Virulence characteristics and epidemiology of Yersinia enterocolitica and Yersiniae other than Y-pseudotuberculosis and Y-pestis isolated from water and sewage

Aims: To determine the species, bio-sero-phagetypes, antimicrobial drug resistance and also the pathogenic potential of 144 strains of Yersinia spp. isolated from water sources and sewage in Brazil.Methods and Results: the 144 Yersinia strains were characterized biochemically, serologically and had their antibiotic resistance and phenotypic virulence markers determined by microbiological and serological standard techniques. The Y. enterocolitica strains related to human diseases were also tested for the presence of virulence genes, by the PCR technique. The isolates were classified as Y. enterocolitica, Y. intermedia, Y. frederiksenii, Y. kristensenii and Yersinia biochemically atypical. The 144 isolates belonged to various bio-serogroups. Half of the strains showed resistance to three or more drugs. The Y. enterocolitica strains related to human diseases exhibited phenotypic virulence characteristics and virulence genes.Conclusions: Water from various sources and sewage are contaminated with Yersinia spp. in Brasil. Among these bacteria, virulent strains of Y. enterocolitica were found, with biotypes and serogroups related to human diseases.Significance and Impact of the Study: This is the first documented description of the occurrence of pathogenic Y. enterocolitica in water sources and sewage in Brazil. The occurrence of virulence strains of Y. enterocolitica shows that the environment is a potential source of human infection by this species in this country.

TNF-alpha, H2O2 and NO response of peritoneal macrophages to Yersinia enterocolitica O : 3 derivatives

In this study, the effect of Yersinia derivatives on nitric oxide (NO), hydrogen peroxide (H2O2) and tumor necrosis factor-alpha (TNF-alpha) production by murine peritoneal macrophages was investigated. Addition of lipopolysaccharide (LPS) to the macrophage culture resulted in NO production that was dose dependent. on the other hand, bacterial cellular extract (CE) and Yersinia outer proteins (Yops) had no effect on NO production. The possible inhibitory effect of Yops on macrophage cultures stimulated with LPS was investigated. Yops partially inhibited NO production (67.4%) when compared with aminoguanidine. The effects of Yersinia derivatives on H2O2 production by macrophages were similar to those on NO production. LPS was the only derivative that stimulated H2O2 release in a dose-dependent manner. All Yersinia derivatives provoked the production of TNF-alpha, but LPS had the strongest effect, as observed for NO production. CE and Yops stimulated TNF-alpha production to a lesser extent than LPS. The results indicate the possibility that in vivo Yops may aid the evasion of the bacteria from the host defense mechanism by impairing the secretion of NO by macrophages. (C) 2003 Elsevier SAS. All rights reserved.

Yersinia enterocolitica O : 3 isolated from patients with or without reactive arthritis induces polyclonal activation of B cells and autoantibody production in vivo

The mechanisms by which arthritis-provoking pathogens such as Yersinia enterocolitica interact with the human immune system to produce inflammatory synovitis are not well known. One of the immunomodulating mechanisms used against these pathogens is the polyclonal activation of lymphocytes. In this study, we investigated the extent of the B-lymphocyte activation induced in mice by a strain of Y. enterocolitica O:3 (FCF 526) isolated from a patient with arthritis, and compared it with two other strains, a virulent one (FCF 397[+]) isolated from a patient without arthritis and its plasmidless isogenic pair (FCF397[-]). Also we investigated the production of autoantibodies in mice infected with these different strains. SPF Swiss mice were infected intravenously with a suspension of Y. enterocolitica . Spleen cells were taken on days 7, 14, 21 and 28 after infection and the number of cells secreting nonspecific and specific antibodies of IgG 1 , IgG 2a , IgG 2b , IgG 3 , IgM and IgA isotypes were determined by the ELISPOT technique. The presence of autoantibodies in mouse serum was investigated by the dot-blot assay. The pattern of infection of the three bacterial strains were almost the same. We observed a general increase in the number of nonspecific Ig-secreting cells with all three strains, and the greatest increases observed were in the IgG 2a and IgG 3 isotypes. Only a small fraction of the immunoglobulins detected were antibacterial, suggesting that the rest resulted from polyclonal B cell activation. The strain isolated from the patient with arthritis (FCF526) induced the greatest production of autoantibodies, coinciding with the period in which the greatest activation of nonspecific B lymphocytes was seen. There were no signs of arthritis or inflammation in the joints of the infected animals. Based on our results, we were unable to determine whether there is an association between the arthritogenic capability of Y. enterocolitica and polyclonal activation of B cells.

Molecular typing and virulence markers of Yersinia enterocolitica strains from human, animal and food origins isolated between 1968 and 2000 in Brazil

Molecular typing and virulence markers were used to evaluate the genetic profiles and virulence potential of 106 Yersinia enterocolitica strains. of these strains, 71 were bio-serotype 4/O: 3, isolated from human and animal clinical material, and 35 were of biotype 1 A or 2 and of diverse serotypes, isolated from food in Brazil between 1968 and 2000. Drug resistance was also investigated. All the strains were resistant to three or more drugs. The isolates showed a virulence-related phenotype in the aesculin, pyrazinamidase and salicin tests, except for the food isolates, only two of which were positive for these tests. For the other phenotypic virulence determinants (autoagglutination, Ca++ dependence and Congo red absorption), the strains showed a diverse behaviour. The inv, ail and ystA genes were detected in all human and animal strains, while all the food isolates were positive for inv, and 3% of them positive for ail and ystA. The presence of virF was variable in the three groups of strains. The strains were better discriminated by PFGE than by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). A higher genomic similarity was observed among the 4/O: 3 strains, isolated from human and animal isolates, than among the food strains, with the exception of two food strains possessing the virulence genes and grouped close to the 4/O: 3 strains by ERIC-PCR. Unusually, the results revealed the virulence potential of a bio-serotype 1 A/O: 10 strain, suggesting that food contaminated with Y. enterocolitica biotype 1 A may cause infection. This also suggests that ERIC-PCR may be used as a tool to reveal clues about the virulence potential of Y. enterocolitica strains. Furthermore, the results also support the hypothesis that animals may act as reservoirs of Y. enterocolitica for human infections in Brazil, an epidemiological aspect that has not been investigated in this country, confirming data from other parts of the world.

EVALUATION OF DIFFERENT TECHNIQUES FOR THE DIFFERENTIATION OF PATHOGENIC AND NON PATHOGENIC STRAINS OF YERSINIA-ENTEROCOLITICA

Different methods and tests have been used to evaluate the pathogenic potential of distinct Y. enterocolitica serotypes and biotypes. We tested a total of 60 Y. enterocolitica strains, being 25 of human origin (serotype O3 biotype 4 and serotype O5 biotype 1); 6 of animal origin (serotype O3 biotype 4); 19 isolated from the environment (serotype O5.27 biotypes 1 and 2); and 8 isolated from food (serotype O5 biotype 1 and serotype 05.27 biotype 1). The methods used were based on plasmid gene expression (autoagglutination, calcium-dependence at 37 degrees C and Congo Red absorption tests), chromosomal gene expression (assays for pyrazinamidase activity, salicin fermentation and esculin hydrolysis), and invasion of HEp-2 cells. All but one of the Y. enterocolitica O3 strains, were found to be potentially pathogenic when submitted to the pyrazinamidase-salicin-esculin tests. In contrast, the results obtained with the assays related to plasmidial gene expression were not so uniform, probably due to plasmid loss. The least homogeneous results were obtained with the HEp-2 cell invasion test. Y. enterocolitica O5 behaved in a uniform manner when tested with the first two groups of tests (based on chromosomal and plasmidial gene expression), but not when tested with the HEp-2 invasion assay. The strains of serotype O5.27 biotype 1 presented a uniform behavior hen submitted to the chromosomic-related tests, showing no pathogenicity. However, they did not provide conclusive results with the tests related to plasmidial gene expression or HEp-2 cell invasion. We conclude that the tests related to chromosomal gene expression (esculin-salicin-pyrazinamidase) are simple and highly effective for the detection of potentially pathogenic Y. enterocolitica isolated from clinical cases.

Kinetics of B cell response during Yersinia enterocolitica infection in resistant and susceptible strains of mice

In this study we analyze the B-cell response in murine yersiniosis. To this end, we determined whether polyclonal activation of B-lymphocytes occurs during infection of susceptible (BALB/c) and resistant (C57BL/6) mice with Y. enterocolitica 0:8 and compared the immunoglobulin (Ig) isotypes produced in response to the infection by the two strains. The number of splenic cells secreting nonspecific and specific immunoglobulins was determined by ELISPOT. The presence of anti-Yersinia antibodies in serum was detected by ELISA. In both strains, the number of specific Ig-secreting cells was relatively low. Polyclonal B-cell activation was observed in both strains of mice, and the greatest activation was observed in the BALB/c mice, mainly for lgG(1)- and IgG(3)- secreting cells. The C57BL/6 mice showed a predominance of IgG(2a)-secreting cells. The peak production of anti-Yersinia IgG antibodies in the sera of BALB/c mice was seen on the 28th day after infection. The greatest increase in IgM occurred on the 14th day. A progressive increase of specific IgG antibodies was observed in C57BL/6 mice up to the 28th day after infection while IgM increased on the 21st day after infection. The production of specific IgA antibodies was not detected in either BALB/c or C57BL/6 mice. We conclude that polyclonal. activation of B lymphocytes occurs in both the Yersinia resistant and Yersinia-susceptible mice and that the more intense activation of B lymphocytes observed in the susceptible BALB/c mice does not enhance their resistance to Y. enterocolitica infection.

AN IMPROVED ENRICHMENT PROCEDURE FOR THE ISOLATION OF YERSINIA-ENTEROCOLITICA AND RELATED SPECIES FROM MILK

A new enrichment procedure is proposed to improve the isolation of Yersinia enterocolitica and related species from milk. This procedure uses tryptic soy broth plus Polymyxin (5 IU/ml) and Novobiocin (10 mug/ml) - TSPN broth - incubated at 18-degrees-C for 3 d. Using raw milk and pasteurized milk inoculated with Yersinia strains, the efficiency of this procedure was compared to that of SB broth (sorbitol bile salts broth) incubated at 4-degrees-C for up to 21 d. Despite of the presence of antibiotics in TSPN broth, there were difficulties in recovering Yersinia organisms. Nevertheless, TSPN broth incubated at 18-degrees-C for 3 d showed better efficiency than that other method. In pasteurized milk samples, TSPN medium at 18-degrees-C for 3 d gave better results than the SB broth at 4-degrees-C for 7 d, showing that the proposed procedure is the preferable one due to the shorter period of incubation.