Differentiation of the genus Listeria from other gram-positive species based on low molecular weight (LMW) RNA profiles.

Low molecular weight RNA (LMW RNA; 5S rRNA and tRNAs) profiles of several Gram-positive species were generated on 9% denaturing polyacrylamide gels. The profiles of five Listeria spp. (L. innocua, L. ivanovii, L. monocytogenes, L. seeligeri and L. welshimeri) were identical when cultured in three different media (APT, BHI and TSBYE), either shaken or statically, both at 30 and 37 degrees C. Twenty-six strains from 15 other species representing seven different genera were also compared. Each species exhibited a characteristic profile. Strain variants of the same species gave identical profiles. The technique represents a simple, reproducible approach to the identification of species and possibly of relationships between species. The taxonomic and phylogenetic implications, particularly with respect to Listeria spp., Brochothrix thermosphacta and the lactic acid bacteria, are considered.     

RAPD typing for distinguishing species and strains in the genus Listeria

The randomly amplified polymorphic DNA (RAPD) technique was employed in the development of a typing protocol for Listeria isolates, particularly Listeria monocytogenes strains. A single strain of L. monocytogenes was used and 200 random decamer primers were screened for their discriminatory abilities by visualizing the amplification products electrophoretically. Three candidate primers displaying potentially useful banding patterns were selected and tested against 52 L. monocytogenes strains, encompassing 11 serotypes, and 12 other strains representing five other Listeria spp. Thirty-four banding profiles were obtained with one particular primer. RAPD analysis allowed differentiation between Listeria spp. and was found to further subdivide strains of the same serotype. Where only one primer was used strains from different serotypes were occasionally found to produce identical banding profiles. RAPD analysis, which in our hands proved to be reproducible, shows much promise as a molecular alternative to traditional L. monocytogenes typing protocols.     

ERIC-PCR技术在李斯特氏菌种、菌株鉴定中的应用

应用肠杆菌基因间重复一致序列聚合酶链反应技术（ERIC-PCR）对李斯特氏菌基因组DNA进行分析,结果显示,李斯特氏菌种间DNA指纹图谱带型差异较大;单核细胞增生性李斯特氏菌株间及相同血清型不同来源的菌株,其DNA指纹图谱带型也有明显差异。在单核细胞增生性李斯特氏菌各株的DNA指纹图谱中发现1600bp的种专一性扩增带。结果表明,ERIC-PCR技术可用于李斯特氏菌种、菌株的鉴定及进一步分型研究。 Abstract:Enterobacteia repetitive intergenic consensus sequences-based PCR(ERIC-PCR) was used to generate DNA fingerprints for Listeria spp.We got the specific profiles with ERIC-PCR technique that enables to identify Listeria species and the L.monocytogenes strains of different sterotype,and the same sterotype of L.monocytogenes from different sources also could be identified.Moreover,the species-spcific 1600bp DNA fragment was obtained from the fingerprint of L.monocytogenes.The study indicates that ERIC-PCR technique can be used in the identification of Listeria species and strains and its further typing,which is simple and quickly.     

Random amplification of polymorphic bacterial DNA: evaluation of 11 oligonucleotides and application to food contaminated with Listeria monocytogenes

CH. NIEDERHAUSER, CH. HÖFELEIN, M. ALLMANN, P. BURKHALTER, J. LÜTHY AND U. CANDRIAN. 1994. The polymerase chain reaction was used to obtain randomly-amplified polymorphic DNA (RAPD) profiles from Listeria spp. and enterobacteria. Eleven different oligonucleotides were evaluated. Only one, HR4 (19mer), generated reproducible and specific profiles for Listeria spp., while results for enterobacteria were controversial. A total of 57 different Listeria strains were subjected to the RAPD analysis and 27 different profiles were recognized. RAPD typing allowed strains of the same serotype to be distinguished but the same profile was obtained from different serotypes of L. monocytogenes in three cases and in one case two different serotypes of L. innocua yielded the same profile. RAPD-typing with HR4 allowed L. monocytogenes contamination in several food outlets to be traced back to a food processing plant. In additional experiments, the general utility of this RAPD system in typing Yersinia enterocolitica, verotoxigenic Escherichia coli and Salmonella enteritidis was evaluated.     

Diversity of Listeria species in urban and natural environments

A total of 442 Listeria isolates, including 234 Listeria seeligeri, 80 L. monocytogenes, 74 L. welshimeri, 50 L. innocua, and 4 L. marthii isolates, were obtained from 1,805 soil, water, and other environmental samples collected over 2 years from four urban areas and four areas representing natural environments. Listeria spp. showed similar prevalences in samples from natural (23.4%) and urban (22.3%) environments. While L. seeligeri and L. welshimeri were significantly associated with natural environments (P ≤ 0.0001), L. innocua and L. monocytogenes were significantly associated with urban environments (P ≤ 0.0001). Sequencing of sigB for all isolates revealed 67 allelic types with a higher level of allelic diversity among isolates from urban environments. Some Listeria spp. and sigB allelic types showed significant associations with specific urban and natural areas. Nearest-neighbor analyses also showed that certain Listeria spp. and sigB allelic types were spatially clustered within both natural and urban environments, and there was evidence that these species and allelic types persisted over time in specific areas. Our data show that members of the genus Listeria not only are common in urban and natural environments but also show species- and subtype-specific associations with different environments and areas. This indicates that Listeria species and subtypes within these species may show distinct ecological preferences, which suggests (i) that molecular source-tracking approaches can be developed for Listeria and (ii) that detection of some Listeria species may not be a good indicator for L. monocytogenes.     

An improved amplified fragment length polymorphism (AFLP) protocol for discrimination of Listeria isolates

Nine restriction enzyme combinations and 108 different primer combinations were initially tested for suitability for amplified fragment length polymorphism (AFLP) analysis of Listeria monocytogenes; the combination of HindIII and HpyCH4IV showed consistently strong signals on gels, amplified an adequate number of DNA fragments and detected polymorphism among closely related strains based on AscI macrorestriction profiles. AFLP also distinguished between L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri and L. grayi species. All Listeria species showed species-specific clusters, with less than 33% similarity between different species. A total of 34 L. monocytogenes strains were characterised by using both AFLP and pulsed-field gel electrophoresis (PFGE). The results of AFLP analysis of L. monocytogenes strains were in concordance with those obtained by PFGE. Both methods identified 29 different genotypes of L. monocytogenes and had a high discrimination index (> 0.999). By combining the results of AFLP and PFGE, subtype discrimination was further improved. Numerical analysis of both AFLP and PFGE profiles yielded three genomic groups of L. monocytogenes strains. AFLP was found to be faster and less labour-intensive than PFGE. We conclude that the AFLP protocol is a highly discriminatory, reproducible and valuable tool in characterisation of Listeria strains and may also be suitable for Listeria species identification.     

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.     

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.     

API Listeria, a new and promising one-day system to identify Listeria isolates.

API Listeria is a new 10-test strip for 24-h biochemical identification of Listeria isolates. With this commercial system, 85% of 646 Listeria strains, including atypical isolates selected for this study, were recognized at the species and subspecies level without a complementary test. A new test differentiates Listeria monocytogenes from L. innocua on the basis of the absence of arylamidase from the former. With this system, 97.7% (252 of 258) of the L. monocytogenes strains tested were correctly identified and differentiated from 99.4% (175 of 176) of the L. innocua strains also tested. Gram-positive bacteria other than Listeria spp. gave quite different biochemical patterns. This system considerably reduced the time needed for conventional identification, since results were available within 18 to 24 h.     

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.     

Rapid molecular identification of Listeria species by use of real-time PCR and high-resolution melting analysis

Identification of Listeria species via a molecular method is critical for food safety and clinical diagnosis. In this study, an assay integrating real-time quantitative PCR (Q-PCR) with high-resolution melting (HRM) curve analysis was developed and assessed for rapid identification of six Listeria species. The ssrA gene, which encodes a transfer-messenger RNA (tmRNA) is conserved and common to all bacterial phyla, contains a variable domain in Listeria spp. Therefore, Q-PCR and a HRM profile were applied to characterize this gene. Fifty-three Listeria species and 45 non-Listeria species were detected using one primer set, with an accuracy of 100% in reference to conventional methods. There was a 93.3% correction rate to 30 artificially contaminated samples. Thus, Q-PCR with melting profiling analysis proved able to identify Listeria species accurately. Consequently, this study demonstrates that the assay we developed is a functional tool for rapidly identifying six Listeria species, and has the potential for discriminating novel species food safety and epidemiological research.     

API Listeria, a new and promising one-day system to identify Listeria isolates.

API Listeria is a new 10-test strip for 24-h biochemical identification of Listeria isolates. With this commercial system, 85% of 646 Listeria strains, including atypical isolates selected for this study, were recognized at the species and subspecies level without a complementary test. A new test differentiates Listeria monocytogenes from L. innocua on the basis of the absence of arylamidase from the former. With this system, 97.7% (252 of 258) of the L. monocytogenes strains tested were correctly identified and differentiated from 99.4% (175 of 176) of the L. innocua strains also tested. Gram-positive bacteria other than Listeria spp. gave quite different biochemical patterns. This system considerably reduced the time needed for conventional identification, since results were available within 18 to 24 h.     

The incidence of Listeria spp. in soft cheeses, butter and raw milk in the province of Bologna

Samples of soft cheese, butter and raw milk were examined for Listeria species. Listeria monocytogenes (serotype 1, haemolytic and virulent for mice) and L. innocua (the only other Listeria sp. isolated) were each found in 2/21 (1.6%) of soft cheese samples. Five per cent of butter samples were contaminated with L. innocua. No Listeria spp. were detected in 40 raw milk samples. The results were compared with similar studies in Italy and abroad.     

Cholesterol oxidase from Rhodococcus equi is likely the major factor involved in the cooperative lytic process (CAMP reaction) with Listeria monocytogenes

J.F. FERNÁNDEZ-GARAYZÁBAL, C. DELGADO, M.M. BLANCO, G. SUÁREZ AND L. DOMÍNGUEZ. 1996. The CAMP reaction between Listeria monocytogenes and Rhodococcus equi was studied by a diffusion assay. Listeria monocytogenes displayed identical cooperative haemolytic effect with supernatant cultures of equi or with commercial cholesterol oxidase (COX). This result, even with enzymes of different sources (commercial COX is obtained from Pseudomonas spp.) suggests that this enzyme secreted by R. equi has a crucial role in the synergistic haemolytic (CAMP) reaction with L. monocytogenes . The mechanism of the cooperative lytic process between L. monocytogenes and R. equi may represent a different and novel mechanism reaction, in which the COX may not act as a conventional second-step factor, and a reaction different to the direct interaction with the cholesterol of the erythrocyte membrane may be involved.     

The incidence of Listeria spp. in soft cheeses, butter and raw milk in the province of Bologna

Samples of soft cheese, butter and raw milk were examined for Listeria species. Listeria monocytogenes (serotype 1, haemolytic and virulent for mice) and L. innocua (the only other Listeria sp. isolated) were each found in 2/21 (1.6%) of soft cheese samples. Five per cent of butter samples were contaminated with L. innocua . No Listeria spp. were detected in 40 raw milk samples. The results were compared with similar studies in Italy and abroad. and accepted 14 June 1989     

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.     

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.     

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.     

Rapid RAPD analysis for distinguishing Listeria species and Listeria monocytogenes serotypes using a capillary air thermal cycler

Using a novel capillary thermal cycler, randomly amplified polymorphic DNA (RAPD) generated DNA fingerprints were obtained in 3 h. The RAPD profiles were produced using a random 10-mer primer (5'-ACCGCCTGCT-3') which discriminated between different Listeria spp. Unique fragment profiles of Listeria monocytogenes serotypes were produced from serotypes 1a, 2, 3a, 4ab, 4a and 4c but serotypes, 1/2a, 4b, 4d and 7 had similar profiles.     

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.