Although quinolone resistance usually results from chromosomal mutations, recent studies indicate that quinolone resistance can also be plasmid mediated. The gene responsible, qnr, is distinct from the known quinolone resistance genes and in previous studies seemed to be restricted to Klebsiella pneumoniae and Escherichia coli isolates from the University of Alabama in Birmingham, where this resistance was discovered. In Shanghai, the frequency of ciprofloxacin resistance in E. coli has exceeded 50% since 1993. Seventy-eight unique ciprofloxacin-resistant clinical isolates of E. coli from Shanghai hospitals were screened for the qnr gene by colony blotting and Southern hybridization of plasmid DNA. Conjugation experiments were done with azide-resistant E. coli J53 as a recipient with selection for plasmid-encoded antimicrobial resistance (chloramphenicol, gentamicin, or tetracycline) and azide counterselection. qnr genes were sequenced, and the structure of the plasmid DNA adjacent to qnr was analyzed by primer walking with a sequential series of outward-facing sequencing primers with plasmid DNA templates purified from transconjugants. Six (7.7%) of 78 strains gave a reproducible hybridization signal with a qnr gene probe on colony blots and yielded strong signals on plasmid DNA preparations. Quinolone resistance was transferred from all six probe-positive strains. Transconjugants had 16-to 250-fold increases in the MICs of ciprofloxacin relative to that of the recipient. All six strains contained qnr with a nucleotide sequence identical to that originally reported, except for a single nucleotide change (CTA3CTG at position 537) encoding the same amino acid. qnr was located in complex In4 family class 1 integrons. Two completely sequenced integrons were designated In36 and In37. Transferable plasmid-mediated quinolone resistance associated with qnr is thus prevalent in quinoloneresistant clinical strains of E. coli from Shanghai and may contribute to the rapid increase in bacterial resistance to quinolones in China.

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Although quinolone resistance commonly results from chromosomal mutation, recent studies indicate that such resistance can also be transferred on plasmids carrying the gene responsible, qnr. One hundred ten ciprofloxacin-resistant clinical isolates of Klebsiella pneumoniae and Escherichia coli from the United States were screened for the qnr gene by PCR and Southern hybridization of plasmid DNA. Conjugation experiments were done with azide-resistant E. coli J53 as the recipient and selection with azide and sulfonamide, a resistance frequently linked to qnr. EcoRI and BamHI digests of qnr-hybridizing plasmids were subjected to electrophoresis on agarose gels and probed with qnr by Southern hybridization. qnr was detected in 8 (11.1%) of 72 K. pneumoniae strains. These eight positive strains were from six states in the United States. qnr was not found in any of the 38 E. coli strains tested. Quinolone resistance was transferred from seven of the eight probe-positive strains. Transconjugants with qnr-hybridizing plasmids had 32-fold increases in ciprofloxacin MICs relative to E. coli J53. For all eight strains, the sequence of qnr was identical to that originally reported. By size and restriction digests, four plasmids were related to the first-reported plasmid, pMG252, and three were different. Five new qnr plasmids encoded FOX-5 ␤-lactamase, as did pMG252, but two others produced SHV-7 extended-spectrum ␤-lactamase. Transferable plasmid-mediated quinolone resistance associated with qnr is now widely distributed in quinolone-resistant clinical strains of K. pneumoniae in the United States. Plasmid-determined quinolone resistance contributes to the increasing quinolone resistance of K. pneumoniae isolates and to the linkage previously observed between resistance to quinolones and the latest ␤-lactam antibiotics.Quinolone resistance is usually caused by chromosomal mutations, but plasmid-mediated quinolone resistance has been discovered recently. The gene responsible for plasmid-mediated resistance, qnr, was found on plasmids varying in size from 54 to Ն180 kb in clinical isolates of Klebsiella pneumoniae and Escherichia coli from which low-level quinolone resistance could be transferred to a sensitive recipient by conjugation (11,19). Although our previous study showed that qnr was present in 8% of quinolone-resistant clinical isolates of E. coli from Shanghai, China (19), surveys for the presence of qnr are few, and in those few surveys qnr has rarely been found in clinical strains from other areas of the world (6, 15). More information on the prevalence of qnr in clinical strains is needed to understand its importance and contribution to the development of quinolone resistance.Our previous study found that qnr is located in complex In4 family class 1 integrons In36 and In37 (19), which are also known as complex sul1-type integrons because of the presence of duplicate qacE⌬1 and sul1genes. This relationship is supported by in vitro susceptibility results on transconjugants containing plasmids carrying qnr, showing...

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Transporters belonging to the chromosomally encoded resistance-nodulation-division (RND) superfamily mediate multidrug resistance in Gram-negative bacteria. However, the cotransfer of large gene clusters encoding RND-type pumps from the chromosome to a plasmid appears infrequent, and no plasmid-mediated RND efflux pump gene cluster has yet been found to confer resistance to tigecycline. Here, we identified a novel RND efflux pump gene cluster, designated tmexCD1-toprJ1, on plasmids from five pandrug-resistant Klebsiella pneumoniae isolates of animal origin. TMexCD1-TOprJ1 increased (by 4- to 32-fold) the MICs of tetracyclines (including tigecycline and eravacycline), quinolones, cephalosporins, and aminoglycosides for K. pneumoniae, Escherichia coli, and Salmonella. TMexCD1-TOprJ1 is closely related (64.5% to 77.8% amino acid identity) to the MexCD-OprJ efflux pump encoded on the chromosome of Pseudomonas aeruginosa. In an IncFIA plasmid, pHNAH8I, the tmexCD1-toprJ1 gene cluster lies adjacent to two genes encoding site-specific integrases, which may have been responsible for its acquisition. Expression of TMexCD1-TOprJ1 in E. coli resulted in increased tigecycline efflux and in K. pneumoniae negated the efficacy of tigecycline in an in vivo infection model. Expression of TMexCD1-TOprJ1 reduced the growth of E. coli and Salmonella but not K. pneumoniae. tmexCD1-toprJ1-positive Enterobacteriaceae isolates were rare in humans (0.08%) but more common in chicken fecal (14.3%) and retail meat (3.4%) samples. Plasmid-borne tmexCD1-toprJ1-like gene clusters were identified in sequences in GenBank from Enterobacteriaceae and Pseudomonas strains from multiple continents. The possibility of further global dissemination of the tmexCD1-toprJ1 gene cluster and its analogues in Enterobacteriaceae via plasmids may be an important consideration for public health planning. IMPORTANCE In an era of increasing concerns about antimicrobial resistance, tigecycline is likely to have a critically important role in the treatment of carbapenem-resistant Enterobacteriaceae, the most problematic pathogens in human clinical settings—especially carbapenem-resistant K. pneumoniae. Here, we identified a new plasmid-borne RND-type tigecycline resistance determinant, TMexCD1-TOprJ1, which is widespread among K. pneumoniae isolates from food animals. tmexCD1-toprJ1 appears to have originated from the chromosome of a Pseudomonas species and may have been transferred onto plasmids by adjacent site-specific integrases. Although tmexCD1-toprJ1 still appears to be rare in human clinical isolates, considering the transferability of the tmexCD1-toprJ1 gene cluster and the broad substrate spectrum of TMexCD1-TOprJ1, further dissemination of this mobile tigecycline resistance determinant is possible. Therefore, from a “One Health” perspective, measures are urgently needed to monitor and control its further spread. The current low prevalence in human clinical isolates provides a precious time window to design and implement measures to tackle this.

Since the discovery of qnrA in 1998, two additional qnr genes, qnrB and qnrS, have been described. These three plasmid-mediated genes contribute to quinolone resistance in gram-negative pathogens worldwide. A clinical strain of Proteus mirabilis was isolated from an outpatient with a urinary tract infection and was susceptible to most antimicrobials but resistant to ampicillin, sulfamethoxazole, and trimethoprim. Plasmid pHS10, harbored by this strain, was transferred to azide-resistant Escherichia coli J53 by conjugation. A transconjugant with pHS10 had low-level quinolone resistance but was negative by PCR for the known qnr genes, aac(6)-Ib-cr and qepA. The ciprofloxacin MIC for the clinical strain and a J53/pHS10 transconjugant was 0.25 g/ml, representing an increase of 32-fold relative to that for the recipient, J53. The plasmid was digested with HindIII, and a 4.4-kb DNA fragment containing the new gene was cloned into pUC18 and transformed into E. coli TOP10. Sequencing showed that the responsible 666-bp gene, designated qnrC, encoded a 221-amino-acid protein, QnrC, which shared 64%, 42%, 59%, and 43% amino acid identity with QnrA1, QnrB1, QnrS1, and QnrD, respectively. Upstream of qnrC there existed a new IS3 family insertion sequence, ISPmi1, which encoded a frameshifted transposase. qnrC could not be detected by PCR, however, in 2,020 strains of Enterobacteriaceae. A new quinolone resistance gene, qnrC, was thus characterized from plasmid pHS10 carried by a clinical isolate of P. mirabilis.Plasmid-mediated quinolone resistance was first described for a ciprofloxacin-resistant strain of Klebsiella pneumoniae in 1998 (15). The responsible gene, qnr (later named qnrA), was located on plasmid pMG252, which encodes multidrug resistance proteins. qnrB and qnrS were discovered in 2005 and 2006, respectively, and mediated similar levels of ciprofloxacin resistance (9, 11). Qnr proteins belong to the pentapeptide repeat protein (PRP) family and protect DNA gyrase and topoisomerase IV from quinolone inhibition (26,27,28). qnr genes show a high level of diversity; there are at least 6 qnrA, 20 qnrB, and 3 qnrS alleles reported, with one or more amino acid alterations within each family (12; http://www.lahey.org /qnrStudies). More recently, qnrD was found in Salmonella isolates (3). qnr genes are widely distributed in clinical Enterobacteriaceae isolates around the world and are usually associated with mobile elements (21). There were also qnr-like genes found on the chromosomes of Vibrio vulnificus, Vibrio parahaemolyticus, Photobacterium profundum, Stenotrophomonas maltophilia, and gram-positive genera such as Enterococcus, Listeria, Clostridium, and Bacillus (1,17,22,24). The wide distribution of qnr genes in different species of Enterobacteriaceae and their high degree of diversity raise the concern that there might be more qnr genes that have not yet been discovered. In this study, a new plasmid-mediated quinolone resistance gene, qnrC, was found on and cloned from a transferable plasmid, pHS10, in a clinical ...

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6Since the plasmid-borne quinolone resistance gene qnr was reported in 1998 (8), many additional qnr alleles have been discovered on plasmids or the bacterial chromosome (reviewed in references 9 and 13). The plasmid-borne qnr genes currently comprise three families, qnrA, qnrB, and qnrS, differing from each other 40% or more in nucleotide sequence. Within each family, minor (Յ10%) variation in sequence has defined a growing number of alleles. For the qnrA and qnrS families, the number of variants has been manageable, with general agreement on allele designations, but lately, the number of qnrB sequences submitted to GenBank has exploded, with the same qnrB allele number claimed for dissimilar sequences by different investigators and the same entry given new allele numbers from week to week.To bring order into the current qnrB numbering chaos, we propose numbering the qnr alleles according to the following rules: (i) priority should be given first to published numbers, then to those in accepted or submitted manuscripts, and finally to the date of submission to GenBank; (ii) only full-length sequences should be assigned allele numbers; (iii) naturally occurring alleles, not those created by mutation, will be numbered; (iv) only nucleotide alterations that result in amino acid changes and not functionally silent substitutions should be taken into account; (v) one or more amino acid alterations define a new allele; (vi) variation in promoter sequences is not considered; (vii) demonstration that an allele in an established family causes reduced susceptibility to nalidixic acid or a fluoroquinolone is desirable but is not required; (viii) a new family (such as qnrC) should differ substantially from existing families (Ն30% difference suggested in nucleotides or derived amino acids) and should be shown to affect quinolone susceptibility; (ix) a database of qnr allele designations will be maintained at http://www.lahey.org/qnrStudies; and (x) further allele numbers will be assigned upon application.Another source of confusion is the presence of two potential in-frame initiation codons for some qnrB alleles. For other qnrB alleles, the first ATG is out of phase with the second. The