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Campylobacter jejuni is one of the most common causes of human bacterial gastroenteritis in the world. Since most C. jejuni infections are sporadic (1), their epidemiology must be clarified in order to design and implement effective intervention strategies. Since methods for high-resolution identification of strains are necessary for epidemiological surveillance and investigation of particular infections, a number of typing methods for C. jejuni have been developed. Recent studies indicate that molecular methods can offer greater potential for C. jejuni strain differentiation than do phenotypic techniques such as serotyping with heat-labile antigens (13), O serotyping based on lipopolysaccharide antigens (18), biotyping (3), and phage typing (6). Genotyping based on molecular methods can evaluate the phylogenetic, as well as epidemiological, relationships among strains that share surface markers, such as those detected by serotype or phage type determinants (5, 10, 14-16).

Detection of restriction fragment length polymorphisms (RFLPs) with either rRNA or specific DNA probes is a relatively simple and reproducible molecular technique and has been useful in the epidemiological characterization of other pathogenic microorganisms (6, 15). Jackson et al., using a C. jejuni cosmid clone as a probe, showed greater discrimination with RFLP than with conventional ribotyping using a 16S rRNA gene probe (11, 12), suggesting the utility of such analyses in Campylobacter epidemiological studies.

The aim of the present study was to evaluate the usefulness of methods based on RFLPs using random chromosomal DNA probes in Southern hybridizations for epidemiological analysis of C. jejuni infection. We analyzed RFLP of C. jejuni DNA after digestion with each of three restriction enzymes (HaeIII, HhaI, and HpaII) in Southern blots, using each of two unrelated cosmid clones containing 30- to 40-kb C. jejuni chromosomal DNA fragments. Examining isolates obtained from patients with sporadic infections and from a defined C. jejuni outbreak showed that we could easily distinguish unrelated and related strains.

Southern hybridization. Purified chromosomal DNAs from these bacteria were prepared by the method of Pitcher et al. (19) with minor modifications, as previously reported (7). The DNAs were digested overnight with each restriction enzyme and were electrophoresed in 0.8% agarose gels with 0.5× TBE (90 mM Tris-borate, 2 mM EDTA) running buffer. After electrophoresis, DNA fragments were transferred to a nylon membrane (Biodyne B; Pall Ultrafine Corp., East Hills, N.Y.) as described previously (9). Membranes then were air dried and the DNA was fixed by baking for 2 h at 80°C. Cosmid clones containing C. jejuni DNA inserts of 30 to 40 kb were used as probes. They were obtained from a genomic library constructed from Sau3AI-digested C. jejuni OH4384 (an isolate from a patient with Guillain-Barré syndrome [2]) chromosomal fragments cloned into the cosmid vector SuperCOS (Stratagene Ltd., Tokyo, Japan). Extraction of purified cosmid DNA was accomplished using the alkaline sodium dodecyl sulfate lysis method (20). The probe (100 ng) and lambda (30 ng) control DNA were labeled with ³²P as described previously (22). Membranes were hybridized with the probes for 18 h at 65°C. After washing, the signal was detected by exposure to X-ray film.

Pattern interpretation. Band matching and estimation of restriction fragment sizes were carried out with the Gel-Pro Analyzer software (Media Cybernetics, Silver Spring, Md.) with HindIII-digested lambda DNA as a control. The designations shown in Table 1 (such as ₁₅H2) represent the probe number (P14 or P15), the restriction enzyme used (A, HaeIII; H, HhaI; P, HpaII), and the RFLP pattern number, respectively. For example, ₁₅H2 indicates RFLP pattern 2 in HhaI-digested samples hybridized with probe A15. The specific patterns used in this study are available from the corresponding author by e-mail.

Selection of restriction enzymes suitable for RFLP analyses and probes. As a first step, we searched for restriction enzymes for optimal analysis using three C. jejuni cosmid clones as DNA probes. The enzymes tested that recognize a five- or six-base sequence were not suitable for analysis, and of those that recognize a four-base sequence, HhaI, HaeIII, and HpaII gave the best discrimination (Fig. 1). A greater number of distinct RFLPs were identified when cosmid clone P14 (30 kb) or P15 (35 kb) was used as the probes than when P2 was used (Fig. 1); therefore, P14 and P15 were chosen for subsequent studies. P14 and P15 did not cross-hybridize, and flaA and 16S ribosomal DNA probes PCR amplified using the primers previously reported (4, 7) did not hybridize to P14 and P15 (data not shown). Partial DNA sequence analysis of these probes and a BLAST search of the Campylobacter database of the Sanger Centre (http://www.medmicro.mds.qmw.ac.uk/campylobacter/) were performed. The sequences of the cosmids were nearly identical to those of clones camp65g8.plc, camp104a12.plt, and camp162g3.qlc for P14 and those of camp5a1.qlt and camp29e2.plc for P15 (data not shown).

View larger version (32K): [in this window] [in a new window]   FIG. 1.   Southern hybridization of HaeIII (lanes 1 to 4)-, HhaI (lanes 5 to 8)-, or HpaII (lanes 9 to 12)-digested C. jejuni DNA using probes P2 (A), P14 (B), and P15 (C). Molecular size markers are HindIII-digested  fragments. Lanes: 1, 5, and 9, strain FSH1; 2, 6, and 10, strain FSH2; 3, 7, and 11, strain FSH3; 4, 8, and 12, strain FSH4.

Stability of RFLPs. In epidemiological analysis, the stability and reproducibility of strain identification systems are critical. To test these, we examined strains that were serially passaged in vitro or passaged in vivo. Strain OH4384 was subcultured serially 12 times on brucella agar plates, and then 10 single colonies were picked and RFLPs were investigated. All of these substrains had identical RFLP profiles. Similarly, strain OH4384 was orally inoculated into a suckling mouse and reisolated from the liver 3 days later. This animal-passaged strain was shown to have the same RFLP patterns as the parental strain (data not shown). These results provide confidence that the genomic regions investigated do not mutate easily and that, although the method proposed cannot detect base substitution mutations except at the recognition sites, the RFLP patterns of C. jejuni strains are sufficiently stable for epidemiological analyses. The stability of RFLP patterns is supported by analyses of isolates from a patient with long-term infection and of strains from patients within a family cluster of infection described below. Therefore, we concluded that RFLP analyses using P14 and P15 as probes and digestion of chromosomal DNA with HaeIII, HhaI, or HpaII are suitable for genotyping of C. jejuni strains.

Application to sporadic and outbreak cases. An outbreak investigation is often used to assess the discriminatory power of a new typing method. In this case, strains from a 1996 school outbreak due to a contaminated lunch were investigated. Ten strains from outbreak-associated patients supplied by the Fukuoka Institute of Health and Environmental Sciences (HKC series; Table 1), belonged to one of three different O serotypes, O12 (one strain), O21 (four strains), or the O4,13,16,43,50 complex (five strains). The O serotypes of the strains were determined using the Campylobacter typing kit (Denkaseiken, Tokyo, Japan), following the manufacturer's instructions. SmaI-digested pulsed-field gel electrophoresis (PFGE) analysis, performed as previously described (8), showed that strains with the same O serotype had identical patterns (Table 1). RFLPs of these strains were analyzed after DNA digestion with HaeIII, HhaI, or HpaII and using P14 or P15 as a probe (Table 1). All five O4,13,16,43,50 complex strains had identical genotypes for P14 (designated ₁₄A1-₁₄H1-₁₄P1) and for P15 (₁₅A1-₁₅H1-₁₅P1). Similarly, the four serogroup O21 strains had identical genotypes (₁₄A2-₁₄H2-₁₄P2 and ₁₅A2-₁₅H2-₁₅P2). The remaining strain (serotype O12) had a different genotype (₁₄A3-₁₄H2-₁₄P2 and ₁₅A3-₁₅H3-₁₅P3). These results indicate that this genotyping technique is feasible for epidemiological analysis of Campylobacter outbreaks.

View larger version (62K): [in this window] [in a new window]   FIG. 2.   PFGE of SmaI-digested C. jejuni chromosomal DNA. The molecular size marker is the  ladder. Lanes: 1 to 3, outbreak strains HKC10, HKC16, and HKC37; 4 to 6, sporadic strains FSH14, FSH15, and FSH16.