Cold Enrichment Research Articles

Methodology for Isolation of Listeria from Foods—A Canadian Perspective

A previously described monoclonal antibody-enzyme immunoassay system for dairy products was examined to determine its potential for detecting Listeria in naturally contaminated ground meats. In addition, a microtiter plate system was developed which has potential in the rapid detection of Listeria species in foods. Experience in Canada with the U.S. Food and Drug Administration procedure for dairy products, the cold enrichment procedure, and the U.S. Department of Agriculture procedure for meats is discussed. Also, the status on attempts to devise improved selective media for the isolation of Listeria species from foods is described.

The presence of Listeria spp. in raw milk in Ontario.

Raw milk samples from bulk tanks in Ontario were analyzed for the presence of Listeria species. The overall incidence of Listeria species in raw milk was 12.4%. Listeria innocua was most frequently isolated and was found in 9.7% (43/445) of the raw milk samples, while L. monocytogenes and L. welshimeri were each found in 1.3% (6/445) of the samples. No other species of Listeria was found. Of five regions in Ontario that were examined, the eastern region had a significantly higher incidence rate of Listeria species than the western, northern, or northwestern regions. There was also a significantly lower incidence rate of Listeria species in winter than in the other three seasons. A comparison of several cold enrichment and shortened enrichment procedures demonstrated that no single procedure was totally satisfactory in isolating Listeria species.

Fate of Listeria monocytogenes in tissues of experimentally infected cattle and in hard salami.

Muscle, organ, and lymphoid tissues of four Holstein cows experimentally inoculated (intravenously) with Listeria monocytogenes were examined 2, 6, or 54 days postinoculation for the presence of the organism by direct plating and cold enrichment procedures. L. monocytogenes was isolated from 66% of the tissues sampled; 38% of the isolations were attributed to the use of cold enrichment. Isolation of the organism from muscle tissue was possible only with animals inoculated 2 days before slaughter. The fate of L. monocytogenes during the manufacture and storage of fermented hard salami made from this meat also was determined. Three sausage treatments were evaluated: (i) uninoculated control sausage, (ii) "naturally" contaminated sausage (NC) made from meat of an experimentally inoculated cow, and (iii) sausage made from beef inoculated with a laboratory culture of L. monocytogenes (I). Initial Listeria levels in NC and I sausage were 10(3) CFU/g in trial 1 and 10(4) CFU/g in trial 2. Numbers of L. monocytogenes decreased by approximately 1 log10 CFU/g during fermentation and decreased further during drying and refrigerated storage. Small numbers (less than or equal to 20 CFU/g) of L. monocytogenes were present in I and NC sausage at the end of 12 weeks of refrigerated storage; recovery of these organisms generally depended on the use of an enrichment procedure. The results indicate that L. monocytogenes does not multiply during the fermentation and drying processes typical of hard salami manufacture but that survival may occur if the organism is initially present at greater than or equal to 10(3) CFU/g.

Loss of Viability by Listeria monocytogenes in Commercial Calf Rennet Extract

Loss of viability by Listeria monocytogenes (strain California) in three lots of commercial calf rennet extract was determined during storage for 56 d at 7°C. Four levels (3 × 103 to 107/ml) of L. monocytogenes were added to the rennet extract, and McBride Listeria Agar was used to determine numbers of survivors. Selected colonies thought to be L. monocytogenes were confirmed biochemically. Samples were also tested during and after completion of cold enrichment (up to 8 weeks at 4°C). Samples of rennet extract inoculated with 103 to 104L. monocytogenes/ml were always free of viable cells of the bacterium after 28 d and sometimes after 14 d, as determined by direct plating and cold enrichment. When the inoculum was 105 to 107/ml, samples of rennet extract usually were free of viable L. monocytogenes (direct plating and cold enrichment) after 42 d and sometimes after 28 d at 7°C. In this work, results of cold enrichment correlated well with those of direct plating. All lots of rennet extract, before inoculation, were free of L. monocytogenes (direct plating and cold enrichment).

Survival of Listeria monocytogenes in raw milk treated in a pilot plant size pasteurizer.

The survival of Listeria monocytogenes in raw milk treated in a pilot plant size pasteurizer was investigated. Raw milk was inoculated with different initial concentrations of L. monocytogenes and heated at temperatures ranging from 69 degrees to 73 degrees C. Listerias were not isolated from any of the milk samples immediately after thermal treatment. They were isolated, however, from 46.6% of heated samples (none from samples heated at 73 degrees C) after variable periods at refrigeration temperature. The results suggest that a low number of listerias survive some thermal treatments, but a cold enrichment is necessary to repair the thermally injured cells and detect these organisms in milk. The importance of the isolation technique in the recovery of listerias from pasteurized milk samples is discussed.

Attempt of Yersinia enterocolitica isolation from human feces in Senegal.

One-hundred-eight stool samples, collected in a fishing village of Senegal from 72 apparently healthy subjects and from 36 patients with gastrointestinal disorders, were examined for the presence of Y. enterocolitica. After 1, 2, 3 weeks of cold enrichment with PBS 1/15M, pH 7.6, plating was performed on MacConkey Agar after use of the alkali method. No Yersinia strains were isolated.

Comparison of methods for the isolation of Yersinia enterocolitica from porcine tonsils and pork

Three enrichment procedures and three plating media were evaluated for their efficiency in isolating Yersinia enterocolitica from porcine tonsils and pork. Cold enrichment in phosphate-sorbitol-bile medium (PBSSB) with alkali treatment before plating resulted in higher isolation rates than enrichment in modified Rappaport broth (MRB) and bile-oxalate-sorbose broth (BOS). Post-enrichment alkali treatment gave a considerable increase in isolation rate with all the enrichment media tested. A sample inoculum of 10 g showed a better recovery than 0.2 g. Cefsulodin-irgasan-novobiocin (CIN) agar was slightly better than MacConkey agar and MacConkey agar + Tween 80 as isolation medium for Yersinia spp. from porcine tonsils. Most of the serotype 0:3 and 0:9 strains were isolated after enrichment in MRB and PBSSB. Enrichment in PBSSB resulted in the isolation of some other potentially virulent Yersinia strains. For the isolation of Yersinia spp. from pork and porcine tonsils an inoculum of 10 g, parallel use of MRB and PBSSB, post-enrichment alkali treatment and isolation on CIN agar is recommended.

Two-Stage Enrichment Procedures for Isolating Listeria monocytogenes from Raw Milk

Recovery of two strains of Listeria monocytogenes (Scott A and V7) inoculated into raw milk, and of strains indigenous to milk was investigated. Isolation of the organisms from the milk was attempted using pre-enrichment broths [nutrient broth no. 2 (NB2) and tryptose broth (TB)] at 1:5 and 1:10 dilutions of milk. The broths were incubated at 4°C for 0, 7, 14, 21, and 28 d. Recoveries were compared by direct plating onto acriflavine-nalidixic acid agar (AN), McBride Listeria agar (MLA) and tryptose agar (TA), and after selective enrichment in thiocyanate-nalidixic acid broth, with and without acriflavine. Favorable recoveries were obtained using a two-stage protocol consisting of cold enrichment of the sample diluted 1:10 in TB, followed by plating 0.1 ml of this pre-enrichment to MLA, and transfer of 1 ml to 9 ml Thio-Nal-Acri broth. Selectively-enriched cultures streaked to MLA and TA yielded optimal isolations after 7–14 of cold enrichment.

Clinical significance of virulence-related assay of Yersinia species.

During the 42-month period from June 1982 through December 1985, 215 fecal specimens from 171 patients were found to be positive for yersiniae by using a combination of CIN agar and cold enrichment. Isolates were tested for markers of virulence including carriage of a plasmid 42 megadaltons in size, calcium dependence, autoagglutination, Congo red uptake, pyrazinamidase activity, fermentation of salicin, and hydrolysis of esculin. The results were correlated to symptoms in patients. A total of 80 Yersinia enterocolitica and 52 Y. enterocolitica-like strains (42 Y. frederiksenii, 8 Y. intermedia, and 2 Y. kristensenii) were examined. Positive virulence-related tests were as follows (for Y. enterocolitica, Y. frederiksenii, Y. intermedia, and Y. kristensenii, respectively): pyrazinamidase negativity, 12.5, 0, 0, and 50%; Congo red positivity, 5, 7.1, 87.5 and 0%; calcium dependence, 3.8, 0, 0, and 0%; autoagglutination positivity, 8.8, 0, 0, and 0%; carriage of the 42-megadalton plasmid, 28.6, 73.2, 5.7, and 0; salicin and esculin negativity, 12.5, 0, 0, and 50%. The isolates recovered from symptomatic patients were characterized in relation to the presenting symptoms. Isolates from 12 of 32 (37.5%) patients with acute-onset diarrhea and 9 of 30 (30.0%) patients with chronic symptoms expressed at least one virulence feature. No individual test or group of tests was consistently associated with onset or either type of symptoms. Routine testing of plasmid carriage, uptake of Congo red, calcium dependence, autoagglutination, and pyrazinamidase activity did not appear to provide information that would link the presence of symptoms with the virulence potential of fecal isolates of yersiniae.

Identification and characterization of Yersinia intermedia isolated from human feces.

Since May 1983, our laboratory has, upon request, cultured stools for Yersinia spp. by using direct plating on cefsulodin-irgasan-novobiocin agar and a 3-week cold enrichment procedure. We isolated bacteria identified as Y. intermedia from six adult patients. All isolates were recovered only by the cold enrichment procedure and misidentified as Y. enterocolitica by the API 20E system (Analytab Products, Plainview, N.Y.). Final identification was made on the basis of results obtained with conventional tube biochemical tests. The isolates were tested for the following characteristics associated with virulence in Y. enterocolitica: lack of pyrazinamidase activity, autoagglutinability, presence of a 40- to 50-megadalton plasmid, production of heat-stable enterotoxin, and mouse lethality. All isolates tested had pyrazinamidase activity, and none were autoagglutinable. However, one isolate possessed a 40-megadalton plasmid. None produced enterotoxin or were lethal for mice. Review of the medical histories of the patients revealed that four of the six had diarrhea; however, none had disease typical of that caused by Y. enterocolitica. Our data confirmed the limited pathogenic potential of Y. intermedia and suggested that its isolation was without clinical significance in our patients. Conventional biochemical tests were required for reliable identification of Y. intermedia.

Comparison of Procedures for Isolating Listeria monocytogenes in Soft, Surface-Ripened Cheese

Ninety samples of soft, surface-ripened cheese from a lot previously identified to contain Listeria were assayed for Listeria monocytogenes by three procedures. These included: (a) cold enrichment, (b) the Food and Drug Administration enrichment procedure, and (c) the selective enrichment procedure of Doyle and Schoeni (Appl. Environ. Microbiol. 15:1127, 1986). L. monocytogenes was isolated from 41 of the 90 cheese samples. The organism was isolated from only 9 of the 41 L. monocytogenes-positive samples by more than one procedure. Most isolations (21) were made by the cold enrichment procedure, with 16 and 13 isolations made by the FDA and Doyle-Schoeni procedures, respectively. In most instances, the organism was isolated from a cheese sample by only one procedure.

Growth and thermal inactivation of Listeria monocytogenes in cabbage and cabbage juice.

Studies were done to determine the interacting effects of pH, NaCl, temperature, and time on growth, survival, and death of two strains of Listeria monocytogenes. Viable population of the organism steadily declined in heat-sterilized cabbage stored at 5 degrees C for 42 days. In contrast, the organism grew on raw cabbage during the first 25 days of a 64-day storage period at 5 degrees C. Growth was observed in heat-sterilized unclarified cabbage juice containing less than or equal to 5% NaCl and tryptic phosphate broth containing less than or equal to 10% NaCl. Rates of thermal inactivation increased as pH of clarified cabbage juice heating medium was decreased from 5.6 to 4.0. At 58 degrees C (pH 5.6), 4 X 10(6) cells/mL were reduced to undetectable levels within 10 min. Thermal inactivation rates in clarified cabbage juice (pH 5.6) were not significantly influenced by the presence of up to 2% NaCl; however, heat-stressed cells had increased sensitivity to NaCl in tryptic soy agar recovery medium. Cold enrichment of heat-stressed cells at 5 degrees C for 21 days enhanced resuscitation. Results indicate that L. monocytogenes can proliferate on refrigerated (5 degrees C) raw cabbage which, in turn, may represent a hazard to health of the consumer. Heat pasteurization treatments normally given to cabbage juice or sauerkraut would be expected to kill any L. monocytogenes cells which may be present.

Faecal carriage rate of Yersinia species.

A total of 1,203 unselected routine faecal samples from 1,006 patients were cultured for Yersinia species by a cold enrichment technique. Seventy-five specimens (6.1%) from 63 patients were culture-positive for Yersinia spp. Fifty-two were Yersinia enterocolitica, 22 Yersinia frederiksenii and 1 Yersinia intermedia. The predominant Y. enterocolitica isolates belonged to biotype 1 - serotype 0:6, 30 or serotype 0:5, 27. Y. frederiksenii strains were non-typable. Forty isolates were recovered from 33 patients with gastroenteritis. During the study period 83 Salmonella spp. from 33 patients, 17 Shigella sonnei from 13 patients and 13 Campylobacter jejuni from 12 patients were cultured. Yersinia spp. was isolated in association with salmonella on three occasions, twice with rotavirus and once each with Shigella sonnei, Campylobacter jejuni and Trichuris trichiura.

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.

Characterization of Yersinia enterocolitica and related species isolated from foods and porcine tonsils in the Netherlands

Yersinia enterocolitica and related species were isolated from a great variety of foods. High isolation rates were found in raw minced pork and beef, porcine tonsils and tongues, egg products, raw vegetables and surface water. Of 310 strains isolated 221 (71%) were identified as Y. enterocolitica and 89 as either Y. frederiksenii (12%), Y. intermedia (8%), Y. kristensenii (5%) or other Yersinia species (4%). The great majority (92%) of these strains were of biotype 1. Only 4% of the Yersinia isolates belonged to serotype 0 : 3 or 0 : 9, which are the main serotypes isolated from patients with yersiniosis in The Netherlands. Based on the results of an autoagglutination virulence assay and a test for calcium dependency at 37°C only the 0 : 3 and 0 : 9 strains isolated from porcine tonsils were potentially virulent. Most of the strains were isolated using cold enrichment in phosphate-buffered saline containing sorbitol and bile salts. However, the potentially virulent strains were all isolated using modified Rappaport broth.

The incidence of Yersinia enterocolitica and Yersinia enterocolitica-like bacteria in goats milk in Northern Ireland

Fifty samples of retail goats milk were analysed for the total number of viable bacteria present and the incidence of Yersinia spp. Both were found to be producerdependent. Overall, 26% of samples contained Yersinia spp. These were mainly Yersinia enterocolitica but Y. intermedia were also isolated. Pre‐enrichment of samples in trypticase‐soy broth (TSB) followed by selective enrichment in a bileoxalate‐sorbose medium was considerably superior to prolonged cold enrichment in TSB for the isolation of Yersinia spp.

Investigation of Yersinia enterocolitica, Yersinia enterocolitica-like bacteria, and thermotolerant campylobacters in Norwegian pork products

Altogether, Yersinia spp. were isolated from 41 (27.0%) of 152 samples of retail pork products. Thermotolerant campylobacters were not detected in this study. Yersinia spp. were recovered from 39 (30.7%) of the 127 raw pork samples and from two (8.0%) of the 25 cooked pork samples investigated. Sixty distinct strains were isolated. Y. enterocolitica comprised 58.3% of the total number, followed by Y. intermedia (20.0%), Y. kristensenii (18.3%), and Y. frederiksenii (3.3%). The serotyping scheme employed enabled us to type 91.7% of the strains. The most frequently isolated serotype was O:5 which comprised 20.0% of the total number, followed by O:7, 8 (11.7%), and O:11 (8.3%). Only one strain of O:3/biotype 4, the predominant human pathogen in Europe, was isolated. This was the only isolate which provoked positive reaction in the autoagglutination virulence assay. A second O:3 strain was classified as Y. kristensenii. Optimal recovery of Yersinia spp. was achieved using three-weeks cold enrichment in addition to a two-step procedure based on pre-enrichment in a low-selectivity medium (8 days, 4°C) followed by selective enrichment in modified Rappaport broth (4 days, 20–25°C). The results indicate that Yersinia spp. are more likely to be isolated from foods with a high level of coliforms than from foods with low coliform counts.

Yersinia enterocolitica and Yersinia enterocolitica-like bacteria in Norwegian slaughter pigs

Yersinia spp. were isolated from the tonsils of 200 (43.4%) of 461 freshly slaughtered pigs. Y. enterocolitica comprised 92.7% of the total number of isolates, while Y. kristensenii, Y. intermedia, and Y. pseudotuberculosis comprised 6.3%, 0.5% and 0.5% respectively. Fifteen different serotypes were recognized. Serotype O:3 comprised 70.9% of the total number, followed by O:5 (6.8%), and O:6,30 (6.3%). Y. enterocolitica O:3/biotype 4, the predominating human pathogen in Europe, was isolated from 31.7% of the pigs examined. 67 (45.9%) of these isolates were potentially virulent as judged by the autoagglutination virulence assay. Three-week cold enrichment in a low-selectivity medium was the most efficient single method for recovery of Y. enterocolitica serotype O:3/biotype 4, Y. enterocolitica biotype 1, and Y. kristensenii. However, optimal recovery was achieved using a multi-method isolation technique, including a two-step procedure based on pre-enrichment followed by selective enrichment in a modified Rappaport broth.

Comparison of Sampling and Isolation Procedures for Recovery of Yersinia Enterocolitica Serotype 0:3 from the Oral Cavity of Slaughter Pigs

Yersinia enterocolitica serotype 0:3/biotype 4 was isolated from the oral cavity of altogether 32 (68.1 %) of 47 freshly eviscerated slaughter pigs. Most efficient recovery was achieved by cultivation of tissue samples from both tongue and tonsils of the same individual. The isolation rate so obtained was significantly higher than that obtained by separate examination of either tonsil swabs or tongue swabs. However, the isolation frequency achieved by combined swabbing of the 2 sites was not significantly different. In general, tonsils were more productive for the recovery of 0:3 strains than were tongues, and tissue samples yielded higher isolation rates than did swabs. Three-week cold enrichment in a low selective medium proved essential for optimal recovery. However, the highest number of isolates was obtained using a combination of methods, including direct plating and selective enrichment in a modified Rappaport broth in addition to cold enrichment.

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+, α-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.