TEXT

Top Abstract Text References

Some bioserotypes of Yersinia enterocolitica correlate with potential pathogenicity in humans (6, 10). Most commonly isolated in gastrointestinal infections are bioserotypes 4/O:3, 2/O:9, and 2-3/O:5-5,27 (5, 9). The Y. enterocolitica-related species can share antigenic reactivities with Y. enterocolitica sensu stricto, but they are avirulent.

Among the biochemical traits that differentially characterize these Y. enterocolitica-related and -sensu stricto species, lack of sucrose fermentation has been a distinct feature of Y. kristensenii (2, 3, 17). Consequently, sucrose-negative Y. enterocolitica-like organisms isolated from clinical specimens have been regarded as avirulent, and in routine practice, they are not characterized further. The present report, however, describes a series of sucrose-negative, Voges-Proskauer (VP)-positive Y. enterocolitica-like isolates that proved, upon thorough characterization, to be fully pathogenic strains of Y. enterocolitica.

Detection of atypical Y. enterocolitica-like clinical isolates. A Yersinia strain, IP22109, was recovered in large numbers on CIN agar base (Difco) from the feces of a 24-year-old man hospitalized at the La Croix St. Simon Hospital, Paris, France, for high fever (39°C), diarrhea, and pain in the right iliac fossa. This case was one of several cases of acute gastroenteritis traced to a common meal, suggesting a food-borne outbreak although this small outbreak was not further investigated. No other potentially pathogenic microorganism was isolated from the stool samples. IP22109 was of serotype O:5. The patient's serum was able to agglutinate the Y. enterocolitica O:5 reference strain as well as its homologous strain, indicating that the latter had triggered an immune response. IP22109 displayed an atypical biochemical profile, since sucrose negativity, typical of Y. kristensenii (3), was associated with a positive VP test which is negative in this species but is positive in Y. enterocolitica (3). The association between sucrose negativity and VP positivity has been occasionally observed in biotype 5 of Y. enterocolitica. This very uncommon biotype, however, is associated with serotype O:2,3, as opposed to serotype O:5 of IP22109.

In vitro and in vivo virulence tests. Pathogenic Yersinia strains carry the 70-kb pYV plasmid, which encodes essential virulence determinants. Four in vitro tests were used to address the presence of pYV in the five atypical isolates. Indirect assessment of the plasmid presence was obtained by (i) plating the strains on magnesium-oxalate agar (MOX test) in search for the growth inhibition triggered by the pYV plasmid under low-calcium conditions at 37°C (11, 16) and (ii) checking bacterial autoagglutination mediated by the pYV-encoded YadA adhesin upon incubation at 37°C in RPMI medium (1, 13). Direct detection of pYV was then performed by colony hybridization with a yadA-specific probe and by plasmid extraction. The MOX and autoagglutination tests were done as described previously (11, 13). The yadA-specific probe used for colony hybridization was obtained by nested PCR of plasmid DNA from Y. enterocolitica IP864 with the pairs of primers 5'-CTGCAAATAAGCTATACCGAT-3' and 5'-ATGCCTGACTAGAACGATAT-3' for the first reaction, and 5'-GTGACTGTAAGTAGTTCGACT-3' and 5'-CCGACACCTGCAGTAAAGTT-3' for the second reaction. PCRs and digoxigenin labeling during the second PCR were performed as published previously (8), except that the first PCR was done with 10 ng of template DNA and with primers at a final concentration of 1 µM each, and in the second PCR, each unlabeled nucleotide was at a final concentration of 40 µM. The first PCR mixture was denatured at 94°C for 2 min and then amplified for 24 cycles at 94°C for 1 min, 54°C for 1 min 30 s, and 72°C for 2 min. The last cycle was followed by a 10-min incubation at 72°C. Amplification products were reamplified with the second primer set and the same PCR parameters as above, except for the priming step, which was carried out at 58°C. Colony transfer and hybridization were performed as published previously (8), and immunological detection of the probe was done according to the manufacturer's instructions (Boehringer). Plasmid extraction was performed according to the method of Birnboim and Doly (4).

View larger version (83K): [in this window] [in a new window]   FIG. 1.   Plasmid fingerprinting of the atypical isolates. EcoRI-restricted plasmids were resolved on a 0.8% agarose gel, ethidium bromide stained, and visualized under UV illumination. Comparison of the plasmid restriction patterns shows that most of the atypical strains harbor the pYV plasmid. Within the O:5 strains (lanes 1 to 6), the only detectable difference in the restriction profiles is an apparent size increase in a low-molecular-weight fragment of strains IP22914 and IP22109 (arrow). Lanes: 1, IP17381; 2, IP19817; 3, IP10664; 4, IP22109; 5, IP22914; 6, IP22228 (control Y. enterocolitica O:5 strain known to harbor pYV). The atypical strain of serotype O:9 (IP25686, lane 8) had a plasmid profile identical to that of the Y. enterocolitica O:9 control strain, IP23073 (lane 7).  and M, /HindIII and 1-kb-ladder molecular weight markers, respectively (GibcoBRL). Sizes are in kilobases.

Species assignment. DNA-DNA hybridizations were performed with the atypical strains IP10664 and IP19817 and the control strains Y. enterocolitica IP22228 and Y. kristensenii IP105, according to the protocol published by Popoff and Coynault (14). DNA labeling of the control strains, assessment of the degree of hybridization, and normalization to the homologous reaction were as described previously (14). As shown in Table 2, the atypical organisms shared a high degree of relatedness with the Y. enterocolitica control strain, IP22228 (>93%), and a low degree of relatedness (49%) with the Y. kristensenii reference strain, IP105. Therefore, the DNA hybridization studies conclusively ascribed the atypical strains to the Y. enterocolitica species.

Detection of an atypical Y. enterocolitica strain of bioserotype 2/O:9. One additional sucrose-negative, VP-positive Yersinia strain, IP25686, was identified in the Yersinia Reference Laboratory collection upon screening of the entire database. This strain was of bioserotype 2/O:9. IP25686 had the virulence characteristics of enteropathogenic Y. enterocolitica, because it belonged to a pathogenic bioserotype, possessed the pYV plasmid (Fig. 1), and lacked detectable pyrazinamidase activity (Table 1).