Novel IncFII plasmid harbouring blaNDM-4 in a carbapenem-resistant Escherichia coli of pig origin, Italy.

OBJECTIVES: To the best of our knowledge, we describe the first evidence in Europe of an MDR, blaNDM-4-positive Escherichia coli isolated from a food-producing animal, harboured by a novel IncFII plasmid of which we report the complete sequence. METHODS: One blaNDM-4-positive E. coli isolated in 2019 from the caecal contents of a fattening pig in Italy was in-depth characterized by combined bioinformatic analysis of Oxford Nanopore long reads and Illumina short reads, for in silico typing, determination of the blaNDM-4 genetic context and full reconstruction of the blaNDM-4-carrying plasmid. RESULTS: The isolate belonged to ST641 and to the genoserotype O108:H23 and tested positive for different virulence genes and plasmid replicons. The MDR phenotype of resistance to all ß-lactams, carbapenems, sulfamethoxazole and trimethoprim was mediated by blaTEM-1B, blaNDM-4, sul1/sul3 and dfrA12, respectively. The blaNDM-4 gene was harboured by a novel 53 043 bp IncFII plasmid (pMOL412_FII) composed of four main genetic regions, including an MDR region (MRR-NDM-4) of 16 kb carrying blaNDM-4 and several antimicrobial resistance genes located in a class 1 integron. pMOL412_FII was closely related to another ∼90.3 kb plasmid (pM109_FII) harbouring blaNDM-4 in an E. coli isolated from a human patient in Myanmar. CONCLUSIONS: To the best of our knowledge, we have identified for the first time in Europe an NDM-producing Enterobacterales in livestock and resolved the complete sequence of the novel pMOL412_FII plasmid harbouring blaNDM-4 in an MRR. A global One Health approach, comparing genomic data from different sources and geographical areas, may help to trace back and control possible plasmid-borne carbapenemase gene transmission between animals and humans and along the food chain at international level.

Evidence of structural rearrangements in ESBL-positive pESI(like) megaplasmids of S.Infantis.

The increasing prevalence of pESI(like)-positive, multidrug-resistant (MDR) S. Infantis in Europe is a cause of major concern. As previously demonstrated, the pESI(like) megaplasmid is not only a carrier of antimicrobial resistant (AMR) genes (at least tet, dfr, and sul genes), but also harbours several virulence and fitness genes, and toxin/antitoxin systems that enhance its persistence in the S. Infantis host. In this study, five prototype pESI(like) plasmids, of either CTX-M-1 or CTX-M-65 ESBL-producing strains, were long-read sequenced using Oxford Nanopore Technology (ONT), and their complete sequences were resolved. Comparison of the structure and gene content of the five sequenced plasmids, and further comparison with previously published pESI(like) sequences, indicated that although the sequence of such pESI(like) 'mosaic' plasmids remains almost identical, their structures appear different and composed of regions inserted or transposed after different events. The results obtained in this study are essential to better understand the plasticity and the evolution of the pESI(like) megaplasmid, and therefore to better address risk management options and policy decisions to fight against AMR and MDR in Salmonella and other food-borne pathogens. Graphical representation of the pESI-like plasmid complete sequence (ID 12037823/11). Block colours indicate the function of the genes: red: repB gene; pink: class I integrons (IntI); yellow; mobile elements; blue: resistance genes; green: toxin/anti-toxin systems; grey: mer operon; light green: genes involve in conjugation.

Genomics insights into a Mycobacterium pinnipedii isolate causing tuberculosis in a captive South American sea lion (Otaria flavescens) from Italy.

Tuberculosis (TB) affects humans and other animals, and it is caused by bacteria within the Mycobacterium tuberculosis complex (MTBC). In this study, we report the characterisation of Mycobacterium pinnipedii that caused a TB case in a sea lion (Otaria flavescens) kept in an Italian zoo. The animal died due to severe, progressive disorders involving the respiratory and gastro-enteric systems and the skin. At necropsy, typical gross lesions referable to a TB generalised form were found. In particular, nodular granulomatous lesions were detected in the lungs and several lymph nodes, and colonies referable to Mycobacterium spp. were isolated from lung, mesenteric, and mediastinal lymph nodes. The isolate was identified by PCR as a MTBC, had a spoligotype SB 1480 ("seal lineage"), and was characterised and characterised by whole-genome sequencing analysis confirming that the MTBC involved was M. pinnipedii. The analysis of the resistome and virulome indicated the presence of macrolide and aminoglycoside resistance genes intrinsic in M. tuberculosis [erm-37 and aac(2')-Ic] and confirmed the presence of the region of difference 1 (RD1), harbouring the esxA and esxB virulence genes, differently from its closest taxon, M. microti. As for other MTCB members, M. pinnipedii infection can spill over into non-pinniped mammalian species; therefore, zoological gardens, veterinary practitioners, and public health officers should be aware of the hazard posed by tuberculosis from marine mammals. Since the isolate under study, as well as all available genomes of M. pinnipedii investigated in this study retains almost all the M. tuberculosis virulence genes, it could indeed cause infection, lesions, and disease in other animal species, including humans.

The hazard of carbapenemase (OXA-181)-producing Escherichia coli spreading in pig and veal calf holdings in Italy in the genomics era: Risk of spill over and spill back between humans and animals.

Carbapenemase-producing Enterobacterales (CPE) are considered a major public health issue. In the frame of the EU Harmonized AMR Monitoring program conducted in Italy in 2021, 21 epidemiological units of fattening pigs (6.98%; 95% CI 4.37-10.47%; 21/301) and four epidemiological units of bovines <12 months (1.29%; 95% CI 0.35-3.27%, 4/310) resulted positive to OXA-48-like-producing E. coli (n = 24 OXA-181, n = 1 OXA-48). Whole Genome Sequencing (WGS) for in-depth characterization, genomics and cluster analysis of OXA-181-(and one OXA-48) producing E. coli isolated, was performed. Tracing-back activities at: (a) the fattening holding of origin of one positive slaughter batch, (b) the breeding holding, and (c) one epidemiologically related dairy cattle holding, allowed detection of OXA-48-like-producing E. coli in different units and comparison of further human isolates from fecal samples of farm workers. The OXA-181-producing isolates were multidrug resistant (MDR), belonged to different Sequence Types (STs), harbored the IncX and IncF plasmid replicons and multiple virulence genes. Bioinformatics analysis of combined Oxford Nanopore Technologies (ONT) long reads and Illumina short reads identified bla OXA-181 as part of a transposon in IncX1, IncX3, and IncFII fully resolved plasmids from 16 selected E. coli, mostly belonging to ST5229, isolated during the survey at slaughter and tracing-back activities. Although human source could be the most likely cause for the introduction of the bla OXA-181-carrying IncX1 plasmid in the breeding holding, concerns arise from carbapenemase OXA-48-like-producing E. coli spreading in 2021 in Italian fattening pigs and, to a lesser extent, in veal calf holdings.

A methicillin-resistant Staphylococcus aureus (MRSA) Sequence Type 8, spa type t11469 causing infection and colonizing horses in Italy.

A Methicillin-resistantStaphylococcus aureus(MRSA) was isolated in Italy from a pathological sample of a mare presenting chronic purulent sinusitis and that had undergone frontal-sinus surgery three months before. Humans, horses, dogs and environmental samples were subsequently collected at the mare's stable and at the Veterinary Hospital, where the mare was operated/hospitalized, and screened for the presence of MRSA that was detected from other horses and from the environment at both sites. All the MRSA isolates belonged to clonal complex (CC)8, ST8-t11469-SCCmec-IVa, and showed similar phenotypic and genetic multidrug resistance patterns and macrorestriction-pulsed-field gel electrophoresis profiles. The only MRSA detected from humans was a CC1, ST1-t127-SCCmec-IVa. This paper represents the first report of a clinical MRSA infection in a horse in Italy. This study also supports the opinion that improper use of antibiotics and hospitalization/surgery can represent risk factors for MRSA colonization/infection in horses, and that the environment is among important sources for exposure.

A New Multilocus Sequence Typing Scheme and Its Application for the Characterization of Photobacterium damselae subsp. damselae Associated with Mortality in Cetaceans.

Photobacterium damselae subsp. damselae (PDD) is a known pathogen of fish, humans and marine mammals. In this study, a Multilocus Sequence Typing (MLST) scheme based on six housekeeping genes (glp, gyrB, metG, pnt, pyrC, and toxR) was developed to better understand the PDD population structure and used to type 73 PDD isolates from cetaceans, mainly striped dolphins (Stenella coeruleoalba) involved in mortality episodes, and from a few marine chelonians. Five reference ATCC strains were also included in the study. Typing allowed the discrimination of groups of PDD strains isolated from different host species, at different times and from different geographic areas, suggesting that a clonal PDD group may have spread in the Tyrrhenian sea at the time of an Unusual Mortality Event (UME) among cetaceans, mainly striped dolphins, occurred in early 2013 along the Italian western coasts.