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Epsilonproteobacteria in humans, New Zealand.

Acute gastrointestinal illness is a major health concern in industrialized countries. In New Zealand, an estimated 4.6 million cases of acute gastrointestinal illness occur every year (1). For many known causes of acute gastrointestinal illness, conventional methods of diagnosis are available; yet, [approximately equal to] 80% of diarrhea cases go undiagnosed (1,2). This lack of data concerning causes of diarrhea hinders the development of intervention strategies.

The class Epsilonproteobacteria is a distinct, diverse bacterial group containing [approximately equal to] 100 taxa (3), including Campylobacter jejuni, recognized as the most frequent bacterial cause of human gastroenteritis worldwide (4,5). Many other epsilonproteobacterial species have been associated with diarrhea, but accurate estimates of the prevalence and role of individual species and proof of a primary pathogenic role have been elusive. Methods commonly used for isolating C. jejuni are not well suited for many other species, and the complex taxonomy of the group makes identification difficult (4). Nevertheless, the body of evidence supporting a causative role for several taxa has grown (5-7). During September 2007-June 2009, we examined fecal samples from healthy volunteers and from patients with diarrhea in New Zealand by using a PCR-denaturing gradient gel electrophoresis (DGGE) method shown to detect and identify Epsilonproteobacteria (8).

The Study

Healthy volunteers were recruited during 2 separate periods in September 2007 and June 2009. The first recruitment period (18 specimens) did not specifically exclude volunteers who had had gastrointestinal disturbances in the 10 days before sampling. The second (31 specimens) were healthy volunteers who had normal bowel habit, no diarrheal disease for [greater than or equal to] 6 weeks, no antimicrobial drug therapy for [greater than or equal to] 4 weeks, and no medication except for asthma inhalers or antihypertensive or contraceptive medication. Volunteers defecated into a bottle suspended in the lavatory bowl with tissue paper to prevent it falling into the water. The Upper South A Regional Ethics Committee (Christchurch, New Zealand) and the multiethics committee of the Ministry of Health, New Zealand (MEC/08/52/EXP), granted ethics approval for the study.

Diarrhea specimens (submitted without patient details during 2008) were distributed among 3 categories, as follows. First were 32 samples in which no causal agent was found; pathogens were excluded by routine examination with conventional diagnostic techniques for bacteria, parasites, and norovirus at Southern Community Laboratories. Second were 57 samples in which a specific causal agent was not found; samples were examined at the Institute of Environmental Science and Research (ESR, Christchurch, New Zealand) reference laboratory by using conventional methods for a specific pathogen at the request of the submitting laboratory. Third were 39 samples in which a known gastrointestinal pathogen had been detected at ESR.

Samples were refrigerated for 24-48 h before DNA extracts were prepared by using the revised protocol described in the ZR Fecal DNA Kit (Zymo Research, Irvine, CA, USA). Fecal DNA extracts were examined with the PCR-DGGE for Epsilonproteobacteria as described (8). After visualization of the PCR-amplified product, individual DNA bands were excised and then DNA was eluted by diffusion into buffer and reexamined by PCR to obtain partial 16S rDNA amplicons for sequencing. Sequences were edited (primer sequences were removed) and subsequently compared with those in GenBank by using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Comparisons were made during April 2011. Assignment of sequences to a taxon was based on the E (expect) values obtained and on expert opinion of the taxonomic distance between the most likely matches obtained. BLAST matches yielded E-values ranging from 7.13e-62 to 2.26e-124.

Of 177 samples from the healthy volunteers and patients with diarrhea, 159 contained Epsilonproteobacteria, of which 20 contained >1 taxa (Table). C. rectus/showae, C. sputorum, C. upsaliensis, Helicobacter pullorum, and H. pylori/heilmannii/nemestrinae were detected in 11 (8.6%) of the 128 diarrhea samples but not in fecal specimens from volunteers. Cryptosporidium spp. also were present in 2 diarrhea samples in which C. rectus/showae were detected. In addition, norovirus was detected in the C. sputorum-positive sample. C. curvus and C. jejuni/coli were found in diarrhea samples examined previously for specific pathogens only, as well as in 1 and 4 samples, respectively, from human volunteers. Sequences of the C. concisus complex, C. ureolyticus, C. hominis, and C. gracilis occurred frequently in samples from all study participants.

We used [chi square] analysis to determine whether the proportion of the 32 diarrhea samples subjected to a complete pathogen screen differed significantly from fecal samples from the second group of 31 volunteers in which these organisms were detected. The pathogen screen contained C. (Bacteroides) ureolyticus, C. concisus complex, C. hominis, or C. gracilis. We found no statistical difference between the proportions detected in these 2 groups of samples.

Conclusions

Although many species belonging to the Epsilonproteobacteria have been associated with gastrointestinal illness for decades, few are proven primary pathogens. By using PCR-DGGE to examine feces from healthy volunteers and patients with diarrhea, we aimed to indicate which taxa might be present as commensal flora and which might have a causal role. C. upsaliensis/ helveticus, H. pullorum, H. pylori/heilmannii/nemestrinae were all detected in diarrhea specimens but not in specimens from healthy volunteers; no other pathogen was found in these diarrhea specimens. C. upsaliensis is presumed to be pathogenic (7). H. pullorum is poorly studied but bears sufficient similarity to diarrheogenic C. jejuni at the molecular-genetic level (9) to support a causative role in gastrointestinal disease, at least for some strains. Poultry harbor H. pullorum (10), and thus represent a vector for foodborne transmission. Use of the PCR-DGGE method (8) on domestic drinking and commercial scald water used in New Zealand chicken production detected H. pullorum in 2 of 5 samples tested (data not shown). Although detection of the taxa H. pylori/heilmannii/nemestrinae might simply represent gastric carriage (the natural environment for these species), perhaps gastric disturbances result in diarrheal sequalae. Even though C. rectus/showae were also detected only in diarrhea samples, 2 specimens also harbored Cryptosporidium spp. In addition, norovirus was detected in the diarrhea sample in which C. sputorum was found.

We detected C. jejuni/coli in 3 samples examined for, but not containing, E. coli O157, which indicates that some cases of campylobacteriosis go undiagnosed. To our surprise, we detected C. jejuni/coli in several fecal samples from healthy volunteers. This detection might represent asymptomatic carriage of C. jejuni/coli, a phenomenon more commonly observed in developing countries where repeated exposure during a prolonged period results in tolerance (11). The high incidence of infection in New Zealand makes this hypothesis credible.

C. concisus was the most frequently encountered species in this study and occurred in participants from both groups. Strains identified as C. concisus with conventional methods might belong to genetically distinct but phenotypically indistinguishable genomospecies differing in their pathogenic potential (12). The PCR-DGGE used here cannot differentiate C. concisus genomospecies; thus strains detected in volunteers and strains found in diarrhea samples might represent distinct genomospecies with different pathogenic potentials.

We detected C. hominis, C. gracilis, and C. ureolyticus in fecal samples of healthy volunteers and patients with diarrhea. C. hominis has long been considered a commensal (13). A molecular study found C. ureolyticus in 83 (23.8%) of 349 Campylobacter spp.-positive diarrhea samples, but no healthy controls were examined (14). Our data suggest these species are unlikely causes of diarrhea.

Our results indicate that certain Epsilonproteobacteria that are not routinely examined for account for a proportion of diarrhea cases of previously unknown etiology. PCRDGGE is a useful tool to study the prevalence and distribution of these bacteria. C. concisus genomospecies are frequently detected in human disease (5,15; this study); elucidation of their pathogenicity should be considered a public health research issue.

This project was funded by the New Zealand Ministry of Research, Science, and Technology through an ESR-administered Capability Fund project.

Ms Cornelius has worked at ESR since 1994 and currently leads the Public Health Laboratory in the Food Programme at ESR in Christchurch. Her research interests focus on the use of molecular methods for the detection and subtyping of pathogens.

References

(1.) Lake RJ, Adlam SB, Perera S, Campbell DM, Baker MG. The disease pyramid for acute gastrointestinal illness in New Zealand. Epidemiol Infect. 2010;138:1468-71. http://dx.doi.org/10.1017/ S0950268810000397

(2.) Lake R, King N, Sexton K, Bridgewater P, Campbell D. Acute gastrointestinal illness in New Zealand: information from a survey of community and hospital laboratories. N Z Med J. 2009;122:48-54.

(3.) Euzeby JP. List of prokaryotic names with standing in nomenclature [cited 2011 May 24]. http://www.bacterio.cict.fr/classifordersclasses. html#Epsilonproteobacteria

(4.) On SLW. Identification methods for campylobacters, helicobacters, and related organisms. Clin Microbiol Rev. 1996;9:405-22.

(5.) Lastovica AJ, Allos BM. Clinical significance of Campylobacter and related species other than Campylobacter jejuni and Campylobacter coli. In: Blaser MJ, Szymanski CM, Nachamkin I, editors. Campylobacter. 3rd ed. Washington: ASM Press; 2008. p. 123-49.

(6.) Vandenberg O, Dediste A, Houf K, Ibekwem S, Souayah H, Cadranel S, et al. Arcobacter species in humans. Emerg Infect Dis. 2004;10:1863-7.

(7.) Labarca JA, Sturgeon J, Borenstein L, Salem N, Harvey SM, Lehnkering E, et al. Campylobacter upsaliensis: another pathogen for consideration in the United States. Clin Infect Dis. 2002;34:E5960. http://dx.doi.org/10.1086/340266

(8.) Petersen RF, Harrington CS, Kortegaard HE, On SLW. A PCRDGGE method for detection and identification of Campylobacter, Helicobacter, Arcobacter and related Epsilobacteria and its application to saliva samples from humans and domestic pets. J Appl Microbiol. 2007;103:2601-15. http://dx.doi.org/10.1111/j.1365 2672.2007.03515.x

(9.) Jervis AJ, Langdon R, Hitchen P, Lawson AJ, Wood A, Fothergill JL, et al. Characterization of N-linked protein glycosylation in Helicobacter pullorum. J Bacteriol. 2010;192:5228-36. http://dx.doi. org/10.1128/JB.00211-10

(10.) Atabay HI, Corry JE, On SLW. Identification of unusual Campylobacter-like isolates from poultry products as Helicobacter pullorum. J Appl Microbiol. 1998;84:1017-24. http://dx.doi.org/10.1046/ j.1365-2672.1998.00438.x

(11.) Oberhelman RA, Taylor DN. Campylobacter infection in developing countries. In: Nachamkin I, Blaser MJ, editors. Campylobacter. 2nd ed. Washington, DC: ASM Press; 2000. p. 139-53.

(12.) Aabenhus R, On SLW, Siemer BL, Permin H, Andersen LP. Delineation of Campylobacter concisus genomospecies by amplified fragment length polymorphism analysis and correlation of results with clinical data. J Clin Microbiol. 2005;43:5091-6. http://dx.doi. org/10.1128/JCM.43.10.5091-5096.2005

(13.) Lawson AJ, On SLW, Logan JMJ, Stanley J. Campylobacter hominis sp. nov. from the human gastrointestinal tract. Int J Syst Evol Microbiol. 2001;51:651-60.

(14.) Bullman S, Corcoran D, O'Leary J, Lucey B, Byrne D, Sleator RD. Campylobacter ureolyticus: an emerging gastrointestinal pathogen? FEMS Immunol Med Microbiol. 2011;61:228-30. http://dx.doi. org/10.1111/j.1574-695X.2010.00760.x

(15.) Engberg J, On SL, Harrington CS, Gerner-Smidt P. Prevalence of Campylobacter, Arcobacter, Helicobacter, and Sutterella spp. in human fecal samples estimated by a reevaluation of isolation methods for campylobacters. J Clin Microbiol. 2000;38:286-91.

Author affiliations: Institute of Environmental Science and Research, Christchurch, New Zealand (A.J. Cornelius, S.M. Brandt, B. Horn, S.L.W. On); Christchurch School of Medicine, Christchurch (S.T. Chambers); and Southern Community Laboratories, Christchurch (J. Aitken)

DOI: http://dx.doi.org/10.3201/eid1803.110875

Address for correspondence: Stephen L.W. On, Institute of Environmental Science and Research-Food Safety Programme, 27 Creyke Rd, Christchurch Ilam PO Box 29 181, New Zealand; email: stephen.on@ esr.cri.nz
Table. Prevalence and distribution of Epsilonproteobacteria taxa
in fecal samples from 49 healthy volunteers and 128 persons
with diarrhea, New Zealand *

Taxa SCL ESR- ESR+ Vol

Campylobacter jejuni/coli 0 3 0 4
complex
C. ureolyticus 3 10 1 12
C. concisus 17 27 16 26
C. curvus 0 1 0 1
C. gracilis 4 10 4 3
C. hominis 4 6 1 8
C. rectus/showae 2 1 2 0
C. sputorum 0 0 1 0
Helicobacter sp. 1 0 0 0
C. upsaliensis/helveticus 0 2 0 0
H. pullorum 0 2 0 0
No Epsiloproteobacterium 6 3 8 1

* Detected by PCR-denaturing gradient gel electrophoresis. SCL,
samples examined by Southern Community Laboratories (Christchurch, New
Zealand) found negative for all common pathogens; ESR-, diarrhea
samples screened for specific pathogens by the Institute of
Environmental Science and Research (Christchurch) at the request of
the submitting laboratory and found negative; ESR+, diarrhea samples
screened for specific pathogens by the Institute of Environmental
Science and Research at the request of the submitting laboratory and
found positive; vol, samples from volunteers with no known recent
history of gastrointestinal illness. Specific pathogens found in ESR+
samples included Cryptosporidium spp., Giardia spp., norovirus,
Bacillus cereus, toxigenic Staphylococcus aureus, and toxigenic
Clostridium perfringens.
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Title Annotation:DISPATCHES
Author:Cornelius, Angela J.; Chambers, Stephen; Aitken, John; Brandt, Stephanie M.; Horn, Beverley; On, Ste
Publication:Emerging Infectious Diseases
Geographic Code:8NEWZ
Date:Mar 1, 2012
Words:2136
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