Abstract
BackgroundY. enterocolitica biotype (BT) 1A strains are often isolated from human clinical samples but their contribution to disease has remained a controversial topic. Variation and the population structure among the clinical Y. enterocolitica BT 1A isolates have been poorly characterized. We used multi-locus sequence typing (MLST), 16S rRNA gene sequencing, PCR for ystA and ystB, lipopolysaccharide analysis, phage typing, human serum complement killing assay and analysis of the symptoms of the patients to characterize 298 clinical Y. enterocolitica BT 1A isolates in order to evaluate their relatedness and pathogenic potential.ResultsA subset of 71 BT 1A strains, selected based on their varying LPS patterns, were subjected to detailed genetic analyses. The MLST on seven house-keeping genes (adk, argA, aroA, glnA, gyrB, thrA, trpE) conducted on 43 of the strains discriminated them into 39 MLST-types. By Bayesian analysis of the population structure (BAPS) the strains clustered conclusively into two distinct lineages, i.e. Genetic groups 1 and 2. The strains of Genetic group 1 were more closely related (97% similarity) to the pathogenic bio/serotype 4/O:3 strains than Genetic group 2 strains (95% similarity). Further comparison of the 16S rRNA genes of the BT 1A strains indicated that altogether 17 of the 71 strains belong to Genetic group 2. On the 16S rRNA analysis, these 17 strains were only 98% similar to the previously identified subspecies of Y. enterocolitica. The strains of Genetic group 2 were uniform in their pathogenecity-related properties: they lacked the ystB gene, belonged to the same LPS subtype or were of rough type, were all resistant to the five tested yersiniophages, were largely resistant to serum complement and did not ferment fucose. The 54 strains in Genetic group 1 showed much more variation in these properties. The most commonly detected LPS types were similar to the LPS types of reference strains with serotypes O:6,30 and O:6,31 (37%), O:7,8 (19%) and O:5 (15%).ConclusionsThe results of the present study strengthen the assertion that strains classified as Y. enterocolitica BT 1A represent more than one subspecies. Especially the BT 1A strains in our Genetic group 2 commonly showed resistance to human serum complement killing, which may indicate pathogenic potential for these strains. However, their virulence mechanisms remain unknown.
Highlights
Y. enterocolitica biotype (BT) 1A strains are often isolated from human clinical samples but their contribution to disease has remained a controversial topic
Genetic population structure and phylogeny In the multi-locus sequence typing (MLST) analysis, a subset of 43 Y. enterocolitica BT 1A strains were discriminated into 39 MLST types and the 10 4/O:3, 3/O:3 or 2/O:9 strains were discriminated into four different MLST types
Bayesian analysis of the MLST sequences divided the BT 1A strains into two distinct genetic clusters, which were clearly separated from the tight cluster formed by the strains of BT’s 2–4 and from the BT 1B strain (8018) (Figure 1)
Summary
Y. enterocolitica biotype (BT) 1A strains are often isolated from human clinical samples but their contribution to disease has remained a controversial topic. Yersinia enterocolitica species has six biotypes (BTs) of which five (1B, 2, 3, 4, 5) contain pathogenic strains. Since BT 1A strains lack most of the classical virulence markers, this biotype is often considered nonpathogenic. A virulence marker commonly found in BT 1A strains is the gene ystB encoding heat-stable Yersinia enterotoxin B whereas they usually lack the ystA gene found from Y. enterocolitica 4/O:3 strains. Weight loss, and death have been detected when young rabbits were infected with a Y. enterocolitica strain that produces heat-stable enterotoxin compared to the infection with a knock-out mutant [12]. A majority of the Y. enterocolitica BT 1A strains possess the ystB gene [13] and some excrete heat-stable YstB enterotoxin at 37°C in experimental conditions corresponding to those found in ileum [14,15]
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