Environmental Surveillance for Salmonella Typhi and its Association With Typhoid Fever Incidence in India and Malawi.

BACKGROUND: Environmental surveillance (ES) for Salmonella Typhi potentially offers a low-cost tool to identify communities with a high burden of typhoid fever. METHODS: We developed standardized protocols for typhoid ES, including sampling site selection, validation, characterization; grab or trap sample collection, concentration; and quantitative PCR targeting Salmonella genes (ttr, staG, and tviB) and a marker of human fecal contamination (HF183). ES was implemented over 12 months in a historically high typhoid fever incidence setting (Vellore, India) and a lower incidence setting (Blantyre, Malawi) during 2021-2022. RESULTS: S. Typhi prevalence in ES samples was higher in Vellore compared with Blantyre; 39/520 (7.5%; 95% confidence interval [CI], 4.4%-12.4%) vs 11/533 (2.1%; 95% CI, 1.1%-4.0%) in grab and 79/517 (15.3%; 95% CI, 9.8%-23.0%) vs 23/594 (3.9%; 95% CI, 1.9%-7.9%) in trap samples. Detection was clustered by ES site and correlated with site catchment population in Vellore but not Blantyre. Incidence of culture-confirmed typhoid in local hospitals was low during the study and zero some months in Vellore despite S. Typhi detection in ES. CONCLUSIONS: ES describes the prevalence and distribution of S. Typhi even in the absence of typhoid cases and could inform vaccine introduction. Expanded implementation and comparison with clinical and serological surveillance will further establish its public health utility.

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.

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.

National outbreak of Pseudomonas aeruginosa associated with an aftercare solution following piercings, July to September 2016, England.

We report a national Pseudomonas aeruginosa outbreak from a common source following piercings between July and September 2016 in England. The multi-agency outbreak investigation included active case finding, microbiological testing of environmental samples and case specimens including Variable Number Tandem Repeat (VNTR) typing and a retrospective cohort study. Overall, 162 outbreak cases (29 confirmed, 14 probable and 119 possible) and 14 non-outbreak cases were identified; all confirmed cases had ear piercings (93% cartilage). Outbreak cases were predominantly female (95%) and had a median age of 18 years (interquartile range: 13-56 years). Nineteen outbreak cases required surgery under general anaesthetic The same outbreak VNTR type (11,3,5,3,3,3,6,4,7) was isolated from bottles of an aftercare solution from a single manufacturer and in specimens from confirmed cases who attended eight different piercing studios supplied with this product. In the cohort study, use of aftercare solution was associated with becoming a case (aOR: 4.60, 95% confidence interval: 1.65-12.90). Environmental, microbiological and epidemiological investigations confirmed that contamination during production of aftercare solution was the source of this national outbreak; highlighting challenges in the regulation of a cosmetic products used in the piercing industry and that guidance on piercing aftercare may need to be reviewed.

Public Health Investigation of Two Outbreaks of Shiga Toxin-Producing Escherichia coli O157 Associated with Consumption of Watercress.

An increase in the number of cases of Shiga toxin-producing Escherichia coli (STEC) O157 phage type 2 (PT2) in England in September 2013 was epidemiologically linked to watercress consumption. Whole-genome sequencing (WGS) identified a phylogenetically related cluster of 22 cases (outbreak 1). The isolates comprising this cluster were not closely related to any other United Kingdom strain in the Public Health England WGS database, suggesting a possible imported source. A second outbreak of STEC O157 PT2 (outbreak 2) was identified epidemiologically following the detection of outbreak 1. Isolates associated with outbreak 2 were phylogenetically distinct from those in outbreak 1. Epidemiologically unrelated isolates on the same branch as the outbreak 2 cluster included those from human cases in England with domestically acquired infection and United Kingdom domestic cattle. Environmental sampling using PCR resulted in the isolation of STEC O157 PT2 from irrigation water at one implicated watercress farm, and WGS showed this isolate belonged to the same phylogenetic cluster as outbreak 2 isolates. Cattle were in close proximity to the watercress bed and were potentially the source of the second outbreak. Transfer of STEC from the field to the watercress bed may have occurred through wildlife entering the watercress farm or via runoff water. During this complex outbreak investigation, epidemiological studies, comprehensive testing of environmental samples, and the use of novel molecular methods proved invaluable in demonstrating that two simultaneous outbreaks of STEC O157 PT2 were both linked to the consumption of watercress but were associated with different sources of contamination.

Investigating One Health risks for human colonisation with extended spectrum ß-lactamase-producing Escherichia coli and Klebsiella pneumoniae in Malawian households: a longitudinal cohort study.

BACKGROUND: Low-income countries have high morbidity and mortality from drug-resistant infections, especially from enteric bacteria such as Escherichia coli. In these settings, sanitation infrastructure is of variable and often inadequate quality, creating risks of extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales transmission. We aimed to describe the prevalence, distribution, and risks of ESBL-producing Enterobacterales colonisation in sub-Saharan Africa using a One Health approach. METHODS: Between April 29, 2019, and Dec 3, 2020, we recruited 300 households in Malawi for this longitudinal cohort study: 100 each in urban, peri-urban, and rural settings. All households underwent a baseline visit and 195 were selected for longitudinal follow-up, comprising up to three additional visits over a 6 month period. Data on human health, antibiotic usage, health-seeking behaviours, structural and behavioural environmental health practices, and animal husbandry were captured alongside human, animal, and environmental samples. Microbiological processing determined the presence of ESBL-producing E coli and Klebsiella pneumoniae, and hierarchical logistic regression was performed to evaluate the risks of human ESBL-producing Enterobacterales colonisation. FINDINGS: A paucity of environmental health infrastructure and materials for safe sanitation was identified across all sites. A total of 11 975 samples were cultured, and ESBL-producing Enterobacterales were isolated from 1190 (41·8%) of 2845 samples of human stool, 290 (29·8%) of 973 samples of animal stool, 339 (66·2%) of 512 samples of river water, and 138 (46·0%) of 300 samples of drain water. Multivariable models illustrated that human ESBL-producing E coli colonisation was associated with the wet season (adjusted odds ratio 1·66, 95% credible interval 1·38-2·00), living in urban areas (2·01, 1·26-3·24), advanced age (1·14, 1·05-1·25), and living in households where animals were observed interacting with food (1·62, 1·17-2·28) or kept inside (1·58, 1·00-2·43). Human ESBL-producing K pneumoniae colonisation was associated with the wet season (2·12, 1·63-2·76). INTERPRETATION: There are extremely high levels of ESBL-producing Enterobacterales colonisation in humans and animals and extensive contamination of the wider environment in southern Malawi. Urbanisation and seasonality are key risks for ESBL-producing Enterobacterales colonisation, probably reflecting environmental drivers. Without adequate efforts to improve environmental health, ESBL-producing Enterobacterales transmission is likely to persist in this setting. FUNDING: Medical Research Council, National Institute for Health and Care Research, and Wellcome Trust. TRANSLATION: For the Chichewa translation of the abstract see Supplementary Materials section.

Drivers of Resistance in Uganda and Malawi (DRUM): a protocol for the evaluation of One-Health drivers of Extended Spectrum Beta Lactamase (ESBL) resistance in Low-Middle Income Countries (LMICs).

In sub-Saharan Africa (sSA), there is high morbidity and mortality from severe bacterial infection and this is compounded by antimicrobial resistance, in particular, resistance to 3rd-generation cephalosporins. This resistance is typically mediated by extended-spectrum beta lactamases (ESBLs). To interrupt ESBL transmission it will be important to investigate how human behaviour, water, sanitation, and hygiene (WASH) practices, environmental contamination, and antibiotic usage in both urban and rural settings interact to contribute to transmission of ESBL E. coli and ESBL K. pneumoniae between humans, animals, and the environment. Here we present the protocol for the Drivers of Resistance in Uganda and Malawi (DRUM) Consortium, in which we will collect demographic, geospatial, clinical, animal husbandry and WASH data from a total of 400 households in Uganda and Malawi. Longitudinal human, animal and environmental sampling at each household will be used to isolate ESBL E. coli and ESBL K. pneumoniae. This will be complimented by a Risks, Attitudes, Norms, Abilities and Self-Regulation (RANAS) survey and structured observations to understand the contextual and psychosocial drivers of regional WASH practices. Bacterial isolates and plate sweeps will be further characterised using a mixture of short-,long-read and metagenomic whole-genome sequencing. These datasets will be integrated into agent-based models to describe the transmission of EBSL resistance in Uganda and Malawi and allow us to inform the design of interventions for interrupting transmission of ESBL-bacteria.

Measuring transfer of human norovirus during sandwich production: Simulating the role of food, food handlers and the environment.

Foodborne outbreaks associated with transmission of norovirus are increasingly becoming a public health concern. Foods can be contaminated with faecal material at the point of production or during food preparation, in both the home and in commercial premises. Transmission of norovirus occurs through the faecal-oral route, either via person-to-person contact or through faecal-contamination of food, water, or environmental surfaces. Understanding the role and pathways of norovirus transmission - either via food handlers' hands, contaminated foods or the environment - remains a key public health priority to reduce the burden of norovirus-associated gastroenteritis. However the proportion of norovirus that is typically transferred remains unknown. Understanding this is necessary to estimate the risk of infection and the burden of gastroenteritis caused by norovirus. In this paper we present a novel method of capture, concentration and molecular detection of norovirus from a wider range of complex food matrices than those demonstrated in existing published methods. We demonstrate that this method can be used as a tool to detect and quantify norovirus from naturally contaminated food, and for monitoring norovirus transfer between food handlers' gloved hands, food or the environment. We measure the effect of introducing contamination at different food production process stages, to the final food product, to determine whether this could cause infection and disease. Between 5.9 and 6.3 Log10 cDNA copies/µl of norovirus GII were inoculated onto food handlers' gloved hands, food or the environment and 1.1-7.4% of norovirus contamination was recovered from all samples tested. When interpreted quantitatively, this percentage equates to levels predicted to be sufficient to cause infection and disease through consumption of the final food product, demonstrating a public health risk. Overall detection and quantification of norovirus from foods, food handlers' gloved hands and the environment, when suspected to be implicated in foodborne transmissions, is paramount for appropriate outbreak investigation.

Microbiological Quality of Cooked Chicken: Results of Monitoring in England (2013-17).

Results from monitoring of the microbiological quality of 2,721 samples of ready-to-eat cooked chicken collected between 2013 to 2017 in England were reviewed: 70% of samples were from retail, catering or manufacture and 30% were imported and collected at English ports. Samples were tested for a range of bacterial pathogens and indicator organisms. Six samples (<1%) had unsatisfactory levels of pathogens which were potentially injurious to health. Neither Salmonella nor Campylobacter were recovered from any sample. Two samples from catering settings contained either an unsatisfactory level of Bacillus cereus (5 x 10 6 CFU/g) or an unsatisfactory level of coagulase positive staphylococci (1.6 x 10 4 CFU/g). Listeria monocytogenes was recovered from 36 samples (one at manufacture, 26 at catering and nine at retail) and in four instances, unsatisfactory levels (≥10 2 CFU/g) were detected (three samples collected at catering and one at retail). For L. monocytogenes there were no significant differences between the rates of contamination with between the samples collected from ports, manufacture, retail supermarkets and other retailers (p = 0.288). There were no differences between the rates of contamination for other potential pathogens detected between samples from different settings. The prevalence of hygiene indicators ( Escherichia coli , Enterobacteriaceae and Aerobic Colony Counts) at import was significantly lower than in samples collected from manufacturers, retail or catering (p < 0.01). Samples collected from catering gave poorer results than all other settings. Regardless of the stage in the food chain, samples from Thailand and from other non-EU countries were of significantly better microbiological quality with respect to indicator organisms than those from the UK or from other EU countries (p = <0.001).

Occurrence of Listeria and Escherichia coli in frozen fruit and vegetables collected from retail and catering premises in England 2018-2019.

Frozen vegetables have previously been associated with outbreaks of listeriosis in both the USA and Europe. An outbreak of Listeria monocytogenes serogroup 4 caused 53 cases in five European countries between 2015 and 2018. Whole genome sequencing (WGS) indicated that frozen sweet corn from a producer in Hungary was the source of illness. However, limited data is available on the prevalence of Listeria in frozen produce. A study of frozen fruit and vegetables from catering and retail premises in England was therefore carried out to assess their microbiological quality with respect to Listeria and Escherichia coli. Between December 2018 and April 2019, 1050 frozen fruit and vegetable samples were collected. Of these, 99% were of a satisfactory or borderline microbiological quality. Eleven samples (1%) contained ≥100 cfu/g of Escherichia coli (considered unsatisfactory in products labelled as ready-to-eat). Listeria monocytogenes or other Listeria species were detected in six samples (2%) of fruit compared to 167 samples (24%) of vegetables and six samples (26%) of fruit and vegetable mixes, but none at a level of ≥100 cfu/g. Characterisation by WGS of 74 L. monocytogenes isolates identified ten genetic clusters indicating a common source. For 8 of the 10 clusters, the isolates came from homogenous food types: four were sweet corn, and there was one cluster each for beans, peas, peppers and broccoli. There were five genetic associations between isolates from frozen vegetables and from clinical cases of listeriosis, including two cultures from frozen beans that were indistinguishable from the 2015-2018 sweet corn outbreak strain. This study indicates that L. monocytogenes was present in 10% of frozen vegetables and even though products are generally not ready-to-eat and are intended to be cooked prior to consumption, these have the potential to cause illness. Clear cooking and handling instructions are therefore required on these products to ensure that the health of consumers is not put at risk, and appropriate Good Manufacturing Practice measures should be followed by all fruit and vegetable freezing plants in order to reduce contamination with Listeria during processing.

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.

Extended-spectrum ß-lactamase-producing Escherichia coli in human-derived and foodchain-derived samples from England, Wales, and Scotland: an epidemiological surveillance and typing study.

BACKGROUND: Extended-spectrum ß-lactamase-producing Escherichia coli isolates (ESBL-E coli) cause more than 5000 cases of bacteraemias annually in the UK. The contribution of the food chain to these infections is debated. We aimed to identify the most important reservoirs of ESBL-E coli that colonise and infect humans to identify strategic intervention points. METHODS: Sampling for ESBL-E coli was done between Aug 1, 2013, and Dec 15, 2014. We used selective media to seek ESBL-E coli in routinely submitted samples from human faeces, and prospectively collected samples from sewage, farm slurry, and retail foodstuffs in London, East Anglia, northwest England, Scotland, and Wales. We sequenced recovered isolates and compared these isolates with 293 bloodstream and 83 veterinary surveillance ESBL-E coli isolates from the same regions. FINDINGS: 2157 (11%) of 20 243 human faeces samples contained ESBL-E coli, including 678 (17%) of 3995 in London. ESBL-E coli also were frequent in sewage and retail chicken (104 [65%] of 159 meat samples), but were rare in other meats and absent from plant-based foods (0 of 400 fruit and vegetable samples). Sequence type (ST) 131 dominated among ESBL-E coli from human blood (188 [64%] of 293 isolates), faeces (128 [36%] of 360), and sewage (14 [22%] of 65) with STs 38 and 648 also widespread; CTX-M-15 was the predominant ESBL in these lineages (319 [77%] of 416). By contrast, STs 602, 23, and 117-mostly with CTX-M-1 ESBL-dominated among food and veterinary isolates (68 [31%] of 218), with only two ST131 organisms recovered. ST10 occurred in both animals and humans, being frequent in surveillance bovines (11 [22%] of 51 cattle) and representing 15 (4%) of 360 human faecal isolates (but only three [1%] of 293 from bacteraemias); however, both human and animal ST10 isolates were diverse in serotype. INTERPRETATION: Most human bacteraemias with ESBL-E coli in the UK involve internationally prevalent human-associated STs, particularly ST131; non-human reservoirs made little contribution to invasive human disease. Any interventions that seek to target food or livestock can affect the numbers of human infections caused by ESBL-E coli; prevention of the spread of resistant lineages among humans is more vital. FUNDING: NIHR Policy Research.

Utility of Whole Genome Sequencing To Describe the Persistence and Evolution of Listeria monocytogenes Strains within Crabmeat Processing Environments Linked to Two Outbreaks of Listeriosis.

This article describes the identification and investigation of two extended outbreaks of listeriosis in which crabmeat was identified as the vehicle of infection. Comparing contemporary and retrospective typing data of Listeria monocytogenes isolates from clinical cases and from food and food processing environments allowed the detection of cases going back several years. This information, combined with the analysis of routinely collected enhanced surveillance data, helped to direct the investigation and identify the vehicle of infection. Retrospective whole genome sequencing (WGS) analysis of isolates provided robust microbiological evidence of links between cases, foods, and the environments in which they were produced and demonstrated that for some cases and foods, identified by fluorescent amplified fragment length polymorphism, the molecular typing method in routine use at the time, were not part of the outbreak. WGS analysis also showed that the strains causing illness had persisted in two food production environments for many years and in one producer had evolved into two strains over a period of around 8 years. This article demonstrates the value of reviewing L. monocytogenes typing data from clinical cases together with that from foods as a means of identifying potential vehicles and sources of infection in outbreaks of listeriosis. It illustrates the importance of reviewing retrospective L. monocytogenes typing alongside enhanced surveillance data to characterize extended outbreaks and inform control measures. Also, this article highlights the advantages of WGS analysis for strain discrimination and clarification of evolutionary relationships that refine outbreak investigations and improve our understanding of L. monocytogenes in the food chain.

Assessment of the Microbiological Quality of Meat Pies from Retail Sale in England 2013.

Outbreaks of foodborne illness caused by Bacillus cereus and Listeria monocytogenes in England associated with meat pie consumption were detected in 2012. To obtain baseline data for pies unrelated to outbreaks, 862 samples of ready-to-eat meat pies were collected at retail or from catering facilities in England in 2013 and examined to enumerate food-poisoning bacteria and indicator organisms using Organization for Standardization (ISO) methods for Listeria spp. including L. monocytogenes (ISO 11290), Clostridium perfringens (ISO 21528), coagulase-positive staphylococci including Staphylococcus aureus (ISO 6888), Bacillus spp. including B. cereus (ISO 1737), Escherichia coli (ISO 16649), Enterobacteriaceae (ISO 21528), and aerobic colony counts (ACCs; ISO 4833). Microbiological quality was satisfactory in 94% of samples, borderline in 5%, and unsatisfactory in 1%. The proportion of pies from markets that were borderline or unsatisfactory significantly increased, and the proportion of borderline or unsatisfactory pies from supermarkets significantly decreased. Among the refrigerated (0 to 15°C) pies, microbiological quality significantly decreased in pies stored at >8°C and further significantly decreased at in pies stored at ambient temperature (>15 to 25°C). Samples collected at 25 to 40°C had the highest proportion of borderline or unsatisfactory results, but results improved in pies stored at >40°C. The most common cause for borderline or unsatisfactory results was elevated ACCs (5% of all samples). Within the individual microbiological parameters, borderline or unsatisfactory results resulted from elevated Enterobacteriaceae or Bacillus levels (10 samples for each), C. perfringens levels (2 samples), and S. aureus or E. coli levels (1 sample each). L. monocytogenes was recovered from one pie at <10 CFU/g. A literature review revealed a range of microbiological hazards responsible for food poisoning and meat pie consumption, and surveillance data from 1992 to 2012 from England indicated that C. perfringens was the most commonly reported cause of outbreaks of foodborne illness.

Assessment of the Microbiological Safety of Precut Fruit from Retail and Catering Premises in the United Kingdom.

Fresh fruit has been associated with a number of foodborne outbreaks in recent years. In particular, a large outbreak of listeriosis in the United States in 2011 was associated with consumption of cantaloupe melon, and an outbreak of Salmonella Newport in the United Kingdom and Europe (also in 2011) was linked to watermelon consumption. A study of precut fruit products from catering and retail premises in the United Kingdom was, therefore, carried out to assess their microbiological safety. Between January and March 2012, samples (1,188) of ready-to-eat precut fruit were collected from retail and catering premises in the United Kingdom, and 99% were of satisfactory microbiological quality. However, four samples (0.3%) were of an unsatisfactory quality (one with 800 CFU/g Listeria monocytogenes and three with >100 CFU/g Escherichia coli), and five samples (0.4%) were of a borderline quality owing to the presence of E. coli (two samples with a level of 20 CFU/g), Staphylococcus aureus (two samples with levels of >50 CFU/g), or L. monocytogenes (one sample with a level of 80 CFU/g). L. monocytogenes or other Listeria species were detected in a further 54 samples (4.5%) at levels below the threshold considered to be borderline or unsatisfactory. A significantly larger proportion of samples from one national supermarket chain was contaminated with L. monocytogenes than other supermarkets, and two types were, in this study, unique to this supermarket. This study shows that overall, the microbiological quality of ready-to-eat precut fruit was good. However, the presence of Listeria species in 5% of samples highlights the need for good hygiene during preparation and satisfactory temperature and time control during storage of these food products.

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.

Interactions of the gastrotropic bacterium Helicobacter pylori with the leukocyte-endothelium adhesion molecules, the selectins--a preliminary report.

The deleterious effects of Helicobacter pylori infection of the stomach are largely the result of a vigorous chronic inflammatory response, and include chronic gastritis, peptic ulceration and gastric cancer. We are exploring the possibility that carbohydrate components on H. pylori contribute to the persistent inflammation through interactions with leukocyte-endothelial adhesion molecules of the host. Lipopolysaccharides of most H. pylori strains contain sequences related to the Lewis (Le(x) or Le(a)) antigens. Carbohydrate sequences of this family encompass ligands for the leukocyte-endothelium adhesion molecules of the host, namely, the E- and P-selectins, which are expressed on inflamed endothelia, and L-selectin, which is constitutively expressed on leukocytes. Here we investigate H. pylori isolates from patients with chronic gastritis, duodenal ulcer and gastric cancer for their interactions with the selectins. Our results provide unequivocal evidence of interactions of isolates from each of the diagnostic groups with E- and L-selectins.

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.

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.