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.

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 β-lactamase (ESBL) activity and/or β-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 β-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 β-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.

Anthrax is a serious infectious disease caused by the gram-positive and spore-forming bacterium Bacillus anthracis. In Italy, anthrax is an endemic disease with sporadic cases each year and few outbreaks, especially in Southern Italy. However, new foci have been discovered in zones without previous history of anthrax. During summer 2016, an outbreak of anthrax caused the death of four goats in the Abruzzo region, where the disease had not been reported before. In order to investigate the outbreak, we sequenced one strain and compared it to 19 Italian B. anthracis genomes. Furthermore, we downloaded 71 whole genome sequences representing the global distribution of canonical SNP lineages and used them to verify the phylogenetic positioning. To this end, we analyzed and compared the genome sequences using canonical SNPs and the whole genome SNP-based analysis. Our results demonstrate that the outbreak strain belonged to the Trans-Eurasian (TEA) group A.Br.011/009, which is the predominant clade in Central-Southern Italy. In conclusion, the high genomic relatedness of the Italian TEA strains suggests their evolution from a common ancestor, while the spread is supposedly driven by trade as well as human and transhumance activities. Here, we demonstrated the capabilities of whole genome sequencing (WGS), which can be used as a tool for outbreak analyses and surveillance activities.

Background: Whole genome sequencing analyzed by core genome multi-locus sequence typing (cgMLST) is widely used in surveillance of the pathogenic bacteriaListeria 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 threeL. monocytogenesreference 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.

Hepatitis E virus (HEV) is an emergent zoonotic pathogen, causing worldwide acute and chronic hepatitis in humans. HEV comprises eight genotypes and several subtypes. HEV genotypes 3 and 4 (HEV3 and HEV4) are zoonotic. In Italy, the most part of HEV infections (80%) is due to autochthonous HEV3 circulation of the virus, and the key role played by wild animals is generally accepted. Abruzzo is an Italian region officially considered an HEV “hot spot”, with subtype HEV3-c being up to now the only one reported among wild boars. During the year 2018–2019, a group of wild boars in Abruzzo were screened for HEV; positive RNA liver samples were subjected to HEV characterization by using the whole genome sequencing (WGS) approach methodology. This represents the first report about the detection of HEV-3 subtypes e and f in the wild boar population in this area. Since in Italy human infections from HEV 3-e and f have been associated with pork meat consumption, our findings deserve more in-depth analysis with the aim of evaluating any potential correlation between wild animals, the pork chain production and HEV human infections.

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.