Genome-wide association of functional traits linked with Campylobacter jejuni survival from farm to fork.

Campylobacter jejuni is a major cause of bacterial gastroenteritis worldwide, primarily associated with the consumption of contaminated poultry. C. jejuni lineages vary in host range and prevalence in human infection, suggesting differences in survival throughout the poultry processing chain. From 7343 MLST-characterised isolates, we sequenced 600 C. jejuni and C. coli isolates from various stages of poultry processing and clinical cases. A genome-wide association study (GWAS) in C. jejuni ST-21 and ST-45 complexes identified genetic elements over-represented in clinical isolates that increased in frequency throughout the poultry processing chain. Disease-associated SNPs were distinct in these complexes, sometimes organised in haplotype blocks. The function of genes containing associated elements was investigated, demonstrating roles for cj1377c in formate metabolism, nuoK in aerobic survival and oxidative respiration, and cj1368-70 in nucleotide salvage. This work demonstrates the utility of GWAS for investigating transmission in natural zoonotic pathogen populations and provides evidence that major C. jejuni lineages have distinct genotypes associated with survival, within the host specific niche, from farm to fork.

Monitoring chicken flock behaviour provides early warning of infection by human pathogen Campylobacter.

Campylobacter is the commonest bacterial cause of gastrointestinal infection in humans, and chicken meat is the major source of infection throughout the world. Strict and expensive on-farm biosecurity measures have been largely unsuccessful in controlling infection and are hampered by the time needed to analyse faecal samples, with the result that Campylobacter status is often known only after a flock has been processed. Our data demonstrate an alternative approach that monitors the behaviour of live chickens with cameras and analyses the 'optical flow' patterns made by flock movements. Campylobacter-free chicken flocks have higher mean and lower kurtosis of optical flow than those testing positive for Campylobacter by microbiological methods. We show that by monitoring behaviour in this way, flocks likely to become positive can be identified within the first 7-10 days of life, much earlier than conventional on-farm microbiological methods. This early warning has the potential to lead to a more targeted approach to Campylobacter control and also provides new insights into possible sources of infection that could transform the control of this globally important food-borne pathogen.

The long-term dynamics of Campylobacter colonizing a free-range broiler breeder flock: an observational study.

A free-range broiler breeder flock was studied in order to determine the natural patterns of Campylobacter colonization over a period of 63 weeks. Campylobacter sequence types (STs) were not mutually exclusive and on average colonized only 17.7% of the birds tested at any time. Campylobacter STs typically reached a peak in prevalence upon initial detection in the flock before tailing off, although the ST and antigenic flaA short variable region in combination were stable over a number of months. There was evidence that, with a couple of exceptions, the ecology of C. jejuni and C. coli differed, with the latter forming a more stable population. Despite being free range, no newly colonizing STs were detected over a 6-week period in autumn and a 10-week period in winter, towards the end of the study. There was limited evidence that those STs identified among broiler chicken flocks on the same farm site were likely to colonize the breeder flock earlier (R(2) 0.16, P 0.01). These results suggest that there is natural control of Campylobacter dynamics within a flock which could potentially be exploited in designing new intervention strategies, and that the two different species should perhaps be considered separately.

Cryptic ecology among host generalist Campylobacter jejuni in domestic animals.

Homologous recombination between bacterial strains is theoretically capable of preventing the separation of daughter clusters, and producing cohesive clouds of genotypes in sequence space. However, numerous barriers to recombination are known. Barriers may be essential such as adaptive incompatibility, or ecological, which is associated with the opportunities for recombination in the natural habitat. Campylobacter jejuni is a gut colonizer of numerous animal species and a major human enteric pathogen. We demonstrate that the two major generalist lineages of C. jejuni do not show evidence of recombination with each other in nature, despite having a high degree of host niche overlap and recombining extensively with specialist lineages. However, transformation experiments show that the generalist lineages readily recombine with one another in vitro. This suggests ecological rather than essential barriers to recombination, caused by a cryptic niche structure within the hosts.

Evidence for phenotypic plasticity among multihost Campylobacter jejuni and C. coli lineages, obtained using ribosomal multilocus sequence typing and Raman spectroscopy.

Closely related bacterial isolates can display divergent phenotypes. This can limit the usefulness of phylogenetic studies for understanding bacterial ecology and evolution. Here, we compare phenotyping based on Raman spectrometric analysis of cellular composition to phylogenetic classification by ribosomal multilocus sequence typing (rMLST) in 108 isolates of the zoonotic pathogens Campylobacter jejuni and C. coli. Automatic relevance determination (ARD) was used to identify informative peaks in the Raman spectra that could be used to distinguish strains in taxonomic and host source groups (species, clade, clonal complex, and isolate source/host). Phenotypic characterization based on Raman spectra showed a degree of agreement with genotypic classification using rMLST, with segregation accuracy between species (83.95%), clade (in C. coli, 98.41%), and, to some extent, clonal complex (86.89% C. jejuni ST-21 and ST-45 complexes) being achieved. This confirmed the utility of Raman spectroscopy for lineage classification and the correlation between genotypic and phenotypic classification. In parallel analysis, relatively distantly related isolates (different clonal complexes) were assigned the correct host origin irrespective of the clonal origin (74.07 to 96.97% accuracy) based upon different Raman peaks. This suggests that the phenotypic characteristics, from which the phenotypic signal is derived, are not fixed by clonal descent but are influenced by the host environment and change as strains move between hosts.

Marked host specificity and lack of phylogeographic population structure of Campylobacter jejuni in wild birds.

Zoonotic pathogens often infect several animal species, and gene flow among populations infecting different host species may affect the biological traits of the pathogen including host specificity, transmissibility and virulence. The bacterium Campylobacter jejuni is a widespread zoonotic multihost pathogen, which frequently causes gastroenteritis in humans. Poultry products are important transmission vehicles to humans, but the bacterium is common in other domestic and wild animals, particularly birds, which are a potential infection source. Population genetic studies of C. jejuni have mainly investigated isolates from humans and domestic animals, so to assess C. jejuni population structure more broadly and investigate host adaptation, 928 wild bird isolates from Europe and Australia were genotyped by multilocus sequencing and compared to the genotypes recovered from 1366 domestic animal and human isolates. Campylobacter jejuni populations from different wild bird species were distinct from each other and from those from domestic animals and humans, and the host species of wild bird was the major determinant of C. jejuni genotype, while geographic origin was of little importance. By comparison, C. jejuni differentiation was restricted between more phylogenetically diverse farm animals, indicating that domesticated animals may represent a novel niche for C. jejuni and thereby driving the evolution of those bacteria as they exploit this niche. Human disease is dominated by isolates from this novel domesticated animal niche.

Progressive genome-wide introgression in agricultural Campylobacter coli.

Hybridization between distantly related organisms can facilitate rapid adaptation to novel environments, but is potentially constrained by epistatic fitness interactions among cell components. The zoonotic pathogens Campylobacter coli and C. jejuni differ from each other by around 15% at the nucleotide level, corresponding to an average of nearly 40 amino acids per protein-coding gene. Using whole genome sequencing, we show that a single C. coli lineage, which has successfully colonized an agricultural niche, has been progressively accumulating C. jejuni DNA. Members of this lineage belong to two groups, the ST-828 and ST-1150 clonal complexes. The ST-1150 complex is less frequently isolated and has undergone a substantially greater amount of introgression leading to replacement of up to 23% of the C. coli core genome as well as import of novel DNA. By contrast, the more commonly isolated ST-828 complex bacteria have 10-11% introgressed DNA, and C. jejuni and nonagricultural C. coli lineages each have <2%. Thus, the C. coli that colonize agriculture, and consequently cause most human disease, have hybrid origin, but this cross-species exchange has so far not had a substantial impact on the gene pools of either C. jejuni or nonagricultural C. coli. These findings also indicate remarkable interchangeability of basic cellular machinery after a prolonged period of independent evolution.

Ribosomal multilocus sequence typing: universal characterization of bacteria from domain to strain.

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.

A longitudinal 6-year study of the molecular epidemiology of clinical campylobacter isolates in Oxfordshire, United kingdom.

Temporal and seasonal trends in Campylobacter genotypes causing human gastroenteritis were investigated in a 6-year study of 3,300 recent isolates from Oxfordshire, United Kingdom. Genotypes (sequence types [ST]) were defined using multilocus sequence typing and assigned to a clonal complex (a cluster of related strains that share four or more identical alleles with a previously defined central genotype). A previously undescribed clonal complex (ST-464) was identified which, together with ST-42, ST-45, and ST-52 complexes, showed increasing incidence. Concurrently, the incidence of ST-574, ST-607, and ST-658 complexes declined. The relative frequencies of three clonal complexes (ST-45, ST-283, and ST-42) peaked during summer and those of two (ST-353 and ST-403) peaked during winter. Nine clonal complexes (ST-22, ST-45, ST-48, ST-61, ST-257, ST-283, ST-403, ST-658, and ST-677) were significantly associated with ciprofloxacin sensitivity (P < 0.05). Seven clonal complexes (ST-49, ST-206, ST-354, ST-446, ST-460, ST-464, and ST-607) were associated with ciprofloxacin resistance (P < 0.05). Clonal complexes exhibited changing incidence and differences in seasonality and antibiotic resistance phenotype. These data also demonstrated that detailed surveillance at a single site captures information which reflects that observed nationally.

Emerging patterns of fluoroquinolone resistance in Campylobacter jejuni in the UK [1998-2018].

Campylobacter jejuni (C.jejuni) is the most common causative agent of bacterial food poisoning worldwide and is known to be genetically highly diverse. C. jejuni is increasingly resistant to fluoroquinolone antibiotics, but very few studies have investigated variant-specific patterns of resistance across time. Here we use statistical modelling and clustering techniques to investigate patterns of fluoroquinolone resistance amongst 10,359 UK isolates from human disease sampled over 20 years. We observed six distinct patterns of fluoroquinolone sensitivity/resistance in C. jejuni across time, grouping by clonal complex (CC). Some CCs were fully resistant, some shifted from susceptible to resistant following a sigmoidal shape, and some remained susceptible over time. Our findings indicate that the fluoroquinolone resistance patterns of C. jejuni are complicated and cannot be analysed as a single species but divided into variant dynamics so that the factors driving resistance can be thoroughly investigated.

Mechanisms of biodiversity between Campylobacter sequence types in a flock of broiler-breeder chickens.

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.

High resolution parallel sequencing reveals multistrain Campylobacter in broiler chicken flocks testing 'negative' by conventional culture methods: implications for control of Campylobacter infection.

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 wk 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 fecal samples collected from broiler flocks aged 23 to 28 d. 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 among flocks that would be determined to be Campylobacter 'positive' by conventional means, but a diverse pattern was seen among 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.

Temporal variation and host association in the Campylobacter population in a longitudinal ruminant farm study.

Campylobacter jejuni and C. coli were quantified and typed, using multilocus sequence typing (MLST), from fecal samples collected from a mixed cattle and sheep farm during summer. Cattle had a significantly higher prevalence than sheep (21.9% [74/338] and 14.0% [30/214], respectively), but both decreased over time. There were no differences in the average Campylobacter concentrations shed by cattle (600 CFU g(-1)) and sheep (820 CFU g(-1)), although sheep did show a significant temporal reduction in the number of Campylobacter organisms shed in their feces. A total of 21 different sequence types (STs) (97.7% C. jejuni, 2.3% C. coli) were isolated from cattle, and 9 different STs were isolated from sheep (40.6% C. jejuni, 59.4% C. coli). The Campylobacter population in cattle was relatively stable, and the frequencies of genotypes isolated showed little temporal variation. However, the composition of subtypes isolated from sheep did show significant temporal differences. The cattle and sheep consistently showed significant differences in their carriage of Campylobacter species, STs, and CCs despite the fact that both were exposed to the same farming environment. This work has highlighted the patterns of a Campylobacter population on a ruminant farm by identifying the existence of both temporal and between-host variations.

Niche segregation and genetic structure of Campylobacter jejuni populations from wild and agricultural host species.

Bacterial populations can display high levels of genetic structuring but the forces that influence this are incompletely understood. Here, by combining modelling approaches with multilocus sequence data for the zoonotic pathogen Campylobacter, we investigated how ecological factors such as niche (host) separation relate to population structure. We analysed seven housekeeping genes from published C. jejuni and C. coli isolate collections from a range of food and wild animal sources as well as abiotic environments. By reconstructing genetic structure and the patterns of ancestry, we quantified C. jejuni host association, inferred ancestral populations, investigated genetic admixture in different hosts and determined the host origin of recombinant C. jejuni alleles found in hybrid C. coli lineages. Phylogenetically distinct C. jejuni lineages were associated with phylogenetically distinct wild birds. However, in the farm environment, phylogenetically distant host animals shared several C. jejuni lineages that could not be segregated according to host origin using these analyses. Furthermore, of the introgressed C. jejuni alleles found in C. coli lineages, 73% were attributed to genotypes associated with food animals. Our results are consistent with an evolutionary scenario where distinct Campylobacter lineages are associated with different host species but the ecological factors that maintain this are different in domestic animals such that phylogenetically distant animals can harbour closely related strains.

Can good broiler flock welfare prevent colonization by Campylobacter?

Using data on rearing and welfare metrics of multiple commercial broiler flocks, we investigate how welfare measures such as hock burn, mortality, 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 directly exacerbated Campylobacter colonization, rather that they were both symptoms of the same parent variable - the managing company. Observed dependency on the management of the flock suggested that yet-undiscovered differences in rearing practice were the principal factor explaining both poor bird welfare and increased risk of Campylobacter, suggesting that action can be taken to improve both these factors simultaneously.

A Mathematical Modeling Approach to Uncover Factors Influencing the Spread of Campylobacter in a Flock of Broiler-Breeder Chickens.

Despite continued efforts to improve biosecurity protocols, Campylobacter continues to be detected in the majority of commercial chicken flocks across Europe. Using an extensive data set of Campylobacter prevalence within a chicken breeder flock for over a year, multiple Bayesian models are presented to explore the dynamics of the spread of Campylobacter in response to seasonal variation, species-specificity, bird health, and total colonization prevalence. These models indicated that birds within the flock varied greatly in their response to bacterial challenge, and that this phenomenon had a large impact on the overall prevalence of different species of Campylobacter. Campylobacter jejuni appeared more frequently in the summer, while Campylobacter coli persisted for a longer duration, amplified by the most susceptible birds in the flock. Our study suggests that strains of Campylobacter that appear most frequently likely possess no demographic advantage, but are instead amplified due to the health of the birds that ingest it.

Parallel sequencing of porA reveals a complex pattern of Campylobacter genotypes that differs between broiler and broiler breeder chickens.

Chicken meat represents an important source of Campylobacter infections of humans world-wide. A better understanding of Campylobacter epidemiology in commercial chicken flocks will facilitate the development of more effective intervention strategies. We developed a gene-specific parallel sequencing approach that efficiently indicated genetic diversity in farm-derived samples and revealed Campylobacter genotypes that would not be detected using microbiological culture. Parallel sequencing of the porA nucleotide fragment identified a different pattern of diversity in broiler flocks compared with broiler-breeder flocks at both individual bird and flock levels. Amongst the flocks tested, broiler flocks and individual birds were dominated by one or two porA fragment types whereas co-dominance with up to six porA fragment types was evident in breeder birds. A high proportion (83.6-93.3%) of porA variants were shared between broiler and breeder flocks. The porA-based diversity profiling could be a useful addition to the repertoire of tools employed to attribute potential sources of contamination for broiler flocks, including the environment, wild animals or other chickens. This approach can be extended to include other loci within Campylobacter and developed for molecular epidemiology studies of other bacterial species.

Domestication of Campylobacter jejuni NCTC 11168.

Reference and type strains of well-known bacteria have been a cornerstone of microbiology research for decades. The sharing of well-characterized isolates among laboratories has run in parallel with research efforts and enhanced the reproducibility of experiments, leading to a wealth of knowledge about trait variation in different species and the underlying genetics. Campylobacter jejuni strain NCTC 11168, deposited at the National Collection of Type Cultures in 1977, has been adopted widely as a reference strain by researchers worldwide and was the first Campylobacter for which the complete genome was published (in 2000). In this study, we collected 23 C. jejuni NCTC 11168 reference isolates from laboratories across the UK and compared variation in simple laboratory phenotypes with genetic variation in sequenced genomes. Putatively identical isolates, identified previously to have aberrant phenotypes, varied by up to 281 SNPs (in 15 genes) compared to the most recent reference strain. Isolates also display considerable phenotype variation in motility, morphology, growth at 37 °C, invasion of chicken and human cell lines, and susceptibility to ampicillin. This study provides evidence of ongoing evolutionary change among C. jejuni isolates as they are cultured in different laboratories and highlights the need for careful consideration of genetic variation within laboratory reference strains. This article contains data hosted by Microreact.

Campylobacter infection of broiler chickens in a free-range environment.

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.