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.     

Optical forward-scattering for detection of Listeria monocytogenes and other Listeria species

We demonstrate here the development of a non-invasive optical forward-scattering system, called 'scatterometer' for rapid identification of bacterial colonies. The system is based on the concept that variations in refractive indices and size, relative to the arrangement of cells in bacterial colonies growing on a semi-solid agar surface will generate different forward-scattering patterns. A 1.2-1.5mm colony size for a 1mm laser beam and brain heart infusion agar as substrate were used as fixed variables. The current study is focused on exploring identification of Listeria monocytogenes and other Listeria species exploiting the known differences in their phenotypic characters. Using diffraction theory, we could model the scattering patterns and explain the appearance of radial spokes and the rings seen in the scattering images of L. monocytogenes. Further, we have also demonstrated development of a suitable software for the extraction of the features (scalar values) calculated from images of the scattering patterns using Zernike moment invariants and principal component analysis and were grouped using K-means clustering. We achieved 91-100% accuracy in detecting different species. It was also observed that substrate variations affect the scattering patterns of Listeria. Finally, a database was constructed based on the scattering patterns from 108 different strains belonging to six species of Listeria. The overall system proved to be simple, non-invasive and virtually reagent-less and has the potential for automated user-friendly application for detection and differentiation of L. monocytogenes and other Listeria species colonies grown on agar plates within 5-10 min analysis time.     

Phosphatidylinositol-specific phospholipase C activity as a marker to distinguish between pathogenic and nonpathogenic Listeria species.

In this study, 468 Listeria strains were checked for the presence of phosphatidylinositol-specific phospholipase C (PI-PLC) activity by using a simple assay that consisted of overlaying colonies formed on agar plates with L-alpha-phosphatidylinositol as substrate. In this assay, PI-PLC-active colonies show turbid halos around the colonies as a result of the release of insoluble diacylglycerol from the substrate. This activity was detected only in the pathogenic species Listeria monocytogenes and was not present in any of the 167 strains of Listeria seeligeri, Listeria welshimeri, Listeria innocua, Listeria murrayi, and Listeria grayi tested. Hence, screening for PI-PLC activity permits discrimination between pathogenic and nonpathogenic Listeria species. In particular, the hemolytic but nonpathogenic species L. seeligeri can now be separated from the hemolytic and pathogenic species L. monocytogenes and L. ivanovii. The use of this assay will improve the specific detection and/or isolation of pathogenic Listeria species from clinical samples or food enrichment cultures.     

Phosphatidylinositol-specific phospholipase C activity as a marker to distinguish between pathogenic and nonpathogenic Listeria species.

In this study, 468 Listeria strains were checked for the presence of phosphatidylinositol-specific phospholipase C (PI-PLC) activity by using a simple assay that consisted of overlaying colonies formed on agar plates with L-alpha-phosphatidylinositol as substrate. In this assay, PI-PLC-active colonies show turbid halos around the colonies as a result of the release of insoluble diacylglycerol from the substrate. This activity was detected only in the pathogenic species Listeria monocytogenes and was not present in any of the 167 strains of Listeria seeligeri, Listeria welshimeri, Listeria innocua, Listeria murrayi, and Listeria grayi tested. Hence, screening for PI-PLC activity permits discrimination between pathogenic and nonpathogenic Listeria species. In particular, the hemolytic but nonpathogenic species L. seeligeri can now be separated from the hemolytic and pathogenic species L. monocytogenes and L. ivanovii. The use of this assay will improve the specific detection and/or isolation of pathogenic Listeria species from clinical samples or food enrichment cultures.     

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 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.     

A COLLABORATIVE STUDY FOR DIRECT ENUMERATION OF LOW LEVELS OF LISTERIA MONOCYTOGENES FROM VARIOUS FOODS

A direct plating method for the enumeration of low levels of foodborne Listeria monocytogenes was evaluated in a collaborative study involving 18 laboratories across Canada. Shrimp, coleslaw, ice cream and wieners were inoculated with low levels (5 × 10² and 10³/g) of L. monocytogenes and shipped to participants. Foods were diluted and then plated onto either lithium chloride phenylethyl and moxa-lactam agar (LPM), Oxford agar (OXA), modified Oxford agar (MOX) or Palcam agar (PAL). Recovery was good for all foods, except coleslaw. Of the four plating media tested, all were more or less equivalent in their ability to recover colonies for enumeration, except that more colonies were enumerated on LPM than on PAL agar. Recovery of L. monocytogenes ranged from <50 to 1250 cfu/g for wieners, <50 to 800 cfu/g for shrimp, <100 to 1440 cfu/g for ice cream and <50 to 700 cfu/g for coleslaw. Results indicate that the direct plating method can be used for the recovery of low levels of Listeria monocytogenes in Category 3 foods, as presently suggested for use in the Canadian Listeria compliance guide.     

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.     

Evaluation of hybridization characteristics of a cloned pRF106 probe for Listeria monocytogenes detection and development of a nonisotopic colony hybridization assay.

An internal fragment (pRF106 fragment, ca. 500 bp) of a gene (msp) coding for a 60-kDa protein of Listeria monocytogenes serotype 1/2a was used to develop a screening method to discriminate between L. monocytogenes and avirulent Listeria spp. on primary isolation plates. The L. monocytogenes-derived probe fragment of pRF106 hybridized to a 13-kb fragment of L. monocytogenes and a 3-kb fragment of one cheese isolate strain of Listeria seeligeri under stringent hybridization conditions (mean thermal denaturation temperature [Tm]-5 degrees C). The probe also hybridized to a 6-kb fragment of Listeria innocua, Listeria ivanovii, and L. seeligeri under less stringent hybridization conditions (Tm-17 degrees C). The pRF106 fragment was labeled with digoxigenin-11-dUTP and used to develop a colony hybridization assay. Colonies from lithium chloride-phenylethanol-moxalactam agar were blotted onto nylon membranes. The cells were pretreated with microwaves before lysis with sodium hydroxide. DNA-DNA hybridization and posthybridization washing were done at high stringency (Tm-7 degrees C). The nonisotopic colony hybridization procedure was specific for L. monocytogenes when evaluated against pure cultures of L. monocytogenes and other Listeria species, excluding the cheese isolate of L. seeligeri. Also, it was specific for L. monocytogenes when evaluated with Listeria-negative food enrichment cultures that were inoculated in the laboratory with Listeria species.     

Evaluation of hybridization characteristics of a cloned pRF106 probe for Listeria monocytogenes detection and development of a nonisotopic colony hybridization assay

An internal fragment (pRF106 fragment, ca. 500 bp) of a gene (msp) coding for a 60-kDa protein of Listeria monocytogenes serotype 1/2a was used to develop a screening method to discriminate between L. monocytogenes and avirulent Listeria spp. on primary isolation plates. The L. monocytogenes-derived probe fragment of pRF106 hybridized to a 13-kb fragment of L. monocytogenes and a 3-kb fragment of one cheese isolate strain of Listeria seeligeri under stringent hybridization conditions (mean thermal denaturation temperature [Tm]-5 degrees C). The probe also hybridized to a 6-kb fragment of Listeria innocua, Listeria ivanovii, and L. seeligeri under less stringent hybridization conditions (Tm-17 degrees C). The pRF106 fragment was labeled with digoxigenin-11-dUTP and used to develop a colony hybridization assay. Colonies from lithium chloride-phenylethanol-moxalactam agar were blotted onto nylon membranes. The cells were pretreated with microwaves before lysis with sodium hydroxide. DNA-DNA hybridization and posthybridization washing were done at high stringency (Tm-7 degrees C). The nonisotopic colony hybridization procedure was specific for L. monocytogenes when evaluated against pure cultures of L. monocytogenes and other Listeria species, excluding the cheese isolate of L. seeligeri. Also, it was specific for L. monocytogenes when evaluated with Listeria-negative food enrichment cultures that were inoculated in the laboratory with Listeria species.     

Replica plating of colonies from Listeria-selective agars to blood agar to improve the isolation of Listeria monocytogenes from foods

Bacterial colonies from Listeria-selective agars were replica plated to sheep blood agar to screen for beta-hemolysis. By using the replica plating method to test for the beta-hemolytic characteristic of all the colonies growing on Listeria-selective agars instead of picking 3 to 10 suspected colonies for further testing, we recovered Listeria monocytogenes from 59 of 142 Listeria-selective agar plates which contained colonies of hemolytic and nonhemolytic Listeria species and were negative when tested by conventional colony picks.     

Replica plating of colonies from Listeria-selective agars to blood agar to improve the isolation of Listeria monocytogenes from foods.

Bacterial colonies from Listeria-selective agars were replica plated to sheep blood agar to screen for beta-hemolysis. By using the replica plating method to test for the beta-hemolytic characteristic of all the colonies growing on Listeria-selective agars instead of picking 3 to 10 suspected colonies for further testing, we recovered Listeria monocytogenes from 59 of 142 Listeria-selective agar plates which contained colonies of hemolytic and nonhemolytic Listeria species and were negative when tested by conventional colony picks.     

Hemolytic activity reevaluation of putative nonpathogenic Listeria monocytogenes strains.

Identification of 12 strains originally characterized as nonpathogenic Listeria monocytogenes was reassured following the evaluation of their hemolytic capability with a newly developed horse blood agar plate. Seven of the strains were observed consistently to be hemolytic and confirmed as L. monocytogenes with the use of two commercial systems: the Gene-Trak L. monocytogenes-specific colorimetric DNA hybridization assay and the API Listeria system. Except for one strain that formed typical smooth colonies, these hemolytic strains formed rough colonies on a selective medium, lithium chloride-ceftazidime agar. The rest of the strains were nonhemolytic and did not hybridize with the DNA probe; they were identified as Listeria innocua on the basis of their API Listeria system biochemical profile. All but one of these nonhemolytic strains formed smooth colonies on lithium chloride-ceftazidime agar.     

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.     

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).     

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.     

Optimization of haemolysis in enhanced haemolysis agar (EHA)_a selective medium for the isolation of Listeria monocytogenes

The presence of Listeria monocytogenes in enrichment media can be masked by faster growth of other Listeria spp. Therefore, enhanced haemolysis agar (EHA) is a good alternative for another isolation media, because the presence of a few L. monocytogenes colonies can be detected in a majority of colonies of other listeriae on the basis of haemolysis. In this study the haemolysis reaction in EHA was optimized. In a collaborative study using reference samples, no significant differences in counts on EHA, Palcam and Oxford agar were shown.     

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.     

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.     

Petite colony formation by Listeria monocytogenes and Listeria species grown on esculin-containing agar

Several strains of Listeria species formed petite-sized colonies from parent stock cultures when grown on agar media containing 0.2-1% (w/v) esculin. This was observed in Listeria monocytogenes (7/22 strains), L. innocua (1/3), L. grayi (1/1), L. seeligeri (1/3), and L. welshimeri (1/1), but not in L. ivanovii (0/1) and L. murrayi (0/1). This phenomenon was only observed on agar media that contained esculin. All petite isolates had biotyping profiles identical to their larger, normal-sized counterpart isolates. Normal and petite-sized isolates from two L. monocytogenes strains, Scott A and V7, were pathogenic to immunosuppressed white mice. On media containing 0.5% (w/v) esculin + ferric iron, Listeria cultures produced colony diameters intermediate in size between those of normal and petite cultures. When pregrown in glucose broth, all petite isolates demonstrated visible beta-glucosidase (esculinase) activity within 5 min, while the normal-sized isolates showed beta-glucosidase activity only after at least 20-70 min. This evidence suggests that cells forming petite colonies are beta-glucosidase constitutive variants within the parent population, while cells that form normal-sized colonies are inducible for beta-glucosidase (esculinase) activity. A possible role for the esculin hydrolysis product, esculetin, in causing petite colony formation is discussed.