Abstract.
Campylobacter is a frequently isolated bacterial pathogen among children with diarrhea. Data are lacking on the distribution and spectrum of disease associated with Campylobacter species and Campylobacter jejuni capsular polysaccharide (CPS) types. This information is essential because current vaccine research seeks to target specific CPS types. An effective CPS-conjugate vaccine will need to cover CPS types that are both common and associated with severe disease. The US Naval Medical Research Unit-3 conducted several prospective cohort studies researching diarrheal disease in Egypt from 1995 to 2003. In total, 1,057 children were enrolled and followed to a maximum age of 36 months. We analyzed Campylobacter-positive stool samples that were collected while subjects were symptomatic, along with corresponding clinical data. Of 441 Campylobacter isolates, 322 represented primary infections (189 C. jejuni, 127 Campylobacter coli, six unspeciated). There were 19 C. jejuni CPS types identified; eight accounted for 63.5% of primary C. jejuni infections. We also screened for the presence of the type-6 secretion system (T6SS), a putative virulence determinant. The T6SS was found in 18.0% of C. coli isolates and 57.6% of C. jejuni isolates (P < 0.001), and was not uniformly distributed among CPS types (P < 0.001). Strains with the T6SS were not associated with more severe disease. Clinical presentations across species and CPS types appeared similar. This study adds to the growing epidemiological data and also provides some analysis of the clinical spectrum associated with infection by specific Campylobacter species, C. jejuni capsule types, and possible virulence determinants.
INTRODUCTION
Campylobacter is a zoonotic disease and a frequent bacterial pathogen seen in children with diarrhea.1,2 Little is known regarding the mechanism(s) of Campylobacter virulence.3,4 Campylobacter jejuni expresses a capsular polysaccharide (CPS),5 but the role of CPS in C. jejuni–associated disease is undefined.4 The classification of C. jejuni historically has relied on a passive slide hemagglutination assay for Penner serotyping, for which CPS is the major serodeterminant. More recently, polymerase chain reaction (PCR) has been used to identify CPS type. Currently there are 47 recognized C. jejuni Penner serotypes, represented by 35 CPS complexes.6 The capsules are characterized by the presence of heptoses in various configurations (e.g., altro, gulo) and by nonstoichiometric modification of the sugars with O-methyl-phosphoramidate (MeOPN).7 Campylobacter jejuni is also one of several gram-negative bacteria known to harbor the type-6 secretion system (T6SS).8,9 The T6SS has been associated with both pathogen–pathogen and pathogen–host interactions among certain diarrheal agents, including Vibrio cholerae.9 Its role in C. jejuni is unclear, although it may be associated with invasive disease.8 The distribution of the T6SS among C. jejuni CPS types and among other Campylobacter species is unknown.
Although symptomatic infections impact children and adults in industrialized nations, in the developing world Campylobacter enteritis is almost exclusively a pediatric disease.2 Seroprevalence of C. jejuni–specific IgG antibodies increases with age and older patients are much more likely to develop infections caused by uncommon CPS types compared with their younger counterparts.10 These findings suggest that prior infection may confer some degree of CPS-specific immunity. CPS has been an effective target for other vaccines against mucosal pathogens including Streptococcus pneumoniae.11 In addition, preliminary studies demonstrate that C. jejuni CPS-conjugate vaccines protect against diarrheal disease in nonhuman primates.7 With 35 known C. jejuni CPS types, an efficacious vaccine will need to target prevalent and disease-associated strains.
The distribution of C. jejuni CPS types along with their contribution to morbidity has not been defined worldwide. A recent meta-analysis evaluated 21,000 sporadic cases of C. jejuni infection, of which nearly 85% came from Europe; there were only 566 samples from Africa. HS4 was the most common C. jejuni CPS type overall (15%) and within Africa (7%). Three C. jejuni CPS types, (HS1/44, HS2, and HS4), accounted for 37% of the total isolates; however, there appeared to be a greater distribution of CPS types in Africa, where HS1/44, HS2, and HS4 combined represented just 20% of the isolates.12
In the current study, we sought to expand the epidemiological data regarding Campylobacter, while evaluating for associations between clinical presentation, Campylobacter species, C. jejuni CPS types, and the T6SS.
MATERIALS AND METHODS
In collaboration with the Egyptian Ministry of Health,13 the Naval Medical Research Unit Number 3 (NAMRU-3) and the National Institutes of Health (NIH) began active surveillance for pediatric diarrhea in Abu Homos, Egypt, in February 1995, as part of an observational pediatric diarrhea cohort study.14 Abu Homos is an agricultural community of approximately 350,000 persons in the Nile Delta in northern Egypt, located 35 kilometers (22 miles) southeast of Alexandria. Before the start of the initial cohort in February 1995, a team of investigators, field workers, and data management personnel mapped and numbered every house in the 16-village complex. A detailed house-by-house census was conducted, with periodic updates to ensure accurate data. With guardian consent, children younger than 24 months of age were enrolled in a prospective study to evaluate pediatric diarrhea in this region. Subsequently, newborns were eligible for enrollment.
From February 1995 through December 2006, four prospective studies of at least 3-year duration were undertaken. Each study was approved by the Institutional Review Board at NAMRU-3 and was overseen by infectious disease physicians and epidemiologists located at NAMRU-3 in collaboration with the Egyptian Ministry of Health. Surveillance methodology was consistent across all studies. Specifically, from enrollment to 36 months of age, each participant was visited twice weekly in the home. Trained field workers administered structured questionnaires soliciting data on the child’s health. Diarrhea was defined as at least three loose or liquid stools or one bloody stool in a 24-hour period. For breast-fed infants, the mother also must have indicated an increase in the frequency or decrease in consistency of stools, in relation to normal stool patterns. A diarrheal episode began on the first diarrheal day following at least three nondiarrheal days, and ended on the last diarrheal day followed by at least three consecutive nondiarrheal days. For all but the last study, in which surveillance was halted after children were 24 months of age, surveillance was continued until the third birthday or until study completion.
Stool samples were inoculated into Cary-Blair transport medium and stored at the project field laboratory for up to 4 days at 4°C before being transported to the laboratory at NAMRU-3. At NAMRU-3, all samples were inoculated on a modified Skirrow’s medium and grown for 48 hours in a microaerophilic environment at 42°C. Colonies that appeared grossly consistent with Campylobacter were confirmed by Gram stain, oxidase and catalase activity, and sensitivity to nalidixic acid. All laboratory-confirmed Campylobacter samples have remained frozen at −80°C.
Isolates were sent to the Naval Medical Research Center in Silver Spring, MD, where they were regrown on Mueller-Hinton agar. DNA was extracted from each sample for PCR analysis. The PCR consists of a C. jejuni CPS multiplex with 35 capsule primer sets spread over four mixes: alpha, beta, gamma, and delta.6 In addition, all strains were tested for a C. jejuni amplicon.15 When negative for C. jejuni, they were additionally tested for a C. coli amplicon.15 Because the hcp gene is a consistent marker of the complete T6SS cluster,8,9 samples were screened for the presence of the hcp gene using a multiplex PCR.16
Infections were categorized as primary (no prior Campylobacter infections isolated) and nonprimary (isolation of any heterologous capsule type following the primary Campylobacter infection or isolation of a homologous capsule type ≥ 90 days from the primary Campylobacter infection). Isolates that were found to be homologous, and obtained from the same subject within 90 days, were excluded from the analysis.
The percentage of infections caused by each Campylobacter species and CPS type was determined along with the corresponding proportions that were positive for the T6SS. The prevalence of the T6SS was estimated by species for all symptomatic infections and limited to primary symptomatic infections. The distribution of Campylobacter species and CPS type, along with the prevalence of each clinical characteristic, was analyzed by age category. The prevalence of clinical disease signs and symptoms was compared by species, CPS type, presence of T6SS, and presence of heptose, deoxyheptose, and MeOPN.
This study protocol was approved by the Naval Medical Research Center Institutional Review Board in compliance with all applicable regulations governing the protection of human subjects. A Mantel–Haenszel χ2 test of homogeneity was used to compare proportions across various categories, whereas Student’s t test was used for comparisons of continuous variables. Confidence intervals (CI) for point estimates of prevalence were calculated using Exact Binomial proportions at the 95% level. All analyses were conducted using Stata V13 (SataCorp, College Station, TX) and statistical significance assessed using a P value = 0.05.
RESULTS
A total of 1,057 children were enrolled and 6,562 stools collected. Campylobacter was isolated in 615 samples from symptomatic subjects; 54 samples were excluded from the analysis based on our criteria, and another 120 samples were no longer viable and so were not analyzed. As shown in Table 1, of the 441 Campylobacter isolates tested, 322 were from primary symptomatic Campylobacter infections, whereas 119 were from nonprimary symptomatic Campylobacter infections. Most isolates (N = 272, 61.7%; 95% CI: 57.0–66.2%) were speciated as C. jejuni, whereas 37.0% (N = 163; 95% CI: 32.4–41.7%) were C. coli and 1.4% (N = 6; 95% CI: 0.5–2.9%) were unable to be speciated. The majority of all Campylobacter isolates came from the primary symptomatic infection (C. jejuni: 69.4%, C. coli: 77.9%, C. non-jejuni/non-coli: 100%).
Table 1.
Distribution of Campylobacter capsular polysaccharide (CPS) types causing diarrheal illness in a pediatric population living in Abu Homos, Egypt, from 1995 to 2006
| Campylobacter species | Campylobacter jejuni capsule type | Primary infection | Nonprimary infection | All infections | hcp*+ |
|---|---|---|---|---|---|
| All | N/A† | 322 | 119 | 441 | 182/424 (42.9%) |
| Untypable | N/A† | 6 | 0 | 6 | 5/6 (83.3%) |
| Campylobacter coli | N/A† | 127 | 36 | 163 | 29/161 (18.0%) |
| C. jejuni | All | 189 | 83 | 272 | 148/257 (57.6%) |
| C. jejuni capsule type | NT‡ | 21 (11.1%) | 11 (13.3%) | 32 (11.8%) | 24/31 (77.4%) |
| HS1/44 | 10 (5.3%) | 5 (6.0%) | 15 (5.5%) | 8/13 (61.5%) | |
| HS2 | 19 (10.1%) | 12 (14.5%) | 31 (11.4%) | 9/27 (33.3%) | |
| HS3 | 22 (11.6%) | 9 (10.8%) | 31 (11.4%) | 26/29 (89.7%) | |
| HS4 | 18 (9.5%) | 4 (4.8%) | 22 (8.1%) | 11/21 (52.3%) | |
| HS5/31 | 9 (4.8%) | 10 (12.0%) | 19 (7.0%) | 7/17 (41.2%) | |
| HS6/7 | 12 (6.3%) | 5 (6.0%) | 17 (6.3%) | 5/17 (29.4%) | |
| HS8/17 | 1 (0.5%) | 0 (0.0%) | 1 (0.4%) | 1/1 (100.0%) | |
| HS9 | 3 (1.6%) | 1 (1.2%) | 4 (1.5%) | 2/2 (100.0%) | |
| HS10 | 12 (6.3%) | 6 (7.2%) | 18 (6.6%) | 12/18 (66.7%) | |
| HS12 | 1 (0.5%) | 0 (0.0%) | 1 (0.4%) | 1/1 (100.0%) | |
| HS15 | 18 (9.5%) | 7 (8.4%) | 25 (9.2%) | 11/25 (44.0%) | |
| HS18 | 1 (0.5%) | 0 (0.0%) | 1 (0.4%) | 1/1 (100.0%) | |
| HS19 | 4 (2.1%) | 0 (0.0%) | 4 (1.5%) | 1/4 (25.0%) | |
| HS22 | 8 (4.2%) | 1 (1.2%) | 9 (3.3%) | 9/9 (100.0%) | |
| HS23/36 | 9 (4.8%) | 2 (2.4%) | 11 (4.0%) | 9/11 (81.8%) | |
| HS41 | 5 (2.6%) | 1 (1.2%) | 6 (2.2%) | 1/6 (16.7%) | |
| HS42 | 8 (4.2%) | 4 (4.8%) | 12 (4.4%) | 2/11 (18.2%) | |
| HS52 | 0 (0.0%) | 1 (1.2%) | 1 (0.4%) | 0/1 (0.0%) | |
| HS53 | 8 (4.2%) | 4 (4.8%) | 12 (4.4%) | 8/12 (66.7%) |
Bold values indicate the two-tailed P value for Chi- square test of homogeneity, comparing C. coli to C. jejuni and untypable to C. jejuni, are both <0.001.
hcp, a genetic marker for the type 6 secretion system (denominator is different because not all isolates were available for hcp testing).
N/A = not applicable (i.e., not C. jejuni).
NT = nontypeable C. jejuni capsule.
Among the primary Campylobacter infections, 103 (31.9%) were co-infected with Shigella (1.6%), rotavirus (13.0%), and/or enterotoxigenic Escherichia coli (19.8%). Of these 103, 57.3% (N = 59) were C. jejuni and 40.8% (N = 42) were C. coli infections. Subjects who were co-infected were 1.5 times more likely to have decreased activity (CI: 1.22–1.93) and were 1.5 times more likely to experience vomiting (CI: 1.02–2.29) than those who were not co-infected. There were no other significant differences in the clinical presentation between those with and without a co-infection.
A CPS type was identified in 88.2% (95% CI: 83.8–91.8%) of all C. jejuni isolates with the most prevalent types being HS2 (11.4%), HS3 (11.4%), HS15 (9.2%), HS4 (8.1%), HS5/31 (7.0%), HS10 (6.6%), HS6/7 (6.3%), and HS1/44 (5.5%). Although those eight capsule types accounted for 65.4% (95% CI: 59.5–71.1%) of the C. jejuni isolates, no other capsule type accounted for more than 5%. In addition, no capsule type was identified in 11.8% (N = 32; 95% CI: 8.2–16.2%) of the C. jejuni isolates. HS5/31 was identified more commonly among nonprimary symptomatic C. jejuni infections (12.0%) than in primary infections (4.8%) (Fisher’s Exact P value: 0.04). There were no other differences in the proportion of C. jejuni capsule types in primary and nonprimary symptomatic C. jejuni infections.
Among the 424 isolates available for hcp analysis, the hcp gene was found in 29 (18.0%; 95% CI: 12.4–24.8%) C. coli isolates compared with 148 (57.6%; 95% CI: 51.3–63.7%) C. jejuni isolates and five (83.3%; 95% CI: 35.9–99.6%) unspeciated isolates (P < 0.001). Among the identified C. jejuni capsules, hcp prevalence varied significantly from 0% to 100%. The most prevalent hcp positive capsule type was HS3 in which 26 of the 29 (89.7%; 95% CI: 72.7–97.8%) tested isolates had the hcp gene.
The mean number of loose stools for each primary C. jejuni–attributed illness was 6.7. In addition, 23.9% experienced vomiting, 22.2% experienced dehydration, 35.2% had a documented fever, 4.5% experienced hematochezia, and just under 50% had parental report of lower than normal activity levels. There were no significant differences in the clinical profile of cases with hcp+ C. jejuni and those with hcp− C. jejuni infections. Primary C. coli–attributed illnesses had a similar clinical presentation to primary C. jejuni–attributed illnesses with no significant differences in the prevalence of signs and symptoms or in the frequency of stool output per day. Campylobacter coli isolates with the hcp gene were associated with a higher number of loose stools than C. coli isolates without the hcp gene (P = 0.001); however, no other significant differences were observed (Table 2).
Table 2.
Clinical features of primary Campylobacter infection by species and presence of the hcp* gene
| Campylobacter jejuni | Campylobacter coli | |||||||
|---|---|---|---|---|---|---|---|---|
| All (N = 176)† | hcp+ (N = 101) | hcp− (N = 75) | P value | All (N = 126)† | hcp+ (N = 22) | hcp− (N = 104) | P value | |
| Number stools/day, mean (std dev) | 6.51 (2.78) | 6.52 (2.57) | 6.49 (3.05) | 0.941 | 6.36 (3.07) | 8.23 (3.60) | 5.96 (2.81) | 0.001 |
| Vomiting, n (%) | 42 (23.9%) | 21 (20.8%) | 21 (28.0%) | 0.267 | 26 (20.6%) | 6 (27.3%) | 20 (19.2%) | 0.397 |
| Dehydration, n (%) | 39 (22.2%) | 27 (26.7%) | 12 (16.0%) | 0.090 | 34 (27.0%) | 7 (31.8%) | 27 (26.0%) | 0.574 |
| Fever, n (%) | 62 (35.2%) | 33 (32.7%) | 29 (38.7%) | 0.410 | 34 (27.0%) | 9 (40.9%) | 25 (24.0%) | 0.105 |
| Hematochezia, n (%) | 8 (4.5%) | 3 (3.0%) | 5 (6.7%) | 0.244 | 1 (0.8%) | 0 (0.0%) | 1 (1.0%) | 0.644 |
| Low activity, n (%) | 85 (48.3%) | 51 (50.5%) | 34 (45.3%) | 0.498 | 56 (44.4%) | 11 (50.0%) | 45 (43.3%) | 0.564 |
hcp, a genetic marker for the type 6 secretion system.
Denominators for total C. jejuni and C. coli are 176 and 126, rather than 189 and 127, respectively, because not all isolates were available for hcp testing.
Among primary C. jejuni symptomatic infections for which we identified a capsule type, low activity was reported more commonly from cases with MeOPN negative C. jejuni capsules (63.2%) compared with MeOPN positive (42.3%) (P = 0.02). A similar trend was seen with heptose, although the difference was not statistically significant (P = 0.06). There appeared to be no difference in the clinical profile of primary C. jejuni infections by strains predicted of expressing CPS containing deoxyheptose (Table 3).
Table 3.
Clinical features of primary Campylobacter jejuni infections by putative CPS* structures
| All CPS types(N = 168) | O-methyl-phosphoramidate (MeOPN) | Heptose | Deoxyheptose | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Present (N = 130) | Absent (N = 38) | P value | Present (N = 130) | Absent (N = 38) | P value | Present (N = 111) | Absent (N = 57) | P value | ||
| Number stools/day, mean (std dev) | 6.72 (2.92) | 6.80 (2.94) | 6.45 (2.85) | 0.51 | 6.60 (2.95) | 7.13 (2.82) | 0.33 | 6.63 (2.99) | 6.89 (2.79) | 0.58 |
| Vomiting, n (%) | 41 (24.4%) | 28 (21.5%) | 13 (34.2%) | 0.11 | 47 (36.2%) | 12 (31.6%) | 0.60 | 25 (22.5%) | 16 (28.1%) | 0.43 |
| Dehydration, n (%) | 38 (22.6%) | 32 (24.6%) | 6 (15.8%) | 0.25 | 27 (20.8%) | 11 (28.9%) | 0.29 | 25 (22.5%) | 13 (22.8%) | 0.97 |
| Fever, n (%) | 59 (35.1%) | 47 (36.2%) | 12 (31.6%) | 0.60 | 47 (36.2%) | 12 (31.6%) | 0.60 | 40 (36.0%) | 19 (33.3%) | 0.73 |
| Hematochezia, n (%) | 7 (4.2%) | 5 (3.8%) | 2 (5.3%) | 0.70 | 7 (5.4%) | 0 (0.0%) | 0.14 | 6 (5.4%) | 1 (1.8%) | 0.26 |
| Low activity, n (%) | 79 (47.0%) | 55 (42.3%) | 24 (63.2%) | 0.02 | 56 (43.1%) | 23 (60.5%) | 0.06 | 49 (44.1%) | 30 (52.6%) | 0.30 |
CPS = capsular polysaccharide.
DISCUSSION
This study describes the distribution of Campylobacter species and C. jejuni CPS types within a birth cohort in rural Egypt. Of the isolated species of Campylobacter, 61.7% were due to C. jejuni, and C. coli accounted for 37.0%. Although the prevalence of C. coli is higher than reported in South Africa (3%),17 Nigeria (17%)18 and other developing countries,19–21 it is similar to prevalence reported by Georges-Courbot et al.22 in Bangui (39%). Only six isolates were not speciated as C. jejuni or C. coli.
Data on C. jejuni CPS-type distribution in pediatric populations within the developing world are limited. Pike et al.12 recently reviewed the published literature on CPS distribution and found that HS1/44, HS2, and HS4 are the CPS types reported worldwide most commonly, isolated in 37% of infections. These three CPS types were identified in 25% (95% CI: 20.0–31.6%) of our C. jejuni isolates. Combined, eight CPS types accounted for 65.4% (95% CI: 59.5–71.1%) of C. jejuni; higher than the 50.4% reported by Pike et al.12 This may be due to the fixed pediatric population or the limited period of study compared with the global distribution over a longer time period and various populations included in the systematic review.12
Similar to a report by Lee et al.,21 we were unable to clinically differentiate C. jejuni and C. coli infections. In addition, our study was limited to the available number of infections identified during active surveillance. As such, although there were variations in the prevalence of specific signs and symptoms across capsule types, our power to detect significant differences was limited. Recent studies suggest that specific C. jejuni capsule structures contribute to virulence. MeOPN appears to enhance rates of colonization of an HS2 strain in piglets23 and loss of a heptose branch from the same HS2 strain has been associated with decreased susceptibility to bile salts and decreased colonization in chickens.24 MeOPN also has been shown to contribute to serum resistance in vitro for both HS2 and HS23/36.25 It is unclear how these structures may impact clinical illness in the host; however, we observed no increase in the severity or frequency of clinical signs and symptoms in subjects infected with strains predicted to express capsules with MeOPN, heptose, or deoxyheptose.
Few reports have investigated the T6SS within Campylobacter. Corcionivoschi et al.26 reported that 56.1% of C. coli and 28.8% of C. jejuni isolated from retail chicken meat were hcp-positive in the United Kingdom. Harrison et al.8 analyzed 181 C. jejuni strains from human, chicken, and environmental sources in the United Kingdom, Vietnam, Pakistan, and Thailand. By region, he observed significant variability in hcp-positivity, from 7.6% in the United Kingdom to 54.4% in Vietnam.8 Similar to the prevalence of hcp-positive strains in Vietnam, we found that 57.6% of our primary C. jejuni isolates were positive for the T6SS, which contrasts with the < 30% prevalence in C. jejuni isolates from retail chicken meat in the United Kingdom. Interestingly, Bleumink-Pluym et al.16 reported that the T6SS was only present in 10% of C. jejuni isolates from animal and human infections but that among the T6SS positive strains, 50% (4/8) had caused bacteremia. Our comparison of clinical signs and symptoms across isolates, however, with and without the T6SS, was largely unrevealing. This variability may be due to the small number of T6SS positive C. jejuni isolates tested by Bleumink-Pluym et al.16 or potential variability across these study populations.
Our study had several inherent limitations. First, this study was not a priori designed to address the research questions addressed here and may have been insufficiently powered to address our stated hypotheses. Such studies, however, are rarely conceived prospectively for a specific enteropathogen. Future studies should use the estimates from this study in framing similar research questions. In addition, approximately 30% of the Campylobacter cases were co-infected with more than one enteropathogen confounding disease attribution. Our methods assumed the presence of the T6SS and molecules composing CPS structure by the presence of a specific gene, which may be an inaccurate assumption. We did not account for asymptomatic infections, unidentified co-pathogens, or nutritional status, all of which may have impacted our results.27,28
We have described the epidemiology of Campylobacter among an Egyptian pediatric population. These data highlight the variability in Campylobacter species as well as C. jejuni CPS diversity in this population. In addition, we observed a high prevalence of the T6SS, particularly among C. jejuni isolates. We found no significant associations between clinical illness and Campylobacter species, C. jejuni CPS types, or suspected virulence factors. Further prospective birth cohort studies are needed to determine the spectrum of disease associated with specific factors and the role of infection in establishing immunity.
Acknowledgments:
We would like to acknowledge Nashiva McDavid and Caroline Tribble for their assistance with laboratory procedures.
Disclaimer: The views expressed in this article are those of the authors and do not reflect the official policy or position of the United States Army, Department of Defense, or the US Government. Title 17 U.S.C 105 provides that “copyright protection under this title is not available for any work of the United States government.” Title 17 U.S.C. 101 defines a United States government work as a work prepared by a military service member or employee of the United States government as part of that person’s official duties. RS, FP, PG, MSR and CP are military service members or civil servants and this work was prepared as a part of their official duties.
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