Introduction
Campylobacter jejuni is recognized as a major pathogen of gastroenteritis worldwide [
1]. As illustrated by the latest epidemiological report,
Campylobacter is the main cause of foodborne illness in Europe and the United States [
1]. Campylobacteriosis cases range from 14 to 57 per 100,000 per year (US and Europe, respectively). In endemic regions, particularly in Southeast Asia, campylobacteriosis cases are estimated to be 10 times higher than in Europe and the USA. This high incidence of
Campylobacter is well documented in children, travelers, and foreign military personnel from Thailand [
2‐
4].
C. jejuni is considered a zoonotic disease. The major source of contamination is through consumption of improperly prepared or stored foods containing poultry, but other sources like unpasteurized milk or water have been documented. The infectious dose can be as low as 500 to 1000 bacteria [
5]. Campylobacteriosis symptoms range from mild abdominal pain and mild to no diarrhea, to severe abdominal cramping, sometimes accompanied with fever, headache, myalgia, and large volumes of mucous and bloody diarrhea that can last for several days [
6].
Despite a worldwide prevalence, there is a paucity of data regarding
C. jejuni virulence factors. Nevertheless, flagella, cytolethal distending toxin (
cdt), fibronectin binding protein (
cadF), lipoprotein (
ceuE) [
7‐
9], and proteins involved in adherence and invasion acquired by plasmids (
pVir) [
10] contribute to virulence. The
Campylobacter polysaccharide capsule (CPS) is the best characterized virulence factor. Mutants deficient in CPS production demonstrated lower adherence and binding in vitro, decreased serum resistance, and reduced colonization of ferret and chicken animal models [
7,
11]. Phenotypic assays can be routinely applied for identification and classification of putative
C. jejuni samples in laboratory settings [
12]. Penner’s serotyping, developed in 1980s, is a well-known serotyping scheme for
C. jejuni and
C. coli that is based on capsular polysaccharide [
13]. Molecular assays have replaced this method for routine
C. jejuni and other
Campylobacter spp. identification. Analyses of different CPS sequences were utilized to develop multiplex PCR assays to distinguish more capsule types among
C. jejuni isolates [
14‐
17]. This capsule typing scheme correlates well to Penner serotypes because the capsule structure is the major serodeterminant [
14].
In addition to the virulence factors aforementioned, additional
C. jejuni isolates were characterized and found to possess the recently described T6SS [
18,
19]. The T6SS apparatus is composed of proteins which structurally and functionally are related to contractile components of bacteriophages [
20]. The hemolysin co-regulated protein (Hcp) in
Pseudomonas aeruginosa T6SS resembles the major component of the T4 phage tail base plate [
21]. The T6SS contributes to bacterial pathogenesis in
Pseudomonas aeruginosa [
22],
Vibrio cholerae [
23],
Salmonella enterica [
24],
Helicobacter hepaticus [
25]
, Edwardsiella spp. [
26],
Burkoderia mallei [
27], as well as in
C. jejuni [
19].
Campylobacter T6SS plays an important roles in host cell adhesion, invasion, and persistent colonization in vivo [
19]. T6SS was reported in
C. jejuni isolated from clinical, poultry, and water sources, and its distribution varied in human and animal sources from different countries [
28‐
30]. However, the distribution and role of T6SS pathogenesis in
C. jejuni from human gastroenteritis has yet to be further determined.
A myriad of post infectious sequelae were linked to
C. jejuni infections. These include autoimmune mediated Guillain–Barré (GBS) and Miller Fisher (MF) syndromes [
31]. These autoimmune diseases are provoked by mimicry of
C. jejuni lipooligosaccharides (LOS) structures that contain
N-acetyl neuraminic (sialic acid), organized into structures that resemble human gangliosides [
31]. Most
C. jejuni strains contain genes for the endogenous synthesis of
N-acetyl neuraminic acid, and there are specific genes involved in the biosynthesis of these ganglioside mimics [
32]. The
cgtA (β-1,4-
N-acetylgalactosyl transferase) and
cgtB (β-1,3-galactosyltransferase) and
cstII (α-2,3 sialyltransferase) are required for molecular mimicry and are associated with human GBS. These genes were used as markers to screen for GBS-related strains of
C. jejuni [
33‐
35]. Multiple variants of
cstII transferase were characterized in the
C. jejuni LOS locus, including
cstIIHS19 and c
stIIHS2 [
33]. The gene
cstII has mono and bi-functional activities (α-2,3 and/or α-2,3/α-2,8-sialyltranferase), and both represent different specificity of enzymes involved in the transfer of the sialic acid residue to the LOS structure [
32,
36]. Genes in LOS biosynthesis loci were studied from different origins of
C. jejuni, and the LOS classes A, B, and C were classified for involvement in human ganglioside mimicry. Recently, LOS classes M and R were reported to possess genes similar to LOS classes A and D, including a gene encoding a sialyltransferase (
cstII) [
37].
The aim of this study was to determine the distribution of genes associated with T6SS and LOS implicated in GBS among different capsule types in clinical C. jejuni isolated from stool samples of travelers and children suffering diarrhea in Thailand.
Discussion
In this study, HS4 and HS2 were the most common
C. jejuni capsule types from traveler and pediatric diarrhea cases, respectively. These two capsule types were the two most common Penner serotypes in global human
C. jejuni isolates, confirming previous reports [
41]. Other capsule types exhibited varied distribution among isolates from travelers and children. The study of traveler’s diarrhea isolates included
C. jejuni, of which > 80% were obtained from stool samples of military personnel attending annual exercises from different locations in Thailand. A point-source of infection of an identical
C. jejuni clone isolated from the same type of food consumed might attribute a distribution of one capsule type over the others (e.g., HS53, HS23/36). The different capsule types seen in travelers may be affected by temporal variations during the exercises of military personnel, and by the contaminated food consumed by travelers at the visiting sites (e.g., resort or hotel), but this was not seen in isolates from children in which samples were collected over a 2 year period. Although
C. jejuni isolates from these two studies were from more than 5 years apart, the five most common capsule types from traveler isolates (HS4, HS2, HS5/31, HS8/17, and HS3) and Thai children isolates (HS2, HS 8/17, HS4, HS5/31, and HS3) were comparable to capsule serotypes of global
C. jejuni isolates reported previously [
41].
T6SS are bacteriophage-like structures which secrete effector molecules into eukaryotic cells or other bacteria [
20,
42]. The presence of a secreted effector, the
hcp needle protein, in culture supernatants suggested contribution of this protein to virulence of
C. jejuni strains [
18,
19]. The
hcp gene was used as a surrogate for the presence of T6SS in
C. jejuni. Using this screening method on different
C. jejuni isolates, heterogeneous results were reported. Harrison et al. [
28] demonstrated the prevalence of
hcp in 60.6% and 33.3% from clinical
C. jejuni isolates in Vietnam and Thailand, respectively. However, only three human
C. jejuni isolates from Thailand were studied. None of the T6SS genes were detected in 34
C. jejuni isolated from human diarrheal cases in Pakistan [
29]. In our study,
hcp was found in 62.6% of the 524 cases, and this proportion was similar to studied isolates in Vietnam (60.6%) and Egypt (57.6%) [
28,
43].
The
hcp gene was associated with 10 capsule types of
C. jejuni isolates whereas a variable
hcp prevalence was reported by others. Sainato et al. [
43] reported a
hcp gene prevalence of 33.3% in HS2 isolates, but up to 86.2% was noted in this study. Moreover, numbers of
hcp-
C. jejuni isolates were more significant in children than travelers (22.2%, 86.7%;
p = 0.0001), especially in HS8/17 isolates. Different distributions of the
hcp gene were also observed among HS4
cpx isolates (28.5% of HS4
cpxB, 92.5% HS4
cpxA, and 100% of HS4
cpxAB). Our study was limited in that we tested for only one gene marker in T6SS, while the T6SS locus composed of at least 13 ORFs [
18]. A fully-functional T6SS was only reported in two
C. jejuni strains in published articles [
18,
19]. Further investigation on more conserved genes in the T6SS locus and expression of their encoded proteins may implicate the contribution of T6SS to
Campylobacter virulence.
Hcp was previously reported to be associated with bloody diarrhea [
28]. We found no significant association between
hcp and
C. jejuni isolates of any specific capsule type. This is similar to the finding by Agnetti et al. [
44], where no significant difference in the clinical manifestations and the course of disease between patient with hcp-positive and -negative
C. jejuni isolates. Similarly, the presence of
hcp gene in
C. jejuni and
C. coli from stool did not have any significant association to clinical outcome in Egyptian children [
43].
Campylobacter jejuni and their LOS classes could be distinguished by different LOS biosynthesis locus, as referenced by others [
32,
33,
39]. Parker et al, reported
orf locus 7ab and 7c to encode for
cstII and
cstIII, respectively. In our study, the primers for four genes, c
gtA,
cgtB,
cstIIHS19 and
cstIIHS2, were used following Nachamkin, et al. [
35]. The amplified products representing clinical
C. jejuni of different capsule types, shown in Fig.
4, were sequenced and analyzed with BLASTn to identify each gene. The alignment matched within locus of
cgtA,
cgtB,
cstII, and cstIII, respectively. In this study, we focused on LOS classes A to C that can cause sialylated LOS synthesis resulting in ganglioside mimicry, which are crucial for ganglioside mimicry from various
C. jejuni strains, as previous reported [
32,
39,
45‐
47]. The GBS genes for LOS classes A, B are predominately amongst
C. jejuni HS19 isolates [
47]. All 524
C. jejuni isolates were associated with gastroenteritis (GI). C
stIIHS19 was previously reported in GBS-HS19 isolates more than in GBS-non HS19 isolates [
35]. In this study, almost 50% (23.3 and 24.4% in Table
2) of the GI-isolates were classified as LOS classes A or B, but only one isolate was HS19 serotype. GBS was commonly associated with HS1/44-, HS2-, and HS4-
C.
jejuni antecedent infections in the Netherlands, whereas HS19-, HS 23/36-, and HS41-
C. jejuni were associated with GBS in Bangladesh, Japan, and South Africa [
47]. Our study demonstrated a high prevalence of HS1/44, HS2, HS23/36, and HS4
cpx, but the HS41 serotype was not detected from the GI-isolates. Distribution of LOS classes A, B, and C in
C. jejuni has been previously demonstrated, as 17%, 32%, and 19%, respectively, but there are data linked to only 5 Penner serotypes from these
C. jejuni isolates [
47]. Parker et al. [
39] reported that 64% of non-GBS associated isolates were in LOS classes (A, B, or C), and this is comparable to our study that 56.9% of
C. jejuni isolated were in LOS classes A, B, and C. A limitation of our study was that the primers used for LOS associated GBS genes could not amplify gene targets to differentiate LOS subclasses A1, A2, B1 and B2, and other LOS loci diversity [
37,
39,
48,
49]. More primers specific to other LOS classes should be included to characterize these
C. jejuni isolates in more depth, especially in 63
C. jejuni isolates with unclassified LOS locus.
Differences in association of CPS and MLST types were also seen among these
C. jejuni strains compared to published studies. Heikema et al. [
47] observed that HS23/36 isolates from GBS patients were associated with a MLST clonal complex (CC)-403, -42, and -206, but most of HS23/36-GI isolates in this study were MLST CC-52 (> 90%). Similarly most of GBS-HS2 isolates in the Heikema study were MLST CC-21, but our GI-HS2 isolates were MLST-CC-464 (83%) and unassigned CC (27%). HS4-GBS and enteritis isolates were MLST CC-48, -61, -206, and -508 (Heikema), while most of GI-isolates in our study were MLST-52, -460, and unassigned CC (data not shown). This suggested there was a selective and divergent clone lineage of
C. jejuni GBS- and GI-strains; however, many other factors likely also contribute to
Campylobacter virulence.
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