No abstract

Examination of 69 strains of Yersinia enterocolitica which represented 20 serotypes and nontypable isolates for HeLa cell infectivity by a roller tube technique that provided a quantitative index of infection showed that infectivity (index, greater than 3.50) was confined to strains of serotypes O:8, O:3, O:5, 27, O:9, O:1, O:1,2,3, O:2,3, O:4,32, and O:21. All strains that were HeLa positive were sucrose positive and negative for salicin, esculin, rhamnose, raffinose, melibiose, alpha-methylglucoside, and citrate. All HeLa-negative strains were either sucrose and salicin positive or were sucrose negative. Twenty-one strains were examined for virulence by the ability to produce guinea pig conjunctivitis and diarrhea in mice. Positive strains were limited to those that were HeLa positive and were autoagglutination positive and calcium dependent at 35 degrees C. There was no association between virulence and the ability to produce enterotoxin measured by the infant mouse assay. Loss of autoagglutinability and calcium dependency was accompanied by loss of virulence, but HeLa cell infectivity was unchanged. The results suggest that at least two properties are necessary for virulence: the presence of the V and W antigens, mediated by the same plasmid as autoagglutination and calcium dependency, and an invasive factor demonstrated in vitro by HeLa cell infectivity. These virulence properties are found in only certain biotypes of Y. enterocolitica.

Two new enrichment media were formulated for the recovery of Yersinia enterocolitica from foods: (i) yeast extract-rose bengal broth for preenrichment at 4 or 10°C; and (ii) bile-oxalate-sorbose broth, a selective enrichment incubated at 22°C. Comparison of these media in a two-step enrichment procedure against cold enrichment and modified Rappaport broth showed improved and more rapid recovery of human strains of Y. enterocolitica from inoculated foods. The use of bile-oxalate-sorbose broth as a selective enrichment also improved the performance of cold enrichment with phosphate-buffered saline. Determination of the best enrichment system for recovery of Y. enterocolitica from samples of retail pork and fresh pork tongues depended on whether the criterion was the number of positive samples, the variety of different serotypes recovered, or the ability to recover the important human serotype 0:3. A single enrichment system with the widest selectivity would include preenrichment at 4°C with either phosphatebuffered saline for 14 days or yeast extract-rose bengal broth for 9 days followed by selective enrichment with bile-oxalate-sorbose broth at 22°C for 5 days.

Yersinia enterocolitica serotypes O:3, O:8, O:9, O:5,27, O:4,32, and O:21 were virulent as determined by autoagglutination and calcium dependency at 35 degrees C and ability to produce guinea pig conjunctivitis and mouse diarrhea.

The in vitro invasive properties of bacteria have frequently been studied by the use of HeLa cell cultures in chamber slides, using microscopic examination to enumerate intracellular bacteria. When this system was used to examine invasive properties of Yersinia enterocolitica, it resulted in rapid internalization of high numbers of bacteria during the infection phase which prevented subsequent discrimination of intracellular multiplication. A modified procedure was developed which standardized the ratio of bacteria to HeLa cells (i.e., multiplicity), the time for the infection phase, and the addition of specific antiserum with gentamicin for restricting bacterial uptake during the intracellular growth phase. Studies with this modified chamber slide system found that strains of human isolates of Y. enterocolitica (serotypes O:3, O:8, O:5,27, and O:6,30) exhibited different degrees of cell infection but did not multiply intracellularly. A second test system was developed that used roller tubes and viable cell counts for the enumeration of intracellular bacteria. This roller tube system confirmed that internalized bacteria did not multiply inside HeLa cells over a 24-h period. The roller tube system with viable cell counts for enumeration is a simplified technique for quantitative comparison of in vitro infectivity of HeLa cells by Y. enterocolitica.