Yersinia enterocolitica strains comprise an important group of bacterial enteropathogens that cause a broad range of gastrointestinal syndromes. Three groups are distinguishable within this bacterial species, namely, the nonpathogenic group (biotype 1A strains), the low-pathogenicity, non-mouse-lethal group (biotypes 2 to 5), and the high-pathogenicity, mouse-lethal group (biotype 1B). To date, the presence of the high-pathogenicity island (HPI), a chromosomal locus that encodes the yersiniabactin system (involved in iron uptake), defines essentially the difference between low-pathogenicity and high-pathogenicity Y. enterocolitica strains, with the low-pathogenicity strains lacking the HPI. Using the powerful tool of representational difference analysis between the nonpathogenic 1A strain, NF-O, and its high-pathogenicity 1B counterpart, WA-314, we have identified a novel type II secretion gene cluster (yts1C-S) occurring exclusively in the high-pathogenicity group. The encoded secreton, designated Yts1 (for Yersinia type II secretion 1) was shown to be important for virulence in mice. A close examination of the almost completed genome sequence of another high-pathogenicity representative, Y. enterocolitica 8081, revealed a second putative type II secretion cluster uniformly distributed among all Y. enterocolitica isolates. This putative species-specific cluster (designated yts2) differed significantly from yts1, while resembling more closely the putative type II cluster present on the genome of Y. pestis. The Yts1 secreton thus appears to have been additionally acquired by the high-pathogenicity assemblage for a virulenceassociated function.The genus Yersinia comprises an important group of bacterial pathogens, with Yersinia enterocolitica, Y. pseudotuberculosis, and Y. pestis representing the species of interest. Y. pestis is the etiologic agent of plague, whereas Y. pseudotuberculosis and Y. enterocolitica are enteropathogens that cause a broad range of gastrointestinal syndromes ranging from acute gastroenteritis to mesenteric lymphadenitis. Central to the pathogenicity of the Yersinia is the presence of a 70-kb pYV virulence plasmid whose products mediate, among other things, the resistance of Yersinia to phagocytosis by polymorphonuclear leukocytes and macrophages (41). In addition, the chromosomally encoded Ail (for attachment invasion loci), Myf (for mucoid Yersinia factor), and Inv (invasin) proteins have been implicated in the virulence of the species to various degrees (3, 38).Two groups have been identified among the Y. enterocolitica strains, which is a focus of the present study: nonpathogenic strains comprising mainly biotype 1A organisms and pathogenic strains carrying the virulence plasmid pYV. The latter group is further subdivided into a low-pathogenicity (LP), nonmouse-lethal group represented by biotypes 2 to 5 and a highpathogenicity (HP), mouse-lethal group exemplified by biotype 1B strains (8). As a mouse virulence determinant, an HP island (HPI) has been identified, which encodes for syn...

Graphical abstract

The unsuitability of the “CFU” parameter and the usefulness of cultivation-independent quantification of Campylobacter on chicken products, reflecting the actual risk for infection, is increasingly becoming obvious. Recently, real-time PCR methods in combination with the use of DNA intercalators, which block DNA amplification from dead bacteria, have seen wide application. However, much confusion exists in the correct interpretation of such assays. Campylobacter is confronted by oxidative and cold stress outside the intestine. Hence, damage caused by oxidative stress probably represents the most frequent natural death of Campylobacter on food products. Treatment of Campylobacter with peroxide led to complete loss of CFU and to significant entry of any tested DNA intercalator, indicating disruption of membrane integrity. When we transiently altered the metabolic state of Campylobacter by abolishing the proton-motive force or by inhibiting active efflux, CFU was constant but enhanced entry of ethidium bromide (EtBr) was observed. Consistently, ethidium monoazide (EMA) also entered viable Campylobacter, in particular when nutrients for bacterial energization were lacking (in PBS) or when the cells were less metabolically active (in stationary phase). In contrast, propidium iodide (PI) and propidium monoazide (PMA) were excluded from viable bacterial cells, irrespective of their metabolic state. As expected for a diffusion-limited process, the extent of signal reduction from dead cells depended on the temperature, incubation time and concentration of the dyes during staining, prior to crosslinking. Consistently, free protein and/or DNA present in varying amounts in the heterogeneous matrix lowered the concentration of the DNA dyes at the bacterial membrane and led to considerable variation of the residual signal from dead cells. In conclusion, we propose an improved approach, taking into account principles of method variability and recommend the implementation of process sample controls for reliable quantification of intact and potentially infectious units (IPIU) of Campylobacter by real-time PCR.

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A real-time PCR procedure targeting the gene of the molecular cochaperon DnaJ (dnaJ) was developed for specific detection of strains belonging to the Enterobacter cloacae group. The inclusivity and exclusivity of the real-time PCR assay were assessed with seven reference strains of E. cloacae, 12 other Enterobacter species and 41 non-Enterobacter strains. Inclusivity as well as exclusivity of the duplex real-time PCR was 100%. In contrast, resolution of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was inadequate for delineation of Enterobacter asburiae, Enterobacter hormaechei, Enterobacter kobei and Enterobacter ludwigii from E. cloacae. Eleven of 56 (20%) clinical isolates of the E. cloacae group could not be clearly identified as a certain species using MALDI-TOF MS. In summary, the combination of MALDI-TOF MS with the E. cloacae-specific duplex real-time PCR is an appropriate method for identification of the six species of the E. cloacae complex.