The quinolone class of antibiotics has become an integral component of the antimicrobial arsenal. However, the presence of plasmid-mediated quinolone resistance genes has been increasing in Enterobacteriaceae since the introduction of quinolones. This study aimed to fill a considerable gap in the literature by assessing the prevalence and type of plasmid-mediated quinolone resistance genes isolated from individuals living in Korea.
To analyze the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants in ciprofloxacin-nonsusceptible Escherichia coli and Klebsiella pneumoniae isolated from patients at a tertiary care hospital in Korea.
A total of 102 nonduplicate isolates of ciprofloxacin-intermediate or ciprofloxacin-resistant E coli (n=80) and K pneumoniae (n=22) from blood cultures were obtained. The qnr (qnrA, qnrB, qnrS), aac(6′)-Ib-cr, qepA and oqxAB genes were detected using polymerase chain reaction (PCR) and confirmed using direct sequencing. To determine whether the PMQR-positive plasmid was horizontally transferable, conjugation experiments were performed.
Of the 102 isolates, 81 (79.4%) had one or more PMQR genes; these consisted of 59 (73.8%) E coli and 22 (100%) K pneumoniae isolates. The qnr genes were present in 15 isolates (14.7%): qnrB4 was detected in 10.8% and qnrS1 was detected in 3.9%. The aac(6′)-Ib-cr, qepA and oqxAB genes were detected in 77.5%, 3.9% and 10.8%, respectively. In conjugation experiments, PMQR genes were successfully transferred from seven (8.6%) isolates. The range of minimum inhibitory concentrations of ciprofloxacin for these seven transconjugants increased to 0.5 mg/L to 1 mg/L, which was 16- to 33-fold that of the recipient E coli J53 bacteria.
PMQR genes were highly prevalent among ciprofloxacin-nonsusceptible E coli and K pneumoniae from blood cultures in the authors’ hospital. Therefore, it is necessary to monitor for the spread of PMQR genes of clinical isolates and to ensure careful antibiotic use in a hospital setting.
aac(6′)-Ib-cr; oqxAB; Plasmid-mediated quinolone resistance genes; qepA; qnr
We have studied by PCR and DNA sequencing the presence of the qnrA, qnrB, qnrS, aac(6′)-Ib-cr, qepA, intI1, and ISCR1 genes in 200 clinical isolates of Enterobacter cloacae (n = 153) and E. aerogenes (n = 47) consecutively collected between January 2004 and October 2005 in two hospitals located in Santander (northern Spain) and Seville (southern Spain). Mutations in the quinolone resistance-determining region of gyrA and parC also were investigated in organisms containing plasmid-mediated quinolone resistance genes. The isolates had different resistant phenotypes, including AmpC hyperproduction, extended-spectrum β-lactamase production, resistance or decreased susceptibility to quinolones, and/or resistance to aminoglycosides. Among the 116 E. cloacae isolates from Santander, qnrS1, qnrB5, qnrB2, and aac(6′)-Ib-cr were detected in 22 (19%), 1 (0.9%), 1 (0.9%), and 3 (2.6%) isolates, respectively. Twenty-one, 17, and 2 qnrS1-positive isolates also contained blaLAP-1, intI1, and ISCR1, respectively. A qnrB7-like gene was detected in one E. aerogenes isolate from Santander. No plasmid-mediated quinolone resistance gene was detected in the isolates from Seville. The qnrS1-containing isolates corresponded to four pulsed-field gel electrophoresis patterns and showed various levels of resistance to quinolones. Six isolates were susceptible to nalidixic acid and presented reduced susceptibility to ciprofloxacin. The qnrS1 gene was contained in a conjugative plasmid of ca. 110 kb, and when the plasmid was transferred to recipient strains that did not have a specific mechanism of quinolone resistance, the ciprofloxacin MICs ranged from 0.047 to 0.125 μg/ml.
Nontyphoidal Salmonella enterica strains with a nonclassical quinolone resistance phenotype were isolated from patients returning from Thailand or Malaysia to Finland. A total of 10 isolates of seven serovars were studied in detail, all of which had reduced susceptibility (MIC ≥ 0.125 μg/ml) to ciprofloxacin but were either susceptible or showed only low-level resistance (MIC ≤ 32 μg/ml) to nalidixic acid. Phenotypic characterization included susceptibility testing by the agar dilution method and investigation of efflux activity. Genotypic characterization included the screening of mutations in the quinolone resistance-determining regions (QRDR) of gyrA, gyrB, parC, and parE by PCR and denaturing high-pressure liquid chromatography and the amplification of plasmid-mediated quinolone resistance (PMQR) genes qnrA, qnrB, qnrS, qnrD, aac(6′)-Ib-cr, and qepA by PCR. PMQR was confirmed by plasmid analysis, Southern hybridization, and plasmid transfer. No mutations in the QRDRs of gyrA, gyrB, parC, or parE were detected with the exception of a Thr57-Ser substitution within ParC seen in all but the S. enterica serovar Typhimurium strains. The qnrA and qnrS genes were the only PMQR determinants detected. Plasmids carrying qnr alleles were transferable in vitro, and the resistance phenotype was reproducible in Escherichia coli DH5α transformants. These data demonstrate the emergence of a highly mobile qnr genotype that, in the absence of mutation within topoisomerase genes, confers the nontypical quinolone resistance phenotype in S. enterica isolates. The qnr resistance mechanism enables bacteria to survive elevated quinolone concentrations, and therefore, strains carrying qnr alleles may be able to expand during fluoroquinolone treatment. This is of concern since nonclassical quinolone resistance is plasmid mediated and therefore mobilizable.
Sixty extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolated from rivers and lakes in Switzerland were screened for individual strains additionally exhibiting a reduced quinolone susceptibility phenotype. Totally, 42 such isolates were found and further characterized for their molecular (fluoro)quinolone resistance mechanisms. PCR and sequence analysis were performed to identify chromosomal mutations in the quinolone resistance-determining regions (QRDR) of gyrA, gyrB, parC and parE and to describe the occurrence of the following plasmid-mediated quinolone resistance genes: qepA, aac-6′-Ib-cr, qnrA, qnrB, qnrC, qnrD and qnrS. The contribution of efflux pumps to the resistance phenotype of selected strains was further determined by the broth microdilution method in the presence and absence of the efflux pump inhibitor phe-arg-β-naphthylamide (PAβN). Almost all strains, except two isolates, showed at least one mutation in the QRDR of gyrA. Ten strains showed only one mutation in gyrA, whereas thirty isolates exhibited up to four mutations in the QRDR of gyrA, parC and/or parE. No mutations were detected in gyrB. Most frequently the amino-acid substitution Ser83→Leu was detected in GyrA followed by Asp87→Asn in GyrA, Ser80→Ile in ParC, Glu84→Val in ParC and Ser458→Ala in ParE. Plasmid-mediated quinolone resistance mechanisms were found in twenty isolates bearing QnrS1 (4/20), AAC-6′-Ib-cr (15/20) and QepA (1/20) determinants, respectively. No qnrA, qnrB, qnrC and qnrD were found. In the presence of PAβN, the MICs of nalidixic acid were decreased 4- to 32-fold. (Fluoro) quinolone resistance is due to various mechanisms frequently associated with ESBL-production in E. coli from surface waters in Switzerland.
OqxAB has recently been identified as one of the mechanisms of plasmid-mediated quinolone resistance (PMQR). Compared to what is observed for other PMQR determinants, there is a paucity of data with regard to the prevalence and epidemiology of OqxAB and its contribution to resistance to different antimicrobials. In this study, the prevalence and dissemination of oqxAB and other PMQR genes in Escherichia coli isolates from animals, farmworkers, and the environment in 2002 in China were investigated. Of the 172 E. coli isolates, 39.0% carried oqxA, while only 4.1%, 2.9%, and 0.6% carried qnr (1 qnrB6 isolate, 5 qnrS1 isolates, and 1 qnrD isolate), qepA, and aac(6′)-Ib-cr, respectively. Among the 33 isolates from farmworkers, 10 (30.3%) were positive for oqxA. oqxAB was associated with IS26 and was carried on the 43- to 115-kb IncF transferable plasmid. Transconjugants carrying oqxAB showed 4- to 16-fold increases in the MICs of quinolones, 16- to 64-fold increases in the MICs of quinoxalines, 8- to 32-fold increases in the MICs of chloramphenicol and trimethoprim-sulfamethoxazole, and 4- to 8-fold increases in the MICs of florfenicol compared to the levels for the recipient. The pulsed-field gel electrophoresis (PFGE) analysis showed that the high levels of prevalence and dissemination of oqxAB in E. coli in animal farms were primarily due to the transmission of plasmids carrying oqxAB, although clonal transmission between human and swine E. coli isolates was observed. It is concluded that oqxAB was widespread in animal farms in China, which may be due to the overuse of quinoxalines in animals. This study warrants the prudent use of quinoxalines in food animals.
We investigated the prevalence of plasmid-mediated quinolone resistance (PMQR) genes in
avian pathogenic Escherichia coli (APEC) strains in Japan. A total of 117
APEC strains collected between 2004 and 2007 were examined for PMQR genes (qnrA,
qnrB, qnrC, qnrD, qnrS,
qepA and oqxAB) by polymerase chain reaction. None of
the APEC strains carried qnrA, qnrB,
qnrC, qnrD, qnrS, qepA or
oqxAB, but one of the isolates was identified as an AAC (6’)-Ib-cr producer.
Phylogenetic grouping, multi-locus sequence typing and serotyping showed that this isolate
belonged to phylogenetic group A, sequence type 167 and untypable serogroup. To our
knowledge, this is the first report of the aac (6’)-Ib-cr gene in
bacteria from food-producing animals in Japan.
aac(6’)-Ib-cr; APEC; PMQR; quinolone resistance
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 μg/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.
Recently, several plasmid-mediated quinolone resistance (PMQR) genes conferring low levels of quinolone resistance have been discovered. To evaluate the temporal change in the prevalence of PMQR genes over a decade in a tertiary hospital in the Republic of Korea, we selected every fifth isolate of Escherichia coli and Klebsiella pneumoniae and every third isolate of Enterobacter cloacae between 1998 and 2001 and between 2005 and 2006 from a collection of blood isolates. Six PMQR genes [qnrA, qnrB, qnrC, qnrS, aac(6′)-Ib-cr, and qepA] were screened by multiplex PCR and then confirmed by direct sequencing, and the aac(6′)-Ib-positive PCR products were digested with BtsCI to identify the aac(6′)-Ib-cr variant. Of 461 isolates, 37 (8%) had one of the six PMQR genes; 13 (5%) of 261 E. coli strains, 13 (10%) of 135 K. pneumoniae strains, and 11 (17%) of 65 E. cloacae strains. qnrB was the most common PMQR gene and was found as early as 1998, whereas qnrS, aac(6′)-Ib-cr, and qepA emerged after 2000. None of the isolates carried qnrA or qnrC. Ciprofloxacin resistance increased over time (P < 0.001), and the overall prevalence of PMQR genes tended to increase (P = 0.20). PMQR-positive isolates had significantly higher ciprofloxacin resistance and multidrug resistance rates (P = 0.005 and P < 0.001, respectively). The increasing frequency of ciprofloxacin resistance in Enterobacteriaceae was associated with an increasing prevalence of PMQR genes, and this change involved an increase in the diversity of the PMQR genes and also an increase in the prevalence of the mutations in gyrA, parC, or both in PMQR-positive strains but not PMQR-negative strains.
Plasmid-mediated quinolone resistance (PMQR) caused by qnr genes has been known for 15 years. Information about global distribution and prevalence of qnr genes is abundant, but clinical information concerning infections produced by these isolates and risk factors for their acquisition is limited.
Klebsiella pneumoniae blood isolates (n = 227) from a 1 year prospective cohort of patients in Taiwan were studied. MICs of quinolones were determined for all isolates, and multiplex PCR for the presence of PMQR genes and DNA gyrase mutations was applied to all 24 isolates with ciprofloxacin MICs ≥0.12 mg/L and a control group of 72 isolates with MICs ≤0.06 mg/L.
All qnr isolates were in the group with ciprofloxacin MICs ≥0.12 mg/L, constituting 9.4% of tested isolates and 3.9% (qnrB 2.6% and qnrS 1.3%) of total isolates. aac(6′)-Ib-cr and qepA were not found. Risk factors for qnr included nosocomial infection, bedridden status, surgery within 3 months, non-K1/K2 serotypes and prior antimicrobial use. Ciprofloxacin MIC ≥0.12 mg/L was associated with prior quinolone use; in contrast, prior cephalosporin use was more closely linked to the presence of qnr. Fourteen-day mortality was similar in patients infected with qnr-positive versus qnr-negative isolates, but there was a trend for increased in-hospital mortality in patients infected with qnr-positive isolates.
In K. pneumoniae blood isolates collected at a hospital in Taiwan, the overall prevalence of qnr genes was 3.9%. Prior quinolone use was linked to increased ciprofloxacin MIC, but not with the prevalence of qnr, which was most strongly linked to exposure to other antimicrobials, especially cephalosporins.
K. pneumoniae; PMQR; resistance
Since its discovery, qnrA has been found in most common Enterobacteriaceae. Ciprofloxacin MICs conferred by different qnrA-positive plasmids could range from 0.1 μg/ml to 2 μg/ml in Escherichia coli J53. The reasons for different ciprofloxacin MICs conferred by qnrA have not been fully clarified. Five hundred forty-one consecutive gram-negative clinical strains that were resistant or intermediate to ciprofloxacin and that were isolated in Shanghai in 2005 were screened for qnrA by PCR. For qnrA-positive isolates, the transferability of quinolone resistance was determined by conjugation and mutations within the quinolone resistance-determining region (QRDR) of gyrA and parC. aac(6′)-Ib-cr was detected and qnrA RNA expression was determined using real-time reverse transcription-PCR for transconjugants with different ciprofloxacin MICs. The qnrA gene was detected in 7 of the 541 clinical isolates. Quinolone resistance was transferred in four strains by conjugation. Mutations in the QRDR of gyrA and parC were detected in five qnrA-positive clinical strains with higher ciprofloxacin MICs. Of four qnrA-bearing plasmids in E. coli J53, pHS4 and pHS5 conferred ciprofloxacin MICs of 0.094 to 0.125 μg/ml; pHS3, which harbored the aac(6′)-Ib-cr gene as well, conferred a ciprofloxacin MIC of 0.25 μg/ml, and pHS6, which had both the aac(6′)-Ib-cr gene and a high expression level of qnrA, had a ciprofloxacin MIC of 1.0 μg/ml. The prevalence of qnrA appeared to be higher in Enterobacter cloacae than in other Enterobacteriaceae. The coexistence of qnrA and aac(6′)-Ib-cr in a single plasmid and increased qnrA expression can account for the different levels of ciprofloxacin resistance seen in transconjugants.
This work describes the characterization of plasmid-mediated quinolone-resistance (PMQR) genes from a multicenter study of ESBL-producing Enterobacteriaceae pediatric clinical isolates in Mexico. The PMQR gene-positive isolates were characterized with respect to ESBLs, and mutations in the GyrA and ParC proteins were determined. The phylogenetic relationship was established by PFGE and the transfer of PMQR genes was determined by mating assays. The prevalence of the PMQR genes was 32.1%, and the rate of qnr-positive isolates was 15.1%; 93.3% of the latter were qnrB and 6.4% were qnrA1. The distribution of isolates in terms of bacterial species was as follows: 23.5% (4/17) corresponded to E. cloacae, 13.7% (7/51) to K. pneumoniae, and 13.6% (6/44) to E. coli. In addition, the prevalence of aac(6’)-Ib-cr and qepA was 15.1% and 1.7%, respectively. The molecular characteristics of qnr- and qepA-positive isolates pointed to extended-spectrum β-lactamase (ESBL) CTX-M-15 as the most prevalent one (70.5%), and to SHV-12 in the case of aac(6’)-Ib-cr-positive isolates. GyrA mutations at codons Ser-83 and Asp-87, and ParC mutations at codons Ser-80 were observed in 41.1% and 35.2% of the qnr-positive isolates, respectively. The analysis of the transconjugants revealed a co-transmission of blaCTX-M-15 with the qnrB alleles. In general, the prevalence of PMQR genes (qnr and aac(6’)-Ib-cr) presented in this work was much lower in the pediatric isolates, in comparison to the adult isolates in Mexico. Also, ESBL CTX-M-15 was the main ESBL identified in the pediatric isolates, whereas in the adult ones, ESBLs corresponded to the CTX-M and the SHV families. In comparison with other studies, among the PMQR-genes identified in this study, the qnrB-alleles and the aac(6’)-Ib-cr gene were the most prevalent, whereas the qnrS1, qnrA1 and qnrB-like alleles were the most prevalent in China and Uruguay.
The association of PMQR and ESBLs in negative-bacteria isolates has been of great concern. The present study was performed to investigate the prevalence of co-transferability of oqxAB and blaCTX-M genes among the 696 Escherichia coli (E. coli) isolates from food-producing animals in South China, and to characterize these plasmids.
The ESBL-encoding genes (blaCTX-M, blaTEM and blaSHV), and PMQR (qnrA, qnrB, qnrS, qnrC, qnrD, aac(6’)-Ib-cr, qepA, and oqxAB) of these 696 isolates were determined by PCR and sequenced directionally. Conjugation, S1 nuclease pulsed-field gel electrophoresis (PFGE) and Southern blotting experiments were performed to investigate the co-transferability and location of oqxAB and blaCTX-M. The EcoRI digestion profiles of the plasmids with oqxAB-blaCTX-M were also analyzed. The clonal relatedness was investigated by PFGE.
Of the 696 isolates, 429 harbored at least one PMQR gene, with oqxAB (328) being the most common type; 191 carried blaCTX-M, with blaCTX-M-14 the most common. We observed a significant higher prevalence of blaCTX-M among the oqxAB-positive isolates (38.7%) than that (17.4%) in the oqxAB-negative isolates. Co-transferability of oqxAB and blaCTX-M was found in 18 of the 127 isolates carrying oqxAB-blaCTX-M. These two genes were located on the same plasmid in all the 18 isolates, with floR being on these plasmids in 13 isolates. The co-dissemination of these genes was mainly mediated by F33:A-: B- and HI2 plasmids with highly similar EcoRI digestion profiles. Diverse PFGE patterns indicated the high prevalence of oqxAB was not caused by clonal dissemination.
blaCTX-M was highly prevalent among the oqxAB-positive isolates. The co-dissemination of oqxAB-blaCTX-M genes in E. coli isolates from food-producing animals is mediated mainly by similar F33:A-: B- and HI2 plasmids. This is the first report of the co-existence of oqxAB, blaCTX-M, and floR on the same plasmids in E. coli.
Resistance to quinolones and fluoroquinolones is being increasingly reported among human but also veterinary isolates during the last two to three decades, very likely as a consequence of the large clinical usage of those antibiotics. Even if the principle mechanisms of resistance to quinolones are chromosome-encoded, due to modifications of molecular targets (DNA gyrase and topoisomerase IV), decreased outer-membrane permeability (porin defect), and overexpression of naturally occurring efflux, the emergence of plasmid-mediated quinolone resistance (PMQR) has been reported since 1998. Although these PMQR determinants confer low-level resistance to quinolones and/or fluoroquinolones, they are a favorable background for selection of additional chromosome-encoded quinolone resistance mechanisms. Different transferable mechanisms have been identified, corresponding to the production of Qnr proteins, of the aminoglycoside acetyltransferase AAC(6′)-Ib-cr, or of the QepA-type or OqxAB-type efflux pumps. Qnr proteins protect target enzymes (DNA gyrase and type IV topoisomerase) from quinolone inhibition. The AAC(6′)-Ib-cr determinant acetylates several fluoroquinolones, such as norfloxacin and ciprofloxacin. Finally, the QepA and OqxAB efflux pumps extrude fluoroquinolones from the bacterial cell. A series of studies have identified the environment to be a reservoir of PMQR genes, with farm animals and aquatic habitats being significantly involved. In addition, the origin of the qnr genes has been identified, corresponding to the waterborne species Shewanella sp. Altogether, the recent observations suggest that the aquatic environment might constitute the original source of PMQR genes, that would secondly spread among animal or human isolates.
Quinolone resistance; Qnr; plasmid; efflux pump
A total of 225 isolates of Salmonella enterica serovar Typhimurium from food-producing animals collected between 2003 and 2007 were examined for the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants, namely qnrA, qnrB, qnrC, qnrD, qnrS, qepA and aac(6')Ib-cr, in Japan. Two isolates (0.8%) of S. Typhimurium DT104 from different dairy cows on a single farm in 2006 and 2007 were found to have qnrS1 on a plasmid of approximately 9.6-kbp. None of the S. Typhimurium isolates had qnrA, qnrB, qnrC, qnrD, qepA and acc(6')-Ib-cr. Currently in Japan, the prevalence of the PMQR genes among S. Typhimurium isolates from food animals may remain low or restricted. The PFGE profile of two S. Typhimurium DT104 isolates without qnrS1 on the farm in 2005 had an identical PFGE profile to those of two S. Typhimurium DT104 isolates with qnrS1. The PFGE analysis suggested that the already existing S. Typhimurium DT104 on the farm fortuitously acquired the qnrS1 plasmid.
Three kinds of plasmid-mediated quinolone resistance (PMQR) determinants have been discovered and have been shown to be widely distributed among clinical isolates: qnr genes, aac(6′)-Ib-cr, and qepA. Few data on the prevalence of these determinants in strains from animals are available. The presence of PMQR genes in isolates from animals was determined by PCR amplification and DNA sequencing. The production of extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases in the strains was detected, and their genotypes were determined. The genetic environment of PMQR determinants in selected plasmids was analyzed. All samples of ceftiofur-resistant (MICs ≥ 8 μg/ml) isolates of the family Enterobacteriaceae were selected from 36 companion animals and 65 food-producing animals in Guangdong Province, China, between November 2003 and April 2007, including 89 Escherichia coli isolates, 9 Klebsiella pneumoniae isolates, and isolates of three other genera. A total of 68.3% (69/101) of the isolates produced ESBLs and/or AmpC β-lactamases, mainly those of the CTX-M and CMY types. Of the 101 strains, PMQR determinants were present in 35 (34.7%) isolates, with qnr, aac(6′)-Ib-cr, and qepA detected alone or in combination in 8 (7.9%), 19 (18.8%), and 16 (15.8%) strains, respectively. The qnr genes detected included one qnrB4 gene, four qnrB6 genes, and three qnrS1 genes. Five strains were positive for both aac(6′)-Ib-cr and qepA, while one strain was positive for qnrS1, aac(6′)-Ib-cr, and qepA. qnrB6 was flanked by two copies of ISCR1 with an intervening dfr gene downstream and sul1 and qacEΔ1 genes upstream. In another plasmid, aac(6′)-Ib-cr followed intI1 and arr-3 was downstream. PMQR determinants are highly prevalent in ceftiofur-resistant Enterobacteriaceae strains isolated from animals in China. This is the first report of the occurrence of PMQR determinants among isolates from companion animals.
Ninety-three Malaysian extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae isolates were investigated for ciprofloxacin resistance. Two mismatch amplification mutation (MAMA) assays were developed and used to facilitate rapid detection of gyrA and parC mutations. The isolates were also screened for plasmid-mediated quinolone resistance (PMQR) genes including aac(6′)-Ib-cr, qepA, and qnr. Ciprofloxacin resistance (MICs 4– ≥ 32 μg/mL) was noted in 34 (37%) isolates, of which 33 isolates had multiple mutations either in gyrA alone (n = 1) or in both gyrA and parC regions (n = 32). aac(6′)-Ib-cr was the most common PMQR gene detected in this study (n = 61), followed by qnrB and qnrS (n = 55 and 1, resp.). Low-level ciprofloxacin resistance (MICs 1-2 μg/mL) was noted in 40 (43%) isolates carrying qnrB accompanied by either aac(6′)-Ib-cr (n = 34) or a single gyrA 83 mutation (n = 6). Ciprofloxacin resistance was significantly associated with the presence of multiple mutations in gyrA and parC regions. While the isolates harbouring gyrA and/or parC alteration were distributed into 11 PFGE clusters, no specific clusters were associated with isolates carrying PMQR genes. The high prevalence of ciprofloxacin resistance amongst the Malaysian ESBL-producing K. pneumoniae isolates suggests the need for more effective infection control measures to limit the spread of these resistant organisms in the hospital.
Fluoroquinolone resistance in members of the Enterobacteriaceae family is mostly due to mutations in the quinolone resistance-determining regions of the topoisomerase genes. However, transferable genes encoding quinolone resistance have recently been described. The current methods for susceptibility testing are not adapted to the detection of new resistance determinants, which confer low levels of resistance. The aim of this study was to compare the ability of the screening of the different quinolones by disk diffusion assays and MIC determinations to detect fluoroquinolone resistance. Sixty-nine Escherichia coli strains and 62 Salmonella strains, including strains fully susceptible to quinolones, nalidixic acid-resistant strains, strains with resistance to fluoroquinolones (resistant to nalidixic acid), and strains showing low-level resistance to fluoroquinolones conferred by transferable quinolone resistance genes, including qnrA, qnrB, qnrS, and aac(6′)Ib-cr, were selected. Disk diffusion assays and MIC determinations by the agar dilution method were performed, according to CLSI standards, with nalidixic acid, flumequine, oxolinic acid, ciprofloxacin, enrofloxacin, marbofloxacin, norfloxacin, ofloxacin, and levofloxacin. The MIC of levofloxacin was determined by an Etest. The results showed a trimodal distribution of the MICs for both E. coli and Salmonella. The MIC distributions for the isolates varied with the compounds tested. Screening for nalidixic acid resistance by MIC testing or disk diffusion assay was not efficient for the detection of some of the isolates carrying qnr and aac(6′)Ib-cr. Transferable resistance genes would best be detected by testing for the MIC of ciprofloxacin or norfloxacin, as testing for the MICs of the other compounds would fail to detect isolates carrying aac(6′)Ib-cr because the enzyme produced is able to reduce the activities of these two compounds only due to their chemical structures. In conclusion, screening with nalidixic acid is efficient for the detection of mutants, but it is not so efficient for the detection of qnr and aac(6′)Ib-cr. Detection would be maximized by screening with either ciprofloxacin or norfloxacin by both MIC determination and disk diffusion assays. Furthermore, a low concentration of ciprofloxacin (1 μg) in the disks seemed to increase the sensitivity of the disk diffusion assay.
In a previous study, four Salmonella isolates from humans in the Henan province of China showed reduced susceptibility to ciprofloxacin (MIC, 0.125 to 0.25 μg/ml) but were susceptible to nalidixic acid (MIC, 4 to 8 μg/ml). All isolates were negative for known qnr genes (A, B, and S), aac(6′)Ib-cr, and mutations in gyrA and parC. Plasmid DNA was extracted from all four isolates and transformed into Escherichia coli TG1 and DH10B cells by electroporation, and transformants were selected on 0.06 μg/ml ciprofloxacin containing brain heart infusion agar plates. Resistance to ciprofloxacin could be transferred by electroporation, and a similar 4,270-bp plasmid was found in all transformants. By sequence analysis, the plasmid was found to carry an open reading frame that had similarities to other qnr genes and that encoded a 214-amino-acid pentapeptide repeat protein. This gene, designated qnrD, showed 48% similarity to qnrA1, 61% similarity to qnrB1, and 41% similarity to qnrS1. Further subcloning of the qnrD coding region into the constitutively expressed tetA gene of vector pBR322 showed that the gene conferred an increase in the MIC of ciprofloxacin by a factor of 32 (from an MIC of 0.002 to an MIC of 0.06 μg/ml). For comparison, qnrA1 and qnrS1 were also subcloned into pBR322 and transformed into DH10B cells, conferring MICs of 0.125 and 0.5 μg/ml, respectively. A phylogenetic analysis of all known qnr sequences was performed and showed that qnrD was more closely related to the qnrB variants but formed an independent cluster. To our knowledge, this is the first description of this qnrD gene.
Three mechanisms for plasmid-mediated quinolone resistance (PMQR) have been discovered since 1998. Plasmid genes qnrA, qnrB, qnrC, qnrD, qnrS, and qnrVC code for proteins of the pentapeptide repeat family that protects DNA gyrase and topoisomerase IV from quinolone inhibition. The qnr genes appear to have been acquired from chromosomal genes in aquatic bacteria, are usually associated with mobilizing or transposable elements on plasmids, and are often incorporated into sul1-type integrons. The second plasmid-mediated mechanism involves acetylation of quinolones with an appropriate amino nitrogen target by a variant of the common aminoglycoside acetyltransferase AAC(6′)-Ib. The third mechanism is enhanced efflux produced by plasmid genes for pumps QepAB and OqxAB. PMQR has been found in clinical and environmental isolates around the world and appears to be spreading. The plasmid-mediated mechanisms provide only low-level resistance that by itself does not exceed the clinical breakpoint for susceptibility but nonetheless facilitates selection of higher-level resistance and makes infection by pathogens containing PMQR harder to treat.
Antimicrobial resistance is under-documented and commensal Escherichia coli can be used as indicator organisms to study the resistance in the community. We sought to determine the prevalence of resistance to broad-spectrum antimicrobials with particular focus on the quinolones, which have recently been introduced in parts of Africa, including Ghana.
Forty (13.7%) of 293 E. coli isolates evaluated were nalidixic acid-resistant. Thirteen (52%) of 2006 and 2007 isolates and 10 (66.7%) of 2008 isolates were also resistant to ciprofloxacin. All but one of the quinolone-resistant isolates were resistant to three or more other antimicrobial classes. Sequencing the quinolone-resistance determining regions of gyrA and parC, which encode quinolone targets, revealed that 28 quinolone-resistant E. coli harboured a substitution at position 83 of the gyrA gene product and 20 of these isolates had other gyrA and/or parC substitutions. Horizontally-acquired quinolone-resistance genes qnrB1, qnrB2, qnrS1 or qepA were detected in 12 of the isolates. In spite of considerable overall diversity among E. coli from Ghana, as evaluated by multilocus sequence typing, 15 quinolone-resistant E. coli belonged to sequence type complex 10. Five of these isolates carried qnrS1 alleles.
Quinolone-resistant E. coli are commonly present in the faecal flora of Accra residents. The isolates have evolved resistance through multiple mechanisms and belong to very few lineages, suggesting clonal expansion. Containment strategies to limit the spread of quinolone-resistant E. coli need to be deployed to conserve quinolone effectiveness and promote alternatives to their use.
Quinolone resistance is an emerging problem in China. To investigate the prevalence of the plasmid-mediated quinolone resistance genes qnr and aac(6′)-Ib-cr, a total of 265 clinical isolates of Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, and Enterobacter cloacae with ciprofloxacin MICs of ≥0.25 μg/ml were screened at nine teaching hospitals in China. The qnrA, qnrB, qnrS, and aac(6′)-Ib genes were detected by PCR. The aac(6′)-Ib-cr gene was further identified by digestion with BtsCI and/or direct sequencing. The qnr gene was present in significantly smaller numbers of isolates with cefotaxime MICs of <2 μg/ml than isolates with higher MICs (≥2.0 μg/ml) (20.6% and 42.1%, respectively; P < 0.05). aac(6′)-Ib-cr was present in 17.0% of the isolates tested, and 7.9% of the isolates carried both the qnr and the aac(6′)-Ib-cr genes. Among the isolates with cefotaxime MICs of ≥2.0 μg/ml, qnr and aac(6′)-Ib-cr were present in 65.7% and 8.6% of E. cloacae isolates, respectively; 65.5% and 21.8% of K. pneumoniae isolates, respectively; 63.3% and 26.7% of C. freundii isolates, respectively; and 6.5% and 16.9% of E. coli isolates, respectively. The 20 transconjugants showed 16- to 128-fold increases in ciprofloxacin MICs, 14 showed 16- to 2,000-fold increases in cefotaxime MICs, and 5 showed 8- to 32-fold increases in cefoxitin MICs relative to those of the recipient due to the cotransmission of blaCTX-M-14, blaCTX-M-3, blaDHA-1, blaSHV-2, and blaSHV-12 with the qnr and aac(6′)-Ib-cr genes. Southern hybridization analysis showed that these genes were located on large plasmids of different sizes (53 to 193 kb). These findings indicate the high prevalence of qnr and aac(6′)-Ib-cr in members of the family Enterobacteriaceae and the widespread dissemination of multidrug resistance in China.
Fluoroquinolone resistance can cause major clinical problems. Here, we investigated fluoroquinolone resistance mechanisms in a clinical Escherichia coli isolate, HUE1, which had no mutations quinolone resistance-determining regions (QRDRs) of DNA gyrase and topoisomerase IV. HUE1 demonstrated MICs that exceeded the breakpoints for ciprofloxacin, levofloxacin, and norfloxacin. HUE1 harbored oqxAB and qnrS1 on distinct plasmids. In addition, it exhibited lower intracellular ciprofloxacin concentrations and higher mRNA expression levels of efflux pumps and their global activators than did reference strains. The genes encoding AcrR (local AcrAB repressor) and MarR (MarA repressor) were disrupted by insertion of the transposon IS3-IS629 and a frameshift mutation, respectively. A series of mutants derived from HUE1 were obtained by plasmid curing and gene knockout using homologous recombination. Compared to the MICs of the parent strain HUE1, the fluoroquinolone MICs of these mutants indicated that qnrS1, oqxAB, acrAB, acrF, acrD, mdtK, mdfA, and tolC contributed to the reduced susceptibility to fluoroquinolone in HUE1. Therefore, fluoroquinolone resistance in HUE1 is caused by concomitant acquisition of QnrS1 and OqxAB and overexpression of AcrAB–TolC and other chromosome-encoded efflux pumps. Thus, we have demonstrated that QRDR mutations are not absolutely necessary for acquiring fluoroquinolone resistance in E. coli.
AcrAB; efflux pump; Escherichia coli; fluoroquinolone resistance; oqxAB; qnrS
We studied a collection of 105 clinical enterobacteria with unusual phenotypes of quinolone susceptibility to analyze the occurrence of plasmid-mediated quinolone resistance (PMQR) and oqx genes and their implications for quinolone susceptibility. The oqxA and oqxB genes were found in 31/34 (91%) Klebsiella pneumoniae and 1/3 Klebsiella oxytoca isolates. However, the oqxA- and oqxB-harboring isolates lacking other known quinolone resistance determinants showed wide ranges of susceptibility to nalidixic acid and ciprofloxacin. Sixty of the 105 isolates (57%) harbored at least one PMQR gene [qnrB19, qnrB10, qnrB2, qnrB1, qnrS1, or aac(6′)-Ib-cr)], belong to 8 enterobacterial species, and were disseminated throughout the country, and most of them were categorized as susceptible by the current clinical quinolone susceptibility breakpoints. We developed a disk diffusion-based method to improve the phenotypic detection of aac(6′)-Ib-cr. The most common PMQR genes in our collection [qnrB19, qnrB10, and aac(6′)-Ib-cr] were differentially distributed among enterobacterial species, and two different epidemiological settings were evident. First, the species associated with community-acquired infections (Salmonella spp. and Escherichia coli) mainly harbored qnrB19 (a unique PMQR gene) located in small ColE1-type plasmids that might constitute its natural reservoirs. qnrB19 was not associated with an extended-spectrum β-lactamase phenotype. Second, the species associated with hospital-acquired infections (Enterobacter spp., Klebsiella spp., and Serratia marcescens) mainly harbored qnrB10 in ISCR1-containing class 1 integrons that may also have aac(6′)-Ib-cr as a cassette within the variable region. These two PMQR genes were strongly associated with an extended-spectrum β-lactamase phenotype. Therefore, this differential distribution of PMQR genes is strongly influenced by their linkage or lack of linkage to integrons.
We investigated the prevalence of plasmid-mediated quinolone resistance and its association with extended-spectrum beta-lactamase (ESBL) and AmpC beta-lactamase in Enterobacteriaceae.
A total of 347 non-duplicated isolates of Enterobacteriaceae were collected between August and October 2006 from 2 hospitals. Qnr determinant screening was conducted using PCR amplification, and all positive results were confirmed by direct sequencing. Qnr-positive strains were determined on the basis of the presence of ESBL and AmpC beta-lactamase genes.
The qnr gene was detected in 47 of 347 clinical Enterobacteriaceae isolates. Among the 47 qnr-positive strains, Klebsiella pneumoniae (N=29) was the most common, followed by Escherichia coli (N=6), Enterobacter cloacae (N=6), Citrobacter freundii (N=5), and Enterobacter aerogenes (N=1). These isolates were identified as qnrA1 (N=6), 8 qnrB subtypes (N=40), and qnrS1 (N=1). At least 1 ESBL was detected in 38 of the 47 qnr-positive strains. Qnr-positive strains also showed high positive rates of ESBL or AmpC beta-lactamase, such as TEM, SHV, CTX-M, and DHA. DHA-1 was detected in 23 of 47 qnr-positive strains, and this was co-produced with 1 qnrA1 and 22 qnrB4. Strains harboring MIR-1T and CMY were also detected among the qnr-positive strains. Antimicrobial-resistance rates of qnr-positive strains to ciprofloxacin, levofloxacin, norfloxacin, nalidixic acid, and moxifloxacin were 51.1%, 46.8%, 46.8%, 74.5%, and 53.2%, respectively.
The qnr genes were highly prevalent in Enterobacteriaceae, primarily the qnrB subtypes. They were closely associated with EBSL and AmpC beta-lactamase.
Qnr; Beta-lactamase; Quinolone; Enterobacteriaceae
Cross-resistance to quinolones and beta-lactams is frequent in Enterobacteriaceae, due to the wide use of these antibiotics clinically and in the food industry. Prescription of one of these categories of antibiotic may consequently select for bacteria resistant to both categories. Genetic mechanisms of resistance may be secondary to a chromosomal mutation located in quinolone resistance determining region of DNA gyrase or topoisomerase IV or to a plasmid acquisition. The insertion sequence ISCR1 is often associated with qnr and may favour its dissemination in Gram-negative bacteria. The aim of this study was to determine the genetic mechanism of quinolone resistance among extended-spectrum beta-lactamase-producing Enterobacteriaceae strains in the Central African Republic.
Among seventeen ESBL-producing Enterobacteriaceae isolated from urine, pus or stool between January 2003 and October 2005 in the Central African Republic, nine were resistant to ciprofloxacin (seven from community patients and two from hospitalized patients). The ESBL were previously characterized as CTX-M-15 and SHV-12. Susceptibility to nalidixic acid, norfloxacin and ciprofloxacin, and the minimal inhibitory concentrations of these drugs were determined by disc diffusion and agar dilution methods, respectively. The presence of plasmid-borne ISCR1-qnrA region was determined by PCR and amplicons, if any, were sent for sequencing. Quinolone resistance determining region of DNA gyrase gyrA gene was amplified by PCR and then sequenced for mutation characterization. We found that all CTX-M-producing strains were resistant to the tested quinolones. All the isolates had the same nucleotide mutation at codon 83 of gyrA. Two Escherichia coli strains with the highest MICs were shown to harbour an ISCR1-qnrA1 sequence. This genetic association might favour dissemination of resistance to quinolone and perhaps other antibiotics among Enterobacteriaceae.
This study shows that at least two mechanisms might explain the emerging resistance of Enterobacteriaceae to quinolones in the CAR. Beside the classical topoisomerase mutation, the cause may be acquisition of a plasmid-borne qnrA1. Clinicians and bacteriologists should be made aware of possible dissemination of ISCR1-qnrA1 among Enterobacteriacae.
Quinolones; Betalactams; Antibiotic resistance; Enterobacteriaceae