Role of antibodies in the opsonization of Yersinia spp

We have determined the opsonic capacity of specific antibodies in patient sera obtained after Yersinia infection. The results indicate that Yersinia antibodies lead to complement activation through the classical pathway, thus overcoming the inhibition of complement-mediated opsonization in the absence of specific antibodies provided by the virulence plasmid in Yersinia enterocolitica and Yersinia pseudotuberculosis. Further, antibodies against plasmid-encoded structures, the Yersinia outer membrane proteins (YOPs), are not necessary in this effect. This is indicated by two facts. (i) Monoclonal antibodies directed against the O polysaccharide of Y. enterocolitica O:3 are capable of opsonizing the plasmid-containing bacteria through C1q binding. (ii) Rabbit antisera show opsonic activity when obtained by immunization both with plasmid-containing Y. enterocolitica expressing the YOPs and a plasmid-cured variant not expressing these proteins.     

Plasmid-mediated surface fibrillae of Yersinia pseudotuberculosis and Yersinia enterocolitica: relationship to the outer membrane protein YOP1 and possible importance for pathogenesis.

The cell surface properties of Yersinia pseudotuberculosis and Yersinia enterocolitica mutants, constructed by insertional inactivation of genes located on the 40- to 50-megadalton virulence plasmid, were examined. Electron microscopy revealed an absolute correlation between expression of four plasmid-dependent, temperature-inducible properties related to the bacterial surface: (i) a fibrillar matrix covering the outer membrane, (ii) outer membrane protein YOP1, (iii) spontaneous autoagglutination, and (iv) mannose-resistant hemagglutination of guinea pig erythrocytes. Immunoelectron microscopy indicated that YOP1 is a structural component of the fibrillae. Experiments demonstrating inhibition of hemagglutination by anti-YOP1 monoclonal antibody suggested a potential role for YOP1 in adhesion. Insertional inactivation of the gene coding for YOP1, with resultant loss of the ability to express fibrillae, led to a significant reduction in the capacity of Y. enterocolitica, but not Y. pseudotuberculosis, to colonize the ileum of orogastrically infected mice. In both Y. enterocolitica and Y. pseudotuberculosis, inactivation of the genes coding for Ca2+ dependency reduced the ability to maintain intestinal colonization, regardless of the ability to express fibrillae. Both surface fibrillae and Ca2+ dependency seem to reflect pathogenic determinants which are required for the establishment of Y. enterocolitica infection. In Y. pseudotuberculosis, however, no clinical significance of the fibrillae has so far been defined.     

Identification and mapping of the temperature-inducible, plasmid-encoded proteins of Yersinia spp.

The structural genes of the outer membrane polypeptides of Yersinia spp. (YOPs) and the V antigen of plasmid pIB1 of Yersinia pseudotuberculosis were recently cloned and mapped (A. Forsberg, I. B?lin, L. Norlander, and H. Wolf-Watz, Microb. Pathogen. 2:123-137, 1987). The corresponding genes were localized on pYV019 and pYV8081 of Yersinia pestis and Yersinia enterocolitica, respectively. No obvious differences were observed on comparison of pIB1 and pYV019, whereas pYV8081 showed intragenic as well as extragenic changes. However, one region of plasmid pYV8081, which coded for the V antigen, YOP3, and YOP4a, was essentially conserved among the three plasmids. Since this region is connected with the Ca2+ region, we suggest that the conserved region of the virulence plasmids of Yersinia spp. be extended to include both of these regions. Low amounts of the YOPs were detected in the membrane fraction at 37 degrees C in the presence of 2.5 mM calcium. Only minor differences were noticed when the individual YOPs of Y. pestis and Y. pseudotuberculosis were compared. Several differences were observed when the YOPs of Y. enterocolitica were included for comparison. All Y. enterocolitica proteins, except YOP1, YOP4b, and the V antigen, exhibited changes in their characteristic molecular sizes. Although these differences were within a range of +/- 2 kilodaltons, the isoelectric point was retained for each protein type.     

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.     

Evaluation of enzyme immunoassay for the detection of pathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis strains.

To determine the virulence plasmid-harboring strains of Yersinia enterocolitica, we prepared antiserum against plasmid-encoded proteins of Y. enterocolitica serotype O3 and carried out an enzyme immunoassay (EIA) against temperature-inducible released proteins. This serum reacted with proteins released from not only a Y. enterocolitica serotype O3 strain but also Y. enterocolitica serotype O5:27, O8, and O9 and Y. pseudotuberculosis serotype 1b, 2a, 2b, 2c, 3, 4a, 4b, 5a, 5b, 6, 7, and 8 strains, which all harbored plasmids. Plasmid-cured Y. enterocolitica and Y. pseudotuberculosis strains did not react in the EIA, nor did nonpathogenic Y. enterocolitica strains or Y. frederiksenii, Y. intermedia, and Y. kristensenii strains. These observations demonstrated that this EIA was useful for determining whether the isolated Yersinia strains were pathogenic or not.     

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.     

Properties of Yersinia enterocolitica and Yersinia pseudotuberculosis in red blood cell concentrate of different ABO groups during 30-day storage at 4 °C

Objective   The present study aimed to investigate the psychrophilic properties of Yersinia enterocolitica and Yersinia pseudotuberculosis as contaminants of donated blood.
Methods   Bags with red blood cell concentrates (RBCCs) of A, B, and AB blood groups were inoculated with a bacterial suspension of Y. enterocolitica (0 : 3 and 0 : 8) and Y. pseudotuberculosis (serovars I and III) and stored at 4 °C for 30 days. Bacterial growth was measured at selected intervals after inoculation. Initial strains and their subcultures (isolated after 30 days' incubation at 4 °C) were tested for glycolytic activity and susceptibility to the bactericidal action of human serum.
Results   It was found that all strains grew well in the RBCCs of A, B, and AB groups. After incubation at 4 °C they increased their glycolytic activity and became more sensitive to the killing ability of human serum.
Conclusions   The prolonged storage of contaminated Y. enterocolitica and Y. pseudotuberculosis RBCCs at 4 °C induces bacterial multiplication to high levels and stimulates glycolytic activity of bacterial cells.     

Plasmid DNA relatedness among different serogroups of Yersinia pseudotuberculosis.

Thirteen different serogroup strains of Yersinia pseudotuberculosis and two strains of Yersinia enterocolitica O:3 were examined for the presence of plasmids and plasmid-mediated properties, calcium growth dependency, and autoagglutination. Two Y. enterocolitica strains and eight serogroup (IA, IIA, IIC, III, IVA, VB, VI, and VIII) strains, except for five serogroups (IB, IIB, IVB, VA, and VII), of Y. pseudotuberculosis harbored plasmids ranging in molecular size from 27 to 115 kilobases. Filter hybridization of restriction endonuclease-digested plasmid DNA from different serogroup strains indicated that all plasmid DNAs conferring calcium growth dependency and autoagglutination shared a high degree of DNA sequence homology, regardless of the different serogroups of Y. pseudotuberculosis and Y. enterocolitica.     

Isolation of Yersinia enterocolitica serovar O8 from free-living small rodents in Japan.

Yersinia species were isolated from 65 of 223 free-living small mammals trapped in 10 regions on Honshu Island in Japan. Of the 65 strains isolated, 1 was Yersinia enterocolitica serovar O3, 8 were Y. enterocolitica O5, 6 were Y. enterocolitica O8, 3 were Y. enterocolitica O9, and 1 was Yersinia pseudotuberculosis 4b. Of the six Y. enterocolitica O8 strains, five were positive for autoagglutination, Ca2+ dependence, and the 45-MDa virulence plasmid and showed high pathogenicity for mice.     

Adhesion protein YadA of Yersinia species mediates binding of bacteria to fibronectin.

The interaction between fibronectin and Yersinia strains was studied. Wild-type Y. enterocolitica strains expressing the virulence-plasmid-encoded adhesion protein YadA adhered strongly to fibronectin-coated coverslips, while their plasmid-cured variants expressed weaker binding. The cloned yadA gene of Y. enterocolitica or Y. pseudotuberculosis conferred fibronectin-binding ability both to Escherichia coli and to Y. psuedotuberculosis strains lacking the YadA protein. The YadA protein did not mediate binding to isolated fragments of fibronectin or to soluble fibronectin.     

Pathogenic Yersinia enterocolitica strains increase the outer membrane permeability in response to environmental stimuli by modulating lipopolysaccharide fluidity and lipid A structure

Pathogenic biotypes of Yersinia enterocolitica (serotypes O:3, O:8, O:9, and O:13), but not environmental biotypes (serotypes O:5, O:6, O:7,8, and O:7,8,13,19), increased their permeability to hydrophobic probes when they were grown at pH 5.5 or in EGTA-supplemented (Ca(2+)-restricted) media at 37 degrees C. A similar observation was also made when representative strains of serotypes O:8 and O:5 were tested after brief contact with human monocytes. The increase in permeability was independent of the virulence plasmid. The role of lipopolysaccharide (LPS) in this phenomenon was examined by using Y. enterocolitica serotype O:8. LPS aggregates of bacteria grown in acidic or EGTA-supplemented broth took up more N-phenylnaphthylamine than LPS aggregates of bacteria grown in standard broth and also showed a marked increase in acyl chain fluidity which correlated with permeability, as determined by measurements obtained in the presence of hydrophobic dyes. No significant changes in O-antigen polymerization were observed, but lipid A acylation changed depending on the growth conditions. In standard medium at 37 degrees C, there were hexa-, penta-, and tetraacyl lipid A forms, and the pentaacyl form was dominant. The amount of tetraacyl lipid A increased in EGTA-supplemented and acidic media, and hexaacyl lipid A almost disappeared under the latter conditions. Our results suggest that pathogenic Y. enterocolitica strains modulate lipid A acylation coordinately with expression of virulence proteins, thus reducing LPS packing and increasing outer membrane permeability. The changes in permeability, LPS acyl chain fluidity, and lipid A acylation in pathogenic Y. enterocolitica strains approximate the characteristics in Yersinia pseudotuberculosis and Yersinia pestis and suggest that there is a common outer membrane pattern associated with pathogenicity.     

Rapid diagnostic test that uses isocitrate lyase activity for identification of Yersinia pestis.

The presence of high levels of isocitrate lyase activity in Yersinia pestis grown on blood agar base medium, as compared with low levels of this enzyme in Yersinia pseudotuberculosis and Yersinia enterocolitica, suggested that the differences in the levels of this enzyme could be used for the presumptive identification of Y. pestis. A modified, semiquantitative assay for isocitrate lyase activity is described which requires no expensive instrumentation, utilizes readily available chemicals and substrates, and requires only 20 min for completion. This test yielded positive results with all 108 isolates of Y. pestis tested and negative results with all strains of Y. pseudotuberculosis (68 isolates) and Y. enterocolitica (202 isolates) tested. Less than 2% of the approximately 1,300 non-Yersinia isolates from the family Enterobacteriaceae and none of the 93 isolates from the family Pseudomonadaceae yielded positive results. We conclude that this test provides for rapid identification of Y. pestis and should be useful in the initial screening of isolates from rodent and flea populations and in the presumptive identification of this organism from suspected cases of human plague.     

Pyrazinamidase activity in Yersinia enterocolitica and related organisms.

Pyrazinamidase activity was tested in 381 Yersinia strains from various ecological and geographical origins and belonging to the following species: Y. enterocolitica (five biogroups), Y. intermedia, Y. frederiksenii, Y. kristensenii, Y. aldovae, Y. pseudotuberculosis, and Y. pestis. The pyrazinamidase test was negative (Pyz-) in all bioserogroups of Y. enterocolitica, in which is usually harbored the virulence plasmid, and was involved in human or animal diseases. Y. pseudotuberculosis and Y. pestis were also Pyz-. The more ubiquitous bioserogroups of Y. enterocolitica, without naturally occurring virulence plasmid, and related species were all Pyz+. Pyrazinamidase activity allowed the separation of the pathogenic North American Y. enterocolitica isolates from other nonpathogenic strains within biogroup 1. Similarly, environmental biogroups 3A and 3B were clearly distinguished from pathogenic biogroup 3. However, the pyrazinamidase test was not linked to the presence of the virulence plasmid itself and should not replace the pathogenicity tests to assess the actual virulence of an individual strain. This test proved to be a valuable tool to distinguish potential pathogenic from nonpathogenic strains of Y. enterocolitica in epidemiological surveillance programs.     

Serum opsonic capacity against Yersinia enterocolitica O:3 in yersiniosis patients with or without reactive arthritis.

The opsonic capacity of 45 sera from patients with reactive arthritis after Yersinia enterocolitica O:3 infection and of 45 matched sera from yersiniosis patients without post-infection complications was studied at 1-3 months, 5-8 months and 12-20 months after the onset of the infection. Antibody-mediated opsonization of virulence-plasmid-containing Y. enterocolitica O:3 was studied by measuring complement-fixation on opsonized bacteria and opsonophagocytic function of the polymorphonuclear leucocytes (PMN). The PMN response against bacteria pre-opsonized by heat-inactivated sera was measured by using a chemiluminescence (CL) assay. The fixation of complement Clq and C3 on bacteria was determined by flow cytometry using fluorescein-conjugated Clq- and C3c-antisera. All the sera were strongly opsonic at the onset of the infection, and this capacity persisted in most of the patients still at the end of the follow-up. No difference was observed in complement-fixing capacity between the sera of the two groups, but the sera from arthritic patients showed stronger augmentation of PMN CL response at the early phase of the infection (P = 0.005 in the presence of complement, P = 0.04 in the absence of complement). These results suggest that enhanced opsonic capacity may play a role in the development of Yersinia-triggered reactive arthritis by leading to strong activation of the PMN and, consequently, to release of inflammatory mediators.     

Mice and moles inhabiting mountainous areas of Shimane Peninsula as sources of infection with Yersinia pseudotuberculosis.

A total of 1,835 Yersinia spp. were isolated from 925 (60.5%) of 1,530 wild mice and from 139 (79.9%) of 174 moles living in mountainous areas of eastern Shimane Prefecture, Japan. The Yersinia spp. included 1,106 Yersinia enterocolitica, 26 Y. enterocolitica-like, 176 Yersinia mollaretii, 149 Yersinia frederiksenii, 70 Yersinia intermedia, 231 Yersinia kristensenii, 5 Yersinia aldovae, and 72 Yersinia pseudotuberculosis. Human pathogenic Y. enterocolitica was not isolated. Y. pseudotuberculosis was divided into 10 virulent 40- to 50-MDa plasmid-positive (P+) strains (serotypes 1b, 4b, and untypeable) and 62 plasmid-negative (P-) strains (serotypes 1b, 2b, 2c, 4a, 5a, 5b, 6, 7, and untypeable). P+ strains of serotypes 1b (two strains), 4b (seven strains), and untypeable (one strain) were isolated from nine Apodemus specious and one Apodemus argenteus. The isolates of Yersinia spp. were more frequently detected in newborn mice and during the breeding season. The P+ Y. pseudotuberculosis strains were recovered at less than 10(4) cells per g of the cecal contents. Thus, the prevalence of Yersinia spp. in small wild animals depends on the newborn animals born during the cold months, and wild mice in mountainous areas are important reservoirs of Y. pseudotuberculosis.     

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.     

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.     

Role of the Yersinia outer membrane protein YadA in adhesion to rabbit intestinal tissue and rabbit intestinal brush border membrane vesicles

The Yersinia virulence plasmid confers on strains of Yersinia pseudotuberculosis and Y. enterocolitica an adhesive potential superior to the one encoded by the chromosome alone. We have evaluated the role of the plasmid-encoded outer membrane protein YadA (formerly called Yopl) in adhesion. Insertional inactivation of the yadA gene (formerly called yopA), which encodes YadA, led to a reduction in the capacity of plasmid-carrying strains of Y. pseudotuberculosis 0:III and Y. enterocolitica 0:9 to adhere to intestinal tissue, brush border membranes and polystyrene surfaces. The adhesive characteristics of the mutants were comparable to those of their plasmid-cured counterparts. When the yadA gene from Y. pseudotuberculosis serotype 0:III or Y. enterocolitica serotype 0:3 or 0:8 was cloned into an Escherichia coli strain, increased ability to adhere to intestinal tissue, brush border membrane vesicles and polystyrene was transferred concomitantly. The introduction of the yadA gene from Y. pestis, which is unable to express YadA due to a one base pair deletion, did not change the adhesive characteristics of E. coli. Expression of YadA in the outer membrane may, therefore, make an important contribution to intestinal adherence of the two enteropathogenic members of the Yersinia species, Y. pseudotuberculosis and Y. enterocolitica.     

Yersinia enterocolitica: Biochemical, Serological, and Gas-Liquid Chromatographic Characterization of Rhamnose-, Raffinose-, Melibiose-, and Citrate-Utilizing Strains

Thirteen atypical Yersinia enterocolitica isolates, all fermenting rhamnose, raffinose, and melibiose and utilizing sodium citrate within 24 to 48 h at 22 degrees C (Y.e.rh+), were examined biochemically-serologically, and by gas-liquid chromatography. These data, as well as cultural, biochemical, and antibiotic susceptibility data gathered from two previous studies involving (i) these same atypical Y.e.rh+ isolates, (ii) Y. enterocolitica serotypes O:1 through O:15 (rhamnose, raffinose, and citrate negative [Y.e.rh-]), (iii) Y. enterocolitica serotype O:16 (rhamnose positive but raffinose and citrate negative), and (iv) Yersinia pseudotuberculosis serogroups I through V were statistically compared. Both preand postabsorption agglutination studies demonstrated the serological distinctiveness of Y.e.rh+ from Y.e.rh- and Y. pseudotuberculosis. At the same time, three immunological groups among the 13 Y.e.rh+ strains were seen; 8 corresponded to Y. enterocolitica serotype O:17; 1 to Y. enterocolitica serotype O:16; and the remaining four were nontypable in antisera against known Y. enterocolitica antigen types. Each of the three Yersinia groups tested chromatographically produced acetic and lactic acids. Both Y.e.rh- and Y.e.rh+ formed propionic acid, but only Y.e.rh+ produced detectable amounts of succinic acid. Based on 49 variables, statistical analysis of the three Yersinia groups studied placed each of the Y.e.rh+ strains in a homogeneous group separate from both Y.e.rh- and Y. pseudotuberculosis. These data, coupled with deoxyribonucleic acid homology studies of Brenner and co-workers (D. J. Brenner, A. G. Steigerwalt, D. F. Falcao, R. E. Weaver, and G. R. Fanning, Int. J. Syst. Bacteriol. 26:180-194, 1976), support the distinctiveness of Y.e.rh+ from typical Y. enterocolitica and Y. pseudotuberculosis.