Hypo- and Hyper-Virulent Listeria monocytogenes Clones Persisting in Two Different Food Processing Plants of Central Italy

A total of 66 Listeria monocytogenes (Lm) isolated from 2013 to 2018 in a small-scale meat processing plant and a dairy facility of Central Italy were studied. Whole Genome Sequencing and bioinformatics analysis were used to assess the genetic relationships between the strains and investigate persistence and virulence abilities. The biofilm forming-ability was assessed in vitro. Cluster analysis grouped the Lm from the meat plant into three main clusters: two of them, both belonging to CC9, persisted for years in the plant and one (CC121) was isolated in the last year of sampling. In the dairy facility, all the strains grouped in a CC2 four-year persistent cluster. All the studied strains carried multidrug efflux-pumps genetic determinants (sugE, mdrl, lde, norM, mepA). CC121 also harbored the Tn6188 specific for tolerance to Benzalkonium Chloride. Only CC9 and CC121 carried a Stress Survival Islet and presented high-level cadmium resistance genes (cadA1C1) carried by different plasmids. They showed a greater biofilm production when compared with CC2. All the CC2 carried a full-length inlA while CC9 and CC121 presented a Premature Stop Codon mutation correlated with less virulence. The hypo-virulent clones CC9 and CC121 appeared the most adapted to food-processing environments; however, even the hyper-virulent clone CC2 warningly persisted for a long time. The identification of the main mechanisms promoting Lm persistence in a specific food processing plant is important to provide recommendations to Food Business Operators (FBOs) in order to remove or reduce resident Lm

Expression of the Jaagsiekte Sheep Retrovirus Envelope Glycoprotein Is Sufficient To Induce Lung Tumors in Sheep

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA). The expression of the JSRV envelope (Env) alone is sufficient to transform a variety of cell lines in vitro and induce lung cancer in immunodeficient mice. In order to determine the role of the JSRV Env in OPA tumorigenesis in sheep, we derived a JSRV replication-defective virus (JS-RD) which expresses env under the control of its own long terminal repeat (LTR). JS-RD was produced by transiently transfecting 293T cells with a two plasmid system, involving (i) a packaging plasmid, with the putative JSRV packaging signal deleted, expressing the structural and enzymatic proteins Gag, Pro, and Pol, and (ii) a plasmid which expresses env in trans for JS-RD particles and provides the genomes necessary to deliver JSRV env upon infection. During the optimization of the JS-RD system we determined that both R-U5 (in the viral 5′ LTR) and the env region are important for JSRV particle production. Two independent experimental transmission studies were carried out with newborn lambs. Four of five lambs inoculated with JS-RD showed OPA lesions in the lungs at various times between 4 and 12 months postinoculation. Abundant expression of JSRV Env was detected in tumor cells of JS-RD-infected animals and PCR assays confirmed the presence of the deleted JS-RD genome. These data strongly suggest that the JSRV Env functions as a dominant oncoprotein in the natural immunocompetent host and that JSRV can induce OPA in the absence of viral spread

Listeria monocytogenes biofilm production on food packaging materials submitted to physical treatment

Listeria monocytogenes (L.m.) is an agent of serious foodborne illness. It is a major concern for the food industry, since microorganism, growing in biofilms is protected against cleaning and disinfection and is difficult to eradicate. Aim of this study was to develop a protocol to assess the ability of two flexible packaging materials, named HGP40 and GND35, submitted to corona discharge treatment, to limit the production of L.m. biofilm at 12±1°C. Two strains were selected for this study: L.m ATCC 7644 and L. m. EURL 12MOB098LM isolated from dairy products. Results suggest that both L.m. strains were able to form biofilm on packaging materials tested. The differences on HGP40 and GND35s treated and not treated surfaces were not statistically significant

Listeria monocytogenes persistence in food processing environments: Whole Genome Sequencing and in vitro assessment of disinfectants resistance and biofilm forming ability

Listeria monocytogenes (Lm) is the causative agent of listeriosis, an invasive disease primarily affecting immunocompromised people, the elderly, children and pregnant women, with high hospitalization (98.6%) and fatality rates (13.8%) 1. The disease is most commonly caused by eating contaminated food, in particular ready-to-eat. The ability of some strains to persist, even for years, in food processing environments can increase the risk of food contamination. Persistence can results from Lm survival after disinfection, thanks to protective biofilm formation, disinfectants and stresses resistance mechanisms or from the repeated reintroduction from raw materials 2 3. The identification of recurring highly genetically related isolates (Whole Genome Sequencing, WGS and core genome MLST, cgMLST) is necessary to define a strain persistent in a plant 4. The aim of this study was to evaluate persistence and resistance to commercial sanitizers commonly used in food processing environments, in Lm strains isolated within the laboratory activity of IZSUM (Istituto Zooprofilattico Sperimentale Umbria e Marche). Our approach was based on both WGS and in vitro assays

A Real-Time PCR Screening Assay for Rapid Detection of Listeria Monocytogenes Outbreak Strains

From January 2015 to March 2016, an outbreak of 23 human cases of listeriosis in the Marche region and one human case in the Umbria region of Italy was caused by Listeria monocytogenes strains showing a new pulsotype never described before in Italy. A total of 37 clinical strains isolated from patients exhibiting listeriosis symptoms and 1374 strains correlated to the outbreak were received by the Italian National Reference Laboratory for L. monocytogenes (It NRL Lm) of Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise (IZSAM) for outbreak investigation. A real-time PCR assay was purposely designed for a rapid screening of the strains related to the outbreak. PCR-positive strains were successively typed through molecular serogrouping, pulsed field gel electrophoresis (PFGE), and Next Generation Sequencing (NGS). Applying the described strategy, based on real-time PCR screening, we were able to considerably reduce time and costs during the outbreak investigation activities

Infection of lung epithelial cells and induction of pulmonary adenocarcinoma is not the most common outcome of naturally occurring JSRV infection during the commercial lifespan of sheep

AbstractJaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA). In this study, we followed over a 31-month period the natural transmission of JSRV in adult sheep and in their offspring. We established groups derived from flocks with either a high or low incidence of OPA and monitored virus transmission, clinical disease and macroscopic/microscopic lung lesions at necropsy. Results obtained show that (i) JSRV infection can occur perinatally or in the first few months of life in lambs and in adult sheep; (ii) only a minority of JSRV-infected animals develop clinical disease during their commercial lifespan; and (iii) JSRV is more readily detectable in peripheral blood leucocytes and lymphoid organs than in the lungs. These data support a model of opportunistic JSRV infection and tumorigenic conversion of type II pneumocytes/Clara cells in the lungs, while lymphoreticular cells serve as the principal virus reservoir

Large-scale phenotypic and genomic characterization of Listeria monocytogenes susceptibility to quaternary ammonium compounds

Listeria monocytogenes is a significant concern for the food industry due to its ability to persist in the food processing environment. Decreased susceptibility to disinfectants is one of the factors that contribute to the persistence of L. monocytogenes. The objective of this study was to explore the diversity of L. monocytogenes susceptibility to quaternary ammonium compounds (QACs) using 1,671 L. monocytogenes isolates. This was used to determine the phenotype-genotype concordance and characterize genomes of the QAC sensitive and tolerant isolates for stress resistance, virulence and plasmid replicon genes. Distribution of QAC tolerance genes among 37,897 publicly available L. monocytogenes genomes were also examined. The minimum inhibitory concentration to QACs was determined by the broth microdilution method and non-sequenced isolates (n=1,244) were whole genome sequenced. Genotype-phenotype concordance was 99% for benzalkonium chloride, DDAC and a commercial QAC based sanitizer. Prevalence of QAC tolerance genes was 23% and 28% in our L. monocytogenes collection and in the global dataset, respectively. qacH was the most prevalent gene in our collection (61%), with 19% prevalence in the global dataset. Notably, bcrABC was most common (72%) globally, while 25% in our collection. Prevalence of emrC and emrE was comparable in both datasets, 7% and 2%, respectively. Replicon genes, indicative of plasmid harborage, were detected in 44% of the isolates and associated with the QAC tolerant phenotype. The presented analysis is based on the biggest L. monocytogenes collection in diversity and quantity for characterization of the L. monocytogenes QAC tolerance at both phenotypic and genomic levels