Performance of a new chromogenic plating medium for the isolation of Listeria monocytogenes from marine environments

AIMS: This study investigated the performance of a new chromogenic plating medium for the detection of Listeria monocytogenes from naturally contaminated samples obtained from marine environments in Morocco in comparison with the conventional plating media PALCAM and Oxford. METHODS: A total of 479 marine samples (sea water, sediment and mussels) were collected from 16 littoral sites in the region of Agadir (western centre of Morocco). They were examined for the presence of L. monocytogenes using a slight modification of the standardized French method (AFNOR V 08-055) for the detection of L. monocytogenes from food and three different isolation media: PALCAM, Oxford and a new chromogenic plating medium. RESULTS AND SIGNIFICANCE OF THE STUDY: The Oxford and the new chromogenic plating media were found relatively more efficient than the PALCAM medium for the isolation of L. monocytogenes (chi-square test, P < 0.05) from marine samples. However, the new chromogenic plating medium was significantly more selective for L. monocytogenes (P < 0.005) than the two other isolation media as 87.5% of the suspect colonies on this medium were indeed confirmed through identification of the isolates vs 12.7% for Oxford and only 3.8% for the PALCAM medium.     

Improvement of the detection of Listeria monocytogenes by the application of ALOA, a diagnostic, chromogenic isolation medium

A new selective agar medium, ALOA, for the selective and differential isolation of Listeria monocytogenes has been evaluated. All stressed cultures of L. monocytogenes serovars tested grew on the medium as bluish colonies surrounded by a distinctive opaque halo and gave a productivity ratio of at least 0.95. Non-pathogenic Listeria sp. produced bluish colonies without a halo as was also the case for some enterococci and bacilli. Special attention must be paid to some Bacillus cereus strains and L. ivanovii since their colony appearance can be misleading. Only some unidentified listeria-like bacteria gave false-positive results. ALOA detected 4. 3% more positives from naturally contaminated dairy and meat samples compared with the ISO procedure when used with GenprobeTM or VidasTM for confirmation of presumptive colonies; 13.9% false negatives were found compared with 38.9% using PALCAM/Oxford. ALOA was also clearly superior to Oxford and PALCAM when samples containing both L. monocytogenes and L. innocua were examined. The introduction of ALOA in standard isolation procedures as an additional medium would enhance the detection ratio and reduce the time and cost of analysis for L. monocytogenes.     

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.     

Comparative study of two plating media (PALCAM and Oxford) for detection of Listeria species in a range of meat products following a variety of enrichment procedures

Fifty-two different varieties of sausage, salami and pâté were examined using Oxford (Oxoid) and PALCAM (Merck) listeria-selective agars. Seven (13%) samples were positive for Listeria monocytogenes and 14 (27%) samples were positive for other Listeria species, while 31 (59%) samples were negative for Listeria species. The effectiveness of PALCAM and Oxford medium for the isolation of L. monocytogenes from the meat samples was compared. PALCAM medium was consistently more effective in suppressing other micro-organisms thus enhancing the possibility of detecting Listeria species present in low numbers. The isolation of Listeria species for identification was also easier using PALCAM medium.     

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 BBL CHROMagar Listeria agar for the isolation and identification of Listeria monocytogenes from food and environmental samples

The performance of BBL CHROMagar Listeria chromogenic agar for the detection of Listeria monocytogenes was evaluated for its ability to isolate and identify L. monocytogenes from food and environmental samples. The medium was compared to non-chromogenic selective agars commonly used for Listeria isolation: Oxford, Modified Oxford, and PALCAM. BBL CHROMagar Listeria had a sensitivity of 99% and 100% for the detection of L. monocytogenes from 200 natural and artificially inoculated food samples, respectively, with a colony confirmation rate of 100%. The sensitivity of non-chromogenic selective media for the detection of L. monocytogenes from these same samples was 97-99% with colony confirmation rates of 65-67.5%. From 93 environmental samples, BBL CHROMagar Listeria agar results correlated 100% with a Listeria spp. visual immunoassay (TECRA) performed on these same samples and the USDA-FSIS standard culture method for the isolation of L. monocytogenes. From environmental samples, the L. monocytogenes confirmation rate was 100% for BBL CHROMagar Listeria as compared to 50% for conventional agars tested. On BBL CHROMagar Listeria, L. monocytogenes forms a translucent white precipitation zone (halo) surrounding blue-pigmented colonies of 2-3 mm in diameter, with an entire border. BBL CHROMagar Listeria offers a high degree of specificity for the confirmation of suspect L. monocytogenes colonies, whereas non-chromogenic selective agars evaluated were not differential for L. monocytogenes from other Listeria species.     

Development of a surface adhesion immunofluorescent technique for the rapid isolation of Listeria monocytogenes and Listeria innocua from meat

The use of a novel surface adhesion technique to isolate Listeria monocytogenes and Listeria innocua from an enrichment meat system was developed. Minced beef samples inoculated with L. monocytogenes (10 cfu g(-1)) were incubated at 30 degrees C for 14-18 h in a suitable enrichment broth. Listeria monocytogenes cells were isolated from the enriched meat sample by surface adhesion onto a polycarbonate membrane which was attached to a glass microscope slide. The Listeria cells on the membrane were subsequently visualized using an immunofluorescent microscopy procedure. The antibody used in this technique reacts with L. monocytogenes and L. innocua. The technique was demonstrated to have a detection level of log10 3.11 cfu ml(-1). There was excellent correlation (r2 = 0.98) between the counts obtained by this surface adhesion immunofluorescent (SAIF) technique and counts obtained using traditional methods, i.e. plate counts on PALCAM. When the regression equation relating the rapid and standard methods was validated using the data from 50 retail beef mince samples, an rsd value of +/- 0.25 was obtained. No false-negative or false-positive results were recorded for L. monocytogenes or L. innocua species using the SAIF technique.     

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.     

Performance testing of six chromogenic ALOA-type media for the detection of Listeria monocytogenes

Aims:  The aim of the study was to test the performance of commercially available chromogenic plating media for detection and enumeration of the food-borne pathogen Listeria monocytogenes . A wide range of chromogenic media similar to Agar Listeria according to Ottaviani and Agosti (ALOA) were compared using PALCAM agar, according to van Netten et al.
Methods and Results:  Six chromogenic media similar to ALOA were challenged for inclusivity and exclusivity. Additionally, the ability of chromogenic agars to facilitate growth of stressed L. monocytogenes strains and mixed cultures with competitive non-Listeria strains was estimated. Finally, we tested the detection and enumeration of L. monocytogenes in artificially inoculated and naturally contaminated food samples. The results of this study indicated that chromogenic media are a good supplementation to PALCAM agar. A single application is not advisable, as the specificity of chromogenic agars is frequently insufficient (50·0–88·9%), particularly in food samples with a complex microflora.
Conclusions:  The competitive flora of food samples is able to overgrow low numbers of L. monocytogenes , especially in half-Fraser enrichment. This might lead to the underestimation of L.   monocytogenes positive samples.
Significance and Impact of the Study:  Although many evaluation studies of chromogenic agar have been published recently, harmonized validation strategies are lacking. This survey provides a new concept for stepwise testing of plating media.     

Evaluation of enhanced haemolysis agar for detection of Listeria spp. and L. monocytogenes from production lines of fresh to cold-smoked fish.

Enhanced haemolysis agar (EHA) was compared to the two conventional Listeria isolation agars Oxford and PALCAM for its ability to detect Listeria spp. from production lines of fresh to cold-smoked fish. The ability of EHA for distinguishing L. monocytogenes colonies from other Listeria spp. was also evaluated.A total of 243 fish and environmental samples were analysed. Overall, 42 samples were found to contain Listeria spp. Only 34 samples were positive simultaneously by the three plating media. Two samples considered to be negative by the two conventional agars were found to be positive after isolation on EHA. All three selective agars were shown to be less effective in recovering Listeria spp. after primary enrichment in half-Fraser broth, compared to secondary enrichment in Fraser broth after 24 and 48 h.From 79 Listeria but presumptive negative L. monocytogenes colonies, EHA identified correctly 76 Listeria spp. and presented three false-negative results_three colonies further identified as L. monocytogenes but showing no noticeable haemolysis on EHA. Twenty-three of the thirty-three L. monocytogenes presumptive positive colonies, were confirmed positive and ten were identified as L. seeligeri.Despite its ability of distinguishing L. monocytogenes from the other Listeria spp., unless it is produced as a commercial medium, EHA cannot be an alternative to time-consuming classical identification because the preparation of this medium is both time and labour intensive.     

The clones of Listeria monocytogenes detected in food depend on the method used

S. LONCAREVIC, W. THAM AND M.-L. DANIELSSON-THAM. 1996. Restriction enzyme analysis (REA) with pulsed-field gel electrophoresis (PFGE) has been used to characterize and compare Listeria monocytogenes strains isolated from foods by two methods, an enrichment procedure and a direct plating procedure. In total 151 isolates from nine foods were investigated. In six of the foods (101 strains investigated) only one clone of L. monocytogenes was found irrespective of the method used. In three foods (50 strains investigated) the direct plating procedure yielded more clones than the enrichment procedure. At the most, five clones were detected in the same food. The results presented here indicate that direct plating from the food reveals more L. monocytogenes clones than revealed by an enrichment procedure.     

SUITABILITY OF SELECTIVE MEDIA FOR RECOVERY AND ENUMERATION OF SUBLETHALLY HEAT- AND ACID-INJURED LISTERIA MONOCYTOGENES

The suitability of PALCAM and modified Oxford (MOX) agars for recovering sublethally heat- and lactic acid-injured Listeria monocytogenes was investigated. L. monocytogenes LM101M, LM103M (meat isolates), and Scott A were suspended in tryptose phosphate broth (TPB), heated for up to 40 min at 54C, and surface plated onto tryptose phosphate agar (TPA), TPA + 4% NaCl (TPAS), PALCAM, and MOX. TPA and TPAS were used to determine total viable and sublethally injured populations, respectively. Heat-injured LM103M was recovered in the highest numbers on all media, followed by Scott A and LM101M (P<0.01). TPA allowed best recovery of all test strains, followed by PALCAMand MOX which were not different, and TPAS (P<0.01). For acid-injury studies, uninjured and heat-injured (54C for 20 min) test strains were suspended in phosphate-buffered TPB + 0.85% lactic acid (bTPBLA) at 25C for up to 24 h and plated as described above. Uninjured and heat-injured L. monocytogenes were recovered better from bTPBLA on MOX than on PALCAM (P<0.05). Heat injured L. monocytogenes LM103M was recovered better than LM101M but similar to Scott A on MOX and PALCAM (P<0.05), whereas Scott A was recovered similarly to LM101M and LM103M on MOX and PALCAM (P>0.05). Acid-injury of L. monocytogenes LM103M was enhanced by prior heat stress.     

Application of a chromogenic medium and the PCR method for the rapid confirmation of L. monocytogenes in foodstuffs

Detection of Listeria monocytogenes in foodstuffs by conventional cultivation methods carried out according to EN ISO guidelines is rather time-consuming. Therefore, two alternative methods were applied for rapid confirmation of L. monocytogenes in foodstuffs. Inoculum from liquid selective broth was plated on PALCAM and OXFORD agar and on chromogenic agar medium RAPID L. mono. Suspect colonies from PALCAM were confirmed according to EN ISO standards and by the multiplex PCR method. In total, 990 samples of foodstuffs were investigated and 63 strains of L. monocytogenes were isolated. The chromogenic medium RAPID L. mono provided results comparable to PCR, it is easier to handle and provides considerable financial savings.     

Atypical colonial morphology and low recoveries of Listeria monocytogenes strains on Oxford, PALCAM, Rapid'L.mono and ALOA solid media

The performance of four commercial media, polymyxin-acriflavine-LiCl-ceftazidime-aesculin-mannitol (PALCAM), Oxford, Rapid'L.mono (Bio-Rad, Marne la Coquette, France) and Agar Listeria according to Ottaviani and Agosti (ALOA: AES Laboratoire, Combourg, France; Biolife, Milan, Italy), used to detect and enumerate 176 Belgian strains of Listeria monocytogenes of human and food origin, was evaluated. Four strains showed a low recovery and/or atypical colonies on one or more media. These results showed that a combination of these media, especially alternative media (Rapid'L.mono and/or ALOA) with esculin-containing media (PALCAM and/or Oxford), should therefore be recommended to detect or enumerate atypical strains of L. monocytogenes. In outbreak case investigation for example, incubation of plates should be extended to at least 96 h if no colonies are typical or growth does not appear after 48 h. This is a cost/benefit calculation that should be done in the context of recent listeriosis risk assessments.     

Listeria monocytogenes isolation from human faecal specimens: experiments with the selective media, PALCAM and L-PALCAMY

The selective media PALCAM and L-PALCAMY were evaluated for their potential ability to detect Listeria monocytogenes in faeces. Recovery on PALCAM was almost total, and similar at 30°C and 35°C with or without CO₂ incubation. Warm enrichment in L-PALCAMY was necessary in order to detect low numbers (<10²/ml faeces). Faeces in excess of 0.25 ml/10 ml L-PALCAMY was inhibitory. The results point to L-PALCAMY and PALCAM as an epidemiological tool.     

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).     

Recovery of different Listeria ribotypes from naturally contaminated, raw refrigerated meat and poultry products with two primary enrichment media.

Isolation rates for Listeria monocytogenes and the other Listeria spp. typically improve when samples are enriched in more than one primary enrichment medium. This study evaluated the abilities of two primary enrichment media, University of Vermont-modified Listeria enrichment broth (UVM) and Listeria repair broth (LRB), to recover different ribotypes of Listeria spp. from raw meat and poultry samples. Forty-five paired 25-g retail samples of ground beef, pork sausage, ground turkey, and chicken (160 samples) underwent primary enrichment in UVM and LRB (30 degrees C for 24 h) followed by secondary enrichment in Fraser broth (35 degrees C for 24 and 40 h) and plating on modified Oxford agar. After 24 h of incubation of 35 degrees C, 608 Listeria colonies from selected positive samples were biochemically confirmed as L. monocytogenes (245 isolates), L innocua (276 isolates), and L. welshimeri (89 isolates) and then ribotyped with the automated Riboprinter microbial characterization system (E. I. du Pont de Nemours & Co., Inc.). Thirty-six different Listeria strains comprising 16 L. monocytogenes (including four known clinical ribotypes), 12 L. innocua, and 8 L. welshimeri ribotypes were identified from selected positive samples (15 samples of each product type; two UVM and two LRB isolates per sample). Twenty-six of 36(13 L. monocytogenes) ribotypes were detected with both UVM and LRB, whereas 3 of 36 (1 L. monocytogenes) and 7 of 36 (3 L. monocytogenes) Listeria ribotypes were observed with only UVM or LRB, respectively. Ground beef, pork sausage, ground turkey, and chicken yielded 22 (8 L. monocytogenes), 21 (12 L. monocytogenes), 20 (9 L. monocytogenes), and 19 (11 L. monocytogenes) different Listeria ribotypes, respectively, with some Listeria ribotypes confined to a particular product. More importantly, major differences in both the number and distribution of Listeria ribotypes, including previously recognized clinical and nonclinical ribotypes of L. monocytogenes, were observed when 10 UVM and 10 LRB isolates from five samples of each product were ribotyped. When a third set of six samples per product type was examined from which two Listeria isolates were obtained by using only one of the two primary enrichment media, UVM and LRB failed to detect L. monocytogenes (both clinical and nonclinical ribotypes) in two and four samples, respectively. These findings stress the importance of using more than one primary enrichment medium and picking a sufficient number of colonies per sample when attempting to isolate specific L. monocytogenes strains during investigations of food-borne listeriosis.     

Comparison of media and methods for detecting and enumerating Listeria monocytogenes in refrigerated cabbage.

Direct plating, selective enrichment, and cold enrichment followed by secondary selective enrichment procedures were compared for detecting and enumerating Listeria monocytogenes in chopped cabbage stored at 5 degrees C for up to 64 days. Addition of Fe3+ to solid media enhanced detection of the organism. Cold enrichment (5 degrees C) in nutrient broth and brain heart infusion broth followed by secondary enrichment (48 h, 30 degrees C) in Trypticase soy-yeast extract-antibiotic broth and thiocyanate-nalidixic acid broth and plating on selective agar media (Doyle and Schoeni selective enrichment agar [minus acriflavin hydrochloride, supplemented with 5 micrograms of Fe3+/ml] and McBride Listeria agar) resulted in the detection of highest populations.     

Comparison of media and methods for detecting and enumerating Listeria monocytogenes in refrigerated cabbage

Direct plating, selective enrichment, and cold enrichment followed by secondary selective enrichment procedures were compared for detecting and enumerating Listeria monocytogenes in chopped cabbage stored at 5 degrees C for up to 64 days. Addition of Fe3+ to solid media enhanced detection of the organism. Cold enrichment (5 degrees C) in nutrient broth and brain heart infusion broth followed by secondary enrichment (48 h, 30 degrees C) in Trypticase soy-yeast extract-antibiotic broth and thiocyanate-nalidixic acid broth and plating on selective agar media (Doyle and Schoeni selective enrichment agar [minus acriflavin hydrochloride, supplemented with 5 micrograms of Fe3+/ml] and McBride Listeria agar) resulted in the detection of highest populations.