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Journal of Clinical Microbiology, October 2004, p. 4840-4842, Vol. 42, No. 10
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.10.4840-4842.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Sequence-Based Typing of flaB Is a More Stable Screening Tool Than Typing of flaA for Monitoring of Campylobacter Populations

Alexander Mellmann,1 Jan Mosters,1 Edda Bartelt,2 Peter Roggentin,3 Andrea Ammon,4 Alexander W. Friedrich,1 Helge Karch,1 and Dag Harmsen1*

Institut für Hygiene, Universitätsklinikum Münster, Münster,1 Institut für Hygiene und Umwelt, Hamburg,3 Bundesinstitut für Risikobewertung,2 Robert Koch-Institut, Berlin, Germany4

Received 17 March 2004/ Returned for modification 20 May 2004/ Accepted 20 June 2004


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ABSTRACT
 
Different typing schemes for Campylobacter spp. were evaluated with 70 outbreak and sporadic isolates. The discriminatory indexes were 0.944 (by pulsed-field gel electrophoresis), 0.920 (by genotyping of the flagellin A gene), 0.902 (by genotyping of flaB), and 0.886 (by multilocus sequence typing). Cross-classification gave 94.77 or 95.82% (PFGE-flaA or PFGE-flaB) concordance. flaA was overdiscriminatory in three cases, most probably due to intragenomic recombination.


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TEXT
 
To differentiate sporadic from epidemiologically related Campylobacter infections, a rapid and discriminatory typing method is required to identify sources of human infection and to determine the routes of infection. Pulsed-field gel electrophoresis (PFGE) has proven to be useful and discriminatory and is therefore considered the present gold standard (17). However, PFGE is laborious, and even with standardized protocols the interlaboratory comparison of PFGE remains difficult. Methods based on DNA sequencing are fast, eliminate experimental variation, and facilitate interlaboratory comparisons (5). Recently, it was shown that the genotyping of the short variable region (SVR) of the flagellin A gene (flaA) provides adequate discrimination in short-term epidemiology (7). However, its use is questioned because of the known intra- and intergenomic recombination within the flagellin genes (1, 6, 16). The flagellum is encoded by two highly homologous genes (flaA and flaB) (Fig. 1) of approximately 1,730 bp joined by an intervening segment of approximately 200 bp (6). The nucleotide sequence of a 321-bp region of the flaA and flaB genes was determined for each isolate. This sequence encompassed the SVR extending from fla nucleotide positions 283 to 603 (inclusive) (3). The flagellin B gene (flaB) is, in contrast to flaA, not essential for motility and associated pathogenicity and is thought to be a genetic reservoir for flaA (1). Therefore, it can be assumed that flaB is a more stable marker. The aim of this study was to evaluate the potential of flaB typing in comparison to that of flaA typing.



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  		FIG. 1. Scheme of the genetic organization of the flagellin genes showing locations of primers used for PCR amplification and sequencing.

(This study was presented in part at the 104th General Meeting of the American Society for Microbiology, New Orleans, La., 23 to 27 May 2004.)

Partial DNA sequences (SVR, 321 bp) of flaA and flaB from 36 Campylobacter isolates (including 3 controls) from three documented outbreaks (Germany in 1997 and 2000 [14, 15] and Kansas in 1988 [12]) were analyzed. The control isolates were matched to each outbreak setting. Also, 34 consecutive strains from 13 sporadic cases of campylobacteriosis isolated in 2002 and 2003 were examined to assess the target's genetic stability. The flaA and flaB sequencing was essentially carried out as previously described (6, 17). Briefly, for amplification of flaA the consensus primers described by Wassenaar et al. (17) were used, and for flaB the primers Bup and A6 were used. For sequencing either the forward primer fla SVR 263f (5'-AAR GCT ATG GAT GAG CAA YTW AAA AT-3') or the reverse primer fla SVR 623r (5'-CCA AGW CCT GTT CCW ACT GAA G -3') were applied for both genes (Fig. 1). The alleles were assigned using the Campylobacter FlaA Variable Region Database (http://phoenix.medawar.ox.ac.uk/flaA/). In addition, the isolates were characterized by PFGE and by multilocus sequence typing (MLST). PFGE was performed according to the electrophoretic conditions previously described by Ribot and colleagues with SmaI as the restriction enzyme (10). Isolates differing at one or more bands were considered to be different. The macrorestriction patterns were arbitrarily designated with capital letters. For MLST, sequencing of seven housekeeping genes (aspA, glnA, gltA, glyA, pgm, tkt, and uncA) and assignment of sequence types (ST) were carried out in agreement with the protocol of Dingle et al. (2). The typing results of 42 C. jejuni isolates for which there was complete data (PFGE, MLST, flaA, and flaB typing results) were compared by assessing the discriminatory index (DI) (4) and the cross-classification results (11).

An overview of the typing results is shown in Table 1, which also includes the epidemiologic information of all Campylobacter isolates analyzed in this study. Mixed infections with two different C. jejuni strains occurred in sporadic cases 8 and 11. In sporadic case 12 even a coinfection with two Campylobacter species was observed. These findings are consistent with the observation that potential sources of Campylobacter infection may be contaminated with more than one strain (9).


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  		TABLE 1. Comparison of the PFGE, MLST, flaA, and flaB sequencing results for sporadic and outbreak-associated Campylobacter isolatesi

Using the primers FLA4F and FLA625RU originally proposed by Meinersmann et al. for amplification of the flaA SVR frequently resulted in sequences with ambiguous bases (7), most probably due to flagellin gene paralogs (8). Changing to the recently published consensus flaA forward and reverse primers gave better results (17). No single ambiguous base was observed when using the Bup and A6 primers for flaB amplification (6). One isolate was resistant to SmaI digestion and therefore was not typeable by PFGE. For the other 42 C. jejuni isolates, PFGE was most (DI, 0.944) and MLST was least discriminatory (DI, 0.886). flaA and flaB typing gave intermediate DI results of 0.920 and 0.902, respectively (Table 2). The DI depends on the number of types and on the homogeneity of frequency distribution of strains into types (13). Therefore, although MLST gave more types than fla typing, the DI was lower because of the inhomogeneous distribution of the STs. Ideally, the DI should be calculated using a test population that includes epidemiologically unrelated strains (13). Obviously, this is not true in our study; therefore, the absolute DI figures should be treated with caution. Nevertheless, the relative ordering of the typing schemes according to the DIs is meaningful.


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  		TABLE 2. Resolving power of PFGE, MLST, flaA, and flaB typing schemes for C. jejuni (n = 42)

The cross-classification of all possible pairs of PFGE and flaA or flaB gave 94.77 and 95.82% concordance. PFGE and MLST results were only 93.96% concordant (Table 3). In two outbreaks, flaA was overdiscriminatory compared to the other employed typing schemes, whereas flaB gave correct typing results. In outbreak 1, two isolates (B01766 and B01768; isolated from independent sources) were different from the others by flaA SVR typing despite their evident epidemiologic implication. These isolates had several nucleotide substitutions (n = 12) distributed throughout a 246-nucleotide region of the SVR, suggesting that the change was due to recombination rather than spontaneous mutation. The same underlying intragenomic recombination (Fig. 1) event between flaB and flaA (5-bp differences in a 150-nucleotide region) could also be shown in another isolate (D2692) with an unstable flaA during outbreak 3. Therefore, the epidemiologic concordance for all 33 confirmed outbreak isolates was 1.00 for flaB typing, whereas for flaA it was only 0.97. Furthermore, false-negative outbreak cross-classification results were not observed for MLST or for flaB. It was not possible to type C. coli by MLST (2), whereas the other typing methods were applicable without modification for the isolates tested (n = 4).


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  		TABLE 3. Cross-classification concordance results between PFGE and MLST, flaA, or flaB (C. jejuni, n = 42; all possible pairs, n = 861)

In conclusion, PFGE remains the most discriminatory typing method for Campylobacter. However, flaB typing is a rapid, reproducible, discriminatory, and stable screening tool. Our data demonstrate that flaB is more stable than flaA, probably due to less selective pressure. Larger studies of epidemiologically well-characterized isolates are needed to prove definitively the superiority of flaB for the monitoring of Campylobacter populations.


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ACKNOWLEDGMENTS
 
This work was supported by a grant from the Bundesministerium für Bildung und Forschung (BMBF, FKZ 01KI0204).

We thank U. Busch and A. Heißenhuber, Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit (Oberschleißheim, Germany), J. Bockemühl, Institut Hygiene und Umwelt (Hamburg, Germany), and J. Steigerwalt and P. Fields, Centers for Disease Control and Prevention (Atlanta, Ga.), for kindly providing us with Campylobacter strains. We are grateful to A. Lagemann, U. Keckevoet (Münster, Germany), and M. Müller (Berlin, Germany) for skillful technical assistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institut für Hygiene, Universitätsklinikum Münster, Robert Koch-Str. 41, D-48149 Münster, Germany. Phone: 49-251-83-5-53-48. Fax: 49-251-83-5-56-88. E-mail: dharmsen{at}uni-muenster.de. Back


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Journal of Clinical Microbiology, October 2004, p. 4840-4842, Vol. 42, No. 10
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.10.4840-4842.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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