Binding of Yersinia enterocolitica to rabbit intestinal brush border membranes, mucus, and mucin.

Mucus and its gel-forming glycoprotein component, mucin, are thought to protect the gastrointestinal tract from enteric pathogens by inhibiting their attachment to enterocytes. In this study, we investigated interactions between Yersinia enterocolitica (isogenic strains of virulent and nonvirulent organisms) and crude mucus, highly purified mucin, and brush border membranes (BBMs) isolated from the upper mid-, and distal small intestine and the proximal colon of the rabbit. Adherence of radiolabeled bacteria was assessed to BBMs, mucus, and mucin immobilized in polystyrene microtiter plate wells. Virulent Y. enterocolitica showed saturable binding to mucus, mucin, and BBMs from all four regions of the intestinal tract, although adherence to BBMs was appreciably greater than that to mucus or mucin. Maximal binding of bacteria was higher to BBMs from the distal small intestine and the proximal colon than to those from the upper and mid-small intestine, which may in part explain why the organism localizes to the ileo-caecal regions of the gut. Adherence of virulent Y. enterocolitica to BBMs was significantly reduced in the presence of homologous mucus or mucin preparations. Binding of virulent bacteria appears to depend on plasmid-encoded proteins located on the outer surface membrane, since (i) the isogenic strain lacking the virulence plasmid showed markedly less binding to all BBM, mucus, and mucin preparations; (ii) growth of the virulent strain at 25 degrees C, which inactivates its plasmid, significantly diminished binding to BBMs, mucus, and mucin; and (iii) mild proteolysis substantially decreased adherence of virulent bacteria to BBMs. Compared with rabbit intestinal and colonic mucins, binding of virulent Y. enterocolitica was significantly greater to purified human intestinal mucin and significantly less to rat intestinal mucin. These findings provide support for the role of mucus and mucin in host defense by preventing adherence of virulent Y. enterocolitica to epithelial cell membranes.     

Adhesion of Yersinia enterocolitica to purified rabbit and human intestinal mucin.

Interactions between Yersinia enterocolitica and purified intestinal mucins from rabbit and humans were investigated. Plasmid-bearing virulent organisms (but not plasmid-free nonvirulent bacteria) bound well to both mucins, suggesting that adherence was controlled by the virulence plasmid. Examination of binding to 14 different preparations of purified human intestinal mucin (8 preparations obtained from normal subjects and 6 samples from patients with cystic fibrosis) revealed no differences between normal and cystic fibrotic mucins in ability to serve as a binding substrate for virulent Y. enterocolitica. Analyses of binding curves suggested the presence of a single type of noninteracting receptor for Y. enterocolitica in both rabbit and human mucins with similar (but not necessarily identical) structures. Virulent bacteria bound to polystyrene through hydrophobic interactions that could be disrupted by treating the organisms with tetramethyl urea. In contrast, binding of plasmid-bearing Y. enterocolitica to intestinal mucin was not susceptible to tetramethyl urea and therefore does not appear to involve hydrophobic interactions. Prior incubation of organisms with mucin significantly inhibited binding to polystyrene, suggesting that mucin can mask hydrophobic adhesins on the bacterial surface. Hapten inhibition studies revealed that the monosaccharides galactose and N-acetylgalactosamine and the disaccharide lactose could markedly reduce (but not abolish) bacterial adherence to mucin but other monosaccharides and the RGD peptide had no effect on mucin binding. We conclude that virulent Y. enterocolitica is capable of interacting with the carbohydrate moiety of intestinal mucin. These interactions appear to be plasmid mediated and not hydrophobic.     

Binding of Yersinia enterocolitica to purified, native small intestinal mucins from rabbits and humans involves interactions with the mucin carbohydrate moiety.

Plasmid-bearing (but not plasmid-cured) Yersinia enterocolitica is known to bind to purified small intestinal mucins from rabbits and humans. This study examined which region(s) of the mucin molecule is important for bacterial adherence. Pronase digestion of mucin and removal of nonglycosylated or poorly glycosylated peptide regions had no effect on bacterial binding, suggesting that plasmid-bearing Y. enterocolitica interacts with mucin carbohydrate. Periodate oxidation also did not alter bacterial adherence, indicating that vicinal hydroxyl groups in the mucin sugars are not important for binding. Boiling of mucin, depolymerization by reduction of disulfide bonds, or removal of noncovalently associated lipid actually enhanced bacterial adherence, suggesting that plasmid-bearing Y. enterocolitica can interact with additional domains in the mucin molecule revealed by these treatments. These domains were destroyed by pronase digestion. In delipidated mucin (but not in reduced or boiled mucin), binding to these domains appeared to be hydrophobic since it could be prevented by treatment of bacteria with tetramethyl urea. Oligosaccharides obtained from both human and rabbit small intestinal mucins were capable of inhibiting attachment of plasmid-bearing (but not plasmid-cured) Y. enterocolitica to mucin. After removal of terminal and backbone sugar residues by treatment of mucin with trifluoromethanesulfonic acid, binding of plasmid-bearing bacteria increased significantly when N-acetylgalactosamine, either alone or with galactose attached, was revealed, indicating that core regions of the sugar side chains are involved in bacterial binding. Adherence of plasmid-cured organisms was unaffected by trifluoromethanesulfonic acid treatment of mucin. We concluded that virulent Y. enterocolitica interacts with the carbohydrate moiety of native small intestinal mucin through a plasmid-mediated process. When mucin becomes denatured, binding of the organism can increase through hydrophobic and nonhydrophobic interactions with (most likely) the mucin protein.     

Expression of antigens encoded by the virulence plasmid of Yersinia enterocolitica under different growth conditions.

The expression of polypeptides of the virulence plasmid of Yersinia enterocolitica serotype O:3 was studied with the immunoblotting technique and specifically absorbed antisera to Y. enterocolitica O:3. At least 16 polypeptides were apparently specified by the virulence plasmid when plasmid-bearing bacterial were grown at 37 degrees C or intraperitoneally in semipermeable capsules. The different growth media used (also with added Ca2+) had quantitatively or qualitatively only a little influence on the expression of these polypeptides, whereas the growth temperature had a stronger influence. The best expression was achieved at 37 degrees C, at 22 degrees C the expression was weak, and at 4 degrees C the plasmid genes were inactive. Two chromosomally encoded polypeptides were expressed only at 37 degrees C, whereas the expression of eight polypeptides expressed at 22 degrees C was repressed at 37 degrees C. The intraperitoneal growth in capsules was used to detect the virulence plasmid-associated polypeptides of Yersinia pestis. Four plasmid-associated polypeptides were detected in Y. pestis with antiserum to Y. enterocolitica virulence plasmid antigens, and three were detected with antiserum to Y. pestis EV76. These results suggested that the virulence plasmid of Y. pestis was activated in the interstitial environment in vivo, where Ca2+ concentration was high, and also that the virulence plasmids of Y. enterocolitica and Y. pestis have three to four immunologically related polypeptides in common.     

Plasmid-associated cell surface charge and hydrophobicity of Yersinia enterocolitica

Virulent strains of Yersinia enterocolitica and their plasmidless, avirulent derivatives were examined for their cell surface properties. Increased surface charge and hydrophobicity of Y. enterocolitica were found to be associated with the possession of a 40- to 48-megadalton plasmid. These surface properties were expressed, as were other plasmid-associated properties, at 37 but not at 22 degrees C. The concentration of calcium in the growth medium had a moderate effect on the expression of the cell surface properties. These cell surface properties were greatly reduced among plasmid-bearing cells grown on tryptic soy agarose regardless of growth temperatures. These properties were also associated with the ability of Y. enterocolitica to colonize the gastrointestinal tract of mice.     

Interaction of Yersinia enterocolitica and Y. pseudotuberculosis with platelets.

Yersinia enterocolitica is a bacterium capable of growth at 4 degrees C in donated blood and has been responsible for many deaths following transfusion. Interaction of Y. enterocolitica with blood cells is of interest in understanding the mechanisms of survival and growth in blood. The closely related organism Y. pseudotuberculosis is known to invade platelets and cause platelet aggregation by a mechanism that involves expression of the chromosomal inv gene. Yersinia isolates were made to express green fluorescent protein (GFP) and their interaction with platelets was studied by flow cytometry, enterocolitica did not cause platelet aggregation or activation, not even when grown at 22 degrees C to maximise inv expression. Attachment of Y. enterocolitica O:9 to platelets occurred with virulence plasmid-bearing (pYV+) strains grown at 37 degrees C but not with pYV- strains nor with strains grown at 22 degrees C. Y. pseudotuberculosis containing inv did cause platelet activation and aggregation when grown at 22 degrees C, as has been shown before, but also showed enhanced attachment to platelets when grown at 37 degrees C. Electron microscopy studies confirmed that inv-expressing Y. pseudotuberculosis invaded platelets but Y. enterocolitica attached only to the outer surface of platelets. Interaction of Y. enterocolitica O:9 with platelets provided a modest protection against bacterial killing by human serum. Interaction of Y. enterocolitica O:9 with platelets does not lead to platelet invasion or activation, and is mediated through plasmid-coded factors, not inv.     

Serum resistance associated with virulence in Yersinia enterocolitica.

Yersinia enterocolitica strains that exhibited a calcium requirement for growth and autoagglutination at 37 degrees C were invariably virulent in rabbits, causing diarrhea and a high degree of lethality, and were capable of colonizing the intestinal lumen and establishing foci of infection on the Peyer's patches of mice. Strains that had lost the properties of calcium dependency and autoagglutinability were totally avirulent in rabbits and were quickly eliminated from the intestinal lumen and tissues of mice. Virulent and avirulent strains were shown to be equally invasive to HeLa cells. However, the virulent strains were resistant to the bactericidal action of normal serum, and this serum resistance was lost with the loss of virulence. Furthermore, the serum resistance of virulent strains was expressed, as were other properties, when strains were grown at 37 degrees C, but not at 27 degrees C. These results suggest that a virulence factor associated with serum resistance plays an essential role in the pathogenicity of Y. enterocolitica.     

Inhibition of human neutrophil chemiluminescence by plasmid-mediated outer membrane proteins of Yersinia enterocolitica.

Recent studies have shown that the cell surface properties of Yersinia enterocolitica are altered by the presence of the virulence plasmid, which mediates temperature-inducible outer membrane proteins (OMP). We investigated the interaction of Y. enterocolitica with human polymorphonuclear leukocytes by monitoring luminol-enhanced chemiluminescence (CL) responses. A plasmid-bearing strain grown at 37 degrees C induced four- to sixfold less CL than did the same strain grown at 25 degrees C or a plasmidless, isogenic strain grown at either temperature. Inhibition of CL responses by whole cells was related to plasmid-mediated expression of OMP. The OMP alone could inhibit the CL response of polymorphonuclear leukocytes stimulated by either opsonized zymosan or whole cells of Y. enterocolitica. Pronase treatment of whole cells, which removed the plasmid-mediated OMP, resulted in partial but significant elimination of CL inhibition by whole cells and by OMP derived from them. Incubation with Y. enterocolitica for 60 min did not affect the viability of polymorphonuclear leukocytes. Our results suggest that the interaction of Y. enterocolitica with human polymorphonuclear leukocytes is directly affected by the plasmid-mediated OMP.     

Modulation of the low-calcium response in Yersinia pestis via plasmid-plasmid interaction

Virulent cells of Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica are known to exhibit a low-calcium response in vitro characterized by restriction of growth and induction of V antigen at 37 degrees C in Ca2+-deficient media (Lcr+). A shared Lcr plasmid mediates these properties and encodes yersiniae outer membrane peptides (Yops) that are expressed in Lcr+ Y. pseudotuberculosis and Y. enterocolitica but not Y. pestis. We present direct evidence here verifying that synthesis of major Yops in the former two species is, like V, repressed by Ca2+ and that these structures are located primarily in the outer membrane. We also verified that rabbits infected with live Lcr+ Y. pestis can raise antibodies against V and Yops. Similar antisera, however, were recovered after immunization with sterile extracts of Ca2+-starved Lcr+ cells of Y. pestis. Results of immunoblots obtained with these antisera showed that restricted Y. pestis produced Yops of about 46 kDa (YopB) and 44 kDa (YopC) after shiftup by addition of Ca2+ at 37 degrees C or reduction of temperature to 26 degrees C. It is established that virulent cells of Y. pestis also possess a unique plasmid known to mediate pesticinogeny (Pst+). Restricted Lcr+, Pst- Y. pestis expressed YopB and YopC plus additional 76 kDa (YopF), 48 kDa (YopH), 36 kDa (YopD), 32.5 kDa (YopJ), and 27 kDa (YopE) outer membrane structures at concentrations comparable to those in Ca2+-starved Y. pseudotuberculosis and Y. enterocolitica. These findings indicate that carriage of the Pst plasmid prevents expression of the Lcr plasmid-mediated Yops in wild type Y. pestis.     

Assay of crystal violet binding for rapid identification of virulent plasmid-bearing clones of Yersinia enterocolitica.

A rapid, reliable, and simple method based on the binding of crystal violet (CV) is described for differentiating virulence-plasmid-bearing strains of Yersinia enterocolitica from their plasmidless derivatives. As with other plasmid-mediated properties of this organism, the binding of CV occurs at 37 degrees C but not at 25 degrees C. The CV-binding technique provides a simple and efficient means of screening Y. enterocolitica for virulence and for identifying individual plasmid-bearing colonies.     

Characterization of common virulence plasmids in Yersinia species and their role in the expression of outer membrane proteins.

The virulence plasmids pYV019, pYV8081, and pIB1 from Yersinia pestis, Yersinia enterocolitica, and Yersinia pseudotuberculosis, respectively, were characterized by restriction endonuclease analysis. The three plasmids exhibited a region of common DNA previously shown to encode determinants which confer Ca2+ dependence. The plasmids from Y. pestis and Y. pseudotuberculosis were similar throughout their genomes. In contrast, a region of the plasmid from Y. enterocolitica which contained an origin of replication differed from the other two plasmids as determined by DNA homology and replication properties. Plasmid-associated outer membrane proteins from all three species of Yersinia were characterized by polyacrylamide gel electrophoresis. There were no differences in the outer membrane protein profiles between plasmid-containing and homogenic strains lacking the plasmid after growth at 28 degrees C. After growth at 37 degrees C, both Y. enterocolitica and Y. pseudotuberculosis showed at least four major plasmid-associated outer membrane proteins. Y. pestis did not show any discernible changes after growth at 37 degrees C. It was shown by using E. coli minicell analysis that the plasmid DNA from all three species of Yersinia contained the coding capacity for production of the novel outer membrane proteins.     

Interactions between Yersinia enterocolitica and rabbit ileal mucus: growth, adhesion, penetration, and subsequent changes in surface hydrophobicity and ability to adhere to ileal brush border membrane vesicles.

Interactions between Yersinia enterocolitica and rabbit ileal mucus were examined. Strains carrying the Yersinia virulence plasmid, pYV, adhered to crude mucus but not to intestinal luminal contents that had been immobilized on polystyrene. Using an Y. enterocolitica O:9 mutant in which the yadA gene (formerly called yopA), encoding the high-molecular-weight outer membrane protein YadA (formerly called protein P1 or Yop1), had been inactivated and an Escherichia coli strain carrying the cloned yadA gene, we demonstrated that the ability to adhere to mucus correlated closely to expression of YadA. Thereafter, we evaluated possible consequences of binding between pYV-carrying Y. enterocolitica O:3 strains and constituents in the mucus layer. pYV-carrying strains were able to multiply at a high rate in mucus but not in luminal contents, and the ability to adhere to mucus could therefore facilitate bacterial colonization of the mucosa. However, we also showed in vitro that mucus acted as a barrier for a mucus-adherent, pYV-carrying Y. enterocolitica strain. Furthermore, penetration through, or preincubation with, mucus reduced subsequent adhesion of the pYV-carrying strain to brush border membrane vesicles without simultaneously causing bacterial aggregation. Preincubation with mucus also changed the bacterial surface of the same strain from hydrophobic to hydrophilic. Immunoglobulins present in mucus did not seem to be of importance for our observations. Interaction of Y. enterocolitica with intestinal mucus may thus reflect a host defense mechanism that reduces the pYV-mediated adhesion to the epithelial cell membrane, possibly by rendering the bacteria less hydrophobic.     

Growth of Yersinia pseudotuberculosis and Yersinia enterocolitica biotype 3B serotype O3 inhibited on cefsulodin-Irgasan-novobiocin agar.

A total of 169 strains of Yersinia spp. were analyzed for their ability to grow on two different kinds of cefsulodin-Irgasan-novobiocin (CIN) agar containing 15 or 4 micrograms of cefsulodin per ml, on salmonella-shigella agar, and on MacConkey agar. CIN media inhibited the growth of Yersinia pseudotuberculosis and Yersinia enterocolitica biotype 3B serotype O3 (3B/O3) but not the growth of the other Yersinia organisms used. Relative to growth on Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.) with 6% yeast extract, 48 and 44% of Y. pseudotuberculosis and Y. enterocolitica 3B/O3 strains, respectively, were inhibited on CIN I agar (low cefsulodin concentration), and 83 and 54%, respectively, were inhibited on CIN II agar (high cefsulodin concentration) after incubation for 24 h at 32 degrees C. The inhibition of Y. pseudotuberculosis growth was significantly more extensive on CIN II agar than on CIN I agar. The MICs of cefsulodin and novobiocin clearly indicated a higher susceptibility for Y. pseudotuberculosis than for the other Yersinia organisms at 32 degrees C. All Y. pseudotuberculosis strains were susceptible to cefsulodin at 15 micrograms/ml (the approximate concentration used in CIN II agar). Y. enterocolitica 3B/O3 strains were resistant to cefsulodin, Irgasan, and novobiocin at the concentrations used in CIN media. These findings show that cefsulodin inhibits the growth of Y. pseudotuberculosis at the concentration used in CIN media and that growth inhibition of Y. enterocolitica 3B/O3 is related to a component of the CIN Base.     

Inhibition of attachment of Escherichia coli RDEC-1 to intestinal microvillus membranes by rabbit ileal mucus and mucin in vitro.

Intestinal mucus is postulated to play a role in preventing colonization of the gastrointestinal tract by microbial pathogens. To evaluate the ability of both crude mucus and purified mucin, a glycoprotein of goblet cell origin, to inhibit mucosal adherence of enteric pathogens, we examined whether mucus and mucin derived from rabbit ileum interact with the rabbit enteropathogen Escherichia coli RDEC-1. We examined the manner in which mucus and mucin inhibited adherence of bacteria to rabbit ileal microvillus membranes (MVMs) in vitro. The purity of the mucin preparation was demonstrated by polyacrylamide gel electrophoresis before and after reduction and by showing that an antiserum raised to the mucin localized to goblet cells in rabbit intestine. Using radioactive labeling of bacteria, we quantitated attachment of RDEC-1 to MVMs, mucus, and mucin that had been immobilized on polystyrene microtiter wells. Binding of RDEC-1 to MVMs was also determined after preincubation of organisms with crude ileal mucus and purified mucin. RDEC-1 bound to both crude mucus and purified mucin when they expressed lectinlike adhesions, previously designated attachment factor rabbit 1 pili. Adherence of piliated RDEC-1 to MVMs, mucus, and mucin was significantly greater than when the bacteria were nonpiliated. Binding of piliated RDEC-1 to MVMs was decreased by preincubation of bacteria with both crude mucus (45.6 +/- 4.2% of control) and purified mucin (50.2 +/- 5.8%). These data indicate that the E. coli enteropathogen RDEC-1 can bind to purified glycoproteins of goblet cell origin and that adherence of these bacteria to mucin is mediated by expression of pili. The findings also support a role for intestinal mucus and its principal organic constituent, mucin, in preventing adherence of a known E. coli enteric pathogen to apical MVMs of enterocytes.     

Evaluation of DNA colony hybridization and other techniques for detection of virulence in Yersinia species.

The virulence of yersiniae varies according to (i) species and biotype and (ii) possession of a 67- to 72-kilobase virulence plasmid. Y. pestis, Y. pseudotuberculosis, and biotypes 1B, 2, 3, 4, and 5 of Y. enterocolitica are inherently virulent but express full virulence only when in possession of a virulence plasmid. Other Yersinia species and biotypes 1A and 3B of Y. enterocolitica are seldom implicated in disease. In this study, we prepared DNA probes from eight nonoverlapping regions of the virulence plasmid of a strain of Y. enterocolitica and from the inv and ail chromosomal loci responsible for the invasive capacity of Y. enterocolitica and Y. pseudotuberculosis. The probes were used in colony hybridization experiments to investigate 156 yersiniae of various species and biotypes and of differing virulence. Probes prepared from the inv gene of Y. pseudotuberculosis hybridized with Y. pseudotuberculosis and Y. pestis only, whereas an analogous probe prepared from Y. enterocolitica hybridized with all species and biotypes of yersiniae (but not with other bacteria) regardless of virulence or potential virulence. Probes prepared from the ail region of Y. enterocolitica reacted almost exclusively with Y. enterocolitica strains of pathogenic biotypes. Probes prepared from the virulence plasmid of a serogroup O:8, biotype 1B isolate of Y. enterocolitica identified virulent yersiniae in all species with a high degree of sensitivity and specificity. These probes did not react with yersiniae of avirulent biotypes or species. Of the other assays of virulence evaluated (calcium dependence, binding of crystal violet, and pyrazinamidase activity), binding of crystal violet provided a simple means for identifying plasmid-bearing strains.     

Influence of a 70 kilobase virulence plasmid on the ability of Yersinia enterocolitica to survive phagocytosis in vitro.

During the course of infection, Yersinia enterocolitica invades tissues where macrophages and polymorphonuclear leucocytes (PMNs) constitute the first line of defence. As expression of virulence in Y. enterocolitica is governed in part by a c. 70 kilobase virulence plasmid (pYV), we investigated the influence of this plasmid on the interaction between Y. enterocolitica and phagocytes in vitro. The results showed that, irrespective of plasmid-carriage, yersiniae survived phagocytosis by macrophages and PMNs. Plasmidless Y. enterocolitica that had grown intracellularly in macrophages, however, were susceptible to killing by PMNs, whereas plasmid-bearing bacteria were resistant. In vitro cultivation of Y. enterocolitica in a Ca(2+)-deficient medium resembling that found within macrophages, did not influence the susceptibility of plasmid-bearing and plasmidless strains to killing by PMNs. These results indicate that passage through macrophages renders plasmidless strains of Y. enterocolitica susceptible to killing by PMNs. This finding may explain some of the differences in the behaviour of plasmid-bearing and plasmidless strains of Yersinia species in vivo.     

Simple assay of calcium dependency for virulent plasmid-bearing clones of Yersinia enterocolitica.

A simplified procedure to detect the calcium dependency of virulent plasmid-bearing strains of Yersinia enterocolitica was developed. A low-calcium, agarose-based medium of brain heat infusion with added magnesium effectively differentiated plasmid-bearing and plasmidless isolates. Further, the expression of calcium dependency in plasmid-bearing strains of Y. enterocolitica as measured by the average colony diameter was proportional to the calcium concentration of the assay.     

Identification of specific outer membrane polypeptides associated with virulent Yersinia enterocolitica

An antiserum (WA-SAA) that agglutinates specifically with mouse virulent but not avirulent strains of Yersinia enterocolitica was used to identify virulence-associated factors by Western blot techniques. Several outer membrane polypeptides were identified only in the virulent strains, which included serotypes O:8, O:3, O:9, O:4,32, O:5,27, and O:21. These included three, and possibly four, major outer membrane polypeptides. The prominent high-molecular-weight species was demonstrated by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot, whereas the others were only revealed by the Western blot technique. Expression of these polypeptides correlated with antiserum agglutination reaction and the presence of a 42- and/or 82-megadalton plasmid. These polypeptides were highly temperature dependent and only slightly affected by the inclusion of 10 mM Ca2+ in the growth medium. These polypeptides were produced during both the logarithmic and stationary phases of growth at 37 degrees C. We suggest that the production of these specific polypeptides and calcium dependency may be coded for by the plasmid(s) but are regulated by independent mechanisms. These polypeptides appear to be novel markers specific for virulent strains of Y. enterocolitica and may be important to the pathogenicity of this organism.     

New selective agar medium for isolation of virulent Yersinia enterocolitica.

A selective agar medium for isolation of virulent Yersinia enterocolitica (VYE agar) was developed for the rapid and accurate isolation of virulent Y. enterocolitica from environmental samples highly contaminated with environmental Yersinia organisms, as well as for isolation from clinical specimens. VYE agar provided a quantitative recovery of 51 different strains of virulent Y. enterocolitica at 32 degrees C after incubation for 24 h. The cefsulodin, irgasan, josamycin, and oleandomycin content of the medium resulted in a high selectivity, and the mannitol and esculin content provided some differentiation. The greatest advantage of VYE agar is that virulent Y. enterocolitica, which forms red colonies, is easily differentiated from most environmental Yersinia organisms and other gram-negative bacteria, which form dark colonies with a dark peripheral zone as a result of esculin hydrolysis. Use of VYE agar led to a high recovery of Y. enterocolitica biotype 3B serotype O:3 strains from experimentally inoculated meat samples, compared with use of CIN agar. Biotype 2 serotypes O:5,27 and O:9 and biotype 1 esculin-negative serotypes O:4,32, O:8, O:13a,13b, O:18, O:20, and O:21 (American types) were readily differentiated from other environmental organisms able to grow on VYE agar. Epidemiological studies on Y. enterocolitica should be greatly facilitated by the use of this selective agar medium.     

Influence of media on temperature-dependent motility test for Yersinia enterocolitica.

The influence of six widely used media on the motility of 100 isolates of Yersinia enterocolitica grown at 25 and 37 degrees C was investigated. Seven isolates were motile at 25 and 37 degrees C in all media, and the presence of flagellated cells was demonstrated by a flagellum stain. Six isolates were nonmotile at both temperatures in all media, and no flagella were observed. Identification schemes for Y. enterocolitica should reflect these possibilities.