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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.)

Integrons 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 coli isolates (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. coli strains 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. coli isolates 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.

Gene cassettes of class 1 integrons may be differently expressed depending on the Pc promoter variant as well as occasionally from a second promoter located downstream of Pc, named P2. So far, the distribution of the variants has only been described in an in silico study. In this study, the prevalence of these variants in vivo was analysed in a population of 85 Escherichia coli strains from a variety of phylogenetic groups isolated from healthy subjects and clinical samples in Spain and France from 2004 to 2007. The weakest variants (PcW and PcH1) prevailed (variants associated with the integrase having the most efficient excision activity), whilst the two strongest variants, PcW(TGN-10) and PcS, were less frequent. Furthermore, a new variant of P2 associated with PcW was characterised in one integron (harbouring the gene cassette bla(OXA-1)-aadA1) from a French strain of a healthy subject. This variant was hereafter named P2m3 and shows a G→A substitution in its -10 element (TACAGT to TACAAT), a mutation that doubled the strength of P2 and approached the level of expression of the strong PcW(TGN-10) variant. When the correlation between the Pc variants and the origin of the strains was analysed, no significant difference (P<0.05) was observed in the Pc variant distribution according to the geographic origin or clinical setting., (Copyright © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Beta-lactamase characterization was carried out in a collection of 18 extended-spectrum beta-lactamase (ESBL)-positive Escherichia coli isolates from blood (n=8) and urine (n=10) obtained in 2007 in a tunisian Hospital. All isolates were clonally unrelated according to PFGE analysis. Seventeen strains presented the bla(CTX-M-)₁₅ gene associated with bla (OXA-)₁ and four of these strains with the (TEM-)₁(b) gene. The remaining ESBL-positive strain contained the bla (CTX-M-)₉ gene associated with the bla (OXA-)₁ and bla (TEM-)₁(b) genes. The orf477 sequence was identified downstream of the bla(CTX-M-)₁₅ gene in all 17 bla(CTX-M-)₁₅-positive strains, and ISEcp1 upstream in 15 of them (in eight cases truncated by IS26). The presence of a class 1 integron was demonstrated in 4 of the 18 ESBL-positive strains (22.2%), with dfrA17 + aadA5 (3 strains) and dfrA12 + orfF + aadA2 (1 strain) being the gene cassettes identified. The variant aac(6´)-Ib-cr was found in 15 bla(CTX-M-)₁₅-containing strains. All 18 ESBL-positive strains were typed as phylogroup B2 and contained at least three of the eight tested virulence genes (fimA, papGIII, hlyA, cnf1, papC, aer, eae and bfp). Six bla(CTX-M-)₁₅-positive strains were included in the serotype O25b and one of them was typed as ST131. Another bla(CTX-M-)₁₅-positive strain serotype-O25 was typed as ST638. The bla(CTX-M-)₁₅, aac(6')- Ib-cr, and aac(3)-II genes were co-transferred by conjugation from 7 donor strains to E. coli CSH26 recipient strain. The bla(CTXM-)₁₅ gene is prevalent among ESBL-positive E. coli strains in the studied hospital, that is frequently found together with aac(6')- Ib-cr, and aac(3)-II genes. The detection of the clone O25b-St131 in a bla(CTX-M-)₁₅ strain corroborates its worldwide dissemination.

Objectives: To study the frequency and diversity of class 1 integrons lacking the 3'-conserved segment (CS) in intI1-positive Escherichia coli isolates of different origins., Methods: The presence of intI1 was previously detected in 84 E. coli isolates of food (21 isolates), animal (32) and healthy-human volunteer (31) origins. The qacEDelta1-sul1 genes were analyzed by PCR and those isolates that lacked these genes were included in this work. The genetic structure of class 1 integrons was determined, using the PCR and sequencing primer-walking strategy. Isolates and plasmids were typed., Results: Class 1 integrons lacking the 3'-CS were found in 13 of the 84 intI1-positive E. coli isolates (15.5%) of food, animal, and human origins. All 13 isolates showed unrelated patterns by REP-PCR. The following gene cassette arrangements were identified inside the class 1 integrons of these 13 strains: dfrA1; dfrA5; dfrA12-orfF-aadA2-cmlA1-aadA1-qacH-IS440-sul3; dfrA12-orfF-aadA2-cmlA1-aadA1-IS440-sul3; estX-psp-aadA2-cmlA1-aadA1-qacH-IS440-sul3; and a new arrangement estX-psp-aadA2-cmlA1Delta-IS1294-DeltacmlA1-aadA1-qacH-IS440-sul3 that contain the IS1294 into the cmlA1 gene (included in GenBank, number EU704128). Complete or truncated mef(B) gene was detected upstream of sul3 gene in this type of integrons. Plasmids were identified in four of the studied strains by PCR-replicon-typing, detecting different combinations of IncY, I1, FIC, FII, FIB plasmids. Non-classic integrons were located into plasmids of 100-150 kb in four studied strains., Conclusions: Occurrence and diversity of class 1 integrons lacking 3'-CS among the studied intI1-positive E. coli isolates of different origins were relatively high. The sul3 gene was detected in most of class 1 integrons lacking 3'-CS., (Copyright 2010 Elsevier B.V. All rights reserved.)

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.)

Introduction: Fecal Escherichia coli isolates showing a phenotype of reduced susceptibility or resistance to extended-spectrum cephalosporins are common among pigs in Spain. The aim of this study was to describe the main beta-lactam resistance mechanisms carried by these strains and their distribution at farm-level., Materials and Methods: Twenty-nine E. coli isolates showing reduced susceptibility or resistance to extended-spectrum cephalosporins were collected from a sampling frame of 80 pig farms distributed over 13 Spanish provinces. The survey was carried out at the slaughterhouse level in 2004., Results: Of the 29 isolates, 21 (72%) met the criteria for a positive phenotypic confirmatory test for extended-spectrum beta-lactamases (ESBL). The following ESBLs were detected: SHV-12 (12 isolates, 41%), CTX-M-1 (three isolates, 10%), CTX-M-9 (three isolates, 10%), and CTX-M-14 (three isolates, 10%). The remaining eight isolates (28%) were phenotypically non-ESBL, with seven of them (24%) showing mutations on the chromosomal ampC gene promoter at positions -42 (C-->T), -18 (G-->A), -1 (C-->T), and +58 (C-->T). A multiplex PCR for detection of plasmidic class C beta-lactamases was negative for all isolates., Conclusion: Different ESBLs and other mechanisms linked to extended-spectrum cephalosporin resistance are widely distributed among fecal E. coli from slaughter pigs in Spain., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Escherichia coli isolates containing the following extended-spectrum beta-lactamases have been detected in 11 of 57 fecal samples (19.3%) in Berlengas Island seagulls: TEM-52 (eight isolates), CTX-M-1 (one isolate), CTX-M-14a (one isolate), and CTX-M-32 (one isolate). Most of the extended-spectrum beta-lactamase-positive isolates harbored class 1 or class 2 integrons, which included different antibiotic resistance gene cassettes.

Characterisation of extended-spectrum beta-lactamase (ESBL) genes and their genetic environments as well as the presence of integrons were analysed in nine Klebsiella pneumoniae and two Escherichia coli ESBL-positive isolates recovered in the Centre of Bone Marrow Transplantation of Tunisia. All strains harboured the bla(CTX-M-15) gene and presented minimum inhibitory concentrations for cefotaxime and ceftazidime of 256-1024 mg L(-1) and 16-512 mg L(-1), respectively, and eight of them showed different pulsed-field gel electrophoresis patterns. The bla(OXA-1) and bla(TEM-1) genes were detected in eight and ten strains, respectively. In addition, bla(SHV-1), bla(SHV-11) and bla(SHV-27) were found in six, one and one K. pneumoniae strains, respectively. The new variant bla(SHV-103) was characterised in one K. pneumoniae strain. The intI1 gene was detected in eight K. pneumoniae strains and the dfrA5+ereA2 and aadA gene cassettes were found in one and five strains, respectively. All strains harboured a 70 kb plasmid, and its transference in addition to bla(CTX-M-15), bla(TEM-1b) and bla(OXA-1) genes was demonstrated from three K. pneumoniae to E. coli. ISEcp1 and orf477 were located upstream and downstream, respectively, of the bla(CTX-M-15) gene in 10 strains. The occurrence of the bla(CTX-M-15) gene in unrelated strains might have originated from the dissemination of mobile genetic elements in which ISEcp1 may have played an important role.