To assess whether flies and slugs acquire strains of Campylobacter jejuni and Campylobacter coli present in local ruminant faeces.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. 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.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.
No single genealogical reconstruction or typing method currently encompasses all levels of bacterial diversity, from domain to strain. We propose ribosomal multilocus sequence typing (rMLST), an approach which indexes variation of the 53 genes encoding the bacterial ribosome protein subunits (rps genes), as a means of integrating microbial genealogy and typing. As with multilocus sequence typing (MLST), rMLST employs curated reference sequences to identify gene variants efficiently and rapidly. The rps loci are ideal targets for a universal characterization scheme as they are: (i) present in all bacteria; (ii) distributed around the chromosome; and (iii) encode proteins which are under stabilizing selection for functional conservation. Collectively, the rps loci exhibit variation that resolves bacteria into groups at all taxonomic and most typing levels, providing significantly more resolution than 16S small subunit rRNA gene phylogenies. A web-accessible expandable database, comprising whole-genome data from more than 1900 bacterial isolates, including 28 draft genomes assembled de novo from the European Bioinformatics Institute (EBI) sequence read archive, has been assembled. The rps gene variation catalogued in this database permits rapid and computationally non-intensive identification of the phylogenetic position of any bacterial sequence at the domain, phylum, class, order, family, genus, species and strain levels. The groupings generated with rMLST data are consistent with current nomenclature schemes and independent of the clustering algorithm used. This approach is applicable to the other domains of life, potentially providing a rational and universal approach to the classification of life that is based on one of its fundamental features, the translation mechanism.
ABSTRACT Contaminated chicken meat is a major source of human Campylobacteriosis and rates of infection remain high, despite efforts to limit the colonisation of broiler (meat) chicken flocks on farms. Using conventional testing methods of culture or qPCR, Campylobacter is typically detected amongst broiler flocks from 3 weeks of age, leading to the assumption that infection is introduced horizontally into chicken rearing houses at this time. In this study, we use parallel sequencing of a fragment of the Campylobacter outer membrane protein, encoded by the porA gene, to test for presence of Campylobacter DNA amongst fresh faecal samples collected from broiler flocks aged 23-28 days. Campylobacter DNA was detected in all of the 290 samples tested using the porA target, and in 48% of samples using 16S bacterial profiling, irrespective of whether or not Campylobacter could be detected using conventional qPCR thresholds. A single porAf2 variant was predominant amongst flocks that would be determined to be Campylobacter ‘ positive’ by conventional means, but a diverse pattern was seen amongst flocks that were Campylobacter ‘negative’. The ability to routinely detect low levels of Campylobacter amongst broiler flocks at a much earlier age than would conventionally be identified requires a re-examination of how and when biosecurity measures are best applied for live birds. In addition, it may be useful to investigate why single Campylobacter variants proliferate in some broiler flocks and not others.
Abstract Robust microbial classification systems are essential, but their definition is complicated by the large size and high diversity of microbial populations combined with a widespread horizontal genetic exchange. Multi-locus approaches that index gene variation without explicit phylogenetic classification mitigates these problems, but reproducibly defining high-level groups remains problematic. We describe a generalisable machine learning approach, 'NeighbourGroups', that reproducibly, robustly, and rapidly classifies multi-locus sequence types with defined precision.
Genetic attribution of bacterial genotypes has become a major tool in the investigation of the epidemiology of campylobacteriosis and has implicated retail chicken meat as the major source of human infection in several countries. To investigate the robustness of this approach to the provenance of the reference data sets used, a collection of 742 Campylobacter jejuni and 261 Campylobacter coli isolates obtained from United Kingdom-sourced chicken meat was established and typed by multilocus sequence typing. Comparative analyses of the data with those from other isolates sourced from a variety of host animals and countries were undertaken by genetic attribution, genealogical, and population genetic approaches. The genotypes from the United Kingdom data set were highly diverse, yet structured into sequence types, clonal complexes, and genealogical groups very similar to those seen in chicken isolates from the Netherlands, the United States, and Senegal, but more distinct from isolates obtained from ruminant, swine, and wild bird sources. Assignment analyses consistently grouped isolates from different host animal sources regardless of geographical source; these associations were more robust than geographic associations across isolates from three continents. We conclude that, notwithstanding the high diversity of these pathogens, there is a strong signal of association of multilocus genotypes with particular hosts, which is greater than the geographic signal. These findings are consistent with local and international transmission of host-associated lineages among food animal species and provide a foundation for further improvements in genetic attribution.
Summary Campylobacter jejuni is the most common cause of bacterial gastroenteritis worldwide, with contaminated chicken meat considered to represent a major source of human infection. Biosecurity measures can reduce C. jejuni shedding rates of housed chickens, but the increasing popularity of free‐range and organic meat raises the question of whether the welfare benefits of extensive production are compatible with food safety. The widespread assumption that the free‐range environment contaminates extensively reared chickens has not been rigorously tested. A year‐long survey of 64 free‐range broiler flocks reared on two sites in Oxfordshire, UK, combining high‐resolution genotyping with behavioural and environmental observations revealed: (i) no evidence of colonization of succeeding flocks by the C. jejuni genotypes shed by preceding flocks, (ii) a high degree of similarity between C. jejuni genotypes from both farm sites, (iii) no association of ranging behaviour with likelihood of Campylobacter shedding, and (iv) higher genetic differentiation between C. jejuni populations from chickens and wild birds on the same farm than between the chicken samples, human disease isolates from the same region and national samples of C. jejuni from chicken meat.
Abstract Commercial poultry flocks frequently harbor the dangerous bacterial pathogen Campylobacter . As exclusion efforts frequently fail, there is interest in potential ecologically informed solutions. A long‐term study of Campylobacter sequence types was used to investigate the competitive framework of the Campylobacter metacommunity and understand how multiple sequence types simultaneously co‐occur in a flock of chickens. A combination of matrix and patch‐occupancy models was used to estimate parameters describing the competition, transmission, and mortality of each sequence type. It was found that Campylobacter sequence types form a strong hierarchical framework within a flock of chickens and occupied a broad spectrum of transmission–mortality trade‐offs. Upon further investigation of how biodiversity is thus maintained within the flock, it was found that the demographic capabilities of Campylobacter , such as mortality and transmission, could not explain the broad biodiversity of sequence types seen, suggesting that external factors such as host‐bird health and seasonality are important elements in maintaining biodiversity of Campylobacter sequence types.
Abstract Campylobacter from contaminated poultry meat is a major source of human gastroenteritis worldwide. To date, attempts to control this zoonotic infection with on-farm biosecurity measures have been inconsistent. A cornerstone of these efforts has been the detection of chicken infection with microbiological culture, which typically does not occur until birds are at least 21 days old. Using molecular methods for detecting Campylobacter presence, 16S microbiome analysis and deep sequencing of the Campylobacter por A gene, Campylobacter can be identified at very low levels in most or all flocks fewer than 8 days old. These young chicks exhibit a much greater diversity of porA types than older birds testing positive for Campylobacter by culture or qPCR. This suggests that, as the bacteria multiply sufficiently to be detected by culture methods, one or two strains, as indicated by porA type, dominate the infection. The findings that (i) most young chicks carry some Campylobacter and (ii) not all flocks become Campylobacter positive by culture, suggests that efforts to control infection should concentrate on how to maintain Campylobacter at low levels by the prevention of the overgrowth of single strains, which ultimately leads to the contamination of food.
ABSTRACT Using data on rearing and welfare metrics of multiple commercial broiler flocks from the last ten years, we investigate how welfare measures such as hock burn, mortality, weight, and pododermatitis, among others, impact the likelihood of a flock becoming colonized by Campylobacter . Using both logistic regression and Bayesian networks, we show that, while some welfare metrics were weakly related to Campylobacter colonization, evidence could not be found to suggest that these metrics actively exacerbated Campylobacter colonization, rather that they were both symptoms of the same underlying cause. Instead, observed dependency on the management of the flock suggested that yet-undiscovered differences in rearing practise were the principal cause of both poor bird welfare and increased risk of Campylobacter , suggesting that action can be taken to improve both these factors simultaneously.
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