Phylogenetic Analysis and Genome-Wide Association Study Applied to an Italian Listeria monocytogenes Outbreak.

From May 2015 to March 2016, a severe outbreak due to Listeria monocytogenes ST7 strain occurred in Central Italy and caused 24 confirmed clinical cases. The epidemic strain was deeply investigated using whole-genome sequencing (WGS) analysis. In the interested area, the foodborne outbreak investigation identified a meat food-producing plant contaminated by the outbreak strain, carried by pork-ready-to-eat products. In the same region, in March 2018, the epidemic strain reemerged causing one listeriosis case in a 10-month-old child. The aim of this study was to investigate the phylogeny of the epidemic and reemergent strains over time and to compare them with a closer ST7 clone, detected during the outbreak and with different pulsed-field gel electrophoresis (PFGE) profiles, in order to identify genomic features linked to the persistence and the reemergence of the outbreak. An approach combining phylogenetic analysis and genome-wide association study (GWAS) revealed that the epidemic and reemergent clones were genetically closer to the ST7 clone with different PFGE profiles and strictly associated with the pork production chain. The repeated detection of both clones was probably correlated with (i) the presence of truly persistent clones and the repeated introduction of new ones and (ii) the contribution of prophage genes in promoting the persistence of the epidemic clones. Despite that no significant genomic differences were detected between the outbreak and the reemergent strain, the two related clones detected during the outbreak can be differentiated by transcriptional factor and phage genes associated with the phage LP-114.

Genetic relationships and biofilm formation of Listeria monocytogenes isolated from the smoked salmon industry.

Among pathogens, L. monocytogenes has the capability to persist on Food Processing Environment (FPE), first of all posing safety issues, then economic impact on productivity. The aim of this work was to determine the influence of biofilm forming-ability and molecular features on the persistence of 19 Listeria monocytogenes isolates obtained from FPE, raw and processed products of a cold-smoked salmon processing plant. To verify the phenotypic and genomic correlations among the isolates, different analyses were employed: serotyping, Clonal Complex (CC), core genome Multi-Locus Sequence Typing (cgMLST) and Single Nucleotide Polymorphisms (SNPs) clustering, and evaluation of the presence of virulence- and persistence-associated genes. From our results, the biofilm formation was significantly higher (*P < 0.05) at 37 °C, compared to 30 and 12 °C, suggesting a temperature-dependent behaviour. Moreover, the biofilm-forming ability showed a strain-specific trend, not correlated with CC or with strains persistence. Instead, the presence of internalin (inL), Stress Survival Islet (SSI) and resistance to erythromycin (ermC) genes was correlated with the ability to produce biofilms. Our data demonstrate that the genetic profile influences the adhesion capacity and persistence of L. monocytogenes in food processing plants and could be the result of environmental adaptation in response to the external selective pressure.

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.

Draft Genome Sequence of Listeria monocytogenes Serovar 1/2a Strain IZSAM_Lm_14-16064, Isolated from an Italian Cooked Ham in 2014.

In this report, the draft genome sequence of Listeria monocytogenes serovar 1/2a strain IZSAM_Lm_14-16064, isolated in Italy from a cooked ham, is announced. The genome is similar to that of a clinical strain isolated in 2014.

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.

Whole-Genome Sequence of Listeria monocytogenes Serovar 1/2a Strain IZSAM_Lm_15_17439_A144, Representative of a Human Outbreak in 2008.

Here, we report the genome sequence of Listeria monocytogenes serovar 1/2a strain IZSAM_Lm_15_17439_A144, isolated in Italy from a patient during a Listeria monocytogenes outbreak in 2008. This strain showed 98.9% sequence identity to a strain isolated in Canada in the same year.

Whole-Genome Sequence of a Reemerging Listeria monocytogenes Serovar 1/2a Strain in Central Italy.

We report the whole-genome sequence of a Listeria monocytogenes strain isolated from a child in central Italy. Interestingly, the sequence showed a difference of only 13 single-nucleotide polymorphisms (SNPs) from a strain responsible for a severe listeriosis outbreak that occurred between January 2015 and March 2016 in the same region.

A severe outbreak of listeriosis in central Italy with a rare pulsotype associated with processed pork products.

PURPOSE: From May 2015 to March 2016, an outbreak due to Listeria monocytogenes serotype 1/2a and clinical pulsotype never previously isolated in Europe occurred in central Italy, involving 24 confirmed clinical cases. The article provides a description of the outbreak and the investigation carried out by a multidisciplinary network. METHODOLOGY: Epidemiological and microbiological surveillance was conducted to confirm the outbreak and to detect the food vehicle of infection. The origin and destination of the implicated food and its ingredients were investigated by tracing-back and -forward investigation. RESULTS: Next-generation sequencing confirmed the unique outbreak strain. On 4 January 2016, a L. monocytogenes strain with pulsotype indistinguishable from that isolated from clinical cases in the outbreak was detected in a sample of hog head cheese purchased from a retail supermarket by one of the patients. The hog head cheese was produced by a small meat processing plant in the Marche region, where microbiological investigation confirmed environmental and food contamination by the outbreak strain. Plant production was suspended and all contaminated batches of the hog head cheese were withdrawn from the market by 19 February by local health authority. We subsequently observed a sharp decline in clinical cases, the last being reported on 11 March 2016. CONCLUSION: The key factor in the timely conclusion of this investigation was intersectoral collaboration among epidemiologists, microbiologists, veterinarians, statisticians and health and food safety authorities at national, regional and local levels.

Whole-Genome Sequences of Two Listeria monocytogenes Serovar 1/2a Strains Responsible for a Severe Listeriosis Outbreak in Central Italy.

We report the whole-genome sequences of two Listeria monocytogenes strains responsible for a severe invasive listeriosis outbreak in central Italy that occurred in 2015 and 2016. These two strains differ by a single band in their pulsed-field gel electrophoresis (PFGE) profiles.

Bactericidal and antibiofilm activity of bactenecin-derivative peptides against the food-pathogen Listeria monocytogenes: New perspectives for food processing industry.

Antimicrobial peptides have received great attention for their potential benefits to extend the shelf-life of food-products. Innate defense regulator peptide-1018 (IDR-1018) represents a promising candidate for such applications, due to its broad-spectrum antimicrobial activity, although food-isolated pathogens have been poorly investigated. Herein, we describe the design and the structural-functional characterization of a new 1018-derivative peptide named 1018-K6, in which the alanine in position 6 was replaced with a lysine. Spectroscopic analysis revealed a noticeable switch from ß-sheet to helical conformations of 1018-K6 respect to IDR-1018, with a faster folding kinetic and increased structural stability. Moreover, 1018-K6 evidenced a significant antibiofilm/bactericidal efficiency specifically against Listeria monocytogenes isolates from food-products and food-processing environments, belonging to serotype 4b involved in the majority of human-listeriosis cases, with EC50 values two- five-fold lower than those measured for IDR-1018. Therefore, a single amino-acid substitution in IDR-1018 sequence produced severe changes in peptide conformation and antimicrobial performances.

Identification and characterization of Yersinia enterocolitica strains isolated from pig tonsils at slaughterhouse in Central Italy.

Yersinia enterocolitica causes foodborne disease in humans and infections are usually acquired from contaminated raw or undercooked pork. Pigs are considered the primary reservoir of human pathogenic bio-serotypes. A total of 376 tonsil tissue samples collected after evisceration and cutting from pig carcasses were tested for Yersinia enterocolitica. Animals came from an abattoir located in the Abruzzo region, Italy. Yersinia enterocolitica was isolated from 35 out of 376 (9.31%) samples. A total of 47 strains were isolated, the prevalent bio-serotype was 4÷O:3 (95.74%), followed by bio-serotype 4÷O:9 (2.13%), and 3÷O:9 (2.13%). When characterized by DNA microarray, all strains clustered into 2 main groups. The bigger group was characterised by the presence of plasmid genes of the secretion apparatus as well as by the genes for the agellum transport machinery, while the smaller group was characterised only by genes for the agellum transport machinery. The high frequency of the pathogenic biotype 4÷O:3 able to infect humans and considered an emerging zoonotic pathogen con rms the role of pigs as natural reservoir. Since there is no official data on Yersinia enterocolitica, it is difficult to assess the implications of this food pathogen for public health. A monitoring program should be implemented for contamination in food in order to assess the risk for the consumer linked to raw or undercooked pork products.

Whole-Genome Sequence of Listeria monocytogenes Serovar 4b Strain IZSAM_Lm_hs2008, Isolated from a Human Infection in Italy.

Listeria monocytogenes is one of the most important foodborne pathogens. In this report, we present the complete and annotated genome of L. monocytogenes sequence type 06 (ST06) serovar 4b strain IZSAM_Lm_hs2008, isolated from an adult immunocompetent patient who developed the disease and died.

Host species barriers to Jaagsiekte sheep retrovirus replication and carcinogenesis.

Understanding the factors governing host species barriers to virus transmission has added significantly to our appreciation of virus pathogenesis. Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA), a transmissible lung cancer of sheep that has rarely been found in goats. In this study, in order to further clarify the pathogenesis of OPA, we investigated whether goats are resistant to JSRV replication and carcinogenesis. We found that JSRV induces lung tumors in goats with macroscopic and histopathological features that dramatically differ from those in sheep. However, the origins of the tumor cells in the two species are identical. Interestingly, in experimentally infected lambs and goat kids, we revealed major differences in the number of virus-infected cells at early stages of infection. These differences were not related to the number of available target cells for virus infection and cell transformation or the presence of a host-specific immune response toward JSRV. Indeed, we also found that goats possess transcriptionally active endogenous retroviruses (enJSRVs) that likely influence the host immune response toward the exogenous JSRV. Overall, these results suggest that goat cells, or at least those cells targeted for viral carcinogenesis, are not permissive to virus replication but can be transformed by JSRV.

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.

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.

Jaagsiekte 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.