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Introduction: The importance of plasmid-mediated quinolone resistance (PMQR) in Enterobacterales and its high incidence has been emphasised many times. However, a clinical strain carrying more than two PMQR genes is rare. This study sequenced plasmid transconjugants from a donor strain carrying four different PMQR genes to establish their genetic locations., Methods: An Escherichia coli clinical strain displayed remarkable quinolone resistance with a ciprofloxacin MIC of 1024 mg/L carrying four PMQR genes: qnrA1, qepA1, aac(6')1b-cr and oqxAB. When outcrossed to Escherichia coli J53 AziR, different PMQR genes were transferred and the resulting strains 7C and 8C were chosen for further characterisation. Plasmids were extracted and sequenced by the Illumina and Oxford Nanopore Technologies platforms. S1 nuclease-PFGE was used to estimate the number and size of plasmids., Results: The parental strain had three plasmid bands, as determined by PFGE. Transconjugant 8C obtained three plasmids: pMG336 (162 647 bp, F18:A-:B1:C4) carrying oqxAB; pMG335 carrying qepA1 (73 874 bp, F2:A-:B-); and pMG334 (59 724 bp, IncN (ST5)) with qnrA1 and aac(6')1b-cr. Interestingly, strain 7C obtained plasmid pMG333 (134 435 bp), which was not present in the parental strain but was an IncN-IncF cointegrate of plasmids pMG334 and pMG335 linked via insertion sequence IS26., Conclusion: This study described the complete nucleotide sequence of three plasmids carrying four PMQR genes in a single strain and the plasmid profile obtained after outcrosses. In addition, it described a cointegrate of two plasmids formed with flanking insertion sequences., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

We examined 13 qnr-positive and 14 qnr-negative clinical isolates of Escherichia coli for mutations previously seen in a qnr-containing laboratory strain exposed to supra minimum inhibitory concentrations (MICs) of ciprofloxacin. Among the qnr-positive strains, those with ciprofloxacin MICs of ≥ 2 µg/mL had at least one mutation in gyrA. Mutations in parC were present in strains with a ciprofloxacin MIC of ≥ 128 µg/mL. The 6 most ciprofloxacin-resistant strains contained additional plasmid-mediated quinolone resistance determinants. aac(6')-Ib-cr was found in 5 of the 6 strains. Eleven of the 13 strains had alterations in MarR, 9 in SoxR, and 5 had mutations in AcrR. All had elevated expression of at least one efflux pump gene, predominantly acrA (92% of the strains), followed by mdtE (54%) and ydhE (46%). Nine had functionally silent alterations in rfa, two had mutations in gmhB, and one of these also had a mutation in surA. An E. coli with ciprofloxacin MIC of 1024 µg/mL contained 4 different plasmid-mediated quinolone resistance determinants as well as gyrA, parC, parE and pump overexpression mutations. Nine of the 14 qnr-negative strains had mutations in topoisomerase genes with a ciprofloxacin MIC of 0.25 to 256 µg/mL. The three most resistant strains also had mutations in parE. Twelve had alterations in MarR, 10 in SoxR and 5 in AcrR. Ten of the 14 strains had elevated expression of efflux pumps with acrA (71.4%), followed by ydhE (50%) and mdtE (14.3%). A diversity of resistance mechanisms occurs in clinical isolates with and without qnr genes., (Copyright © 2018 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

The antimicrobial resistance phenotype and genotype, the flanking regions of sulphonamide resistance genes and the integrons were analyzed in 166 Escherichia coli isolates recovered from poultry meat in Tunisia. High percentages of resistance were detected to ampicillin, streptomycin, nalidixic acid, sulphonamide and tetracycline (66-95%), and lower percentages to gentamicin, amoxicillin-clavulanic acid and cefoxitin (1-4%). The bla(TEM), tet(A)/tet(B), aph(3')-Ia, aac(6')-Ib-cr, aac(3)-II and cmlA genes were identified in 92, 82, 29, 2, 2 and 7 isolates, respectively. Class 1 and/or class 2 integrons were detected in 52% of E. coli isolates and five different gene cassette arrangements were identified in the variable regions of class 1 integrons, which included antimicrobial resistance determinants. Sixty-eight isolates contained the sul1 gene and 37 of them presented this gene into a class 1 integron structure. The sul3 gene was detected associated with non-classic class 1 integrons in 4 out of 46 sul3-positive isolates. The sul2 gene was detected in 66 isolates, 51 of them were linked to strA/B genes in seven different genetic structures. Seventy-three-per-cent of integron-positive isolates presented resistance to at least five different antimicrobial families versus 38.7% of integron-negative isolates. Our study highlights the role of commensal E. coli isolates from poultry meat as an important reservoir for sulphonamide resistance genes and integrons carrying antimicrobial resistance genes., (Copyright © 2010 Elsevier B.V. All rights reserved.)

There is a great concern by the emergence and the wide dissemination of extended-spectrum beta-lactamases (ESBLs) among animal Escherichia coli isolates. We intended to determinate the carriage level and type of ESBLs in E. coli obtained 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. coli recovery. 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. coli isolates 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 aadA or strA-strB genes 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. coli isolates, especially of the CTX-M-1 type.

The main aim of this study was to determine the frequency of antibiotic resistance among Escherichia coli isolates recovered in Levine agar plates from 54 fecal samples of captive ostriches from a farm in the South of Portugal. Fifty-four nonselected E. coli isolates 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 bla(TEM) gene 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 ampC promoter region were identified in one cefoxitin-resistant isolate. Further, the occurrence of extended-spectrum beta-lactamase (ESBL)-producing E. coli isolates was estimated in the 54 fecal samples of ostriches using cefotaxime-supplemented Levine agar plates for ESBL-positive E. coli recovery. Three samples contained ESBL-positive E. coli isolates of which one isolate/sample was characterized, leading to the detection of the following beta-lactamases: bla(CTX-M-14a) + bla(TEM-1b) (two isolates) and bla(TEM-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. coli isolates in different ecosystems, including captive ostriches, that could be transferred to humans through the food chain.

The prevalence and characterisation of integrons and the genetic environment of sulphonamide resistance genes were studied in 135 Escherichia coli isolates recovered from blood cultures in a Spanish hospital during 2007. Class 1 and 2 integrons were identified in 54 isolates (intI1, 52 isolates; intI2, 1 isolate; and intI1+intI2, 1 isolate). Of the 53 intI1-positive isolates, 36 (67.9%) contained the classic class 1 integron including the qacEDelta1-sul1 region, and 11 different gene cassette arrangements were demonstrated in 33 of these isolates. Seventeen intI1-positive isolates lacked the qacEDelta1-sul1 region, and 8 gene cassette arrangements were demonstrated in 12 of these isolates. Seventy-one isolates showed a sulphonamide-resistant phenotype, 63 of which contained sul genes. The sul1 gene was associated with intI1 in 36 of 42 sul1-positive isolates, and the sul3 gene was associated with non-classic class 1 integrons in 5 of 7 sul3-positive isolates. Finally, sul2 was found associated with strA-strB genes in 32 of 35 sul2-positive isolates, identifying 11 genetic structures, 1 of them presenting the IS150 element disrupting the strB gene; this structure was included in GenBank with accession no. FJ705354. Almost one-half of the E. coli isolates from blood cultures contained integrons and sul genes. Moreover, sul genes were detected in different structures, one of them new, and could be important determinants in antibiotic resistance dissemination., (Copyright 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Fifty-five Escherichia coli isolates 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. bla(TEM) gene was found in 22 of 25 ampicillin-resistant isolates, and 1 isolate harbored bla(OXA-1) gene. Tetracycline resistance was predominately mediated by the tetA gene. The sul1, sul2, and sul3 genes, alone or combined, were detected in 46 of 48 sulfonamide-resistant isolates, and sul1 and sul3 were 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-orfX gene 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 IS26 inside the aadA1 gene (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. coli isolates of poultry origin could be a reservoir of antimicrobial-resistance genes and of integrons, and its evolution should be tracked in the future.

Objectives: The aim was to study the oral carriage of Enterococcus and Escherichia coli isolates and their content in antimicrobial-resistance and virulence genes in patients with fixed appliances and in healthy volunteers., Study Design: Samples from supragingival plaques/tooth surfaces/fixed orthodontic appliances were taken in patients with fixed appliances (n = 46) and in healthy volunteers (n = 55). Samples were seeded on specific media for enterococcal and E. coli recovery, and 1 isolate of each type per sample was selected. Antimicrobial susceptibility and the presence of genes encoding antimicrobial resistance, bacteriocins, and virulence factors were checked by polymerase chain reaction., Results: Enterococci or E. coli were not recovered from healthy volunteers. Nevertheless, 10 isolates (5 E. faecium, 3 E. faecalis, and 2 E. coli) were obtained from 19.5% of patients with fixed appliances, and poor oral hygiene was evidenced in all of the these patients. Percentages of antimicrobial resistance and the resistance genes detected among the enterococci were: erythromycin: 100%, erm(B); kanamycin: 75%, aph(3')-IIIa; tetracycline: 50%, tet(L) with/without tet(M); streptomycin: 37%, ant(6)-Ia; chloramphenicol: 12%, catA. One E. coli isolate showed a phenotype of multiresistance containing 5 resistance genes and class 1 and 2 integrons. All enterococci produced gelatinase, and 4 isolates contained genes encoding enterocins L50A/B and P. The esp virulence gene was found in 1 multiresistant E. faecalis isolate., Conclusions: Poor or improper oral hygiene in individuals with fixed appliances favors the oral carriage of antimicrobial-resistant E. coli and enterococci. Additional investigations are needed to assess its implication in human health.

Fifty-three faecal samples from yellow-legged gulls (Larus cachinnans) at the Berlengas nature reserve in Portugal were cultured on Levine agar plates not supplemented with antimicrobial agents, and one Escherichia coli colony was isolated and identified from each sample. The percentages of resistant isolates for each of the drugs were ampicillin (43.4 per cent), tetracycline (39.6 per cent), nalidixic acid (34.0 per cent), streptomycin (32.1 per cent), trimethoprim-sulfamethoxazole (SXT) (26.4 per cent), ciprofloxacin (18.9 per cent), chloramphenicol (18.9 per cent), gentamicin (7.5 per cent), tobramycin (7.5 per cent) amikacin (5.7 per cent) and amoxicillin-clavulanic acid (1.9 per cent). All the isolates were susceptible to cefoxitin, ceftazidime, cefotaxime, aztreonam and imipenem. The following resistance genes were detected: bla(TEM) (17 of 23 ampicillin-resistant isolates), tet(A) and/or tet(B) (18 of 21 tetracycline-resistant isolates), aadA (12 of 17 streptomycin-resistant isolates), cmlA (all chloramphenicol-resistant isolates), aac(3)-II with or without aac(3)-IV (all four gentamicin-resistant isolates), and sul1 and/or sul2 and/or sul3 (all 14 SXT-resistant isolates). The intI1 gene was detected in 10 of 14 SXT-resistant isolates, and three of them also contained class 2 integrons; four different gene cassette arrangements were identified among class 1 integrons (aadA, dfrA1+aadA1, dfrA12+orfF+aadA2 and sat+psp+aadA2) and one among the class 2 integrons (dfrA1+sat+aadA1). Ninety per cent of the isolates were included in the A or B1 phylogenetic groups.

Objectives: To analyse the prevalence and diversity of integrons in faecal Escherichia coli isolates from healthy humans in Spain., Methods: One hundred E. coli isolates were obtained in Levine agar plates from faecal samples of 100 healthy humans during March to October 2007. Susceptibility to 16 antimicrobial agents was determined by the disc diffusion method. The presence and characterization of class 1, 2 and 3 integrons, as well as the presence of other antimicrobial resistance genes, were performed by PCR and DNA sequencing., Results: Integrases associated with class 1 and/or class 2 integrons were identified in 29 E. coli isolates (intI1 gene in 26 isolates, intI2 in 1 isolate and intI1 + intI2 in 2 isolates), the remaining 71 isolates being free of these integrons. Seven different gene cassette arrangements were demonstrated in 27 of the 28 intI1-positive isolates and were as follows (number of isolates): dfrA1 + aadA1 (12), aadA (8), dfrA17 + aadA5 (3), dfrA7 (1), dfrA5 (1), dfrA1 (1) and dfrA12 + orfF + aadA2 (1). Four isolates presented defective class 1 integrons lacking the 3'-conserved region. The three isolates containing class 2 integrons harboured the dfrA1 + sat + aadA1 gene cassette array in their variable region. Integron-positive isolates showed higher percentages of resistance to streptomycin, ampicillin, tetracycline, trimethoprim, sulfamethoxazole, chloramphenicol and nalidixic acid than integron-negative isolates. Sixty-five percent of the integron-positive isolates belonged to phylogenetic groups A or D., Conclusions: A high prevalence of integrons was detected in faecal E. coli of healthy humans. Individuals in the community could be a reservoir of integron-containing E. coli isolates.

Faecal samples of healthy dogs (n=39) and cats (n=36) obtained in Northern Portugal were seeded on Levine agar plates, and two Escherichia coli isolates per sample were recovered (78 of dogs and 66 of cats). The susceptibility to 16 antimicrobial agents was tested in this series of 144 E. coli isolates. Almost 20% of them showed tetracycline resistance and 12 and 15% presented ampicillin or streptomycin resistance, respectively. The percentage of resistance to the other antimicrobial agents was in all cases below 4%, and no resistant isolates were detected for ceftazidime, imipenem, cefoxitin or amikacin. Two isolates (from one dog) showed cefotaxime-resistance and harboured both the CTX-M-1 and OXA-30 beta-lactamases. A bla(TEM) gene was detected in 12 of 17 ampicillin-resistant isolates, the aac(3)-II gene in the three gentamicin-resistant isolates, aadA in 7 of 22 streptomycin-resistant isolates, and tet(A) and/or tet(B) gene in all 28 tetracycline-resistant isolates. The gene encoding class 1 integrase was detected in six E. coli isolates, including the four trimethoprim-sulfamethoxazole-resistant isolates and those two harbouring CTX-M-1 and OXA-30 beta-lactamases; different gene cassette arrangements were identified: dfrA1+aadA1 (two isolates), dfrA12+orfF+aadA2 (two isolates) and bla(OXA30)+aadA1 (two isolates). One amino acid change in GyrA protein (Ser83Leu or Asp87Tyr) was detected in four nalidixic acid-resistant and ciprofloxacin-susceptible isolates and two amino acid changes in GyrA (Ser83Leu+Asp87Asn) and one in ParC (Ser80Ile) were identified in one nalidixic acid- and ciprofloxacin-resistant isolate. Faecal E. coli isolates of healthy pets could be a reservoir of antimicrobial resistance genes.