Background and aimPlasmid-mediated colistin resistance mechanisms have been identified worldwide in the past years. A multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes (mcr-1 to mcr-5, and variants) in Enterobacteriaceae was developed for surveillance or research purposes. Methods: We designed four new primer pairs to amplify mcr-1, mcr-2, mcr-3 and mcr-4 gene products and used the originally described primers for mcr-5 to obtain a stepwise separation of ca 200 bp between amplicons. The primer pairs and amplification conditions allow for single or multiple detection of all currently described mcr genes and their variants present in Enterobacteriaceae. The protocol was validated testing 49 European Escherichia coli and Salmonella isolates of animal origin. Results: Multiplex PCR results in bovine and porcine isolates from Spain, Germany, France and Italy showed full concordance with whole genome sequence data. The method was able to detect mcr-1, mcr-3 and mcr-4 as singletons or in different combinations as they were present in the test isolates. One new mcr-4 variant, mcr-4.3, was also identified. Conclusions: This method allows rapid identification of mcr-positive bacteria and overcomes the challenges of phenotypic detection of colistin resistance. The multiplex PCR should be particularly interesting in settings or laboratories with limited resources for performing genetic analysis as it provides information on the mechanism of colistin resistance without requiring genome sequencing.

We report the spread of a clone of multidrug-resistant (MDR), ESBL-producing (bla CTX-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 bla CTX-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.

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 investigated the evolution and epidemiology of a novel livestock-associated methicillin-resistant Staphylococcus aureus strain, which colonizes and infects urban-dwelling Danes even without a Danish animal reservoir. Genetic evidence suggests both poultry and human adaptation, with poultry meat implicated as a probable source.

S taphylococcus aureus is a major pathogen causing considerable human morbidity and mortality worldwide, and it is a leading cause of infections of some economically important livestock species and, as a prominent bacterial cause of contagious bovine mastitis, a major economic burden for the dairy cattle industry (1). Methicillin-resistant S. aureus (MRSA) lineages are causes of health care-and community-associated infections, which are a major burden of disease on a global scale (2). In the last decade, MRSA sequence type 398 (ST398) has found an ecological niche in the pig, cattle, and poultry industries, although other MRSA lineages (e.g., ST1, ST5, ST9, ST97, ST130, and ST433) have been identified in farmed animals worldwide (3) All these lineages are currently termed "livestock-associated MRSA" (LA-MRSA).The MRSA clonal complex 97 (CC97) lineage has been reported as the second most prevalent MRSA lineage in the Italian pig industry, where spa types t1730 and t4795, both belonging to ST97, were estimated to have colonized 11% of finishing holdings (4). In Italy, in the last 5 years, the CC97 MRSA lineage has also been increasingly detected in cattle primary production, either from mastitis in dairy cattle or from bulk tank milk (5-7). Occasionally, CC97 MRSA isolates have been detected in cattle in Germany and Spain.CC97 is one of the major S. aureus clonal complexes in bovines (8), and recently, a livestock origin of the human pandemic CC97 MRSA strains has been demonstrated, resulting in two emergent human epidemic CC97 community-acquired/associated MRSA (CA-MRSA) clones (8).The aim of this study was to determine the genetic relatedness of MRSA and methicillin-susceptible S. aureus (MSSA) CC97 isolates from pigs and dairy cattle in Italy in the last few years (2008 to 2012) for epidemiological and risk assessment purposes.