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.     

Unidentified Listeria-like bacteria isolated from cheese

Unidentified Listeria -like bacteria, which lack only one of the phenotypic characteristics used to confirm Listeria spp., were isolated from cheese during routine analysis for Listeria monocytogenes . The VIDAS Listeria assay and the Listeria specific PCR or DNA probe assays used did not identify these strains as Listeria species. This group of bacteria was studied for its homogeneity using rep-PCR and PFGE. Sequence analysis of the 16S rRNA gene showed a homology of 94% to established Listeria spp., implicating a closer relationship than that between Listeria spp. and Brochothrix spp.     

EFFECT of OXYRASE ON GROWTH of SALMONELLA spp. and LISTERIA MONOCYTOGENES IN the "UNIVERSAL PREENRICHMENT MEDIUM"

The importance of Salmonella and Listeria monocytogenes in food safety is well-known. Recovery of these organisms is usually done in different types of enrichment broths. Recently Bailey and Cox (1992) reported the development of a "Universal Preenrichment Medium" capable of recovering both Salmonella and Listeria monocytogenes , thus eliminating the need of using two broths to enrich these important foodborne pathogens. In our laboratory we have ascertained that Oxyrase, an oxygen scavenger, is able to allow the rapid growth of Listeria monocytogenes and other Listeria spp. (Yu and Fung 1991a) as well as other facultative anaerobic foodborne pathogens (Yu and Fung 1991b). It seems reasonable that Oxyrase will be able to stimulate growth of Salmonella spp. and Listeria monocytogenes in the "Universal Preenrichment Medium." the purpose of this investigation was to ascertain the stimulatory capability of Oxyrase on Salmonella spp. and Listeria monocytogenes in the "Universal Preenrichment Medium."     

The microbial ecology of high-risk, chilled food factories; evidence for persistent Listeria spp. and Escherichia coli strains

AIMS: The intention of this study was to provide evidence of any Listeria spp. or Escherichia coli strain persistence, and to identify whether strains of these organisms adapt to specific environmental or product niches in food factories. METHODS AND RESULTS: A 3-year assessment of the microbial ecology of four, ready-to-eat food-processing factories was undertaken in which approx. 196 000 and 75 000 product and environmental samples were examined for Escherichia coli and Listeria spp. respectively. A total of 152 E. coli isolates (44 environmental and 108 product in 62 ribogroups) and 260 Listeria spp. isolates (174 environmental and 86 product in 30 ribogroups) were identified and ribotyped. The overall prevalence of E. coli (0.08%), all Listeria spp. (0.35%) and L. monocytogenes (0.23%) was very low. Some 10 E. coli ribogroups and 14 Listeria spp. ribogroups showed evidence for persistence, defined as the isolation of the same strain, from the same site, over a prolonged time period. The majority of E. coli strains were product niche oriented whilst the majority of Listeria spp. strains were environmental niche oriented. CONCLUSION: Current UK high-risk food factory designs, personnel hygiene and cleaning and disinfection regimes are sufficient to control Listeria spp. and E. coli to very low levels. SIGNIFICANCE AND IMPACT OF THE STUDY: Persistent strains of these organisms, however, can remain within factory high-risk production areas over considerable time periods, warranting an examination of the strain persistence mechanisms and alternative hygiene controls.     

Evaluation of a new chromogenic agar for the detection of Listeria in food

AIM: Rapid identification of Listeria in food is important in protecting consumers from infection. The development of chromogenic media such as agar Listeria according to Ottaviani and Agosti (ALOA) has allowed more rapid detection of Listeria monocytogenes, with presumptive identification of this pathogenic species after only 24 h of incubation. The aim of this study was to evaluate Oxoid chromogenic Listeria agar (OCLA) in comparison with ALOA and a traditional, nonchromogenic medium, Oxford agar. METHODS AND RESULTS: Media were compared using pure cultures, spiked food samples and naturally contaminated samples. Whilst development of typical colony morphology took 48 h on Oxford agar, Listeria spp. were frequently detected after 24 h of incubation on OCLA and ALOA. There was no significant difference in recovery between the two chromogenic media. CONCLUSIONS: Results indicate that OCLA gives equivalent recovery of Listeria spp. compared with ALOA. Whilst L. monocytogenes was frequently detected after 24 h of incubation, a 48-h incubation time was necessary to ensure detection of both L. monocytogenes and other Listeria spp. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown that a commercially available chromogenic medium other than ALOA is appropriate for use in the international standard method. The commercial availability of more than one medium will facilitate the more widespread use of the method, thus increasing confidence in the ability to detect L. monocytogenes in food in the presence of other Listeria spp.     

Antimicrobial activity of Enterococcus faecium against Listeria spp.

Listeria spp. have been isolated from vegetation, silage, the intestinal tracts of animals and foods such as milk and cheese. Lisleria spp. are taxonomically related to lactobacilli (Seeliger & Jones 1986) and some bacteriocins produced by lactic acid bacteria will inhibit growth of Listeria spp. Bacteriocins such as nisin from Lactococcus lactis and pediocin A from Pediococcus pentosoreus, are active against microorganisms from several Gram-positive genera, and will inhibit L. monocytogenes. Bacteriocins (e.g. helveticin J and lactacin F) which only inhibit strains closely related to the producing micro-organism are not effective against L. monocytogenes     

Incidence of Listeria spp. and Listeria monocytogenes in a Poultry Processing Environment and in Poultry Products and Their Rapid Confirmation by Multiplex PCR

Volume 60, no. 12, p. 4602: Fig. 1 should appear as shown below. FIG. 1. Incidence of L. monocytogenes and other Listeria spp. in a poultry processing environment and in raw and cooked poultry products from March to September 1992. (A) Raw (R) and cooked (C) areas within the processing environment. (B) Raw and cooked poultry products. (box), L. monocytogenes; (box), Listeria spp. (not L. monocytogenes); (symbl), no Listeria spp. [This corrects the article on p. 4600 in vol. 60.].     

Evaluation of the MicroFoss system for the detection of Listeria species in environmental samples

The MicroFoss system was evaluated for its ability to detect Listeria species in environmental samples. The sensitivity and specificity of the MicroFoss were determined in relation to a standard culture method for Listeria detection. The sensitivities of both the MicroFoss and standard culture methods were similar (88.4%-MicroFoss, 90.7%-Culture) based on the total number of positive results obtained by both methods. The MicroFoss system detected Listeria spp. in 12 samples, which were not detected by culture, and the culture method detected Listeria spp. in 15 samples, which were not detected by the MicroFoss method. This was likely due to uneven distribution of low levels of Listeria organisms in the split sponge samples used to assess the performance of these test methods. The specificity value determined for the MicroFoss system was 92.7%. The majority of microbes causing false positive results in the MicroFoss system were Bacillus species, which were readily distinguishable from Listeria species by a simple Gram stain and morphological features. Listeria monocytogenes (89.4%-MicroFoss, 88.0%-Culture) and Listeria innocua (8.8%-MicroFoss, 7.7%-Culture) were the most common isolates of Listeria detected by the two test methods, with L. monocytogenes being the most predominant isolate detected. The highly comparable results and rapid nature of the MicroFoss system demonstrate its effectiveness as a detection system for species of Listeria in environmental samples. The fact that the sensitivity of the MicroFoss system was similar to that of the culture method and the Listeria results were obtained within 48 h of testing, support the use of the MicroFoss as an alternative rapid method for screening large numbers of environmental samples for Listeria spp.     

Development and evaluation of a 24 well microtitre plate method for isolation of Listeria spp. or Listeria monocytogenes from foods

A rapid and simple method (24M) using 24 well microtitre plates was developed to determine the presence of Listeria monocytogenes or Listeria spp. in food samples. The 24M was composed of two 24 well microtitre plates connected with a yellow tip. The 24M was evaluated with pathogen cocktails and ground beef samples and compared with the conventional method for presumptive identification of Listeria spp. Only food-borne pathogen cocktails and ground beef samples containing L. monocytogenes or Listeria spp. showed a positive reaction in 24M after 24 h incubation at 35 degrees C. Test results were the same with the conventional method and the 24M method and showed high efficiency for recovery of Listeria spp. from foods. This new, convenient and economical method can isolate Listeria spp. simultaneously from 24 different food samples.     

Comparison of lithium chloride-phenylethanol-moxalactam and modified Vogel Johnson agars for detection of Listeria spp. in retail-level meats, poultry, and seafood

The effectiveness of Modified Vogel Johnson agar and lithium chloride-phenylethanol-moxalactam agar for detection of Listeria spp. in foods was compared by using the media to analyze retail-level meat, poultry, and seafood both by direct plating and in conjunction with a three-tube most-probable-number enrichment. The most-probable-number protocol detected Listeria species, including Listeria monocytogenes, in a substantial portion of the fresh meat and seafood samples. In most instances the Listeria levels were less than 2 CFU/g, which precluded detection by direct plating. Modified Vogel Johnson agar performed as well as did lithium chloride-phenylethanol-moxalactam agar and was considerably easier to use because of its ability to differentiate Listeria spp. from other microorganisms.     

Comparison of lithium chloride-phenylethanol-moxalactam and modified Vogel Johnson agars for detection of Listeria spp. in retail-level meats, poultry, and seafood.

The effectiveness of Modified Vogel Johnson agar and lithium chloride-phenylethanol-moxalactam agar for detection of Listeria spp. in foods was compared by using the media to analyze retail-level meat, poultry, and seafood both by direct plating and in conjunction with a three-tube most-probable-number enrichment. The most-probable-number protocol detected Listeria species, including Listeria monocytogenes, in a substantial portion of the fresh meat and seafood samples. In most instances the Listeria levels were less than 2 CFU/g, which precluded detection by direct plating. Modified Vogel Johnson agar performed as well as did lithium chloride-phenylethanol-moxalactam agar and was considerably easier to use because of its ability to differentiate Listeria spp. from other microorganisms.     

Antimicrobial resistance of Listeria spp. recovered from processed bison

AIMS: The current study examined the antimicrobial susceptibility of 86 Listeria spp. isolated from processed bison carcasses. MATERIALS AND METhods: Susceptibility to 25 antimicrobial agents was determined using E-test and National Antimicrobial Resistance Monitoring System (NARMS) panels. Most Listeria isolates (88-98%) exhibited resistance to bacitracin, oxacillin, cefotaxime, and fosfomycin. Resistance to tetracycline (18.6%) was also common. Of the 16 tetracycline-resistant Listeria isolates, 15 carried tetM and 2 contained integrase of Tn1545 transposons. Rifampicin and trimethoprim-sulfamethoxazole were the most active antimicrobial agents against Listeria spp., with a MIC(90) of 0.38 microg ml(-1). Ampicillin, erythromycin, penicillin, gentamicin, and tobramycin also exhibited good activity against Listeria spp., with MIC(90) not exceeding 1 microg ml(-1). Differences in resistance among Listeria spp. was displayed, as Listeria innocua strains were more resistant than other Listeria species. CONCLUSIONS: The study showed that Listeria monocytogenes strains from bison were susceptible to the antibiotics most commonly used to treat human listeriosis. However, the presence of antimicrobial resistance in L. innocua indicates the potential for transfer of resistance and a conjugative transposon to L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of our study will provide useful information for the development of public health policy in the use of antimicrobials in food animal production.     

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 .     

Specific detection and quantitative enumeration of Listeria spp. using fluorescent in situ hybridization in combination with filter cultivation (FISHFC)

Aims:  To design a rapid specific method for enumeration of viable Listeria spp. using the fluorescence in situ hybridization with filter cultivation (FISHFC) method.
Methods and Results:  The probe, Lis-1400, was designed from the 23S rRNA region of the Listeria genome, and labelled with 5'-carboxy-tetramethyl-rhodamine- N - hydroxy-succinimide-ester. Fluorescence was observed for all Listeria species but not for any organisms from the other genera, suggesting Lis-1400 is highly specific for Listeria spp. For purposes of filter cultivation prior to hybridization, hydrophilic polypropylene membrane filters gave better contrast between fluorescing colonies and background fluorescence. This was because of a high S/N ratio (fluorescence intensity of each microcolony/fluorescence intensity of background noise) after FISH treatment. Results were achievable in 14 h using Lis-1400-aided FISHFC as compared with 4–7 days required for confirmation of Listeria spp. by conventional plate count methods. Moreover, viable Listeria counts in selected food samples showed no significant differences between Lis-1400-aided FISHFC and conventional methods.
Conclusions:  The Lis-1400-aided FISHFC method is more efficient than conventional methods for enumeration of viable Listeria spp. in food samples.
Significance and Impact of the Study:  For enumeration of Listeria spp., Lis-1400-aided FISHFC method is equally accurate yet faster than conventional plate count methods, and can be valuable in the control of listeriosis.     

Direct identification in food samples of Listeria spp. and Listeria monocytogenes by molecular methods

A new molecular approach for the detection and identification of Listeria spp. and Listeria monocytogenes in food is presented here. The method is based on the PCR amplification of a fragment of the iap gene from the five species belonging to the genus and on the analysis of the PCR products obtained by denaturing gradient gel electrophoresis (DGGE). The protocol was first optimized by using strains from international collections. Based on the differences present in the sequences amplified, it was possible to obtain species-specific DGGE migration that allowed fast and easy identification of L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri, and L. ivanovii. Moreover, for L. monocytogenes serotypes, partial differentiation was possible. The optimized protocol was used for identification of Listeria strains traditionally isolated from food and for direct detection and identification of Listeria members in food after an overnight enrichment. Identification of 48 food isolates and direct detection of Listeria spp. in 73 food samples show the potential of the method that can be used as a fast screening test to investigate the presence of Listeria spp. and L. monocytogenes in food.     

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.     

Incidence of Listeria spp. and Listeria monocytogenes in a poultry processing environment and in poultry products and their rapid confirmation by multiplex PCR.

The incidence of Listeria spp. and Listeria monocytogenes in a poultry processing plant and in raw and cooked poultry products was determined over a 6-month period. Within the raw and cooked poultry processing environments, 46% (36 of 79) and 29% (51 of 173) of the samples contained Listeria spp. while 26% (21 of 79) and 15% (27 of 173) contained L. monocytogenes, respectively. Various sites within the processing environment were found to be consistently positive for L. monocytogenes throughout the entire sampling period. Of the raw and cooked products tested, 91% (53 of 58) and 8% (8 of 96) were found to contain Listeria spp. while 59% (34 of 58) and 0% (0 of 96) contained L. monocytogenes, respectively. Although L. monocytogenes was not detected in the cooked products examined, the presence of other Listeria spp. highlights the potential which exists for postprocessing contamination. Multiplex PCR proved to be a convenient and time-saving technique for rapid confirmation of Listeria spp. and L. monocytogenes in a single reaction.     

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.     

Design and evaluation of 16S rRNA-targeted peptide nucleic acid probes for whole-cell detection of members of the genus Listeria

Six fluorescein-labeled peptide nucleic acid oligomers targeting Listeria-specific sequences on the 16S ribosomal subunit were evaluated for their abilities to hybridize to whole cells by fluorescence in situ hybridization (FISH). Four of these probes yielded weak or no fluorescent signals after hybridization and were not investigated further. The remaining two FISH-compatible probes, LisUn-3 and LisUn-11, were evaluated for their reactivities against 22 Listeria strains and 17 other bacterial strains belonging to 10 closely related genera. Hybridization with BacUni-1, a domain-specific eubacterial probe, was used as a positive control for target accessibility in both Listeria spp. and nontarget cells. RNase T1 treatment of select cell types was used to confirm that positive fluorescence responses were rRNA dependent and to examine the extent of nonspecific staining of nontarget cells. Both LisUn-3 and LisUn-11 yielded rapid, bright, and genus-specific hybridizations at probe concentrations of approximately 100 pmol ml(-1). LisUn-11 was the brightest probe and stained all six Listeria species. LisUn-3 hybridized with all Listeria spp. except for L. grayi, for which it had two mismatched bases. A simple ethanolic fixation yielded superior results with Listeria spp. compared to fixation in 10% buffered formalin and was applicable to all cell types studied. This study highlights the advantages of peptide nucleic acid probes for FISH-based detection of gram-positive bacteria and provides new tools for the rapid detection of Listeria spp. These probes may be useful for the routine monitoring of food production environments in support of efforts to control L. monocytogenes.     

Simultaneous quantitative detection of Listeria spp. and Listeria monocytogenes using a duplex real-time PCR-based assay

We report a duplex real-time PCR-based assay for the simultaneous quantitative detection of Listeria spp. and the food-borne pathogen Listeria monocytogenes. The targets of this single tube reaction were the 23S rDNA and hly genes of Listeria spp. and L. monocytogenes, respectively. Our assay was efficient, 100% selective (i.e., it allowed accurate simultaneous identification of 52 L. monocytogenes and 120 Listeria spp. strains through the FAM-labelled hly and the VIC-labelled 23S rDNA probes, respectively); and had a detection limit of one target molecule in 100% (23S rDNA) and 56% (hly) of the reactions. Simultaneous quantification was possible along a 5-log dynamic range, with an upper limit of 30 target molecules and R2 values > 0.995 in both cases. Our results indicate that this assay based on the amplification of the 23S rDNA gene can accurately quantify any mixture of Listeria species and simultaneously unambiguously quantify L. monocytogenes.