atypical campylobacters associated with gastroenteritis electrophoretic profile studies (6), and...
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JOURNAL OF CLINICAL MICROBIOLOGY, JUlY 1987, p. 1248-1252 0095-1137/87/071248-05$02.00/0 Copyright © 1987, American Society for Microbiology
Atypical Campylobacters Associated with Gastroenteritis WEE TEE,'* BRUCE N. ANDERSON,2 BRUCE C. ROSS,2 AND BRIAN DWYER'
Campylobacter Laboratory, Department of Clinical Pathology,1 and Virology Laboratory,2 Fairfield Hospital, Victoria 3078, Australia
Received 29 December 1986/Accepted 17 March 1987
Nine strains of Campylobacter species other than Campylobacter jejuni, Campylobacter coli, and Campylo- bacter laridis were isolated from patients with acute diarrhea. Ali nine strains showed preferred growth at 37°C under microaerophilic conditions. Conventional microbiological tests and DNA-DNA dot blotting were used to identify these strains. Three of the nine Campylobacter strains hydrolyzed hippurate, reduced nitrate, produced catalase, were resistant to cephalothin, and were shown to be highly related to C. jejuni type strains. Two strains had negative or weak catalase activity and were hippurate negative. Three other strains had characteristics similar to those of Campylobacter cinaedi. The ninth strain, isolated from a homosexual man with antibodies to human immunodeficiency virus (human T-cell lymphotropic virus type III), showed unique features different from those of all the known campylobacters used in this study. This strain grew well at 25 and 37°C and was catalase and nitrate positive, hippurate negative, and resistant to cephalothin.
Enteric pathogens of the genus Campylobacter have been increasingly well defined in recent years. Campylobacter jejuni, Campylobacter coli, and Campylobacter laridis are well established etiologic agents of human gastroenteritis. Of these, C. jejuni is most commonly encountered. Campylo- bacter fetus is primarily a cause of systemic illness in the malnourished or immunosuppressed, although it is occasion- ally implicated as a cause of bacterial diarrhea (7). The identification and differentiation of clinical Campylo-
bacter isolates is based on a limited number of morphologi- cal and biochemical criteria, and misidentification may result from atypical reactions. Alternative approaches to strain differentiation include gas-liquid chromatographic analysis of cellular fatty acid composition (11), whole cell protein electrophoretic profile studies (6), and determination of DNA base composition (16, 24). The recent emergence of new strains, such as Campylo-
bacter fennellae, Campylobacter cinaedi (5), Campylobac- ter pyloridis (14), and atypical Campylobacter strains iso- lated from children with gastroenteritis in Central and South Australia (21), and the increasing appearance of reports of unusual biochemical reactions of known campylobacters (4, 7) necessitate the use of more sophisticated methods for species differentiation and identification. With the introduction of two temperatures, 37 and 43°C,
for routine culture of campylobacters from patients' fecal specimens, we have isolated a number of atypical strains. In this study, we used DNA-DNA hybridization dot blotting to determine whether these atypical strains belong to unique species or are variants of previously described Campylobac- ter species.
MATERIALS AND METHODS
Bacterial strains. The reference strains used in this study were C. jejuni NCTC 11351, C. coli NCTC 11366, C. laridis NCTC 11352, C. fetus NCTC 10842, "Campylobacter faecalis" NCTC 11415, Campylobacter venerealis NCTC 10352, Campylobacter sputorum subsp. mucosalis NCTC 11000, Campylobacter concisus NCTC 11485, Campylobac- ter hyointestinalis NCTC 11608, C. fennellae 906, C. cinaedi
* Corresponding author.
255, N03-negative strain 093, catalase-negative or -weak- positive (CNW)-like Campylobacter sp. strain 267, and C. pyloridis 101. The reference strains (NCTC-designated strains) were obtained from the National Collection of Type Cultures, London, England. C. cinaedi and C. fennellae were kindly supplied by Cynthia Fennell, Seattle, Wash., and C. pyloridis was recovered from an antral biopsy of a patient with a duodenal ulcer. Two other strains, CNW-like Campylobacter sp. strain 267 and N03-negative Campylo- bacter sp. strain 093, were kindly supplied by Trevor Steele of the Institute of Medical and Veterinary Science, Adelaide, South Australia, Australia. The origin and designation of each of the nine atypical Campylobacter strains under inves- tigation are shown in Table 1. The nine test strains were isolated from the feces of patients with acute diarrheal gastroenteritis admitted to this hospital. Of the nine patients, four were returned travelers from Southeast Asia, two were children under 3 years old, two were otherwise asympto- matic homosexual males with antibodies to human immuno- deficiency virus (human T-cell lymphotropic virus type III), and one had no apparent risk factors.
Isolation and identification. All nine strains recovered from patients' feces were isolated on 6% horse blood agar (Co- lumbia agar base) containing vancomycin (10 ,uglml), poly- myxin B (1,250 IU/ml), and trimethoprim (10 ,ug/ml). A drop of fecal suspension (1 g of feces in 10 ml of saline agitated into suspension with glass beads) was plated in duplicate directly onto selective plates. The plates were incubated at 37°C (7 days) and 43°C (4 days) in 10% C02-6% 02-84% N2. All the cultures were examined daily. Oxidase-positive, gram-negative, motile, spiral or curved rods which did not grow in air at 37°C were subjected to further characteriza- tion.
Physiological and biochemical characterization. Tests for nitrate reduction, catalase production, H2S production in the presence and absence of 1% cysteine hydrochloride (lead- acetate strip method using brucella broth as base), growth in 2.5% NaCI, growth in 1% glycine, susceptibility to naladixic acid (30-,ug disk) and cephalothin (30-,ug disk), selenite reduction, and tolerance to 0.04% triphenyltetrazolium chlo- ride were performed by methods previously described (3, 8,
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ATYPICAL CAMPYLOBACTERS AND GASTROENTERITIS
TABLE 1. Description of bacterial strains
Patient Test strain Age Sex Occupation Overseas
Sex ~~~~~~~~~~~travel C899 ? Female Not given (friend of donor India
of C900 isolate) C900 28 yr Male Unemployed teacher India C380 22 yr Female College student India C033 1 yr Female Child C790 13 mo Male Child C948 29 yr Female Missionary worker Indonesia C902 35 yr Male' Teacher C767 29 yr Maleb Unemployed C004 37 yr Male Geologist
a All strains were isolated from stools. b Homosexuals.
12). Hydrolysis of sodium hippurate was assessed by the method of Hwang and Ederer (9).
Extraction and purification of Campylobacter DNA. All the strains were subcultured onto 6% horse blood agar (Colum- bia) and incubated at 37°C microaerophilically for 6 days. Bacterial cells from 20 plates were harvested with cotton swabs and suspended in 20 ml of TE buffer (10 mM Tris, 1 mM EDTA [pH 8]). The isolation and purification of the DNA was accomplished by following closely the method of Totten et al. (23), except that DNA was precipitated with cold ethanol. The DNA pellet was dissolved in 0.42 M NaCi in TE. The purity and approximate concentration of the isolated DNA was assessed by comparing the electropho- retic patterns with those displayed by standard DNA prep- arations on agarose gels.
Preparation of DNA for hybridization. A series of eight doubling dilutions of each DNA preparation was made in 1 M ammonium acetate, and 20-,ul samples of each dilution were transferred to nitrocellulose filters housed in a Bio-dot apparatus (Bio-Rad Laboratories, Richmond, Calif.) using the conditions specified by the manufacturer. The filters were dried in air and then baked at 80°C for 2 h before hybridization.
Preparation of DNA probes. For each strain under study, approximately 500 ng of DNA was labeled with 10 ,uCi of [32P]dATP by nick translation (22), and unincorporated [32P]dATP was removed by column chromatography through Sephadex G75 (Pharmacia, Uppsala, Sweden). Labeled DNAs to be used as probes in hybridization reactions were denatured by boiling for 5 min. DNA-DNA hybridization. We hybridized the DNA by the
method of Maniatis et al. (13). Baked nitrocellulose filters were prehybridized at 42°C for 4 h in 50 pI of prehybridiza- tion solution (18) per cm2 before the addition of the 32p_ labeled DNA probe. Hybridization was performed overnight at 42°C, and the filters were then washed twice at room temperature for 5 min each in double-strength SSC (l x SSC is 0.15 M NaCI plus 0.015 M sodium citrate) with 0.1% sodium dodecyl sulfate and then twice in lx SSC-0.1% sodium dodecyl sulfate for 30 min at 50°C (13). The filters were dried in air and exposed to X-ray film between inten- sifying screens at -70°C for 1 to 2 days. The film was developed and examined for specific hybridization of the probe to the test samples bound to the nitrocellulose filters. Probes prepared from all strains were then examined in a
checkerboard fashion against DNA preparations from the same organisms. We undertook a series of cross-hybrid- ization studies using these strain-specific DNAs labeled with
32 as probes of nitrocellulose filters spotted with serial dilutions of target DNA from the same 23 strains.
RESULTS From February 1985