Osmoregulation-dependent expression of Yersinia enterocolitica virulence factors.

The effects of hyperosmotic stress on expression of plasmid coded Yop and Yad A proteins--virulence factors of Y.enterocolitica serotype 0:9 were characterized. When cells were shifted to high osmolarity and cultured at 37 degrees C in medium without Ca2+ the production of Yops was inhibited. In contrast, the amount of Yad A protein was unaffected. Addition of glycine betaine to this culture alleviated the effect of high osmolarity. It was also found that hyperosmotic stress causes increased negative supercoiling of DNA in Y. enterocolitica 0:9. Changes in DNA supercoiling coincided with expression of Yop proteins. These results suggest that in high osmolarity the expression of yop genes may be regulated by DNA supercoiling.     

Evaluation of usefulness for selected virulence markers for identifying pathogenic Yersinia enterocolitica strains. IV. Genes myfA and ureC

We check by polymerase chain reaction (PCR) the presence of gene ureC and myfA, encoding subunits of urease and Myf fimbriae, among clinical and food-originated strains of Yersinia to determine their usefulness as molecular virulence markers of Y. enterocolitica. The examinations were done on 130 clinical strains of Y. enterocolitica O:3/4 isolated in Poland from humans. All strains were obtained from stool and possessed the virulence plasmid pYV. In addition 40 isogenic, plasmid-cured strains were tested. The 52 strains including Y. enterocolitica (biotype 1A, 4, 2 and 1B), Y. pseudotuberculosis, Y. intermedia, Y. frederiksenii, Y. kristensenii, E. coli, Citrobatcer, Shigella and Salmonella were used as controls. The PCR assay resulted in detection of genes: ureC and myfA in genomic DNA of all 130 tested clinical strains of Y. enterocolitica pYV+, as well as in plasmid cured strains. Furthermore, ureC was found in all tested strains of Y. enterocolitica biotype A1 and in one strain of Y. intermedia and Y. kristensenii. In contrast to ureC, myfA was detected only in strains of Y. enterocolitica considered as pathogenic. Obtained results show, gene myfA seems to be the reliable virulence marker of Y. enterocolitica, whereas ureC is not recommended for identification of pathogenic strains of this species.     

Yersinia enterocolitica immunodominant 60 kDa antigen, common to a broad range of bacteria, is a heat-shock protein

Monoclonal antibodies (mAbs) against the Yersinia enterocolitica immunodominant 60 kDa antigen, termed cross-reacting protein antigen (CRPA), were obtained by fusion of spleen cells from mice immunized with CRPA with murine myeloma cells. The reactivities of the mAbs were examined by Western blotting against extracts of Y. enterocolitica and 23 other species of Gram-positive and Gram-negative bacteria. Cross-reactions were recognized with a wide range of bacteria, but not with Gram-positive cocci. The reactivities were different for each mAb, suggesting that both species-specific and multiple cross-reactive epitopes were present on the CRPA molecule. CRPA was produced under heat-shock conditions in Y. enterocolitica and was shown to correspond immunologically to the GroEL protein in Escherichia coli, a protein involved in the morphogenesis of coliphage. In addition to CRPA, at least nine other major heat-shock proteins were detected by two-dimensional gel electrophoresis of extracts of heat-shocked Y. enterocolitica.     

The osmotic regulator OmpR is involved in the response of Yersinia enterocolitica O:9 to environmental stresses and survival within macrophages

Various environmental signals control the expression of the virulence factors in pathogenic Yersinia enterocolitica strains. The role of the osmotic regulator OmpR protein in controlling the production of Yop proteins, virulence determinants in Y. enterocolitica O:9 (European type) has been studied. An ompR deletion mutant was constructed via allelic exchange with an ompR gene of Y. enterocolitica mutagenized in vitro by a reverse genetic polymerase chain reaction (PCR)-based strategy. The ompR mutant showed a reduced ability to survive under conditions of various environmental stresses in vitro. In particular, low pH stress resulted in increased cell mortality levels. Under conditions of high osmolarity, the wild strain's Yop protein production was reduced, whereas protein levels from the mutant strain remained constant regardless of osmolarity variance. In J774A.1 macrophage cell culture survival of the ompR mutant was decidedly lower than that of the wild-type strain, suggesting that the OmpR protein may play a significant role in protecting cells against intracellular conditions associated with macrophage phagocytosis.     

Structure and function of pore-forming proteins from bacteria of the genus Yersinia: I. Isolation and a comparison of physicochemical properties and functional activity of Yersinia porins

The molecular organization and functional activity of porins isolated from the outer membrane (OM) of the Yersinia enterocolitica and three phylogenetically close nonpathogenic Yersinia species (Y. intermedia, Y. kristensenii, and Y. frederiksenii) cultured at 6-8 degrees C were comparatively studied for the first time. The proteins were isolated in two molecular forms (trimeric and monomeric), and their spatial structures were characterized by the methods of optical spectroscopy, CD and intrinsic protein fluorescence. The studied porins were shown to belong to the beta-structural proteins (they have 59-96% total beta structures and 0-17% alpha helices). The spatial structures of the proteins were demonstrated to depend on the nature of the detergent used for solubilization. Unlike the enterobacterial pore-forming proteins, the porin trimers are less stable to sodium dodecyl sulfate (SDS). The spatial structures of the porins become more compact after the substitution of octyl beta-D-glucoside for SDS: the content of beta structures increases and the accessibility of Trp residues to solvent decreases. It was established with the use of the technique of bilayer lipid membranes that the functional properties of the porins are similar to those of the OmpF proteins of Gram-negative bacteria. Trimers are functionally active forms of the porins. Special features of the pore-forming activity of the Yersinia porins were revealed to depend on the microorganism species and the value of the membrane potential.     

Antagonism betweenYersinia intermedia andYersinia enterocolitica in water

One strain of Yersinia enterocolitica and one strain of Y. intermedia were grown in peptone water at 25 or 37 degrees C, or in ground water at 15 degrees C. Similar growth rates were observed when these strains were cultivated separately in the same media and at the same temperature. Mixed cultures at 37 degrees C displayed equivalent growth rates. In contrast, mixed cultures incubated at 15 or 25 degrees C were regularly unfavourable to Y. enterocolitica, whereas they did not modify the growth of Y. intermedia. A bacteriophage active on Y. enterocolitica and not on Y. intermedia was characterized from the filtrate of mixed cultures at low temperatures. This phage produced by the lysogenic Y. intermedia strain might be a potential factor responsible for the inhibition of Y. enterocolitica, since no additional antibacterial factor or nutritional competition between Y. intermedia and Y. enterocolitica were found in the mixed cultures.     

Characterization of outer membrane proteins of Yersinia pestis and Yersinia pseudotuberculosis strains isolated from India

The majority of virulence factors including the 12 Yersinia outer membrane proteins (Yops), 29 Yop secretion proteins (Ysc) and few specific Yop chaperone (Syc) are contributed by the 70 kb LCR middle plasmid of Yersinia pestis. Yersinia pestis isolates recovered during 1994 plague outbreak and rodent surveillance samples of Southern states of India were studied for the presence of important Yops by the conventional procedure of partially purifying outer membrane proteins (Omps) after cultivation in calcium deficient media. Prominent bands numbering 4-5 between 34-42 kDa region corresponding to important Yops were seen in all the isolates as well as in other Yersinia and non-Yersinia species by SDS-PAGE. Western blotting with the polyclonal antisera raised against these Omp preparations revealed few immuno-reactive bands that appeared to be shared among Y. pestis, Y. pseudotruberculosis, Y. enterocolitica, Y. fredrocksenii, Y. intermedia, Y. kristensenii and E. coli. Three recombinant Yop proteins namely, YopM, YopB and LcrV were produced and antisera to these proteins could reveal presence of these Yops in the Y. pestis Omp preparations. In order to further characterize the important Yops among Omps, attempts were made to generate monoclonal antibodies against Omp preparation. Three of the 4 stable reactive clones that were obtained, when tested, had extensive cross-reactions among pathogenic Yersinia species, Y. pestis and Y. pseudotuberculosis isolates, other Yersinia species and the members of Enterobacteriaceae in dot-ELISA and Western blotting. One of the monoclonal antibodies, YP1, exhibited reaction to all the pathogenic Yersinia species and the isolates, with restricted cross-reactivity to Y. intermedia, Y. kristensenii, K. pneumoniae. None of the 4 monoclonal antibodies had reactions with the 3 recombinant Yop proteins. It appears that under low calcium response, the Y. pestis not only activates secretion of Yops but also a large number of other proteins, which as per the present observations are cross-reactive within the family Enterobacteriaceae.     

Comparative study of temperate bacteriophages isolated from Yersinia

170 Yersinia strains belonging to various species were investigated for the presence of temperate bacteriophages. By induction with mitomycin C seven phages were isolated from Y. enterocolitica strains and one phage from a Y. frederiksenii strain. The phages were characterized on the basis of their morphology, host range, genome size, DNA homology, and protein composition. They belong to different phage families and reveal narrow to moderate wide host ranges. Some of the isolated phages were able to infect pathogenic as well as nonpathogenic strains of Y. enterocolitica. The genomes of all isolated phages were found to be composed of double stranded DNA ranging from about 40 to 60 kb. In addition to the analysed phages, a number of putative phages were induced in strains of Y. frederiksenii, Y. kristensenii, Y. intermedia, and Y. mollaretii. The putative phages were identified by isolation of phage DNA from cell free lysates but could not be propagated on indicator strains. Southern hybridization experiments revealed relationships between phages belonging to different families. Moreover, DNA homologies were observed between phages isolated from nonpathogenic Yersinia strains and a phage which was isolated from a pathogenic Y. enterocolitica serogroup O:3 strain.     

Contribution of trimeric autotransporter C-terminal domains of oligomeric coiled-coil adhesin (Oca) family members YadA, UspA1, EibA, and Hia to translocation of the YadA passenger domain and virulence of Yersinia enterocolitica

The Oca family is a novel class of autotransporter-adhesins with highest structural similarity in their C-terminal transmembrane region, which supposedly builds a beta-barrel pore in the outer membrane (OM). The prototype of the Oca family is YadA, an adhesin of Yersinia enterocolitica and Yersinia pseudotuberculosis. YadA forms a homotrimeric lollipop-like structure on the bacterial surface. The C-terminal regions of three YadA monomers form a barrel in the OM and translocate the trimeric N-terminal passenger domain, consisting of stalk, neck, and head region to the exterior. To elucidate the structural and functional role of the C-terminal translocator domain (TLD) and to assess its promiscuous capability with respect to transport of related passenger domains, we constructed chimeric YadA proteins, which consist of the N-terminal YadA passenger domain and C-terminal TLDs of Oca family members UspA1 (Moraxella catarrhalis), EibA (Escherichia coli), and Hia (Haemophilus influenzae). These constructs were expressed in Y. enterocolitica and compared for OM localization, surface exposure, oligomerization, adhesion properties, serum resistance, and mouse virulence. We demonstrate that all chimeric YadA proteins translocated the YadA passenger domain across the OM. Y. enterocolitica strains producing YadA chimeras or wild-type YadA showed comparable binding to collagen and epithelial cells. However, strains producing YadA chimeras were attenuated in serum resistance and mouse virulence. These results demonstrate for the first time that TLDs of Oca proteins of different origin are efficient translocators of the YadA passenger domain and that the cognate TLD of YadA is essential for bacterial survival in human serum and mouse virulence.     

Generation and Characterization of Murine Monoclonal Antibodies to Recombinant YopM, YopB and LcrV Proteins of Yersinia pestis

Summary YopM, an effector, YopB, a translator, and LcrV, a regulator, are proteins forming important componants of type III secretion system of Yersinia pestis. Recombinant truncated YopM of 32 kDa, YopB of 28 kDa and LcrV of 31 kDa sizes were utilized for priming BALB/c mice for the generation of monoclonal antibodies following standard poly-ethylene glycol (PEG) fusion protocol. Nine, 10 and 6 stabilized hybridoma cell lines could be generated against YopM, YopB and LcrV proteins, respectively. All these monoclonal antibodies were found reactive to Y. pestis strain A1122 and did not show any cross-reactivity to Y. enterocolitica, Y. pseudotuberculosis, Y. kristensenii, Y. frederiksenii, Y. intermedia, Klebsiella pneumoniae, Escherichia coli, Salmonella typhi, Salmonella abortus-equi and Staphylococcus aureus tested by ELISA and Western blotting. Monoclonal antibodies also exhibited reactivity to their corressponding native protein antigens in Y. pestis i.e. 42 kDa for YopM, 41 kDa for YopB and 37 kDa for LcrV in immunoblotting. Reactivity of monoclonal antibodies was further assessed on 26 Y. pestis isolates including 18 from 1994 plague outbreak regions (11 from pneumonic patients, 7 from rodents) and 8 from rodents of Deccan plateau of Southern India by Western blotting as well as by sandwich ELISA. The monoclonal antibodies could specifically locate the expression of yopM, yopB and lcrV genes among these Indian Y. pestis strains as well. Results obtained with sandwich ELISA and Western blot were identical to those observed by PCR. Monoclonal antibodies to Yops, therefore, can be employed for an early and reliable identification of virulent Y. pestis strains.     

Genotyping of human and porcine Yersinia enterocolitica, Yersinia intermedia, and Yersinia bercovieri strains from Switzerland by amplified fragment length polymorphism analysis

In this study, 231 strains of Yersinia enterocolitica, 25 strains of Y. intermedia, and 10 strains of Y. bercovieri from human and porcine sources (including reference strains) were analyzed using amplified fragment length polymorphism (AFLP), a whole-genome fingerprinting method for subtyping bacterial isolates. AFLP typing distinguished the different Yersinia species examined. Representatives of Y. enterocolitica biotypes 1A, 1B, 2, 3, and 4 belonged to biotype-related AFLP clusters and were clearly distinguished from each other. Y. enterocolitica biotypes 2, 3, and 4 appeared to be more closely related to each other (83% similarity) than to biotypes 1A (11%) and 1B (47%). Biotype 1A strains exhibited the greatest genetic heterogeneity of the biotypes studied. The biotype 1A genotypes were distributed among four major clusters, each containing strains from both human and porcine sources, confirming the zoonotic potential of this organism. The AFLP technique is a valuable genotypic method for identification and typing of Y. enterocolitica and other Yersinia spp.     

A heat-modifiable outer membrane protein carries an antigen specific for the species Yersinia enterocolitica and Yersinia pseudotuberculosis

The outer membranes of gram-negative bacteria are considered to be of importance in host-bacteria interaction, in protective immunity, and occasionally in subclassification within a species. In this study, the outer membranes of several strains of Yersinia enterocolitica and Y. pseudotuberculosis were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It was found that the appearance of the major proteins depended on the temperature at which they were solubilized in SDS. A protein was identified with the use of two-dimensional gels and preparative SDS-PAGE, which was equivalent to the "heat-modifiable protein" (protein II) of other Enterobacteriaceae species. A monoclonal antibody, 4G1, was generated against an isolated preparation of this Y. enterocolitica protein. This antibody was tested with whole cell bacterial antigens of 46 individual bacterial strains. The reactive strains included only Y. enterocolitica and Y. pseudotuberculosis. In addition, the reactivity of the 4G1 monoclonal antibody preparation could be absorbed only with Y. enterocolitica and Y. pseudotuberculosis, and not with other strains of bacteria. The reactivity of this 4G1 monoclonal antibody was also tested by the Western Blot technique. Six individual strains were tested: a Y. enterocolitica serotype 0:3, a Y. enterocolitica serotype 0:9, an Escherichia coli, a Salmonella typhimurium, a Shigella flexneri, and a Klebsiella pneumoniae. The 4G1 antibody reacted with only the proteins of the two Y. enterocolitica strains. In conclusion, the equivalent of the heat-modifiable protein was present in Y. enterocolitica and Y. pseudotuberculosis. Moreover, this protein also carried a species-specific antigenic determinant.     

Protein phosphorylation in rat liver mitochondria

Summary Incubation of rat liver mitochondria in the presence of either [³²P] Pi or ₃₂ ^y -P] ATP resulted in a phosphorylation of four proteins with Mr 50, 47, 44 and 36 kDa, respectively. The endogenous phosphorylation of these proteins in the presence of [³²P] Pi was markedly influenced by the osmolarity of the incubation medium and differentially affected by various effectors of mitochondrial functions, such as Ca²⁺, oligomycin, FCCP, arsenite and dichloroacetate. In particular, the 36 kDa protein, unlike the other proteins, appears to be phosphorylated also by direct incorporation of [³²P], independently of respiratory chain-linked ATP synthesis. The four proteins, located in the mitoplasts, seem to be phosphorylated by diiferent protein kinases, as suggested by the observation that the endogenous phosphorylation of 36 kDa protein resulted selectively increased by addition of exogenous protein kinases, such as casein kinases S and TS. A tentative identification of these phosphorylatable protein is discussed.     

Purification of cross-reacting protein antigen shared by Yersinia enterocolitica and other gram-negative bacteria with monoclonal antibody

A monoclonal antibody against the Yersinia enterocolitica 60-kilodalton (kDa) antigen, designated cross-reacting protein antigen (CRPA), was obtained by cell fusion. The CRPA common to gram-negative bacteria was purified from Y. enterocolitica by the affinity chromatography with the monoclonal antibody (IgG1) thus obtained. The purified CRPA showed a single band of 60 kDa in SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and reacted with rabbit antisera against Y. enterocolitica, Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa, and Shigella sonnei in Western blot analysis. The monoclonal antibody, however, reacted with a 60 kDa peptide from Y. enterocolitica, but not with the antigens from other gram-negative bacteria such as V. cholerae, E. coli, S. sonnei, Salmonella enteritidis, Serratia marcescens, Klebsiella pneumoniae, Proteus mirabilis, and P. aeruginosa. The results suggested that both species-specific and cross-reactive epitopes were present on a CRPA molecule.     

Monoclonal antibodies directed against plasmid-encoded released proteins of enteropathogenic Yersinia

Abstract Yersinia enterocolitica of serotypes O:3, O:8, O:9 and O:5,27 and Yersinia pseudotuberculosis of serotypes I and III release plasmid-encoded proteins into calcium-deficient medium. Mouse monoclonal antibodies were elicited against plasmid-encoded released proteins of Y. enterocolitica of serotype O:9. As shown by immunoblot analysis the monoclonal antibody Mab9–200 recognized the 46-kDa protein of Y. enterocolitica of serotypes O:3, O:9 and O:5,27, the 58-kDa protein of Y. enterocolitica of serotype O:8 and the 67-kDa protein of Y. pseudotuberculosis of serotypes I and III. Mab9–15 reacted with the 36-kDa protein of Y. enterocolitica of serotypes O:9, O:3 and O:8, and the 34-kd protein of Y. enterocolitica of serotype O:5,27 and Y. pseudotuberculosis of serotypes I and III. The 25-kDa proteins of Y. enterocolitica of serotypes O:3, O:9, O:8 and O:5,27, but not those of Y. pseudotuberculosis were recognized by the monoclonal antibody Mab-128. This species-specific recognition of epitopes could not be achieved by mouse polyclonal antibodies.     

Electrophoretic mobility and immune response of outer membrane proteins of Vibrio cholerae O139

Abstract The outer membrane (OM) protein components of a Vibrio cholerae O1 and four V. cholerae O139 strains, collected from cholera patients, were analysed by SDS-PAGE. A protein of 69 kDa molecular mass was observed only when the OMPs were prepared from strains grown in synthetic broth. As a result of passage in the rabbit ileal loop (RIL), virulence was enhanced, and a protein component around 18 kDa of the V. cholerae O139 OM became the major protein component. On immunoblot analysis with rabbit antiserum against V. cholerae O139 OM, it was shown that, apart from the major protein component of V. cholerae O1 OM of around 45 kDa and that of V. cholerae O139 OM of around 38 kDa, all other minor protein components were cross-reactive between the two serogroups. In immunoblot assays with convalescent sera obtained from V. cholerae O139-infected patients, it was observed that in addition to the lipopolysaccharide (LPS)-induced antibody, only the 38 kDa major protein component elicited considerable levels of antibody in the pateint. Minor OM components of 18 kDa were detected in the immunoblot analysis by LPS-directed antibody, however, as the OM proteins are known to be associated with LPS.     

virF-positive Yersinia pseudotuberculosis and Yersinia enterocolitica found in migratory birds in Sweden

During spring and autumn migrations, 468 fecal samples from 57 different species of migratory birds were collected in Sweden. In total, Yersinia spp. were isolated from 12.8% of collected samples. The most commonly found species was Yersinia enterocolitica, which was isolated from 5.6% of all collected samples, followed by Y. intermedia (3.8%), Y. frederiksenii (3.0%), Y. kristensenii (0.9%), Y. pseudotuberculosis (0.6%), and Y. rohdei (0.4%). The pathogenic, virF-positive Y. pseudotuberculosis strains were recovered from three thrushes. These strains belonged to the same bioserotype, 1/O:2, but had two different profiles as determined by pulsed-field gel electrophoresis with NotI and SpeI enzymes. In addition, 10 Y. enterocolitica strains, all from barnacle geese, belonged to bioserotype 3/O:3, which is associated with human disease. Two of the strains were pathogenic, carrying the virF gene on their plasmids. All pathogenic Y. pseudotuberculosis and Y. enterocolitica strains were recovered during the spring, and as the birds were caught during active migration they likely became infected at an earlier stage of the migration, thus potentially transporting these bacterial pathogens over long geographical distances.     

Analysis of bacteriocinogenic properties of Yersinia enterocolitica strains

The aim of the study was the investigation of bacteriocinogenic properties of 102 Yersinia enterocolitica strains. The influence of selected factors on the production of bacteriocins by Y. enterocolitica and properties of jersiniacin 44JPSBKOH were also investigated. Bacteriocinogenic properties of Y. enterocolitica strains were tested by using the delayed cross-streaking method. It was found that the production of bacteriocins by Y. enterocolitica depended on the type of media on which the producer and indicator strains were grown. It turned out that some strains of Y. enterocolitica showed bacteriocinogenic properties at 25 degrees C, 30 degrees C and 37 degrees C irrespective of the presence of manganese ions in medium. In the presence of iron ions these strains showed bacteriocinogenic properties only at 25 degrees C. Y. enterocolitica strains which required Mn2+ or Mn7+ ions for bacteriocins production showed this activity only at 25 degrees C but in presence of Fe3+ ions they had no bacteriocinogenic properties. The partially purified jersiniacin 44JPSBKOH is a protein, its molecular weight was estimated to be 40 kDa. Yersiniacin 44JPSBKOH was active in the pH range of 3 to 9. Its bactericidal activity was rapidly lost when heated to 100 degrees C and treated with proteolytic enzymes. Yersiniacin 44JPSBKOH showed bactericidal activity against other Y. enterocolitica strains and some strains of Pseudomonas aeruginosa isolated from humans.     

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.     

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 degrees 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 degrees C followed by selective enrichment in bile-oxalate-sorbose (BOS) medium for 5 d at 22 degrees C and plating on CIN agar (48 h at 25 degrees C) allowed the greatest increase in the numbers of Yersinia spp. and maximum inhibition of the competing microflora.