Detection of Salmonella Reservoirs in Birds of Prey Hosted in an Italian Wildlife Centre: Molecular and Antimicrobial Resistance Characterisation.

In the European Union, salmonellosis is one of the most important zoonoses reported. Poultry meat and egg products are the most common food matrices associated with Salmonella presence. Moreover, wild and domestic animals could represent an important reservoir that could favour the direct and indirect transmission of pathogens to humans. Salmonella spp. can infect carnivorous or omnivorous wild birds that regularly ingest food and water exposed to faecal contamination. Birds kept in captivity can act as reservoirs of Salmonella spp. following ingestion of infected prey or feed. In this paper, we describe the isolation of different Salmonella serovars in several species of raptors hosted in aviaries in an Italian wildlife centre and in the raw chicken necks used as their feed but intended for human consumption. Characterisations of strains were carried out by integrating classical methods and whole genome sequencing analysis. The strains of S. bredeney isolated in poultry meat and birds belonged to the same cluster, with some of them being multidrug-resistant (MDR) and carrying the Col(pHAD28) plasmid-borne qnrB19 (fluoro)quinolone resistance gene, thus confirming the source of infection. Differently, the S. infantis found in feed and raptors were all MDR, carried a plasmid of emerging S. infantis (pESI)-like plasmid and belonged to different clusters, possibly suggesting a long-lasting infection or the presence of additional undetected sources. Due to the high risk of fuelling a reservoir of human pathogens, the control and treatment of feed for captive species are crucial.

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.

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.

Emergence of a Clonal Lineage of Multidrug-Resistant ESBL-Producing Salmonella Infantis Transmitted from Broilers and Broiler Meat to Humans in Italy between 2011 and 2014.

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.

Livestock-Associated Methicillin Resistant and Methicillin Susceptible Staphylococcus aureus Sequence Type (CC)1 in European Farmed Animals: High Genetic Relatedness of Isolates from Italian Cattle Herds and Humans.

Methicillin-resistant Staphylococcus aureus (MRSA) Sequence Type (ST)1, Clonal Complex(CC)1, SCCmec V is one of the major Livestock-Associated (LA-) lineages in pig farming industry in Italy and is associated with pigs in other European countries. Recently, it has been increasingly detected in Italian dairy cattle herds. The aim of this study was to analyse the differences between ST1 MRSA and methicillin-susceptible S. aureus (MSSA) from cattle and pig herds in Italy and Europe and human isolates. Sixty-tree animal isolates from different holdings and 20 human isolates were characterized by pulsed-field gel electrophoresis (PFGE), spa-typing, SCCmec typing, and by micro-array analysis for several virulence, antimicrobial resistance, and strain/host-specific marker genes. Three major PFGE clusters were detected. The bovine isolates shared a high (≥90% to 100%) similarity with human isolates and carried the same SCCmec type IVa. They often showed genetic features typical of human adaptation or present in human-associated CC1: Immune evasion cluster (IEC) genes sak and scn, or sea; sat and aphA3-mediated aminoglycoside resistance. Contrary, typical markers of porcine origin in Italy and Spain, like erm(A) mediated macrolide-lincosamide-streptograminB, and of vga(A)-mediated pleuromutilin resistance were always absent in human and bovine isolates. Most of ST(CC)1 MRSA from dairy cattle were multidrug-resistant and contained virulence and immunomodulatory genes associated with full capability of colonizing humans. As such, these strains may represent a greater human hazard than the porcine strains. The zoonotic capacity of CC1 LA-MRSA from livestock must be taken seriously and measures should be implemented at farm-level to prevent spill-over.

Extended-spectrum-beta-lactamases, AmpC beta-lactamases and plasmid mediated quinolone resistance in klebsiella spp. from companion animals in Italy.

We report the genetic characterization of 15 Klebsiella pneumoniae (KP) and 4 isolates of K. oxytoca (KO) from clinical cases in dogs and cats and showing extended-spectrum cephalosporin (ESC) resistance. Extended spectrum beta-lactamase (ESBL) and AmpC genes, plasmid-mediated quinolone resistance (PMQR) and co-resistances were investigated. Among KP isolates, ST101 clone was predominant (8/15, 53%), followed by ST15 (4/15, 27%). ST11 and ST340, belonging to Clonal Complex (CC)11, were detected in 2012 (3/15, 20%). MLST on KP isolates corresponded well with PFGE results, with 11 different PFGE patterns observed, including two clusters of two (ST340) and four (ST101) indistinguishable isolates, respectively. All isolates harbored at least one ESBL or AmpC gene, all carried on transferable plasmids (IncR, IncFII, IncI1, IncN), and 16/19 were positive for PMQR genes (qnr family or aac(6')-Ib-cr). The most frequent ESBL was CTX-M-15 (11/19, 58%), detected in all KP ST101, in one KP ST15 and in both KP ST340. blaCTX-M-15 was carried on IncR plasmids in all but one KP isolate. All KP ST15 isolates harbored different ESC resistance genes and different plasmids, and presented the non-transferable blaSHV-28 gene, in association with blaCTX-M-15, blaCTX-M-1 (on IncR, or on IncN), blaSHV-2a (on IncR) or blaCMY-2 genes (on IncI1). KO isolates were positive for blaCTX-M-9 gene (on IncHI2), or for the blaSHV-12 and blaDHA-1 genes (on IncL/M). They were all positive for qnr genes, and one also for the aac(6')-Ib-cr gene. All Klebsiella isolates showed multiresistance towards aminoglycosides, sulfonamides, tetracyclines, trimethoprim and amphenicols, mediated by strA/B, aadA2, aadB, ant (2")-Ia, aac(6')-Ib, sul, tet, dfr and cat genes in various combinations. The emergence in pets of multidrug-resistant Klebsiella with ESBL, AmpC and PMQR determinants, poses further and serious challenges in companion animal therapy and raise concerns for possible bi-directional transmission between pets and humans, especially at household level.

Extended-spectrum beta-lactamases in Escherichia coli isolated from dogs and cats in Rome, Italy, from 2001 to 2003.

We report expanded-spectrum cephalosporin resistance in Escherichia coli from dogs and cats in Rome, Italy. Three major beta-lactamases (CMY-2, SHV-12, and CTX-M-1) are reported for the first time in E. coli from sick and healthy dogs and cats. Molecular characterization suggests the presence of several combinations of beta-lactamase genes in E. coli from companion animals.