RESUMO
A combination of four qualitative SYBR®Green qPCR screening assays targeting two levels of discrimination: Listeria genus (except Listeria grayi) and Listeria monocytogenes, is presented. These assays have been developed to be run simultaneously using the same polymerase chain reaction (PCR) programme. The paper also proposes a new validation procedure to specifically validate qPCR assays applied to food microbiology according to two guidelines: the ISO 22118 norm and the "Definition of minimum performance requirements for analytical methods of GMO testing". The developed assays target the iap, prs and hlyA genes that belong to or neighbour the virulence cluster of Listeria spp. The selected primers were designed to amplify short fragments (60 to 103 bp) in order to obtain optimal PCR efficiency (between 97 and 107 % efficiency). The limit of detection of the SYBR®Green qPCR assays is two to five copies of target genes per qPCR reaction. These assays are highly accurate (98.08 and 100 % accuracy for the Listeria spp. and L. monocytogenes assays, respectively).
Assuntos
Microbiologia de Alimentos/métodos , Listeria monocytogenes/genética , Listeria/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Listeria/isolamento & purificação , Listeria monocytogenes/isolamento & purificaçãoRESUMO
In the present study, three Listeria monocytogenes strains and one Listeria innocua strain out of a collection of 241 Listeria isolates from human and food-processing sources were found to display resistance to tetracycline (TC) due to the presence of the tet(M) gene. Through sequence analysis, it was shown that tet(M) genes in two of the isolates belong to sequence homology group (SHG) II, a group comprising chromosomally encoded tet(M) genes previously found in Staphylococcus aureus and in lactobacilli. The tet(M) genes of the two other L. monocytogenes strains were associated with a member of the Tn916-Tn1545 family of conjugative transposons and were closely related to SHG III, which harbours enterococcal tet(M) genes associated with Tn916. One of these transposon-containing strains was able to transfer the tet(M) gene to Enterococcus faecalis recipient strain JH2-2. Collectively, these sequence and conjugation data indicate that the acquisition of tet(M) by Listeria strains may be triggered by successive transfers between other Gram-positive organisms.
Assuntos
Microbiologia de Alimentos , Listeria/efeitos dos fármacos , Listeria/genética , Sequência de Bases , Bélgica , Primers do DNA , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , França , Humanos , Listeria/classificação , Listeria/isolamento & purificação , Listeriose/epidemiologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Tetraciclina/farmacologia , Resistência a Tetraciclina/genéticaRESUMO
The Belgian Listeria Reference Centre receives between 30 and 50 human clinical strains of Listeria monocytogenes per year. In general, epidemiological data are absent or incomplete, preventing recognition of episodes of listeriosis. However, data on a clonal relationship between strains can indirectly give an idea of the occurrence of episodes. Human isolates of L. monocytogenes from 2001 were serotyped, their arsenic-cadmium resistance profiles were determined, and they were pulsotyped with the application of pulsed-field gel electrophoresis using AscI and ApaI restriction endonucleases. On five occasions, two or more strains presented the same serovar, metal-resistance profile and pulsovar, suggesting a clonal relationship. This is the first report to identify accurately potential listeriosis episodes occurring in Belgium.