High-frequency endonuclease (REA) typing: results from the WHO collaborative study group on subtyping of Listeria monocytogenes.
ABSTRACT Eighty isolates of Listeria monocytogenes were typed by high-frequency restriction endonuclease analysis. Two laboratories participated in the study with two restriction enzymes each. The enzymes used were EcoRI, Hae1II, HhaI. and its isoschizomer Cfo1. EcoRI was less discriminatory than the other enzymes. The profiles generated by Hha1 and CfoI were not fully stable for some closely related isolates. The size and the number of restriction bands generated by Hha1 in one laboratory and its isoschizomer Cfo1 in another laboratory were directly comparable. This indicates that REA-typing may be used as a definitive typing method for Listeria monocytogenes if the typing procedure is standardized. The stability of the REA-types needs further elucidation in order to establish firm differentiation criteria for comparison of isolates.
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ABSTRACT: Large epidemics of diarrhoea associated with seafood consumption and Vibrio parahaemolyticus occurred during the austral summers of 2004 and 2005 in the environs of Puerto Montt, Chile (41 ° ° ° ° 29 ′ ′ ′ ′ S 72 ° ° ° ° 24 ′ ′ ′ ′ W). There are no reports of V. parahaemolyticus infections before 2004 in this region, their absence being explained by the low ocean temperatures which seldom reach 16 ° ° ° ° C. We analysed V. parahaemolyticus obtained from shellfish and clinical samples during epidemics. Isolates were examined using con-ventional protocols and an improved method for restriction enzyme analysis using total bacterial DNA which permits direct genome restriction enzyme anal-ysis by conventional gel electrophoresis (DGREA) with a similar discrimination index as restriction frag-ment length polymorphism-pulsed field gel electro-phoresis (RFLP-PFGE). Analysis of clinical samples showed that the epidemics were caused by the V. parahaemolyticus O3:K6 pandemic clonal group. On the other hand, analysis of shellfish samples dur-ing both epidemics showed that 53% contained V. parahaemolyticus (3–93 g − − − − 1). Detailed analysis of 50 positive shellfish samples showed that only three contained detectable levels of the pandemic clone. Most V. parahaemolyticus isolates obtained from shellfish corresponded to non-pandemic clones dif-ferentiated into 14 groups by DGREA. In summary, the causative agent during epidemics was only a minor component of a small but diverse population of V. parahaemolyticus in shellfish.
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ABSTRACT: In most developed countries a sharp increase in foodborne intoxications occurs since the last decade. Amongst the bacterial pathogens, the incidence rate is highest for Campylobacter jejuni and Salmonella spp. Nucleid acid based identification and detection methods have been developed for nearly all bacterial pathogens, based on probes in hybridisation assays or primers in PCR, NASBA or RT-PCR assays. As targets for molecular identification, virulence genes, the rRNA gene region or other specific sequences can be used and several commercialised systems are already available. For the (direct) detection of pathogens in food products, several problems may be encountered: PCR inhibition by food components, contamination in sensitive PCR assays, detection of living as well as dead cells. The latter problem can be solved by using mRNA as amplification target, but for routine applications the combination of a short culturing period with a less sensitive PCR is more suitable. Direct quantification of pathogens is possible with quantitative competitive PCR using an internal standard or with kinetic quantitative PCR (TaqMan or LightCycler commercial system). In bacterial typing, distinct types, strains or clones within a pathogenic bacterial species are differentiated which is important in epidemiological studies of foodborne outbreaks but also in the “from stable to table” investigation of the whole food production chain. Compared to the classical phenotypic typing techniques, molecular typing techniques have several advantages such as general applicability and a high discriminatory power. The currently available molecular techniques can be classified according to their working principle in PCR-mediated typing techniques (RAPD, rep-PCR), typing techniques combining PCR with restriction analysis (e.g.flaA typing of C. jejuni), typing techniques based on chromosomal restriction fragment length polymorphisms (e.g. ribotyping, pulsed field gel electrophoresis or PFGE), typing techniques combining restriction digestion with selective amplification (AFLP), and plasmid analysis. Both PFGE and AFLP are proposed as likely candidates for a uniform definite molecular typing approach using appropriate software for cluster analysis and database storing of the fingerprints. For Salmonella, two typing levels can be proposed: the first important level corresponds with the serovar level and the second level can be performed by classical phage typing or molecular typing revealing clonal lineage or strain level. Several molecular techniques have a serovar dependent discriminatory power with the greatest challenge presented by the highly clonal serovar Salmonella enteritidis.12/2001: pages 193-238;
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ABSTRACT: Listeria spp. are considered of interest in public health since their presence indicates the potencial existence of L. monocytogenes. Total cellular proteins and DNA from four strains of L. monocytogenes serotype 4, four strains of L. monocytogenes belonging to serotype 1, twelve strains of L. innocua, four strains of L. seeligeri and two strains of L. welshimeri isolated from ready–to–eat food were studied by SDS–PAGE and restriction endonuclease digestion. SDS–PAGE protein profiles obtained were species specific and could be evaluated by visual comparison. Enzyme for restriction endonuclease analysis was EcoRI, discriminating L. monocytogenes from other Listeria spp. These methodologies might be a helpful tool and a good alternative for epidemiological tracking of listeriosis in laboratories, where other methods are not available.Food Research International 01/2004; DOI:10.1016/j.foodres.2004.06.011 · 3.05 Impact Factor