Background
Campylobacter is one of the most common causes of bacterial gastroenteritis worldwide, outnumbering
Salmonella, toxigenic
Escherichia coli and
Listeria combined [
1,
2]. In the United Kingdom (UK), it is estimated that more than 299,000 cases occur annually [
3,
4]. In most cases, infections are self-limiting, however, some cases result in persistent or invasive infections where antimicrobial therapy may be necessary [
5‐
7]. Diagnosis is usually made by identification using PCR-based rapid detection assays or culture-based isolation of a single presumptive
Campylobacter colony from a stool specimen [
8], which does not identify co-infection with different
Campylobacter species nor the presence of multiple sequence types [
9]. With an estimated minimum infective dose of between 500 and 10,000 organisms,
C. jejuni is responsible for 90% of known human campylobacteriosis infections [
10‐
12]. Intraspecific recombination within
C. jejuni is frequent [
13]. Due to this, genetic exchange events are frequently overestimated in SNP analyses that compare strains at the nucleotide level, which significantly reduces the signals of
C. jejuni population structure [
14]. Other analytical approaches have demonstrated some
C. jejuni populations have undergone clonal proliferation that exhibit a multi-host profile and may account for a large proportion of clinical strains [
15]. A cg/wgMLST genotyping approach demonstrated a lineage of
C. jejuni (ST2254-9-1) that had low genetic variability compared to other lineages of
C. jejuni [
15]. However, typing schemes available for
C. jejuni strain classification continue to be challenging. Diversity profiling using a fragment of
porA gene in
Campylobacter also identified wide diversity within broiler breeder and broiler flocks, indicating a diverse population of
Campylobacter has the potential to transmit through the poultry meat production route [
16].
Poultry meat has been the predominant source attributable to human campylobacteriosis cases [
16,
17]. Further back from the direct exposure to consumers, genetic diversity of
C. jejuni isolated from chicken carcasses at a slaughter plant included multiple genotypes that are associated with strains found in human infections [
18].
Failure to capture the genetic diversity of a
C. jejuni population within a single human case stool specimen may confound source attribution investigations [
9]. Moreover, the replacement of culture-based testing by PCR-based analysis in diagnostic laboratories is eliminating the availability of
C. jejuni isolates, making epidemiological tracking for outbreak investigation near impossible [
19‐
21].
Currently, the most discriminatory method for investigating strain diversity is by using whole genome sequencing (WGS) and analysing the complete genome [
22,
23]. Cao et al. [
24] estimated that the
C. jejuni pangenome consisted of 900 core genes and 4621 accessory genes, based on 173
C. jejuni strains, whilst Rossi et al
. identified 678 core and 2117 accessory genes based on 6526
C. jejuni isolates [
25]. The different sizes of the pangenomes between these studies can be attributed to the different genomes, software and cut-offs used, but both highlight that
C. jejuni has a relatively small core genome compared to its large accessory genome. The genetic variation of the genome within the species is thought to be linked to some strains carrying genes associated with increased pathogenicity in human infection [
24]. Pathogenicity genes associated with the organism’s ability to survive in adverse conditions and possible host specificity have been reported [
24]. The overall diversity of
C. jejuni therefore requires that a large proportion of the population is analysed in epidemiological investigations [
24,
26].
Antimicrobial resistant infections are more difficult to treat, can last longer, and can cause further complications. This increases the costs of healthcare expenses and may further disseminate resistant
Campylobacter in the community [
25,
27]. The WHO has categorised fluoroquinolone resistant
Campylobacter as a priority list pathogen and classified it as a public health threat [
28]. Moreover, in recent years,
C. jejuni derived from human and chicken specimens have been found to contain resistance to β-lactam and tetracycline antibiotics, which are widely used in human medicine [
29,
30]. Since
Campylobacter is known to exchange genetic material [
36], including antimicrobial resistance genes (ARG), the inclusion of resistance determinants is another indicator of intraspecies diversity.
Genetic diversity among multiple isolates can also be described by mapping DNA sequences to a reference genome of the same species to identify variable sites that display single nucleotide polymorphisms (SNPs) [
31]. However, SNP analysis for
C. jejuni has drawbacks as this strategy treats horizontal genetic exchange, locally grouped SNPs acquired in a single event, in the same way as dispersed repeats acquired by multiple events [
32]. Horizontal gene exchange is common between
C. jejuni strains [
33] and so standard SNP analysis without removing putative recombinations is likely to overestimate genetic distance between isolates.
Campylobacter have high frameshift rates that can contribute to genetic diversity and host adaptation through phase variable gene expression [
34].
The aims of this study were to investigate the intraspecies genetic variation of a C. jejuni population at the pangenome level within patients that presented with gastroenteritis and evaluate whether or not this diversity could have been accumulated since the estimated onset of campylobacteriosis.
Discussion
In this study we report the genomic diversity of ninety-two C. jejuni isolates from four clinical stool specimens at the pangenome level. The C. jejuni were cultured using two methods: direct culturing and filtration. For one patient, C. jejuni was only isolated using the filtration method, whilst for the other three patients, C. jejuni was isolated using a combination of direct and filtration methods. For the three stool specimens where C. jejuni were cultured using both methods, no genes or mutations were found to be associated with method of detection. This demonstrates that using the two methodologies increased the chances of culturing Campylobacter but did not have an associated effect on the diversity observed.
In this study, we found the maximum number of core non-recombinant SNPs amongst
C. jejuni isolates belonging to the same sequence type and from the same specimen was 12–43 SNPs. Since
Campylobacter co-infection is known to occur [
9] and genomic diversity generated within a patient through mutation and horizontal gene exchange is frequent [
35], outbreak investigations using single colonies are unlikely to capture the genetic diversity of isolates within patients, which could lead to false conclusions [
35]. Our modelling of SNPs suggests that to capture 95% of the core non-recombinant SNPs from specimens, up to 80 isolates would need to be collected.
In most cases, human campylobacteriosis is self-limiting, however a significant minority of invasive or chronic infections may require antimicrobial therapy [
5,
6].
Campylobacter isolates from humans and chickens have evolved resistance to β-lactam and tetracycline antimicrobials [
29,
36]. In this study, antimicrobial resistance determinants were associated with β-lactam, tetracycline and quinolone resistance. Previous studies have reported 50–61% of
C. jejuni isolates with ampicillin resistance [
37], 50–100% with tetracycline resistance [
38,
39], and 11–40.5% with quinolone resistance [
40,
41]. All of
C. jejuni isolates from patients 1, 3 and 4 contained the
blaOXA-61 gene, responsible for the production of β-lactamase [
29] and associated with ampicillin resistance [
36]. All
C. jejuni isolates collected from patients 2 and 4 and the outlier ST-354 in patient 3, contained the chromosomal T86I mutation in
gyrA associated with quinolone resistance. The single-step T86I amino acid change in the
gyrA gene found in ST-21, ST-354 and ST-2066 of our study is one of the most prevalent resistance mutations on the chromosome associated with decreased
Campylobacter susceptibility to fluoroquinolones [
42] and so this was an expected finding. There is worldwide concern around quinolone resistance [
43‐
45] threatening the treatment of severe
Campylobacter infections in humans [
46,
47], but transmission routes are not clear—understanding the diversity in a single patient will help us to track the movement of resistance.
Campylobacter collected from human patients have been shown to vary in genetic diversity. Dunn et al
. [
48] investigated two
Campylobacter isolates collected from the same patient from separate stool specimens on subsequent days and identified a single SNP difference between them, whilst Cody et al. [
49] investigated twenty patients, comparing two
Campylobacter isolates collected from separate stool specimens and found three patients with isolates belonging to different sequence types and 17 with isolates belonging to the same sequence type but that differed at 3–14 loci (SNP or frameshift differences). In our study, we isolated two sequence types from one of the patients, and a maximum number of 12–43 core non-recombinant SNPs and 0–20 frameshifts amongst isolates belonging to the same sequence type from the same patient. These results indicate more diverse populations of
Campylobacter than in the patient described by Dunn et al
. [
48] and some of the patients described by Cody et al. [
49]. However, many of the patients described by Cody et al
. [
49] had
Campylobacter populations with similar diversity measurements as those described in this study, suggesting that only collecting two isolates from a patient is often unable to capture the diversity of
Campylobacter from patients. Bloomfield et al. [
50] and Baker et al. [
7] investigated
C. jejuni collected from the same patients over several years and found a maximum number of 53–84 core non-recombinant SNPs and 18–19 frameshifts amongst the isolates collected from the two patients, and these mutations were associated with genes involved in cell motility and signal transduction. These associations were not observed in this study, suggesting the selection pressures identified by Bloomfield et al. and Baker et al
. may occur in persistent infections over a longer time period. Frameshifts often occur in genes involved in phase variation and can rapidly accumulate in
C. jejuni populations. Because of their genetic instability it has been argued that these frameshifts should not be used for public health investigations [
22]. However, we also identified core non-recombinant SNPs that are more genetically stable. Bloomfield et al. [
50] and Baker et al. [
7] both used phylogenetic analysis on core non-recombinant SNPs to determine the date of common ancestor for the
Campylobacter collected from each patient to estimate when the patients were initially colonised. However, these estimates assume the long-term patients were not colonised with a heterogenous population. Based on the results from this study they may have overestimated the length of time the long-term patients were colonised with
Campylobacter.
The most distantly related isolates belonging to the same sequence type from each patient shared 12–43 core non-recombinant SNPs. The SNP modelling suggests that we did not detect all SNPs from isolates from the same specimen. Since
C. jejuni has a substitution rate of 1.5–4.5 × 10
–6 substitutions site
−1 year
−1 [
51], that equates to 2–8 SNPs per year, suggesting the isolates may have shared a common ancestor years before the specimens were collected. The exact length of time between when the patient became infected with
C. jejuni and the collection of stool specimens was unavailable for analysis in this study. In previous studies, patients excreted
Campylobacter in their stool for up to 2 months post exposure [
52]. However, the substitution rate estimates were based on long-term colonisation of human patients, and the substitution rate may be higher for
C. jejuni during initial infections. Also, those long-term patients were all colonised with ST-45 and it has been proposed that substitution rates may differ significantly between different lineages of
C. jejuni [
53]. Regardless, we believe there is sufficient genetic diversity demonstrated between isolates in this study collected from the same patient to suggest that all patients were colonised with a genetically diverse population of
C. jejuni. In the case of patient 3, isolates belonging to two sequence types, and in the case of patients 1, 2 and 4, isolates belonging to the same sequence types but genetically diverse in terms of core non-recombinant SNPs. The populations may have become more genetically diverse between infection and specimen collection, but it is unlikely that they accumulated the genetic diversity observed in this study after infection. It is also possible that the patients were exposed to
C. jejuni on multiple occasions, and since three of the patient specimens contained isolates belonging to the same sequence type, multiple exposures to the same source type may be another exposure scenario. The level of diversity amongst the multiple isolates within a patient described here suggests an infection with a genetically diverse population of
C. jejuni through a single source or repeated infections from different sources containing different strains of
C. jejuni, which have persisted in the human host.
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