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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.)

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.)

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.)

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.