Azithromycin resistance in Escherichia coli and Salmonella from food-producing animals and meat in Europe.

OBJECTIVES: To characterize the genetic basis of azithromycin resistance in Escherichia coli and Salmonella collected within the EU harmonized antimicrobial resistance (AMR) surveillance programme in 2014-18 and the Danish AMR surveillance programme in 2016-19. METHODS: WGS data of 1007 E. coli [165 azithromycin resistant (MIC > 16 mg/L)] and 269 Salmonella [29 azithromycin resistant (MIC > 16 mg/L)] were screened for acquired macrolide resistance genes and mutations in rplDV, 23S rRNA and acrB genes using ResFinder v4.0, AMRFinder Plus and custom scripts. Genotype-phenotype concordance was determined for all isolates. Transferability of mef(C)-mph(G)-carrying plasmids was assessed by conjugation experiments. RESULTS: mph(A), mph(B), mef(B), erm(B) and mef(C)-mph(G) were detected in E. coli and Salmonella, whereas erm(C), erm(42), ere(A) and mph(E)-msr(E) were detected in E. coli only. The presence of macrolide resistance genes, alone or in combination, was concordant with the azithromycin-resistant phenotype in 69% of isolates. Distinct mph(A) operon structures were observed in azithromycin-susceptible (n = 50) and -resistant (n = 136) isolates. mef(C)-mph(G) were detected in porcine and bovine E. coli and in porcine Salmonella enterica serovar Derby and Salmonella enterica 1,4, [5],12:i:-, flanked downstream by ISCR2 or TnAs1 and associated with IncIγ and IncFII plasmids. CONCLUSIONS: Diverse azithromycin resistance genes were detected in E. coli and Salmonella from food-producing animals and meat in Europe. Azithromycin resistance genes mef(C)-mph(G) and erm(42) appear to be emerging primarily in porcine E. coli isolates. The identification of distinct mph(A) operon structures in susceptible and resistant isolates increases the predictive power of WGS-based methods for in silico detection of azithromycin resistance in Enterobacterales.

ResFinder 4.0 for predictions of phenotypes from genotypes.

OBJECTIVES: WGS-based antimicrobial susceptibility testing (AST) is as reliable as phenotypic AST for several antimicrobial/bacterial species combinations. However, routine use of WGS-based AST is hindered by the need for bioinformatics skills and knowledge of antimicrobial resistance (AMR) determinants to operate the vast majority of tools developed to date. By leveraging on ResFinder and PointFinder, two freely accessible tools that can also assist users without bioinformatics skills, we aimed at increasing their speed and providing an easily interpretable antibiogram as output. METHODS: The ResFinder code was re-written to process raw reads and use Kmer-based alignment. The existing ResFinder and PointFinder databases were revised and expanded. Additional databases were developed including a genotype-to-phenotype key associating each AMR determinant with a phenotype at the antimicrobial compound level, and species-specific panels for in silico antibiograms. ResFinder 4.0 was validated using Escherichia coli (n = 584), Salmonella spp. (n = 1081), Campylobacter jejuni (n = 239), Enterococcus faecium (n = 106), Enterococcus faecalis (n = 50) and Staphylococcus aureus (n = 163) exhibiting different AST profiles, and from different human and animal sources and geographical origins. RESULTS: Genotype-phenotype concordance was ≥95% for 46/51 and 25/32 of the antimicrobial/species combinations evaluated for Gram-negative and Gram-positive bacteria, respectively. When genotype-phenotype concordance was <95%, discrepancies were mainly linked to criteria for interpretation of phenotypic tests and suboptimal sequence quality, and not to ResFinder 4.0 performance. CONCLUSIONS: WGS-based AST using ResFinder 4.0 provides in silico antibiograms as reliable as those obtained by phenotypic AST at least for the bacterial species/antimicrobial agents of major public health relevance considered.

Genetic Diversity of Salmonella enterica subsp. enterica Serovar Enteritidis from Human and Non-Human Sources in Portugal.

Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) is one of the leading causes of foodborne infections associated with broilers and laying hens. Portugal has had the lowest notification rates of salmonellosis in recent years, due to the vaccinations of layer and breeder flocks and strict compliance with biosecurity measures. However, data about the genetic diversity of S. Enteritidis in Portugal are scarce. In this study, 102 S. Enteritidis isolates selected from human (n = 63) and non-human sources (n = 39) were characterized by serotyping, antimicrobial susceptibility, and whole genome sequencing. The S. Enteritidis population was mainly resistant to fluoroquinolones, and a sole isolate showed resistance to extended-spectrum cephalosporins. ST11 was the most frequent sequence type, and three novel STs from human isolates (ST9236, ST4457, and ST9995) were assigned. Several Salmonella pathogenic islands (SPI) and Putative SPI were present in the genomes, namely SPI-1, 2, 3, 4, 5, 9, 10, 12, 13, and 14, C63PI, CS54_island, and 170 virulence genes were identified. The phylogenetic analysis revealed that strains from Portugal are genetically heterogeneous regarding sample type, collection date, and genetic content. This study increases the available data, essential to a better characterization of strains in a global context.

Plasmid-Mediated Colistin Resistance Genes mcr-1 and mcr-4 in Multidrug-Resistant Escherichia coli Strains Isolated from a Healthy Pig in Portugal.

Antimicrobial resistance encoded by mobile colistin resistance (mcr) genes is a global and emergent threat. In this study, we report the occurrence of two different populations of colistin-resistant Escherichia coli harboring mcr-1 and mcr-4 variants in the intestinal microbiome of a healthy pig. Following antimicrobial susceptibility determination, the presence of mcr genes in two E. coli strains, isolated according to different selective microbiological procedures, was screened by PCR. Whole-genome sequencing confirmed that both strains were multidrug-resistant; INIAV_002EC was an AmpC producer carrying blaCMY-2, blaTEM-1B, qnrS1, mcr-1.1 genes, and INIAV_001EC carrying blaTEM-1A, tetB, and mcr-4.1 genes, along with mutations in quinolone resistance-determining regions. In addition, both strains harbored sul3, dfrA, and aadA1 determinants. Further genome analysis revealed different plasmid replicons associated with the mcr genes, IncX4 associated with mcr-1.1, and ColE10 with mcr-4.1. In addition, other replicons, including IncFIA, IncI1-Iγ, IncX1, IncY, in INIAV_002EC, and IncX1, IncI1, and p0111, in INIAV_001EC, were identified. Furthermore, both strains belonged to ST215 serotype O68:H12 and ST156 serotype O25:H28, respectively. This finding highlights the pig gut flora as a potential reservoir of mobile colistin resistance genes and reports the presence of the mcr-4.1 gene found for the first time in Portugal.

First comparative genomic characterization of the MSSA ST398 lineage detected in aquaculture and other reservoirs.

Staphylococcus aureus ST398 can cause diseases in several different animals. In this study we analyzed ten S. aureus ST398 previously collected in three different reservoirs in Portugal (humans, gilthead seabream from aquaculture and dolphin from a zoo). Strains tested against sixteen antibiotics, by disk diffusion or minimum inhibitory concentration, showed decreased susceptibility to benzylpenicillin (all strains from gilthead seabream and dolphin) and to erythromycin with an iMLSB phenotype (nine strains), and susceptibility to cefoxitin (methicillin-susceptible S. aureus, MSSA). All strains from aquaculture belonged to the same spa type, t2383, whereas strains from the dolphin and humans belonged to spa type t571. A more detailed analysis using single nucleotide polymorphisms (SNPs)-based tree and a heat map, showed that all strains from aquaculture origin were highly related with each other and the strains from dolphin and humans were more distinct, although they were very similar in ARG, VF and MGE content. Mutations F3I and A100V in glpT gene and D278E and E291D in murA gene were identified in nine fosfomycin susceptible strains. The blaZ gene was also detected in six of the seven animal strains. The study of the genetic environment of erm(T)-type (found in nine S. aureus strains) allowed the identification of MGE (rep13-type plasmids and IS431R-type), presumably involved in the mobilization of this gene. All strains showed genes encoding efflux pumps from major facilitator superfamily (e.g., arlR, lmrS-type and norA/B-type), ATP-binding cassettes (ABC; mgrA) and multidrug and toxic compound extrusion (MATE; mepA/R-type) families, all associated to decreased susceptibility to antibiotics/disinfectants. Moreover, genes related with tolerance to heavy metals (cadD), and several VF (e.g., scn, aur, hlgA/B/C and hlb) were also identified. Insertion sequences, prophages, and plasmids made up the mobilome, some of them associated with ARG, VF and genes related with tolerance to heavy metals. This study highlights that S. aureus ST398 can be a reservoir of several ARG, heavy metals resistance genes and VF, which are essential in the adaption and survival of the bacterium in the different environments and an active agent in its dissemination. It makes an important contribution to understanding the extent of the spread of antimicrobial resistance, as well as the virulome, mobilome and resistome of this dangerous lineage.

Building an International One Health Strain Level Database to Characterise the Epidemiology of AMR Threats: ESBL-AmpC Producing E. coli as An Example-Challenges and Perspectives.

Antimicrobial resistance (AMR) is one of the top public health threats nowadays. Among the most important AMR pathogens, Escherichia coli resistant to extended spectrum cephalosporins (ESC-EC) is a perfect example of the One Health problem due to its global distribution in animal, human, and environmental sources and its resistant phenotype, derived from the carriage of plasmid-borne extended-spectrum and AmpC ß-lactamases, which limits the choice of effective antimicrobial therapies. The epidemiology of ESC-EC infection is complex as a result of the multiple possible sources involved in its transmission, and its study would require databases ideally comprising information from animal (livestock, companion, wildlife), human, and environmental sources. Here, we present the steps taken to assemble a database with phenotypic and genetic information on 10,763 ESC-EC isolates retrieved from multiple sources provided by 13 partners located in eight European countries, in the frame of the DiSCoVeR Joint Research project funded by the One Health European Joint Programme (OH-EJP), along with its strengths and limitations. This database represents a first step to help in the assessment of different geographical and temporal trends and transmission dynamics in animals and humans. The work performed highlights aspects that should be considered in future international efforts, such as the one presented here.

Antimicrobial Susceptibility of Enterococcus Isolates from Cattle and Pigs in Portugal: Linezolid Resistance Genes optrA and poxtA.

Enterococci are part of the commensal gut microbiota of mammals, with Enterococcus faecalis and Enterococcus faecium being the most clinically relevant species. This study assesses the prevalence and diversity of enterococcal species in cattle (n = 201) and pig (n = 249) cecal samples collected in 2017. Antimicrobial susceptibility profiles of E. faecium (n = 48) and E. faecalis (n = 84) were assessed by agar and microdilution methods. Resistance genes were screened through PCR and nine strains were analyzed by Whole Genome Sequencing. A wide range of enterococci species was found colonizing the intestines of pigs and cattle. Overall, the prevalence of resistance to critically important antibiotics was low (except for erythromycin), and no glycopeptide-resistant isolates were identified. Two daptomycin-resistant E. faecalis ST58 and ST93 were found. Linezolid-resistant strains of E. faecalis (n = 3) and E. faecium (n = 1) were detected. Moreover, oxazolidinone resistance determinants optrA (n = 8) and poxtA (n = 2) were found in E. faecalis (ST16, ST58, ST207, ST474, ST1178) and E. faecium (ST22, ST2138). Multiple variants of optrA were found in different genetic contexts, either in the chromosome or plasmids. We highlight the importance of animals as reservoirs of resistance genes to critically important antibiotics.

Antibiotic Susceptibility Profiles and Resistance Mechanisms to ß-Lactams and Polymyxins of Escherichia coli from Broilers Raised under Intensive and Extensive Production Systems.

The intensive and extensive broiler production systems imply different veterinary interventions, including the use of antimicrobials. This study aimed to compare the antimicrobial susceptibility profiles of Escherichia coli isolated from both systems, characterize resistance mechanisms to ß-lactams and polymyxins, and identify genetic elements such as integrons. E. coli isolates recovered from broiler cecal samples were assayed for antimicrobial susceptibility through the broth microdilution technique. The molecular characterization of acquired resistance mechanisms to ß-lactams and colistin and the detection of integrons was performed by a multiplex PCR. For most antibiotics tested, the prevalence of reduced susceptibility is higher in commensal and extended-spectrum ß-lactamases (ESBL)/AmpC producers from broilers raised in the intensive system, compared with those raised under extensive conditions. SHV-12 was the most common ESBL enzyme found in both production systems. Other ESBL variants such as CTX-M-1, CTX-M-55, CTX-M-14, CTX-M-32, CTX-M-9, TEM-52, and plasmid-encoded AmpC enzyme CMY-2 were also present. MCR-1 was identified in a colistin-resistant isolate from broilers raised under the intensive system. This study highlights the differences in E. coli antibiotic susceptibility from both production types and emphasizes that a great deal of work remains to decrease consumption and antimicrobial resistance levels.

Emergence of Cfr-Mediated Linezolid Resistance among Livestock-Associated Methicillin-Resistant Staphylococcus aureus (LA-MRSA) from Healthy Pigs in Portugal.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) ST398 is mainly found in Europe and North America, colonizing the nasal cavity of pigs. This study characterized the MRSA isolates recovered from pig nasal swabs (n = 171) by evaluating the antimicrobial susceptibility profile by broth microdilution and characterizing the genetic lineages by spa-typing. Three linezolid-resistant isolates were subjected to Whole-Genome Sequencing (WGS). All strains harbored the mecA gene and were resistant to tetracycline and susceptible to vancomycin. A high frequency of multidrug resistance (97.6%) was evidenced, with 55 different multidrug resistance profiles identified. The MRSA strains were found to belong to 17 spa-types, three being novel. The linezolid-resistant strains appeared to belong to the ST398 type, spa-type t011, and SCCmec_type_Vc and to harbor the cfr, fexA, blaZ, mecA, tetM, and tetK genes. The cfr gene was predicted to be carried in the plasmid, flanked by ISSau9 and the transposon TnpR. MRSA from Portuguese fattening pigs present a high diversity of genetic lineages. The presence of cfr-positive LA-MRSA may represent a risk of transmission to humans, mainly to those in contact with livestock.

Prevalence and Characterization of ESBL/AmpC Producing Escherichia coli from Fresh Meat in Portugal.

The present study aimed to characterize the extended-spectrum ß-lactamases and plasmid-mediated AmpC ß-lactamases (ESBL/PMAß) among Escherichia coli producers isolated from beef, pork, and poultry meat collected at retail, in Portugal. A total of 638 meat samples were collected and inoculated on selective medium for the search of E. coli resistant to 3rd generation cephalosporins. Isolates were characterized by antimicrobial susceptibility testing, molecular assays targeting ESBL/AmpC, plasmid-mediated quinolone resistance (PMQR), and plasmid-mediated colistin resistance (PMCR) encoding genes. The highest frequency of E. coli non-wild type to 3rd generation cephalosporins and fluoroquinolones was observed in broiler meat (30.3% and 93.3%, respectively). Overall, a diversity of acquired resistance mechanisms, were detected: blaESBL [blaCTX-M-1 (n = 19), blaCTX-M-15 (n = 4), blaCTX-M-32 (n = 12), blaCTX-M-55 (n = 8), blaCTX-M-65 (n = 4), blaCTX-M-27 (n = 2), blaCTX-M-9 (n = 1), blaCTX-M-14 (n = 11), blaSHV-12 (n = 27), blaTEM-52 (n = 1)], blaPMAß [blaCMY-2 (n = 8)], PMQR [qnrB (n = 27), qnrS (n = 21) and aac(6')-Ib-type (n = 4)] and PMCR [mcr-1 (n = 8)]. Our study highlights that consumers may be exposed through the food chain to multidrug-resistant E. coli carrying diverse plasmid-mediated antimicrobial resistance genes, posing a great hazard to food safety and a public health risk.

Occurrence of a Rare Multidrug Resistant Escherichia coli Coharboring blaCTX-M-32 and blaCTX-M-2 Genes in a Bovine.

In this study, a rare strain of Escherichia coli co-harboring blaCTX-M-32 and blaCTX-M-2 genes was characterized by whole genome sequencing. Escherichia coli strain was recovered from a bovine cecal sample collected at slaughter. The strain was resistant to third and fourth generation cephalosporins, fluoroquinolones, aminoglycosides, tetracycline and sulfamethoxazole, which was in concordance with the genotype: blaCTX-M-32 and blaCTX-M-2, aac(3)-Via and tet(A) and sul1 genes, respectively. The E. coli strain was characterized as pathogenic, and belongs to the ST226 (CC226), serotype O141ab/ac:H5, phylogenetic group A and fumC27/fimH41 types. No virulence genes were found in the genome. According to the plasmid analysis, IncFIB, IncHI2 and p0111 were present. The analysis of the contigs revealed that the blaCTX-M-32 was located in the chromosome, upstream IS5-like/ISKpn26 and IS1380-like/ISEc9 sequences, whereas blaCTX-M-2 was in the p0111 plasmid, within an integron class 1, and flanked by Orf3/qacEΔ1 and IS91/ISCR1. To our knowledge, this is the first report of a multidrug resistant Escherichia coli strain co-harboring two CTX-M enzymes in a healthy bovine from Portugal. This finding allows a better understanding on the dissemination of these resistance mechanism among bacteria of animal origin.

Emergence and Clonal Spread of CTX-M-65-Producing Escherichia coli From Retail Meat in Portugal.

The emergence and dissemination of resistance to third- and fourth-generation cephalosporins among Enterobacteriaceae from different sources impose a global public health threat. Here, we characterized by whole-genome sequencing four Escherichia coli strains harboring the bla CTX-M-65 gene identified among 49 isolates from beef and pork collected at retail. The genomic content was determined using the Center for Genomic Epidemiology web tools. Additionally, the prediction and reconstruction of plasmids were conducted, the genetic platform of the bla CTX-M-65 genes was investigated, and phylogenetic analysis was carried out using 17 other genomes with the same sequence type and harboring the bla CTX-M-65 gene. All strains harbored bla CTX-M-65, bla OXA-1, and bla TEM-1B, and one also carried the bla SHV-12 gene. Other resistance genes, namely, qnrS2, aac(6')-Ib-c, dfrA14, sul2, tetA, and mphA, were present in all the genomes; the mcr-1.1 gene was identified in the colistin-resistant strains. They belong to sequence type 2179, phylogenetic group B1, and serotype O9:H9 and carried plasmids IncI, IncFIC(FII), and IncFIB. All strains share an identical genetic environment with IS903 and ISEcp1 flanking the bla CTX-M-65 gene. It seems likely that the bla CTX-M-65 gene is located in the chromosome in all isolates based on deep in silico analysis. Our findings showed that the strains are clonally related and belong to two sub-lineages. This study reports the emergence of CTX-M-65-producing E. coli in Portugal in food products of animal origin. The chromosomal location of the bla CTX-M-65 gene may ensure a stable spread of resistance in the absence of selective pressure.

Genetic Relatedness and Diversity of Staphylococcus aureus from Different Reservoirs: Humans and Animals of Livestock, Poultry, Zoo, and Aquaculture.

The main aim of this study was the characterization of antibiotic resistance mechanisms in 82 Staphylococcus aureus strains isolated from humans and animals. Antibiotic susceptibility testing was performed on all S. aureus isolates accordingly, and antibiotic-resistant genes were investigated by genotypic methods. The genetic diversity of S. aureus was studied through spa, multilocus sequence typing (MLST), and agr typing methods. The majority of S. aureus from human sources were resistant to cefoxitin (and harbor the mecA gene) and fluoroquinolones, whereas only four strains of S. aureus from animal sources revealed resistance to ciprofloxacin. In the set of S. aureus isolated from humans, the most frequent spa, MLST, and agr group were t032, ST22, and I, respectively. In strains from animal origin the most common spa, MLST, and agr group found were t2383, ST398, and III/not typable, respectively. S. aureus from humans and animals were identified either in clonal complexes CC5, CC30, and CC398, suggesting that they have the same putative founder in their evolution. Considering the three CCs encompassing strains from human and animal reservoirs with different spa-types, we can hypothesize that this might reflect an adaptation to different phylogenetic lineages in those reservoirs (host species) probably associated to genetic diversification of pre-existing strains.

IncX4 Plasmid Carrying the New mcr-1.9 Gene Variant in a CTX-M-8-Producing Escherichia coli Isolate Recovered From Swine.

We studied a commensal colistin-resistant Escherichia coli isolated from a swine cecum sample collected at a slaughter, in Portugal. Antimicrobial susceptibility phenotype of E. coli LV23529 showed resistance to colistin at a minimum inhibitory concentration of 4 mg/L. Whole genome of E. coli LV23529 was sequenced using a MiSeq system and the assembled contigs were analyzed for the presence of antibiotic resistance and plasmid replicon types using bioinformatics tools. We report a novel mcr-1 gene variant (mcr-1.9), carried by an IncX4 plasmid, where one-point mutation at nucleotide T1238C leads to Val413Ala substitution. The mcr-1.9 genetic context was characterized by an IS26 element upstream of the mcr-pap2 element and by the absence of ISApl1. Bioinformatic analysis also revealed genes conferring resistance to ß-lactams, sulphamethoxazole, trimethoprim, chloramphenicol and colistin, corresponding to the phenotype noticed. Moreover, we highlight the presence of mcr-1.9 plus bla CTX-M-8, a bla ESBL gene rarely detected in Europe in isolates of animal origin; these two genes were located on different plasmids with 33,303 and 89,458 bp, respectively. MCR-1.9-harboring plasmid showed high identity to other X4-type mcr-1-harboring plasmids characterized worldwide, which strongly suggests that the presence of PMCR-encoding genes in food-producing animals, such as MCR-1.9, represent a potential threat to humans, as it is located in mobile genetic elements that have the potential to spread horizontally.

Revealing mcr-1-positive ESBL-producing Escherichia coli strains among Enterobacteriaceae from food-producing animals (bovine, swine and poultry) and meat (bovine and swine), Portugal, 2010-2015.

We screened 1840 Enterobacteriaceae isolates from food-producing animals, meat, meat products and animal feed, for the detection of plasmid-mediated colistin resistance, during 2010-2015. The mcr-1 gene was detected in 8.0% (97/1206) Escherichia coli and in 0.47% (3/634) Salmonella enterica isolates, with a high number of mcr-1 positive E. coli isolates (45.7%) being extended-spectrum ß-lactamase or plasmid-mediated AmpC ß-lactamase co-producers. No mcr-2 gene was detected. Our findings highlight the spread of mcr-1 genes within a wide-ranging sample of food-producing animals and meat, in Portugal.

New insights into resistance to colistin and third-generation cephalosporins of Escherichia coli in poultry, Portugal: Novel blaCTX-M-166 and blaESAC genes.

The increasing incidence of intestinal colonization with extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae and Gram negative organisms that has been observed in food animals such as poultry, cattle and pigs, are suggestive that animals, food and environment are potential sources of ESBL-producing bacteria. Hence, the aim of this study was to characterized commensal E. coli obtained from healthy broiler and turkey flocks at slaughter for the presence of penicillinases-, ESBL-, extended-spectrum AmpC (ESAC)-, plasmid-mediated quinolone resistance- and MCR-encoding genes. Study of clonal relatedness showed genetic diversity among CTX-M-type, SHV-12 and TEM-52 producing isolates with human isolates of the same type, was also assessed. We detected that eleven (5.4%, 11/202) and forty-five (2.2%, 45/185) E. coli isolates from broilers and turkeys, respectively, carried blaESBL or blaESAC genes and two isolates from turkeys carried mcr-1 gene. A new variant blaCTX-M-166 was reported in a multidrug resistant isolate from a broiler flock. Overall, we detected a diversity of resistance mechanisms among E. coli from food-producing animals, all of them with high importance at a public health level.

Draft Genome Sequence of an Escherichia coli Strain Isolated from a Gallus gallus Broiler Producing the Novel CTX-M-166 Variant.

We report here the draft genome sequence of the CTX-M-166-harboring O6:H16 sequence type 48 (ST48)-fimH34 Escherichia coli strain recovered from a Gallus gallus broiler. Sequence analyses revealed the presence of an IncI1/ST103-ISEcp1-blaCTX-M-166-orf477 plasmid region and of diverse antibiotic resistance and virulence-acquired genes.

Draft Genomic Analysis of an Avian Multidrug Resistant Morganella morganii Isolate Carrying qnrD1.

Morganella morganii is a commensal bacterium and opportunistic pathogen often present in the gut of humans and animals. We report the 4.3 Mbp draft genome sequence of a M. morganii isolated in association with an Escherichia coli from broilers in Portugal that showed macroscopic lesions consistent with colisepticemia. The analysis of the genome matched the multidrug resistance phenotype and enabled the identification of several clinically important and potentially mobile acquired antibiotic resistance genes, including the plasmid-mediated quinolone resistance determinant qnrD1. Mobile genetic elements, prophages, and pathogenicity factors were also detected, improving our understanding toward this human and animal opportunistic pathogen.

QnrS1- and Aac(6')-Ib-cr-Producing Escherichia coli among Isolates from Animals of Different Sources: Susceptibility and Genomic Characterization.

Salmonella enterica and Escherichia coli can inhabit humans and animals from multiple origins. These bacteria are often associated with gastroenteritis in animals, being a frequent cause of resistant zoonotic infections. In fact, bacteria from animals can be transmitted to humans through the food chain and direct contact. In this study, we aimed to assess the antibiotic susceptibility of a collection of S. enterica and E. coli recovered from animals of different sources, performing a genomic comparison of the plasmid-mediated quinolone resistance (PMQR)-producing isolates detected. Antibiotic susceptibility testing revealed a high number of non-wild-type isolates for fluoroquinolones among S. enterica recovered from poultry isolates. In turn, the frequency of non-wild-type E. coli to nalidixic acid and ciprofloxacin was higher in food-producing animals than in companion or zoo animals. Globally, we detected two qnrS1 and two aac(6')-Ib-cr in E. coli isolates recovered from animals of different origins. The genomic characterization of QnrS1-producing E. coli showed high genomic similarity (O86:H12 and ST2297), although they have been recovered from a healthy turtle dove from a Zoo Park, and from a dog showing symptoms of infection. The qnrS1 gene was encoded in a IncN plasmid, also carrying bla TEM-1-containing Tn3. Isolates harboring aac(6')-Ib-cr were detected in two captive bottlenose dolphins, within a time span of two years. The additional antibiotic resistance genes of the two aac(6')-Ib-cr-positive isolates (bla OXA-1, bla TEM-1,bla CTX-M-15, catB3, aac(3)-IIa, and tetA) were enclosed in IncFIA plasmids that differed in a single transposase and 60 single nucleotide variants. The isolates could be assigned to the same genetic sublineage-ST131 fimH30-Rx (O25:H4), confirming clonal spread. PMQR-producing isolates were associated with symptomatic and asymptomatic hosts, which highlight the aptitude of E. coli to act as silent vehicles, allowing the accumulation of antibiotic resistance genes, mobile genetic elements and other relevant pathogenicity determinants. Continuous monitoring of health and sick animals toward the presence of PMQR should be strongly encouraged in order to restrain the clonal spread of these antibiotic resistant strains.