Genomics Insight into cfr-Mediated Linezolid-Resistant LA-MRSA in Italian Pig Holdings.

The cfr genes encode for a 23S rRNA methyltransferase, conferring a multiresistance phenotype to phenicol, lincosamide, oxazolidinone, pleuromutilin, and streptogramin A antibiotics. These genes have been described in staphylococci, including methicillin-resistant Staphylococcus aureus (MRSA). In this study, we retrospectively performed an in-depth genomic characterisation of three cfr-positive, multidrug-resistant (MDR) livestock-associated (LA) MRSA clonal complexes (CCs) 1 and 398 detected in different Italian pig holdings (2008-2011) during population studies on Italian livestock (2008-2014). We used a combined Illumina and Oxford Nanopore Technologies (ONT) whole genome sequencing (WGS) approach on two isolates (the 2008 CC1 and the 2010 CC398 isolates, but not the 2011 CC1 isolate). Interestingly, the three isolates presented different cfr variants, with only one displaying a linezolid-resistant phenotype. In isolate 2008 CC1, the cfr gene was identified within a Tn558 composite transposon-like structure flanked by IS elements located on a novel 44,826 bp plasmid. This represents the first report of CC1 LA-MRSA harbouring the cfr gene in its functional variant. Differently, cfr was chromosomally located in isolate 2010 CC398. Our findings have significant public health implications, confirm the need for the continuous genomic surveillance of cfr-positive zoonotic LA-MRSA, and backdate cfr presence in LA-MRSA from Italian pigs to at least 2008.

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.

Multicentre evaluation of a selective isolation protocol for detection of mcr-positive E. coli and Salmonella spp. in food-producing animals and meat.

This study was conducted to evaluate the performance of a screening protocol to detect and isolate mcr-positive Escherichia coli and Salmonella spp. from animal caecal content and meat samples. We used a multicentre approach involving 12 laboratories from nine European countries. All participants applied the same methodology combining a multiplex PCR performed on DNA extracted from a pre-enrichment step, followed by a selective culture step on three commercially available chromogenic agar plates. The test panel was composed of two negative samples and four samples artificially contaminated with E. coli and Salmonella spp. respectively harbouring mcr-1 or mcr-3 and mcr-4 or mcr-5 genes. PCR screening resulted in a specificity of 100% and a sensitivity of 83%. Sensitivity of each agar medium to detect mcr-positive colistin-resistant E. coli or Salmonella spp. strains was 86% for CHROMID® Colistin R, 75% for CHROMagarTM COL-APSE and 70% for COLISTIGRAM. This combined method was effective to detect and isolate most of the E. coli or Salmonella spp. strains harbouring different mcr genes from food-producing animals and food products and might thus be used as a harmonized protocol for the screening of mcr genes in food-producing animals and food products in Europe.

A European multicenter evaluation study to investigate the performance on commercially available selective agar plates for the detection of carbapenemase producing Enterobacteriaceae.

The European Food Safety Authority (EFSA) advised to prioritize monitoring carbapenemase producing Enterobacteriaceae (CPE) in food producing animals. Therefore, this study evaluated the performance of different commercially available selective agars for the detection of CPE using spiked pig caecal and turkey meat samples and the proposed EFSA cultivation protocol. Eleven laboratories from nine countries received eight samples (four caecal and four meat samples). For each matrix, three samples contained approximately 100 CFU/g CPE, and one sample lacked CPE. After overnight enrichment in buffered peptone water, broths were spread upon Brilliance™ CRE Agar (1), CHROMID® CARBA (2), CHROMagar™ mSuperCARBA™ (3), Chromatic™ CRE (4), CHROMID® OXA-48 (5) and Chromatic™ OXA-48 (6). From plates with suspected growth, one to three colonies were selected for species identification, confirmation of carbapenem resistance and detection of carbapenemase encoding genes, by methods available at participating laboratories. Of the eleven participating laboratories, seven reported species identification, susceptibility tests and genotyping on isolates from all selective agar plates. Agars 2, 4 and 5 performed best, with 100% sensitivity. For agar 3, a sensitivity of 96% was recorded, while agar 1 and 6 performed with 75% and 43% sensitivity, respectively. More background flora was noticed for turkey meat samples than pig caecal samples. Based on this limited set of samples, most commercially available agars performed adequately. The results indicate, however, that OXA-48-like and non-OXA-48-like producers perform very differently, and one should consider which CPE strains are of interest to culture when choosing agar type.

Emergence of IncHI2 Plasmids With Mobilized Colistin Resistance (mcr)-9 Gene in ESBL-Producing, Multidrug-Resistant Salmonella Typhimurium and Its Monophasic Variant ST34 From Food-Producing Animals in Italy.

A collection of 177 genomes of Salmonella Typhimurium and its monophasic variant isolated in 2014-2019 from Italian poultry/livestock (n = 165) and foodstuff (n = 12), previously screened for antimicrobial susceptibility and assigned to ST34 and single-locus variants, were studied in-depth to check the presence of the novel mcr-9 gene and to investigate their genetic relatedness by whole genome sequencing (WGS). The study of accessory resistance genes revealed the presence of mcr-9.1 in 11 ST34 isolates, displaying elevated colistin minimum inhibitory concentration values up to 2 mg/L and also a multidrug-resistant (MDR) profile toward up to seven antimicrobial classes. Five of them were also extended-spectrum beta-lactamases producers (bla SHV - 12 type), mediated by the corresponding antimicrobial resistance (AMR) accessory genes. All mcr-9-positive isolates harbored IncHI2-ST1 plasmids. From the results of the Mash analysis performed on all 177 genomes, the 11 mcr-9-positive isolates fell together in the same subcluster and were all closely related. This subcluster included also two mcr-9-negative isolates, and other eight mcr-9-negative ST34 isolates were present within the same parental branch. All the 21 isolates within this branch presented an IncHI2/2A plasmid and a similar MDR gene pattern. In three representative mcr-9-positive isolates, mcr-9 was demonstrated to be located on different IncHI2/IncHI2A large-size (∼277-297 kb) plasmids, using a combined Illumina-Oxford Nanopore WGS approach. These plasmids were also compared by BLAST analysis with publicly available IncHI2 plasmid sequences harboring mcr-9. In our plasmids, mcr-9 was located in a ∼30-kb region lacking different genetic elements of the typical core structure of mcr-9 cassettes. In this region were also identified different genes involved in heavy metal metabolism. Our results underline how genomics and WGS-based surveillance are increasingly indispensable to achieve better insights into the genetic environment and features of plasmid-mediated AMR, as in the case of such IncHI2 plasmids harboring other MDR genes beside mcr-9, that can be transferred horizontally also to other major Salmonella serovars spreading along the food chain.

Carbapenemase IncF-borne blaNDM-5 gene in the E. coli ST167 high-risk clone from canine clinical infection, Italy.

The blaNDM-5-producing E. coli Sequence Type (ST)167 high-risk clone is emerging worldwide in human clinical cases, while its presence in companion animals is sporadic and has never been described in Italy. Using a combined Oxford Nanopore (ONT) long-reads and Illumina short-reads sequencing approach, an E. coli ST167 isolated from a hospitalized dog, was in-depth characterized by WGS and the plasmid containing blaNDM-5 was fully reconstructed. The complete sequence of the pMOL008 mosaic plasmid (F36:F31:A4:B1; pMOL008) harbouring blaNDM-5, was resolved and characterized. Moreover, a (pro)phage and IncFII, containing blaCMY-2 and ermB, and IncI2 plasmid types were also identified. pMOL008 was almost identical to blaNDM-5-containing plasmids from E. coli ST167 isolated from Italian human clinical cases and from a Swiss dog and colonized humans. blaNDM-5 was located in a class 1 integron together with aadA2, aac(3)-IIa, mph(A), sul1, tet(A) and dfrA12. The risk of spill-over and spill-back transmission of carbapenem-resistance genes, related plasmids and strains between humans and dogs, represents a Public Health threat and highlights the importance of the One Health approach for the AMR surveillance.

Livestock-Associated Methicillin-Resistant Staphylococcus aureus and Related Risk Factors in Holdings of Veal Calves in Northwest Italy.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) have emerged worldwide as zoonotic pathogens. Data on LA-MRSA in veal calf production in Italy are lacking; the aim of this survey was to fill current knowledge gaps in its prevalence and characteristics. Between February 2012 and January 2013 nasal swabs were taken from 1650 three- to six-month-old veal calves on 55 farms in Piedmont (northwest Italy), including gathering-related epidemiological data. S. aureus were screened for methicillin resistance by phenotypic and molecular (mecA gene detection) methods. MRSA were further genotyped by multilocus sequence typing. About 30% of the herds tested positive for MRSA: three different clonal complexes (CC398, CC97, and CC1) and staphylococcal cassette chromosome mec types (IVa, IVb, and V) were detected. Multilevel logistic regression model indicated poor cleaning, importation from Austria, and animal age as risk factors and coagulase-negative staphylococci colonization as a predictive factor for the occurrence of MRSA. The detection of CCs circulating in pigs and dairy cattle in Italy underscores the ability of the LA-MRSA clones to spread among animal production systems. In addition to maintaining preventive control measures for human health, better cleaning procedures need to be implemented, especially after new calves have been introduced into the herd.

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.

Molecular epidemiology of methicillin-resistant Staphylococcus aureus from dairy farms in North-eastern Italy.

Transmission of Staphylococcus aureus along the dairy production chain is an emerging public health problem with human, veterinary, and food safety issues. The prevalence of multidrug-resistant, particularly methicillin-resistant S. aureus (MRSA), has steadily increased in several European countries. In this study, the prevalence of S. aureus in raw cow milk and farm workers was investigated, and the trajectories of MRSA transmission at the primary stage of the dairy chain were assessed. To this purpose, a longitudinal survey was conducted in 618 dairy farms in two contiguous regions with high livestock density in North-eastern Italy. S. aureus contamination of bulk tank milk (BTM) was observed in more than 80% of farms, while MRSA prevalence was 3.6% and 15.9% in BTM and farm workers, respectively. The majority of MRSA isolates from both BTM and farm workers were assigned to ST398, and showed a worrisome multidrug-resistant phenotype. Enterotoxin and Panton-Valentine leukocidin genes were detected in 11.5% and 4.9% of MRSA isolates from both sources. Nearly all MRSA isolates from workers belonged to the same epidemiological type as BTM isolates from the corresponding farm, denoting a bidirectional MRSA transmission pattern. A focus on the ST398 spa type t899 MRSA lineage in the Italian livestock system highlighted the presence of two major clusters whose dissemination was likely facilitated by the selective pressure imposed by antimicrobial use in animal farming. Our findings emphasize the need for continuous monitoring of MRSA along the dairy production chain, not only to avoid transmission between animals and exposed workers, but also to contain the risk of raw milk and dairy product contamination by multidrug resistant and toxigenic strain.

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.

Antimicrobial Usage and Resistance in Companion Animals: A Cross-Sectional Study in Three European Countries.

Companion animals have been described as potential reservoirs of antimicrobial resistance (AMR), however data remain scarce. Therefore, the objectives were to describe antimicrobial usage (AMU) in dogs and cats in three European countries (Belgium, Italy, and The Netherlands) and to investigate phenotypic AMR. A questionnaire and one fecal sample per animal (n = 303) were collected over one year and AMU was quantified using treatment incidence (TI). Phenotypic resistance profiles of 282 Escherichia coli isolates were determined. Nineteen percent of the animals received at least one antimicrobial treatment six months preceding sampling. On average, cats and dogs were treated with a standard daily dose of antimicrobials for 1.8 and 3.3 days over one year, respectively. The most frequently used antimicrobial was amoxicillin-clavulanate (27%). Broad-spectrum antimicrobials and critically important antimicrobials for human medicine represented 83% and 71% of the total number of treatments, respectively. Resistance of E. coli to at least one antimicrobial agent was found in 27% of the isolates. The most common resistance was to ampicillin (18%). Thirteen percent was identified as multidrug resistant isolates. No association between AMU and AMR was found in the investigated samples. The issue to address, regarding AMU in companion animal, lies within the quality of use, not the quantity. Especially from a One-Health perspective, companion animals might be a source of transmission of resistance genes and/or resistant bacteria to humans.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) spa type t127, Sequence Type (ST)1, quickly spreads and persists among young pigs.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) clones other than Clonal Complex (CC)398, as CC1, have been isolated in pigs in some countries, and appeared to be prevalent in Italy. The aim of this study was to evaluate the capability of Sequence Type (ST)1, CC1, LA-MRSA clone to colonize and to be transmitted among piglets. Eighteen caesarean-derived/colostrum-deprived piglets of 35 days of age were assigned randomly to three groups: four seeder piglets were contaminated with a spa type t127, ST1, SCCmec V, MRSA (Group A), 10 MRSA-negative piglets were exposed to Group A after 2 days post-contamination, dpc (Group B) and 4 piglets were used as control group (Group C). Piglets were evaluated until 44 dpc (Group A) or at 42 days post-exposure, dpe (Group B) and then euthanized and necropsied. All nasal and skin cultures of Group A resulted MRSA-positive throughout the experiment starting from two dpc, while Group C tested always MRSA-negative. At first sampling, all Group B piglets became positive and remained positive throughout the experiment. This is the first colonization/transmission study with a CC1 LA-MRSA in pigs. The results add further knowledge on the ability of CC1 LA-MRSA to colonize pigs, and on colonization/transmission patterns, both suggesting good host adaptation.

Detection and isolation of Shiga Toxin-producing Escherichia coli (STEC) strains in caecal samples from pigs at slaughter in Italy.

Shiga toxin-producing Escherichia coli (STEC) strains are food-borne pathogens of public health concern. Despite ruminants are the most important reservoir, STEC human infections have also been attributed to pigs. We examined for the presence of STEC in 234 samples of swine caecal content collected during the year 2015 at Italian abattoirs in the framework of the harmonized monitoring of antimicrobial resistance (Decision 2013/652/EU). The presence of stx genes was detected in 122 (52.1%) samples, which were subsequently subjected to STEC isolation and characterization. The analysis of the 66 isolated STEC strains showed that the majority of the isolates (74.2%) possessed the stx2a gene subtype, in a few cases (16.7%) in combination with stx2b or stx2c. Only 25.8% of isolates possessed the stx2e subtype, typical of swine-adapted STEC. None of the isolates possessed the intimin-coding eae gene and the majority of them did not belong to serogroups commonly associated with human infections. The results of this study suggest that pigs can be considered as potential reservoir of certain STEC types.

Systemic tuberculosis by MYCOBACTERIUM BOVIS in a free-ranging MARSICAN brown bear (URSUS ARCTOS MARSICANUS): a Case report.

BACKGROUND: Mycobacterium bovis is known to have a wide host range and has been isolated from numerous free-ranging wildlife species, carnivores included. In bears, M. bovis has been previously reported only from a culture of pooled lymph nodes of a black bear (Ursus americanus) in the absence of lesions. The aims of this study were to describe gross and microscopic pathological findings of M. bovis tuberculosis in a deceased Marsican brown bear (Ursus arctos marsicanus). CASE PRESENTATION: In March 2014, an adult female Marsican brown bear was found in the Abruzzo, Lazio and Molise National Park (Italy) showing severe non-specific clinical signs. The animal died soon after its discovery and the carcass was submitted to post-mortem examination to identify the cause of death. The bear was diagnosed with a severe Mycobacterium bovis infection, with both pathological and microbiological aspects suggesting ongoing generalization. A presumptive diagnosis of mycobacterial infection was initially made based on gross findings. Histopathology showed the presence of acid-fast bacilli in all sampled tissues along with poorly organized granulomatous lesions. Slow-growing Mycobacterium sp. was isolated from multiple organs (intestine, mesenteric lymph nodes, liver, spleen, lung and kidneys). The PCR and sequencing algorithm identified the Mycobacterium sp. isolate as M. bovis. Spoligotyping demonstrated that the M. bovis isolate belonged to spoligotype SB0120. CONCLUSIONS: This is the first report of lethal M. bovis tuberculosis infection in a free-ranging brown bear. This pathogen could have serious adverse effects in an endangered relic population such as the Marsican brown bear. Stricter application of health regulations in force, surveillance of M. bovis infections in wild ungulates and carnivore scavengers, along with dismissal of supplementary feeding points intended for cattle or wildlife, are warranted to control the presence of bovine tuberculosis in wild and domestic animals in protected areas.

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.

Non-toxigenic Corynebacterium ulcerans sequence types 325 and 339 isolated from two dogs with ulcerative lesions in Italy.

Corynebacterium ulcerans, an emerging pathogen related to C. diphtheriae and C. pseudotuberculosis, is able to cause disease in both human and animal hosts. C. ulcerans may harbor acquired virulence factors such as dermonecrotic exotoxin phospholipase D (PLD) and the prophage-encoded diphtheria toxin (DT). Infections typically occur in persons reporting close contact with animals. In pets, C. ulcerans has been isolated from both asymptomatic carriers and clinically affected dogs and cats. We describe the isolation and characterization of C. ulcerans strains from 2 pet dogs with ulcerative lesions in Italy. The 2 isolates tested negative for both DT genes, but were PLD-producers and belonged to sequence types (STs) 325 and 339. These 2 cases highlight that C. ulcerans cutaneous infections might be underestimated in pets, given that many veterinary laboratories do not routinely consider and/or identify Corynebacterium species from cutaneous samples. Early detection and molecular typing of C. ulcerans is essential in order to implement effective treatment and to prevent diffusion and possible zoonotic transmission of certain STs.