A study of the growth kinetics of Yersinia enterocolitica serotype O:3 in pure and meat culture systems

The growth kinetics of a virulence plasmid-bearing (P+) and a plasmid-cured (P−) strain of Yersinia enterocolitica serotype O:3 in pure and meat culture were investigated. Growth studies were carried out at 25 and 37 °C in supplemented phosphate-buffered saline, buffered peptone water , cefsulodin-irgasan-novobiocin broth base or supplemented broth base (CIN). The lag phase durations and growth rates under these conditions were determined by linear regression analysis. In pure culture, under most sets of equivalent conditions, P+ and P− strains had similar lag phase durations. However, under one set of conditions, i.e. CIN broth at 37 °C, the lag phase duration of the P+ strain was significantly longer than P−. In all but the most selective medium, P+ strains had slower growth rates than P− strains at 37 °C, probably due to the increased metabolic burden entailed in the maintenance of the virulence plasmid. In the most selective medium, i.e. CIN broth, P+ strains grew significantly faster than P−. This finding suggests that possession of virulence plasmid confers an enhanced ability to grow in the presence of selective agents. In meat cultures, both strains had longer lag phases than in equivalent pure cultures, with longer lag phases noted at 37 than at 25 °C. No significant differences were observed between the length of lag phases of P+ and P− strains in meat culture. Both strains of Y. enterocolitica displayed faster growth rates in meat cultures than in pure cultures, indicating that one or more components of meat enhanced the growth of this organism. The effects and interaction of incubation temperature, enrichment broth and meat on the growth kinetics of plasmid-bearing and plasmid-cured Y. enterocolitica strains are discussed.     

Growth studies of plasmid bearing and plasmid cured Yersinia enterocolitica GER O:3 in the presence of cefsulodin, irgasan and novobiocin at 25 and 37 degrees C

AIM: In this study, the growth characteristics of Yersinia enterocolitica biotype 4, GER O:3 plasmid bearing (P+) and plasmid cured (P-) strain types were evaluated in brain heart infusion broth supplemented with cefsulodin, irgasan, and novobiocin alone or in combination. METHODS AND RESULTS: Growth curves were obtained for the two strain types in broth supplemented with selective agents at 25 or 37 degrees C for 32 h to obtain data on the lag phase durations and growth rates of the strains. Generally, the lag times and growth rates of the P+ and P- strains were similar for cultures incubated at 25 degrees C regardless of the selective agent added and where plasmid replication and expression were not under any significant burden. However, where the lag times and growth rates of the strains were examined at 37 degrees C, significant differences were observed in the lag phase durations of the plasmid bearing strain type compared the plasmid cured strain, an effect that was due to the burden of the plasmid and the influence of selective agents. Generally, when two or more agents were present, lag phase durations were longer for the plasmid bearing strain. Some exceptions noted where in the presence of irgasan or full selective agent (CIN) the opposite case was observed. When growth rates were compared, the plasmidless strain type was typically faster than the plasmid bearing strain in the presence of most selective agents at 37 degrees C and the growth rates of both strain types at 25 degrees C were similar where the temperature appeared to negate the effects of plasmid. CONCLUSIONS: The data obtained in these studies suggest that selective agents (in particular irgasan) and incubation temperature play a significant role in influencing the growth characteristics of plasmid bearing and plasmid cured strains of Y. enterocolitica. SIGNIFICANCE AND IMPACT OF THE STUDY: This data presented in this study has significant implications for enrichment methods used in the detection or recovery of plasmid bearing Y. enterocolitica strains from food, environmental or clinical samples.     

Evaluation of the usefulness of selected virulence markers for identification of virulent Yersinia enterocolitica strains. II. Genotypic markers associated with the pYV plasmid

Pathogenic strains of Yersinia enterocolitica bear virulence associated plasmid pYV. Unfortunately plasmid pYV is easily lost by these bacteria incubated at elevated temperatures (37 degrees C) or long stored at room temperatures. This sometimes makes difficult the detection of the virulence plasmid, especially by its isolation or biochemical tests. On the other hand, observations done by some authors suggest that polymerase chain reaction (PCR) could be useful for demonstration of the pYV plasmid of Yersinia strains. Accordingly to this observation the aim of the presented study was to check the usefulness of plasmid-localised genes virF and yadA, detected by PCR, for the identification of the virulent strains of Y. enterocolitica. In the presented study one hundred and fifty two clinical strains of Y. enterocolitica belonging to serogroup O3 were investigated by the PCR for the presence of genes virF and yadA. Bacterial strains were first tested for the presence of pYV plasmid. In addition the phenotypic features: calcium dependence, Congo red binding and autoagglutination were determined. In this way the virulence plasmid was found in 130 of 152 examined strains. For PCR studies also forty plasmid-cured strains of Y. enterocolitica and 32 non-Y. enterocolitica, Enterobacteriaceae strains were included. The obtained results show that the tested genes were present only in Yersinia strains possessing the pYV plasmid and no one non-specific PCR product was observed. The detection level of these genes in nested PCR permits to detect pathogenic Y. enterocolitica in suspension composed of 1 x 10(3) CFU/ml of pYV+ bacilli and 3 x 10(9) CFU/ml plasmid-cured, isogenic bacteria. In the study it was shown that genes virF and yadA were useful virulence markers, which could be helpful in clinical studies for the detection of the virulence plasmid in Y. enterocolitica strains long stored or incubated at elevated temperatures.     

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.     

Isolation of Yersinia enterocolitica from well water and growth in distilled water.

Yersinia enterocolitica was recovered from well water during a large water-borne outbreak of gastrointestinal illness. Isolates were predominantly Nilehn biotype 1, of which 57% were serologically nontypable. Isolation and enumeration of these Y. enterocolitica strains were made on M-Endo broth. Laboratory studies were conducted on selected isolates to establish the growth of Y. enterocolitica in distilled water and the competitive growth of this organism in various enteric media. Growth was obtained in sterile distilled water without added nutrients at 4, 25, and 37 degrees C. M-Endo medium gave equal or better recovery of Y. enterocolitica in competitive growth studies than did other commonly used enteric media using the membrane filter technique and incubating at 35 degrees C. All well water isolates were confirmed biochemically at 25 and 35 degrees C and serotyped, and antibiotic susceptibility tests were performed.     

Virulence plasmid (pYV)-associated susceptibility of Yersinia enterocolitica to chlorine and heavy metals

This study was aimed at elucidating the role of virulence plasmid (pYV) in the susceptibility of Yersinia enterocolitica to bactericidal agents such as chlorine and heavy metals. Plasmid-bearing (pYV+) Y. enterocolitica was less susceptible to the antimicrobial action of chlorine and heavy metals compared with the isogenic plasmidless (pYV-) derivative. This difference was, however, observed only with bacteria cultured at 25 degrees C. pYV-associated susceptibility apart, cells cultured at 37 degrees C were also found to be less susceptible to the antimicrobial action of these agents. The results indicate that the susceptibility of Y. enterocolitica to these agents was influenced both by the presence of the virulence plasmid and the temperature at which the cells were cultured.     

Use of a single procedure for selective enrichment, isolation, and identification of plasmid-bearing virulent Yersinia enterocolitica of various serotypes from pork samples.

Many selective enrichment methods for the isolation of Yersinia enterocolitica from foods have been described. However, no single isolation procedure has been described for the recovery and identification of various plasmid-bearing serotypes. A single improved procedure for selective enrichment, isolation, identification, and maintenance of plasmid-bearing virulent serotypes of Y. enterocolitica from pork samples was developed. Enrichment at 12 degrees C in Trypticase soy broth containing yeast extract, bile salts, and Irgasan was found to be an efficient medium for the recovery of plasmid-bearing virulent strains of Y. enterocolitica representing O:3; O:8; O:TACOMA; O:5, O:27; and O:13 serotypes. MacConkey agar proved to be a reliable medium for the isolation of presumptive colonies, which were subsequently confirmed as plasmid-bearing virulent strains by Congo red binding and low calcium response. Further confirmation by multiplex PCR employed primers directed at the chromosomal ail and plasmid-borne virF genes, which are present only in pathogenic strains. The method was applied to pig slaughterhouse samples and was effective in isolating plasmid-bearing virulent strains of Y. enterocolitica from naturally contaminated porcine tongues. Strains isolated from ground pork and tongue expressed plasmid-associated phenotypes and mouse pathogenicity.     

A comparison of media and methods for the recovery of Yersinia enterocolitica and Yersinia enterocolitica-like bacteria from milk containing simulated raw milk microfloras

A number of plating and enrichment media proposed for the isolation of Yersinia enterocolitica from foodstuffs were examined for their ability to recover the type strains of Y. enterocolitica sensu stricto, Y. intermedia, Y. frederiksenii and Y. kristensenii. Nine selective plating media were evaluated for the quantitative recovery of the type strains in pure culture, and their inhibition of other organisms typical of both milk and enteric microfloras. Cefsulodin-irgasan-novobiocin (CIN) agar, incubated for 48 h at 25 degrees C, allowed a high recovery of all the Yersinia spp. and was the most selective medium. The same four type strains were added to UHT milk that had been previously inoculated with bacteria to simulate either freshly drawn or cold stored milk microfloras. Twenty-six enrichment procedures (including cold enrichment, selective enrichment at higher temperatures, two-step procedures and a post-enrichment alkali treatment) were assessed for the efficiency of recovery of the Yersinia spp. Pre-enrichment in trypticase-soy broth (TSB) for 24 h at 22 degrees C followed by selective enrichment in bile-oxalate-sorbose (BOS) medium for 5 d at 22 degrees C and plating on CIN agar (48 h at 25 degrees C) allowed the greatest increase in the numbers of Yersinia spp. and maximum inhibition of the competing microflora.     

Production of bacteriocin-like substances byYersinia frederiksenii,Y. kristensenii,andY. intermedia strains

The production of bacteriocin-like substances by strains of Yersinia frederiksenii, Y. kristensenii and Y. intermedia in broth culture was established. These substances showed a selective activity against Y. enterocolitica, Y. frederiksenii, Y. kristensenii and Y. intermedia strains. Electron micrographs revealed the presence of phage tails in culture media. The production of these substances was detected in cultures grown at 25 degrees C but not in those grown at 37 degrees C, while these bacteriocin-like substances were active at 25 and 37 degrees C. Y. enterocolitica serogroups 0:3 and 0:9 were more susceptible to these bacterin-like substances than strains of Yersinia isolated from environmental sources.     

Synthesis of a selective agar medium for Yersinia enterocolitica

A new agar medium for isolation of Yersinia enterocolitica was formulated based on growth studies which defined an optimum basal, and the evaluation of selective chemical agents, dyes, and antibiotics. The final formulation, designated cefsulodin-irgasan-novobiocin(CIN) agar, provided quantitative recovery of 40 different strains of Y. enterocolitica in 24 h using incubation at 32 degrees C or with 48 h of incubation at 22 degrees C. The medium was highly selective, especially against Pseudomonas aeruginosa. Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis. Colony morphology coupled with a differential reaction resulting from mannitol fermentation permitted discrimination of Y. enterocolitica from most of those Gram-negative bacteria that were able to grow on the medium. Recovery and selective characteristics of CIN agar were stable during storage at room temperature for 9 days. CIN agar gave a higher recovery of Y. enterocolitica from feces both direct and with cold enrichment (0.4/1.5%) than Salmonella-Shigella (0.0/0.7%) and MacConkey (0.0/0.9%) agars while significantly reducing the level of background organisms.     

The effect of pH and culture system on the attachment of plasmid-bearing and plasmid-cured Yersinia enterocolitica to a polycarbonate membrane in a surface adhesion immunofluorescent technique

A rapid surface adhesion based immunofluorescence technique was used to isolate and detect Yersinia enterocolitica from inoculated enriched culture systems. The pathogen was isolated by surface adhesion to a polycarbonate membrane which was mounted on a glass slide and immersed in the enriched culture for 15 min. The pathogen was detected using a fluorescent labelled (FITC) monoclonal antibody which was specific for Y. enterocolitica serotype O:3 and then viewed using fluorescent microscopy. The effect of culture type (broth, meat homogenate and minced meat) and pH (5.00, 7.00, 9.00 and 11.00) on the adhesion of plasmid-bearing and plasmid-cured Y. enterocolitica to the polycarbonate membrane in this technique was determined. The pH had a significant effect (P < 0.05) in broth and meat homogenate cultures, with enhanced attachment of Y. enterocolitica (P+ and P-) at pH 9.00 than at pH 5, 7 or 11. Culture type was also important, with differences observed in the numbers of Yersinia adhering to membranes immersed in broth, meat homogenate and minced beef. Differences in attachment were noted between plasmid-bearing and plasmid-cured Y. enterocolitica isolated from similar cultural environments. The reasons for these observed differences, and their implications for the surface adhesion immunofluorescent rapid method, are discussed.     

The cultural properties and virulence of Yersinia]

Study of the cultivation properties of 82 enterobacterial strains has revealed that the colonies of virulent Y. enterocolitica (serovars O3, O9) and Y. pseudotuberculosis (serovar I) are temperature-sensitive. This sign, closely connected with the presence and expression of the virulence plasmid with a molecular weight of 44-48 MD, is not characteristic of other strains. Virulent Yersinia grown in nutrient agar for 48 hours at 37 degrees C form colonies which are smaller in diameter than those formed during cultivation at 26 degrees C (with the significance of differences t greater than or equal to 4), their diameter at 37 degrees C not exceeding 1.0 mm. The test for the determination of the temperature-sensitive morphology of Yersinia colonies, along with the tests for other virulence markers, is probably suitable for the detection of the causative agents of yersiniosis or pseudotuberculosis.     

Isolation of Yersinia enterocolitica from well water and growth in distilled water.

Yersinia enterocolitica was recovered from well water during a large water-borne outbreak of gastrointestinal illness. Isolates were predominantly Nilehn biotype 1, of which 57% were serologically nontypable. Isolation and enumeration of these Y. enterocolitica strains were made on M-Endo broth. Laboratory studies were conducted on selected isolates to establish the growth of Y. enterocolitica in distilled water and the competitive growth of this organism in various enteric media. Growth was obtained in sterile distilled water without added nutrients at 4, 25, and 37 degrees C. M-Endo medium gave equal or better recovery of Y. enterocolitica in competitive growth studies than did other commonly used enteric media using the membrane filter technique and incubating at 35 degrees C. All well water isolates were confirmed biochemically at 25 and 35 degrees C and serotyped, and antibiotic susceptibility tests were performed.     

Conjugative 40-megadalton plasmid in Streptococcus lactis subsp. diacetylactis DRC3 is associated with resistance to nisin and bacteriophage

Streptococcus lactis subsp. diacetylactis DRC3 was examined for plasmid DNA and found to contain a previously unreported plasmid of 40 X 10(6) daltons. This plasmid, designated pNP40, was conjugally transferred to a plasmid-cured derivative of S. lactis C2. Transconjugants containing pNP40 acquired resistance to nisin produced by strains of S. lactis and to commercially available nisin when assay plates were incubated at 21, 32, and 37 degrees C. In addition, c2 phage growth was completely restricted in transconjugants containing pNP40 at 21 and 32 degrees C, but not at 37 degrees C. This result suggests that pNP40 may be coding for a temperature-sensitive enzyme that restricts phage growth at 21 and 32 degrees C, but not at 37 degrees C. Eight consecutive transfers of a transconjugant containing pNP40 in Elliker broth at 37 degrees C resulted in 100% loss of resistance to c2 phage when colonies were tested at 32 degrees C. These phage-sensitive isolates had lost pNP40 and had also become sensitive to nisin. This result suggests that pNP40 may also be thermosensitive in its replication. The finding of a phage resistance determinant located on a conjugative plasmid should prove useful in constructing phage-resistant variants for dairy fermentation processes.     

Conjugative 40-megadalton plasmid in Streptococcus lactis subsp. diacetylactis DRC3 is associated with resistance to nisin and bacteriophage.

Streptococcus lactis subsp. diacetylactis DRC3 was examined for plasmid DNA and found to contain a previously unreported plasmid of 40 X 10(6) daltons. This plasmid, designated pNP40, was conjugally transferred to a plasmid-cured derivative of S. lactis C2. Transconjugants containing pNP40 acquired resistance to nisin produced by strains of S. lactis and to commercially available nisin when assay plates were incubated at 21, 32, and 37 degrees C. In addition, c2 phage growth was completely restricted in transconjugants containing pNP40 at 21 and 32 degrees C, but not at 37 degrees C. This result suggests that pNP40 may be coding for a temperature-sensitive enzyme that restricts phage growth at 21 and 32 degrees C, but not at 37 degrees C. Eight consecutive transfers of a transconjugant containing pNP40 in Elliker broth at 37 degrees C resulted in 100% loss of resistance to c2 phage when colonies were tested at 32 degrees C. These phage-sensitive isolates had lost pNP40 and had also become sensitive to nisin. This result suggests that pNP40 may also be thermosensitive in its replication. The finding of a phage resistance determinant located on a conjugative plasmid should prove useful in constructing phage-resistant variants for dairy fermentation processes.     

Evaluation of the usefulness of selected virulence markers for the identification of virulent Yersinia enterocolitica strains. I. Phenotypic markders associated with Plasmid pYV

The species Yersinia enterocolitica includes either pathogenic or non-pathogenic strains. Therefore it is necessary to differentiate virulent bacilli from other. It is well known that pathogenic strains of Y. enterocolitica bearing virulence associated plasmid called pYV, which could be demonstrated by its isolation or detected by the presence of specific, phenotypic properties directly related with this plasmid. The aim of the presented paper was to check the ability of some phenotypic virulence markers associated with pYV, to detection of pathogenic Y. enterocolitica strains. In the presented work 152 (130 carrying pYV) clinical strains of Y. enterocolitica O3 isolated mainly from stool were examined for the presence of phenotypic virulence markers such as: calcium dependency, Congo-red binding, autoagglutination and agglutination with Mangifera indica extract. Both first features were detected parallel, on the same plate, using CRMOX (Congo-red, Magnesium Oxalate) agar. The detection of the tested markers in the examined strains was compared with the presence of virulence plasmid. The obtained results confirmed the observations done by other authors that Y. enterocolitica strains, in which bacilli bearing the virulence plasmid predominate, exhibit all tested phenotypic properties whereas the plasmid-cured isogenic strains show no one of these features. Therefore all the tested markers could be useful for detection of virulent Y. enterocolitica strains directly isolated from patients. The most useful virulence markers in bacteriological study seems to be calcium dependency and Congo-red binding, examined together by the use of CRMOX agar, because they confirm the presence of the virulence plasmid by parallel detection of two physiologically different features associated with this plasmid. In addition CRMOX agar allows for the examination rough strains while agglutination tests do not.     

The Yersinia enterocolitica pYV Virulence Plasmid Contains Multiple Intrinsic DNA Bends Which Melt at 37°C

Temperature has a pleiotropic effect on Yersinia enterocolitica gene expression. Temperature-dependent phenotypes include the switching between two type III protein secretion systems, flagellum biosynthesis (相似文献   

Evaluation of the usefulness of selected virulence markers for identification of virulent strains of Yersinia enterocolitica strains. III. Chromosome markers of virulence

It is well known that virulent strains of Y. enterocolitica bear the virulence-associated plasmid pYV. Moreover some authors consider that the pathogenic strains of these bacteria have chromosome encoded phenotypic and genotypic features such as: genes ail and yst which could be used as virulence markers. The virulent strains of Y. enterocolitica do not produce pyrasinamidase and are not able to ferment salicin and cannot hydrolyse esculin. In addition these strains produce thermostable enterotoxin called YST and protein Ail (attachment invasion locus). In contrast to phenotypic virulence makers the biological function of proteins Ail, YST and nucleotide sequence of genes ail and yst is well described. In the presented study one hundred thirty virulence plasmid bearing Y. enterocolitica strains belonging to serogroup O3 were examined for the presence of genes ail, yst, and were tested for their inability to pyrasinamidase production, salicin fermentation and esculin hydrolysis. In addition forty pYV plasmid-cured isogenic strains were included in to the study. Genes ail and yst were detected by polymerase chain reaction (PCR). The obtained results indicate that all tested 130 pYV+ Y. enterocolitica strains as well as 40 plasmid-cured isogenic strains have carried ail and yst genes. All tested strains did not produce pyrasinamidase, hydrolyse esculin and ferment salicin. This generally was in agreement with the observations done by other authors and suggest that the chromosomal virulence markers, especially well described genes ail and yst, could be useful for excluding the potential virulence of Y. enterocolitica strains, which had lost pYV plasmid and have no ail or yst genes. Therefore, in clinical studies, Y. enterocolitica strains isolated directly from patients should be primarily tested for the presence of the virulence plasmid and secondarily, the negative ones could be examined for the presence of the chromosomal virulence markers.     

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.     

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.