Application of Yersinia kristensenii bacteriocin as a specific marker for the rapid identification of suspected isolates of Yersinia enterocolitica

Out of 143 suspected foods and clinical isolates of the genus Yersinia , 77 isolates were bacteriocin-sensitive and 66 bacteriocin non-sensitive. Of the 77 bacteriocin-sensitive isolates, 72 (93.5%) were identified as Y. enterocolitica and 5 (6.49%) as Y. frederiksenii. Out of 66 bacteriocin non-sensitive suspected isolates, 22 (33.3%) were identified as Yersinia spp. other than Y. enterocolitica and 43 (65.1%) were other Gram-negative bacteria with the exception of one (1.5%) isolate identified as Y. enterocolitica.     

Partial purification and characterization of bacteriocin from Yersinia kristensenii

A raw milk bacterial isolate, identified as Yersinia kristensenii was found to produce a bacteriocin which was inhibitory to Yersinia enterocolitica but not to other selected species of Yersinia or Gram-negative bacteria. Maximum production of bacteriocin was obtained when the organism was grown in shake culture at 28°C. Mitomycin C at a concentration of 0.5 μg ml^-1 induced bacteriocin production. The bacteriocin was partially purified and characterized by ammonium sulphate fractionation and gel filtration. The bacteriocin was completely inactivated when treated with proteolytic enzymes (trypsin and chymotrypsin). Bacteriocin activity was heat-resistant and it retained some of its activity after 5 min at boiling temperature. A total of 15 bacteriocin sensitive-suspected food isolates were further identified biochemically as Yersinia enterocolitica and a non-sensitive isolate was identified as Yersinia intermedia.     

Characterization of enterocoliticin, a phage tail-like bacteriocin, and its effect on pathogenic Yersinia enterocolitica strains.

Yersinia enterocolitica 29930 (biogroup 1A; serogroup O:7,8) produces a bacteriocin, designated enterocoliticin, that shows inhibitory activity against enteropathogenic strains of Y. enterocolitica belonging to serogroups O:3, O:5,27 and O:9. Enterocoliticin was purified, and electron micrographs of enterocoliticin preparations revealed the presence of phage tail-like particles. The particles did not contain nucleic acids and showed contraction upon contact with susceptible bacteria. Enterocoliticin addition to logarithmic-phase cultures of susceptible bacterial strains led to a rapid dose-dependent reduction in CFU. Calorimetric measurements of the heat output of cultures of sensitive bacteria showed a complete loss of cellular metabolic activity immediately upon addition of enterocoliticin. Furthermore, a dose-dependent efflux of K(+) ions into the medium was determined, indicating that enterocoliticin has channel-forming activity.     

Detection of pathogenic Yersinia enterocolitica in environmental waters by PCR

The isolation of pathogenic strains of Yersinia enterocolitica from food and water samples by culture is time-consuming and unreliable. A two-step PCR procedure has been developed which, after a period of bacterial enrichment, can detect and confirm the presence of pathogenic Y. enterocolitica within a single day. This PCR method works effectively for a range of environmental water types, including reticulated waters, reservoirs and creeks. A survey of environmental waters in Victoria, Australia, showed that the PCR method detected pathogenic Y. enterocolitica in water sampled from four separate sites (two creeks and two reservoirs). Repeat samplings of the two reservoirs yielded PCR-positive results on all but one occasion. Culture analysis of the same samples detected pathogenic Y. entrocolitica in only one sample, indicating that the PCR can detect pathogenic Y. enterocolitica which are undetectable by culture. Results from this study confirm that potentially pathogenic strains of Y. enterocolitica can exist in environmental waters.     

Thermal inactivation of Yersinia enterocolitica in milk.

Three strains of Yersinia enterocolitica isolated from milk had D values at 62.8 degrees C from 0.24 to 0.96 min and z values of 5.11 to 5.78 degrees C. Since the pasteurization processes for dairy products recommended by the Food and Drug Administration are adequate to destroy large concentrations of these organisms, Y. enterocolitica in pasteurized milk probably results from substandard processing or recontamination after pasteurization.     

Piglets are a source of pathogenic Yersinia enterocolitica on fattening-pig farms

To study the origin and spread of Yersinia enterocolitica among pigs, fecal and blood samples were repeatedly taken on a fattening farm. A few piglets were found to be already infected on breeding farms. After the piglets were mixed, the infection spread through the whole unit. Eventually, all the pigs excreted the pathogen.     

Recognition of pathogenic Yersinia enterocolitica by crystal violet binding and polymerase chain reaction

Cefsulodin-Irgasan-Novobiocin (CIN) agar is used for the selective isolation and enumeration of Yersinia enterocolitica from clinical specimens and food. The medium contains crystal violet and about 1 mmol l^-1 calcium and can be used for the phenotypic characterization of strains that carry a virulence plasmid. At 32°C, irrespective of pathogenicity, colonies are translucent with a pale pink centre surrounded by a transparent border ('bullseye'), while at 37°C pathogenic strains grow as calcium-dependent microcolonies which, because of crystal violet binding, are intensely coloured. These results were confirmed by the polymerase chain reaction with primers directed at the vir F gene, which is present only in pathogenic strains of Y. enterocolitica. Pathogenic strains of Y. enterocolitica can be recognized by growth at 37°C on Yersinia selective agar.     

Purification of lipopolysaccharide from strains of Yersinia enterocolitica belonging to serogroups 03 and 09

Lipopolysaccharide (LPS) was purified from strains of Yersinia enterocolitica belonging to serogroups 03 and 09, by three methods, and analysed by SDS-PAGE and silver staining for carbohydrate. SDS-PAGE of LPS prepared from whole-cells by digestion with proteinase-K, produced profiles containing high molecular mass LPS and a lower molecular mass region migrating as discrete bands. LPS prepared from strains belonging to serogroup 03, using a hot-phenol procedure alone was found to contain cellular proteins, and LPS prepared from strains of serogroup 09, by this method, did not contain high molecular mass carbohydrate. A novel method of preparing LPS by digesting bacterial outer membranes with proteinase-K prior to hot-phenol extraction produced protein-free LPS from strain of Y. enterocolitica 03 and high molecular mass LPS from strains belonging to serogroup 09.     

Identification and characterization of pathogenic Yersinia enterocolitica isolates by PCR and pulsed-field gel electrophoresis

Approximately 550 to 600 yersiniosis patients are reported annually in Sweden. Although pigs are thought to be the main reservoir of food-borne pathogenic Yersinia enterocolitica, the role of pork meat as a vehicle for transmission to humans is still unclear. Pork meat collected from refrigerators and local shops frequented by yersiniosis patients (n=48) were examined for the presence of pathogenic Yersinia spp. A combined culture and PCR method was used for detection, and a multiplex PCR was developed and evaluated as a tool for efficient identification of pathogenic food and patient isolates. The results obtained with the multiplex PCR were compared to phenotypic test results and confirmed by pulsed-field gel electrophoresis (PFGE). In all, 118 pork products (91 raw and 27 ready-to-eat) were collected. Pathogenic Yersinia spp. were detected by PCR in 10% (9 of 91) of the raw pork samples (loin of pork, fillet of pork, pork chop, ham, and minced meat) but in none of the ready-to-eat products. Isolates of Y. enterocolitica bioserotype 4/O:3 were recovered from six of the PCR-positive raw pork samples; all harbored the virulence plasmid. All isolates were recovered from food collected in shops and, thus, none were from the patients' home. When subjected to PFGE, the six isolates displayed four different NotI profiles. The same four NotI profiles were also present among isolates recovered from the yersiniosis patients. The application of a multiplex PCR was shown to be an efficient tool for identification of pathogenic Y. enterocolitica isolates in naturally contaminated raw pork.     

Characterization of enterotoxin produced by four Yersinia enterocolitica strains of pig origin

All four isolates of Yersinia enterocolitica and one isolate of Y. frederiksenii from pigs were found to be enterotoxigenic. Whole-cell preparations of Y. enterocolitica isolates did not induce any change in the rabbit ligated gut test after 6 and 18 h of inoculation, but Y. frederiksenii on the other hand showed a positive gut response at 18 h. Cell-free supernatant (CFS) of all five isolates induced dilatation in the rabbit gut up to 6 h, after which Y. enterocolitica became negative, while Y. frederiksenii continued to show a reaction up to 18 h. CFS of all five isolates were also found positive with the infant mouse test. Of the five isolates of Yersinia, three gave a positive reaction for the permeability factor on rabbit skin. Yersinia enterotoxin could be concentrated by methanol extraction. It was stable at 100°C for 20 min and at 120°C for 15 min. However, its activity was lost at low (2.0) and high pH (10.0). Enterotoxic preparations of Y. enterocolitica lost part of their enterotoxic activity upon dialysis.     

Isolation, primary structure and synthesis of heat-stable enterotoxin produced by Yersinia enterocolitica

Five heat-stable enterotoxins were isolated from the culture supernatant of Yersinia enterocolitica and purified to homogeneity by DEAE-Sephacel and high-performance liquid chromatographies. They caused acute fluid accumulation in the intestine of suckling mice. The amino acid sequence of one of the enterotoxins was determined to be Ser-Ser-Asp-Trp-Asp-Cys-Cys-Asp-Val-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys, by Edman degradation of its pyridylethylated derivative and a combination of fast atom bombardment mass spectrometry and carboxypeptidase B digestion. This structure was unambiguously confirmed by chemical synthesis. The other enterotoxins had longer or shorter amino acid sequences at their N termini, but the same sequence at their C termini. The six half-cystine residues formed intramolecular disulfide linkages, as shown by measurement of the molecular masses of the enterotoxins by fast atom bombardment mass spectrometry. The sequence of 13 amino acid residues at the C terminus showed similarity to those of heat-stable enterotoxins isolated from enterotoxigenic Escherichia coli [Aimoto, S. et al. (1982) Eur. J. Biochem. 129, 257-263; Takao, T. et al. (1983) FEBS Lett. 152, 1-5] suggesting that these similar sequences are related to the common biological and immunological properties of enterotoxins produced by Y. enterocolitica and enterotoxigenic E. coli.     

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.     

Multiple-locus variable-number tandem-repeat analysis of pathogenic Yersinia enterocolitica in China

The predominant bioserotypes of pathogenic Yersinia enterocolitica in China are 2/O: 9 and 3/O: 3; no pathogenic O: 8 strains have been found to date. Multiple-Locus Variable-Number Tandem-Repeat Analysis (MLVA) based on seven loci was able to distinguish 104 genotypes among 218 pathogenic Y. enterocolitica isolates in China and from abroad, showing a high resolution. The major pathogenic serogroups in China, O: 3 and O: 9, were divided into two clusters based on MLVA genotyping. The different distribution of Y. enterocolitica MLVA genotypes maybe due to the recent dissemination of specific clones of 2/O: 9 and 3/O: 3 strains in China. MLVA was a helpful tool for bacterial pathogen surveillance and investigation of pathogenic Y. enterocolitica outbreaks.     

Physical and structural characterization of yersiniophore, a siderophore produced by clinical isolates of Yersinia enterocolitica

Clinical isolates of Yersinia enterocolitca, which belong to mouse-lethal serotypes, produce the siderophore yersiniophore. Siderophore production was shown to be iron regulated and to reach maximum production in late log phase. Yersiniophore is a fluorescent siderophore with maximum excitation at 270 nm and a major emission peak at 428 nm. Absorption maxima were seen at 210 and 250 nm with a low broad peak from 280 to 320 nm. Purification of unchelated yersiniophore for structural analysis was made difficult by low yields (1–2 mg mg^-1), and susceptibility to acid hydrolysis, oxidation and possibly polymerization. Yersinophore was therefore purified as an Al³⁺ chelate, which was found to be stable in solution for several weeks. To purify Al³⁺-yersiniophore, unchelated yersiniophore was first extracted from culture supernatants with dichloromethane, concentrated by rotary evaporation and adsorbed to a DEAE-sephacel column. Al³⁺-yersiniophore was eluted with 0.01 m AlCl₃ and further purified by HPLC. The structure was established by a combination of elemental analysis, high resolution mass spectrometry and two-dimensional NMR experiments. Yersiniophore is a phenolate-thiazole siderophore with the formula C₂₁H₂₄N₃O₄S₃Al and a molecular weight of 505.07404 when chelated to Al³⁺. The structure of yersiniophore was determined to be closely related to the structures of pyochelin, produced by Pseudomonas aeruginosa, and anguibactin, produced by Vibrio anguillarum.     

Real-time PCR method for detection of pathogenic Yersinia enterocolitica in food

The current methods for the detection of pathogenic Yersinia enterocolitica bacteria in food are time consuming and inefficient. Therefore, we have developed and evaluated in-house a TaqMan probe-based real-time PCR method for the detection of this pathogen. The complete method comprises overnight enrichment, DNA extraction, and real-time PCR amplification. Also included in the method is an internal amplification control. The selected primer-probe set was designed to use a 163-bp amplicon from the chromosomally located gene ail (attachment and invasion locus). The selectivity of the PCR method was tested with a diverse range (n = 152) of related and unrelated strains, and no false-negative or false-positive PCR results were obtained. The sensitivity of the PCR amplification was 85 fg purified genomic DNA, equivalent to 10 cells per PCR tube. Following the enrichment of 10 g of various food samples (milk, minced beef, cold-smoked sausage, fish, and carrots), the sensitivity ranged from 0.5 to 55 CFU Y. enterocolitica. Good precision, robustness, and efficiency of the PCR amplification were also established. In addition, the method was tested on naturally contaminated food; in all, 18 out of 125 samples were positive for the ail gene. Since no conventional culture method could be used as a reference method, the PCR products amplified from these samples were positively verified by using conventional PCR and sequencing of the amplicons. A rapid and specific real-time PCR method for the detection of pathogenic Y. enterocolitica bacteria in food, as presented here, provides a superior alternative to the currently available detection methods and makes it possible to identify the foods at risk for Y. enterocolitica contamination.     

Amino acid sequence of a novel heat-stable enterotoxin produced by a yst gene-negative strain of Yersinia enterocolitica

Summary A novel heat-stable enterotoxin (designated Y-STb) was isolated and purified to homogeneity from the culture supernatant of a pathogenic but yst gene-negative strain of Yersinia enterocolitica. The amino acid sequence of the toxin was determined to be Lys-Ala-Cys-Asp-Thr-Gln-Thr-Pro-Ser-Pro-Ser-Glu-Glu-Asn-Asp-Trp-Cys-Cys-Glu- Val-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys. Y-STb was 20-fold more potent (minimum effective dose in the suckling mouse assay was 0.35 pmol) than the previously documented heat-stable enterotoxin (Y-STa) which is produced by yst gene-positive strains of Y. enterocolitica and has a minimum effective dose of 7.8 pmol. The sequence of Y-STb is different from that of Y-STa in the N-terminal half (1–17), but quite similar in the C-terminal half (18–30). To elucidate the effect of 13 amino acid substitutions in Y-STb on enhancing the toxicity, several short analogs of Y-STb were synthesized and their toxicities were compared in the suckling mouse assay. The enhanced enterotoxicity could be ascribed to the addition of a tryptophan residue at the N-terminus of the ST toxic domain which is the minimum structure essential for toxic activity; the presence of an aspartic acid residue at the same position caused a decrease in toxicity.     

Biological activity of synthetic peptides analogous to heat-stable enterotoxin produced by Yersinia enterocolitica

Abstract 3 peptides were synthesized chemically by following the primary structure of heat-stable enterotoxin (ST) produced by Yersinia enterocolitica . A peptide 1–30, having the whole sequence of 30 amino-acid residues, showed a ST activity similar to that of analogue peptide 15–30 composed of the C-terminal 16 amino acid residues. The c-GMP levels of L cells increased through an interaction with peptide 1–30 but not with peptide 15–30, while membranes isolated from broken L cells responded to both. Peptide 1–11, composed of the N-terminal 11 amino-acid residues, showed no biological activity.     

Detection of pathogenic Yersinia enterocolitica using a digoxigenin labelled probe targeting the yst gene

A 145 base pair digoxigenin-d-UTP-labelled probe, specific for pathogenic Yersinia enterocolitica heat-stable enterotoxin yst gene, was prepared by PCR. The probe was used in DNA-DNA colony hybridization and dot-blot hybridization assays. The specificity of the probe was confirmed using 52 strains representing all Yersinia spp., except Y. pestis . Out of a total of 25 Y. enterocolitica strains screened, the probe correctly identified all 18 pathogenic strains. Among the other Yersinia spp. screened, only one strain of Y. kristensenii was positively detected by the yst probe but could be differentiated by its weak signal response as compared with that obtained by pathogenic strains of Y. enterocolitica .     

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.