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.

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.

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.

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.

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.