Characterization of antibiotic resistance mechanisms in gram negative bacteria isolated from animals and food products of animal origin

Tese de Doutoramento em Ciências Veterinárias. Especialidade de Sanidade AnimalABSTRACT - Antibiotics were a truly innovative option in medical therapy for the treatment of diseases caused by microbial agents, having largely contributed for the decrease levels of human and animal morbidity and mortality. Therefore, the overuse and misuse of these drugs in human clinical therapy and in the veterinary medicine, including animal production, contributed for the emergence and dissemination of antibiotic resistant microorganisms, which are a serious threat to human and animal health, and to the ecosystem. The aim of the present thesis was to search the main acquired antibiotic resistance mechanisms to β-lactams, fluoroquinolones and polymixins in Gram negative bacteria recovered from different animal species and matrices, and to investigate the most important mobile genetic elements involved in the dissemination. Thus, the studies concerning antibiotic susceptibility and molecular characterization were performed in collections of bacterial isolates belonging to Enterobacteriaceae family (mainly Escherichia coli and Salmonella enterica). Both bacterial species were associated to antibiotic resistant determinants of clinical relevance in human and veterinary medicine, namely, blaCTX-M-1, blaCTX-M-14, blaCTX-M-15, blaCTXM- 32, blaCMY-2, qnrS1, aac(6’)-Ib-cr, mcr-1. The diversity of detected mobile genetic elements, e.g., IncI1, IncF and IncX4 plasmids, insertion sequences ISEcp1, as well as integrons of class 1 and 2, suggest their involvement in the dissemination of resistance genes interspecies, and movement within the bacterial cell. Genomic analysis of two isolates (Morganella morganii and Salmonella Enteritidis), highlighted the potencial of omic technologies, as an additional tool to the phenotypic and genotypic characterization of antibiotic resistance. The results obtained throughout this thesis highlight the importance of the different animal species as reservoirs of antibiotic resistant bacteria. In addition, it was reinforced the need of a permanent research and monitoring of antibiotic resistance in the different ecological niches, and the use of genomic approaches, which had an important role in the understanding of the complex problem represented by the dynamic of antibiotic resistance.RESUMO - Os antibióticos constituíram uma opção verdadeiramente inovadora na terapêutica medicamentosa para o tratamento de doenças provocadas por agentes microbianos, tendo contribuído largamente para a diminuição das taxas de morbilidade e mortalidade humana e animal. Porém, a utilização abusiva e inadequada destes fármacos na prática clínica humana e na medicina veterinária, incluindo a produção animal, contribuiu para a emergência e disseminação de microrganismos resistentes, os quais constituem uma grave ameaça à saúde humana e animal, e para o ecossistema. A presente dissertação teve como objetivo central investigar os principais mecanismos de resistência adquirida aos antibióticos β-lactâmicos, fluoroquinolonas e polimixinas em bactérias de Gram negativo isoladas de diferentes espécies animais e matrizes, bem como os principais elementos genéticos móveis responsáveis pela sua disseminação. Assim, os estudos de suscetibilidade aos antibióticos e caracterização molecular foram realizados em coleções de estirpes bacterianas pertencentes à família Enterobacteriaceae (maioritariamente Escherichia coli e Salmonella enterica). Ambas as espécies bacterianas estavam associadas a determinantes de resistência de relevância clínica humana e veterinária, nomeadamente blaCTX-M-1, blaCTX-M-14, blaCTX-M-15, blaCTX-M-32, blaCMY-2, qnrS1, aac(6’)-Ib-cr, mcr-1. A diversidade de elementos genéticos móveis detetados, e.g. plasmídeos IncI1, IncF e IncX4, sequências de inserção ISEcp1, bem como integrões de classes 1 e 2, sugere o seu envolvimento na disseminação de genes de resistência aos antibióticos entre espécies, tal como a sua movimentação dentro da própria bactéria. A análise do genoma de duas estirpes (Morganella morganii e Salmonella Enteritidis) realçou o potencial das tecnologias ómicas, como ferramenta adicional na caracterização fenotípica e genotípica da resistência aos antibióticos. Os resultados obtidos salientam a importância que as várias espécies animais representam como reservatórios de bactérias resistentes aos antibióticos. Foi igualmente reforçada a necessidade de uma permanente investigação e monitorização da resistência aos antibióticos nos vários nichos ecológicos, e do uso de abordagens genómicas, as quais tiveram um papel importante na compreensão do complexo problema que representa a dinâmica da resistência aos antibióticos.N/

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.VS has her Ph.D. fellowship granted by FCT (Fundação para a Ciência e a Tecnologia) with the reference SFRH/BD/133100/2017 co-financed by European Social Fund and the Operational Program for Human Capital (POCH), Portugal. This work was financial supported with funding from FCT/MCTES (UIDB/00211/2020) through national funds.info:eu-repo/semantics/publishedVersio

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 β-lactams, sulphamethoxazole, trimethoprim, chloramphenicol and colistin, corresponding to the phenotype noticed. Moreover, we highlight the presence of mcr-1.9 plus blaCTX-M-8, a blaESBL 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.VM was supported by Fundação para a Ciência e a Tecnologia (FCT) fellowship (Grant No. SFRH/BPD/77486/2011), financed by the European Social Funds (COMPETE-FEDER), and National Funds of the Portuguese Ministry of Education and Science (POPH-QREN). The authors thank to FCT for the project grant UID/MULTI/00211/2013.info:eu-repo/semantics/publishedVersio

Antimicrobial susceptibility of Salmonella enterica isolated from food-producing animals, animal feed and food products of animal origin, in Portugal - Genetic analysis of isolates with reduced susceptibility/resistance to third generation cephalosporins and cephamycins

Salmonella is a widely distributed foodborne pathogen and one of the most common causes of bacterial foodborne illnesses in humans. An epidemiologic study was conducted on 1600 Salmonella spp isolates recovered from poultry, swine, other animal species, animal feed and food products of animal origin, over the period of 2009-2013, to determine their serotype and antimicrobial susceptibility to a panel of ten antimicrobials (ampicillin, cefotaxime, ciprofloxacin, nalidixic acid, trimethoprim, sulfamethoxazole, chloramphenicol, streptomycine, gentamicine and tetracycline), through the determination of Minimum Inhibitory Concentration (MIC), using the agar dilution technique. Molecular characterization of isolates showing a non-wild type MIC to cefotaxime was performed, to determine extended spectrum β-lactamases (ESBL), plasmid mediated AmpC (PMAβ), plasmid mediated quinolone (PMQR) resistance determinants and mobile genetic elements involved in the dissemination of resistance genes. In live poultry (breeders, broilers, layers) of the 843 isolates recovered, 27.9% comprised S. Enteritidis, 23,5% Salmonella Havana and 14.1% Salmonella Mbandaka; in turkeys, Salmonella Derby was the most common serovar isolated (44%), followed by Salmonella I 4,[5],12:i:- (16%). In swine, of 101 isolates 21.8% comprised Salmonella Rissen and Salmonella Typhimurium, 10.9% Salmonella Derby and Salmonella London. In other animal species, Salmonella Typhimurium was the prevalent serovar with 65.6% of the isolates, followed by Salmonella I 4,[5],12:i:- (9.8%). Overall, S. 4,[5],12:i:- was the most common serotype recovered from food products (25.8%), followed by S. Typhimurium (19.2%) and Salmonella Rissen (18.4%). S. Enteritidis was the most frequent serotype in poultry products (36.3%). Susceptibility profiles differed according with the serotype and the origin of the isolates. A higher frequency of multidrug resistant isolates was recovered from food of swine and bovine origin, with 62.6% and 59.4%, respectively. Polymerase chain reaction and sequencing of the amplicons confirmed the presence of blaCTX-M-type (n=8), blaSHV-type (n=2), blaTEM-type (n=2) and plasmid-mediated AmpC β-lactamases (PMAβ) genes (n=2). No plasmid mediated quinolone resistance-encoding genes were detected. Six isolates( three S. I 4,[5],12:i:-, two S. Havana and one S. Enteritidis) carried class 1 integrons and one S. I 4,[5],12:i:- isolate harboured a class 2 integron. In conclusion, the growing concern of the emergence of bacterial strains bearing ESBL in food-producing animals highlights the importance of continuous monitoring.info:eu-repo/semantics/publishedVersio

Occurrence of extended-spectrum beta-lactamases in Salmonella enterica strains isolated from broilers and food of animal origin in Portugal

Salmonella enterica is a zoonotic bacteria transmitted through the food chain and isolates harbouring extended-spectrum beta-lactamases (ESBLs) have emerged worldwide during the last decade, with the CTX-M group being particularly important. The aim of the present study was to determine the antimicrobial susceptibility of S. enterica strains isolated from broilers and food of animal origin and to characterize ESBLs producers. On the scope of the national antimicrobial resistance surveillance programme on Salmonella, a total of 283 strains isolated from broilers (n=100) and food of animal origin (n=183), were received at the National Laboratory of Veterinary Research in 2011. The minimum inhibitory concentration (MIC) of 11 antimicrobials (nalidixic acid, ciprofloxacin, ampicillin, cefotaxime, chloramphenicol, florfenicol, streptomycin, gentamicin, tetracycline, sulphamethoxazole and trimethoprim) for all isolates was determined by agar dilution method. Susceptibility towards cefoxitin was determined through disk diffusion method. Breakpoints were interpreted accordingly to EUCAST epidemiological cut-off values. ‘Non-wild type’ (‘NWT’) isolates for cefotaxime (MIC>0.5mg/L) and cefoxitin (<19mm) were screened for the presence of ESBL- (blaTEM, blaOXA, blaSHV, blaCTX) and PMA_-encoding genes, using PCR method. Sequencing was applied to fully identify beta-lactamases. Among broilers, we identified 62% of ‘NWT’ isolates for ciprofloxacin, 57% for nalidixic acid and 28% for sulphamethoxazole, whereas in isolates from food of animal origin, 71%, 63% and 56% were ‘NWT’ isolates for tetracycline, sulphamethoxazole and ampicillin, respectively. Among all, 5/283 (1.8%) strains presented ‘NWT’ MICs for cefotaxime and were multidrug resistant: 2 Salmonella Havana isolated from broilers and 3 Salmonella S. 4,[5],12:i:- isolated from food of animal origin (swine); these isolates had one blaCTX-M-type gene, and 2 from food of animal origin presented 1 blaTEM-type gene and 1 blaSHV-type gene, respectively; they were ‘wild type’ for cefoxitin and no PMAB-encoding gene was detected. To our knowledge, this is the first time in Portugal that ESBL-encoding genes, particularly from blaCTXM family, were detected in isolates of Salmonella Havana, a very common serotype isolated from our broiler population. It should also be emphasised that third generation cephalosporins are not allowed in the national poultry production, contrary to the large animal production, which may explain the detection of ESBL-encoding genes in our strains from swine origin. Horizontal gene transfer may be responsible for the coresistance of strains to non-beta-lactam antibiotics. This study shows that national animal health monitoring systems play an important role and should be improved in an international level

Diversity of β-lactamase-encoding genes in Escherichia coli strains isolated from food-producing, companion and zoo animals in Portugal

A rapid development of plasmid-mediated resistance to extended-spectrum cephalosporins has been observed in Enterobacteriaceae worldwide, predominantly due to the dissemination of extended-spectrum beta-lactamases (ESBL) and plasmid-mediated AmpC beta-lactamases (PMAB). The aim of the present study was to evaluate the extension of ESBL- and PMAB-producing E. coli strains isolated from different animal origins in Portugal. For surveillance purposes, 376 E. coli isolates identified at National Laboratory of Veterinary Research (2009-2011) were submitted to antimicrobial susceptibility testing: 123, 51 and 202 were isolated from food-producing, companion and zoo animals, respectively. Minimum Inhibitory Concentrations (MIC) of 11 antimicrobials for all isolates was determined through agar dilution method. Susceptibility towards cefoxitina was determined through disk diffusion method. Breakpoints were interpreted accordingly to EUCAST epidemiological cut-off values. ‘Non-wild type’ (NWT) isolates for cefotaxime (MIC>0.25mg/L) and/or cefoxitina (<19mm) were screened for the presence of ESBL (blaTEM, blaOXA, blaSHV, blaCTX) and PMAB encoding genes, using PCR method. Sequencing was applied to fully identify beta-lactamases. Seventeen isolates (4.5%) were ‘NWT’ strains for cefotaxime, being 5 (29.4%) from companion animals, 4 (23.5%) from food-producing animals and 8 (47.1%) from zoo animals. We identified blaCTX-M-14 (n=1) in a dog and blaCTX-M-15-type genes (n=9) in 6 zoo animals and 3 in food-producing animals. We also identified blaCMY-type genes (n=3) in ‘NWT’ isolates for cefoxitin, one from each animal category. Other beta-lactamase encoding genes were identified: blaOXA in 5 strains (29.4%) isolated from dolphins, blaTEM in 7 strains (41.2%) isolated from 3 companion animals, 2 food-producing and 2 zoo animals, and blaSHV identified in one isolate (5.9%) from a zoo animal; 13 beta-lactamase-producing isolates (76.5%) were multidrug resistant. Among ‘NWT’ E. coli isolates for cefotaxime, we identified an important diversity of ESBL encoding genes, belonging to different families, being blaCTX-M-15-type gene the predominant. The spread of ESBL-producing bacteria among species from different origins, such as food-producing, companion and zoo animals, is a concern at public health level. Thus, it should be a priority to monitor and identify the reservoirs of antimicrobial resistance, contributing to a single health for all

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 β-lactams, sulphamethoxazole, trimethoprim, chloramphenicol and colistin, corresponding to the phenotype noticed. Moreover, we highlight the presence of mcr-1.9 plus blaCTX-M-8, a blaESBL 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

Epidemiology and zoonotic potential of Escherichia coli CTX-M-15-producing isolates in Portugal

Background: The recent spread of plasmid-located CTX-M-ESBL-encoding genes is a serious threat to the clinical efficacy of expanded-spectrum cephalosporins. This study proposes to identify the epidemiology of plasmid-mediated CTX-M-encoding genes between an Escherichia coli strain isolated from a dolphin and several E. coli strains of human origin and, explain the responsible mechanism and reservoirs of current spread of CTX-M-type enzymes. Molecular typing of these strains establishes the linkage of dissemination between human and animal isolates. Methods: Sixty two ESBL-positive E. coli strains isolated from different clinical specimens in seven hospitals (2004 to 2009), from four different geographic regions, were screened for the presence of CTX-M encoding genes. An E. coli isolated from a respiratory exsudate in a dolphin in 2009, at the National Laboratory of Veterinary Research (LNIV) and characterized as CTX-M-producer, was also included in this study. Antimicrobial susceptibility was performed by broth-microdilution method. PCR and sequencing were used to screen and identify bla genes. Genetic relatedness among all isolates was examined by PFGE using XbaI enzyme. MLST was performed among the clinical epidemic human isolates clustering together and the isolate from the dolphin, according to the MLST database. Results: Forty eight human clinical isolates (77%) were CTX-M producers. Susceptibility towards beta-lactams confirmed all isolates as ESBL producers and also suggesting CTX-M enzymes expression. We detected blaCTX-M-15 (n=34), blaCTX-M-1 (n=4), blaCTX-M-3 (n=3), blaCTX-M-32 (n=3), blaCTX-M-14 (n=4), blaTEM-1 (n=39), and blaSHV-12 (n=8) genes; the dolphin isolate presented the blaCTX-M-15 gene. Genetic relatedness analysis by PFGE revealed one major cluster corresponding to a single epidemic clone A, which included 22 (35%) of all human isolates and the dolphin isolate, which clustered together with this clone A and, exhibited the same combination of MLST alleles across the seven sequenced loci, corresponding to the ST131. Twenty-one clinical isolates corresponding to the CTX-M-15-positive clone A were multidrug-resistant (95%), as well as the dolphin isolate. Conclusions: This study illustrated that genetic relatedness between human and animal E. coli CTX-M-15-producing clone strengthens its zoonotic potential. It may also explain the current spread of CTX-M-type enzymes worldwide among species from different origins and highlights the importance to identify antimicrobial resistance reservoirs, contributing to a single health for all

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

(This article belongs to the Special Issue Epidemiology of ESBL-Producing Enterobacteriaceae)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 β-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.This research was funded by Promoting One Health in Europe through joint actions on foodborne zoonoses, antimicrobial resistance, and emerging microbiological hazards–One Health EJP, grant number 773830 (DiSCoVeR). Research at the German Federal Institute for Risk Assessment, Germany, was partially supported by the internal project BfR-BIOS-08-43-001. Research at the National Institute for Agrarian and Veterinary Research (INIAV) was partially supported by the project PTDC/CVT-CVT/28469/2017 financed by the “Fundação para a Ciência e Tecnologia” (FCT), Portugal. Research at the National Veterinary Research Institute (PIWet), Poland, was also partially supported by the Polish Ministry of Education and Science from the funds for science in the years 2018–2022 allocated for the implementation of a co-financed international project. The environmental isolates from Ireland were collected as part of the AREST project, which is jointly funded by the Environmental Protection Agency, under the EPA Research Programme 2014–2020, and the Health Service Executive (2017-HW-LS-1). The isolates collected from pig farms in Ireland were collected as part of a Walsh Scholarship project funded by Teagasc (ref 2018027). Research at the VISAVET Health Surveillance Centre (Spain) was partially supported by the project Antimicrobial resistance transmission dynamics in the human-animal interface: Shaping the risk posed by epidemic plasmids (PID2021-125136OB-I00, Ministerio de Ciencia, Innovación y Universidades, MICINN).info:eu-repo/semantics/publishedVersio

Establishing Streptomycin Epidemiological Cut-Off Values for Salmonella and Escherichia coli

This study was conducted to elucidate the accuracy of the current streptomycin epidemiological cut-off value (ECOFF) for Escherichia coli and Salmonella spp. A total of 236 Salmonella enterica and 208 E. coli isolates exhibiting MICs between 4 and 32¿mg/L were selected from 12 countries. Isolates were investigated by polymerase chain reaction for aadA, strA, and strB streptomycin resistance genes. Out of 236 Salmonella isolates, 32 (13.5%) yielded amplicons for aadA (n¿=¿23), strA (n¿=¿9), and strB (n¿=¿11). None of the 60 Salmonella isolates exhibiting MIC 4¿mg/L harbored resistance genes. Of the Salmonella isolates exhibiting MICs 8¿mg/L, 16¿mg/L, and 32¿mg/L, 1.6%, 15%, and 39%, respectively, tested positive for one or more genes. For most monitoring programs, the streptomycin ECOFF for Salmonella is wild type (WT) =32 or =16¿mg/L. A cut-off value of WT =32¿mg/L would have misclassified 13.5% of the strains as belonging to the WT population, since this proportion of strains harbored resistance genes and exhibited MICs =32¿mg/L. Out of 208 E. coli strains, 80 (38.5%) tested positive for aadA (n¿=¿69), strA (n¿=¿18), and strB (n¿=¿31). Of the E. coli isolates exhibiting MICs of 4¿mg/L, 8¿mg/L, 16¿mg/L, and 32¿mg/L, 3.6%, 17.6%, 53%, and 82.3%, respectively, harbored any of the three genes. Based on the European Committee on Antimicrobial Susceptibility Testing guidelines (ECOFF =16¿mg/L), 25% of the E. coli strains presenting MIC =16¿mg/L would have been incorrectly categorized as belonging to the WT population. The authors recommend an ECOFF value of WT =16¿mg/L for Salmonella and WT =8¿mg/L for E. coli