Listeria monocytogenes in poultry: Detection and strain characterization along an integrated production chain in Italy.

In this study, thirteen batches of broiler chicken from an integrated Italian poultry company were investigated for the detection of Listeria monocytogenes. The prevalence was evaluated in faeces samples at farm level and after transport, caecal contents and carcass neck skin from 2 slaughterhouses (M1 and M2), for a total of 2080 samples, throughout a 27-month period. No positive results were recorded in faeces, while the overall prevalence of contamination in carcass neck skin was 26.7%. Then, 123 isolates out of 139 positive skin samples, with the prevalent serotypes 4b (76%) and 1/2b (94%) from slaughterhouses M1 and M2 respectively, were PFGE characterized, showing the presence of 18 different pulsotypes and 8 genetic clusters. The same pulsotypes were found in carcasses from different farms, but slaughtered in the same abattoir, highlighting the environmental origin of contamination. The persistence of the pathogen over long time seemed to be very likely, considering that undistinguishable pulsotypes were found in carcasses slaughtered in the same slaughterhouse after periods up to 18 months long. The implementation of cleaning and sanitation at slaughterhouse level could represent the main factor for the control of such pathogen in the poultry meat processing line.

Genetic diversity of Listeria monocytogenes strains contaminating food and food producing environment as single based sample in Italy (retrospective study).

Human listeriosis outbreaks are often associated with food products, which could be contaminated, at the same time, also by different clones of Listeria monocytogenes. This emphasize the need to type more than one L.monocytogenes isolate found in a single food or environmental sample. The purpose of the present study was to evaluate the presence of different L.monocytogenes strains in food and food production environment in order to understand if there is need to type more isolates from the same sample in case of presence of L.monocytogenes. Between 2011 and 2015, at the Italian National Reference Laboratory for L.monocytogenes, for each positive sample, from two to twenty-three isolates of L.monocytogenes were collected. All the isolates were characterized by conventional serotyping and pulsed field gel electrophoresis (PFGE). Moreover, isolates from the same sample, having indistinguishable PFGE profile, were subjected to whole genome sequencing in order to perform core genome Multi Locus Sequence Typing (cgMLST). Within each sample, more than one serotype and one pulsotype were found in 11.9% and 27.5%, respectively. For indistinguishable PFGE patterns the cgMLST analysis showed 96.2% of concordance demonstrating the added value of new sequencing technologies. This study has demonstrated the need to select and type more than one L.monocytogenes colony in one food or food environmental sample to detect the diversity of L.monocytogenes strains and facilitate downstream investigations and effective source attribution in foodborne outbreak inquiry.

Co-Infection of L. monocytogenes and Toxoplasma gondii in a Sheep Flock Causing Abortion and Lamb Deaths.

Abortion in livestock is a public health burden, and the cause of economic losses for farmers. Abortion can be multifactorial, and a deep diagnostic investigation is important to reduce the spread of zoonotic disease and public health prevention. In our study, a multidisciplinary investigation was conducted to address the cause of increased abortion and lamb mortality on a farm, which detected a co-infection of Listeria monocytogenes and Toxoplasma gondii. Hence, it was possible to conclude that this was the reason for a reduced flock health status and the cause of an increased abortion rate. Furthermore, the investigation work and identification of the L. monocytogenes infection root allowed the reduction of economic loss.

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.

Intensive Environmental Surveillance Plan for Listeria monocytogenes in Food Producing Plants and Retail Stores of Central Italy: Prevalence and Genetic Diversity.

Listeria monocytogenes (Lm) can persist in food processing environments (FPEs), surviving environmental stresses and disinfectants. We described an intensive environmental monitoring plan performed in Central Italy and involving food producing plants (FPPs) and retail grocery stores (RSs). The aim of the study was to provide a snapshot of the Lm circulation in different FPEs during a severe listeriosis outbreak, using whole genome sequencing (WGS) to investigate the genetic diversity of the Lm isolated, evaluating their virulence and stress resistance profiles. A total of 1217 samples were collected in 86 FPEs with 12.0% of positive surfaces at FPPs level and 7.5% at RSs level; 133 Lm isolates were typed by multilocus sequencing typing (MLST) and core genome MLST (cgMLST). Clonal complex (CC) 121 (25.6%), CC9 (22.6%), CC1 (11.3%), CC3 (10.5%), CC191 (4.5%), CC7 (4.5%) and CC31 (3.8%) were the most frequent MLST clones. Among the 26 cgMLST clusters obtained, 5 of them persisted after sanitization and were re-isolated during the follow-up sampling. All the CC121 harboured the Tn6188_qac gene for tolerance to benzalkonium chloride and the stress survival islet SSI-2. The CC3, CC7, CC9, CC31 and CC191 carried the SSI-1. All the CC9 and CC121 strains presented a premature stop codon in the inlA gene. In addition to the Lm Pathogenicity Island 1 (LIPI-1), CC1, CC3 and CC191 harboured the LIPI-3. The application of intensive environmental sampling plans for the detection and WGS analysis of Lm isolates could improve surveillance and early detection of outbreaks.

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.

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 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.

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.

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.

Listeria monocytogenes in Smoked Salmon and Other Smoked Fish at Retail in Italy: Frequency of Contamination and Strain Characterization in Products from Different Manufacturers.

Seven hundred seventy-eight samples of packaged smoked fish (774 smoked salmon and 4 smoked swordfish) on sale in Italy, from 50 different manufacturers located in 12 European Union countries, were purchased from the Italian market between May and December 2011. The surface temperatures of the samples on sale ranged from 0 to 13°C (3.4 ± 1.5°C, mean ± SD). Six hundred eighty (87.4%) of 778 samples were stored at ≤4°C. One hundred fifty-seven samples (20.2%, 95% confidence interval 17.5 to 23.1%) were contaminated by Listeria monocytogenes , with 26 samples (3.3%, 95% confidence interval 2.3 to 4.8%) at levels >100 CFU/g. The maximum level of contamination was 1.3 ×106 CFU/g. The differences in the level of contamination of smoked fish between countries (χ2 = 91.54, P < 0.05) and manufacturers (χ2 = 193.22, P < 0.05) were significant. The frequency of detection for products from different manufacturing premises ranged from 0 to 76.9%. Serotyping by serological agglutination revealed that the main serotypes detected were 1/2a (65.3%) and 1/2b (22.4%). Pulsed-field gel electrophoresis typing with restriction enzymes AscI and ApaI yielded 36 pulsotypes from 144 isolates, clustering into 17 groups. Eight main pulsotypes accounted for 70.8% of the isolates. Three of the main pulsotypes were exclusively from products of a single manufacturer. In general, products from the same manufacturer showed genetic homogeneity, with one strongly prevalent pulsotype. Different manufacturers usually showed very different levels of contamination of the final product, confirming the importance of the management of process hygiene for controlling L. monocytogenes contamination.

Comparison of Multiple­Locus Variable number tandem repeat Analysis and Pulsed Field Gel Electrophoresis in molecular subtyping of Listeria monocytogenes isolates from Italian cheese.

Subtyping of Listeria monocytogenes strains is an essential epidemiological tool to trace contamination and determine evolutionary relationships among different strains. Pulsed field gel electrophoresis (PFGE) is the current gold standard method for Listeria characterization. Multiple-Locus Variable-number tandem repeat Analysis (MLVA) is a rapid subtyping method based on Polymerase Chain Reaction (PCR) amplification that has been successfully developed for subtyping bacterial genera. The purpose of this study was to evaluate MLVA for subtyping L. monocytogenes strains isolated from Italian cheese and to compare it with PFGE. The type ability and discriminatory power of MLVA was determined on a collection of 90 isolates corresponding to 5 serotypes and 29 pulsotypes with enzymes AscI and ApaI. A panel of 5 variable-number tandem repeat loci was used. MLVA and PFGE showed very similar discriminatory power (Simpson's Index of diversity 0.840 with 95% Confidence Interval [CI] 0.780/0.899 and 0.837 with 95% CI 0.768/0.906, respectively). MLVA is an easy test to perform. It is relatively fast, reproducible and could be implemented in any molecular laboratory but, according to the performed protocol, it is not sufficient for discriminating the L. monocytogenes strains isolated from cheese. This method could be combined with PFGE to increase the discrimination in molecular subtyping of these strains.

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.