Comparison of seven plating media for enumeration of Listeria spp.

The suitability of seven media for the enumeration of Listeria spp. was evaluated at 30 degrees C for 48 h. The media tested were (i) the original McBride Listeria agar formulation (with glycine); (ii) modified McBride agar containing glycine anhydride; (iii) LiCl-phenylethanol-moxalactam (LPM) agar; (iv) acriflavine-ceftazidime agar; (v) Rodriguez isolation agar (RISA); (vi) modified Vogel-Johnson (MVJ) agar; (vii) cyclohexanedione-nalidixic acid-phenylethanol agar; and tryptose agar as control. A total of 66 organisms were used including 11 Listeria monocytogenes strains and 5 other Listeria spp. For L. monocytogenes strains only, all media performed highly similarly. Of the other Listeria spp., only two grew on MVJ agar and three each grew on LPM and RISA. Only LPM agar inhibited the 50 non-listeriae, including five yeasts, while MVJ agar inhibited all but one yeast. The McBride Listeria agar formulation that contained glycine anhydride was less selective than the original. When pure cultures of 10 bacteria (including one L. monocytogenes strain) were combined and plated on four media, L. monocytogenes colonies were easiest to enumerate on MVJ agar, followed by LPM and RISA. These media ranked in the same order when plated with homogenates of various foods to which was added L. monocytogenes Scott A, but LPM agar was the best overall since Scott A was inhibited by MVJ. Upon microscopic examination of listerial colonies from the plating media, atypical cell morphology was noted with cells being about twofold in size on LPM, MVJ, and acriflavine-ceftazidime agars. Overall, LPM agar was the most suitable of the media tested even though it was inhibitory to Listeria grayi and Listeria murrayi.     

Evaluation of chromogenic media for the detection of Listeria species in food

AIMS: The aim of this study was to evaluate the performance of chromogenic agars, Agar Listeria according to Ottaviani and Agosti (ALOA) and Rapid L. mono agar, compared with Oxford agar for the enumeration and detection of Listeria species in food. METHODS AND RESULTS: A total of 170 food samples were examined using the three plating media. Listeria species were isolated from 63 samples. In contrast to Oxford agar, detection of Listeria colonies on chromogenic media was as good after 24 h of incubation of plates as after 48 h. While there was no significant difference in recovery of Listeria monocytogenes on the three media, recovery of other Listeria species was significantly poorer on Rapid L. mono agar compared with Oxford and ALOA agars. Recovery of species other than L. monocytogenes was significantly improved by including a secondary enrichment stage in the detection method. CONCLUSIONS: Using chromogenic agars, presumptive identification of L. monocytogenes is possible after 24 h, compared with 3-4 days using Oxford agar. However, the poor detection of species other than L. monocytogenes on Rapid L. mono agar is a disadvantage of this medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new information regarding the isolation of Listeria species other than L. monocytogenes from food using chromogenic plating media. This is important, as non-pathogenic Listeria species act as markers for the likelihood of presence of L. monocytogenes and allow preventive action to be taken to avoid its presence.     

A 20–24 H MICROCOLONY-IMMUNOBLOT TECHNIQUE to DIRECTLY DETECT and ENUMERATE LISTERIA MONOCYTOGENES INOCULATED INTO FOODS

A microcolony-immunoblot technique (MCIBI) was developed to directly enumerate, in less than 24 h, very low numbers of Listeria monocytogenes (8–12 colony forming units: CFU/g or mL) inoculated into foods. Four meat and poultry and two dairy products were artificially inoculated with L. monocytogenes V7 diluted and plated on Oxford agar medium. Each plate was overlaid with an Immobilon-P membrane and incubated for 18–20 h at 37C. Blot-transferred colonies on these membranes were probed with C11E9 monoclonal antibody (MAb) and developed using peroxidase conjugated goat antimo use Ig G and a water insoluble substrate (3,3-diaminobenzidin tetrahydmchloride; (DAB-HCI), Nickel chloride and H₂O₂). the MCIBT gave L. monocytogenes counts that were not significantly lower than direct colony counts on selective agars. This technique allowed the recovery of 94–100% of L. monocytogenes cells inoculated into foods containing natural background flora counts of 3 × 10⁴ to 8 × 10⁶ CFU/g or mL. Using a 2 h resuscitation period on nonselective agar before overlay with Oxford media, the MCIBT allowed detection of sublethally heat injured cells of strain V7.     

Selective plating medium for quantitative recovery of food-borne Listeria monocytogenes.

A new plating medium (lithium chloride-ceftazidime agar [LCA]) was designed to quantitatively recover food-borne Listeria monocytogenes in the form of large colonies while inhibiting most other food-borne microorganisms. This medium included brain heart infusion agar as the nutritive agar base and a combination of selective agents (LiCl, glycine anhydride, and ceftazidime). Comparison of LCA and lithium chloride-phenylethanol-moxalactam agar (LPM) indicated that both were equally effective for the enumeration of the cold-tolerant pathogen in artificially and naturally contaminated foods. However, LCA was more effective than LPM in the recovery of sublethally heat-injured cells. Moreover, Listeria colonies on LCA exhibited a more distinct bluish hue than those on LPM when viewed by the Henry oblique transillumination technique.     

Selective plating medium for quantitative recovery of food-borne Listeria monocytogenes

A new plating medium (lithium chloride-ceftazidime agar [LCA]) was designed to quantitatively recover food-borne Listeria monocytogenes in the form of large colonies while inhibiting most other food-borne microorganisms. This medium included brain heart infusion agar as the nutritive agar base and a combination of selective agents (LiCl, glycine anhydride, and ceftazidime). Comparison of LCA and lithium chloride-phenylethanol-moxalactam agar (LPM) indicated that both were equally effective for the enumeration of the cold-tolerant pathogen in artificially and naturally contaminated foods. However, LCA was more effective than LPM in the recovery of sublethally heat-injured cells. Moreover, Listeria colonies on LCA exhibited a more distinct bluish hue than those on LPM when viewed by the Henry oblique transillumination technique.     

Evaluation of selective direct plating media for their suitability to recover uninjured, heat-injured, and freeze-injured Listeria monocytogenes from foods.

Six direct plating media were evaluated for their suitability to recover uninjured, heat-injured, and freeze-injured cells of four strains of Listeria monocytogenes from four foods. Cells were inoculated into foods to achieve ca. 10(2) to 10(3), 10(4) to 10(5), or 10(5) to 10(6) viable cells per ml or g (low, medium, and high populations, respectively). No appreciable differences in recovery of the four test strains within a treatment were observed. Generally, recovery on all test media was similar and not markedly affected by freeze treatment. Modified Despierres agar and modified McBride Listeria agar yielded poorer recovery of heat-injured cells than did McBride Listeria agar and gum base-nalidixic acid-tryptone soya agar. Overall, gum base-nalidixic acid-tryptone soya agar was best for recovering L. monocytogenes from pasteurized milk and chocolate ice cream mix. Enumeration was complicated by the growth of background microflora present in Brie cheese and cabbage, especially at the low inoculum. Dominguez Rodriguez isolation agar was superior for recovering L. monocytogenes from Brie cheese, whereas modified Despierres agar was best for recovering the organism from cabbage. Direct plating procedures can successfully be utilized for recovering healthy and injured L. monocytogenes from foods containing low populations of background microflora.     

Evaluation of selective direct plating media for their suitability to recover uninjured, heat-injured, and freeze-injured Listeria monocytogenes from foods

Six direct plating media were evaluated for their suitability to recover uninjured, heat-injured, and freeze-injured cells of four strains of Listeria monocytogenes from four foods. Cells were inoculated into foods to achieve ca. 10(2) to 10(3), 10(4) to 10(5), or 10(5) to 10(6) viable cells per ml or g (low, medium, and high populations, respectively). No appreciable differences in recovery of the four test strains within a treatment were observed. Generally, recovery on all test media was similar and not markedly affected by freeze treatment. Modified Despierres agar and modified McBride Listeria agar yielded poorer recovery of heat-injured cells than did McBride Listeria agar and gum base-nalidixic acid-tryptone soya agar. Overall, gum base-nalidixic acid-tryptone soya agar was best for recovering L. monocytogenes from pasteurized milk and chocolate ice cream mix. Enumeration was complicated by the growth of background microflora present in Brie cheese and cabbage, especially at the low inoculum. Dominguez Rodriguez isolation agar was superior for recovering L. monocytogenes from Brie cheese, whereas modified Despierres agar was best for recovering the organism from cabbage. Direct plating procedures can successfully be utilized for recovering healthy and injured L. monocytogenes from foods containing low populations of background microflora.     

A combined PCR and selective enrichment method for rapid detection of Listeria monocytogenes

Development of a routine detection assay for Listeria monocytogenes in foods that uses the polymerase chain reaction (PCR) and enrichment cultures was investigated. Oligonucleotide primers were chosen to amplify a 3'region of L. monocytogenes hly A gene spanning a conserved Hin dIII site. PCR detection sensitivity for L. monocytogenes in dilutions of pure enrichment cultures was between 50 and 500 colony forming units. A short enrichment period before PCR amplification allowed detection of the organisms in a range of complex foods contaminated with 10⁴ cfu/g. Detection sensitivity for the assay in the presence of chicken skin and soft cheese was determined at 10–100 cfu/g. Utilization of enrichment cultures and PCR allowed identification of the organism within 24 h or 2 days.     

Occurrence of Listeria species in raw milk and dairy products produced in Northern Ireland.

The overall incidence of Listeria spp. in raw milk samples surveyed was found to be 25.0% (Listeria monocytogenes 15.3%), with the incidence in samples from processing centres 54.0% (L. monocytogenes 33.3%); this was higher than that in samples from dairy farms (Listeria spp. 8.8%; L. monocytogenes 5.3%). The FDA enrichment procedure was much more productive than cold enrichment and Oxford agar was superior to modified McBride agar for isolation of Listeria. Listeria monocytogenes was never isolated by direct plating of raw milk samples on Oxford agar at a detection level of 1.0 cfu/ml. Listeria spp. were isolated from 1 of 95 pasteurized milk samples (L. monocytogenes) and 1 of 33 soft cheese samples (L. seeligeri). Restriction fragment length polymorphism was more useful than sero- or phage-typing for typing of L. monocytogenes strains, and results suggest that specific L. monocytogenes strains may persist in both farm and processing environments.     

Fate of Listeria monocytogenes during manufacture and ripening of semi-hard cheese

Semi-hard cheeses were experimentally elaborated with pasteurized milk from sheep, goat and cow (15: 35: 50) and inoculated to contain 1.9 times 10⁵ Listeria monocytogenes /ml in cheeses 1 and 2 and 4 times 10³ L. monocytogenes /ml in cheeses 3 and 4. Counts of L. monocytogenes were determined by direct surface plating of samples on listeria selective agar medium. The results show the substantial survival of L. monocytogenes present in milk during manufacture and ripening of this type of cheese.     

COMPARISON OF THREE POLYMERASE CHAIN REACTION-BASED METHODS FOR THE DETECTION OF LISTERIA MONOCYTOGENES IN FOOD

Three PCR-based methods for the detection of Listeria monocytogenes in food (BAX for Screening, Probelia and a method according to Kaclíková et al. (2003) were compared on the basis of the determination of detection limits for 15 artificially contaminated food products. Detection limits of all methods for all samples were 10⁰ cfu per 10 g, with the exception of three cheese samples which did not produce valid results because of the inhibition of Probelia PCR. Detection limits for nonviable L. monocytogenes cells were sufficiently high ( 10⁹ cfu per 10 g) for BAX and the method according to Kaclíková et al. (2003), but considerably low ( 10⁶ cfu per 10 g) for Probelia. The results demonstrate that BAX for Screening as well as the Kaclíková et al. (2001) method fulfill the sensitivity requirements for a rapid alternative method for the detection of L. monocytogenes in food, which would be equivalent to the standard method EN ISO 11290–1.     

A technique for the direct identification of haemolytic-pathogenic listeria on selective plating media

A technique based on the addition of a red cells top layer to a selective plating medium after listeria growth is proposed in order to detect directly the haemolytic activity of pathogenic listeria colonies. It was applied to different selective plating media (modified McBride agar, lithium chloride-phenylethanol-moxalactam, listeria selective medium–Oxford formulation, polymyxin-acriflavine-lithium chloride-ceftazidime-aesculin-mannitol and LSAMM). The haemolytic activity of listeria colonies was more easily detected with the top layer than when red cells were incorporated in the selective plating medium. The LSAMM was the best medium for the recovery and identification of Listeria monocytogenes colonies by this technique (three Listeria monocytogenes colonies were distinguished among 2520 Listeria innocua colonies in raw milk).     

Occurrence of Listeria species in raw milk and dairy products produced in Northern Ireland

J. HARVEY AND A. GILMOUR. 1992. The overall incidence of Listeria spp. in raw milk samples surveyed was found to be 25.0% ( Listeria monocytogenes 15.3%), with the incidence in samples from processing centres 54.0% ( L. monocytogenes 33.3%); this was higher than that in samples from dairy farms ( Listeria spp. 8.8% L. monocytogenes 5.3%). The FDA enrichment procedure was much more productive than cold enrichment and Oxford agar was superior to modified McBride agar for isolation of Listeria. Listeria monocytogenes was never isolated by direct plating of raw milk samples on Oxford agar at a detection level of 1.0 cfu/ml. Listeria spp. were isolated from 1 of 95 pasteurized milk samples ( L. monocytogenes ) and 1 of 33 soft cheese samples ( L. seeligeri ). Restriction fragment length polymorphism was more useful than sero- or phage-typing for typing of L. monocytogenes strains, and results suggest that specific L. monocytogenes strains may persist in both farm and processing environments.     

Evaluation of three newly developed direct plating media to enumerate Listeria monocytogenes in foods

LiCl-phenylethanol-moxalactam Agar (LPMA), ARS Modified McBride Agar, and Modified Vogel Johnson Agar were compared with previously tested plating media in the enumeration of Listeria monocytogenes from pasteurized whole milk, chocolate ice cream mix, Brie cheese, and raw cabbage. LPMA was most suitable for analyzing Brie cheese and cabbage. Gum base-nalidixic acid-tryptone-soya medium (previously tested) was most suitable for analyzing milk and chocolate ice cream mix.     

Evaluation of three newly developed direct plating media to enumerate Listeria monocytogenes in foods.

LiCl-phenylethanol-moxalactam Agar (LPMA), ARS Modified McBride Agar, and Modified Vogel Johnson Agar were compared with previously tested plating media in the enumeration of Listeria monocytogenes from pasteurized whole milk, chocolate ice cream mix, Brie cheese, and raw cabbage. LPMA was most suitable for analyzing Brie cheese and cabbage. Gum base-nalidixic acid-tryptone-soya medium (previously tested) was most suitable for analyzing milk and chocolate ice cream mix.     

Improved detection of Listeria monocytogenes in soft mould-ripened cheese

In comparison with standard methods, enrichment in half-Fraser broth for 24 h at 30 degrees C, followed by plating out onto Listeria monocytogenes blood agar (LMBA) and PALCAM medium combined with an additional streak proved to be the most rapid and specific method for the detection of indigenous L. monocytogenes populations from soft mould-ripened cheese. This procedure, with a high sensitivity (93%) and a low detection limit (1-10 cfu 25 g-1), provided negative and presumptive positive results within 2-3 d. Differences between LMBA, PALCAM and Oxford medium turned out to be highly significant (at 99% significance level); plating on LMBA after standard enrichment protocols giving the best overall results. An improvement in detection was also obtained by modifying the confirmation procedure. A loopful of culture (an additional streak) from PALCAM or Oxford medium was streaked on non-selective medium in addition to streaking only separate colonies as specified in the standards.     

Susceptibility and resistance of anaerobic rumen fungi to antimicrobial feed additives

Listeria monocytogenes survived in meat, cheese and egg ravioli stored at 5°C for 14 d. Ravioli were considered edible for the first 9 d of storage. Initial L. monocytogenes populations of 3 × 10⁵ cfu/g of ravioli were reduced to non-detectable levels after heat treatment simulating that which would be used by the consumer.     

USE OF UNIVERSAL PREENRICHMENT MEDIUM SUPPLEMENTED WITH OXYRASETM FOR THE SIMULTANEOUS RECOVERY OF ESCHERICHIA COLI O157:H7 AND YERSINIA ENTEROCOLITICA

Universal Preenrichment (UP) medium was used successfully for the simultaneous recovery of two strains each of Escherichia coli O157:H7 and Yersinia enterocolitica in the presence of Listeria monocytogenes and Salmonella typhimurium. E. coli O157:H7 and Y. enterocolitica populations reached ca. 10⁸ CFU/ml in UP medium in 18 h from an initial level ofca. 10² CFU/ml. Addition of Oxyrase_TM enhanced the growth of both E. coli O157:H7 strains and one strain of Y. enterocolitica. These three strains were able to recover from heat injury by 6 h when 24-h cultures were tested, but not when 18-h cultures were used. Injured and noninjured E. coli O157:H7 could be recovered from artificially inoculated food samples (shredded cheddar cheese, turkey ham, hot dogs, mayonnaise, and ground beef) in UP medium supplemented with Oxyrase^TM (UPO) by 18 h using immunoblotting. Y. enterocolitica could be recovered from turkey ham, hog dogs, and mayonnaise by direct plating on CIN agar from UPO medium. However, recovery of Y. enterocolitica from shredded cheddar cheese and ground beef required subsequent selective enrichment in sorbitol bile broth and isolation on Cefsulodin Irgasan Novobiocin agar (CIN). UPO medium can be used for simultaneous detection of E. coli O157:H7 and Y. enterocolitica from foods. However, subsequent selective enrichment and isolation on selective plating media are required for isolation of Y. enterocolitca from raw foods containing high population levels of background microflora.     

Effect of heat treatment on Listeria monocytogenes and Gram-negative bacteria in sheep, cow and goat milks

Sheep milk, compared with cow and goat milk, had a protective effect on Gram-negative bacteria and Listeria spp. heated at 65°C in a test-tube method. This effect was not solely due to fat content as cow milk artificially reconstituted to 10% homologous fat was not as protective. Listeria monocytogenes in whole sheep, cow and goat milks at an inoculum level of 1 times 10⁶ cfu ml^-1 was heated at 68°C for 15 s in the plate pasteurizer and survival was only detected in whole sheep milk after heating. Whole sheep, cow and goat milks containing high levels of L. monocytogenes (1 times 10⁶ cfu ml^-1) could not survive the current HTST plate pasteurization protocol.     

Use of the polymerase chain reaction for direct detection of Listeria monocytogenes in soft cheese

The polymerase chain reaction (PCR) amplification technique was investigated as a tool for direct detection of Listeria monocytogenes in soft cheeses. Different sets of oligonucleotide primers were used, and parts of the L. monocytogenes Dth18-gene could be amplified specifically when either a plasmid vector carrying the cloned gene or chromosomal DNA was used as a template. The detection limit for L. monocytogenes in dilutions of pure cultures was between 1 and 10 colony-forming units. In extracts from soft cheeses containing L. monocytogenes DNA, the amplification was strongly inhibited. This inhibition could be reduced by an additional purification step. Despite this the detection limit showed a large variation, depending on the brand of cheese used. In some cheeses 10³ cfu/0.5 g could be visualized whereas in others the presence of 10⁸ cfu/0.5 g did not yield a detectable quantity of amplified product.