers of amoxicillin-resistant commensal Campylobacter except for one flock where amoxicillin-resistant Campylobacter temporarily dominated. Amoxicillin therapy did not select amoxicillin-resistant isolates from a previous susceptible strain. Co-amoxiclav remained active against amoxicillin-resistant isolates.

Identification of Campylobacter infection in chickens from volatile faecal emissions.

Volatile organic compounds from chicken faeces were investigated as biomarkers for Campylobacter infection. Campylobacter are major poultry-borne zoonotic pathogens, colonizing the avian intestinal tract. Chicken faeces are the principal source of contamination of carcasses. Fresh faeces were collected on farm sites, and Campylobacter status established microbiologically. Volatile organic compounds were pre-concentrated from the headspace above 71 separate faecal samples using solid-phase microextraction and separated and identified by gas chromatography/mass spectrometry. A Campylobacter-specific profile was identified using six of the extracted volatile organic compounds. The model developed reliably identified the presence or absence of Campylobacter in >95% of chickens. The volatile biomarker identification approach for assessing avian infection is a novel approach to enhancing biosecurity in the poultry industry and should reduce the risk of disease transmission to humans.

Real-time PCR detection and frequency of 16S rDNA mutations associated with resistance and reduced susceptibility to tetracycline in Helicobacter pylori from England and Wales.

OBJECTIVES: To investigate the occurrence of 16S rDNA mutations associated with resistance or reduced susceptibility to tetracycline in Helicobacter pylori isolated in England and Wales, and to develop a real-time PCR assay to detect these DNA polymorphisms from culture and gastric biopsies. METHODS: Tetracycline susceptibility was determined by disc diffusion. The MIC of isolates with reduced susceptibility was determined by Etest and agar dilution methods. The 16S rDNA of these isolates was sequenced and resistance-associated mutations identified. A LightCycler assay developed to detect these mutations was applied to DNA extracted from culture and gastric biopsies. RESULTS: From 1006 isolates of H. pylori examined, 18 showed reduced susceptibility to tetracycline. Of these, three were resistant (>or=4 mg/L). Mutations in 16S rDNA were detected in 10 of the reduced susceptibility isolates: one double base mutation of A926T/A928C, one A926C, one A928C; and seven A926G. The first two polymorphisms were novel and had not been reported from clinical isolates previously. The LightCycler assay identified each of the 10 isolates with 16S rDNA mutations, but did not detect polymorphisms in 100 tetracycline-susceptible H. pylori isolates. The assay correctly determined the tetracycline susceptibility of H. pylori in 20 gastric biopsy samples. CONCLUSIONS: Mutations in 16S rDNA were detected in H. pylori isolated in England and Wales with reduced susceptibility to tetracycline, but resistance to this antibiotic was uncommon. We show molecular-based susceptibility testing for tetracycline is possible direct from biopsy material.

Helicobacter pylori antibiotic-resistance patterns and risk factors in adult dyspeptic patients from ethnically diverse populations in central and south London during 2000.

Surveillance of Helicobacter pylori antibiotic susceptibility from patients in London, the largest metropolitan area in the UK, is limited, despite resistance being a key factor in treatment failure. A two-centre survey was performed over 12 months (1999-2000) to determine antibiotic-resistance rates of isolates from dyspeptic patients attending endoscopy clinics serving two ethnically diverse central and south London communities. The in vitro antibiotic susceptibilities were determined from disc diffusion and epsilometer (E) tests on 101 H. pylori isolates. Overall resistance rates were 59% for metronidazole and 11% for clarithromycin, with 8 % resistance to both antibiotics. Corresponding primary resistance rates were 50% and 7%, respectively. High-level-resistance was a feature of 82% of the metronidazole (MIC > or = 256 mg l(-1)) -resistant and 55% of the clarithromycin (MIC > or = 32 mg l(-1)) -resistant strains. All isolates were susceptible to amoxycillin and tetracycline. No associations between resistance and either the gender or the age of the patients were detected. The main risk for resistance to metronidazole was non-UK birth as comparative rates were 68% for non-UK vs. 40% for UK-born individuals. Resistant isolates were genotypically diverse with respect to cagA/vacA type. Four 23S rDNA nucleotide polymorphisms were associated with clarithromycin resistance, mostly (9/11) at A2143G. In conclusion, the high overall metronidazole-resistance rate of 59% for H. pylori from inner London was twice the rate found in other UK-based studies and was attributed to the higher risk of resistant strains infecting individuals born outside the UK. The need for continued resistance surveillance is indicated to monitor the effects of the 'test and treat' strategy for H. pylori eradication, particularly of isolates from at-risk individuals.

Prevalence and subtypes of ciprofloxacin-resistant Campylobacter spp. in commercial poultry flocks before, during, and after treatment with fluoroquinolones.

Five commercial broiler chicken flocks were treated with either difloxacin or enrofloxacin for a clinically relevant infection, as instructed by a veterinarian. Campylobacters were isolated from individual fecal samples and from samples associated with the broiler environment before, during, and after treatment. Ciprofloxacin-resistant Campylobacter jejuni and/or C. coli strains were detected pretreatment in four flocks, but they constituted a very small proportion of the campylobacters present. When the broilers were treated with a fluoroquinolone, a rapid increase in the proportion of ciprofloxacin-resistant campylobacters was observed. During treatment nearly 100% of campylobacters were resistant, and in some flocks a high proportion of resistant strains persisted for up to 4 weeks after treatment. Prior to treatment a variety of campylobacter subtypes were present. During and after treatment considerable changes in both species and subtype prevalence were observed, but no single fluoroquinolone-resistant clone became dominant. Instead, resistant C. coli strains or a mixture of resistant C. coli and C. jejuni strains became dominant, whereas susceptible C. jejuni strains had usually been dominant prior to treatment. The resistant subtypes which emerged and became dominant were not always the same as those detected pretreatment. The persistence of resistant strains for up to 4 weeks posttreatment has important implications for any strategy designed to avoid the introduction of such strains into the food chain.

Helicobacter pylori antibiotic resistance patterns and genotypes in adult dyspeptic patients from a regional population in North Wales.

OBJECTIVE: Surveillance data on Helicobacter pylori antibiotic susceptibilities in Wales are limited, despite resistance being a key factor in treatment failure. A single-centre survey was undertaken over 3 years to determine local antibiotic resistance rates on isolates from dyspeptic patients in Bangor, Gwynedd (North Wales). METHODS: Susceptibilities were determined for 363 isolates by disc diffusion and the Etest. Isolates were also genotyped (cagA presence and vacA allelic types). RESULTS: Overall in vitro resistance rates were 24% for metronidazole and 7% for clarithromycin, with 4% resistant to both antibiotics. Resistant strains typically had high MICs of >256 mg/L. Tetracycline resistance was identified in only one isolate whereas no isolates showed resistance to amoxicillin. There was a two-fold increase in resistance over the study period. No gender and age associations with resistance were detected. Resistant and susceptible isolates were genotypically diverse with respect to cagA/vacA type but the vacA s1m2 form was a feature of all clarithromycin-resistant isolates compared with 56% of the susceptible isolates. CONCLUSION: Although the overall antibiotic resistance rates of H. pylori from North Wales were low compared with many other regions in Europe, continued surveillance, particularly of high-level resistance (MIC >256 mg/L), is recommended to monitor the effects of the 'test and treat' strategy for H. pylori eradication.

Application of 3'-mismatched reverse primer PCR compared with real-time PCR and PCR-RFLP for the rapid detection of 23S rDNA mutations associated with clarithromycin resistance in Helicobacter pylori.

Helicobacter pylori clarithromycin (Cla) resistance dramatically reduces efficacy of eradication therapy. In this study, 3'-mismatched reverse primer PCR (3M-PCR), real-time PCR (LightCycler), and PCR-RFLP assays were investigated to determine their sensitivity for detecting clarithromycin resistance associated with 23S rDNA mutations (A2142G, A2142C, and A2143G). For 84.8% (123/145) of isolates, the same allelic type was detected by each method although methods differed in efficiency of detecting mutations in cultures either containing mixtures of two alleles (24 isolates), or that were dual allelic variants (two isolates). The novel 3M-PCR assay format was the most sensitive, detecting all alleles at > or =0.02 ng/microl in DNA mixtures, and thus provides more precise information to guide clinical management of patients at risk of treatment failure.