flagellin gene profiling were used to discriminate 44 isolates of Campylobacter jejuni Penner heat stable (HS) serotypes 9, 38 and 63 from sporadic human ...
Letters in Applied Microbiology 1998, 26, 179–182
Subtyping of Campylobacter jejuni Penner serotypes 9, 38 and 63 from human infections, animals and water by pulsed field gel electrophoresis and flagellin gene analysis E. Lorenz1, A. Lastovica2 and R.J. Owen1 1
Laboratory of Enteric Pathogens, Central Public Health Laboratory, London, UK, and 2Red Cross War Memorial Children’s Hospital, Cape Town, South Africa 1574/97: received 23 June 1997 and accepted 6 October 1997
Pulsed field gel electrophoresis and PCR-RFLP flagellin gene profiling were used to discriminate 44 isolates of Campylobacter jejuni Penner heat stable (HS) serotypes 9, 38 and 63 from sporadic human infections and other sources. Genomic similarities between HS9 and HS38 strains were demonstrated. HS63 and HS1 strains of Camp. jejuni ssp. jejuni were similar but were genomically distinct from Camp. jejuni ssp. doylei HS63. The molecular analyses provided a basis for assessing associations between cross-agglutinating strains of Camp. jejuni and for subtyping within those serogroups. E . L OR E NZ , A . LA ST O VI CA A ND R. J . O WE N . 1998.
INTRODUCTION
Campylobacter jejuni is a major cause of diarrhoeal disease in human populations throughout the world (Skirrow and Blaser 1992). Strains causing infections are serologically diverse and some 47 heat stable (HS) serotypes are currently recognized (Penner and Hennessey 1980). A feature of the scheme, which uses unabsorbed antisera, is that strains may cross-react in a number of different antisera; for example, the HS4 complex is an established group of strains with associated antigens (HS4, HS13, HS16, HS43 and HS50). In this study, DNA macrorestriction analysis by pulsed field gel electrophoresis (PFGE), and PCR–RFLP analysis of the flagellin (fla) A gene, were applied to investigate similarities amongst epidemiologically distinct but serologically associated isolates of Camp. jejuni.
UK). Strains were preserved at – 70 °C on glass beads in Nutrient broth no. 2 (Oxoid CM67; Basingstoke, UK) containing 10% (v/v) glycerol. Strains were serotyped by standard procedures (Penner and Hennessey 1980; Fitzgerald et al. 1996).
Pulsed field gel electrophoresis
Preparation of Camp. jejuni genomic DNA and digestion with SmaI for PFGE was done as described previously (Gibson et al. 1994). Electrophoresis was carried out for 22 h at 200 V and 14 °C constant temperature in a CHEF-DR II unit (BioRad, Hemel Hempstead, UK) with pulse times ramped from 10 to 35 s.
Flagellin gene profiling MATERIALS AND METHODS
Forty-four isolates of Camp. jejuni serotypes HS9, HS38 and HS63 (Table 1) were selected from our strain collection. All were reported on serologically or genomically in previous studies (Owen et al. 1995; Fitzgerald et al. 1996; Gibson et al. 1996). The bacteria were cultivated at 37 °C for 48 h on 5% (v/v) defibrinated horse blood agar under microaerophilic conditions (5% O2, 5% CO2, 2% H2, 88% N2) in a Variable Atmosphere Incubator (Don Whitley Scientific Ltd, Shipley, Correspondence to: R.J. Owen, Laboratory of Enteric Pathogens, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT, UK. © 1998 The Society for Applied Microbiology
Cell suspensions in sterile distilled water were used as DNA template material in a standard PCR mixture as previously described (Santesteban et al. 1996). For DNAse producing strains (Lior biotypes II and IV), suspensions were boiled first in sterile distilled water. Briefly the primers used were the 18 bp primer pg50 [5?-ATGGGATTTCGTATTAAC3?] as the forward primer and the 26 bp mixed synthesis primer RAA19 [5?-GCACC(CT)TTAAG(AT)GT(AG) GTTACACCCTGC-3?] as the reverse primer. These primers were predicted to amplify a 1·448 kbp fragment representing flaA of Camp. jejuni (Alm et al. 1993). The amplification cycle consisted of an initial denaturation of
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Table 1 Characterization of Campylobacter jejuni isolates by HS serotyping, PFGE and flagellin gene profiling –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — Molecular profile — ––––––––––––––––––––––––––––––––––––– Serotype SmaI FlaA Isolate Host or source (HS) PFGE RFLP Ribotypea –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — HS9 and 38 group (C. jejuni ssp. jejuni) NCTC 12508 NKb 9 E1 10 4:18 C94/93 chicken 9 E1 10 4:18 C444/93 river water 9 E1 10 4:18 C492/93 human 9 E1 10 4:18 NCTC 12540 NK 38 E1 10 4:18 C1132/94 human 38 E1 10 NT C166/94 chicken 38 E1 2 4:18 C1/96, C2/96, C3/96, C4/96, C5/96, C6/96 chicken 38 E1 2 NT C778/94 human 38,63 E3 1 4:18 C10/94 human 38,63 E3 10 4:18 C700/94 human 38,63 E3 10 NT C992/94 human 38,63 E1 10 4:18 C792/93 human 38,63 E2 8 NT
HS63 group (C. jejuni ssp. jejuni) NCTC 12556 C408/93 C678/93 A735/92 C807/93 C833/93 C1111/93 C293/93 C600/93 A738/92 C352/94 C390/94 C417/94 C187/94
NK cow human human (SA) human human human cow human human (SA) chicken human human bovine
63 63 63 63 63 63 63 63 63 63 63,2 63,2 63,2 63,5
D A A A A2 A3 A4 B2 B2 F1 A1 B1 B C
12 7 2 2 2 7 2 2 2 11 2 2 2 10
26:33 NT 1:1 NT 1:1 NT 1:1 1:1 NT NT 1:1 NT 1:1 NT
HS63 group (C. jejuni ssp. doylei) A734/92, A736/92, A741/92 human (SA) 63 NC/1c d1d NT A740/92 human (SA) 63 NC/* d2 NT A744/92 human (SA) 63 NC/* d2 NT A742/92 human (SA) 63 E d4 NT A1078/92 human (SA) 63 E4 d2 NT A739/92 human (SA) 63 E5 d3 NT A743/92 human (SA) 63 E5 d2 NT A1079/92, A1080/92 human (SA) 63 E6 d3 NT A1077/92 human (SA) 63 F d5 NT –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– — a Data from previous studies (Fitzgerald et al. 1996; Owen et al. 1995). b Abbreviations used: NK, not known, NC, not cut; NT, not tested; SA, South Africa. c Results with KpnI *, unique profile. d Prefix d indicates a doylei profile.
© 1998 The Society for Applied Microbiology, Letters in Applied Microbiology 26, 179–182
S UB TY P IN G O F CA MP . JE JU N I H S9 , 38 AN D 63 181
target DNA at 94 °C for 2 min followed by denaturation at 94 °C for 15 s, primer annealing at 45 °C for 15 s and extension at 72 °C for 30 s. Samples were amplified through 25 cycles. RFLP analysis of amplicons was then performed with PstI and EcoRI in a mixed digest according to previously described methods to give patterns of two to five bands (Alm et al. 1993; Santestaban et al. 1996). RESULTS AND D ISCUSSION
The 44 strains of Camp. jejuni showed a variety of molecular patterns (Table 1). The three HS9 isolates had identical SmaI PFGE and flaA-profiles, as well as the identical ribopatterns reported previously (Fitzgerald et al. 1996). Both human and chicken isolates of HS9 were identical to the HS9 seroreference strain (NCTC 12508). Two SmaI PFGE profiles were obtained for the eight isolates of HS38 (no serological cross-reactions) and the HS38 sero-reference strain (NCTC 12540). The profiles each contained up to seven bands sized between 72 and 620 Kbp (Fig. 1) and were given the arbitrary designations listed in Table 1. These strains of Camp. jejuni
had relatively conserved profiles and differed mainly in the larger sized bands (notably in the 315–370 kbp band range). The strains also differed in their flaA-RFLP profiles (Table 1) and the associations did not always correspond to similarities based on the SmaI PFGE profiles. The HS38 sero-reference strain (NCTC 12540) was identical to the HS9 sero-reference strain (NCTC 12508) in both kinds of molecular profile. Similarities were also evident in molecular profiling results for the eight HS38 field strains and for a further five strains that cross-agglutinated in both the HS63 and HS38 antisera; three of these latter strains had similar SmaI PFGE profiles. Overall results on the 18 strains of the HS9/HS38 group showed that the predominant SmaI PFGE profile was type E (with subtypes 1–3) which was usually found to be in combination with flaA-type10. The strains with similar types were from humans (faeces) as well as from chickens (carcasses). Molecular typing results (Table 1) for 14 strains of Camp. jejuni ssp. jejuni HS63 were compared with results on 12 strains of Camp. jejuni ssp. doylei HS63, which were all human clinical isolates from South Africa. The SmaI PFGE profiles
Fig.1 Diagrammatic representation of different PFGE SmaI profiles of Campylobacter jejuni ssp. jejuni types A-E3 and Camp. jejuni ssp. doylei E4-F1. Profiles were given arbitrary designations (A-F) for this study according to overall band patterns. Further strain details are given in Table 1 © 1998 The Society for Applied Microbiology, Letters in Applied Microbiology 26, 179–182
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of the subspecies jejuni HS63 strains were diverse, irrespective of occasional cross-reactions with HS2 and HS5 antisera. Eleven different SmaI PFGE profiles were detected amongst the 14 strains, and three strains from diverse sources had the same profile (A). Diversity was evident to a lesser degree in the flaA-profiles, with most being type 2. Overall, the subspecies jejuni HS63 strains had SmaI PFGE profiles that were most similar to the HS1 and HS2 strains, e.g. the A and B profiles matched the typical HS1 or HS2 SmaI PFGE profiles described previously (Owen et al. 1995; Gibson et al. 1995). These genomic similarities with HS1 and HS2 were consistent with the previously published ribotyping data on the strains (Owen et al. 1995; Fitzgerald et al. 1996). Both the HS63 sero-reference strain (NCTC 12556) and strain A738/92 had unique molecular profiles, which possibly reflected geographical variation as they originated from South Africa, whereas the other strains were all of UK origin. The strains of Camp. jejuni ssp. doylei HS63 were also genomically diverse. Seven of these gave seven-band SmaI PFGE profiles, which were mostly unique and different from the profiles of Camp. jejuni ssp. jejuni HS63 (Fig. 1). DNA of five other Camp. jejuni ssp. doylei HS63 strains was not cut by SmaI but was cut with KpnI to give different yet related profiles. Previous ribotyping analyses demonstrated associations between the two subspecies of Camp. jejuni (Owen et al. 1995), although the subspecies doylei results were on different strains to those in the present study. The SmaI PFGE and flaAprofile results highlighted the lack of fine detail genomic overlap between strains of the two subspecies, despite their apparent serological similarities in the HS scheme. In conclusion, this study has shown that the HS9 and HS38 strains of Camp. jejuni are closely related and it would appear that low titre cross-reactions with the HS63 antiserum were non-specific and were not reflected in any genotypic associations. There was a core genotype amongst strains within the HS9/HS38 serogroup (SmaI PFGE type E1, flaA type 10, ribotype 4:18) although other strains could be further discriminated by PFGE and flaA-profiling. Likewise, PFGE and/or flaA-profiling provided a good basis for subtyping strains of Camp. jejuni ssp. jejuni HS63, including some giving low titre cross-reactions with the HS2 antiserum. These findings were consistent with the overall genomic similarities of these strains to HS1 and HS2 strains of subspecies jejuni but not to HS63 strains of Camp. jejuni ssp. doylei. Our study
highlights associations between different serotypes in the Penner scheme which should be recognized and taken into account in future when interpreting Camp. jejuni serotyping data. ACKNOWLEDGEMENT
This work was supported in part by a grant from the Department of Health (London). REFERENCES Alm, R.A., Guerry, P. and Trust, T. (1993) Distribution and polymorphism of the flagellin genes from isolates of Campylobacter coli and Campylobacter jejuni. Journal of Bacteriology 175, 3051– 3057. Fitzgerald, C., Owen, R.J. and Stanley, J. (1996) Comprehensive ribotyping scheme for heat-stable serotypes of Campylobacter jejuni. Journal of Clinical Microbiology 34, 265–269. Gibson, J.R., Fitzgerald, C. and Owen, R.J. (1995) Comparison of PFGE, ribotyping and phage-typing in the epidemiological analysis of Campylobacter jejuni serotype HS2 infections. Epidemiology and Infection 115, 215–225. Gibson, J.R., Sutherland, K. and Owen, R.J. (1994) Inhibition of DNAse activity in PFGE analysis of DNA from Campylobacter jejuni. Letters in Applied Microbiology 19, 357–358. Owen, R.J., Desai, M. and Garcia, S. (1993) Molecular typing of thermotolerant species of Campylobacter with ribosomal RNA gene patterns. Research in Microbiology 144, 709–720. Gibson, J., Lorenz, E. and Owen, R.J. (1996) Lineages within Campylobacter jejuni defined by numerical analysis of pulsed-field gel electrophoretic DNA profiles. Journal of Medical Microbiology 45, 1–7. Owen, R.J., Sutherland, K., Fitzgerald, C., Gibson, J., Borman, P. and Stanley, J. (1995) Molecular subtyping scheme for serotypes HS1 and HS4 of Campylobacter jejuni. Journal of Clinical Microbiology 33, 872–877. Penner, J.L. and Hennessey, J.N. (1980) Passive hemagglutination technique for serotyping Campylobacter fetus subsp. jejuni on the basis of soluble heat-stable antigens. Journal of Clinical Microbiology 12, 265–269. Santesteban, E., Gibson, J. and Owen, R.J. (1996) Flagellin gene profiling of Campylobacter jejuni heat-stable serotype 1 and 4 complex. Research in Microbiology 147, 641–649. Skirrow, M.B. and Blaser, M.J. (1992) Clinical and epidemiologic considerations. In Campylobacter jejuni: Current Status and Future Trends ed. Nachamkin, I., Blaser, M. J. and Tompkins, L. S. pp. 3–8. Washington DC: American Society for Microbiology.
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