Rapid duplex PCR assay for the detection of pathogenic Yersinia enterocolitica strains

For the detection of pathogenic Yersinia enterocolitica strains, a duplex PCR has been developed based on differences observed between the fingerprint profiles of pathogenic and non-pathogenic strains. The profiles were obtained by using a primer derived from the Enterobacterial Repetitive Intergenic Consensus (ERIC) sequences. From the sequence of one pathogen-specific amplified fragment, a discriminative primer has been designed bridging the sequence of the highly conserved core region and 3' end of the ERIC element. In combination with three other primers, all located within the detected open reading frame that resembled the sequence of the bipA gene, this primer was applied in a duplex PCR assay to simultaneously detect Y. enterocolitica and to discriminate between pathogenic and non-pathogenic strains. The same primer combinations were used in an on line rapid cycling real-time PCR assay. The used SYBR Green I format allowed for the easy translation of the PCR conditions and confirmation of the resulting amplicons. The time of analysis was reduced to approximately 60 min.     

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.     

Lack of correlation between the presence of plasmids and fimbriae in Yersinia enterocolitica and Yersinia pseudotuberculosis

S kurnik , M. 1984. Lack of correlation between the presence of plasmids and fimbriae in Yersinia enterocolitica and Yersinia pseudotuberculosis. Journal of Applied Bacteriology 56 , 355–363.
Thirty-nine strains of Yersinia enterocolitica and 10 strains of Yersinia pseudoluber-culosis were studied for the presence of fimbriae, plasmids and two plasmid associated phenotypic expressions, autoagglutination and Ca²⁺ dependent growth at 37C. All of the strains studied became fimbriated, which was confirmed by electron microscopy and haemagglutination tests. Fimbriation was not correlated with the presence or absence of plasmids.     

Lack of correlation between the presence of plasmids and fimbriae in Yersinia enterocolitica and Yersinia pseudotuberculosis

Thirty-nine strains of Yersinia enterocolitica and 10 strains of Yersinia pseudotuberculosis were studied for the presence of fimbriae, plasmids and two plasmid associated phenotypic expressions, autoagglutination and Ca2+ dependent growth at 37 degrees C. All of the strains studied became fimbriated, which was confirmed by electron microscopy and haemagglutination tests. Fimbriation was not correlated with the presence or absence of plasmids.     

Analysis of bacteriocinogenic properties of Yersinia enterocolitica strains

The aim of the study was the investigation of bacteriocinogenic properties of 102 Yersinia enterocolitica strains. The influence of selected factors on the production of bacteriocins by Y. enterocolitica and properties of jersiniacin 44JPSBKOH were also investigated. Bacteriocinogenic properties of Y. enterocolitica strains were tested by using the delayed cross-streaking method. It was found that the production of bacteriocins by Y. enterocolitica depended on the type of media on which the producer and indicator strains were grown. It turned out that some strains of Y. enterocolitica showed bacteriocinogenic properties at 25 degrees C, 30 degrees C and 37 degrees C irrespective of the presence of manganese ions in medium. In the presence of iron ions these strains showed bacteriocinogenic properties only at 25 degrees C. Y. enterocolitica strains which required Mn2+ or Mn7+ ions for bacteriocins production showed this activity only at 25 degrees C but in presence of Fe3+ ions they had no bacteriocinogenic properties. The partially purified jersiniacin 44JPSBKOH is a protein, its molecular weight was estimated to be 40 kDa. Yersiniacin 44JPSBKOH was active in the pH range of 3 to 9. Its bactericidal activity was rapidly lost when heated to 100 degrees C and treated with proteolytic enzymes. Yersiniacin 44JPSBKOH showed bactericidal activity against other Y. enterocolitica strains and some strains of Pseudomonas aeruginosa isolated from humans.     

Sequences related to the major subunit gene fedA of F107 fimbriae in porcine Escherichia coli strains that express adhesive fimbriae

Abstract Porcine Escherichia coli strains isolated from cases fo postweaning diarrhea or edema disease were analysed for the presence of fedA , the major subunit gene of F107 fimbriae. The E. coli isolates were known to contain colonisation factor '8813', or to express F107, 2134P or other fimbriae, different from F4, F5, F6, and F41. PCR with fedA -specific primers, restriction enzyme digestion of the PCR product, and nucleotide sequence analysis demonstrated that 2134P pili, colonisation factor '8813' and fimbriae identified on Australian strains of the O141 serotype belong to one family of F107 fimbrial antigens.     

The major heat-modifiable outer membrane protein CD is highly conserved among strains of Branhamella catarrhalis

The outer membrane of Branhamella catarrhalis contains a major, heat-modifiable outer membrane protein called CD which has epitopes on the surface of the intact bacterium. The gene encoding CD was cloned and expressed in Escherichia coli. The protein migrates in gels as a doublet, indicating that CD is encoded by single gene whose gene product has two stable conformations. The nucleotide sequence of the gene encoding CD was determined and shows homology with the OprF outer membrane protein of Pseudomonas species. The CD protein contains a proline-rich region, which appears to account for its aberrant migration in gels. Restriction fragment-length analysis of 30 isolates of B. catarrhalis with oligonucleotide probes corresponding to sequences in the CD gene produced identical patterns in Southern blot assays. The major heat-modifiable outer membrane protein CD shares homology with the OprF protein and is highly conserved among strains of B. catarrhalis.     

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.     

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.     

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.     

The polymerase chain reaction: an epidemiological tool to differentiate between two clusters of pathogenic Yersinia enterocolitica strains

A primer set designed to amplify the enterotoxin (yst) gene of pathogenic Yersinia enterocolitica strains generated two different electrophoretic profiles of the target sequence when a collection of strains of worldwide origin was screened. Serovars O:1,3; O:2a,3; O:3; O:5,27 and O:9, known as European strains, produced a 200-bp fragment that matched the size of the target sequence. However, serovars O:4,32; O:8; O:13a,13b; O:20 and O:21, known as American strains, generated two fragments of 1.4 and 1.6 kb. The amplified products of one American strain were sequenced and the presence of the yst gene was confirmed in both fragments. Thus, the potential of the polymerase chain reaction to be used as an epidemiological tool in differentiation between the two clusters of pathogenic strains of Y. enterocolitica could be demonstrated.     

Biochemical characteristics of Yersinia enterocolitica strains isolated in the Maritime Territory

The detailed study of 179 strains, considered to be typical and atypical representatives of Y. enterocolitica upon their isolation, has been carried out. Of these, 129 strains have been found to belong to Y. enterocolitica with their typical biochemical properties and 50 strains, to new Yersinia species. The ecological sources of all the isolated strains are indicated. The necessity of the thorough epidemiological, clinical and laboratory study of the etiological role of Yersinia in acute intestinal diseases in humans is pointed out.     

小肠结肠炎耶尔耶尔森氏菌血清群的研究：Ⅱ.我国小肠...

Since 1980, we have collected 1120 strains of Yersinia enterocolitica, from the different parts of China. These strains have been obtained from various sources in man, animals and natural environment accompanied by their clinical or ecological information of Yersinia enterocolitica. The results of our tests have shown that the 747 strains have exhibited the clinical morphological and biochemical characteristics of Yersinia enterocolitica. Through comparing under the same conditions, out of the 747 strains 335 have been selected out with better antigenicity and have been produced antisera from their representative strains. This set of antisera is very satisfactory for its potency and specificity. This set of antisera is ready to supply and have good efficacy and application facilitated for control strains on identifying strains and their epidemiologic observation.     

The major subunit of Escherichia coli type 1 fimbriae is not required for D-mannose-specific adhesion

Type 1 fimbriae are surface organelles on Escherichia coli, which mediate specific binding to D-mannose-containing structures. These fimbriae are heteropolymers composed of a major building element, the FimA protein, and small amounts of the FimF, FimG and FimH proteins. The FimH protein is uniquely responsible for the D-mannose receptor binding. In this work data are presented which indicate that the major subunit of type 1 fimbriae is dispensable for D-mannose-specific binding. A recombinant strain was studied which harboured an insertional deletion in the fimA gene, and was thereby unable to produce type 1 fimbriae; however, it was still able to express a D-mannose-binding phenotype. However, the deletion resulted in a 25-fold reduction of the adhesive potential, as measured by binding to D-mannose-coated Sepharose beads. Serological and specific receptor binding evidence is presented that suggests that the FimH adhesion is capable of being exposed on the bacterial surface without being an integral part of the fimbriae.     

Identification of Yersinia enterocolitica within the genus Yersinia

In this report we describe a PCR strategy for the unambigous identification of biochemically presumptive typed Yersinia (Y.) enterocolitica. A total of 269 isolates belonging to ten species of the genus Yersinia were investigated. In a first PCR only isolates classified as Y. enterocolitica (n = 113) gave rise to a specific amplification resulting in a sensitivity and a specificity of 100%. By sequencing the 269 amplicons of a second pan-Yersinia PCR spanning a distinct 16S rRNA gene region, 20 different sequence clusters could be identified within the genus. By this, Y. enterocolitica isolates of American and European origin could be distinguished safely and already described sequence clusters of the species Y. frederiksenii were confirmed. New 16S rRNA gene sequence clusters were detected for the species Y. frederiksenii, Y. intermedia, Y. mollaretii, Y. aldovae, Y. kristensenii, and Y. rohdei.     

Genotyping of human and porcine Yersinia enterocolitica, Yersinia intermedia, and Yersinia bercovieri strains from Switzerland by amplified fragment length polymorphism analysis

In this study, 231 strains of Yersinia enterocolitica, 25 strains of Y. intermedia, and 10 strains of Y. bercovieri from human and porcine sources (including reference strains) were analyzed using amplified fragment length polymorphism (AFLP), a whole-genome fingerprinting method for subtyping bacterial isolates. AFLP typing distinguished the different Yersinia species examined. Representatives of Y. enterocolitica biotypes 1A, 1B, 2, 3, and 4 belonged to biotype-related AFLP clusters and were clearly distinguished from each other. Y. enterocolitica biotypes 2, 3, and 4 appeared to be more closely related to each other (83% similarity) than to biotypes 1A (11%) and 1B (47%). Biotype 1A strains exhibited the greatest genetic heterogeneity of the biotypes studied. The biotype 1A genotypes were distributed among four major clusters, each containing strains from both human and porcine sources, confirming the zoonotic potential of this organism. The AFLP technique is a valuable genotypic method for identification and typing of Y. enterocolitica and other Yersinia spp.     

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.