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.     

An improved selective isolation medium for the recovery of Listeria monocytogenes from smoked fish

AIMS: The aim of this study was to improve the selective isolation of Listeria monocytogenes from smoked haddock fillets. METHODS AND RESULTS: Listeria selective agar (LSA)--Oxford formulation was supplemented with 25 microg x ml(-1) of colistin sulphate and 30 microg x ml(-1) of nalidixic acid. Inocula from four smoked haddock fillets produced colonies (approx. 2-13 bacteria x g(-1)), identified as L. monocytogenes, on LSA supplemented with antimicrobial compounds (MLSA). Moreover, there was only negligible evidence of bacteria which were not L. monocytogenes on MLSA. In contrast, LSA supported dense bacterial growth, which was not equated with L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The modified medium permitted the recovery of L. monocytogenes from smoked haddock fillets and reduced the growth of contaminating bacteria.     

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.     

Detection and prevalence of Listeria monocytogenes in the agricultural ecosystem

The sensitivity of four different enrichment procedures to detect Listeria monocytogenes in the presence of high levels of Streptococcus faecalis was investigated. Defined mixed cultures of Strep. faecalis and L. monocytogenes gave better results with one-stage enrichment techniques. For manure samples, however, two-stage enrichment techniques gave the best performance. The so-called cold enrichment techniques were found to be unsatisfactory for samples from natural environments. The following materials were examined for the presence of L. monocytogenes: fresh pig faeces (16% positive), fresh cattle faeces (20% positive), stored liquid manure (0% positive), manured soil samples (0% positive) and ground water samples (5% positive). After 3 weeks of storage L. monocytogenes could be detected in only one of the initially nine positive fresh faeces samples. Two months after inoculation of stored liquid pig manure, stored liquid cattle manure and soil with L. monocytogenes, this bacterium could not be traced in any of these materials. Radishes (Raphanus sativus) and carrots (Daucus carota), sown in soil inoculated with L. monocytogenes, were gathered after 3 months and examined for the presence of L. monocytogenes. Three of six radish samples were found to be positive. Remarkably, however, all carrot samples (six) were free of L. monocytogenes.     

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.     

Assessment of the Accuprobe Listeria monocytogenes culture identification reagent kit for rapid colony confirmation and its application in various enrichment broths.

The Accuprobe Listeria monocytogenes Culture Identification Reagent Kit, a nonradioactive probe, was evaluated as a colony confirmation test and in different selective or nonselective enrichment broths. The probe was 100% sensitive and 100% specific when applied to isolated colonies. The minimal detection limit in physiological saline was established to be about 10(5) CFU of L. monocytogenes. Hybridization done directly in broths seeded with L. monocytogenes showed variable results. Three nonselective broths (Todd-Hewitt broth, brain heart infusion broth, and tryptic soy broth) and one selective broth (FDA) gave positive reactions at an inoculum of 5 x 10(6) CFU, whereas two other selective broths (UVM, and PALCAMY) gave negative reactions with up to 10(8) and 10(9) CFU. In FDA broth, the level of detection of L. monocytogenes was not modified by the presence of other organisms in mixed cultures.     

Assessment of the Accuprobe Listeria monocytogenes culture identification reagent kit for rapid colony confirmation and its application in various enrichment broths.

The Accuprobe Listeria monocytogenes Culture Identification Reagent Kit, a nonradioactive probe, was evaluated as a colony confirmation test and in different selective or nonselective enrichment broths. The probe was 100% sensitive and 100% specific when applied to isolated colonies. The minimal detection limit in physiological saline was established to be about 10(5) CFU of L. monocytogenes. Hybridization done directly in broths seeded with L. monocytogenes showed variable results. Three nonselective broths (Todd-Hewitt broth, brain heart infusion broth, and tryptic soy broth) and one selective broth (FDA) gave positive reactions at an inoculum of 5 x 10(6) CFU, whereas two other selective broths (UVM, and PALCAMY) gave negative reactions with up to 10(8) and 10(9) CFU. In FDA broth, the level of detection of L. monocytogenes was not modified by the presence of other organisms in mixed cultures.     

Method for flow cytometric detection of Listeria monocytogenes in milk

This report describes a method for the detection of Listeria monocytogenes in raw milk by flow cytometric analysis of fluorescently labeled bacterial populations. The use of immunofluorescence in combination with measures of DNA content by propidium iodide labeling and size by light scattering enabled specific identification of L. monocytogenes from Streptococcus faecalis, Streptococcus agalactiae, Streptococcus uberis, Staphylococcus epidermidis, and Staphylococcus hyicus. Additional specific resolution of L. monocytogenes populations was achieved through selective enrichment of raw milk in Listeria enrichment broth. These procedures should permit the rapid screening of milk and other food samples for L. monocytogenes and eliminate many of the short-comings associated with conventional fluorescent-antibody procedures.     

Method for flow cytometric detection of Listeria monocytogenes in milk.

This report describes a method for the detection of Listeria monocytogenes in raw milk by flow cytometric analysis of fluorescently labeled bacterial populations. The use of immunofluorescence in combination with measures of DNA content by propidium iodide labeling and size by light scattering enabled specific identification of L. monocytogenes from Streptococcus faecalis, Streptococcus agalactiae, Streptococcus uberis, Staphylococcus epidermidis, and Staphylococcus hyicus. Additional specific resolution of L. monocytogenes populations was achieved through selective enrichment of raw milk in Listeria enrichment broth. These procedures should permit the rapid screening of milk and other food samples for L. monocytogenes and eliminate many of the short-comings associated with conventional fluorescent-antibody procedures.     

Competitive fitness of Listeria monocytogenes serotype 1/2a and 4b strains in mixed cultures with and without food in the U.S. Food and Drug Administration enrichment protocol

Thirteen different serotypes of the food-borne pathogen Listeria monocytogenes have been described. Serotype 4b strains are most often associated with illness, and serotype 1/2a strains are most often isolated from foods and processing plants. Different abilities to respond to stresses have been described for serotype 4b and 1/2a strains. One of the common enrichment protocols used to test foods for the presence L. monocytogenes is described in the U.S. Food and Drug Administration (FDA) Bacterial Analytical Manual (BAM). We compared three strains of L. monocytogenes serotype 4b and five strains of serotype 1/2a in direct competition with each other in two-strain mixed cultures by using the FDA BAM enrichment protocol, which includes both enrichment broth and selective agar, with and without added food to mimic the conditions that occur during attempts to isolate Listeria species from contaminated foods. Using a colony immunoblot procedure and analyzing over 112,000 colonies, we observed differences in strain fitness, but these differences were not attributable to serotype or genetic lineage.     

Detection and prevalence of Listeria monocytogenes in the agricultural ecosystem

B. VAN RENTERGHEM, F. HUYSMAN, R. RYGOLE AND W. VERSTRAETE. 1991. The sensitivity of four different enrichment procedures to detect Listeria monocytogenes in the presence of high levels of Streptococcus faecalis was investigated. Defined mixed cultures of Strep. faecalis and L. monocytogenes gave better results with one-stage enrichment techniques. For manure samples, however, two-stage enrichment techniques gave the best performance. The so-called cold enrichment techniques were found to be unsatisfactory for samples from natural environments. The following materials were examined for the presence of L. monocytogenes: fresh pig faeces (16% positive), fresh cattle faeces (20% positive), stored liquid manure (0% positive), manured soil samples (0% positive) and ground water samples (5% positive). After 3 weeks of storage L. monocytogenes could be detected in only one of the initially nine positive fresh facces samples. Two months after inoculation of stored liquid pig manure, stored liquid cattle manure and soil with L. monocytogenes , this bacterium could not be traced in any of these materials. Radishes ( Raphanus sativus ) and carrots ( Daucus carota ), sown in soil inoculated with L. monocytogenes , were gathered after 3 months and examined for the presence of L. monocytogenes. Three of six radish samples were found to be positive. Remarkably, however, all carrot samples (six) were free of L. monocytogenes.     

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.     

Binding to faeces and influence on human anaerobes of antimicrobial agents used for selective decontamination

The degree of binding of ampicillin, cephradine, co-trimoxazole, gentamicin, nalidixic acid, neomycin, polymyxin B and tobramycin by faecal substance as well as the influence of these antibiotics on human intestinal obligate anaerobes was investigated. In contrast to ampicillin, cephradine, co-trimoxazole and nalidixic acid, the nonabsorbable antibiotics polymyxin B and neomycin were bound to a considerable degree by human faeces. The binding of tobramycin and gentamicin to the solid part of faeces was less effective. The inhibitory effect of co-trimoxazole, gentamicin, nalidixic acid, neomycin, polymyxin B and tobramycin on the human obligate anaerobes was weak as compared with ampicillin and cephradine. Drugs which effectively eliminate Enterobacteriaceae from the gastrointestinal tract and which have a moderate effect on obligate anaerobes, like polymyxin B, are particularly suitable for selective decontamination of the gastrointestinal tract. The strong inactivating binding of aminoglycosides and polymyxin B to faeces accounts for the relatively high oral dose needed for a suitable faecal concentration.     

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.     

Bias in the Listeria monocytogenes enrichment procedure: lineage 2 strains outcompete lineage 1 strains in University of Vermont selective enrichments

Listeria monocytogenes can be isolated from a range of food products and may cause food-borne outbreaks or sporadic cases of listeriosis. L. monocytogenes is divided into three genetic lineages and 13 serotypes. Strains of three serotypes (1/2a, 1/2b, and 4b) are associated with most human cases of listeriosis. Of these, strains of serotypes 1/2b and 4b belong to lineage 1, whereas strains of serotype 1/2a and many other strains isolated from foods belong to lineage 2. L. monocytogenes is isolated from foods by selective enrichment procedures and from patients by nonselective methods. The aim of the present study was to investigate if the selective enrichment procedure results in a true representation of the subtypes of L. monocytogenes present in a sample. Eight L. monocytogenes strains (four lineage 1 strains and four lineage 2 strains) and one Listeria innocua strain grew with identical growth rates in the nonselective medium brain heart infusion (BHI), but differed in their growth rate in the selective medium University of Vermont medium I (UVM I). When coinoculated in UVM I, some strains completely outgrew other strains. This outcome was dependent on the lineage of L. monocytogenes rather than the individual growth rate of the strains. When inoculated at identical cell densities in UVM I, L. innocua outcompeted L. monocytogenes lineage 1 strains but not lineage 2 strains. In addition, lineage 2 L. monocytogenes strains outcompeted lineage 1 L. monocytogenes strains in all combinations tested, indicating a bias in strains selected by the enrichment procedures. Bias also occurred when coinoculating two lineage 2 or lineage 1 strains; however, it did not appear to correlate with origin (clinical versus food). Identical coinoculation experiments in BHI suggested that the selective compounds in UVM I and II influenced this bias. The results of the present study demonstrate that the selective procedures used for isolation of L. monocytogenes may not allow a true representation of the types present in foods. Our results could have a significant impact on epidemiological studies, as lineage 1 strains, which are often isolated from clinical cases of listeriosis, may be suppressed during enrichment by other L. monocytogenes lineages present in a food sample.     

Comparison of selective and non-selective enrichment media in the detection of Listeria monocytogenes from meat containing Listeria innocua

AIMS: This study investigated whether the higher incidence of recovery from meat of Listeria innocua compared with L. monocytogenes could be due to the laboratory media used, leading to an artificially lower detection of the pathogenic species, L. monocytogenes. METHODS AND RESULTS: Minced beef was inoculated with L. monocytogenes, L. innocua, or a mixture of these species, and stored at 0 or 10 degrees C under vacuum or aerobic conditions for up to 28 days. Listeria were recovered from the minced beef using selective (University of Vermont Medium, UVM) and non-selective (Buffered Peptone Water, BPW) enrichment broths after 0, 14, and 28 days of storage. In general, there were no significant differences (P < 0.05) between the numbers of L. monocytogenes recovered from minced beef samples after 24 h enrichment in BPW and the numbers recovered using UVM. In addition, the presence of L. innocua in meat samples containing L. monocytogenes did not significantly (P < 0.05) affect the numbers of L. monocytogenes recovered using either enrichment broth. In most cases there were no significant differences (P < 0.05) between the numbers of L. innocua recovered from minced beef samples after 24 h enrichment in BPW compared with numbers recovered using UVM. CONCLUSION: Listeria innocua was found to have no significant competitive advantage over L. monocytogenes in selective or non-selective enrichment media. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that, in some instances, the use of a selective enrichment broth offers no advantage over a non-selective enrichment broth for the recovery of Listeria species from minced beef.     

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.     

Detection and identification of Listeria monocytogenes in cooked sausage products and in milk by in vitro amplification of haemolysin gene fragments

Recent outbreaks of listeriosis have emphasized the urgent need for rapid and reliable detection methods for Listeria spp., especially in food. Haemolysin production is a major factor in the pathogenesis of listeriosis and the polymerase chain reaction (PCR) was used to amplify two specific DNA fragments of the alpha- and the beta-haemolysin genes. The amplification system specifically recognized L. monocytogenes strains. The detection limit determined with pure cultures was 10 bacteria when estimated with alpha-haemolysin primers. In the analysis of 50 samples of cooked sausage products, bacterial colonies suspected to be Listeria spp. were isolated by conventional methods from six samples. PCR analysis identified three of six as L. monocytogenes. Subsequent serotyping showed perfect agreement with the PCR results. Since enrichment is the most time consuming step in conventional methods a PCR procedure which allows the direct detection of L. monocytogenes in milk was developed. Pasteurized milk was artificially contaminated with various levels of L. monocytogenes. The detection limit was determined to be 10 bacteria/10 ml milk and direct detection and identification of L. monocytogenes took less than two working days. These results show that this haemolysin gene amplification system is very rapid and reliable and therefore avoids cumbersome and lengthy cultivation steps.     

A PCR detection method for rapid identification of Paenibacillus larvae

American foulbrood is a disease of larval honeybees (Apis mellifera) caused by the bacterium Paenibacillus larvae. Over the years attempts have been made to develop a selective medium for the detection of P. larvae spores from honey samples. The most successful of these is a semiselective medium containing nalidixic acid and pipermedic acid. Although this medium allows the growth of P. larvae and prevents the growth of most other bacterial species, the false-positive colonies that grow on it prevent the rapid confirmation of the presence of P. larvae. Here we describe a PCR detection method which can be used on the colonies that grow on this semiselective medium and thereby allows the rapid confirmation of the presence of P. larvae. The PCR primers were designed on the basis of the 16S rRNA gene of P. larvae and selectively amplify a 973-bp amplicon. The PCR amplicon was confirmed as originating from P. larvae by sequencing in both directions. Detection was specific for P. larvae, and the primers did not hybridize with DNA from closely related bacterial species.