A comparison of media and methods for the recovery of Yersinia enterocolitica and Yersinia enterocolitica-like bacteria from milk containing simulated raw milk microfloras

A number of plating and enrichment media proposed for the isolation of Yersinia enterocolitica from foodstuffs were examined for their ability to recover the type strains of Y. enterocolitica sensu stricto, Y. intermedia, Y. frederiksenii and Y. kristensenii. Nine selective plating media were evaluated for the quantitative recovery of the type strains in pure culture, and their inhibition of other organisms typical of both milk and enteric microfloras. Cefsulodin-irgasan-novobiocin (CIN) agar, incubated for 48 h at 25°C, allowed a high recovery of all the Yersinia spp. and was the most selective medium. The same four type strains were added to UHT milk that had been previously inoculated with bacteria to simulate either freshly drawn or cold stored milk microfloras. Twenty-six enrichment procedures (including cold enrichment, selective enrichment at higher temperatures, two-step procedures and a post-enrichment alkali treatment) were assessed for the efficiency of recovery of the Yersinia spp. Pre-enrichment in trypticase-soy broth (TSB) for 24 h at 22°C followed by selective enrichment in bile-oxalate-sorbose (BOS) medium for 5 d at 22°C and plating on CIN agar (48 h at 25°C) allowed the greatest increase in the numbers of Yersinia spp. and maximum inhibition of the competing microflora.     

Heat resistance of Campylobacter and Yersinia strains by three methods

Two methods of determining the heat resistance of bacteria, a glass cup and a test tube method, were compared with a method using capillary tubes. Three strains of Yersinia enterocolitica , one of Campylobacter jejuni and two of C. coli were tested in physiological saline. The differences between the results obtained by the glass cup method and the reference method were not statistically significant for five strains and were small also for the other, a Yersinia strain. D values obtained by the glass cup method at 58, 60 and 62°C were 1.4–1.8, 0.40–0.51 and 0.15–0.19 min ( z values 4.00–4.52°C) for the Yersinia strains, and 0.42, 0.13 and 0.07 min ( z value 5.07°C) for one C. coli strain. For the other Campylobacter strains, D values of 0.71–0.78, 0.24–0.28 and 0.12–0.14 min ( z values 4.94 and 5.60°C) were recorded at 56, 58 and 60°C. D values obtained at 60°C by the test tube method were 2.7–5.0 min and were considered to be unrealistic.     

Partial purification and characterization of phospholipase C from Yersinia enterocolitica

About 34% of the strains of Yersinia enterocolitica isolated from raw milk were found to produce lecithinase. A selected strain produced phospholipase C at 22°C and 37°C; production was optimum at 37°C in the stationary phase (14–16 h). A decrease in phospholipase C activity at various storage temperatures (—5°C, 4°C, 37°C) was also observed, although the enzyme was active over a wide range of temperature (5–65°C) and pH (3mD5–7mD5). The phospholipase C was partially purified by ammonium sulphate precipitation and Sephadex column chromatography, and characterized.     

Autoaggregating Yersinia enterocolitica express surface fimbriae with high surface hydrophobicity

For 13 strains of Yersinia enterocolitica, there was a good correlation between the production of the broad-spectrum, mannose-resistant Yersinia haemagglutinin (MR/Y HA), the presence of fimbriae and high surface hydrophobicity. Each of these characters was expressed in cultures grown at low (less than 32 degrees C) but not at high (Greater than 35 degrees C) temperatures.     

Inhibition and inactivation of pathogenic serogroups of Yersinia enterocolitica by a bacteriocin produced by Yersinia kristensenii

Growth of Yersinia enterocolitica strains representing serogroups O: 3, O: 5, 27, O:6, 30, O:8, O:9 (human isolates) and O:6, 31 (food isolate) were inhibited in the presence of a bacteriocin produced by Yersinia kristensenii at high initial cell count of 10⁶ ml^-1. Complete (100%) inactivation of most Y. enterocolitica cells of different serotypes was observed within 24 h at low initial cell counts of 10⁴ ml^-1. Complete injury of the cells was observed within 4–8 h, with all the serotypes at 10°C and 28°C. The degree of susceptibility to the injury and the recovery of cells from the injury varied from serogroup to serogroup.     

The use of a surface adhesion immunofluorescent (SAIF) method for the rapid detection of Yersinia enterocolitica serotype O:3 in meat

This study examined the attachment kinetics of Yersinia enterocolitica serotype O:3 to determine the optimum conditions for its isolation from meat enrichment systems using a novel surface adhesion technique. Minced beef was inoculated with Y. enterocolitica at an initial level of 10 cfu g⁻¹ and incubated at 25 °C in an enrichment broth. Yersinia was recovered from enriched samples on polycarbonate membranes by surface adhesion and enumerated using immunofluorescence microscopy. The surface adhesion immunofluorescence technique (SAIF) had a minimum detection limit of approximately 4·0–4·5 log₁₀ cfu ml⁻¹ and provided good correlation between the estimation of the numbers of Yersinia in the enrichment broth derived from plate counts on Yersinia Selective agar (CIN) and those determined by SAIF ( r ² ₌ 0·94; rsd ₌ ± 0·21). A derived regression equation of the SAIF count vs plate counts was used to predict Y. enterocolitica numbers in spiked meat samples stored at 0 °C for up to 20 d. The numbers as predicted by the SAIF method showed good correlation with counts derived by plating techniques ( r ² ₌ 0·78; rsd ₌ ± 0·42). The application of the SAIF technique for the rapid detection of Y. enterocolitica serotype O:3 from meat is discussed.     

Recognition of pathogenic Yersinia enterocolitica by crystal violet binding and polymerase chain reaction

Cefsulodin-Irgasan-Novobiocin (CIN) agar is used for the selective isolation and enumeration of Yersinia enterocolitica from clinical specimens and food. The medium contains crystal violet and about 1 mmol l^-1 calcium and can be used for the phenotypic characterization of strains that carry a virulence plasmid. At 32°C, irrespective of pathogenicity, colonies are translucent with a pale pink centre surrounded by a transparent border ('bullseye'), while at 37°C pathogenic strains grow as calcium-dependent microcolonies which, because of crystal violet binding, are intensely coloured. These results were confirmed by the polymerase chain reaction with primers directed at the vir F gene, which is present only in pathogenic strains of Y. enterocolitica. Pathogenic strains of Y. enterocolitica can be recognized by growth at 37°C on Yersinia selective agar.     

Influence of nutrient-limited growth on pathogenesis-associated outer membrane proteins of Yersinia enterocolitica

From studies based on batch culture, it has been postulated that the expression of the virulence-associated proteins of Yersinia spp. is controlled by temperature and Ca²⁺, such that these proteins are synthesized only at the higher temperature (37°C) and calcium-scarce conditions of the intracellular environment. It was found, however, that in Yersinia enterocolitica one of these proteins (140 kDa) is not synthesized at submaximal growth rates under any of the relevant conditions, and that another of the implicated proteins (34 kDa), is synthesized even at 28°C during nutrient-limited growth. Thus, temperature and Ca²⁺ influence the synthesis of these proteins differently under growth conditions that better approximate the natural environments than do batch cultures.     

Isolation of Yersinia enterocolitica and related species from the faeces of cows

A total of 203 samples of faeces from 124 cows was examined for the presence of Yersinia enterocolitica and related species by a variety of isolation procedures. Cold enrichment at 5°C for three weeks, followed by plating on cefsulodin-irgasannovobiocin agar yielded Yersinia species most frequently. Yersinia enterocolitica or related species were isolated from 50% of the cows.     

Partial purification and characterization of bacteriocin from Yersinia kristensenii

A raw milk bacterial isolate, identified as Yersinia kristensenii was found to produce a bacteriocin which was inhibitory to Yersinia enterocolitica but not to other selected species of Yersinia or Gram-negative bacteria. Maximum production of bacteriocin was obtained when the organism was grown in shake culture at 28°C. Mitomycin C at a concentration of 0.5 μg ml^-1 induced bacteriocin production. The bacteriocin was partially purified and characterized by ammonium sulphate fractionation and gel filtration. The bacteriocin was completely inactivated when treated with proteolytic enzymes (trypsin and chymotrypsin). Bacteriocin activity was heat-resistant and it retained some of its activity after 5 min at boiling temperature. A total of 15 bacteriocin sensitive-suspected food isolates were further identified biochemically as Yersinia enterocolitica and a non-sensitive isolate was identified as Yersinia intermedia.     

Growth rate and physiology of Yersinia enterocolitica; influence of temperature and presence of the virulence plasmid

The effect of temperature on the growth rate, protein pattern and fatty acid composition of Yersinia enterocolitica strain W22703 pYV⁺, its plasmidless isogenic derivative W22703 pYV^- and four recent field isolates was examined.
The growth rate was clearly influenced by presence or absence of the virulence plasmid: pYV^- strains grew consistently faster than pYV⁺ strains. This difference in growth rate was high at 30–35°C, moderate at 1–10°C and 25°C, but hardly significant at 15–20°C.
Increasing the growth temperature above 25°C resulted in the induction of the 220 kDa virulence plasmid-encoded Yop1 protein. In the 1–20°C range no obvious temperature- or plasmid-related differences in protein patterns could be detected.
The fatty acid composition showed a clear temperature-dependent change: with all strains the degree of saturation was low at 1°C and gradually increased with raising temperatures. All strains had similar fatty acid patterns, except one of the field isolates which showed aberrant C16 : 1 and cyclic fatty acid contents in the 5–25°C and 15°C ranges respectively. With strain W22703, the presence or absence of the virulence plasmid did not significantly alter the fatty acid pattern.     

Isolation of Yersinia enterocolitica-resembling Organisms and Alteromonas putrefaciens from Vacuum-Packed Chilled Beef Cuts

Yersinia enterocolitica -resembling organisms were found at levels of 10⁷/g on a high pH (pH ≧ 6·0) vacuum-packaged beef striploin held for 6 weeks at 0·2°C, but did not exceed 10⁵/g on normal pH (pH < 6·0) striploins held for 10 weeks. Gram negative bacteria that produced H₂S on peptone iron agar were isolated from high pH vacuum packed striploins. These organisms were identified as Alteromonas putrefaciens . They attained levels of about 10⁷/g in 6 weeks at 0–2°C, at which time greening of the fat surface and 'drip'had occurred. On meat of normal pH, counts of A. putrefaciens were less than 10⁴/g after 6 weeks and no greening was evident.     

The psychrotrophic bacterium Yersinia enterocolitica requires expression of pnp, the gene for polynucleotide phosphorylase, for growth at low temperature (5°C)

The psychrotrophic bacterium Yersinia enterocolitica is characterized by temperature-dependent adaptations. To investigate Y . enterocolitica genes involved in cold adaptation, a mutant restricted in its ability to grow at 5°C was isolated from a transposon mutant library. The transposon insertion site in this psychrotrophy-defective (PD) mutant mapped 16 bp upstream of an open reading frame whose predicted amino acid sequence showed 93% similarity with the Escherichia coli exoribonuclease polynucleotide phosphorylase (PNPase), encoded by pnp . Expression of this gene was blocked in the PD mutant. However, the introduction of a second copy of pnp , including 0.33 kbp sequences upstream of its coding region, into the chromosome of the PD mutant restored pnp expression as well as the ability to grow at 5°C. Furthermore, the expression of pnp appeared to be temperature dependent: in the parental Y . enterocolitica strain, the levels of both pnp mRNA and PNPase were 1.6-fold higher at 5°C compared with 30°C. A similarly enhanced level of PNPase at 5°C was observed in the merodiploid recombinant strain, which indicates that the 0.33 kbp region upstream of pnp harboured a cold-inducible promoter. A putative cold shock promoter motif (ATTGG) was observed in this region.     

Microculture model studies on the effect of various gas atmospheres on microbial growth at different temperatures

A microculture technique, employing 96-well tissue culture plates in plastic bags, was used to test the effect of different gas atmospheres (vacuum, air, nitrogen, and carbon dioxide) on the growth of Escherichia coli, Bacillus macerans, Salmonella typhimurium, Candida albicans, Lactobacillus plantarum, Pseudomonas Acinetobacter/Moraxella -group, Brochothrix thermosphacta and Yersinia enterocolitica at 2, 6, and 20°C. In general, carbon dioxide was the most effective inhibitor. The inhibition increased with decreasing temperature. Only the combination of carbon dioxide and 2°C provided complete inhibition of Broch. thermosphacta and Y. enterocolitica.     

Expanded response surface model for predicting the effects of temperatures, pH, sodium chloride contents and sodium nitrite concentrations on the growth rate of Yersinia enterocolitica

The previously reported data set for the low temperature (5, 12 and 19°C) of Yersinia enterocolitica was expanded to include higher abusive temperature (28, 37 and 42°C). In addition to temperature, the data set included the effects and interactions of pH (4.5–8.5), sodium chloride (0.5-5%) and sodium nitrite (0-200 μg ml^-1) on the aerobic growth of Y. enterocolitica in brain heart infusion broth. Growth curves were modeled by fitting viable count data to the Gompertz equation. Quadratic models of natural logarithm transformations of the Gompertz B and M values and the derived values for lag phase durations and generation times were obtained using response surface analyses. Predictions based on the models for B and M values were comparable to predictions based on the derived values. These revised models provide an expanded means for rapidly estimating how the bacterium is likely to respond to any combination of the four variables within the specified ranges.     

Haemagglutinins and Fimbriae in Different Serotypes and Biotypes of Yersinia enterocolitica

When cultured in static broth at 20°C, 46 of 115 strains of Yersinia enterocolitica , diverse in biotype and serotype, produced a broad-spectrum mannose-resistant (MR) adhesin that agglutinated the erythrocytes of all of 10 animal species examined. The production of haemagglutinin (HA) was associated with the presence of fimbriae of S nm diameter. Culture of HA+ strains at 37° resulted in the disappearance of haemagglutinating ability and loss of fimbrial production. Strains of Y. enterocolitica with K1 antigen produced an MR adhesin that agglutinated only fowl erythrocytes and was associated with fimbriae of 4–4.5 nm diameter. None of 14 strains of Y. pseudotuberculosis was haemagglutinating.     

The effect of carbon dioxide on the growth of Yersinia enterocolitica in a simulated milk medium

Carbon dioxide (30 mmol/l) was shown to inhibit the growth of four strains of Yersinia enterocolitica grown separately in a simulated milk medium at 7°C and 60 rev/min for 4 d. This indicates that addition of CO₂ to refrigerated raw milk supplies is likely to be a safe process with respect to that organism.     

A novel non-fimbrial mannose-sensitive and eluting (MSE) haemagglutinin of Yersinia frederiksenii strains

Abstract An unusual, mannose-sensitive and eluting haemagglutinin (MSEHA), not previously described in Yersinia species, was produced by some Yersinia frederiksenii strains. The MSEHA, which was formed best in broth-grown cultures at 18°C, agglutinated erythrocytes of guinea pig, fowl, horse and pig in haemagglutination tests made in the absence of α-methylmannoside and in the cold. The MSEHA, which was labile to proteolytic enzymes, heat, alcohol and acid, seemed to be a non-fimbrial, cell-bound protein. This unusual kind of non-fimbrial MSEHA has not previously been described in Enterobacteriaceae.