Identifying the seasonal origins of human campylobacteriosis.

Human campylobacteriosis exhibits a distinctive seasonality in temperate regions. This paper aims to identify the origins of this seasonality. Clinical isolates [typed by multi-locus sequence typing (MLST)] and epidemiological data were collected from Scotland. Young rural children were found to have an increased burden of disease in the late spring due to strains of non-chicken origin (e.g. ruminant and wild bird strains from environmental sources). In contrast the adult population had an extended summer peak associated with chicken strains. Travel abroad and UK mainland travel were associated with up to 17% and 18% of cases, respectively. International strains were associated with chicken, had a higher diversity than indigenous strains and a different spectrum of MLST types representative of these countries. Integrating empirical epidemiology and molecular subtyping can successfully elucidate the seasonal components of human campylobacteriosis. The findings will enable public health officials to focus strategies to reduce the disease burden.

Combining risk assessment and epidemiological risk factors to elucidate the sources of human E. coli O157 infection.

E. coli O157 can be transmitted to humans by three primary (foodborne, environmental, waterborne) and one secondary (person-to-person transmission) pathways. A regression model and quantitative microbiological risk assessments (QMRAs) were applied to determine the relative importance of the primary transmission pathways in NE Scotland. Both approaches indicated that waterborne infection was the least important but it was unclear whether food or the environment was the main source of infection. The QMRAs over-predicted the number of cases by a factor of 30 and this could be because all E. coli O157 strains may not be equally infective and/or the level of infectivity in the dose-response model was too high. The efficacy of potential risk mitigation strategies to reduce human exposure to E. coli O157 using QMRAs was simulated. Risk mitigation strategies focusing on food and environment are likely to have the biggest impact on infection figures.

Putative household outbreaks of campylobacteriosis typically comprise single MLST genotypes.

During a 15-month period in Scotland a small but important number of human Campylobacter cases (3·2%) arose from 91 putative household outbreaks. Of the 26 outbreaks with known strain composition, 89% were composed of the same MLST which supports the potential use of MLST in public health epidemiology. The number of cases associated with household outbreaks is much larger than general outbreaks and there is some evidence to indicate that there may be secondary transmission, although this is relatively rare.

Multi-locus sequence types of Campylobacter carried by flies and slugs acquired from local ruminant faeces.

AIMS: To assess whether flies and slugs acquire strains of Campylobacter jejuni and Campylobacter coli present in local ruminant faeces. METHODS AND RESULTS: Campylobacter was cultured from flies, slugs and ruminant faeces that were collected from a single farm in Scotland over a 19-week period. The isolates were typed using multi-locus sequence typing (MLST) and compared with isolates from cattle and sheep faeces. Campylobacter jejuni and Camp. coli were isolated from 5·8% (n=155, average of 75 flies per pool) and 13·3% (n=15, average of 8·5 slugs per pool) of pooled fly and slug samples, respectively. The most common sequence type (ST) in flies was Camp. coli ST-962 (approx. 40%) regardless of the prevalence in local cattle (2·3%) or sheep (25·0%) faeces. Two positive slug pools generated the same ST that has not been reported elsewhere. CONCLUSIONS: Despite their low carriage rate, flies are able to acquire Campylobacter STs that are locally present, although the subset carried may be biased when compared to local source. Slugs were shown to carry a previously unreported Campylobacter ST. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has demonstrated that flies carry viable Campylobacter and may contribute to the transfer of STs within and between groups of animals on farms. Further, they may therefore present a risk to human health via their contact with ready-to-eat foods or surfaces.

Campylobacter immunity and coinfection following a large outbreak in a farming community.

An outbreak of campylobacteriosis affected approximately one-half of 165 people attending an annual farmers' dance in Montrose, Scotland, in November 2005. Epidemiological investigations, including a cohort study (n = 164), identified chicken liver paté as the most likely vehicle of infection. Paté preparation involved deliberate undercooking of chicken livers by flash-frying, followed by mechanical homogenization. Typing of 32 Campylobacter strains (isolated from submitted stools) by multilocus sequence typing identified four distinct clades of Campylobacter jejuni. There was good agreement when isolates were typed by Penner serotyping, pulsed-field gel electrophoresis, and flaA short variable region sequencing but poorer agreement with phage and antibiotic susceptibility testing. At least three attendees were coinfected with two Campylobacter strains each. The outbreak was probably due to several livers contributing Campylobacter strains that survived undercooking and were dispersed throughout the paté. The study highlights improper culinary procedures as a potential human health risk and provides a striking counterexample to the "dominant outbreak strain" view of point source outbreaks of food-borne infections. It also demonstrates that previous exposure to biologically plausible sources of Campylobacter may confer protection against subsequent infection.

Diversity of Escherichia coli O157 in a longitudinal farm study using multiple-locus variable-number tandem-repeat analysis.

AIMS: To perform a longitudinal study of the diversity of Escherichia coli O157 from a ruminant pasture/stream environment using multiple-locus variable-number tandem-repeat analysis (MLVA). METHODS AND RESULTS: Samples of faecal droppings from grazing ruminants and from an adjacent stream were tested longitudinally for E. coli O157 by enrichment and immunomagnetic separation (IMS). Using MLVA, 24 different profiles were identified from a total of 231 E. coli O157 isolates, of which 80 were included in a similarity analysis. Four main clusters with several subclusters were observed. Although there was close contact between sheep and cattle during the study period, E. coli O157 was surprisingly not detected from cattle faeces. CONCLUSIONS: The cluster analysis indicated both unrelated and closely related E. coli O157 strains. The choice of loci to target in MLVA is important for the subtyping result, as loci with high diversities are essential for discriminating between closely related isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: There is a lack of data available on the use of MLVA to describe E. coli O157 diversity and changes over time in the animal reservoirs and the environment. Such data are needed in order to further develop MLVA as a typing method.

A sensitive microsphere coagulation ELISA for Escherichia coli O157:H7 using Russell's viper venom.

A microsphere coagulation enzyme-linked immunosorbent assay (MC-ELISA) using Russell's viper venom factor X activator (RVV-XA) is described for the detection of Escherichia coli O157:H7. This microtitre plate assay comprises a standard sandwich immunoassay incorporating RVV-XA as the enzyme label. Coagulation substrate together with polystyrene microspheres are added to the wells of the microtitre plate. RVV-XA initiates the coagulation cascade causing formation of an artificial clot of polystyrene microspheres bound together with fibrin. As few as 10(2) E. coli O157 in a well (10(3) per ml) can be detected within 3 h. The assay is two orders of magnitude more sensitive than a standard ELISA and is a generic technique with the potential for widespread use in sandwich immunoassays.

Modelling the vector pathway and infection of humans in an environmental outbreak of Escherichia coli O157.

Quantifying the transfer of Escherichia coli O157 from the environment to humans is essential for understanding outbreaks, establishing the infectious dose of the organism and proposing safeguards. We modelled the pathogen loading shed onto a field by sheep immediately prior to a scout camp where 18 scouts and two adults were infected with E. coli O157. We estimated the dose ingested (4-24 organisms) which is in agreement with the low infective dose reported previously for this organism in food outbreaks. These data closely fit a surrogate Shigella dose-response model which can be used as a basis for risk assessment.

A conductance medium to distinguish between Salmonella and Citrobacter spp.

Published methods for detecting Salmonella by conductance methods yield some Citrobacter spp. as false positives. A medium is described which, when used in conjunction with selenite-cystine/trimethylamine oxide/mannitol (SC/T/M) resolves this difficulty. It is based on the ability of Salmonella to decarboxylate lysine in the presence of selenite (4 g/l), after pre-enrichment in buffered peptone water supplemented with glucose and lysine for 7-17 h. Salmonella produced a conductance change of 250-400 microS in 24 h with a maximum rate of conductance change of 10-15 microS/10 min. 299 samples were tested and the only false positives were due to Hafnia alvei which gave no conductance response in the SC/T/M medium. Using both media in a two tube system would lead to a rapid clearance of Salmonella negative samples.

Modelling the growth, survival and death of microorganisms in foods: the UK food micromodel approach.

Techniques for the development of mathematical models in the area of predictive microbiology have greatly improved recently, allowing better and more accurate descriptions of microbial responses to particular environmental conditions, thus enabling predictions of those responses to be made with greater confidence. Recognising the potential value of applying these techniques in the food industry, the Ministry of Agriculture, Fisheries and Food (MAFF) initiated a nationally coordinated five-year programme of research into the growth and survival of microorganisms in foods, with the aim of developing a computerised Predictive Microbiology Database in the UK. This initiative has resulted in the systematic generation of data, through protocols which ensure consistency of methodology, so that data in the database are truly comparable and compatible, and lead to reliable predictive models. The approaches taken by scientists involved in this programme are described and the various stages in the development of mathematical models summarized. It is hoped that this initiative and others being developed in the USA, Australia, Canada and other countries, will encourage a more integrated approach to food safety which will influence all stages of food production and, eventually, result in the development of an International Predictive Microbiology Database.

An interlaboratory study to find an alternative to the MPN technique for enumerating Escherichia coli in shellfish.

Nine laboratories in eight countries tested 16 batches of common mussels (Mytilus edulis) over a 32 week period in order to find an alternative to the Most Probable Number (MPN) technique to enumerate E. coli. The alternatives investigated included the 3M Petrifilm system, the Merck Chromocult agar method and a Malthus conductance technique. The Petrifilm was found to be unsuitable and was subsequently dropped from the trial. After 669 analyses, a correlation of 0.83 was observed for log E. coli counts between the MPN and Chromocult methods and there was no significant evidence that either method tended to give higher readings than the other. The MPN was slightly better than the Chromocult method for repeatability but the Chromocult was slightly better for reproducibility. However, the observed differences are probably too small to be of practical importance. On the basis of these data therefore, the two methods appear equally suitable for E. coli enumeration in shellfish. There were poor correlations between these methods and the Malthus technique. A small but significant number of samples tested positive on the Malthus instrument but were recorded negative on the MPN and Chromocult tests. Subsequent analysis positively identified E. coli from these Malthus assays. After statistical analysis, errors were noted in both the MPN and Chromocult methods but it was found that there would be no statistical differences if the Chromocult agar were used as an alternative to the MPN technique.

The synergistic effect of excimer and low-pressure mercury lamps on the disinfection of flowing water.

Microorganisms in flowing water were disinfected by UV radiation from two excimer (excited dimer) lamps (emitting at 172 and 222 nm) in combination with two low-pressure mercury lamps (emitting at 254 nm). Synergies were investigated among the three types of radiation in the treatment of water spiked in turn with Escherichia coli, Listeria innocua, Shewanella putrefaciens, and spores of Bacillus subtilis and Bacillus cereus. Synergy was demonstrated between radiations at 222 and 254 nm in the treatment of E. coli, L. innocua, and S. putrefaciens, but little or no synergy was observed in the treatment of B. subtilis and B. cereus. At maximum flow rates (60 liters/min), 5-log reductions in E. coli were achieved at 254 nm, although at 222 nm, less than 1-log reductions were observed. No bacterial kill was observed with 172-nm radiation alone, despite increasing exposure time by reducing flow rates to less than 3 liters/min.

A comparison of three identification kits for the confirmation of Aeromonas spp.

A comparative study was made of the ability of three commercial identification kits to confirm the identity of motile aeromonads isolated from foods. The kits included the API 20E, API 20NE and Microbact 24E. The results showed that both the API 20NE and Microbact 24E correctly identified 97.5% of isolates but the API 20E only 72.5%.

Source attribution, prevalence and enumeration of Campylobacter spp. from retail liver.

Campylobacter prevalence from retail liver (chicken, cattle, pig and sheep) was found to be 81%, 69%, 79% and 78% respectively. Molecular source attribution demonstrated that strains from chicken liver were most similar to those found commonly in humans. This provides further evidence of liver being a probable source of human infection.

Hierarchical dose response of E. coli O157:H7 from human outbreaks incorporating heterogeneity in exposure.

The infectivity of pathogenic microorganisms is a key factor in the transmission of an infectious disease in a susceptible population. Microbial infectivity is generally estimated from dose-response studies in human volunteers. This can only be done with mildly pathogenic organisms. Here a hierarchical Beta-Poisson dose-response model is developed utilizing data from human outbreaks. On the lowest level each outbreak is modelled separately and these are then combined at a second level to produce a group dose-response relation. The distribution of foodborne pathogens often shows strong heterogeneity and this is incorporated by introducing an additional parameter to the dose-response model, accounting for the degree of overdispersion relative to Poisson distribution. It was found that heterogeneity considerably influences the shape of the dose-response relationship and increases uncertainty in predicted risk. This uncertainty is greater than previously reported surrogate and outbreak models using a single level of analysis. Monte Carlo parameter samples (alpha, beta of the Beta-Poisson model) can be readily incorporated in risk assessment models built using tools such as S-plus and @ Risk.

Sexual dimorphism in campylobacteriosis.

Sexual dimorphism in infectious diseases whereby disease incidence is more prevalent in one gender has been reported repeatedly in the scientific literature. Both behavioural and physiological differences have been suggested as a cause of this gender bias but there is a paucity of data to support either of these viewpoints. Here it is hypothesized that for campylobacteriosis physiological factors play an important role in the higher incidence in males. We demonstrate in the human population (from several countries in three continents) that this bias exists in young children (<1 year) where behavioural differences between genders are likely to be minimal. Further we demonstrate this difference in an animal model where both infection rates and shedding rates of the organism are greater in male mice.

Use of multilocus sequence typing to investigate the association between the presence of Campylobacter spp. in broiler drinking water and Campylobacter colonization in broilers.

The presence of campylobacters in broiler chickens and throughout the broiler water delivery systems of 12 farms in northeastern Scotland was investigated by sensitive enrichment methods and large-volume filtration. Campylobacter presence was independent of the water source and whether the water was treated. The genotypes of Campylobacter jejuni isolates recovered from chickens and various locations within the water delivery systems were compared by multilocus sequence typing. Matching strains in shed header tanks and birds were found at 1 of the 12 farms investigated. However, the sequence of contamination or whether the source was within or outside the shed was not determined. Nevertheless, these data provide evidence that drinking water could be associated with broiler infection by campylobacters.

Concentration and prevalence of Escherichia coli O157 in sheep faeces at pasture in Scotland.

AIMS: To study the presence, numbers and virulence profiles of Escherichia coli O157 in sheep faeces and validate the microbiological methods used to attain these data. METHODS AND RESULTS: Flock level prevalence was found to be 40% (six from 15) and 6.5% of faecal samples tested were found to be positive. Two farms gave samples defined as high shedding (>10(4) CFU g(-1)), one of which comprised 91% positive samples with 13/33 at the high shedding level. CONCLUSIONS: These data confirmed that sheep are an important reservoir of E. coli O157. SIGNIFICANCE AND IMPACT OF THE STUDY: Sheep play a significant role in the maintenance and dispersal of E. coli O157 in the farming environment and are an important source of human infection.

Use of conductance methods to predict bacterial counts in fish.

Conductance methods to measure bacterial growth are more rapid than conventional methods for assessing the load of spoilage bacteria in fish. With the correct choice of medium, an estimate of the count can be obtained within 24 h which shows a very good correlation with the conventional methods. Moreover, the conductance changes correlate better with counts of those organisms thought to be responsible for spoilage. The Malthus conductance instrument provides an automated system capable of the simultaneous monitoring of 128 different samples, resulting in considerable savings of time and effort over traditional plate counting techniques.