1 Servicio de Microbiología, Hospital Universitario Marqués de Valdecilla, Avenida de Valdecilla s/n, 39008-Santander, Spain
2 Departamento de Biología Molecular, Facultad de Medicina, Universidad de Cantabria, Centro Asociado al CIB, CSIC, Cardenal Herrera Oria s/n, 39011, Santander, Spain
Correspondence
Juan M. García Lobo
jmglobo{at}unican.es
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ABSTRACT |
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INTRODUCTION |
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Yersinia enterocolitica, a pathogenic bacterium responsible for gastrointestinal infections carried by contaminated food, has a well-established typing system based on a combination of serological, biochemical and phage susceptibility tests. In addition, several molecular techniques have proved to be adequate to type Y. enterocolitica (Iteman et al., 1996), including the analysis of polymorphisms associated with repeated sequences (Hallanvuo et al., 2002
). However, the isolates coming from many outbreaks, and those prevalent in a given geographical area, often share the same phenotype and genomic fingerprint using the currently available methods. Therefore, the availability of new markers in Y. enterocolitica with additional discriminatory power would constitute a very useful epidemiological tool.
Here we describe the identification and characterization of a VNTR sequence in Y. enterocolitica. This locus was polymorphic in a sample of clinical isolates belonging to the same biotype and serotype, but not related epidemiologically. The locus was also polymorphic in strains with the same pulsotype. Our preliminary results suggest that the analysis of this VNTR may provide a new method to further discriminate among Y. enterocolitica isolates found to be identical with other epidemiological tools.
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METHODS |
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DNA isolation, amplification and analysis.
Total DNA from bacteria for PCR was prepared with the InstaGene matrix (Bio-Rad). DNA obtained by this method from some strains was readily degraded, presumably by the nucleases present in the preparation. In these cases DNA was prepared with the Roche high pure PCR template preparation kit.
Around 10 ng total DNA was used as template in PCR with the primers described in the text. The size of the amplification products was evaluated by analytical agarose gel electrophoresis using 3 % gels and a 50 bp ladder (Life Technologies) as a size marker. For DNA sequencing the appropriate bands were excised and purified from agarose gels using the Qiagen band purification kit. DNA sequencing was performed with a Perkin Elmer automatic sequencer at the Centro de Investigaciones Biológicas, Madrid, Spain.
Sequence comparison with the unfinished Yersinia pseudotuberculosis genome was performed at the web server of the Biology and Biotechnology Research Program of the Lawrence Livermore National Laboratory, CA, USA (http://bbrp.llnl.gov/bbrp/bin/y.pseudotuberculosis_blast).
The unfinished Y. enterocolitica 8081 genome was accessed at the Sanger Centre web server at http://www.sanger.ac.uk/Projects/Y_enterocolitica/blast_server.shtml
PFGE.
The genomic DNA of the different strains embedded in agarose plugs was prepared essentially as described by Saken et al. (1994). DNA in plugs was digested with NotI for 16 h at 37 °C. The samples were electrophoresed by the contour-clamped homogeneous electric field technique in a CHEF-DR III system (Bio-Rad) in 1 % (w/v) agarose gels made in 0·5x TBE buffer at 14 °C at 6 V cm-1. Time pulses were ramped from 8 to 23 s in 20 h.
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RESULTS |
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In some cases we sequenced several strains with the same amplicon size, as determined by gel electrophoresis, and obtained the same sequence. Accordingly, we assumed for the remaining strains that the number of repeated copies could be directly deduced from their amplicon size (Table 1).
The serotype and biotype of the strains used was determined as indicated in Methods. All the strains belonged to serotype O : 3, biotype 4 (except strain YHV52 which was non-serotypable and belonged to biotype 1A) and produced the Myf factor (Leyva et al., 1995)
Analysis of the CCAGCA repeat in other Yersinia strains
We also determined the status of the locus in some Y. enterocolitica strains from our collection belonging to other biotypes (Fig. 3). Two strains of biotype 1A produced the same amplicon corresponding in size to five copies of the repeat. Two strains of biotype 1B gave bands corresponding to four repeat copies. Two strains of biotype 2 presented, respectively, eleven and four copies of the repeat. Finally, two strains belonging to biotype 4, serogroup 3, lysotype IXb contained nine and five repeat copies.
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We investigated the occurrence of other repeated CCAGCA tracts (n>2) in the Y. enterocolitica 8081 unpublished sequence. An ORF of 1320 nt (1 107 3471 106 028), encoding a polypeptide of 439 aa containing a relaxase domain, contained 23 repeated CCAGCA copies, which corresponded to 23 PA repeats in the polypeptide. A further case of a gene product containing multiple PA repeats was observed in an uncharacterized membrane protein (3 035 7753 036 578), which contained 11 PA repeats in its carboxy end. The proline residue in this locus was encoded by a CCT codon, resulting in a CCTGCA hexanucleotide repeat. These two loci were either absent or non-repeated in Y. pestis and Y. pseudotuberculosis.
Analysis by PFGE
To determine whether the isolates with different numbers of CCAGCA units in the orf528 locus, belonging to the same serotype and biotype, could be distinguished by other molecular typing methods, we analysed by PFGE their chromosomes digested with the restriction endonuclease NotI. Eight strains representative of the different orf528 alleles were found to produce four different restriction patterns (Fig. 4). This indicated that the analysis of the CCAGCA repeat in orf528 had more discriminating power than PFGE. Furthermore, we observed that eight strains with the same PFGE pattern presented seven different orf528 alleles (data not shown), demonstrating that the two analyses were independent and that the study of the orf528 locus can improve greatly the discriminatory ability of the PFGE.
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DISCUSSION |
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Polymorphisms in VNTR loci inside an ORF usually lead to a change in protein function. Group B streptococci escape from phagocytosis by this mechanism (Madoff et al., 1996) and Listeria controls actin-mediated intracellular motility (Smith et al., 1996
). Even more spectacular is the expansion of a TCT triplet in the ahpC gene of E. coli resulting in the conversion of a peroxiredoxin into a disulfide reductase (Ritz et al., 2001
). The different alleles of orf528 will probably produce proteins with some functional difference. We do not have any evidence indicating that this polymorphism could be subjected to environmental selection, but this aspect should be further investigated to validate the study of this locus as an epidemiological tool (van Belkum, 1999
).
Further analysis of the sequences around the orf528 locus, revealed several differences in gene order between Y. enterocolitica strains Y56 and 8081, suggesting that this locus can represent a hotspot for gene rearrangement in this species.
Analysis of the locus in our collection of clinical isolates and other Yersinia strains indicated that at least 14 alleles of orf528 were possible, containing from two to fifteen hexanucleotide copies. The already characterized strain Y56, with 15 repeat copies showed the more expanded locus. Y. enterocolitica strain 8081 contained 10 copies and the two sequenced strains of Y. pestis contained two copies of the repeat. Only eight of these possible alleles were represented in the sample analysed (Table 1). The allele with 13 copies was the most frequent among our clinical Y. enterocolitica isolates (15 out of 55 YHV strains analysed), followed in frequency by the eight- and seven-repeat alleles (nine occurrences each).
The 55 strains used in this study were characterized after the classical serogrouping and biotyping scheme of Y. enterocolitica and were found to belong (except one) to serotype O : 3 biotype 4. All were pathogenic as they produced the Myf factor. This result agrees with previous data and confirmed that the Y. enterocolica strains of biotype 4, serotype O : 3 were prevalent in our area. Our results showed that the analysis of the VNTR locus described here in a sample of 54 Y. enterocolica clinical strains, which were identical according to classical typing schemes, allowed the identification of eight different alleles. Furthermore, the analysis of the locus in Y. enterocolitica strains belonging to other biotypes and serotypes showed that the locus was always present and that it could also be polymorphic.
The analysis of the completed sequence of Y. enterocolitica 8081 revealed the existence of a second locus with 23 CCAGCA repeats. This locus is probably also polymorphic and its analysis, in combination with the VNTR in orf528 could be exploited for Y. enterocolitica typing, as has been recently done with a VNTR found at eight different loci in the genus Brucella (Bricker et al., 2003).
A sample of the strains representing each one of the different alleles was also analysed by PFGE after genomic digestion with NotI. The eight strains presented four different pulsotypes, indicating that the two systems of analysis were independent and showing that the study of the VNTR in orf528 had a discriminatory power greater than PFGE. Furthermore, we have observed that eight strains belonging to the same biotype and serotype and presenting the same pulsotype after digestion with NotI, presented seven different allelic forms of orf528.
In summary, we conclude that the analysis of the VNTR locus described herein may provide a valuable method to discriminate among strains of Y. enterocolitica that seem to be identical according to other typing methods.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 11 July 2003;
revised 30 September 2003;
accepted 15 October 2003.
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