The ail Gene Is Present in Some Yersinia enterocolitica Biotype 1A Strains

Introduction

Y ersinia enterocolitica is a causative agent of gastroenteritis and a variety of postinfectious sequelae. Y. enterocolitica strains are divided into six biotypes (BT), of which five (1B, 2, 3, 4, 5) include pathogenic strains. BT 1A strains are generally regarded as nonpathogenic, because they lack both the Yersinia virulence plasmid (pYV) and most of the chromosomally encoded virulence markers. The outer membrane protein Ail promotes attachment and invasion of Y. enterocolitica to cells and contributes to serum resistance (Bliska and Falkow, 1992). A high correlation has been found between the presence of the ail gene and the virulence of Y. enterocolitica (Miller et al., 1989). As pYV curing has been reported during laboratory cultivation of Yersinia strains, detection methods for pathogenic Y. enterocolitica that include chromosomal virulence targets are preferred. For instance, a sensitive real-time method for the detection of pathogenic Y. enterocolitica based on the ail gene has been developed (Lambertz et al., 2008). The European Committee for Standardization, in collaboration with the International Organization for Standardization, is currently preparing a standard for the probe-based real-time polymerase chain reaction (PCR) detection of pathogenic Y. enterocolitica. The method is based on the detection of the ail locus in food samples enriched overnight. Here, we report two BT 1A strains that were positive in conventional ail-specific PCR, which suggests that the ail gene alone is an insufficient virulence marker for detecting pathogenic Y. enterocolitica strains.

Methods

Screening for the chromosomal gene ail and the lcrE gene located in pYV was done for 299 Y. enterocolitica BT 1A strains isolated in a study on the prevalence of Y. enterocolitica bio/serotypes in clinical specimens (Sihvonen et al., 2009) and one BT 1A environmental strain obtained from MTT Agrifood Research Finland. The ail PCR was performed using primers ail9A and ail10A with conditions previously described (Thisted Lambertz et al., 1996). The primers for lcrE PCR were AMV-4 and AMV-5, and the PCR protocol was that previously published (Viitanen et al., 1991). The 16S rRNA genes were amplified with primers FD1mod (Kotilainen et al., 1998) and r533 (Jalava et al., 1995) as previously described (Sihvonen et al., 2009). The 16S rRNA and ail gene sequences obtained were deposited in GenBank/EMBL/DDBJ under accession numbers FN812721, FN812722, and FN812734–FN812738.

Results and Discussion

All the Y. enterocolitica BT 1A strains were lcrE negative. Two strains, FE 81228 and FE 94338, were positive for the ail gene. The clinical strain FE 81228 originated from a stool sample from a 56-year-old woman with stomach pain and diarrhea and without a travel history. The ail-positive strain FE 94338 was isolated from a carrot sample. The partial ail gene nucleotide sequences of the strains were identical and 99.7% similar (1 difference per 394 bp) to the ail gene sequence of the Y. enterocolitica BT 1B serotype O:8 strain 8081 and 96.2% similar (15 differences per 394 bp) to the serotype O:3 ail sequence (Fig. 1). Partial 16S rRNA gene sequences confirmed that the strains belonged to Y. enterocolitica ssp. palearctica.

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FIG. 1.

Alignment of the nucleotide sequences (394 bp) showing the differences between the ail genes of Yersinia enterocolitica BT 1B/O:8 strains 8081 (GenBank accession number: M29945), BT 1A FE 81228 (FN812732), and BT 4/O:3 NCTC 11176 (FN812737).

FE 81228 was the only ail-positive strain among the 299 screened clinical Y. enterocolitica BT 1A strains isolated in Finland in 2006. Thus, the presence of the ail gene in the BT 1A strain was not a common finding. There have been previous observations wherein sporadic BT 1A strains were positive in ail-specific PCRs (Thoerner, 2003; Falcao et al., 2006; Zheng et al., 2008). In a recent study of river water in Ontario, Canada, three BT 1A/O:5 strains with the ail gene have been isolated (Cheyne et al., 2010). Not much attention has been paid to these observations of ail-positive BT 1A strains. However, if the ail gene alone is used to estimate the amount of pathogenic Yersinia in food or environmental samples, there is a risk of misjudgment.

The ail locus is flanked on both sides by insertion element and transposase sequences (GenBank accession numbers: AJ605740 and NC_008800), making it prone to horizontal gene transfer. The ail gene sequences of BT 1A strains detected in the present study were highly similar to those of Y. enterocolitica BT 1B strains. However, highly pathogenic BT 1B strains have never been isolated in Finland, which suggests that the almost identical ail gene in Finnish BT 1A strains originates from other sources. It may even be that the ail gene sequences in BT 1A strains are relics of a transposition event in the past. Whether the ail gene in BT 1A strains is functional remains to be studied.

Conclusion

When using methods based on the detection of the ail gene, it is important to know that the usually nonpathogenic BT 1A strains may carry the ail gene. Therefore, besides using ail-based detection methods for Yersinia, either the strain should be isolated for further characterization or at least the presence of other virulence markers should be confirmed for verification of the presence of potentially pathogenic Y. enterocolitica.