The incidence of Yersinia enterocolitica and Yersinia enterocolitica-like organisms in raw and pasteurized milk in Northern Ireland

Yersinia enterocolitica and Y. enterocolitica-like bacteria were frequently isolated from samples of both raw bulked milk (34/150) and farm bottled (raw) milk (5/20). These bacteria were also found to contaminate creamery pasteurized milk (6/100 samples) and farm pasteurized milk (4/50 samples). Although Y. enterocolitica was the most commonly isolated species, Y. intermedia and Y. frederiksenii were also frequently obtained (52, 31 and 15% of isolates, respectively). Also, one atypical strain was identified as Y. aldovae. The Y. enterocolitica strains were largely biotype 1 (20/27) including five strains which could ferment lactose. One third of the Y. enterocolitica strains were not typable, but of those which were, the serotypes were 0:34 (18.5%), 0:5.27 (18.5%), 0:6.3 (15%), 0:4 (11%) and 0:7 (4%). Pre-enrichment in trypticase-soy broth (TSB) (at 22 degrees C for 24 h) followed by selective enrichment in bile-oxalate-sorbose broth (at 22 degrees C for 6 d) allowed the recovery of 92.3% of all isolates, as compared with 15.4% using cold enrichment in TSB at 4 degrees C for 21 d.     

The incidence of Yersinia enterocolitica and Yersinia enterocolitica-like organisms in raw and pasteurized milk in Northern Ireland

Yersinia enterocolitica and Y. enterocolitica -like bacteria were frequently isolated from samples of both raw bulked milk (34/150) and farm bottled (raw) milk (5/20). These bacteria were also found to contaminate creamery pasteurized milk (6/100 samples) and farm pasteurized milk (4/50 samples). Although Y. enterocolitica was the most commonly isolated species, Y. intermedia and Y. frederiksenii were also frequently obtained (52, 31 and 15% of isolates, respectively). Also, one atypical strain was identified as Y. aldovae . The Y. enterocolitica strains were largely biotype 1 (20/27) including five strains which could ferment lactose. One third of the Y. enterocolitica strains were not typable, but of those which were, the serotypes were 0:34 (18.5%), 0:5,27 (18.5%), 0:6,30 (15%), 0:4 (11%) and 0:7 (4%). Pre-enrichment in trypticase-soy broth (TSB) (at 22°C for 24 h) followed by selective enrichment in bile-oxalate-sorbose broth (at 22°C for 6 d) allowed the recovery of 92.3% of all isolates, as compared with 15.4% using cold enrichment in TSB at 4°C for 21 d.     

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.     

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°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°C followed by selective enrichment in bile-oxalate-sorbose (BOS) medium for 5 d at 22°C and plating on CIN agar (48 h at 25°C) allowed the greatest increase in the numbers of Yersinia spp. and maximum inhibition of the competing microflora.     

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.     

Isolation and characterization of staphylococci from goats milk produced in Northern Ireland

Goats milk was examined for total viable bacteria and staphylococci (Baird-Parker medium, Schleifer & Kramer's (SK) medium, SK medium with a reduced sodium azide content (SKR) and SK and SKR with 5% added sheep blood). Staphylococcus aureus, Staph. epidermidis and Staph. simulans were the predominant species isolated overall. SK medium proved inhibitory with respect to the isolation of Staph. caprae and Staph. chromogenes. This was reduced by plating on the modified SK media. Representative strains of each species isolated were examined for production of enterotoxins A-E. Enterotoxin C alone was produced by 35% of the Staph. aureus strains tested. None of the other staphylococcal species examined produced any of the known enterotoxins.     

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.     

Yersinia enterocolitica in raw goat's milk.

Biochemical and serological data are presented for 35 isolates of Yersinia enterocolitica from raw goat's milk produced in New South Wales, Australia. Strains resembled biotype I or 2, but the majority (25 of 35) fermented rhamnose and some showed other atypical reactions.     

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.     

Isolation of Yersinia enterocolitica from raw milk

Four enrichment procedures were used for examining 131 raw milk samples for the presence of Yersinia enterocolitica. Forty-two isolations were obtained from 19 pooled- (31.1 percent positive) and 10 individual-producer samples (14.3 percent positive). Enrichment by Butterfields phosphate buffer incubated at 4 degrees C for 14 days and then inoculation of modified Rappaport broth incubated at 23 degrees C for 5 days produced the greatest number of isolations. The majority of isolates were biotype 1, and many were atypical from clinical isolates in being rhamnose positive (47.6 percent), citrate positive (16.7 percent), and lactose positive (26.2 percent). Thirteen isolates were serotypable, belonging to seven different O serotypes, with O:5 occurring most frequently.     

Isolation of Yersinia enterocolitica from raw milk.

Four enrichment procedures were used for examining 131 raw milk samples for the presence of Yersinia enterocolitica. Forty-two isolations were obtained from 19 pooled- (31.1 percent positive) and 10 individual-producer samples (14.3 percent positive). Enrichment by Butterfields phosphate buffer incubated at 4 degrees C for 14 days and then inoculation of modified Rappaport broth incubated at 23 degrees C for 5 days produced the greatest number of isolations. The majority of isolates were biotype 1, and many were atypical from clinical isolates in being rhamnose positive (47.6 percent), citrate positive (16.7 percent), and lactose positive (26.2 percent). Thirteen isolates were serotypable, belonging to seven different O serotypes, with O:5 occurring most frequently.     

Enterotoxin production and thermal resistance of Yersinia enterocolitica in milk.

Three of 36 raw milk isolates of Yersinia enterocolitica produced enterotoxin in milk at 25 degrees C, but not at 4 degrees C. No strain tested could survive pasteurization.     

Incidence of Yersinia enterocolitica in raw milk in eastern France.

A total of 75 raw milk samples collected from a central dairy or from retailers in Alsace, France, were analyzed for the presence of Yersinia enterocolitica. Three procedures were used: enrichment at 4 degrees C for 1 month; enrichment in modified Rappaport medium at room temperature for 72 h after a preenrichment at 4 degrees C for 1 month; and enrichment in a new medium containing sucrose, tris(hydroxymethyl)aminomethane, sodium azide, and ampicillin (PSTA) at 28 degrees C for 48 h after a preenrichment at 4 degrees C for 1 month. Isolation of Y. enterocolitica was made on Hektoen medium plus ampicillin. Sixty-one samples were positive (81.4%), but the PSTA medium produced the greatest number of isolates. Biochemical, serological, and phage typing of 40 isolates showed that chemotype 1 and serogroup O:5 were predominant. In seven cases, two different strains were obtained from the same samples. Most of the 66 isolates tested for their antimicrobial susceptibility were resistant to ampicillin and carbenicillin, and all were sensitive to tetracycline, chloramphenicol, streptomycin, sulfonamides, and mercuric ions.     

Immunomodulation by Yersinia enterocolitica: comparison of live and heat-killed bacteria

This study compared the immunomodulating properties of viable and killed Yersinia enterocolitica O9 in BALB/c mice. At 10 days after infection by the intragastric route, ex vivo assays showed a suppression of spleen cell proliferation in response to Salmonella lipopolysaccharide, concanavalin A and heat-killed yersiniae. Mice infected with Y. enterocolitica O9 for 10 days resisted the challenge with a lethal dose of Listeria monocytogenes. In contrast, intravenous administration of heat-killed yersiniae did not modify the ability of spleen cells to proliferate in response to lipopolysaccharide or concanavalin A, and proliferation in response to killed yersiniae was significantly increased. By 3 days after administration of a single dose of heat-killed yersiniae, the resistance of mice to L. monocytogenes challenge was significantly increased. Our findings show profound differences in immunomodulation by viable and heat-killed yersiniae, but suggest that killed yersiniae retain interesting immunomodulating properties.     

Antagonism by gram-negative bacteria to growth of Yersinia enterocolitica in mixed cultures

The inhibition of the growth of Yersinia enterocolitica by other gram-negative bacteria in mixed cultures at 32 degrees C was not the consequence of a depletion in essential nutrients, an unfavorable change in pH or oxygen tension or the production of toxic metabolic products. The inability of Y. enterocolitica to attain its potential maximum population in mixed cultures appeared instead to result from "metabolic crowding," which occurred when the faster-growing antagonistic organism reached stationary-phase density. Lowering the incubation temperature, a technique commonly used in "cold" enrichment for isolation of Y. enterocolitica, tended to equalize growth rates and thereby allowed Y. enterocolitica to achieve a higher population.     

The Myf fibrillae of Yersinia enterocolitica

The Myf antigen produced by Yersinia enterocolitica appeared as a proteic polymer composed of 21 kDa subunits. By transposon mutagenesis we isolated Myf-defective mutants. Those allowed us to clone and sequence a 4.4 kb chromosomal locus involved in Myf production. This region was found to contain three genes that we called myfA, myfB and myfC. Genes myfB and myfC encode an assembly machine related to those involved in the synthesis of many fimbriae; MyfB, the putative chaperone, possesses the consensus residues of the PapD family and MyfC encodes a putative outer-membrane protein. MyfA, the major subunit, was found to be 44% identical to the pH 6 antigen of Y.pestis. Myf is thus the K enterocolitica counterpart of this antigen, but it is by far not so well conserved as the other virulence determinants such as the Yops, suggesting that Myf and pH 6 antigen do not necessarily play the same role in Y. enterocolitica and Y. pestis. The study of the prevalence of myfA in various species of Yersinia reveaied that, like the yst enterotoxin gene, its presence is restricted to the pathogenic serotypes of Y. enterocolitica. By immuno-gold labelling, Myf appeared as a layer of extracellular material extending locally 2μm from the bacterial surface, indicative of a fibrillar structure.     

Incidence of pathogenic bacteria in raw milk in Ireland

Raw milk from 70 farms was sampled over 13 months for salmonellas, listerias, Escherichia coli, Staphylococcus aureus and mastitic streptococci; total bacterial counts (TBC), coliforms and somatic cells were also counted. TBC ≤30000/ml were obtained in 63% of samples. High count milks were found mainly during the winter months: 13% of samples had > 10⁴ mastitis pathogens/ml of milk. The mean somatic cell count varied from 4.0 × 10⁵ to 8.0 × 10⁵/ml throughout the year with highest counts during the late lactation period. Coliforms were present in all samples, but 65–71% of samples had < 100 coliforms/ml. Up to 60% of supplies had ≤10 E. coli /ml. One of the 589 samples tested (0.1%) was positive for salmonellas. Yersinia enterocolitica and Y. enterocolitica -like organisms were isolated from 39% of samples with up to 68% of samples positive at some sampling periods. A total of 222 strains of yersinias were isolated; Y. enterocolitica (59%) was the most common strain followed by Y. fredriksenii (35%), Y. kristensenii (1.0%), Y. intermedia (4.5%) and Y. aldovae (0.5%). Listerias were isolated from 8.3% of samples tested; 4.9% were Listeria monocytogenes and 3.4% were L. innocua. There was a significant rise in the isolation rate between December and April from a base line of 0–5% during the spring and summer to 35–37% during the winter months while the cows were indoors. Of 66 silage samples tested from the farms involved in the survey 9% of samples were positive for listerias; 3% of these were L. monocytogenes and 6% were L. innocua. Only half of the farms feeding contaminated silage produced milk containing listerias.     

The serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosis

The techniques of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were evaluated for the serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosis. Lipopolysaccharide (LPS) was prepared from strains comprising four serogroups of Y. enterocolitica and five serogroups of Y. pseudotuberculosis, tested against 200 sera submitted to the Laboratory of Enteric Pathogens for routine serodiagnosis, and shown to contain antibodies to Yersinia LPS by agglutination. Forty four sera were found to contain antibodies that bound to one of the LPS preparations used in the immunoassay. Thirty five of the sera contained antibodies to the LPS of Y. enterocolitica O3, whilst three contained antibodies to the LPS of Y. enterocolitica O5, 27 and Y. enterocolitica O9 LPS respectively. Two sera had antibodies to the LPS of Y. pseudotuberculosis II and a single serum contained antibodies to Y. pseudotuberculosis IV. The SDS-PAGE-immunoblotting procedure described proved to be a reliable procedure for the serodiagnosis of infections with Y. enterocolitica and Y. pseudotuberculosis.