Search

ABSTRACTPlasmid-mediated qnrgenes provide only a modest decrease in quinolone susceptibility but facilitate the selection of higher-level resistance. In Escherichia colistrain J53 without qnr, ciprofloxacin resistance often involves mutations in the GyrA subunit of DNA gyrase. Mutations in gyrAwere absent, however, when 43 mutants with decreased ciprofloxacin susceptibility were selected from J53(pMG252) with qnrA1. Instead, in 13 mutants, individual and whole-genome sequencing identified mutations in marRand soxRassociated with increased expression of marAand soxSand, through them, increased expression of the AcrAB pump, which effluxes quinolones. Nine mutants had increased expression of the MdtE efflux pump, and six demonstrated increased expression of the ydhEpump gene. Many efflux mutants also had increased resistance to novobiocin, another pump substrate, but other mutants were novobiocin hypersusceptible. Mutations in rfaDand rfaEin the pathway for inner core lipopolysaccharide (LPS) biosynthesis were identified in five such strains. Many of the pump and LPS mutants had decreased expression of OmpF, the major porin channel for ciprofloxacin entry. Three mutants had increased expression of qnrAthat persisted when pMG252 from these strains was outcrossed. gyrAmutations were also rare when mutants with decreased ciprofloxacin susceptibility were selected from E. coliJ53 with aac(6′)-Ib-cror qepA. We suggest that multiple genes conferring low-level resistance contribute to enhanced ciprofloxacin resistance selected from an E. colistrain carrying qnrA1, aac(6′)-Ib-cr, or qepAbecause these determinants decrease the effective ciprofloxacin concentration and allow more common but lower-resistance mutations than those in gyrAto predominate.

Nineteen extended-spectrum β-lactamase (ESBL)-positive Escherichia colistrains recovered from food samples in Tunisia were characterized by multilocus sequence typing and phylogenetic typing, and the virulence gene and plasmid content were also determined. These strains presented unrelated pulsed-field gel electrophoresis patterns and contained genes coding for the following ESBLs (the number of strains is in parentheses): CTX-M-1 (15), CTX-M-14 (2), CTX-M-8 (1), and SHV-5 (1). Twelve different sequence types (STs) were identified among the 19 ESBL-positive strains, which included two new STs (ST2022 in 2 blaCTX-M-14-containing strains and ST1970 in 2 blaCTX-M-1–containing strains). ST155 and ST602 were detected in four and three blaCTX-M-1–containing strains, respectively, and ST405 was detected in one blaCTX-M-8-producing strain. All ESBL-positive strains were ascribed to the phylogenetic groups A and B1. Most of the blaCTX-M-1–containing strains harbored an IncI1 plasmid, except for the four blaCTX-M-1–positive strains of beef origin and ST155, which harbored an IncN plasmid. The two blaCTX-M-14-containing strains contained an IncI1 plasmid. The virulence gene fimAwas detected in all strains. Most strains also carried the aergene, and six strains carried the eaegene. All strains were negative for the virulence genes sxt, papG-III, papC, hly, cnf1, and bfp.We conclude that ESBL-producing E. colistrains of food origin in Tunisia show high diversity and that plasmids harboring ESBL genes could be implicated in the dissemination of this resistance phenotype.

AbstractThere is a great concern by the emergence and the wide dissemination of extended-spectrum beta-lactamases (ESBLs) among animal Escherichia coliisolates. We intended to determinate the carriage level and type of ESBLs in E. coliobtained from fecal samples from pigs raised on an intensive pig farm in Portugal; further to characterize other associated resistance genes and their plasmid content, the phylogenetic groups, and the clonal relationship of ESBL-positive isolates. Sixty-five fecal samples were seeded in Levine media supplemented with cefotaxime for E. colirecovery. Susceptibility to 16 antimicrobial agents was performed by disk diffusion agar. ESBL-phenotypic detection was carried out by double-disk test; and the presence of the genes encoding TEM, OXA, SHV, and CTX-M type beta-lactamases was studied by polymerase chain reaction and sequencing. Other mechanisms of antimicrobial resistance and phylogenetic groups were also determined. Clonal relationship was performed by pulsed-field gel electrophoresis. ESBL-producing E. coliisolates were detected in 16 fecal samples, and one isolate per sample was studied. The CTX-M-1 type ESBL was detected in the 16 isolates. The gene encoding TEM-1 was identified to be associated with eight CTX-M-1-positive isolates. The tet(A) gene was found in 12 of 14 tetracycline-resistant isolates, and the aadAor strA-strBgenes were found in the streptomycin-resistant isolates. Fourteen and two ESBL-containing isolates belonged to A and B1 phylogenetic groups, respectively. Clonal relationship of ESBL-containing isolates identified seven unrelated patterns. Swine represent an important reservoir of ESBL-containing E. coliisolates, especially of the CTX-M-1 type.

AbstractThe main aim of this study was to determine the frequency of antibiotic resistance among Escherichia coliisolates recovered in Levine agar plates from 54 fecal samples of captive ostriches from a farm in the South of Portugal. Fifty-four nonselected E. coliisolates were obtained (one/sample) and the phenotypes and genotypes of antibiotic resistance were characterized. The following numbers of isolates showed antibiotic resistance: ampicillin (nine), tetracycline (seven), streptomycin (three), amoxicillin–clavulanic acid, cefoxitin, or gentamicin (one), and cefotaxime, ceftazidime, azthreonam, imipenem, nalidixic acid, ciprofloxacin, and trimethoprim/sulfamethoxazole (zero). The blaTEMgene was identified in six out of nine ampicillin-resistant isolates, and the tet(A) or tet(B) genes in five out of seven tetracycline-resistant isolates. Mutations at positions −42, −18, −1, and +58 of ampCpromoter region were identified in one cefoxitin-resistant isolate. Further, the occurrence of extended-spectrum beta-lactamase (ESBL)–producing E. coliisolates was estimated in the 54 fecal samples of ostriches using cefotaxime-supplemented Levine agar plates for ESBL-positive E. colirecovery. Three samples contained ESBL-positive E. coliisolates of which one isolate/sample was characterized, leading to the detection of the following beta-lactamases: blaCTX-M-14a+ blaTEM-1b(two isolates) and blaTEM-52c(one isolate). The three ESBL-positive isolates were classified into the phylogroup B1, and contained class 1 integrons with the gene cassettes dfrA17+ aadA5(one isolate) and aadA1(two isolates). This study adds to our knowledge about the wide dissemination of ESBL-producing E. coliisolates in different ecosystems, including captive ostriches, that could be transferred to humans through the food chain.

AbstractFifty-five Escherichia coliisolates were acquired from chicken and turkey meat obtained from two slaughterhouses in Tunis. Eighty-nine percent, 80%, 78%, 67%, 45%, 27%, 7%, 4%, and 2% of these isolates showed resistance to tetracycline, trimethoprim/sulfamethoxazole, streptomycin, nalidixic acid, ampicillin, chloramphenicol, ciprofloxacin, colistine, and gentamicin, respectively. No resistance was detected to cefotaxime, ceftazidime, or amikacin. blaTEMgene was found in 22 of 25 ampicillin-resistant isolates, and 1 isolate harbored blaOXA-1gene. Tetracycline resistance was predominately mediated by the tetAgene. The sul1, sul2, and sul3genes, alone or combined, were detected in 46 of 48 sulfonamide-resistant isolates, and sul1and sul3were included in class 1 integrons in some cases. Sixty percent of isolates harbored integrons (class 1, 30 isolates; class 2, 5 isolates). Class 2 integrons contained in all cases the dfrA1-sat1-aadA1-orfXgene cassette arrangement. Nine gene cassette arrangements have been detected among class 1 integrons, containing different alleles of dfrA(five alleles) and aadA(2 alleles) genes, which encode trimethoprim and streptomycin resistance, respectively. An uncommon gene cassette array (sat-psp-aadA2-cmlA1-aadA1-qacH-IS440-sul3) has been identified in three class 1 integron-positive isolates, and one additional isolate had this same structure with the insertion of IS26inside the aadA1gene (included in GenBank with accession no. FJ160769). The 55 studied isolates belong to the four phylogenic groups of E. coli, and phylogroups A and D were the most prevalent ones. At least one virulence-associated gene (fimA, papC, or aer) was detected in 44 of the 55 (80%) studied isolates. E. coliisolates of poultry origin could be a reservoir of antimicrobial-resistance genes and of integrons, and its evolution should be tracked in the future.

Phenotypic and genotypic characterization of antimicrobial resistance was conducted for 98 Escherichia coliisolates recovered from 40 food samples of animal origin (poultry, sheep, beef, fish, and others) obtained in supermarkets and local butcheries in Tunis during 2004 and 2005. Susceptibility to 15 antimicrobial agents was tested by disk diffusion and agar dilution methods, the mechanisms of resistance were evaluated using PCR and sequencing methods, and the clonal relationship among isolates was evaluated using pulsed-field gel electrophoresis. High resistance was detected to tetracycline, sulphonamides, nalidixic acid, ampicillin, streptomycin, and trimethoprim-sulfamethoxazole (29 to 43% of isolates), but all isolates were susceptible to cefotaxime, ceftazidime, cefoxitin, azthreonam, and amikacin. One-third of the isolates had multiresistant phenotypes (resistance to at least five different families of antimicrobial agents). Different variants of blaTEM, tet, sul, dfrA, aadA, and aac(3)genes were detected in most of the strains resistant to ampicillin, tetracycline, sulphonamide, trimethoprim, streptomycin, and gentamicin, respectively. The presence of class 1 and class 2 integrons was studied in 15 sulphonamide-resistant unrelated E. colistrains, and 14 of these strains harbored class 1 integrons with five different arrangements of gene cassettes, and a class 2 integron with the dfrA1+ sat+ aadA1arrangement was found in one strain. This study revealed the high diversity of antimicrobial resistance genes, some of them included in integrons, in E. coliisolates of food origin.

AbstractIntegrons are important genetic elements implicated in acquisition and expression of antimicrobial resistance genes. Gene cassettes of class 1 integrons may be differently expressed depending on the Pc promoter variant. Thirty-four Escherichia coliisolates (carrying 38 class 1 integrons), recovered from poultry meat in previous studies in Tunisia and selected by their specific traits, were further characterized in this study. Integron promoter variants and the pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) of isolates were determined. Three types of promoter variants were identified among the 38 class 1 integrons (PcW, PcH1, and PcS); the weak promoters were the most predominant. A high clonal diversity of the E. colistrains was demonstrated by PFGE or by MLST. Fifteen PFGE profiles were detected among 19 integron-positive isolates of phylogroup B2, and 12 different sequence types were identified by MLST among the remaining 15 isolates (ST48, ST88, ST101, ST117, ST155, ST189, ST351, ST359, ST410, ST641, ST665, and one new ST). These data reflect that the presence of integrons in these isolates is not due to the clonal dispersion but to dissemination of genetic structures carrying integrons in different clones. To the best of our knowledge, this is the first report examining the gene cassette promoter variants in class 1 integrons of E. coliisolates from poultry meat origin. The predominance of promoters implicated in weak expression/high excision activity of gene cassette arrays is of interest because they could theoretically enhance the capacity of integrons to adapt to antibiotic pressure.