Identification of Virulence-Associated Characteristics in Clinical Isolates of Yersinia enterocolitica Lacking Classical Virulence Markers

Yersinia enterocolitica is an important enteric pathogen which has well-defined virulence determinants that allow the bacteria to become established in their hosts and overcome host defenses. A number of strains obtained from patients with diarrhea, however, lack these genes. Accordingly, the mechanisms by which they cause disease are uncertain. Most of these isolates belong to biotype 1A. Strains of this biotype are also frequently isolated from a variety of nonclinical sources, such as food, soil, water, and healthy animals, and there is evidence that some of these strains are avirulent. In this study we investigated 111 strains of Y. enterocolitica biotype 1A, 79 from symptomatic humans and 32 from nonclinical sources, for virulence-associated characteristics. DNA hybridization studies showed that none of the strains carried sequences homologous with pYV, the ∼70-kb Yersinia virulence plasmid. Some strains hybridized with DNA probes for one of the following chromosomal virulence-associated genes: ail (7.2%), myfA (11.7%), ystA (0.9%), and ystB (85%). In addition, 33 strains (29.7%) produced an enterotoxin that was reactive in infant mice. However, the frequencies of these virulence-associated properties in clinical and nonclinical isolates were similar. Clinical isolates invaded HEp-2 cells and Chinese hamster ovary cells to a significantly greater extent than nonclinical strains (P ≤ 0.002). In addition, clinical strains colonized the intestinal tracts of perorally inoculated mice for significantly longer periods than nonclinical isolates (P ≤ 0.01). Light and electron microscopic examination of tissue culture cells incubated with invasive yersiniae revealed that the bacteria invaded selected cells in large numbers but spared others, suggesting that biotype-1A strains of Y. enterocolitica may invade cells by a novel mechanism. These results indicate that some clinical isolates of Y. enterocolitica which lack classical virulence markers may be able to cause disease via virulence mechanisms which differ from those previously characterized in enteropathogenic Yersinia species.Yersinia enterocolitica is an important human pathogen which causes a variety of disorders, ranging from nonspecific diarrhea to invasive disease such as mesenteric lymphadenitis, hepatosplenic abscesses, and septicemia (5, 10, 37). The heterogenous nature of Y. enterocolitica, including differences in virulence, has led to the division of the species into subgroups based upon biochemical behavior and lipopolysaccharide O antigens (5, 37). At present, six biotypes are recognized, of which biotype 1B and biotypes 2 through 5 are regarded as including primary pathogens (5, 22, 26, 37, 40). The primary pathogenic strains of Y. enterocolitica are recognized in part by their ability to invade tissue culture cells in large numbers (7, 27, 29, 35). Genes which contribute this ability include the inv and ail genes on the bacterial chromosome and yadA, which is borne on a ca. 70-kb virulence plasmid known as pYV (4, 7, 27). Interestingly, however, other pYV-borne genes impede bacterial penetration of epithelial cells and macrophages, with the result that Y. enterocolitica is located extracellularly in infected animals (9, 15). Chromosomal genes other than inv and ail which may also contribute to virulence include yst (also known as ystA), which encodes a heat-stable enterotoxin (YST-a), myf, which encodes the production of fibrillae (Myf), and the urease gene complex (11, 12, 21).Biotype-1A strains of Y. enterocolitica, which generally are considered to be avirulent, are highly heterogenous, and include a large number of O serogroups (5, 37). They occur throughout the world in a wide range of environments and generally lack the genotypic and phenotypic markers associated with virulence of classical invasive strains of Y. enterocolitica, such as pYV, ail, myf, ystA, or a functional inv gene (12, 21, 28, 35, 40, 41, 45). Moreover, biotype-1A strains of environmental origin do not colonize the gastrointestinal tracts of experimentally inoculated animals (33, 42, 48). Despite these observations, some biotype-1A strains have been implicated as a cause of gastrointestinal disease. For example, a nosocomial outbreak of gastroenteritis in Canada involving nine patients was attributed to a strain of Y. enterocolitica biotype 1A, serogroup O:5 (36). In several countries, moreover, including Australia, Canada, The Netherlands, New Zealand, the Republic of Georgia, South Africa, Switzerland, and the United States of America, a significant proportion of Y. enterocolitica isolates obtained from patients with diarrhea belong to biotype 1A (3, 6, 17, 32, 34, 39, 46, 47). In addition, a prospective case control study with Chilean children showed that biotype-1A strains were significantly associated with diarrhea (30), and a clinical study in Switzerland demonstrated that the illness associated with biotype-1A strains of Y. enterocolitica was indistinguishable from that due to classical virulent biotypes (6).If biotype-1A strains of Y. enterocolitica are able to cause disease, their pathogenic mechanisms are not clear because they lack the well-established virulence markers of primary pathogenic strains of Y. enterocolitica. Some Y. enterocolitica strains produce variants of YST-a, known as YST-b and YST-c (20, 52, 53), but their prevalence and contribution to disease are not known. In addition, a biotype-1A strain of serogroup O:6 was reported to produce a novel heat-stable enterotoxin, termed YST-II. This toxin differs from YST-a in a number of respects, including its mechanism of action, which does not appear to involve activation of guanylate cyclase (41). Other putative virulence determinants in this or other biotype-1A strains have not been investigated or reported.As biotype-1A strains of Y. enterocolitica are so heterogenous and occupy such a diverse range of environmental niches, we hypothesized that there may be a subset of these bacteria which are capable of causing disease but which lack the classical virulence markers of Yersinia species and therefore cannot be identified by assays for these markers. The aim of this study was to test this hypothesis by examining a collection of biotype-1A strains of clinical and nonclinical origins for virulence-associated determinants.