Severe Streptococcus equi Subspecies zooepidemicus Outbreak from Unpasteurized Dairy Product Consumption, Italy.

During November 2021-May 2022, we identified 37 clinical cases of Streptococcus equi subspecies zooepidemicus infections in central Italy. Epidemiologic investigations and whole-genome sequencing showed unpasteurized fresh dairy products were the outbreak source. Early diagnosis by using sequencing technology prevented the spread of life-threatening S. equi subsp. zooepidemicus infections.

In vitro and in silico parameters for precise cgMLST typing of Listeria monocytogenes.

BACKGROUND: Whole genome sequencing analyzed by core genome multi-locus sequence typing (cgMLST) is widely used in surveillance of the pathogenic bacteria Listeria monocytogenes. Given the heterogeneity of available bioinformatics tools to define cgMLST alleles, our aim was to identify parameters influencing the precision of cgMLST profiles. METHODS: We used three L. monocytogenes reference genomes from different phylogenetic lineages and assessed the impact of in vitro (i.e. tested genomes, successive platings, replicates of DNA extraction and sequencing) and in silico parameters (i.e. targeted depth of coverage, depth of coverage, breadth of coverage, assembly metrics, cgMLST workflows, cgMLST completeness) on cgMLST precision made of 1748 core loci. Six cgMLST workflows were tested, comprising assembly-based (BIGSdb, INNUENDO, GENPAT, SeqSphere and BioNumerics) and assembly-free (i.e. kmer-based MentaLiST) allele callers. Principal component analyses and generalized linear models were used to identify the most impactful parameters on cgMLST precision. RESULTS: The isolate's genetic background, cgMLST workflows, cgMLST completeness, as well as depth and breadth of coverage were the parameters that impacted most on cgMLST precision (i.e. identical alleles against reference circular genomes). All workflows performed well at ≥40X of depth of coverage, with high loci detection (> 99.54% for all, except for BioNumerics with 97.78%) and showed consistent cluster definitions using the reference cut-off of ≤7 allele differences. CONCLUSIONS: This highlights that bioinformatics workflows dedicated to cgMLST allele calling are largely robust when paired-end reads are of high quality and when the sequencing depth is ≥40X.

A Whole-Genome-Based Gene-by-Gene Typing System for Standardized High-Resolution Strain Typing of Bacillus anthracis.

Whole-genome sequencing (WGS) has been established for bacterial subtyping and is regularly used to study pathogen transmission, to investigate outbreaks, and to perform routine surveillance. Core-genome multilocus sequence typing (cgMLST) is a bacterial subtyping method that uses WGS data to provide a high-resolution strain characterization. This study aimed at developing a novel cgMLST scheme for Bacillus anthracis, a notorious pathogen that causes anthrax in livestock and humans worldwide. The scheme comprises 3,803 genes that were conserved in 57 B. anthracis genomes spanning the whole phylogeny. The scheme has been evaluated and applied to 584 genomes from 50 countries. On average, 99.5% of the cgMLST targets were detected. The cgMLST results confirmed the classical canonical single-nucleotide-polymorphism (SNP) grouping of B. anthracis into major clades and subclades. Genetic distances calculated based on cgMLST were comparable to distances from whole-genome-based SNP analysis with similar phylogenetic topology and comparable discriminatory power. Additionally, the application of the cgMLST scheme to anthrax outbreaks from Germany and Italy led to a definition of a cutoff threshold of five allele differences to trace epidemiologically linked strains for cluster typing and transmission analysis. Finally, the association of two clusters of B. anthracis with human cases of injectional anthrax in four European countries was confirmed using cgMLST. In summary, this study presents a novel cgMLST scheme that provides high-resolution strain genotyping for B. anthracis. This scheme can be used in parallel with SNP typing methods to facilitate rapid and harmonized interlaboratory comparisons, essential for global surveillance and outbreak analysis. The scheme is publicly available for application by users, including those with little bioinformatics knowledge.

First report of a bla NDM-producing extensively drug resistant Klebsiella pneumoniae ST437 in Italy.

Carbapenemase-producing Klebsiella pneumoniae strains (CP-Kps) have recently been observed to spread rapidly worldwide. New Delhi metallo-ß-lactamase (NDM) producing clones of Klebsiella pneumoniae (K. pneumoniae) cause a significant healthcare burden, particularly in Indian sub-continent, where this clone is circulating widely. However, in Italy, data on the incidence of these new clones is limited, and an ST437 NDM-producing K. pneumoniae strain has not been reported to date. A sacral ulcer infection caused by a K. pneumoniae strain was identified in an 85-year-old Italian male patient with several comorbidities. Antimicrobial susceptibility testing revealed an extensive resistance to a wide range of antimicrobials, including novel agents such as cefiderocol and ceftazidime/avibactam. Genomic analysis identified the pathogen as an ST437 K. pneumoniae strain harboring bla NDM-5, bla OXA-232 and bla CTX-M-15 genes. Following the identification of this first case, several infection control measures were implemented in healthcare settings, including direct precautions and reinforcement of standard cross-transmission control measures. The emergence of pathogenic microbial clones carrying new genetic determinants, particularly in a little city, requires prompt diagnosis and therapeutic protocols. An effective infection control system for the early detection and/or control of the transmission of NDM-producing Enterobacteriaceae is also needed. Further investigations are required to better understand the potential transmission routes and evolution of these clones.

Diversity, functional classification and genotyping of SHV ß-lactamases in Klebsiella pneumoniae.

Interpreting the phenotypes of bla SHV alleles in Klebsiella pneumoniae genomes is complex. Whilst all strains are expected to carry a chromosomal copy conferring resistance to ampicillin, they may also carry mutations in chromosomal bla SHV alleles or additional plasmid-borne bla SHV alleles that have extended-spectrum ß-lactamase (ESBL) activity and/or ß-lactamase inhibitor (BLI) resistance activity. In addition, the role of individual mutations/a changes is not completely documented or understood. This has led to confusion in the literature and in antimicrobial resistance (AMR) gene databases [e.g. the National Center for Biotechnology Information (NCBI) Reference Gene Catalog and the ß-lactamase database (BLDB)] over the specific functionality of individual sulfhydryl variable (SHV) protein variants. Therefore, the identification of ESBL-producing strains from K. pneumoniae genome data is complicated. Here, we reviewed the experimental evidence for the expansion of SHV enzyme function associated with specific aa substitutions. We then systematically assigned SHV alleles to functional classes (WT, ESBL and BLI resistant) based on the presence of these mutations. This resulted in the re-classification of 37 SHV alleles compared with the current assignments in the NCBI's Reference Gene Catalog and/or BLDB (21 to WT, 12 to ESBL and 4 to BLI resistant). Phylogenetic and comparative genomic analyses support that (i) SHV-1 (encoded by bla SHV-1) is the ancestral chromosomal variant, (ii) ESBL- and BLI-resistant variants have evolved multiple times through parallel substitution mutations, (iii) ESBL variants are mostly mobilized to plasmids and (iv) BLI-resistant variants mostly result from mutations in chromosomal bla SHV. We used matched genome-phenotype data from the KlebNET-GSP AMR Genotype-Phenotype Group to identify 3999 K. pneumoniae isolates carrying one or more bla SHV alleles but no other acquired ß-lactamases to assess genotype-phenotype relationships for bla SHV. This collection includes human, animal and environmental isolates collected between 2001 and 2021 from 24 countries. Our analysis supports that mutations at Ambler sites 238 and 179 confer ESBL activity, whilst most omega-loop substitutions do not. Our data also provide support for the WT assignment of 67 protein variants, including 8 that were noted in public databases as ESBL. These eight variants were reclassified as WT because they lack ESBL-associated mutations, and our phenotype data support susceptibility to third-generation cephalosporins (SHV-27, SHV-38, SHV-40, SHV-41, SHV-42, SHV-65, SHV-164 and SHV-187). The approach and results outlined here have been implemented in Kleborate v2.4.1 (a software tool for genotyping K. pneumoniae), whereby known and novel bla SHV alleles are classified based on causative mutations. Kleborate v2.4.1 was updated to include ten novel protein variants from the KlebNET-GSP dataset and all alleles in public databases as of November 2023. This study demonstrates the power of sharing AMR phenotypes alongside genome data to improve the understanding of resistance mechanisms.

Multi-approach methods to predict cryptic carbapenem resistance mechanisms in Klebsiella pneumoniae detected in Central Italy.

The rapid emergence of carbapenem-resistant Klebsiella pneumoniae (Kp) strains in diverse environmental niches, even outside of the clinical setting, poses a challenge for the detection and the real-time monitoring of novel antimicrobial resistance trends using molecular and whole genome sequencing-based methods. The aim of our study was to understand cryptic resistance determinants responsible for the phenotypic carbapenem resistance observed in strains circulating in Italy by using a combined approach involving whole genome sequencing (WGS) and genome-wide association study (GWAS). In this study, we collected 303 Kp strains from inside and outside clinical settings between 2018-2022 in the Abruzzo region of Italy. The antimicrobial resistance profile of all isolates was assessed using both phenotypic and bioinformatic methods. We identified 11 strains resistant to carbapenems, which did not carry any known genetic determinants explaining their phenotype. The GWAS results showed that incongruent carbapenem-resistant phenotype was associated specifically with strains with two capsular types, KL13 and KL116 including genes involved in the capsule synthesis, encoding proteins involved in the assembly of the capsule biosynthesis apparatus, capsule-specific sugar synthesis, processing and export, polysaccharide pyruvyl transferase, and lipopolysaccharide biosynthesis protein. These preliminary results confirmed the potential of GWAS in identifying genetic variants present in KL13 and KL116 that could be associated with carbapenem resistance traits in Kp. The implementation of advanced methods, such as GWAS with increased antimicrobial resistance surveillance will potentially improve Kp infection treatment and patient outcomes.

The Slaughterhouse as Hotspot of CC1 and CC6 Listeria monocytogenes Strains with Hypervirulent Profiles in an Integrated Poultry Chain of Italy.

In Europe, very few studies are available regarding the diversity of Listeria monocytogenes (L. monocytogenes) clonal complexes (CCs) and sequence types (ST) in poultry and on the related typing of isolates using whole genome sequencing (WGS). In this study, we used a WGS approach to type 122 L. monocytogenes strains isolated from chicken neck skin samples collected in two different slaughterhouses of an integrated Italian poultry company. The studied strains were classified into five CCs: CC1-ST1 (21.3%), CC6-ST6 (22.9%), CC9-ST9 (44.2%), CC121-ST121 (10.6%) and CC193-ST193 (0.8%). CC1 and CC6 strains presented a virulence gene profile composed of 60 virulence genes and including the Listeria Pathogenicity Island 3, aut_IVb, gltA and gltB. According to cgMLST and SNPs analysis, long-term persistent clusters belonging to CC1 and CC6 were found in one of the two slaughterhouses. The reasons mediating the persistence of these CCs (up to 20 months) remain to be elucidated, and may involve the presence and the expression of stress response and environmental adaptation genes including heavy metals resistance genes (cadAC, arsBC, CsoR-copA-copZ), multidrug efflux pumps (mrpABCEF, EmrB, mepA, bmrA, bmr3, norm), cold-shock tolerance (cspD) and biofilm-formation determinants (lmo0673, lmo2504, luxS, recO). These findings indicated a serious risk of poultry finished products contamination with hypervirulent L. monocytogenes clones and raised concern for the consumer health. In addition to the AMR genes norB, mprF, lin and fosX, ubiquitous in L. monocytogenes strains, we also identified parC for quinolones, msrA for macrolides and tetA for tetracyclines. Although the phenotypical expression of these AMR genes was not tested, none of them is known to confer resistance to the primary antibiotics used to treat listeriosis The obtained results increase the data on the L. monocytogenes clones circulating in Italy and in particular in the poultry chain.

Listeria monocytogenes in ready to eat meat products from Zambia: phenotypical and genomic characterization of isolates.

The contamination of ready to eat foods (RTE) products due to Listeria monocytogenes could compromise the products safety becoming a great risk for the consumers. The high presence of L. monocytogenes in RTE products has been described worldwide, but few data are available about these products from African countries. The aims of this study were to report the presence of L. monocytogenes in Zambian RTE products, providing genomic characterization and data on similarity with African circulating strains using whole genome sequencing (WGS). A total of 304 RTE products, produced by different Zambian manufacturers, were purchased at retail, from major supermarkets located in Lusaka, Zambia, comprising 130 dairy and 174 meat products. L. monocytogenes was detected only in 18 (10.3%) RTE meat products of the 174 samples tested. The MLST analysis grouped the 18 L. monocytogenes isolates in 7 clonal complexes (CCs): CC1 (n = 5), CC2 (n = 4), CC9 (n = 4), CC5 (n = 2), CC121 (n = 1), CC155 (n = 1), and CC3 (n = 1). According to the cgMLST results, several clusters were detected, in particular belonging to hyper-virulent clones CC1 and CC2. Regarding the virulence factors, a complete L. monocytogenes Pathogenicity Island 3 (LIPI-3) was present both in the CC1 and CC3, in addition to LIPI-1. Several resistance genes and mobile genetic elements were detected, including Stress Islands, the bcrABC cassette and Tn6188_qac transposon, plasmids and intact prophages. Despite being a first preliminary work with a limited number of samples and isolates, this study helped to increase existing knowledge on contaminated RTE products in Zambia, confirming the presence of hyper-virulent L. monocytogenes CCs, which could play an important role in human diseases, posing a public health concern for consumers.

Identification of the Novel Streptococcus equi subsp. zooepidemicus Sequence Type 525 in Donkeys of Abruzzo Region, Italy.

Streptococcus equi sub. zooepidemicus (SEZ) is described as a commensal bacterium of several animal species, including humans. Growing evidence supports the potential role of SEZ in the onset and progression of severe clinical manifestations of diseases in horses and other animals. In the present communication, we describe the diagnostic procedure applied to characterize the streptococcal infections caused by a novel SEZ sequence type (ST525) in donkeys raised on a farm in Abruzzo, Italy. The diagnostic process began with anamnesis and anatomopathological analysis, which revealed a severe bacterial suppurative bronchopneumonia associated with systemic vascular damage and haemorrhages. Then, SEZ infection was confirmed by applying an integrative diagnostic strategy that included standard bacterial isolation techniques, analytical tools for bacteria identification (MALDI-TOF MS), and molecular analysis (qPCR). Furthermore, the application of the whole-genome sequencing approach helped us to identify the bacterial strains and the virulence factors involved in animal diseases. The novel SEZ-ST525 was identified in two cases of the disease. This new sequence type was isolated from the lung, liver, and spleen in Case 1, and from retropharyngeal lymph nodes in Case 2. Moreover, the presence of the virulence gene mf2, a virulence factor carried by prophages in Streptococcus pyogenes, was also found for the first time in an SEZ strain. The results of the present study highlight the need to apply an integrated diagnostic approach for the identification and tracking of pathogenic strains of SEZ, shedding new light on the re-evaluation of these bacteria as a causative agent of disease in animals and humans.

Atypical Serogroup IVb-v1 of Listeria monocytogenes Assigned to New ST2801, Widely Spread and Persistent in the Environment of a Pork-Meat Producing Plant of Central Italy.

In this study, we characterized 84 Listeria monocytogenes (Lm) strains having an atypical IVb-v1 profile and isolated in a meat producing plant of Central Italy. They were assigned to the new MLST type ST2801 (CC218). The new ST was widespread in the food-producing environment where it was able to persist for over a year even after cleaning and sanitation. Cluster analysis identified three main clusters genetically close to each other (0-22 allelic differences and 0-28 SNPs) from two different cgMLST types, suggesting a common source. The coexistence of closely related clusters over time could be the result of a different evolution path starting from a common ancestor first introduced in the plant and/or the consequence of the repetitive reintroduction of closely related clones probably by raw materials. All the strains presented several determinants for heavy metals resistance, stress response, biofilm production, and multidrug efflux pumps with no significant differences among the clusters. A total of 53 strains carried pLI100 and the j1776 plasmids, while in one strain, the pLM33 was found in addition to pLI100. Only the strains carrying plasmids presented cadA and cadC for cadmium resistance and the mco gene encoding a multicopper oxidase and gerN for an additional Na+/H+-K+ antiporter. All the strains presented a virulence profile including a full-length inlA gene and the additional LIPI-3. The isolation of a new ST with a large pattern of stress-adaptation genes and able to persist is an important contribution to deepening the current knowledge on the uncommon IVb-v1 and in general on the genomic diversity of Lm.

Phenotypic and Genetic Characterization of Klebsiella pneumoniae Isolates from Wild Animals in Central Italy.

Despite Klebsiella pneumoniae being widely recognized as a nosocomial pathogen, there is a critical lack in defining its reservoirs and sources of infections. Most studies on risk factors have focused on multidrug-resistant (MDR) isolates and clinically-oriented questions. Over a two-year period, we sampled 131 wild animals including mammal and bird species from three regions of Central Italy. All typical colonies isolated from the analytical portions were confirmed by real-time PCR and identified by MALDI-TOF mass spectrometry (MALDI-TOF MS). All confirmed K. pneumoniae isolates were tested for antimicrobial susceptibility to 29 antimicrobials and subjected to whole genome sequencing. Typical colonies were detected in 17 samples (13%), which were identified as K. pneumoniae (n = 16) and as K. quasipneumoniae (n = 1) by MALDI-TOF MS. The antimicrobial susceptibility profile showed that all the isolates were resistant to ß-lactams (ceftobiprole, cloxacillin, cefazolin) and tetracycline; resistance to ertapenem and trimethoprim was observed and nine out of 16 K. pneumoniae isolates (56.2%) were classified as MDR. Genomic characterization allowed the detection of fluoroquinolone resistance-associated efflux pumps, fosfomycin and ß-lactamase resistance genes, and virulence genes in the overall dataset. The cluster analysis of two isolates detected from wild boar with available clinical genomes showed the closest similarity. This study highlights the link between humans, domestic animals, and wildlife, showing that the current knowledge on this ecological context is lacking and that the potential health risks are underestimated.

The Current Landscape of Antibiotic Resistance of Salmonella Infantis in Italy: The Expansion of Extended-Spectrum Beta-Lactamase Producers on a Local Scale.

Salmonella enterica serovar Infantis is one of the five main causes of human salmonellosis in the European Union (EU) and in recent years, has been increasingly reported to carry multiple antimicrobial resistance determinants, including extended-spectrum beta-lactamase (ESBL) genes. In our study, we used WGS-based tools to characterize S. Infantis strains circulating in the Abruzzo and Molise regions of Italy between 2017 and 2020 and compared this local dataset to the S. Infantis population present in Italy over the last two decades. Phylogenetic analyses demonstrated that the majority of strains isolated from poultry and turkeys from Abruzzo and Molise were closely related and belonged to one of the two main genetic clusters present in Italy, which were grouped predominantly as ESBL-producing strains that harbored pESI-like plasmid. We showed that 60% of the local strains carried multiple antibiotic resistance genes, including ESBL gene bla CTX-M-1 as well as aadA1, dfrA1, dfrA14, sul1, and tet(A) genes present on the pESI-like megaplasmid. The analysis of strains from Abruzzo and Molise and the publicly available Italian S. Infantis sequences revealed a dramatic increase in the number of identified AMR genes in the strains isolated after 2011. Moreover, the number of strains resistant to five or more antibiotic classes increased from 20-80% in the last decade likely due to the acquisition of the megaplasmid. The persistence of the ESBL-producing and the multidrug-resistant (MDR) clone of S. Infantis in poultry populations in Italy and in Europe requires rapid and efficient intervention strategies to prevent further expansion of the clone.

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.

Assessment of a new microsatellites panel for traceability in Italian inbreed pigs using parentage test

The origin of meat and meat products can be traced by verifying the identity of an offspring from its parents' genotypes. Although there are many microsatellite panels applicable to swine population, efficiency of parental testing decreases when the population consists of consanguineous animals. The aims of the present study were to develop a new microsatellite panel for traceability using parentage test in inbreed pig population and to assess how hybridization can influence the efficiency of parental testing. A new genotyping assay, based on 20­microsatellite assay, was performed in 304 individuals consisting of related and unrelated animals. The results showed that the microsatellites used in this study display high level of polymorphism ensuring a parentage assignment of 100%. This genotyping panel can be a useful tool to test a 'parent­to­fork' traceability system based on 20 microsatellite loci and can overcome technical limitations in inbreed population.

Hyper-Virulent Listeria monocytogenes Strains Associated With Respiratory Infections in Central Italy.

Listeria monocytogenes (Lm) is a foodborne pathogen causing listeriosis. Invasive forms of the disease mainly manifest as septicaemia, meningitis and maternal-neonatal infections. Lm-associated respiratory infections are very rare and little known. We reported two Lm respiratory infection cases occurred in Central Italy during the summer of 2020, in the midst of the SARS-CoV2 pandemic. In addition to collect the epidemiological and clinical characteristics of the patients, we used Whole Genome Sequencing to study the genomes of the Lm isolates investigating their virulence and antimicrobial profiles and the presence of genetic mobile elements. Both the strains belonged to hypervirulent MLST clonal complexes (CC). In addition to the Listeria Pathogenicity Island 1 (LIPI-1), the CC1 strain also carried LIPI-3 and the CC4 both LIPI-3 and LIPI-4. Genetic determinants for antimicrobial and disinfectants resistance were found. The CC1 genome presented prophage sequences but they did not interrupt the comK gene, involved in the phagosomal escape of Lm. None of the strains carried plasmids. Lm is an important, although rare, opportunistic pathogen for respiratory tract and lung infections. To avoid dangerous diagnostic delays of these severe clinical forms, it is important to sensitize hospital laboratories to this rare manifestation of listeriosis considering Lm in the differential diagnosis of respiratory infections.

Draft Genome Sequence of Serratia rubidaea, a Potential Opportunistic Pathogen Isolated from Food in Italy.

Serratia rubidaea has emerged in recent years as an opportunistic nosocomial pathogen. Here, we present the draft genome sequence of an isolate derived from an industrial meat food product purchased in a large-scale retail store that revealed fluoroquinolone, ß-lactam, and aminoglycoside resistance genes and two different host-unspecific prophages.

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.

Occurrence of Listeria monocytogenes and Escherichia coli in Raw Sheep's Milk from Farm Bulk Tanks in Central Italy.

ABSTRACT: For milk hygiene and safety, the milking phase is a critical moment because it is a probable pathway for the introduction of unwanted microorganisms in the dairy chain. In particular, Listeria monocytogenes and Escherichia coli are known as possible microbial contaminants of raw sheep's milk, although extensive knowledge regarding their contamination dynamics on sheep farms is still lacking. This study aimed to examine the occurrence and concentration of these microorganisms in milk samples collected from farm bulk tanks in the region of Lazio (Central Italy) and to investigate the related risk factors. Over a period of 1 year, we collected 372 milk samples from 87 sheep farms and administered a questionnaire to acquire information regarding relevant farm management variables. L. monocytogenes was not found in any of the samples, which indicates a low occurrence of this pathogen in sheep's bulk tank milk. In contrast, E. coli was found in almost two-thirds of milk samples (61%) but at levels below 102 CFU/mL in most of them (approximately 75%). Statistical analysis indicated that, during the warmest seasons, E. coli presence is more probable and counts are significantly higher. Unexpectedly, milk collected by hand milking had a lower level of contamination. Although further studies are necessary to clarify some aspects, the reported data add to the knowledge about the occurrence of L. monocytogenes and E. coli in raw sheep's milk and will be useful for future risk assessments.