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.

Molecular epidemiology of Salmonella Infantis in Europe: insights into the success of the bacterial host and its parasitic pESI-like megaplasmid.

Salmonella Infantis is one of the five serovars most frequently causing human salmonellosis in Europe, mainly associated with poultry. A clone harbouring a conjugative plasmid of emerging S. Infantis (pESI)-like megaplasmid, carrying multidrug resistant (MDR) and extended-spectrum beta-lactamases (ESBL) genes, has spread in the Italian broiler chicken industry also causing human illness. This work is aimed at elucidating the molecular epidemiology of S. Infantis and pESI-like in Europe using whole-genome sequencing and bioinformatics analysis, and to investigate the genetic relatedness of S. Infantis clones and pESI-like from animals, meat, feed and humans provided by institutions of nine European countries. Two genotyping approaches were used: chromosome or plasmid SNP-based analysis and the minimum spanning tree (MST) algorithm based on core-genome multilocus sequence typing (cgMLST). The European S. Infantis population appeared heterogeneous, with different genetic clusters defined at core-genome level. However, pESI-like variants present in 64.1 % of the isolates were more genetically homogeneous and capable of infecting different clonal lineages in most of the countries. Two different pESI-like with ESBL genes (n=82) were observed: blaCTX-M-1-positive in European isolates and blaCTX-M-65-positive in American isolates (study outgroup). Both variants had toxin-antitoxin systems, resistance genes towards tetracyclines, trimethoprim, sulphonamides and aminoglycosides, heavy metals (merA) and disinfectants (qacEΔ). Worryingly, 66 % of the total isolates studied presented different gyrA chromosomal point mutations associated with (fluoro)quinolone resistance (MIC range 0.125-0.5 mg/L), while 18 % displayed transferable macrolide resistance mediated by mph, mef and erm(B) genes. Proper intervention strategies are needed to prevent further dissemination/transmission of MDR S. Infantis and pESI-like along the food chain in Europe.

Corrigendum: Colistin Resistance Mediated by mcr-1 in ESBL-Producing, Multidrug Resistant Salmonella Infantis in Broiler Chicken Industry, Italy (2016-2017).

[This corrects the article DOI: 10.3389/fmicb.2018.01880.].

Colistin Resistance Mediated by mcr-1 in ESBL-Producing, Multidrug Resistant Salmonella Infantis in Broiler Chicken Industry, Italy (2016-2017).

Colistin-resistance mediated by mobilisable and plasmid-borne mcr genes has emerged worldwide, threatening the efficacy of colistin, a last resort antibiotic increasingly used for treating human invasive infections by multidrug-resistant or extensively drug-resistant Enterobacteriaceae. In this study, we report the first evidence of mcr-1-mediated colistin resistance in four multidrug resistant (MDR) out of 324 Salmonella infantis from the Italian antimicrobial resistance (AMR) monitoring (2001-2017) in broilers and broiler meat. Two were also Extended Spectrum Beta-Lactamases (ESBL)-producing isolates. Characterization by whole genome sequencing (WGS), located mcr-1.1 on an incX4 plasmid. Phylogenetic analysis of these isolates with selected Italian S. Infantis previously isolated from animals, meat and human clinical cases with unknown epidemiological relationship, demonstrated that ESBL-producing, mcr-1-positive isolates belonged to the emerging pESI-like-positive-ESBL-producing clone described in Italy in 2015.

Molecular Epidemiology of mcr-Encoded Colistin Resistance in Enterobacteriaceae From Food-Producing Animals in Italy Revealed Through the EU Harmonized Antimicrobial Resistance Monitoring.

Colistin resistance by mobilisable mcr genes has been described in bacteria of food-animal origin worldwide, which has raised public health concerns about its potential foodborne transmission to human pathogenic bacteria. Here we provide baseline information on the molecular epidemiology of colistin-resistant, mcr-positive Escherichia coli and Salmonella isolates in food-producing animals in Italy in 2014-2015. A total 678, 861 and 236 indicator E. coli, Extended Spectrum Beta-Lactamase (ESBL)/AmpC-producing E. coli, and Salmonella isolates, respectively, were tested for colistin susceptibility. These isolates were collected according to the EU harmonized antimicrobial resistance monitoring program and are representative of at least 90 and 80% of the Italian poultry (broiler chickens and turkeys) and livestock (pigs and bovines < 12 months) production, respectively. Whole genome sequencing by Illumina technology and bioinformatics (Center for Genomic Epidemiology pipeline) were used to type 42 mcr-positive isolates by PCR. Colistin resistance was mainly observed in the ESBL/AmpC E. coli population, and was present in 25.9, 5.3, 6.5, and 3.9% of such isolates in turkeys, broilers, pigs, and bovines, respectively. Most colistin-resistant isolates (141/161, 87.5%) harbored genes of the mcr-1 group. mcr-1 was also detected in a small proportion of Salmonella isolates (3/146, 2.0%) in turkeys. Additional mcr types were mcr-3 in four ESBL-producing E. coli from bovines, and two mcr-4 in ESBL (n = 1) and indicator E. coli (n = 1) from pigs and bovines. We describe notable diversity of mcr variants with predominance of mcr-1.1 and mcr-1.2 on conjugative IncX4 plasmids in E. coli and in Salmonella serovars Typhimurium, Newport, Blockley from turkey. A new variant, mcr-1.13 was detected in the chromosome in E. coli in turkey and pig isolates. Additionally, we describe mcr-3.2 and mcr-4.3 in E. coli from bovines, and mcr-4.2 in E. coli from pigs. These findings elucidate the epidemiology of colistin resistance in food-producing animals in Italy along with its genetic background, and highlight the likelihood of mcr horizontal transfer between commensal bacteria and major food-borne pathogens (Salmonella) within the same type of productions. Thorough action and strategies are needed in order to mitigate the risk of mcr transfer to humans, in a "One Health" perspective.

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.

A Livestock-Associated, Multidrug-Resistant, Methicillin-Resistant Staphylococcus aureus Clonal Complex 97 Lineage Spreading in Dairy Cattle and Pigs in Italy.

Pandemic methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 97 (CC97) lineages originated from livestock-to-human host jumps. In recent years, CC97 has become one of the major MRSA lineages detected in Italian farmed animals. The aim of this study was to characterize and analyze differences in MRSA and methicillin-susceptible S. aureus (MSSA) mainly of swine and bovine origins. Forty-seven CC97 isolates, 35 MRSA isolates, and 6 MSSA isolates from different Italian pig and cattle holdings; 5 pig MRSA isolates from Germany; and 1 human MSSA isolate from Spain were characterized by macrorestriction pulsed-field gel electrophoresis (PFGE) analysis, multilocus sequence typing (MLST), spa typing, staphylococcal cassette chromosome mec (SCCmec) typing, and antimicrobial resistance pattern analysis. Virulence and resistance genes were investigated by PCR and microarray analysis. Most of the isolates were of SCCmec type V (SCCmec V), except for two German MRSA isolates (SCCmec III). Five main clusters were identified by PFGE, with the German isolates (clusters I and II) showing 60.5% similarity with the Italian isolates, most of which (68.1%) grouped into cluster V. All CC97 isolates were Panton-Valentine leukocidin (PVL) negative, and a few (n = 7) tested positive for sak or scn. All MRSA isolates were multidrug resistant (MDR), and the main features were erm(B)- or erm(C)-mediated (n = 18) macrolide-lincosamide-streptogramin B resistance, vga(A)-mediated (n = 37) pleuromutilin resistance, fluoroquinolone resistance (n = 33), tet(K) in 32/37 tet(M)-positive isolates, and blaZ in almost all MRSA isolates. Few host-associated differences were detected among CC97 MRSA isolates: their extensive MDR nature in both pigs and dairy cattle may be a consequence of a spillback from pigs of a MRSA lineage that originated in cattle as MSSA and needs further investigation. Measures should be implemented at the farm level to prevent spillover to humans in intensive farming areas.

Molecular characterization of spa type t127, sequence type 1 methicillin-resistant Staphylococcus aureus from pigs.

OBJECTIVES: The aim of this study was to provide molecular characterization of methicillin-resistant Staphylococcus aureus (MRSA) spa type t127, sequence type (ST) 1 isolates, detected in a European baseline survey in holdings of breeding pigs, to determine phenotypic and genotypic drug resistance and to compare the results with those obtained from a collection of t127, ST1 MRSA and methicillin-susceptible S. aureus (MSSA) clinical isolates. METHODS: Twenty-four t127, ST1 MRSA from dust sampled in different breeding holdings in Italy, Spain and Cyprus were studied, along with 2 t127, ST1 MRSA from fattening pigs and 11 human t127, ST1 MRSA and MSSA. Genotyping was performed using multilocus sequence typing (MLST), spa typing and PFGE. SCCmec elements were characterized by multiplex-PCR and resistance and pathogenicity genes by PCR and microarray. RESULTS: PFGE patterns separated a porcine cluster (PC) from a human cluster (HC), with 75% similarity. The PC carried SCCmec cassette type V, while all isolates of the HC carried SCCmec cassette type IVa. Kanamycin resistance mediated by aadD, fluoroquinolone and erm(A)-mediated macrolide resistance and the absence of the sakA gene were features of the PC only. All isolates of both clusters were positive for LukE-LukD and LuF-LukS-HlgA leukotoxin genes and one human MSSA harboured Panton-Valentine leucocidin genes. CONCLUSIONS: Despite differences in the host-specific genetic features, the possibility of PC transmission to humans cannot be excluded. MRSA spa type t127, ST1 from pigs possesses several virulence and resistance genes towards major classes of antimicrobials and may represent a serious therapeutic challenge in case of invasive infections in humans.