Affiliation:
1. Laboratoire de Microbiologie, Faculté de Pharmacie, Université de Bordeaux 2, Bordeaux
2. Laboratoire d'Ecologie Moléculaire, Université de Pau et des Pays de l'Adour, Pau, France
Abstract
ABSTRACT
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
Xmn
I or
Cfo
I and, if needed, S1 was digested by either
Alu
I or
Cla
I. 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.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
49 articles.
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