Serotyping and bacteriophage typing have traditionally been used for intraspecies differentiation of L. monocyto-genes isolates and epidemiological survey of listeriosis. An improved discrimination between L. monocytogenes isolates has been obtained following the development of multilocus enzyme electrophoresis, which differentiates isolates according to the mobility of a great number of housekeeping enzymes. Several DNA-based typing methods such as restriction enzyme analysis (REA) of chromosomal DNA, plasmid profiling, restriction fragment length polymorphism (RFLP), and PCR-based fingerprinting techniques have also been developed for this purpose. Some standardized protocols have been defined to allow comparisons between laboratories and to favor the recognition of potential outbreaks as quickly as possible. For that aim, ribotyping, an RFLP technique that allows the analysis of the rRNA gene restriction patterns after hybridization with a rRNA gene, has been automatized (Riboprinter Qualicon, Willmingtonn, DE). A standardized protocol of PFGE has also been developed by a national network of public health and food regulatory laboratories in the United States (PulseNet). PFGE patterns identified by PulseNet are stored in a national electronic database maintained at the Centers for Disease Control and Prevention (Atlanta, GA). Fingerprinting techniques based on PCR amplification of DNA segments flanking either undetermined sequences [RAPD and arbitrarily primed PCR (AP-PCR)] or defined and conserved sequences [rRNA genes (PCR ribotyping), the repetitive elements REP and ERIC (rep-PCR) and infrequent DNA restriction fragments (IRS-PCR and AFLP)] have also been used to study the genetic structure
and epidemiology of L. monocytogenes. , ] Restriction endonuclease analysis of PCR-amplified virulence-associated genes (RE-PCR) has also been devised for that aim.[30,31] More recently, DNA sequence-based subtyping strategies were developed to reduce interpretation ambiguity and to make easier the exchange and comparison of subtype data among laboratories. These methods to determine allelic variations include pyrosequencing of a short sequence of the inlB gene, multilocus sequence typing (MLST) of several housekeeping genes, and sequencing of multiple genes with various evolving speeds (virulence genes, stress response genes, intergenic region, and housekeeping genes).[32-34] Finally, DNA microarrays were found to be promising tools for differentiating among closely related L. monocytogenes isolates.[35,36]
Studies of the genetic diversity and population structure of L. monocytogenes by using many of the above methods revealed that this species is composed of two distant phylogenetic divisions of strains, which are correlated with the flagellar antigen groups, division (division I, which is composed of strains of serotypes 1/ 2a and 1/2c, and division II, which is composed of strains of serotypes 1/2b and 4b). An additional division has also been proposed based on the sequence of several genes involved in virulence. This latter division, consisting of serotypes 4a and 4c strains, has recently been shown to be a branch of the first division. A mismatch amplification mutation (MAMA-PCR) assay targeting the hemolysin gene has been developed to characterize L. monocytogenes isolates with regard to these lineage types.
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