Widespread occurrence of the restriction endonuclease YenI, an isoschizomer of PstI, in Yersinia enterocolitica serotype O8.

The cold-active restriction endonuclease YenI, an isoschizomer of PstI, was found in 12 of 14 Yersinia enterocolitica serotype O8 strains of different origins, but not in other serotypes of Y. enterocolitica, Yersinia pseudotuberculosis, or Yersinia pestis. In spite of the limited number of strains tested, the result suggests that the detection of YenI endonuclease or the gene might result in more rapid determination of the prominently pathogenic serotype of Y. enterocolitica.     

Characterization of a novel unique restriction-modification system from Yersinia enterocolitica O:8 1B

Genetic manipulations with enteropathogenic Yersinia enterocolitica O:8 are complicated by the presence of an efficient PstI-like YenI restriction-modification (R-M) system. We have characterized the YenI R-M system in Y. enterocolitica O:8, biotype 1B. A 5039 bp DNA fragment of the pSAK2 recombinant plasmid carrying the yenI locus was used to determine the nucleotide sequence. DNA sequence analysis identified a single 2481 bp open reading frame (ORF) that encodes an 826 amino acid large polypeptide having an apparent molecular mass of 93 kDa. The N-terminal part of the YenI ORF has 45 and 40% identity to PstI and BsuI methyltransferases (MTases), respectively; while the C-terminal part depicts 55 and 45% identity to endonucleases (ENases) of both isoschyzomeric enzymes. The yenI gene was cloned into pT7-5 plasmid and has been shown to encode a single polypeptide of expected molecular mass. A specific recognition sequence, typical to the type II R-M systems and single peptide organization, typical to type IV R-M systems, make YenI unique among known restriction-modification systems. We have constructed a truncated recombinant variant of YenI enzyme, which conserved only MTase activity, and that can be applied to YenI methylation of the DNA to be transformed into Y. enterocolitica O:8 biotype 1B strains.     

Isolation of an R- M+ mutant of Yersinia enterocolitica serotype O:8 and its application in construction of rough mutants utilizing mini-Tn5 derivatives and lipopolysaccharide-specific phage.

A generally applicable procedure was used to isolate a spontaneous restriction-deficient mutant of Yersinia enterocolitica serotype O:8. Transposition frequency in the mutant strain 8081-res was approximately 6.7 x 10(-6) per recipient, while it was practically zero in the wild-type strain 8081-c. Mobilization frequency into 8081-res was 10(5) times higher than that into the wild-type strain. The mutant had lost the ability to express the YenI restriction endonuclease activity present in serotype O:8 strains. This allowed the construction of a transposon library in 8081-res. Insertion mutants with transposons in the genes of the rfa region were selected from this library.     

Chromosomal-encoded siderophores are required for mouse virulence of enteropathogenic Yersinia species

Abstract Yersinia enterocolitica and Y. pseudotuberculosis are enteropathogenic for humans. Essential virulence functions of these pathogens are determined by a 40-mDa plasmid. Plasmid-bearing Y. pseudotuberculosis strains and Y. enterocolitica strains of serotypes 0 : 8, 0 : 13, 0 : 20 and 0 : 40 are lethal for mice. In contrast, human pathogenic Y. enterocolitica strains of serotype 0 : 3, 0 : 9 and 0 : 5.27 are not mouse-lethal. Using a sensitive siderophore-indicator CAS-agar, we were able to detect siderophore production in all mouse-lethal Y. enterocolitica and Y. pseudotuberculosis strains mentioned above. By Tn5-transposon insertions into the chromosome of a serotype 0 : 8 strain we obtained two siderophore-deficient mutants. Introduction of the virulence plasmid did not render these mutants mouse-lethal, indicating that siderophore production is an essential virulence factor. The human nonpathogenic, aerobactin-producing strains of Y. intermedia, Y. kristensenii and Y. frederiksenii remained avirulent for mice after receiving the virulence plasmid of Y. enterocolitica . Obviously the siderophore aerobactin does not contribute to virulence in the genus Yersinia .     

Direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis from meat

Studies were done to determine the usefulness of dilute alkali (KOH) treatment of meat samples for direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis, without enrichment. Virulent Y. enterocolitica and Y. pseudotuberculosis in pork contaminated with 10(2), 10(3), and 10(4) cells per g survived the direct KOH treatment and were never recovered by using KOH postenrichment treatment. From 6 (4.8%) of 125 samples of retail ground pork, four biotype 4 serotype O3 and one biotype 3B serotype O3 strains of Y. enterocolitica and one Y. pseudotuberculosis serotype 4b strain were recovered by using direct KOH treatment without enrichment. As these isolations were attained without using enrichment cultural procedures, they represent an important time-saving alternative to simplify and speed isolation of Yersinia spp. from meat.     

Direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis from meat.

Studies were done to determine the usefulness of dilute alkali (KOH) treatment of meat samples for direct isolation of Yersinia enterocolitica and Yersinia pseudotuberculosis, without enrichment. Virulent Y. enterocolitica and Y. pseudotuberculosis in pork contaminated with 10(2), 10(3), and 10(4) cells per g survived the direct KOH treatment and were never recovered by using KOH postenrichment treatment. From 6 (4.8%) of 125 samples of retail ground pork, four biotype 4 serotype O3 and one biotype 3B serotype O3 strains of Y. enterocolitica and one Y. pseudotuberculosis serotype 4b strain were recovered by using direct KOH treatment without enrichment. As these isolations were attained without using enrichment cultural procedures, they represent an important time-saving alternative to simplify and speed isolation of Yersinia spp. from meat.     

Inhibition of Yersinia enterocolitica serotype O3 by natural microflora of pork

Yersinia enterocolitica serotype O3 did not grow but did survive in inoculated raw ground pork kept at 6 and 25 degrees C. The antagonistic effect of microbial flora, especially Hafnia alvei and environmental Yersinia organisms, on the growth of Y. enterocolitica serotype O3 in raw ground pork was evident. These results were supported by evidence of the inhibition of growth of Y. enterocolitica serotype O3 by Enterobacteriaceae, especially H. alvei and environmental Yersinia organisms, in mixed cultures at 6 and 25 degrees C. We suggest that naturally contaminated pork is a source of human infection, since Y. enterocolitica serotype O3 was capable of surviving in the raw pork for a long time.     

Modulation of complement activityin Vitro andin Vivo byYersinia wild and mutant strains

The ability of released proteins (Yops) and surface lipopolysaccharides (LPS) from the wild-type strain Yersinia enterocolitica 8081-L2, serotype 0:8 to influence the complement activity was determined. Yops and LPS from wild-type and mutant strains showed different ability to affect the classical pathway (CP) functional complement activity in vitro. The serum CP activity was inhibited during the infection induced with six Y. enterocolitica and three Y. pseudotuberculosis strains in rabbits. The changed complement activity might be of importance for the course of Yersinia infections.     

Inhibition of Yersinia enterocolitica serotype O3 by natural microflora of pork.

Yersinia enterocolitica serotype O3 did not grow but did survive in inoculated raw ground pork kept at 6 and 25 degrees C. The antagonistic effect of microbial flora, especially Hafnia alvei and environmental Yersinia organisms, on the growth of Y. enterocolitica serotype O3 in raw ground pork was evident. These results were supported by evidence of the inhibition of growth of Y. enterocolitica serotype O3 by Enterobacteriaceae, especially H. alvei and environmental Yersinia organisms, in mixed cultures at 6 and 25 degrees C. We suggest that naturally contaminated pork is a source of human infection, since Y. enterocolitica serotype O3 was capable of surviving in the raw pork for a long time.     

Recovery of Yersinia enterocolitica from streams and lakes of California.

Stream and lake water from the Mammoth Lakes region of California was sampled for Yersinia enterocolitica. From 10 of the 34 sites examined, organisms were isolated that were biochemically identified as Y. enterocolitica. Only one of the ten strains could be serologically confirmed. This strain was identified as Y. enterocolitica serotype 16. Although an outbreak of enteritis in the area prompted this study, no correlation with gastrointestinal disease could be established since the majority of the strains were untypeable.     

Unusual Yersinia enterocolitica Isolates Not Associated with Mesenteric Lymphadenitis

Thirteen Yersinia enterocolitica were recovered from a variety of clinical sources. Of these, only one was associated with mesenteric lymphadenitis and belonged to serotype 8. The 12 remaining strains were isolated from nonmesenteric sources and belonged to serotype 17. All strains exhibited the main characteristics of Y. enterocolitica which differentiated them from other Enterobacteriaceae, i.e., motility at 22 C but not at 37 C, positive urease and ornithine decarboxylase activities, and negative phenylalanine deaminase. These 12 strains differed, however, from other Y. enterocolitica previously described in the United States in that they fermented rhamnose and raffinose at 22 C, and failed to grow on Salmonella-Shigella and Hektoen-Enteric agars.     

Characteristics of Yersinia spp. isolated from wild and zoo animals

Thirteen strains of Yersinia spp. were isolated at the Rome zoo and at Castelporziano, a game preserve near Rome. The strains were tested for calcium dependency, autoagglutination, heat-stable toxin production, 50% minimum lethal dose in mice (LD50), pyrazinamidase activity and content of plasmids by electrophoresis in agarose gel. The former three tests were negative for all strains, the LD50 was always greater than or equal to 1 X 10(7.6) CFU/ml and pyrazinamidase activity was positive for all strains. Electrophoresis revealed the presence of two plasmids of 27 and 66 megadaltons (MDa) in the two strains of Y. enterocolitica of serotype 027 isolated from animals in the zoo. The two strains of the same species and serotype, isolated from wild animals harboured a 42-MDa plasmid. A small plasmid of 2 MDa was found in two strains of Y. enterocolitica of serotype 07.8 from two subsequent samples of a zoo animal.     

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.     

Yersinia enterocolitica as a causative agent of septicaemia.

The authors describe the first case of isolation of Yersinia enterocolitica from blood in a septic infection in Czechoslovakia. They also isolated a strain of Y. enterocolitica from an inguinal tumour in the same patient. Both strains belonged to biotype 4, serotype 0:3 and phagotype 8 and were non-pathogenic for guinea pigs and hares.     

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.     

Immunomodulation in mice by experimental infection with Yersinia enterocolitica

Intraperitoneal infection of mice with two strains of Yersinia enterocolitica resulted in an inflammatory response and immunomodulation which appeared to be related to the invasive properties of the bacteria. The primary antibody response to sheep erythrocytes was enhanced by noninvasive cultures of Y. enterocolitica (serotype O:4-33 grown at 22 C and at 37 C, and serotype O:3 grown at 37 C), when given at the same time or two days after the antigen (invasiveness was tested on HeLa cells). In contrast, invasive cultures of serotype O:3 grown at 22 C, injected three days before the antigen suppressed the antibody response; enhancement was caused by these cultures only when given on the day of immunization. Delayed-type hypersensitivity to sheep erythrocytes was also suppressed by invasive cultures of Y. enterocolitica. These data indicate that the temperature of growth as well as some serotype-linked factors play a role in immunomodulation by Y. enterocolitica.     

Survey on the Incidence of Yersinia enterocolitica Infection in Canada

Data pertaining to 278 cultures of Yersinia enterocolitica isolated in Canada are summarized in this paper. Of this amount, 256 were isolated from humans, whereas 22 were of nonhuman sources. The typing of these cultures is presented together with their geographical location. Y. enterocolitica serotype O:3 biotype 4, phage type 9b, was practically the only serotype present in the Province of Quebec. This serotype O:3 was also predominant in Ontario, followed by serotypes O:5,27 and O:6,30; other serotypes were seldom isolated. In the central and western areas of Canada, Y. enterocolitica was occasionally isolated; the strains were indole-positive, serotypes O:5,27, O:8, and O:4,32.     

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.     

Detection and enumeration of virulent Yersinia enterocolitica in food by DNA colony hybridization.

A portion of a 44-megadalton plasmid found in Yersinia enterocolitica 8081 was used as a genetic probe to differentiate virulent and nonvirulent strains of the species. A DNA colony hybridization technique was employed. Three BamHI restriction endonuclease fragments labeled with 32P by nick translation were hybridized to lysed colonies of pure cultures, mixtures of virulent and nonvirulent cells, and portions of a food sample artificially contaminated with virulent Y. enterocolitica. The results of the colony hybridization test for virulence were the same as those obtained by the autoagglutination and suckling mouse tests. DNA colony hybridization detects pathogenic Y. enterocolitica in foods without the need for enrichment or pure cultures.     

The high-pathogenicity island of Yersinia pseudotuberculosis can be inserted into any of the three chromosomal asn tRNA genes

Pathogenicity islands (PAIs) have been identified in several bacterial species. A PAI called high-pathogenicity island (HPI) and carrying genes involved in iron acquisition (yersiniabactin system) has been previously identified in Yersinia enterocolitica and Yersinia pestis . In this study, the HPI of the third species of Yersinia pathogenic for humans, Y. pseudotuberculosis , has been characterized. We demonstrate that the HPI of strain IP32637 has a physical and genetic map identical to that of Y. pestis . A gene homologous to the bacteriophage P4 integrase gene is located downstream of the asn tRNA locus that borders the HPI of strain IP32637. This int gene is at the same position on the HPI of all three pathogenic Yersinia species. However, in contrast to Y. pestis 6/69, the HPI of Y. pseudotuberculosis IP32637 is not invariably adjacent to the pigmentation segment and can be inserted at a distance ≥ 190 kb from this segment. Also, in contrast to Y. pestis and Y. enterocolitica , the HPI of Y. pseudotuberculosis IP32637 can precisely excise from the chromosome, and, strikingly, it can be found inserted in any of the three asn tRNA loci present on the chromosome of this species, one of which is adjacent to the pigmentation segment. The pigmentation segment, which is present in Y. pestis but not in Y. enterocolitica , is also present and well conserved in all strains of Y. pseudotuberculosis studied. In contrast, the presence and size of the HPIs vary depending on the serotype of the strain: an entire HPI is found in strains of serotypes I only, a HPI with a 9 kb truncation in its left-hand part that carries the IS 100 sequence and the psn and ybtE genes characterizes the strains of serotype III, and no HPI is found in strains of serotypes II, IV and V.