Role of the fly in the transport of Yersinia enterocolitica.

Yersinia enterocolitica were isolated from flies collected from a piggery and a kitchen of farm and from ham hung in a piggery. The cultures were identified as Y. enterocolitica biovar 4 and serovar 3 by biochemical and serological characteristics. From these results it is suggested that flies may play an important role in food contamination by Y. enterocolitica. In this study, the probable donors of Y. enterocolitica to the flies were swine.     

The application of PCR fingerprinting to the differentiation of Yersinia enterocolitica strains isolated from humans and pigs

The distribution of different genotypes of Yersinia enterocolitica strains recovered from humans and from healthy pigs was investigated using PCR fingerprinting. The thirty six strains of Y. enterocolitica from humans, thirty five strains from pigs and Y. enterocolitica ATCC 9610 strain were included in this study. The tested strains of Y. enterocolitica belonged to O3 and O9 serogroups. The PCR fingerprinting using EAE5 primer (5' CTT AAT CTC AGT AAT GCT GGC CTT GG) made it possible to form five groups among the tested Y. enterocolitica strains. Two groups were very numerously represented by the tested strains. The thirty of Y. enterocolitica O3 strains from humans (thirty one of tested) and eighteen of Y. enterocolitica O3 strains from pigs (twenty of tested) belonged to one group. This group also included Y. enterocolitica ATCC9610 strain and four Y. enterocolitica O9 strains from pigs. All investigated Y. enterocolitica O9 strains from humans and the majority of Y. enterocolitica O9 strains isolated from pigs created a second, numerous group. The third genotype was created by two strains O9 from pigs, and the remaining two strains, isolated from pigs, belonging to O3 and O9 serogroups showed different binding patterns revealed by gel electrophoresis and created two other genotypes. The tested Y. enterocolitica strains which were isolated from humans formed only two groups but Y. enterocolitica strains isolated from pigs were found in five groups but such as the Y. enterocolitica strains from humans, the majority of strains from pigs were in first and second group. The Y. enterocolitica O3 strains regardless of their origin mostly represented the same PCR fingerprinting profile. The tested Y. enterocolitica O9 strains were more genetically diverse and represented four PCR fingerprinting profiles.     

Factors contributing to the reduced invasiveness of chlorine-injured Yersinia enterocolitica

The invasion of epithelial cells in vitro and in vivo by chlorine-injured Yersinia enterocolitica was assessed by direct microscopic observations. These experiments showed that injury by chlorine inhibited invasiveness of virulent Y. enterocolitica. Two requirements appeared to be necessary for invasiveness: the organism must be viable and metabolically active, and the organism must have certain surface components to initiate engulfment. Inhibition of RNA synthesis by rifampin and protein synthesis by chloramphenicol, tetracycline, and spectinomycin inhibited the invasiveness but not the attachment of Y. enterocolitica to epithelial cells. Membrane preparations from untreated and antimicrobial-agent-treated Y. enterocolitica blocked the invasiveness of virulent Y. enterocolitica, whereas membranes from chlorinated cells were unable to block invasiveness. Chlorine did not change the hydrophobicity or surface charge of injured Y. enterocolitica. The results indicate that invasion was more than simple association of the bacterium with the epithelial cell and involved a specific trigger to stimulate engulfment.     

Factors contributing to the reduced invasiveness of chlorine-injured Yersinia enterocolitica.

The invasion of epithelial cells in vitro and in vivo by chlorine-injured Yersinia enterocolitica was assessed by direct microscopic observations. These experiments showed that injury by chlorine inhibited invasiveness of virulent Y. enterocolitica. Two requirements appeared to be necessary for invasiveness: the organism must be viable and metabolically active, and the organism must have certain surface components to initiate engulfment. Inhibition of RNA synthesis by rifampin and protein synthesis by chloramphenicol, tetracycline, and spectinomycin inhibited the invasiveness but not the attachment of Y. enterocolitica to epithelial cells. Membrane preparations from untreated and antimicrobial-agent-treated Y. enterocolitica blocked the invasiveness of virulent Y. enterocolitica, whereas membranes from chlorinated cells were unable to block invasiveness. Chlorine did not change the hydrophobicity or surface charge of injured Y. enterocolitica. The results indicate that invasion was more than simple association of the bacterium with the epithelial cell and involved a specific trigger to stimulate engulfment.     

Studies on the interactions of immunostimulated macrophages and Yersinia enterocolitica O:8

Immunological and electron microscopy investigations of the phagocytic and killing activities of peritoneal macrophages from rats and mice against Yersinia enterocolitica serotype O:8 cells were performed. The effect of in vivo application of cytoplasmic membranes (CM) from the stable Escherichia coli WF+ L-form on macrophage activity was also studied. It was established that rat macrophages more actively phagocytosed the plasmidless pYV(-) Y. enterocolitica cells, compared to the plasmid-bearing pYV(+) Y. enterocolitica cells. The killing ability against both variants of the Y. enterocolitica strain was significantly enhanced in macrophages from CM-treated rats after 2 h, 4 h, and 24 h incubation. The CM treatment enhanced the phagocytic activity of the macrophages. The in vitro interaction of normal and immunostimulated rat macrophages with both pYV(+) and pYV(-) variants of Y. enterocolitica did not lead to any additional apoptotic and necrotic changes in macrophages compared to control macrophages, which were cultivated without Y. enterocolitica. Electron-microscopic investigation showed that mouse macrophages eliminated Y. enterocolitica pYV(+) cells in vivo after 24 h. No engulfed or digested bacterial cells were observed. Activation of cell surfaces and vacuolization of macrophage cytoplasm, both of CM-treated non-infected and infected mice, were observed. The experimental results showed that Y. enterocolitica pYV(+) cells could be eliminated by peritoneal macrophages.     

Nucleotide-binding oligomerization domain 2 (Nod2) is dispensable for the innate immune responses of macrophages against Yersinia enterocolitica

Nucleotide-binding oligomerization domain 2 (Nod2) is a cytosolic sensor for muramyl dipeptide, a component of bacterial peptidoglycan. In this study, we have examined whether Nod2 mediates the immune response of macrophages against Yersinia enterocolitica. Bone-marrow-derived macrophages (BMDMs) were isolated from WT and Nod2-deficient mice and were infected with various strains of Y. enterocolitica. ELISA showed that the production of IL-6 and TNF-α in BMDMs infected with Y. enterocolitica was not affected by the Nod2 deficiency. iNOS mRNA expression was induced in both WT and Nod2-deficienct BMDMs in response to Y. enterocolitica, beginning 2 h after infection. Nitric oxide (NO) production by Y. enterocolitica did not differ between WT and Nod2-deficient BMDMs. Western blot analysis revealed that Y. enterocolitica induces activation of NF-κB, p38, and ERK MAPK through a Nod2-independent pathway. Neither LDH release by Y. enterocolitica nor the phagocytic activity of the macrophages was altered by Nod2 deficiency. An in vivo experiment showed that bacterial clearance ability and production of IL-6 and KC in serum were comparable in WT and Nod2-deficient mice infected with Y. enterocolitica. These findings suggest that Nod2 may not be critical for initiating the innate immune response of macrophages against Yersinia infection.     

Association of Yersinia enterocolitica with the manufacture of cheese and occurrence in pasteurized milk.

Raw milk in southern Ontario frequently contains Yersinia enterocolitica. The potential for transmission of this organism by cheese manufactured from unpasteurized milk was evaluated by examination of milk and cheese curd samples from cheese manufacturing plants and finished cheddar and Italian cheeses. The incidence of Y. enterocolitica was lower in cheese curd samples (9.2%) than in raw milk (18.2%). Most of the curd samples showed a positive phosphatase test, indicating production from raw milk. One curd sample yielded Y. enterocolitica after 4 weeks of storage at 4 degrees C but was negative after 8 weeks. All samples of cheddar and Italian cheeses, most of which showed a positive phosphatase test, were negative for Y. enterocolitica. One out of 265 samples (0.4%) of pasteurized fluid dairy products contained Y. enterocolitica.     

House flies (Musca domestica) as possible vectors of Campylobacter fetus subsp. jejuni.

A total of 161 strains of Campylobacter fetus subsp. jejuni were isolated from house flies (Musca domestica). The carrier rates detected were 50.7% in flies captured on a chicken farm and 43.2% in flies from a piggery. The relative prevalences of Campylobacter coli, C. jejuni, and nalidixic acid-resistant thermophilic campylobacters were 90.1, 6.2, and 3.7%, respectively. The results indicate that flies may play a linking role in the epidemiology of Campylobacter infection in humans by transmitting campylobacters from animals to human food.     

A new chromogenic agar medium for detection of potentially virulent Yersinia enterocolitica

Several outbreaks of foodborne yersiniosis have been documented and this disease continues to be source of infections transmitted through foods. The selective agars most commonly used to isolate Yersinia enterocolitica in clinical, food and environmental samples, cefsulodin-irgasan-novobiocin (CIN) and MacConkey (MAC) agars, lack the ability to differentiate potentially virulent Y. enterocolitica from other Yersinia that may be present as well as some other bacterial spp. This study proposes the use of an agar medium, Y. enterocolitica chromogenic medium (YeCM), for isolation of potentially virulent Y. enterocolitica. This agar contains cellobiose as the fermentable sugar, a chromogenic substrate and selective inhibitors for suppression of colony formation by many competing bacteria. All strains of potentially virulent Yersinia of biotypes 1B, and biotypes 2-5 formed convex, red bulls-eye colonies on YeCM that were very similar to those described for CIN agar. However, Y. enterocolitica biotype 1A and other related Yersinia formed colonies that were purple/blue on YeCM while they formed typical red bulls-eye colonies on CIN agar. When a mixture of potentially virulent Y. enterocolitica biotype 1B, Y. enterocolitica biotype 1A and 5 other bacterial species was used to artificially contaminate tofu and then spread-plated on three selective agars, Y. enterocolitica biotype 1B colonies were easily distinguished from other strains on YeCM. However, Y. enterocolitica biotype 1B colonies were indistinguishable from many other colonies on CIN and only distinguishable from those of C. freundii on MAC. When colonies were picked and identified from these agars, typical colonies from YeCM were confirmed only as Y. enterocolitica biotype 1B. Typical colonies on CIN and MAC were found to belong to several competing species and biotypes.     

Recognition of Yersinia enterocolitica multiple strain infection in twin infants using PCR-based DNA fingerprinting

AIMS: Yersinia enterocolitica causes several syndromes in humans. The most common presentation is enterocolitis in children, presenting as fever and diarrhoea. A Y. enterocolitica multiple strain infection in twin infants was investigated. METHODS AND RESULTS: One isolate was recovered from one patient and two morphologically-different isolates were recovered from the other infant. Biochemically, all isolates were identified as Y. enterocolitica group. The genomic DNA from each strain was purified and DNA fingerprinting was performed. The banding patterns observed for Y. enterocolitica isolates 2 and 3, from patients 1 and 2, respectively, were identical when comparing the presence or absence of major bands. However, Y. enterocolitica isolate 1, from patient 1, showed a distinctive banding pattern from isolates 2 and 3. CONCLUSION: The findings indicate that one infant was colonized by more than one strain of Y. enterocolitica, demonstrating that multiple strains can colonize and invade a patient. SIGNIFICANCE AND IMPACT OF THE STUDY: Recognition of multiple strain infections can be important in diagnosis, treatment and prognosis of Y. enterocolitica infections, as well as in disease epidemiology. The technique described here offers a straightforward method for strain comparison.     

Effect of dyes on the quantitative recovery of Yersinia enterocolitica.

A total of 69 dyes were incorporated separately at different concentrations into an agar medium for evaluation of their effects on the quantitative recovery of five serotypes of Yersinia enterocolitica. One strain of Pseudomonas aeruginosa and one strain of Bacillus cereus were included for comparative purposes. Certain dyes were evaluated further for their selective properties with five additional serotypes of Y. enterocolitica, three strains of P. aeruginosa, and two of Engerobacter spp. Metanil yellow was the only dye which was tolerated bettr by Y. enterocolitica than by P. aeruginosa. Dye sensitivity was variable amond strains of the same serotype of Y. enterocolitica. In general, Y. enterocolitica showed a tolerance to dyes greater than that of gram-positive bacteria and similar to that of other gram-negative bacteria.     

The serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosis

The techniques of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were evaluated for the serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosis. Lipopolysaccharide (LPS) was prepared from strains comprising four serogroups of Y. enterocolitica and five serogroups of Y. pseudotuberculosis, tested against 200 sera submitted to the Laboratory of Enteric Pathogens for routine serodiagnosis, and shown to contain antibodies to Yersinia LPS by agglutination. Forty four sera were found to contain antibodies that bound to one of the LPS preparations used in the immunoassay. Thirty five of the sera contained antibodies to the LPS of Y. enterocolitica O3, whilst three contained antibodies to the LPS of Y. enterocolitica O5, 27 and Y. enterocolitica O9 LPS respectively. Two sera had antibodies to the LPS of Y. pseudotuberculosis II and a single serum contained antibodies to Y. pseudotuberculosis IV. The SDS-PAGE-immunoblotting procedure described proved to be a reliable procedure for the serodiagnosis of infections with Y. enterocolitica and Y. pseudotuberculosis.     

Effect of dyes on the quantitative recovery of Yersinia enterocolitica.

A total of 69 dyes were incorporated separately at different concentrations into an agar medium for evaluation of their effects on the quantitative recovery of five serotypes of Yersinia enterocolitica. One strain of Pseudomonas aeruginosa and one strain of Bacillus cereus were included for comparative purposes. Certain dyes were evaluated further for their selective properties with five additional serotypes of Y. enterocolitica, three strains of P. aeruginosa, and two of Engerobacter spp. Metanil yellow was the only dye which was tolerated bettr by Y. enterocolitica than by P. aeruginosa. Dye sensitivity was variable amond strains of the same serotype of Y. enterocolitica. In general, Y. enterocolitica showed a tolerance to dyes greater than that of gram-positive bacteria and similar to that of other gram-negative bacteria.     

The role of the Yersinia plasmid of calcium-dependency in the formation of the plague immunity

It was shown that viable variants of the Y. enterocolitica microorganisms containing the plasmid of Ca-dependency (native one or from EV/ I Y. pestis cells) were more immunogenic to plague-infected guinea pigs than isogenic non-plasmid variant. As a result of studying the protective properties of the bacteria Y. enterocolitica sub-cellular fractions it was determined that the largest quantity of immunogen per unit of protein was accumulated in the cultures of bacteria's plasmid variant. The aggregate of the obtained experimental data suggested that there was a non-identified protective antigen (or antigens) for guinea pigs to be encoded by chromosome's DNA. Besides, the Ca-dependency plasmid gene products take part in its excretion and, as the factors of pathogenicity, provide survivability of the Y. enterocolitica microorganisms in the host organism, increasing thus the immunogen accumulation in it.     

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.     

Relevance of N-acyl-L-homoserine lactone production by Yersinia enterocolitica in fresh foods

AIMS: Determination of the food matrix impact on the potential for N-acyl-L-homoserine lactones (AHLs) production by Yersinia enterocolitica. METHODS AND RESULTS: Induction and inhibition of a sensor strain and a fluorescent assay were used to investigate Y. enterocolitica AHL production in artificial media, as well as in different food extracts. All Y. enterocolitica strains tested produced AHLs in artificial media. Thin Layer Chromatography analysis of Y. enterocolitica strains indicated the production of 3-oxo-hexanoyl homoserine lactone and hexanoyl homoserine lactone. Yersinia enterocolitica produced AHL principally in fish and meat extracts. CONCLUSIONS: AHL production by Y. enterocolitica was observed in products of animal origin, but were inhibited by some vegetables extracts. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that quorum sensing systems in Y. enterocolitica is significant in foods but depends upon the type of food. Determination of physiological functions in Y. enterocolitica which are regulated by quorum sensing and their relation to the production of AHLs in foods need to be further assessed.     

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.     

Note: Isolation, characterization and epidemiology of Yersinia enterocolitica from humans and animals

During an 11-year period (1983 to 1994), 51 strains of Yersinia enterocolitica were isolated from humans and animals. Specimens were collected from a total of 3601 sources consisting of 956 patients with enteritis, 300 patients with urinary tract infection, 1564 healthy humans, 510 swine, 38 guinea-pigs, 118 rats and 115 rabbits. Five strains of Y. enterocolitica , bio/serogroups 2/O:9 and 4/O:3, virulence positive, were recovered from patients. Forty-two variants of Y. enterocolitica belonging to pathogenic serogroup O:3, Voges-Proskauer-negative biogroup 3 were recovered from swine, rats and rabbits. The rate of isolation of Y. enterocolitica from diarrhoeal swine was apparently greater than those from healthy swine. The incidence of human infections due to Y. enterocolitica was very low and bioserogroups of isolates were different from the strains which were isolated from animals. There was no evidence to suggest that swine were the source of Y. enterocolitica in humans.     

Characterization of IS1541-like elements in Yersinia enterocolitica and Yersinia pseudotuberculosis

We characterized Yersinia enterocolitica and Yersinia pseudotuberculosis insertion sequences related to insertion sequence 1541, recently identified in Yersinia pestis. For each of the two species, two insertion sequence copies were cloned and sequenced. Genetic elements from Y. pseudotuberculosis were almost identical to insertion sequence 1541, whereas these from Y. enterocolitica were less related. Phylogenetic analysis of the putative transposases encoded by insertion sequences from the three pathogenic members of the genus Yersinia showed that they clustered with those encoded by Escherichia coli and Salmonella enterica elements belonging to the insertion sequence 200/insertion sequence 605 group. Insertion sequences originating from Y. pestis and Y. pseudotuberculosis constitute a monophyletic lineage distinct from that of Y. enterocolitica.