Isolation ofYersinia enterocolitica andYersinia enterocolitica-like organisms from raw milk in Italy

Thirty samples of raw milk, originating from individual producers in the Turin area, were examined for the presence ofYersinia enterocolitica. A cold enrichment method with phosphate-buffered saline (PBS) 1/15M, pH 7.6, and sorbitol-bile-salts broth (SB) was used. After 7, 14, or 21 days at 4°–5°C, plating was performed on selective agar media directly (MacConkey agar andSalmonella-Shigella agar) after the alkali method was used. Six strains ofY. enterocolitica (biotype 1) and 32 strainsY. enterocolitica-like (threeY. fredericksenii; nineYersinia rhamnose-, melibiose+, -methyl-d-glucoside+, raffinose+, probablyYersinia intermedia biotype rhamnose-; and 20Y. intermedia) were isolated.Yersinia strains were found in 11 samples of raw milk, andY. enterocolitica in four samples.     

A new isolation medium for the revovery ofYersinia enterocolitica from environmental sources

Yersinia enterocolitica has been isolated from a wide variety of sources throughout the world. The isolation of this organism requires special awareness from bacteriologists. To ease this procedure, we have developed a new isolation medium—BABY 4—meant to be used for environmental studies. Compared with previously recommended media, such as SS agar, SS-D agar, and urea-novobiocin agar, it allows easy detection ofY. enterocolitica in various types of waters. It counterselects Enterobacteriaceae species fromY. enterocolitica in a ratio of 10^–1 to 10^–5 and makes quantitative studies of environmental sources possible. This medium also allows the detection after 3–4 days' incubation of as few as 10³ Y. enterocolitica per gram of stool.     

Evaluation of isolation methods for pathogenic Yersinia enterocolitica from pig intestinal content

Aims: The aim of this study was to evaluate the efficiency of four isolation methods for the detection of pathogenic Yersinia enterocolitica from pig intestinal content. Methods and Results: The four methods comprised of 15 isolation steps using selective enrichments (irgasan–ticarcillin–potassium chlorate and modified Rappaport broth) and mildly selective enrichments at 4 or 25°C. Salmonella–Shigella‐desoxycholate–calcium chloride agar, cefsulodin–irgasan–novobiocin agar were used as plating media. The most sensitive method detected 78% (53/68) of the positive samples. Individual isolation steps using cold enrichment as the only enrichment or as a pre‐enrichment step with further selective enrichment showed the highest sensitivities (55–66%). All isolation methods resulted in high numbers of suspected colonies not confirmed as pathogenic Y. enterocolitica. Conclusions: Cold enrichment should be used in the detection of pathogenic Y. enterocolitica from pig intestinal contents. In addition, more than one parallel isolation step is needed. Significance and Impact of the Study: The study shows that depending on the isolation method used for Y. enterocolitica, the detected prevalence of Y. enterocolitica in pig intestinal contents varies greatly. More selective and sensitive isolation methods need to be developed for pathogenic Y. enterocolitica.     

Enterotoxin production at refrigeration temperature byYersinia enterocolitica andYersinia enterocolitica-like bacteria

Enterotoxin production after cultivation for 7 days in a refrigerator (3–6°C) was indicated for 4 of 20 strains ofYersinia enterocolitica andY. enterocolitica-like bacteria, by use of the infant mouse assay. These four strains were isolated from wild-living small mammals and water. Three of these isolates (Y. kristensenii, serogroups 11 and 28) were enterotoxigenic at 22 and 37°C as well as at refrigeration temperature. The remaining strain (Y. enterocolitica sensu stricto, serogroup 6) produced enterotoxin only at refrigeration temperature and at 22°C. The results indicate thatY. enterocolitica andY. enterocolitica-like bacteria may be capable of causing food intoxication after food storage at refrigeration temperature. A potential clinical significance of theY. enterocolitica enterotoxin in cold-blooded animals such as fish is suggested.     

Anti-microbial susceptibility of Yersinia pseudotuberculosis and Yersinia enterocolitica under different cultural conditions

Generally, Yersinia pseudotuberculosis and Y. enterocolitica grown at 37°C had increased susceptibility to antibiotics than when grown at 25°C. The susceptibility to kanamycin, cephalothin, tetracycline and chloramphenicol of Yersinia was also influenced by the growth medium and gas composition.N. Markova, T. Radoucheva, L. Ilieva and D. Veljanov are with the Institute of Microbiology, Bulgarian Academy of Science, 1113 Sofia, Bulgaria.     

Increase of the antimicrobial susceptibility ofYersinia enterocolitica associated with the expression of the virulence plasmid

Virulent strains ofYersinia enterocolitica incubated in RPMI 1640 medium with 25 mM HEPES at 37°C were more susceptible to several antibiotics than their plasmid-free isogenic derivatives. The enumeration of viable bacteria in RPMI 1640 agar at 37°C to discriminate between plasmid-bearing and spontaneously derived, plasmid-free bacteria made it possible to show that the plasmid presence was associated with a fourfold decrease of minimal inhibitory concentration of ampicillin, streptomycin, gentamicin, chloramphenicol, and oxytetracycline. SinceY. enterocolitica is an intracellular pathogen and RPMI 1640 medium mimics the intracellular milieu, the plasmid-associated increase of susceptibility to antibiotics that are concentrated by animal cells may be of clinical relevance.     

A simplified method for detecting pathogenic Yersinia enterocolitica in slaughtered pig tonsils

The aim of this study was to collect preliminary data on the carriage of pathogenic Yersinia enterocolitica in slaughtered pigs in France and to test a simplified method for detecting these strains from tonsils. From January to March 2009, 900 tonsil swabs were taken from pigs at one slaughterhouse in Brittany, France. The swabs were vortexed in 10 ml PSB broth, then 1 ml was added to 9 ml ITC broth. The media were incubated for 48 h at 25 °C. The PSB enrichment broth was streaked on CIN plates and the ITC enrichment broth on SSDC plates. In addition to the ISO 10273 method, we also streaked ITC enrichment broth on CIN plates. The plates were incubated for 24 h at 30 °C, and we then streaked a maximum of four typical colonies per plate onto a plate containing chromogenic medium (YeCM), for the isolation of pathogenic Y. enterocolitica isolates. In parallel, biochemical assays were carried out to confirm the identification of the isolates as Yersinia and to determine biotype.After passage on a YeCM plate and biochemical tests, 380 strains were confirmed to be pathogenic Y. enterocolitica. Finally, with the ISO 10273 method, 9.1% (CI_95% [5.8-12.4]) of tonsil swabs and 60% (CI_95% [45.4-74.6]) of the batches were positive. With the ITC-CIN method, 14.0% (CI_95% [10.7-17.3]) of the tonsil swabs and 68.9% (CI_95% [54.3-83.5]) of the batches were positive. Identification as pathogenic Y. enterocolitica was confirmed for 97.0% of the typical colonies obtained on the chromogenic medium, YeCM. The most prevalent biotype was biotype 4 (80.5% of the isolates), followed by biotype 3.This study demonstrates that the ITC-CIN method, followed by streaking on YeCM, may be an effective approach to the isolation of pathogenic Y. enterocolitica from tonsil swabs and the recovery of positive samples. This method is less time-consuming than the ISO 10273 method and reduces the number of biochemical tests required for the confirmation of Yersinia identification, through the use of YeCM.     

Induction of sporulation in food-spoilage yeasts

Four yeasts, Hansenula anomala, Kluyveromyces fragilis, Lodderomyces elongisporus and Saccharomyces cerevisiae, were cultured in two presporulation media at 30 ° C. Media consisted of yeast extract — peptone — acetate and yeast extract — peptone — dextrose broths. Except for K. fragilis, the test yeasts reached a high degree of sporulation when transferred to acetate- and ethanol-supplemented sporulation media. The percentage of S. cerevisiae cells forming asci was as high as 79% after 24 h incubation. H. anomala and L. elongisporus sporulated more rapidly in ethanol- compared to acetate-containing medium. Within test parameters, the concentration of acetate or ethanol, _pH, and incubation temperature (25 ° C and 30 ° C) did not substantially influence the extent of sporulation.     

Abscisic acid and mefluidide in the regulation of cold hardiness development in Solanum tuberosum L. potato

Plants of Solanum tuberosum L. potato do not cold acclimate when exposed to low temperature such as 5°C, day/night. When ABA (45 M) was added to the culture medium, stem-cultured plantlets of S. tuberosum, cv. Red Pontiac, either grown at 20°C/15°C, day/night, or at 5°C, increased in cold hardiness from –2°C (killing temperature) to –4.5°C. The increase in cold hardiness could be inhibited in both temperature regimes if cycloheximide (70 M) was added to the culture medium at the inception of ABA treatment. Cycloheximide did not inhibit cold hardiness development, however, when it was added to the culture medium 3 days after ABA treatment.When pot-grown plants were foliar sprayed with mefluidide (50 M), ABA content increased from 10 nmol to 30 nmol g^–1 dry weight and plants increased in cold hardiness from –2°C to about –3.5°C. The increases in free ABA and cold hardiness occurred only in plants grown at 20°C/15°C; neither ABA nor cold hardiness increased in plants grown at 5°C.The results suggest that an increase in ABA and a subsequent de novo synthesis of proteins are required for the development of cold hardiness in S. tuberosum regardless of temperature regime, and that the inability to synthesize ABA at low temperature, rather than protein synthesis, appears to be the reason why S. tuberosum does not cold acclimate.     

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.     

Spontaneous binding ofYersinia enterocolitica to murine leukocytes

Twelve strains ofYersinia enterocolitica were examined for their ability to bind spontaneously to murine leukocytes. Each of eight HeLa cell invasive strains exhibited nonselective binding to peritoneal leukocytes, lymph node leukocytes, and thymocytes, whereas four noninvasive strains lacked binding properties. Like the HeLa cell invasion, the binding ofY. enterocolitica to leukocytes was much less efficient for bacteria grown at 37°C than for bacteria grown at 22°C. The binding properties were not influenced by the virulence plasmid that codes for Vwa⁺ phenotype. This leukocyte binding test is proposed as a simple assay for invasive properties ofY. enterocolitica.     

The Effect of the Cultivation Method and the Growth Phase on the Lipopolysaccharide Composition of Yersinia pseudotuberculosis

Effects of the cultivation method (suspension cultures in a liquid nutrient broth or colonies on a solid agarized medium) and the growth phase on the lipopolysaccharide (LPS) composition of Yersinia pseudotuberculosis(O : Ib serovar, strain KS 3058) grown in cold (5°C) were studied. The amount of the LPS synthesized by cells depended on the bacteria growth phase for both media. The LPS acylation degree was constant, whereas the length of the O-specific polysaccharide chain varied with the culture age and for both media achieved maximum in the stationary growth phase. The bacteria cultivation on the nutrient agar stimulated more intensive synthesis of LPS, which were extracted more easily, had longer polysaccharide O-chains, and were more toxic than LPS of the bacteria cultivated in the liquid medium. It was proposed that the cultivation of Yersinia pseudotuberculosisin cold as colonies on the agar surface increases the bacterial virulence.     

Some extracellular enzymes associated with two tomato fruit spoilage molds

The production of amylolytic, cellulolytic and pectinolytic enzymes by Aspergillus flavus and A. fumigatus was investigated. The two fungi were cultured on wheat offal and liquid crystalline carboxymethylcellulose media. A. flavus produced amylases on basal and starch containing media while A. fumigatus could only produce amylases on starch medium. The cellulolytic activities of filtrates from culture or infected fruits showed that A. flavus produced lesser quantities of cellulolytic enzymes than A. fumigatus. At 25 °C and at a pH range of 6–8, A. flavus best produces amylases and cellulases, while A. fumigatus showed highest activities of the two enzymes at 35–40 °C and at pH 7.0. Two pectinolytic enzymes — polymethylgalacturonase and pectinmethyltrans-eliminase — were identified in vivo with the two molds. An endopolygalacturonase in addition to these two pectinolytic enzymes was well associated with A. fumigatus.     

Maximizing Plasmid Stability and Production of Released Proteins in Yersinia enterocolitica

Virulent serotypes of Yersinia enterocolitica carry a plasmid (pYV) encoding a family of proteins that are released into the medium and whose expression is temperature and calcium regulated. The plasmid is easily lost from cells during their growth in the laboratory. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with a monoclonal antibody (3.2C) that is specific for a 25-kDa released protein to show that 32°C is the lowest temperature at which plasmid-encoded proteins are expressed in quantity. The highest calcium concentration allowing full expression of these proteins was 445 to 545 μM at 32°C. Calcium concentrations of 745 μM and above at 37°C completely prevented the loss of pYV during multiple subcultures, while at 32°C, calcium concentrations of 245 μM and greater were sufficient to stabilize the plasmid. Growth of Y. enterocolitica at pH 5.5 was slower than at neutral pH values, but it also resulted in greatly increased stability of pYV. These studies showed that bacterial growth, retention of pYV, and expression of plasmid-encoded proteins may be maximized at 32°C with 445 μM calcium and that pYV stability is enhanced by growth at low pH. These observations suggest new approaches for isolation of plasmid-bearing virulent strains of Y. enterocolitica from samples contaminated with this organism and also may improve our understanding of pYV retention in vivo.     

Behavior of Yersinia enterocolitica in the Presence of the Bacterivorous Acanthamoeba castellanii

Free-living protozoa play an important role in the ecology and epidemiology of human-pathogenic bacteria. In the present study, the interaction between Yersinia enterocolitica, an important food-borne pathogen, and the free-living amoeba Acanthamoeba castellanii was studied. Several cocultivation assays were set up to assess the resistance of Y. enterocolitica to A. castellanii predation and the impact of environmental factors and bacterial strain-specific characteristics. Results showed that all Y. enterocolitica strains persist in association with A. castellanii for at least 14 days, and associations with A. castellanii enhanced survival of Yersinia under nutrient-rich conditions at 25°C and under nutrient-poor conditions at 37°C. Amoebae cultivated in the supernatant of one Yersinia strain showed temperature- and time-dependent permeabilization. Intraprotozoan survival of Y. enterocolitica depended on nutrient availability and temperature, with up to 2.8 log CFU/ml bacteria displaying intracellular survival at 7°C for at least 4 days in nutrient-rich medium. Transmission electron microscopy was performed to locate the Yersinia cells inside the amoebae. As Yersinia and Acanthamoeba share similar ecological niches, this interaction identifies a role of free-living protozoa in the ecology and epidemiology of Y. enterocolitica.     

Characterization of the bacterial flora of mass cultivated Brachionus plicatilis

Bacterial density and composition in association of mass cultivated rotifers (Brachionus plicatilis, SINTEF-strain) was investigated, during experimental conditions identical to the procedures used for preparing rotifers as live food for marine cold water fish larvae. These procedures include cultivation, enrichment with squid meal and acclimation to low temperature by storage of the rotifer culture at 6 °C. Large variations were observed in the number of rotifer associated (1.8–7.6 · 10³ colony forming units per rotifer^–1) and free-living (0.6–25 10⁷ cells·ml^–1) bacteria. An increase of 50–150% in the bacterial number was normally observed after feeding the rotifer with squid meal, but after three days of acclimation at 6 °C, the bacterial numbers decreased to the initial level.After enrichment of the cultures with squid meal, the similarity in the composition of the bacterial flora between the rotifers and water was reduced. However, acclimation of the culture at 6 °C resulted in better agreement of the rotifer associated flora and that in water. Enrichment of the cultures induced a shift in the bacterial composition from Cytophaga/Flavobacterium dominance to Pseudomonas/Alcaligenes dominance. The bacterial flora of the rotifer cultures are dominated by presumably opportunistic species after enrichment, which may have detrimental effects when rotifers are fed as live food to marine fish larvae.     

Cold storage ofPhytoseiulus persimilis (Phytoseiidae)

Phytoseiulus persimilis is commerelally mass-reared for use as a biological control agent for spider mites, primarilyTetranychus urticae, and cold storage is a potentially valuable aspect of mass-production. Cold storage ofP. persimilis in empty containers was found to be unsatisfactory, but provision of moisture during cold storage greatly increased survival. Provision of food further increased survival even though the mites were stored at temperatures below their threshold for development of 11°C. When food was provided, survival at 7.5°C was 97% after 4 weeks and 80% after 6 weeks. Subsequent longevity and fecundity of mites that survived 8 weeks at 7.5°C was comparable to mites taken directly from mass-rearing cultures. Survival of mites packaged in bran or vermiculite and held at 6°C for 10 days ranged from 75% to 85% and was not decreased by agitating the containers to simulate shipping. However, survival of mites held in bran or vermiculite at 5°C or 8°C for 4 weeks was poor, ranging from 0–19%, due to growth of mould in the media.Phytoseiulus persimilis can be successfully stored for 4 to 6 weeks in containers provisioned only with food and moisture; granular media used for distribution of the mites should be added just prior to shipping.     

Growth phase and low pH affect the thermal regulation of the Yersinia enterocolitica inv gene

The inv gene encodes the protein invasin, which is the primary invasion factor for Yersinia enterocolitica in vitro and in vivo. Previous studies of Yersinia species have shown that inv expression and entry into mammalian cells are temperature regulated. Invasin production is reduced at the host temperature of 37°C as compared to production at ambient temperature; consequently, this study was initiated to determine whether other host environmental signals might induce inv expression at 37°C. An inv::phoA translational fusion was recombined on to the Y. enterocolitica chromosome by allelic exchange to monitor inv expression. Molecular characterization of expression of the wild-type inv gene and the inv phoA fusion showed that invasin is not produced until early stationary phase in bacteria grown at 23°C. Y. enterocolitica grown at 37°C and pH 5.5 showed levels of inv expression comparable to those observed in bacteria grown at 23 C. An increase in Na⁺ ions caused a slight increase in expression at 37 C. However, expression at 37°C was unaffected by anaerobiosis, growth’medium, calcium levels, or iron levels. Additionally, Y. enterocolitica expressed invasin in Peyer's patches two days after being introduced intragastrically into BALB/c mice. These results suggest that invasin expression in K enterocolitica may remain elevated eariy during interaction with the intestinal epithelium, a site at which invasin was shown to be necessary.     

Assessment of virulence determinants in Yersinia enterocolitica 1A,O:5 strains isolated from chicken carcasses

The virulence potential associated with 42–45 MDa plasmid pYV, of three Yersinia enterocolitica, 1A, O:5, strains isolated previously from chicken carcasses was tested by plasmid extraction, and by four in vitro tests: (1) Auto-agglutination; (2) Calcium dependence for growth at 37 °C; (3) Congo Red and Crystal Violet binding; and (4) Resistance to the bactericidal effect of serum. The in vitro tests were negative in all strains tested, but from strain PP131 it was possible to extract a plasmid which was quite similar in size to the wild type pYV, but heavier and not observed in any other isolated strains. These contradictions between the presence of a plasmid of approximately 42–45 MDa and the in vitro tests negative results may suggest that there is no homology between the PP131 plasmid and pYV. From these results we conclude that the plasmid found in PP131 is not pYV and that the three Y. enterocolitica 1A, O:5, strains are non-pathogenic on pYV classical mechanism basis, which do not exclude the possibility of them becoming infectious by a different mechanism, especially in children or immune-depressed individuals.     

Growth studies on xenic cultures of Entamoeba gingivalis using established media

Wantland's egg medium, modified Shaffer-Frye (MSF) medium and Tryptose-Trypticase-Yeast Extract-Serum-Blood (TTY-SB) medium were compared with variations of the latter two media for their ability to support xenic growth of Entamoeba gingivalis. Wantland's egg medium was unsuitable for growth of E. gingivalis. Accompanying bacteria became resistant to penicillin and streptomycin, overwhelming the amoeba culture. MSF medium was also unsuitable for the cultivation of E. gingivalis. Bacterial growth was heavy and protozoan growth sparse. MSF medium without mercaptosuccinic acid, but with rice starch, dextran or levan substituted for glucose and with Yersinia enterocolitica added, supported limited growth of the amoeba. Unmodified TTY-SB medium did not sustain growth of E. gingivalis. However, when rice starch suspension was substituted for glucose, l-cysteine HCl was deleted, and a Crithidia sp. was added to the E. gingivalis culture grown xenically, enhanced growth of the oral amoeba resulted in this modified TTY-SB medium. E. gingivalis is very sensitive to changes in incubation temperature. Optimum growth was found to be in the narrow range from 34.5 to 35°C for all media tested.