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We tested two summer protocols for oxytetracycline (OTC) administration on beehives to evaluate: toxicity for the bees, residues in honey and presence of genes encoding for OTC antibiotic resistance in bacteria isolated from the gut of adult bees. In July 2020, eighteen healthy colonies were divided into three groups. Two OTC treatment protocols - long and short, were devised, and one group was left untreated. We did not detect any toxicity on adult bees or brood but found OTC residues in the honey of the nest up to seven months after both treatments (210.3±221.2 µg/kg after long protocol; 216.2±238.4 µg/kg after short protocol), even in the untreated group (up to the mean 75.8±173.2 µg/kg). Antibiotic resistance genes were found, even in the untreated group, possibly due to the long exposure to antibiotics. More in-depth studies should be performed to verify how the long-term persistence of antibiotics impacted the beehive during the inactive season.

Culex pipiens is a major vector of pathogens, including West Nile and Usutu viruses, that poses a significant public health risk. Monitoring pyrethroid resistance in mosquito populations is essential for effective vector control. This study aims to evaluate four DNA extraction protocols—QIAsymphony, DNAzol® Direct reagent, PrepMan® Ultra Sample Preparation Reagent (USPR), and Chelex® 100—to identify an optimal method to extract DNA from individual Culex pipiens, as part of a high-throughput surveillance of pyrethroid resistance using Real-Time Genotyping PCR. The target is the L1014F mutation in the voltage-sensitive sodium channel (VSSC) gene, which confers knockdown (kdr) resistance to pyrethroids. Mosquitoes were collected from wintering and summer habitats in Lazio and Tuscany, Italy, and DNA was extracted using the four methods. The quality, quantity, extraction time, and cost of the DNA were compared among the various methods. The PrepMan® USPR protocol was the most efficient, providing high-quality DNA with a 260/280 purity ratio within the optimal range at the lowest cost and in a short time. This method also demonstrated the highest amplification success rate (77%) in subsequent real-time PCR assays, making it the preferred protocol for large-scale genotyping studies. [ABSTRACT FROM AUTHOR]

Mycolicibacterium hassiacum (homotypic synonym: Mycobacterium hassiacum) represents an ungrouped thermotolerant rapidly growing mycobacteria (RGM) species occasionally associated with infections and disease in humans. In this report, we describe a case of pyogranulomatous dermatitis and panniculitis due to M. hassiacum in an immunocompetent adult cat. To the best of our knowledge, this represents the first report of M. hassiacum infection in animals. We also report the results of the in-depth genome characterization of the isolate using a combined short- and long-read whole-genome sequencing (WGS) approach. We observed the lack of acquired-resistance genes and no evidence of mutations in housekeeping genes associated with resistance to rifampicin and isoniazid. We detected some virulence factors in our isolate, such as some associated with the interaction of mycobacteria with host cells, and the presence of multiple copies of heavy metal resistance genes (arsB, arsR, and arsL/cadL). In conclusion, M. hassiacum should be included among the RGM species associated with feline subcutaneous atypical mycobacteriosis (SAM). A reliable and fast RGM laboratory identification and characterization is important not only for an accurate etiological diagnosis but also for a correct approach to SAM treatment options. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: The raccoon (Procyon lotor) is a carnivore native to North and Central America, gradually introduced into Asia and Europe, including Italy. It is an important carrier of multiple endoparasites, some of them being zoonotic. The aim of this study was to investigate the endoparasites of the non-native raccoon population of Central Italy. The results showed the presence of a number of different Protozoa and Nematodes. Among them, we identified Baylisascaris procyonis, an emerging helminthic zoonotic agent of serious concern for public and animal health, given the possibility of its transmission to paratenic hosts, including humans and pets, at times with severe clinical consequences. The role of the raccoon as a multi-parasite carrier highlights the importance of the eradication/control of this alien species and the need to implement related disease surveillance programs. The raccoon (Procyon lotor) is a carnivore native to North and Central America, gradually introduced into Asia and Europe, including Italy. It is an important carrier of multiple endoparasites, both Protozoa and Helminths, some of them being zoonotic. The aim of this study was to investigate the endoparasites of the non-native raccoon population of Central Italy. Sixty-two raccoons were collected by local competent authorities (sixty trapped and euthanized, two found dead) and subjected to necroscopic examination. Carcasses underwent a broad parasitological investigation, including coprological techniques (macroscopic examination of the gastrointestinal tract, lungs, trachea, and heart, Flotac®, Baermann test, and immunofluorescence for Giardia duodenalis and Cryptosporidium spp.), research on respiratory/urinary capillariosis and artificial digestion for Trichinella spp. larvae, and a histopathological examination of the ileum. Ascarid parasites were further identified at the species level using a next-generation sequencing-based amplicon sequencing approach. The results showed the presence of different Protozoa and Nematodes: Baylisascaris procyonis (26/62; 41.9%), Pearsonema sp. (6/62; 9.6%), Capillariidae (6/62; 9.6%), Eimeria sp. (2/62; 3.2%), Cryptosporidium sp. (2/62; 3.2%), and Ancylostomatidae (2/62; 3.2%). B. procyonis is an emerging helminthic zoonotic agent considered a serious concern for public and animal health, given the possibility of its transmission to paratenic hosts, including humans and pets. The demonstrated role of the raccoon as a multi-parasite carrier should be an incentive to continuing the eradication/control of this alien species, and supports the need to implement related disease surveillance programs. [ABSTRACT FROM AUTHOR]

Contrary to humans, candidiasis is a rare infection in animals. However, in reptiles, candidiasis can cause gastrointestinal, cutaneous, or rarely systemic infections in stressed animals. The infections due to Yarrowia lipolytica have been increasingly described in human medicine, and hundreds of cases are reported, comprised of granulomatous lung lesions. Herein, granulomatous pneumonia of a spectacled caiman, Caiman crocodilus, was described, and the presence of Y. lipolytica in the lesion was confirmed through histopathology, microbiologic cultures, and molecular methods. The cause of death of the spectacled caiman was ascribed to bacterial shock septicemia consequentially to a traumatic lesion. However, in the right lung, several nodules containing white exudate were evidenced. At mycological and molecular analyses, Y. lipolytica was evidenced, and the histological finding confirmed the presence of a Candida infection in the lung granulomatous lesions. The comparison of ITS sequences with 11 Yarrowia spp. isolates, recently described in green sea turtles, and with a human strain was conducted, and the whole genome of a strain isolated in the spectacled caiman was sequenced. Even though Y. lipolytica is considered a non-pathogenic yeast and has been rarely described in animals, it seems to cause granulomatous lesions in reptiles as in humans. [ABSTRACT FROM AUTHOR]

This paper reported a case of a metastrongyloid nematode Angiostrongylus vasorum infection in a fennec (Vulpes zerda) kept in a zoo in central Italy. The fennec had shown paralysis of the hind limbs, anorexia, weakness and respiratory signs before death. Cardiomegaly and granulomatous pneumonia were the major anatomopathological findings. Inflammatory lesions associated with parasitic larvae were observed in the lungs, brain, liver, heart, spinal cord and kidney of the fennec at histology. A. vasorum diagnosis was confirmed by both morphological and molecular identification of adult worms recovered at necropsy. Fennecs are active predators and maintain their hunting behaviour in captivity. Hence, it is likely that the animal was exposed to infection by preying on parasitised gastropods, intermediate hosts of A. vasorum, entering zoo enclosures from the surrounding environment. This is the first report of A. vasorum systemic infection in a captive fennec (V. zerda) in a zoo in Italy. [ABSTRACT FROM AUTHOR]

To the Editor: Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has been identified in livestock animals (particularly pigs), veterinarians, and animal farm workers (1,2). CA-MRSA strains from pigs have been classified most [...]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We report the spread of a clone of multidrug-resistant (MDR), ESBL-producing (blaCTX-M-1) Salmonella enterica subsp. enterica serovar Infantis, in the Italian broiler chicken industry and along the food-chain. This was first detected in Italy in 2011 and led to human infection in Italy in 2013–2014.A set (n = 49) of extended-spectrum cephalosporin (ESC)-resistant (R) isolates of S. Infantis (2011–2014) from humans, food-producing animals and meat thereof, were studied along with a selected set of earlier and more recent ESC-susceptible (ESC-S) isolates (n = 42, 2001–2014). They were characterized by macrorestriction-PFGE analysis and genetic environment of ESC-resistance. Isolates representative of PFGE-patterns and origin were submitted to Whole Genome Sequencing. The emerging ESC-R clone, detected mainly from broiler chickens, broiler meat and humans, showed a minimum pattern of clinical resistance to cefotaxime, tetracycline, sulfonamides, and trimethoprim, beside ciprofloxacin microbiological resistance (MIC 0.25 mg/L). All isolates of this clone harbored a conjugative megaplasmid (~ 280–320 Kb), similar to that described in ESC-susceptible S. Infantis in Israel (pESI-like) in 2014. This megaplasmid carried the ESBL gene blaCTX-M-1, and additional genes [tet(A), sul1, dfrA1 and dfrA14] mediating cefotaxime, tetracycline, sulfonamide, and trimethoprim resistance. It also contained genes conferring enhanced colonization capability, virulence (fimbriae, yersiniabactin), resistance and fitness (qacE1, mer) in the intensive-farming environment. This emerging clone of S. Infantis has been causing infections in humans, most likely through the broiler industry. Since S. Infantis is among major serovars causing human infections in Europe and is an emerging non-typhoidal Salmonella globally, further spread of this lineage in primary productions deserves quick and thorough risk-management strategies. [ABSTRACT FROM AUTHOR]

Avian malaria is a worldwide distributed, vector-born disease of birds caused by parasites of the order Haemosporida. There is a lack of knowledge about the presence and pathogenetic role of Haemosporida in Psittacidae. Here we report a case of avian malaria infection in lovebirds (Agapornis roseicollis), with the genetic characterization of the Plasmodium species involved. The birds were hosted in a zoo located in Italy, where avian malaria cases in African penguins (Spheniscus demersus) were already reported. Animals (n = 11) were submitted for necropsy after sudden death and were subjected to further analyses including histopathology, bacteriology, and PCR for the research of haemosporidians. Clinical history, gross lesions and histopathological observation of schizonts, together with positive PCR results for Plasmodium spp., demonstrated that avian malaria was the cause of death for one animal and the possible cause of death for the other nine. The sequences obtained were compared using BLAST and analyzed for similarity to sequences available at the MalAvi database. Genetic analyses demonstrated a 100% nucleotide identity to Plasmodium matutinum LINN1 for all the obtained sequences. To our knowledge, this is the first report describing avian malaria in lovebirds. [ABSTRACT FROM AUTHOR]

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, SCC mec 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. [ABSTRACT FROM AUTHOR]

Salmonella enterica serovar Infantis represents the most frequent serovar detected in broilers and broiler meat and one of the top five serovars involved in human infections in Europe. The increasing incidence of S. infantis infections may be complicated by the spread of multidrug-resistant (MDR) strains, such as the recent spread in Europe of MDR, Extended Spectrum Beta-Lactamases (ESBL)-producing S. infantis harbouring a conjugative pESI-like megaplasmid in broilers, broiler meat and humans. In this study we report for the first time the isolation and in-depth characterization by whole genome sequencing (WGS) of four, mcr-1-positive, MDR S. infantis isolated from broilers and broiler meat samples in the frame of the Italian antimicrobial resistance monitoring (2016-2017). Phylogenetic relationships with previously characterized Italian S. Infantis, belonging to the pESI-like positive, (ESBL)-producing clone, were also assessed. All isolates presented the same gyrA point mutation associated with fluoroquinolones resistance and harboured: a. a pESI-like plasmid containing genes coding for different toxin-antitoxin systems and specific markers associated with virulence, enhanced colonization capability and enhanced fitness, previously described in pESI-likepositive S. infantis in Italy; b. an incX4 plasmid harbouring mcr-1.1 and the genes coding for the HicAB toxin-antitoxin complex. Additionally, two isolates were also ESBL-producers (CTX-M-1 type) and within the same cluster that included isolates from broiler chicken, broiler meat and human clinical cases with unknown epidemiological relationship, all belonging to the ESBL-pESI-like-positive S. infantis clone previously described in Italy. Overall, these findings are of great concern, since this clone, has genetic traits mediated by the pESI-like plasmid of enhanced virulence, MDR and fitness in the intensive farming system, and it is often ESBL-producing. Additionally, we have demonstrated that this clone has the attitude to acquire additional transferable resistance to last-resort drugs like colistin. These characteristics inevitably lead to a further reduction of therapeutic options for Salmonella invasive infections in humans, transmitted through the food-chain. Our results highlight the need of implementation of risk-management strategies and actions to be taken in order to drastically reduce the amount of colistin used in broilers in Italy and reduce the overall prevalence of S. Infantis, within the Italian broiler chicken industry. [ABSTRACT FROM AUTHOR]

• First evidence in Italy of a NDM-5-producing E. coli in companion animals. • Full reconstruction of the bla NDM-5 –carrying mosaic plasmid. • Evidence of transmission of bla NDM-5 E. coli from humans to animals and vice-versa. 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 bla NDM-5 was fully reconstructed. The complete sequence of the pMOL008 mosaic plasmid (F36:F31:A4:B1; pMOL008) harbouring bla NDM-5 , was resolved and characterized. Moreover, a (pro)phage and IncFII, containing bla CMY-2 and ermB , and IncI2 plasmid types were also identified. pMOL008 was almost identical to bla NDM-5 -containing plasmids from E. coli ST167 isolated from Italian human clinical cases and from a Swiss dog and colonized humans. bla NDM-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. [ABSTRACT FROM AUTHOR]

Avian malaria is a parasitic disease of birds caused by protozoa belonging to the genus Plasmodium , within the order Haemosporida. Penguins are considered particularly susceptible, and outbreaks in captive populations can lead to high mortality. We used a multidisciplinary approach to investigate the death due to avian malaria, occurred between 2015 and 2019, in eight African penguins ( Spheniscus demersus ) kept in two Italian zoos located in central Italy, and situated about 30 km apart. We also provided information about the presence and circulation of Plasmodium spp. in mosquitoes in central Italy by sampling mosquitoes in both zoos where penguin mortalities occurred. In the eight dead penguins, gross and histopathological lesions were consistent with those previously observed by other authors in avian malaria outbreaks. Organs from dead penguins and mosquitoes collected in both zoos were tested for avian malaria parasites by using a PCR assay targeting the partial mitochondrial conserved region of the cytochrome b gene. Identification at species level was performed by sequencing analysis. Plasmodium matutinum was detected in both dead penguins and in mosquitoes ( Culex pipiens ), while Plasmodium vaughani in Culex pipiens only. Parasites were not found in any of the PCR tested Aedes albopictus samples. Based on our phylogenetic analysis, we detected three previously characterized lineages: Plasmodium matutinum LINN1 and AFTRU5, P. vaughani SYAT05. In Culex pipiens we also identified two novel lineages, CXPIP32 (inferred morphospecies Plasmodium matutinum ) and CXPIP33 (inferred morphospecies P. vaughani ). Significantly, LINN1 and AFTRU5 were found to be associated to penguin deaths, although only LINN1 was detected both in penguins (along the years of the study) and in Culex pipiens , while AFTRU5 was detected in a single penguin dead in 2017. In conclusion, in our study Plasmodium matutinum was found to cause avian malaria in captive penguins kept in Europe, with Culex pipiens being its most probable vector. Our results are in agreement with previous studies suggesting that Culex pipiens is one of the main vectors of Plasmodium spp. in Europe and the Northern Hemisphere. Zoos maintaining captive penguins in temperate areas where Culex pipiens is abundant should be well aware of the risks of avian malaria, and should put every effort to prevent outbreaks, in particular during the periods when the number of vectors is higher., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Iurescia, Romiti, Cocumelli, Diaconu, Stravino, Onorati, Alba, Friedrich, Maggi, Magliano, Ermenegildi, Carfora, Caprioli, De Liberato and Battisti.)

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: bla CTX-M-1 -positive in European isolates and bla CTX-M-65 -positive in American isolates (study outgroup). Both variants had toxin-antitoxin systems, resistance genes towards tetracyclines, trimethoprim, sulphonamides and aminoglycosides, heavy metals ( mer A) and disinfectants ( qac EΔ). Worryingly, 66 % of the total isolates studied presented different gyr A 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.

We report the first diagnostic test for the identification of Staphylococcus pseudintermedius involving a simple PCR-restriction fragment length polymorphism approach. The method allows discrimination of S. pseudintermedius from the closely related members of the Staphylococcus intermedius group and other important staphylococcal pathogens of humans and dogs.