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 the efficacy of different techniques, culture media, and sources of blood in determining the hemolytic activity of Listeria spp.

Hemolytic activity is a fundamental criterion for the differentiation of Listeria species; therefore, a simple and inexpensive procedure to clearly distinguish hemolytic strains from each other and from nonhemolytic strains would be of great aid. We compared the efficacy of several techniques, culture media, and types of blood in demonstrating the hemolysis of Listeria spp. The hemolytic activities of Listeria monocytogenes and Listeria seeligeri were more easily detected with a red blood cell top-layer (RBCTL) technique and with a microplate technique than when the strains were streaked on blood agar (BA). Listeria ivanovii produced a marked hemolysis regardless of the technique employed. In general, the hemolytic activity of these three species was stronger on media containing brain heart infusion (BHI) agar and (or) potassium tellurite (PT). However, Listeria innocua produced questionable hemolytic reactions when nonselective culture media with BHI and PT were utilized, limiting the advantages gained by employing the two compounds. The RBCTL and the BA techniques disclosed greater hemolytic activity for L. monocytogenes, L. seeligeri, and L. ivanovii with sheep and guinea pig blood than with horse and human blood. When the microplate technique was used, all four kinds of blood were equally effective.     

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.     

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.     

The type strain(s) of Listeria monocytogenes: a source of continuing difficulties

The type strain of Listeria monocytogenes differs from wild-type L. monocytogenes strains in more characteristics than just the previously reported deficiency in hemolytic activity and virulence in the murine infection model. The type strain from the American Type Culture Collection (strain ATCC 15313) produces lecithinase, is hemolytic on rabbit (but not sheep) blood agar, lacks motility, and shows limited cytopathogenic effects on Caco-2 monolayers, whereas the type strain from the Special Listeria Culture Collection (strain SLCC 53) is unable to produce lecithinase, is nonhemolytic on rabbit or sheep blood agar, is motile, and shows no cytopathogenic effects on Caco-2 monolayers.     

Evaluation of the Organon-Teknika MICRO-ID LISTERIA system.

The MICRO-ID LISTERIA system, designed to identify Listeria isolates to species level within 24 h, was compared with conventional biochemical identification. MICRO-ID LISTERIA used in combination with the CAMP test correctly identified 409 (98.8%) of 414 strains isolated from human, animal, food, and environmental sources belonging to the seven species currently defined within the genus Listeria. The kit was easy to use and simple to interpret. However, 8 of the 15 tests (i.e., phenylalanine deaminase, hydrogen sulfide, indole, ornithine decarboxylase, lysine decarboxylase, malonate, urease, and o-nitrophenyl-beta-D-galactopyranoside) were considered superfluous for the differentiation of Listeria spp. The CAMP test was indispensable when using the MICRO-ID LISTERIA system, in particular to differentiate CAMP test-positive L. monocytogenes from the nonhemolytic, rhamnose-positive L. innocua. The hemolytic L. seeligeri and L. ivanovii strains and the nonhemolytic, non-rhamnose-acidifying L. welshimeri strains could also be differentiated from one another only on the basis of their CAMP test results. The very few strains of L. grayi and L. murrayi were easily differentiated from the other nonhemolytic species. Catalase-negative cocci should not be tested, because 12 out of 19 catalase-negative strains (all enterococci) in our test were misidentified as Listeria spp. The MICRO-ID LISTERIA system identified strains within 18 to 24 h and is thus less time-consuming than conventional tests. The system could, therefore, be used together with correctly done CAMP tests for the rapid identification of Listeria isolates, especially food and environmental isolates, for which rapid species differentiation is important.     

Evaluation of the Organon-Teknika MICRO-ID LISTERIA system.

The MICRO-ID LISTERIA system, designed to identify Listeria isolates to species level within 24 h, was compared with conventional biochemical identification. MICRO-ID LISTERIA used in combination with the CAMP test correctly identified 409 (98.8%) of 414 strains isolated from human, animal, food, and environmental sources belonging to the seven species currently defined within the genus Listeria. The kit was easy to use and simple to interpret. However, 8 of the 15 tests (i.e., phenylalanine deaminase, hydrogen sulfide, indole, ornithine decarboxylase, lysine decarboxylase, malonate, urease, and o-nitrophenyl-beta-D-galactopyranoside) were considered superfluous for the differentiation of Listeria spp. The CAMP test was indispensable when using the MICRO-ID LISTERIA system, in particular to differentiate CAMP test-positive L. monocytogenes from the nonhemolytic, rhamnose-positive L. innocua. The hemolytic L. seeligeri and L. ivanovii strains and the nonhemolytic, non-rhamnose-acidifying L. welshimeri strains could also be differentiated from one another only on the basis of their CAMP test results. The very few strains of L. grayi and L. murrayi were easily differentiated from the other nonhemolytic species. Catalase-negative cocci should not be tested, because 12 out of 19 catalase-negative strains (all enterococci) in our test were misidentified as Listeria spp. The MICRO-ID LISTERIA system identified strains within 18 to 24 h and is thus less time-consuming than conventional tests. The system could, therefore, be used together with correctly done CAMP tests for the rapid identification of Listeria isolates, especially food and environmental isolates, for which rapid species differentiation is important.     

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.     

Tn916-induced mutations in the hemolysin determinant affecting virulence of Listeria monocytogenes.

A genetic determinant essential for hemolysin production by Listeria monocytogenes has been inactivated by insertion of transposon Tn916 into L. monocytogenes DNA. The transposon was transferred by means of conjugation of a streptomycin-resistant L. monocytogenes recipient strain with Streptococcus faecalis CG110 on membrane filters. Among the tetracycline-resistant transconjugants, mutants were detected which had lost hemolytic activity. When tested in a mouse model, these mutants appeared to have lost the virulence that characterizes the parental strain. An extracellular protein of 58,000 apparent molecular weight was eliminated in the nonhemolytic mutants. In some of the mutants, the decrease in the production of the 58,000-dalton protein was accompanied by the production of a new protein of 49,000 apparent molecular weight. Hemolytic revertants regained the hemolytic phenotype and virulence and produced the extracellular protein that characterizes the recipient strain. Hybridization studies with Tn916 DNA indicated that the transposon is present in EcoRI and HindIII fragments of the nonhemolytic mutants. Single copies of Tn916 were detected in the chromosomal DNA of two of the three nonhemolytic mutants that were studied in detail. In hemolytic, tetracycline-sensitive revertants Tn916 appeared to be completely excised from the chromosome.     

Rapid identification of Listeria species by using restriction fragment length polymorphism of PCR-amplified 23S rRNA gene fragments

A molecular method based on restriction fragment length polymorphism (RFLP) of PCR-amplified fragments of the 23S rRNA gene was designed to rapidly identify Listeria strains to the species level. Two fragments (S1, 460 bp, and S2, 890 bp) were amplified from boiled DNA. S2 was cut with the restriction enzymes XmnI or CfoI and, if needed, S1 was digested by either AluI or ClaI. This method was first optimized with six reference strains and then applied to 182 isolates collected from effluents of treatment plants. All isolates were also identified by the API Listeria kit, hemolysis, and phosphatidylinositol-specific phospholipase C production (PI-PLC) on ALOA medium. The PCR-RFLP method unambiguously identified 160 environmental strains, including 131 in concordance with the API system, and revealed that 22 isolates were mixed cultures of Listeria monocytogenes and Listeria innocua. Discrepant results were resolved by a multiplex PCR on the iap gene, which confirmed the PCR-RFLP data for 49 of the 51 discordances, including the 22 mixed cultures. Sequencing of the 16S rRNA gene for 12 selected strains and reconstruction of a phylogenetic tree validated the molecular methods, except for two unclassifiable strains. The 158 single identifiable isolates were 92 L. monocytogenes (including seven nonhemolytic and PI-PLC-negative strains), 61 L. innocua, 4 Listeria seeligeri, and 1 Listeria welshimeri strain. The PCR-RFLP method proposed here provides rapid, easy-to-use, inexpensive, and reliable identification of the six Listeria species. Moreover, it can detect mixtures of Listeria species and thus is particularly adapted to environmental and food microbiology.     

Characteristics of maltose transporter activity in an ale and larger strain of the yeast Saccharomyces cerevisiae

R.R. BEUMER, M.C. TE GIFFEL, M.T.C. KOK AND F.M. ROMBOUTS. 1996. All confirmation and identification methods used in this study can be used for the screening of suspected colonies on isolation media for Listeria spp. In traditional enrichment procedures the Microscreen Listeria latex test gives fast results. The DNA probes (Accuprobe and Gene-Trak) are very specific in detecting Listeria monocytogenes . For identification of Listeria spp. both tests (API and Micro-ID) performed equally well. Preference may be given to the API test, since differentiation of L. monocytogenes from L. innocua is based on the absence of arylamidase, through which tests for haemolytic activity and/or CAMP reactions can be omitted. However, the use of Enhanced Haemolysis Agar as isolation medium makes further testing essentially superfluous, since L. monocytogenes strains can be differentiated from L. innocua .     

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.     

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.     

Cloning of the listeriolysin O gene and development of specific gene probes for Listeria monocytogenes

A clone containing 3.1 kb of Listeria DNA was selected from a gene library of Listeria monocytogenes Scott A strain. The Escherichia coli clone produced hemolysin on sheep blood agar and in sonicated extracts but very little in the culture supernatant. This 3.1-kb DNA fragment and a 650-bp HindIII fragment located within the listeriolysin gene were used as probes in a colony hybridization assay. Both probes were specific for L. monocytogenes and did not hybridize with any other Listeria strains at high stringency. Two synthetic probes, one from the 650-bp HindIII fragment and one from the carboxy-terminal region of the protein, were also specific for L. monocytogenes.     

Cloning of the listeriolysin O gene and development of specific gene probes for Listeria monocytogenes.

A clone containing 3.1 kb of Listeria DNA was selected from a gene library of Listeria monocytogenes Scott A strain. The Escherichia coli clone produced hemolysin on sheep blood agar and in sonicated extracts but very little in the culture supernatant. This 3.1-kb DNA fragment and a 650-bp HindIII fragment located within the listeriolysin gene were used as probes in a colony hybridization assay. Both probes were specific for L. monocytogenes and did not hybridize with any other Listeria strains at high stringency. Two synthetic probes, one from the 650-bp HindIII fragment and one from the carboxy-terminal region of the protein, were also specific for L. monocytogenes.     

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.     

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.