Epidemiologia molecolare di Listeria monocytogenes: genotipizzazione, sierotipizzazione molecolare e analisi dei profili di espressione di geni implicati nei meccanismi di patogenesi e virulenza

Listeria monocytogenes is a intracellular bacterium widely distributed in the environment that causes severe diseases with a high fatality rate especially in immunocompromised individuals. Several molecular techniques have been developed for strains characterization because significant differences in the virulence profile of isolates have been demonstrated. In this study, L. monocytogenes strains (n=250) from different sources were analysed by single Enzyme Amplified Fragment Length Polymorphism (sAFLP). A representative subgroup was selected to compare the traditional gel electrophoresis with a chip-based microfluidic system (ExperionTM). Moreover, additional Listeria spp. isolates (n=109) were selected to standardize a new assay for serogroup identification in Real-time by using two different TaqMan-based Triplex-PCR and the results were compared with molecular reference method. Furthermore, by qRT-PCR, the expression levels of stress response and virulence genes were evaluated in isolates (n=20) from foods and human listeriosis. Particularly, the effects of sub-lethal concentration of benzalkonium chloride (10 ppm) on the expression profiles of sigB (stress-response sigma factor), mdrL and lde (efflux pumps), and orfA (putative mdrL repressor) were assessed. The antimicrobial activity of benzalkonium chloride (BC) was also investigated according to UNI EN 1276/2000. Finally, the expression levels of genes encoding the internalins A and B (inlA, inlB), Listeriolysin O (hly), the LIPI-1 transcriptional regulator (prfA), and bile salt hydrolase (bsh) were determined. Genotyping by sAFLP allowed to identify 13 clusters, included in two well defined groups and in agreement to the Lineage classification and epidemiological data on the prevalence of most common serotypes involved in human illness and foodstuffs contamination. The sAFLP has proved to be an accurate method with high discriminatory power for L. monocytogenes typing and its epidemiological usefulness was further confirmed in combination with automated microfluidic electrophoresis system, requiring lower samples volume and time analysis. Molecular serogrouping by Real-time PCR confirmed previous classification into four groups and allowed a rapid discrimination of 1/2a, 1/2b, 1/2c, and 4b serotypes, recognized in more than 90% of food and patients isolates. Therefore, the new approach can improve routine surveillance of listeriosis and epidemiological investigations. The benzalkonium chloride showed the antimicrobial activity in 70% of strains, particularly in human isolates and affected the target genes expression. Adaptation to BC determined a significant (p<0,05) overexpression of mdrl and sigB. All less sensitive food isolates showed the highest sigB, mdrL, and lde transcription levels. The efflux pumps could play a role in BC-resistance and the sub-lethal exposure to antimicrobial agent can lead cross-resistance between antibiotics and disinfectants. The expression levels of inlB, hly, prfA and bsh were higher in clinical than in food strains, except for inlA. However, the maximum levels of inlA were found in two human strains which also showed high levels of all virulence genes. A significant correlation between inlA and inlB expression was observed as between prfA and bsh, according to other studies. Furthermore, relevant differences (≤0,001) between Lineage I (1/2b, 4b) and II (1/2a, 3a, 1/2c) strains were found in mdrL, lde, inlA, prfA, and bsh melting temperature, suggesting the presence of mutations in genomic sequence. The characterization of more virulent strains and the comprehension of molecular mechanisms are essential for the epidemiological surveillance and the risk assessment of human listeriosis.