BACKGROUND
intestinal infections by campylobacter jejuni and campylobacter coli are, jointly with salmonella enterica infections, the most frequent bacterial causes of diarrheal diseases of humans worldwide  <cit> .
c. jejuni causes the majority  of human symptomatic campylobacter infections  <cit> .
campylobacter ssp.
are transmitted mainly by contaminated food of animal origin  <cit> .
animals are frequently infected persistently with campylobacters and, in most cases, show no symptoms, contrary to the acute and self-limiting intestinal infection caused in humans.
the cause of these discrepant outcomes of infection in different hosts is not clear, although previous work has suggested that host as well as bacterial factors may contribute  <cit> .
numerous c. coli and c. jejuni subtypes with similar morphology and biochemistry are found in different mammalian and bird hosts, with the possibility of zoonotic transmission to humans.
in addition, campylobacter species display extensive intraspecies variation.
these properties require sophisticated molecular typing methods in order to differentiate species and subtypes, and to elucidate infection sources.
multi locus sequence typing  analyses have proven to be a molecular method of choice and previously have provided clear evidence that genetically related strains of either c. jejuni or c. coli can colonize different hosts  <cit> .
these strains frequently are part of large clonal complexes, whose members share most of the mlst alleles.
however, mlst studies rely solely on the analysis of seven housekeeping genes, belonging to the core genome  <cit> , and disregard the rest of the ~ <dig>  mb genome  <cit> , which includes virulence-associated genes as well as possible strain-specific genes or gene variants.
thus, the question remains open, whether different strain subtypes infecting humans differ from animal-colonizing and food-associated types within one closely related genetic grouping .
little is known about the mechanisms of host-specific colonization, virulence and pathogenicity factors of campylobacter species.
with few exceptions  <cit> , genome sequences currently available in databases for campylobacter species  have been derived from human isolates, therefore genome information about strains from other sources is still scarce.
in order to complement these recent advances, it may be relevant to gather more information about host association and animal-associated strains and subtypes, some of which may be more prone to be transmitted or cause severe complications during human infections.
prophylactic measures are required to prevent transmission from food and animals to humans, which also require a better knowledge of strain-to-strain variability and adaptation potential to different hosts and environments such as food.
this study was designed to characterize the genetic relationships and population structure of c. jejuni and c. coli currently prevalent in humans, domestic animals and food sources in germany.
furthermore, a major aim was to establish the extent of diversity within dominant phylogenetic groups of c. jejuni from this collection, which are present in diverse hosts, such as st- <dig>  with regard to their different sources.
to that end, we collected  <dig> c. jejuni and c. coli isolates from humans, as well as animal , food, and environmental isolates between  <dig> and  <dig> in germany.
mlst analysis and extensive molecular typing of the isolates was performed, which confirmed c. jejuni st- <dig> as one of the dominant "generalist" variants occurring in all sources.
further phenotypic characterization and whole genome analyses of closely related st- <dig> c. jejuni strains from different sources provided little evidence for host or source-associated traits.
nevertheless, st- <dig> isolates from different sources displayed a high genomic and phenotypic diversity and flexibility.
domestic circulating c. jejuni sts such as st- <dig> appear to use mechanisms such as inter-strain recombination, metabolic and surface-associated genetic modules and contingency genes to permit a generalist lifestyle and the flexible expression of fitness factors in diverse environments.
RESULTS
multi locus sequence typing of c. coli and c. jejuni isolates from diverse isolation sources in humans, animals and food sources
we collected  <dig> campylobacter strains  from animal hosts , human patients, and food sources over a five-year period  in several geographically separate centers in germany.
human patient samples were collected in three university hospitals ; the majority of non-patient isolates were from domestic animals and food samples, while few strains originated from wild-living animals.
molecular typing of the whole strain collection was performed by mlst  <cit> .
<dig> different sequence types  were identified, of which  <dig> were new sts not previously represented in the pubmlst database.
these new sts include  <dig> that are novel combinations of previously known alleles, and  <dig> that included  <dig> novel alleles .
the st distribution was highly diverse, with  <dig> sts occurring only once, and the ten most common sts comprising only  <dig> % and  <dig> % of isolates for c. jejuni and c. coli, respectively.
of the  <dig> sts represented more than once,  <dig> were found in humans and, in addition, in either animals or food or in both of the latter, suggesting a host range consistent with zoonotic potential.
all sts contained either only c. jejuni or c. coli strains.
only two alleles were found to occur in both species, pgm <dig>  present in  <dig> c. coli and  <dig> c. jejuni strains, and unca <dig>  a very common allele present in  <dig> c. coli and  <dig> c. jejuni strains.
minimal spanning trees were generated from the mlst data illustrating the phylogenetic relationships of the strains .
in confirmation and extension of previous results, mlst revealed dominant sequence types within the strains .
the dominant sequence types in the fbi-zoo collection partially overlapped with dominant sequence types reported for campylobacter samplings in the united kingdom, the netherlands, the u.s., canada, denmark and finland , which provide the majority of mlst types previously deposited in the public mlst database , and in some other countries.
this result suggests that campylobacter sequence types prevalent in many geographic areas of the world represent similar dominant strain types and clonal complexes.
the five most dominant mlst sequence types for c. jejuni in the strain collection were st- <dig>  st- <dig>  st- <dig>  st- <dig>  st- <dig>  and for c. coli st- <dig>  st- <dig>  st- <dig>  st- <dig>  and st- <dig>  many common mlst types, such as st- <dig>  st- <dig>  st- <dig>  were distributed rather evenly between strains isolated from domestic animal, human and food sources .
some, mostly rarer, strain types were only found either in animals, food sources or in humans.
c. coli strains were more closely related to each other than the c. jejuni strains , in concordance with previous studies  <cit> .
the standardized index of association,   <cit> , was calculated for the complete set of allelic profiles and separately for c. coli and c. jejuni strains, respectively.
sia was  <dig>  for the complete dataset and the c. jejuni dataset, indicating a limited amount of recombination not sufficient to completely destroy linkage between alleles.
by contrast, the sia for the c. coli dataset was  <dig> , a value close to the expected value for linkage equilibrium .
extended genetic pathotyping of campylobacter jejuni
due to the restriction of the campylobacter mlst system to seven housekeeping genes, potential host-specific genes, genes providing strain-specific means of enhanced survival and transmission, or predictive markers for infection-associated severe disease may only be detected by looking at an enhanced set of proposed virulence genes, or at the complete genomes of strains.
we therefore selected a subgroup of c. jejuni strains  from the fbi-zoo collection that contained representative isolates of all dominant mlst sequence types, and included strains from all major available isolation sources  for all dominant sts.
each strain in this subset was subjected to pcr-based typing to detect the presence or absence of a panel of  <dig> genes previously proposed to be involved in pathogenesis, metabolism or host specificity .
three additional genes were included for the typing of  <dig> c. jejuni strains belonging to ten different sts .
a few tested genes were present in all strains, such as cj <dig>  flac, flaa, cadf, pglb, and flpa .
most of the tested genes were only detectable in a subset of the strains, including ggt , secreted ansb , dmsa, cdtb, fucp, ciab, capa  and neuc <dig>  genes  <cit> .
both the ggt gene and the secreted ansb  allelic variant were previously proposed to provide niche or host specificity  <cit> .
interestingly, the proportion of strains possessing ggt or ansbs genes was overall very low in our strain collection .
in addition, in isogenic mlst groups containing strains from all three major isolation sources , the presence of the neuc <dig>  ggt or ansbs genes did not correlate with isolation source.
nor did the presence of any other gene in the pathotyping matrix  correlate with isolation source.
however, close correlation with mlst type was detected for the proposed virulence genes fspa <dig> and fspa <dig>  <cit> , except for st- <dig>  suggesting that these gene types are usually coinherited in phylogenetically closely related strain subtypes.
this result corroborates recent data for fspa <dig> obtained from finnish strains  <cit> .
overall, taking into account phylogenetic relatedness of strains within st groups, no correlation of presence of individual genes with the isolation source was observed.
additional genes involved in metabolic functions and iron uptake  were tested for presence or absence in a subset of the  <dig> strains from different sts and isolation sources and did not show a host-specific distribution .
for some of the genes which were present in all or most strains  and some non-ubiquitous genes , we performed sequencing of the genes on a subset of pcr-typed strains .
sequence diversity between the strains was found in these genes to a different extent .
in summary, none of the tested genes or allelic variants taken singly or in combination were correlated with isolation source.
evidence of phenotypic differences between c. jejuni st- <dig> strains from different sources
the data described above suggested that the identification of any genes, gene variants or traits potentially providing host specificity to c. jejuni might require more sophisticated methodological approaches.
we therefore decided to perform in-depth investigations of the phenotypic and genetic traits of eight selected c. jejuni strains from one single exemplary mlst sequence type , a common and dominant st worldwide.
the selected st- <dig> strains  were isolated from human patients, domestic animals  and food sources; pcr typing of strain-variable genes  had provided evidence that some genes differed between these strains.
this prompted us to first address the question whether these strains show any other phenotypic variation which would warrant further characterization with regard to the diversification of closely related strains.
we tested the phenotypic variation of these strains using the following criteria: metabolic variation , energy harvest, motility, morphology, protein expression patterns and cell interaction.
some of the experiments were performed at 42°c and at 37°c.
all of these freshly isolated strains possessed very high motility except for the milk-derived strain  <dig>  the bacterial morphology in transmission electron microscopy was very similar.
cell interaction of the strains was comparatively tested in adherence assays with the human intestinal epithelial cell line caco <dig>  the chicken macrophage-like cell line hd- <dig>  the ipec-j <dig> swine intestinal epithelial cell line  and human epithelial hela cells .
in addition, proinflammatory cytokine production  and cell activation were tested for eight st- <dig> strains on the same cell lines containing an nf-kb-driven luciferase reporter .
the tested st- <dig> strains showed differences in cell adherence, cytokine production and nf-kb activation , indicating bacterial phenotypic differences.
next, we used rabbit-derived and chicken sera reactive against c. jejuni in order to compare the patterns of immunogenic proteins between different strains of the same sequence type in western blots.
differential protein patterns were detected in closely related strains when cultivated at both 37°c and 42°c .
likewise, mlst-isogenic strains differed in their metabolic properties and energy harvest as determined by biolog analysis and intrabacterial atp levels of eight st- <dig> strains .
interestingly, using the biolog characteristics, the group st- <dig> strains could be largely subdivided into two phenotypic variants with characteristic patterns of properties.
these two variants  were represented with equal frequency in the eight strains  tested by biolog, and did not show any correlation to a specific host or source.
the common characteristics of half of these strains  were the metabolic use of three specific glycyl-l-dipeptides , l-galactonic acid g-lactone, tyramine, m/p-hydroxy-phenylacetic acid , while the biotype  <dig> strains did not use those.
these metabolic trait differences had not been described before for closely related c. jejuni strains.
these results were very reproducible and not temperature-dependent .
when nine additional strains from other sts  were tested in the same biolog system, distinct metabolic fingerprints were observed for each st. surprisingly, some of the tested strains also displayed increased electron chain activity with one hexose sugar  and one phosphate sugar .
in summary, these results provided clear evidence for phenotypic differences between st- <dig> strains, with no indication of host specificity.
complete genome analysis of genetically closely related c. jejuni st- <dig> strains reveals interstrain variation
in order to test the hypothesis of a genetic basis for the observed phenotypic diversity within the st- <dig> group, whole genome sequencing was performed for five st- <dig> strains from bovine, chicken, human and food sources .
this approach was also aimed at answering the question, whether the observed phenotypic diversity was due to unique genomic events which could not be identified with pathotyping of known genes, and whether it was somehow correlated with association to a specific host.
the genome sequences revealed relatively little variation in gene content  but a high level of polymorphism within shared genes, contingency genes and intergenic regions .
those included single nucleotide  and clustered nucleotide polymorphisms  .
sequence length differences were mostly found in intergenic regions .
four of the five sequenced strains had acquired phage gene clusters .
four selected phage genes were tested by specific pcr for their presence in a wider array of st- <dig> strains and found to represent single events , uncorrelated with any host or source.
the human isolate xy <dig> possessed a deletion in a gene responsible for surface or flagellar glycosylation , which was not found in other human strains.
the bovine strain  <dig> lacked the gene equivalent to cj <dig> , which is a gene of unknown function in a metabolic gene cluster relating to a pyridine nucleotide-disulphide oxidoreductase.
the allelic diversity between the strains comprised a large number of synonymous and non-synonymous nucleotide substitutions in coding regions , many of which originated from homologous recombination, and some deletions and insertions in both coding and intergenic regions of all strains .
repeat length differences probably originating from slipped strand mispairing mutagenesis were evident in all strains.
this concerned eight genes of the glycosylation gene clusters leading to frameshifts in coding regions.
we confirmed these repeat length differences in a larger number of st- <dig> strains using sanger sequencing .
by this extended sequence determination, the repeat length differences obtained by  <dig> sequencing were confirmed, but none of the tested nucleotide polymorphisms was stably associated with the source of the strain.
only a limited number of single nucleotide polymorphisms and repeat differences could be investigated by this labour-intensive approach.
aligning the five genome sequences  revealed features of genome plasticity in c. jejuni, with a highly conserved overall synteny of the genomes.
we detected that frequent homologous recombination events had happened since the divergence of the genomes from a common ancestor , with an average imported length of  <dig> bp .
the genome regions containing clustered polymorphisms caused by recombination are summarized for all five st- <dig> genomes in additional file 2: table s <dig>  they contain genes of various functional categories .
none of the functional categories showed significantly higher percentages for having undergone recombination than the average of all functional categories .
when the st- <dig> genomes were aligned to previously sequenced c. jejuni genomes from unrelated phylogenetic groups  in addition to nucleotide polymorphisms, notable differences in gene content were observed .
correlating phenotypic variation with genetic differences in st- <dig> strains
both genotypic and phenotypic differences were observed for st- <dig> and other c. jejuni strains, and the genotypic differences might be a basis for a potential adaptive phenotype.
from the biolog assays, there was no evidence for a correlation of the two different metabolic biotypes within st- <dig> with any specific source.
nevertheless, it is worthwhile investigating the gene variants potentially providing phenotypic differences, including those of metabolic capacities detected in the biolog assay.
we next asked if genetic polymorphisms within st- <dig> that had been identified by the genome sequence analysis could be correlated with the metabolic biotypes.
as already described previously, strains of the st- <dig> lineage are able to use fucose, due to the presence of a genomic island  <cit> .
as a proof of concept, use of fucose was confirmed for all tested st- <dig> strains  using the biolog system .
within the other tested sts, st- <dig> and st- <dig> did not display fucose utilization, as expected from previous studies  <cit> .
one of the st- <dig> strains selected for whole genome sequencing, the bovine isolate  <dig>  belonged to metabolic biotype  <dig> and did not metabolize the three dipeptides, tyramine, and m/p-hydroxy-phenylacetic acid.
although several genetic polymorphisms found in the  <dig> genome were investigated in all st- <dig> strains belonging to biotype  <dig>  and compared with the corresponding loci of biotype  <dig> strains, a specific genetic variant which could be responsible for the metabolic phenotype was not identified.
several genes and intergenic regions of potential relevance for metabolic activities varied between st- <dig> strains .
these included two peptidase genes , msra , cmeb  , sdha , frda , dcub  , ansb , and args  .
these allelic or intergenic differences were resequenced or tested with allele-specific primers for further isolates of st- <dig> and other sts from various sources.
none of them could be affirmed to be source-associated or related to metabolic biotype for carbon sources.
in addition, rna-based real time pcr was performed for six st- <dig> strains , two of biotype  <dig> and three of biotype  <dig> , to elucidate whether genes were differentially transcribed, in particular genes of global regulators.
these analyses yielded clear differences in transcript abundance between the strains  which were uncorrelated with metabolic biotype or source .
most conspicuously, strain  <dig>  appeared to have a largely reduced rpon transcript amount, which also seemed to negatively affect luxs transcript, and matched the low observed in vitro motility of this strain.
chicken colonization by two genetically closely related st- <dig> c. jejuni strains from human and chicken
chicken colonization studies have helped previously to determine whether certain c. jejuni variants were infection-competent, including studies that tested strains with presumed adaptation to other hosts that were no longer able to infect chickens  <cit> .
we infected three weeks-old white leghorn chicks with two of the genome-sequenced closely related st- <dig> strains, xy <dig>  and rb <dig> .
all inoculated animals were colonized by both test strains, which had been verified in advance to possess full motility, since motility is a major prerequisite for successful colonization of campylobacters in any host and subject to strong phase variation mechanisms  <cit> .
during the two-weeks' infection period, the chicken-derived strain colonized chickens slightly better  than the human-derived strain .
however, the human strain was fully competent to colonize all animals persistently.
discussion
in order to curb the high numbers of human campylobacter infections, zoonotic transmission to humans needs to be reduced.
hence, the identification of specific groups of strains or strain properties which are associated with the colonization of humans or specific animals may help to provide targets for such measures.
in the present study, an in-depth genetic and phenotypic characterization of a diverse array of campylobacter strains was initiated, including in particular closely genetically related strains of st- <dig> isolated from diverse habitats and isolation sources.
from  <dig> to  <dig>  campylobacter isolates were collected in the framework of the fbi-zoo network from humans, diverse animals and food sources in germany.
the complete collection of  <dig> strains was subjected to mlst analysis, making this one of the most comprehensive mlst analyses of campylobacter from one country outside of the uk.
approximately one third of c. jejuni and one fourth of c. coli isolates belonged to five dominant sts, each representing at least 3% of the isolates.
many dominant sts are also very prevalent in other geographical regions ; as in other european regions, st- <dig>  which is prevalent in new zealand , was not identified in germany.
for c. jejuni, strains of all dominant sts were isolated from both humans and animals/food samples, indicating a broad host range and zoonotic potential.
in contrast, approximately two thirds of c. jejuni strains were very diverse and represent rare sts, so that the low number of isolates per st made it impossible to draw valid conclusions about their host association.
these and previous data suggest that all dominant sts found in a variety of sources have a "generalist" phenotype, enabling them to colonize both humans and domestic animals.
from these hosts, they can then be transmitted to humans either directly or via food.
a previous whole genome-based study including  <dig> strains  <cit>  suggested, that a host-specific campylobacter subtype, or host-specific campylobacter genes, may not exist, particularly in the dominant mlst sequence types, which are mainly derived from domestic sources that may have evolved under conditions of ample recent admixture  <cit> .
this does not rule out that some of the rarer sequence types, such as isolates from wild animals which rarely mix with other niches, have irreversibly adapted to a single host species.
evidence for fixed host adaptation in a rare st has been provided recently for sequence type st- <dig>  <cit> , a c. jejuni variant from bank voles, which failed to colonize chickens.
while strong evidence of admixture between c. coli and c. jejuni has been presented before  <cit> , and is supported by the existence of alleles occurring in both c. jejuni and c. coli, the overall frequency of such shared alleles was very low in our collection .
calculations of the standardized index of association and other tests for recombination were in agreement with previous studies demonstrating an important role of recombination in shaping the population structure of campylobacter species.
notably, the sia of c. coli was much lower than that of c. jejuni, suggesting more efficient exchange of alleles between c. coli strains than c. jejuni strains.
c. jejuni and c. coli have been shown to be versatile and diverse species, harbouring numerous genes related to host interactions or metabolism that are not present in all strains, or are subject to phase variation  <cit> .
we have performed an extensive analysis of such genes in a large subset of our collection, and additional reference isolates.
<dig> c. jejuni strains were studied for the absence/presence and sequence variability of  <dig> genes with putative roles in phenotypes relevant to host interaction.
these included genes coding for surface proteins, los, protein glycosylation and metabolic functions which have been suggested to play an important part in niche- or host-associated colonization abilities  <cit> .
the present data have not provided evidence that any of the genes tested was robustly associated with isolation source.
by contrast, pathotyping profiles were in many cases closely correlated with phylogenetic grouping by sequence types, suggesting that campylobacter have diversified their complements of in vivo essential genes.
apparently, the host interaction gene complement of dominant sts was selected to provide equal fitness advantages in both domestic  animals and humans, permitting strain circulation between humans and domestic animals, making them "generalists".
these generalist strains could then make use of different genetic features including reversible frame-shifting of contingency genes, intra-genomic and inter-genomic recombination to fine-tune their genetic and phenotypic capacities, which may benefit their survival in individual hosts or changing environments without preventing their generalist adaptation potential.
st- <dig> is one of the most prevalent sequence types of c. jejuni present in a broad range of hosts  <cit> .
we have selected st- <dig> to analyze the genetic and phenotypic diversity of c. jejuni within a group of closely related isolates, which appear to have generalist properties with respect to domestic hosts.
phenotypic characterization and metabolic profiling was performed for eight st- <dig> isolates from different sources , and the genomes of five of these strains were sequenced.
metabolic profiling identified two characteristic biotypes within st- <dig>  with distinctive differences in their ability to metabolize certain carbon sources, including some dipeptides.
similar to the pathotyping results, none of these biotypes was associated with a specific isolation source, suggesting that both biotypes are compatible with a generalist lifestyle.
in addition to differences in their metabolic capacities, the same strains also showed phenotypic diversity in their interaction with eukaryotic cells, independently of their isolation source.
a further nine human c. jejuni strains from other rare and frequent sts  that were compared using the biolog system showed metabolic fingerprints distinct from each other and also different from all st- <dig> strains, revealing the metabolic fingerprint as a distinctive feature between and even within phylogenetic subtypes of the species.
however, our study did not provide any evidence for a common metabolic fingerprint of human-derived strains.
the genome analysis of five st- <dig> strains confirmed their high genetic relatedness, with long stretches of complete sequence identity and generally conserved synteny.
however, pairwise and multiple comparisons of all sequences provided evidence of recombination that has caused the exchange of chromosome fragments of varying length, up to ~ <dig> kbp.
these genome mosaicisms are reminiscent of the situation in the human gastric pathogen helicobacter pylori, where genome comparisons of sequentially isolated strains from one single individual have yielded very similar mosaic patterns  <cit>  to the ones observed here.
in particular, the average length of imported fragments in the st- <dig> genomes was  <dig>  bp, between two and six times more than a previous estimate based on mlst data only  <cit> , which could reflect the fact that mlst genes are under strong purifying selection  <cit> .
the import length is about 3- <dig> times longer than the average length of imports calculated from h. pylori genome comparisons  with the same analytical model.
mutation and recombination were estimated to have occurred at similar rates, but recombination introduced  <dig>  times more polymorphisms than mutation as it often substitutes several sites simultaneously  <cit> .
despite the strong evidence for recombination, the gene content in the five st- <dig> genomes was very highly conserved.
only few strain-specific or unique genes were identified, most of which were part of phage gene clusters.
these phage acquisitions represented almost the entire set of gene content differences between the sequenced st- <dig> strains.
some phage genes were so far unknown; three of the phages  were similar to previously described campylobacter phages , largely caused by extensive inter-strain recombination and contingency genes.
this corroborated earlier results from microarray comparisons which claimed that c. jejuni strains possess a high level of genome diversity, even within a single sequence type  <cit> .
extended testing for variant alleles of certain candidate genes in other dominant or rare sts or in specific groups of strains from specific host or source did not reveal any fixed host relation.
some of these variants may indicate a pressure for phenotypic diversification, for example due to bacteriophage resistance, or might still affect fitness in specific hosts or under certain colonization conditions.
we propose that the observed microdiversity may allow these bacteria to flexibly and reversibly adapt to diverse habitats and to propagate their generalist phenotypes.
experimental infection of two genome-sequenced st- <dig> strains in chickens revealed that a st- <dig> strain isolated from a human was fully competent to colonize chickens, albeit to a slightly lower density than a chicken isolate inoculated in a parallel experiment.
this suggests that no general colonization barrier for human-derived st- <dig> strains exists in chicken.
extensive genetic analyses of  <dig> whole genomes of c. coli and c. jejuni  have recently revealed that genetically distantly related isolates show considerable variability in gene content  <cit> .
the authors of this study, however, did not find evidence of statistical association between gene content and type of host.
additional work suggested that the observed genetic diversity between c. jejuni isolates were instead closely associated with phylogenetic origin of the strains  <cit> .
in a recent study, a human c. jejuni strain was inoculated in parallel into outbred chickens and inbred genetically modified mice  <cit> , resulting in a much lower variation in the output pool from the mice compared to that of chickens.
in a second experimental system of strain diversification, one specific c. jejuni variant in a contingency gene was strongly enriched by serial mouse passage  <cit> .
this may suggest that, in contrast to a host population of low genetic variability such as inbred mice, campylobacter requires genetic flexibility in a host population consisting of phenotypically and immunologically diverse individuals, including factors such as resident microbiota and bacteriophage abundance.
given these findings, it seems plausible that abundant campylobacter types profit in many different ways from their established genome flexibility, even within a single host species.
CONCLUSIONS
while our findings do not rule out the existence of phenotypic and genotypic determinants of campylobacter tissue tropism and host specificity, our results from st- <dig> genomic and phenotypic comparisons rather support a view where all st- <dig> strains are characterized by a generalist genome, phenotype and lifestyle, which support their ability to colonize both humans and food animals.
the same might be true for other dominant c. jejuni sts and should be investigated further.
this view is also supported by an extended mlst study of campylobacter isolates in the uk  <cit> .
these frequent types may possess a clear fitness advantage under diverse conditions.
we propose that future efforts should be directed towards defining the molecular determinants of this phenotypic flexibility and generalist capacity, which might be useful targets for interventions into the transmission cycle, and hence ultimately reduce campylobacter-associated diseases.
for their high numbers of human infections caused world wide, and their very broad transmission capacities, the dominant campylobacter sts deserve intensive attention.
if the hypothesis of generalist and specialist strain types can be confirmed in larger campylobacter strain collections, it would suggest to study host-specificity or the predictive value of molecular subtyping methods separately for the dominant and the rare st groups.

 intestinal infections by campylobacter jejuni and campylobacter coli are, jointly with salmonella enterica infections, the most frequent bacterial causes of diarrheal diseases of humans worldwide. c. jejuni causes the majority of human symptomatic campylobacter infections. campylobacter ssp. are transmitted mainly by contaminated food of animal origin. animals are frequently infected persistently with campylobacters and, in most cases, show no symptoms, contrary to the acute and self-limiting intestinal infection caused in humans. the cause of these discrepant outcomes of infection in different hosts is not clear, although previous work has suggested that host as well as bacterial factors may contribute. numerous c. coli and c. jejuni subtypes with similar morphology and biochemistry are found in different mammalian and bird hosts, with the possibility of zoonotic transmission to humans. in addition, campylobacter species display extensive intraspecies variation. these properties require sophisticated molecular typing methods in order to differentiate species and subtypes, and to elucidate infection sources. multi locus sequence typing analyses have proven to be a molecular method of choice and previously have provided clear evidence that genetically related strains of either c. jejuni or c. coli can colonize different hosts. these strains frequently are part of large clonal complexes, whose members share most of the mlst alleles. however, mlst studies rely solely on the analysis of seven housekeeping genes, belonging to the core genome, and disregard the rest of the ~ mb genome, which includes virulence-associated genes as well as possible strain-specific genes or gene variants. thus, the question remains open, whether different strain subtypes infecting humans differ from animal-colonizing and food-associated types within one closely related genetic grouping. little is known about the mechanisms of host-specific colonization, virulence and pathogenicity factors of campylobacter species. with few exceptions, genome sequences currently available in databases for campylobacter species have been derived from human isolates, therefore genome information about strains from other sources is still scarce. in order to complement these recent advances, it may be relevant to gather more information about host association and animal-associated strains and subtypes, some of which may be more prone to be transmitted or cause severe complications during human infections. prophylactic measures are required to prevent transmission from food and animals to humans, which also require a better knowledge of strain-to-strain variability and adaptation potential to different hosts and environments such as food. this study was designed to characterize the genetic relationships and population structure of c. jejuni and c. coli currently prevalent in humans, domestic animals and food sources in germany. furthermore, a major aim was to establish the extent of diversity within dominant phylogenetic groups of c. jejuni from this collection, which are present in diverse hosts, such as st- with regard to their different sources. to that end, we collected c. jejuni and c. coli isolates from humans, as well as animal, food, and environmental isolates between and in germany. mlst analysis and extensive molecular typing of the isolates was performed, which confirmed c. jejuni st- as one of the dominant "generalist" variants occurring in all sources. further phenotypic characterization and whole genome analyses of closely related st- c. jejuni strains from different sources provided little evidence for host or source-associated traits. nevertheless, st- isolates from different sources displayed a high genomic and phenotypic diversity and flexibility. domestic circulating c. jejuni sts such as st- appear to use mechanisms such as inter-strain recombination, metabolic and surface-associated genetic modules and contingency genes to permit a generalist lifestyle and the flexible expression of fitness factors in diverse environments.  multi locus sequence typing of c. coli and c. jejuni isolates from diverse isolation sources in humans, animals and food sources we collected campylobacter strains from animal hosts, human patients, and food sources over a five-year period in several geographically separate centers in germany. human patient samples were collected in three university hospitals; the majority of non-patient isolates were from domestic animals and food samples, while few strains originated from wild-living animals. molecular typing of the whole strain collection was performed by mlst. different sequence types were identified, of which were new sts not previously represented in the pubmlst database. these new sts include that are novel combinations of previously known alleles, and that included novel alleles. the st distribution was highly diverse, with sts occurring only once, and the ten most common sts comprising only % and % of isolates for c. jejuni and c. coli, respectively. of the sts represented more than once, were found in humans and, in addition, in either animals or food or in both of the latter, suggesting a host range consistent with zoonotic potential. all sts contained either only c. jejuni or c. coli strains. only two alleles were found to occur in both species, pgm present in c. coli and c. jejuni strains, and unca a very common allele present in c. coli and c. jejuni strains. minimal spanning trees were generated from the mlst data illustrating the phylogenetic relationships of the strains. in confirmation and extension of previous results, mlst revealed dominant sequence types within the strains. the dominant sequence types in the fbi-zoo collection partially overlapped with dominant sequence types reported for campylobacter samplings in the united kingdom, the netherlands, the u.s., canada, denmark and finland, which provide the majority of mlst types previously deposited in the public mlst database, and in some other countries. this result suggests that campylobacter sequence types prevalent in many geographic areas of the world represent similar dominant strain types and clonal complexes. the five most dominant mlst sequence types for c. jejuni in the strain collection were st- st- st- st- st- and for c. coli st- st- st- st- and st- many common mlst types, such as st- st- st- were distributed rather evenly between strains isolated from domestic animal, human and food sources. some, mostly rarer, strain types were only found either in animals, food sources or in humans. c. coli strains were more closely related to each other than the c. jejuni strains, in concordance with previous studies. the standardized index of association,, was calculated for the complete set of allelic profiles and separately for c. coli and c. jejuni strains, respectively. sia was for the complete dataset and the c. jejuni dataset, indicating a limited amount of recombination not sufficient to completely destroy linkage between alleles. by contrast, the sia for the c. coli dataset was, a value close to the expected value for linkage equilibrium. extended genetic pathotyping of campylobacter jejuni due to the restriction of the campylobacter mlst system to seven housekeeping genes, potential host-specific genes, genes providing strain-specific means of enhanced survival and transmission, or predictive markers for infection-associated severe disease may only be detected by looking at an enhanced set of proposed virulence genes, or at the complete genomes of strains. we therefore selected a subgroup of c. jejuni strains from the fbi-zoo collection that contained representative isolates of all dominant mlst sequence types, and included strains from all major available isolation sources for all dominant sts. each strain in this subset was subjected to pcr-based typing to detect the presence or absence of a panel of genes previously proposed to be involved in pathogenesis, metabolism or host specificity. three additional genes were included for the typing of c. jejuni strains belonging to ten different sts. a few tested genes were present in all strains, such as cj flac, flaa, cadf, pglb, and flpa. most of the tested genes were only detectable in a subset of the strains, including ggt, secreted ansb, dmsa, cdtb, fucp, ciab, capa and neuc genes. both the ggt gene and the secreted ansb allelic variant were previously proposed to provide niche or host specificity. interestingly, the proportion of strains possessing ggt or ansbs genes was overall very low in our strain collection. in addition, in isogenic mlst groups containing strains from all three major isolation sources, the presence of the neuc ggt or ansbs genes did not correlate with isolation source. nor did the presence of any other gene in the pathotyping matrix correlate with isolation source. however, close correlation with mlst type was detected for the proposed virulence genes fspa and fspa, except for st- suggesting that these gene types are usually coinherited in phylogenetically closely related strain subtypes. this result corroborates recent data for fspa obtained from finnish strains. overall, taking into account phylogenetic relatedness of strains within st groups, no correlation of presence of individual genes with the isolation source was observed. additional genes involved in metabolic functions and iron uptake were tested for presence or absence in a subset of the strains from different sts and isolation sources and did not show a host-specific distribution. for some of the genes which were present in all or most strains and some non-ubiquitous genes, we performed sequencing of the genes on a subset of pcr-typed strains. sequence diversity between the strains was found in these genes to a different extent. in summary, none of the tested genes or allelic variants taken singly or in combination were correlated with isolation source. evidence of phenotypic differences between c. jejuni st- strains from different sources the data described above suggested that the identification of any genes, gene variants or traits potentially providing host specificity to c. jejuni might require more sophisticated methodological approaches. we therefore decided to perform in-depth investigations of the phenotypic and genetic traits of eight selected c. jejuni strains from one single exemplary mlst sequence type, a common and dominant st worldwide. the selected st- strains were isolated from human patients, domestic animals and food sources; pcr typing of strain-variable genes had provided evidence that some genes differed between these strains. this prompted us to first address the question whether these strains show any other phenotypic variation which would warrant further characterization with regard to the diversification of closely related strains. we tested the phenotypic variation of these strains using the following criteria: metabolic variation, energy harvest, motility, morphology, protein expression patterns and cell interaction. some of the experiments were performed at 42°c and at 37°c. all of these freshly isolated strains possessed very high motility except for the milk-derived strain the bacterial morphology in transmission electron microscopy was very similar. cell interaction of the strains was comparatively tested in adherence assays with the human intestinal epithelial cell line caco the chicken macrophage-like cell line hd- the ipec-j swine intestinal epithelial cell line and human epithelial hela cells. in addition, proinflammatory cytokine production and cell activation were tested for eight st- strains on the same cell lines containing an nf-kb-driven luciferase reporter. the tested st- strains showed differences in cell adherence, cytokine production and nf-kb activation, indicating bacterial phenotypic differences. next, we used rabbit-derived and chicken sera reactive against c. jejuni in order to compare the patterns of immunogenic proteins between different strains of the same sequence type in western blots. differential protein patterns were detected in closely related strains when cultivated at both 37°c and 42°c. likewise, mlst-isogenic strains differed in their metabolic properties and energy harvest as determined by biolog analysis and intrabacterial atp levels of eight st- strains. interestingly, using the biolog characteristics, the group st- strains could be largely subdivided into two phenotypic variants with characteristic patterns of properties. these two variants were represented with equal frequency in the eight strains tested by biolog, and did not show any correlation to a specific host or source. the common characteristics of half of these strains were the metabolic use of three specific glycyl-l-dipeptides, l-galactonic acid g-lactone, tyramine, m/p-hydroxy-phenylacetic acid, while the biotype strains did not use those. these metabolic trait differences had not been described before for closely related c. jejuni strains. these results were very reproducible and not temperature-dependent. when nine additional strains from other sts were tested in the same biolog system, distinct metabolic fingerprints were observed for each st. surprisingly, some of the tested strains also displayed increased electron chain activity with one hexose sugar and one phosphate sugar. in summary, these results provided clear evidence for phenotypic differences between st- strains, with no indication of host specificity. complete genome analysis of genetically closely related c. jejuni st- strains reveals interstrain variation in order to test the hypothesis of a genetic basis for the observed phenotypic diversity within the st- group, whole genome sequencing was performed for five st- strains from bovine, chicken, human and food sources. this approach was also aimed at answering the question, whether the observed phenotypic diversity was due to unique genomic events which could not be identified with pathotyping of known genes, and whether it was somehow correlated with association to a specific host. the genome sequences revealed relatively little variation in gene content but a high level of polymorphism within shared genes, contingency genes and intergenic regions. those included single nucleotide and clustered nucleotide polymorphisms. sequence length differences were mostly found in intergenic regions. four of the five sequenced strains had acquired phage gene clusters. four selected phage genes were tested by specific pcr for their presence in a wider array of st- strains and found to represent single events, uncorrelated with any host or source. the human isolate xy possessed a deletion in a gene responsible for surface or flagellar glycosylation, which was not found in other human strains. the bovine strain lacked the gene equivalent to cj, which is a gene of unknown function in a metabolic gene cluster relating to a pyridine nucleotide-disulphide oxidoreductase. the allelic diversity between the strains comprised a large number of synonymous and non-synonymous nucleotide substitutions in coding regions, many of which originated from homologous recombination, and some deletions and insertions in both coding and intergenic regions of all strains. repeat length differences probably originating from slipped strand mispairing mutagenesis were evident in all strains. this concerned eight genes of the glycosylation gene clusters leading to frameshifts in coding regions. we confirmed these repeat length differences in a larger number of st- strains using sanger sequencing. by this extended sequence determination, the repeat length differences obtained by sequencing were confirmed, but none of the tested nucleotide polymorphisms was stably associated with the source of the strain. only a limited number of single nucleotide polymorphisms and repeat differences could be investigated by this labour-intensive approach. aligning the five genome sequences revealed features of genome plasticity in c. jejuni, with a highly conserved overall synteny of the genomes. we detected that frequent homologous recombination events had happened since the divergence of the genomes from a common ancestor, with an average imported length of bp. the genome regions containing clustered polymorphisms caused by recombination are summarized for all five st- genomes in additional file 2: table s they contain genes of various functional categories. none of the functional categories showed significantly higher percentages for having undergone recombination than the average of all functional categories. when the st- genomes were aligned to previously sequenced c. jejuni genomes from unrelated phylogenetic groups in addition to nucleotide polymorphisms, notable differences in gene content were observed. correlating phenotypic variation with genetic differences in st- strains both genotypic and phenotypic differences were observed for st- and other c. jejuni strains, and the genotypic differences might be a basis for a potential adaptive phenotype. from the biolog assays, there was no evidence for a correlation of the two different metabolic biotypes within st- with any specific source. nevertheless, it is worthwhile investigating the gene variants potentially providing phenotypic differences, including those of metabolic capacities detected in the biolog assay. we next asked if genetic polymorphisms within st- that had been identified by the genome sequence analysis could be correlated with the metabolic biotypes. as already described previously, strains of the st- lineage are able to use fucose, due to the presence of a genomic island. as a proof of concept, use of fucose was confirmed for all tested st- strains using the biolog system. within the other tested sts, st- and st- did not display fucose utilization, as expected from previous studies. one of the st- strains selected for whole genome sequencing, the bovine isolate belonged to metabolic biotype and did not metabolize the three dipeptides, tyramine, and m/p-hydroxy-phenylacetic acid. although several genetic polymorphisms found in the genome were investigated in all st- strains belonging to biotype and compared with the corresponding loci of biotype strains, a specific genetic variant which could be responsible for the metabolic phenotype was not identified. several genes and intergenic regions of potential relevance for metabolic activities varied between st- strains. these included two peptidase genes, msra, cmeb, sdha, frda, dcub, ansb, and args. these allelic or intergenic differences were resequenced or tested with allele-specific primers for further isolates of st- and other sts from various sources. none of them could be affirmed to be source-associated or related to metabolic biotype for carbon sources. in addition, rna-based real time pcr was performed for six st- strains, two of biotype and three of biotype, to elucidate whether genes were differentially transcribed, in particular genes of global regulators. these analyses yielded clear differences in transcript abundance between the strains which were uncorrelated with metabolic biotype or source. most conspicuously, strain appeared to have a largely reduced rpon transcript amount, which also seemed to negatively affect luxs transcript, and matched the low observed in vitro motility of this strain. chicken colonization by two genetically closely related st- c. jejuni strains from human and chicken chicken colonization studies have helped previously to determine whether certain c. jejuni variants were infection-competent, including studies that tested strains with presumed adaptation to other hosts that were no longer able to infect chickens. we infected three weeks-old white leghorn chicks with two of the genome-sequenced closely related st- strains, xy and rb. all inoculated animals were colonized by both test strains, which had been verified in advance to possess full motility, since motility is a major prerequisite for successful colonization of campylobacters in any host and subject to strong phase variation mechanisms. during the two-weeks' infection period, the chicken-derived strain colonized chickens slightly better than the human-derived strain. however, the human strain was fully competent to colonize all animals persistently. discussion in order to curb the high numbers of human campylobacter infections, zoonotic transmission to humans needs to be reduced. hence, the identification of specific groups of strains or strain properties which are associated with the colonization of humans or specific animals may help to provide targets for such measures. in the present study, an in-depth genetic and phenotypic characterization of a diverse array of campylobacter strains was initiated, including in particular closely genetically related strains of st- isolated from diverse habitats and isolation sources. from to campylobacter isolates were collected in the framework of the fbi-zoo network from humans, diverse animals and food sources in germany. the complete collection of strains was subjected to mlst analysis, making this one of the most comprehensive mlst analyses of campylobacter from one country outside of the uk. approximately one third of c. jejuni and one fourth of c. coli isolates belonged to five dominant sts, each representing at least 3% of the isolates. many dominant sts are also very prevalent in other geographical regions; as in other european regions, st- which is prevalent in new zealand, was not identified in germany. for c. jejuni, strains of all dominant sts were isolated from both humans and animals/food samples, indicating a broad host range and zoonotic potential. in contrast, approximately two thirds of c. jejuni strains were very diverse and represent rare sts, so that the low number of isolates per st made it impossible to draw valid conclusions about their host association. these and previous data suggest that all dominant sts found in a variety of sources have a "generalist" phenotype, enabling them to colonize both humans and domestic animals. from these hosts, they can then be transmitted to humans either directly or via food. a previous whole genome-based study including strains suggested, that a host-specific campylobacter subtype, or host-specific campylobacter genes, may not exist, particularly in the dominant mlst sequence types, which are mainly derived from domestic sources that may have evolved under conditions of ample recent admixture. this does not rule out that some of the rarer sequence types, such as isolates from wild animals which rarely mix with other niches, have irreversibly adapted to a single host species. evidence for fixed host adaptation in a rare st has been provided recently for sequence type st-, a c. jejuni variant from bank voles, which failed to colonize chickens. while strong evidence of admixture between c. coli and c. jejuni has been presented before, and is supported by the existence of alleles occurring in both c. jejuni and c. coli, the overall frequency of such shared alleles was very low in our collection. calculations of the standardized index of association and other tests for recombination were in agreement with previous studies demonstrating an important role of recombination in shaping the population structure of campylobacter species. notably, the sia of c. coli was much lower than that of c. jejuni, suggesting more efficient exchange of alleles between c. coli strains than c. jejuni strains. c. jejuni and c. coli have been shown to be versatile and diverse species, harbouring numerous genes related to host interactions or metabolism that are not present in all strains, or are subject to phase variation. we have performed an extensive analysis of such genes in a large subset of our collection, and additional reference isolates. c. jejuni strains were studied for the absence/presence and sequence variability of genes with putative roles in phenotypes relevant to host interaction. these included genes coding for surface proteins, los, protein glycosylation and metabolic functions which have been suggested to play an important part in niche- or host-associated colonization abilities. the present data have not provided evidence that any of the genes tested was robustly associated with isolation source. by contrast, pathotyping profiles were in many cases closely correlated with phylogenetic grouping by sequence types, suggesting that campylobacter have diversified their complements of in vivo essential genes. apparently, the host interaction gene complement of dominant sts was selected to provide equal fitness advantages in both domestic animals and humans, permitting strain circulation between humans and domestic animals, making them "generalists". these generalist strains could then make use of different genetic features including reversible frame-shifting of contingency genes, intra-genomic and inter-genomic recombination to fine-tune their genetic and phenotypic capacities, which may benefit their survival in individual hosts or changing environments without preventing their generalist adaptation potential. st- is one of the most prevalent sequence types of c. jejuni present in a broad range of hosts. we have selected st- to analyze the genetic and phenotypic diversity of c. jejuni within a group of closely related isolates, which appear to have generalist properties with respect to domestic hosts. phenotypic characterization and metabolic profiling was performed for eight st- isolates from different sources, and the genomes of five of these strains were sequenced. metabolic profiling identified two characteristic biotypes within st- with distinctive differences in their ability to metabolize certain carbon sources, including some dipeptides. similar to the pathotyping results, none of these biotypes was associated with a specific isolation source, suggesting that both biotypes are compatible with a generalist lifestyle. in addition to differences in their metabolic capacities, the same strains also showed phenotypic diversity in their interaction with eukaryotic cells, independently of their isolation source. a further nine human c. jejuni strains from other rare and frequent sts that were compared using the biolog system showed metabolic fingerprints distinct from each other and also different from all st- strains, revealing the metabolic fingerprint as a distinctive feature between and even within phylogenetic subtypes of the species. however, our study did not provide any evidence for a common metabolic fingerprint of human-derived strains. the genome analysis of five st- strains confirmed their high genetic relatedness, with long stretches of complete sequence identity and generally conserved synteny. however, pairwise and multiple comparisons of all sequences provided evidence of recombination that has caused the exchange of chromosome fragments of varying length, up to ~ kbp. these genome mosaicisms are reminiscent of the situation in the human gastric pathogen helicobacter pylori, where genome comparisons of sequentially isolated strains from one single individual have yielded very similar mosaic patterns to the ones observed here. in particular, the average length of imported fragments in the st- genomes was bp, between two and six times more than a previous estimate based on mlst data only, which could reflect the fact that mlst genes are under strong purifying selection. the import length is about 3- times longer than the average length of imports calculated from h. pylori genome comparisons with the same analytical model. mutation and recombination were estimated to have occurred at similar rates, but recombination introduced times more polymorphisms than mutation as it often substitutes several sites simultaneously. despite the strong evidence for recombination, the gene content in the five st- genomes was very highly conserved. only few strain-specific or unique genes were identified, most of which were part of phage gene clusters. these phage acquisitions represented almost the entire set of gene content differences between the sequenced st- strains. some phage genes were so far unknown; three of the phages were similar to previously described campylobacter phages, largely caused by extensive inter-strain recombination and contingency genes. this corroborated earlier results from microarray comparisons which claimed that c. jejuni strains possess a high level of genome diversity, even within a single sequence type. extended testing for variant alleles of certain candidate genes in other dominant or rare sts or in specific groups of strains from specific host or source did not reveal any fixed host relation. some of these variants may indicate a pressure for phenotypic diversification, for example due to bacteriophage resistance, or might still affect fitness in specific hosts or under certain colonization conditions. we propose that the observed microdiversity may allow these bacteria to flexibly and reversibly adapt to diverse habitats and to propagate their generalist phenotypes. experimental infection of two genome-sequenced st- strains in chickens revealed that a st- strain isolated from a human was fully competent to colonize chickens, albeit to a slightly lower density than a chicken isolate inoculated in a parallel experiment. this suggests that no general colonization barrier for human-derived st- strains exists in chicken. extensive genetic analyses of whole genomes of c. coli and c. jejuni have recently revealed that genetically distantly related isolates show considerable variability in gene content. the authors of this study, however, did not find evidence of statistical association between gene content and type of host. additional work suggested that the observed genetic diversity between c. jejuni isolates were instead closely associated with phylogenetic origin of the strains. in a recent study, a human c. jejuni strain was inoculated in parallel into outbred chickens and inbred genetically modified mice, resulting in a much lower variation in the output pool from the mice compared to that of chickens. in a second experimental system of strain diversification, one specific c. jejuni variant in a contingency gene was strongly enriched by serial mouse passage. this may suggest that, in contrast to a host population of low genetic variability such as inbred mice, campylobacter requires genetic flexibility in a host population consisting of phenotypically and immunologically diverse individuals, including factors such as resident microbiota and bacteriophage abundance. given these findings, it seems plausible that abundant campylobacter types profit in many different ways from their established genome flexibility, even within a single host species.  while our findings do not rule out the existence of phenotypic and genotypic determinants of campylobacter tissue tropism and host specificity, our results from st- genomic and phenotypic comparisons rather support a view where all st- strains are characterized by a generalist genome, phenotype and lifestyle, which support their ability to colonize both humans and food animals. the same might be true for other dominant c. jejuni sts and should be investigated further. this view is also supported by an extended mlst study of campylobacter isolates in the uk. these frequent types may possess a clear fitness advantage under diverse conditions. we propose that future efforts should be directed towards defining the molecular determinants of this phenotypic flexibility and generalist capacity, which might be useful targets for interventions into the transmission cycle, and hence ultimately reduce campylobacter-associated diseases. for their high numbers of human infections caused world wide, and their very broad transmission capacities, the dominant campylobacter sts deserve intensive attention. if the hypothesis of generalist and specialist strain types can be confirmed in larger campylobacter strain collections, it would suggest to study host-specificity or the predictive value of molecular subtyping methods separately for the dominant and the rare st groups. 
a subset of closely related c. jejuni strains of the highly prevalent sequence type st- was selected from different hosts and isolation sources. whole genome sequencing was performed for five st- strains from chicken, human, bovine, and food sources, in order to gain insight into st- genome diversity. the results demonstrated remarkable phenotypic diversity within the st- group, which however did not correlate with isolation source. furthermore, phenotypic variation of these strains was tested using the following criteria: metabolic variation, protein expression patterns, and eukaryotic cell interaction. the genome information and phenotypic data obtained in vitro and in a chicken infection model provided little evidence of fixed adaptation to a specific host. here, we investigate putative host-specificity using phenotypic characterization and genome-wide analysis of genetically closely related c. jejuni strains from different sources. by contrast, no genomic features associated with isolation source or host were identified. campylobacter jejuni and campylobacter coli are human intestinal pathogens of global importance. instead, the dominant c. jejuni st- appeared to be characterized by phenotypic flexibility and high genetic microdiversity, revealing properties of a generalist. high genetic flexibility might allow generalist variants of c. jejuni to reversibly express diverse fitness factors in changing environments. zoonotic transmission from livestock animals or animal-derived food is the likely cause for most of these infections. however, little is known about their general and host-specific mechanisms of colonization, or virulence and pathogenicity factors. pcr typing of strain-variable genes provided evidence that some genes differed between these strains.
 campylobacter jejuni and campylobacter coli are human intestinal pathogens of global importance. zoonotic transmission from livestock animals or animal-derived food is the likely cause for most of these infections. however, little is known about their general and host-specific mechanisms of colonization, or virulence and pathogenicity factors. in certain hosts, campylobacter species colonize persistently and do not cause disease, while they cause acute intestinal disease in humans.  here, we investigate putative host-specificity using phenotypic characterization and genome-wide analysis of genetically closely related c. jejuni strains from different sources. a collection of fresh campylobacter isolates from germany was assembled between and and characterized using mlst. a subset of closely related c. jejuni strains of the highly prevalent sequence type st- was selected from different hosts and isolation sources. pcr typing of strain-variable genes provided evidence that some genes differed between these strains. furthermore, phenotypic variation of these strains was tested using the following criteria: metabolic variation, protein expression patterns, and eukaryotic cell interaction. the results demonstrated remarkable phenotypic diversity within the st- group, which however did not correlate with isolation source. whole genome sequencing was performed for five st- strains from chicken, human, bovine, and food sources, in order to gain insight into st- genome diversity. the comparisons showed extensive genomic diversity, primarily due to recombination and gain of phage-related genes. by contrast, no genomic features associated with isolation source or host were identified.  the genome information and phenotypic data obtained in vitro and in a chicken infection model provided little evidence of fixed adaptation to a specific host. instead, the dominant c. jejuni st- appeared to be characterized by phenotypic flexibility and high genetic microdiversity, revealing properties of a generalist. high genetic flexibility might allow generalist variants of c. jejuni to reversibly express diverse fitness factors in changing environments. 