Virulence-associated plasmids from Yersinia enterocolitica and Yersinia pestis.

A 44-megadalton plasmid associated with virulence and Ca2+ dependence from Yersinia enterocolitica 8081 was compared at the molecular level with a 47-megadalton plasmid associated with Ca2+ dependence from Yersinia pestis EV76. The plasmids were found to share 55% deoxyribonucleic acid sequence homology distributed over approximately 80% of the plasmid genomes. One region in which the plasmids differed was found to contain sequences concerned with essential plasmid functions. Forty-five mutants of Y. pestis were isolated which had spontaneously acquired the ability to grow on calcium-free medium (Ca2+ independence). Of these mutants, 21 were cured of their 47-megadalton plasmid, whereas the remaining had either suffered a major deletion of the plasmid or had a 2.2-kilobase insertion located in one of two adjacent BamHI restriction fragments encompassing approximately 9 kilobases. The inserted sequence was found at numerous sites on the Y. pestis chromosome and on all three plasmids in the strain and may represent a Y. pestis insertion sequence element.     

Direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis from meat.

Studies were done to determine the usefulness of dilute alkali (KOH) treatment of meat samples for direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis, without enrichment. Virulent Y. enterocolitica and Y. pseudotuberculosis in pork contaminated with 10(2), 10(3), and 10(4) cells per g survived the direct KOH treatment and were never recovered by using KOH postenrichment treatment. From 6 (4.8%) of 125 samples of retail ground pork, four biotype 4 serotype O3 and one biotype 3B serotype O3 strains of Y. enterocolitica and one Y. pseudotuberculosis serotype 4b strain were recovered by using direct KOH treatment without enrichment. As these isolations were attained without using enrichment cultural procedures, they represent an important time-saving alternative to simplify and speed isolation of Yersinia spp. from meat.     

Isolation of Yersinia enterocolitica from raw milk.

Four enrichment procedures were used for examining 131 raw milk samples for the presence of Yersinia enterocolitica. Forty-two isolations were obtained from 19 pooled- (31.1 percent positive) and 10 individual-producer samples (14.3 percent positive). Enrichment by Butterfields phosphate buffer incubated at 4 degrees C for 14 days and then inoculation of modified Rappaport broth incubated at 23 degrees C for 5 days produced the greatest number of isolations. The majority of isolates were biotype 1, and many were atypical from clinical isolates in being rhamnose positive (47.6 percent), citrate positive (16.7 percent), and lactose positive (26.2 percent). Thirteen isolates were serotypable, belonging to seven different O serotypes, with O:5 occurring most frequently.     

Isolation of virulent Yersinia enterocolitica from porcine tongues.

Thirty-one tongues from apparently normal, freshly slaughtered pigs were assayed for the presence of Yersinia enterocolitica by different enrichment and postenrichment techniques. Sixteen different isolates were recovered, including six of serotype O:8, four of serotype O:6,30, two of serotype O:3 phage type IXb, and one each of serotypes O:13,7, O:18, and O:46. One isolate was not typable. Cold enrichment in phosphate-buffered saline followed by treatment with dilute KOH or subsequent enrichment in modified Rappaport broth recovered 12 and 7 isolates, respectively. Four of the same isolates were recovered from the same tongues by both procedures. Cold enrichment without a selective postenrichment treatment recovered two isolates. Direct enrichment in modified Rappaport broth or modified selenite broth was ineffective in recovering yersiniae, as no isolates were obtained by either method. All of the serotype O:8 isolates were virulent to mice, causing the death of adults after oral challenge. This is the first report that associates Y. enterocolitica serotype O:8 with a natural reservoir.     

Thermal injury of Yersinia enterocolitica.

Procedures were developed to evaluate thermal injury to three strains of Yersinia enterocolitica (serotypes 0:3, 0:8, and 0:17). Serotype 0:17 (atypical strain) was more sensitive to bile salts no. 3 (BS) and to sublethal heat treatment than the typical strains, 0:3 and 0:8. When the 0:3, 0:8, and 0:17 serotypes were thermally stressed in 0.1 M PO4 buffer, pH 7.0, at 47 degrees C for 70, 60, and 12 min, respectively, greater than 99% of the total viable cell population was injured. Injury was determined by the ability of cells to form colonies on brain heart infusion (BHI) agar, but not on Trypticase soy agar (TSA) plus 0.6% BS for serotypes 0:3 and 0:8 and TSA plus 0.16% BS for 0:17. Heat injury of serotype 0:17 cells for 15 min in 0.1 M PO4 buffer caused an approximate 1,000-fold reduction in cell numbers on selective media as compared with cells heated in pork infusion (PI), BHI broth, and 10% nonfat dry milk (NFDM). The extended lag and resuscitation period in BHI broth was 2.5 times greater for 0:17 cells injured in 0.1 M PO4 than for cells injured in BHI or PI. The rate and extent of repair of Y. enterocolitica 0:17 cells in three recovery media were directly related to the heating menstruum used for injury. The use of metabolic inhibitors demonstrated that ribonucleic acid synthesis was required for repair, whereas deoxyribonucleic, cell wall, and protein synthesis were not necessary for recovery of 0:17 cells injured in 0.1 M PO4 buffer, BHI, or PI. Inhibition of respiration by 2,4-dinitrophenol slowed repair only for 0:17 cells injured in 0.1 M PO4 buffer, not for cells injured in PI or BHI.     

Yersinia enterocolitica produces superantigenic activity.

We have recently observed that antigenic preparations from Yersinia enterocolitica are capable of inducing strong proliferative responses in normal murine spleen cell cultures. As a consequence of this observation, we evaluated whether Yersinia-derived Ag possess superantigenic activity. Stimulatory activity can be found in culture supernatants, as well as membrane and cytoplasmic fractions of Y. enterocolitica. Cell depletion studies indicate that the primary responding cell is a CD4+ T cell, which requires the presence of APC for responsiveness to Y. enterocolitica Ag. Furthermore, these APC must express MHC class II Ag, as evidenced by the fact that either antibody depletion of class II+ APC or addition of anti-class II antibodies (that block class II Ag on the surface of APC) eliminates the proliferative response. Evaluation of TCR usage by BALB/c T cells responsive to Y. enterocolitica revealed that those T cells bearing V beta 3, 6, and 11 and possibly 7 and 9 were expanded after exposure to Y. enterocolitica Ag preparations. By using a panel of T cell hybridomas, we have shown that hybridomas bearing V beta 3, 7, 8.1, 9, and 11 but not 2, 8.2, 8.3, and 13 respond to Yersinia. When cytoplasmic fractions of Y. enterocolitica were subjected to column chromatography, proliferative activity was enriched approximately 27-fold, and the elution characteristics of the active material suggest that it possesses hydrophobic regions and is, therefore, probably membrane associated. These data indicate that Y. enterocolitica produces antigenic material that has properties consistent with those of T cell superantigens.     

Direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis from meat

Studies were done to determine the usefulness of dilute alkali (KOH) treatment of meat samples for direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis, without enrichment. Virulent Y. enterocolitica and Y. pseudotuberculosis in pork contaminated with 10(2), 10(3), and 10(4) cells per g survived the direct KOH treatment and were never recovered by using KOH postenrichment treatment. From 6 (4.8%) of 125 samples of retail ground pork, four biotype 4 serotype O3 and one biotype 3B serotype O3 strains of Y. enterocolitica and one Y. pseudotuberculosis serotype 4b strain were recovered by using direct KOH treatment without enrichment. As these isolations were attained without using enrichment cultural procedures, they represent an important time-saving alternative to simplify and speed isolation of Yersinia spp. from meat.     

Alkali method for rapid recovery of Yersinia enterocolitica and Yersinia pseudotuberculosis from foods.

A new culture method employing a potassium hydroxide treatment was compared with the conventional cold enrichment method for efficacy in recovering Yersinia sp. from naturally and artificially contaminated food. The new method increased the yield of Yersinia sp. fourfold and the sensitivity 100-fold, shortened the incubation period, and appreciably decreased the growth of non-Yersinia bacteria from a variety of meats, shellfish, and vegetables.     

Improved procedure for recovery of Yersinia enterocolitica from meats.

A 1- to 3-day enrichment-KOH postenrichment procedure was evaluated and found to be as effective in recovering Yersinia enterocolitica from meats as a 14- to 21-day cold enrichment procedure, with or without KOH postenrichment. The shortened procedure consists of enriching 1.0- and 25-g samples of meat in phosphate-buffered saline (pH 7.2) at 25 degrees C. After incubation (48 and 72 h for 1.0-g samples and 24 and 48 h for 25-g samples); 0.5-ml portions of enrichment culture were treated with 4.5 ml of 0.25% KOH-0.5% NaCl for 2 min and 0.5% KOH-0.5% NaCl for 15 s, and 0.1-ml portions of treated culture were plated onto MacConkey or CIN agars or both. The procedure effectively recovered 2 to 12 cells of a number of both mouse-virulent and avirulent strains per g of ground beef with aerobic plate counts of approximately 10(6) to 10(7) CFU/g. Similarly, the procedure isolated both likely virulent and avirulent strains from porcine tongues (aerobic plate counts of 10(5) to 10(7) CFU/g) naturally contaminated with Y. enterocolitica. The organism was isolated from the tongues at similar rates by both shortened enrichment and cold enrichment procedures. Eight tongues were positive for serotype O:5,27 strains that agglutinate with WA-specific absorbed antiserum, an antiserum specific for mouse-virulent Y. enterocolitica (Doyle et al., Infect. Immun. 37:1234-1240, 1982), indicating that the oral cavity of swine is a reservoir of likely virulent serotype O:5,27 strains.     

Biological Activities of Endotoxins from Yersinia enterocolitica

The chemical properties and the general biological activities of lipopolysaccharide (LPS) and Boivin-type endotoxin obtained respectively by phenol-water and trichloroacetic acid extraction from Yersinia enterocolitica serotypes 03 and 09 were studied. The yield of LPS from the O9 strain was about 10% of the O3 strain possibly because of the lower solubility of O9-LPS in aqueous phase. However, the chemical composition of O9-LPS was similar to that of O3-LPS in the proportions of reducing sugar, glucosamine, heptose, KDO, and lipid A. In pyrogenicity and local Shwartzman reactivity in rabbits and lethality for mice, there was also no difference between O3 and O9-LPS. The anthrone-positive carbohydrate and lipid A contents of Boivin-type endotoxin from O3 were higher than those of the endotoxin from O9. The biological activities of Boivin-type endotoxin from O3 were also remarkably higher than those of the endotoxin from O9. It seems that endotoxin of Y. enterocolitica serotype O3 may play an important role in infection by this organism.     

Isolation of Yersinia enterocolitica from raw milk

Four enrichment procedures were used for examining 131 raw milk samples for the presence of Yersinia enterocolitica. Forty-two isolations were obtained from 19 pooled- (31.1 percent positive) and 10 individual-producer samples (14.3 percent positive). Enrichment by Butterfields phosphate buffer incubated at 4 degrees C for 14 days and then inoculation of modified Rappaport broth incubated at 23 degrees C for 5 days produced the greatest number of isolations. The majority of isolates were biotype 1, and many were atypical from clinical isolates in being rhamnose positive (47.6 percent), citrate positive (16.7 percent), and lactose positive (26.2 percent). Thirteen isolates were serotypable, belonging to seven different O serotypes, with O:5 occurring most frequently.     

Yersinia enterocolitica in raw goat's milk.

Biochemical and serological data are presented for 35 isolates of Yersinia enterocolitica from raw goat's milk produced in New South Wales, Australia. Strains resembled biotype I or 2, but the majority (25 of 35) fermented rhamnose and some showed other atypical reactions.