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).

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.

Listeriosis is a rare but severe disease, mainly caused by Listeria monocytogenes. This study shows the results of the laboratory-based surveillance of Listeriosis in Belgium over the period 1985–2014. Besides the incidence and some demographic data we present also more detailed microbiological and molecular characteristics of human strains isolated since 2000. The strains from the latter period were compared to food and animal strains from the same period. Our study shows that different food matrices were commonly contaminated with L. monocytogenes presenting the same PFGE profile as in patient’s isolates. Since 1985, we observed a significant decrease in incidence of the Materno-Neonatal cases (from 0.15 to 0.04 cases /100,000 inhabitants-year), which is probably to be attributed to active prevention campaigns targeting pregnant women. Despite the strengthening of different control measures by the food industry, the incidence of non-Materno-Neonatal listeriosis increased in Belgium (from 0.3 to 0.7 cases /100,000 inhabitants-year), probably due to the rise of highly susceptible patients in an aging population. This significant increase found in non-Materno-Neonatal cases (slope coefficient 7.42%/year, P<0.0001) can be attributed to significant increase in incidence of isolates belonging to serovars 1/2a (n = 393, slope coefficient 6.62%/year, P<0.0001). Although resistance to antimicrobials is rare among L. monocytogenes isolates, a trend to increasing MIC values is evident with chloramphenicol, amoxicillin, tetracycline and ciprofloxacin. We show that fluoroquinolone resistance is not linked to chromosomal mutations, but caused by a variety of efflux pumps. Our study also shows that huge majority of known underlying pathologies (426 out of 785 cases) were cancers (185/426, 43.1%) and haematological malignancies (75/185, 40.5%). Moreover the risk population is susceptible to low levels of contamination in food stressing the need of prevention campaigns specifically targeting these persons.

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, metalresistance profile and pulsovar, suggesting a clonal relationship. This is the first report to identify accurately potential listeriosis episodes occurring in Belgium.