Detection of plasmid-mediated quinolone resistance determinants and qnrS expression in Enterobacteriaceae clinical isolates

Introduction: Rapid dissemination of plasmid-mediated quinolone resistance (PMQR) has been reported in clinical isolates. Methodology: A total of 149 clinical isolates of Enterobacteriaceae were collected in Beijing and screened for PMQR genes usingpolymerase chain reaction (PCR). Real-time quantitative PCR was used to study the expression of qnrS. Results: The rates of qnr and aac(6’)-Ib-cr genes were 7.4% and 8.1%, respectively. The higher basal expression of qnrS was observed in transconjugant strains, which had higher minimum inhibitory concentrations (MICs) of quinolones. Furthermore, qnrS expression levels increased in all three isolates when a quinolone was present. Conclusions: Our data suggest that the level of qnrS expression was associated with quinolone resistance.


Introduction
Resistance to quinolones has increased globally in recent years, particularly in members of theEnterobacteriaceae [1][2].Two main resistance mechanisms to quinolones are recognized: mutations in the quinoloneresistance-determining regions (QRDRs) of the chromosomal gyrA and parC genes, and decreased accumulation inside the bacteria due to impermeability of the membrane and/or an overexpression of efflux pump systems [3,4].However, plasmid-mediated quinolone resistance (PMQR) has been reported since 1998 [5].The plasmid-borne qnr genes currently comprise five families:qnrA, qnrB, qnrC, qnrD, and qnrS, which encode Qnr proteins that protect the DNA gyrase from the activity of quinolones [6].Additionally, the aac(6')-Ib-cr gene encodes a variant aminoglycoside acetyltransferase that can modify quinolones with a piperazinyl substituent [7].Quinolone induction of qnr genes independent of the SOS system has been observed in previous studies [8][9].Although PMQR determinants confer low-level resistance to quinolones, they are a favorable background for selection of additional chromosome-encoded quinolone resistance mechanisms, which makes the clinical therapy more difficult.In this study, we investigated the presence of PMQR genes in clinical isolates of Enterobacteriaceae, identified mutations within the QRDRs, and examined the expression of qnrS in transconjugants obtained from different donor isolates.

Methodology
One hundred and forty-nineclinical isolates were obtained from two hospitals in Beijing, China, between March and July 2011.These included 86 strains of E. coli, 35 strains of P. mirabilis, and 28 strains of K. pneumoniae.All isolates were screened for the qnr (qnrA, qnrB, qnrC, qnrD, and qnrS) and aac(6')-Ib genes by polymerase chain reaction (PCR) amplification using the primers described previously [10][11][12][13].Mutations in the gyrA and parC genes were identified by DNA sequencing of their PCR products [14].The transfer of quinolone resistance was studied by performing conjugation experiments with Escherichia coli J53Az r (resistant to sodium azide) as the recipient, as described previously [10].Transconjugants wereselected on trypticase soy agar (TSA; Land Bridge Technology Co. Ltd., Beijing, China) plates containing sodium azide (150 µg mL -1 ; Sigma-Aldrich, St. Louis, USA) and ciprofloxacin (0.25 µgmL -1 ; Sigma-Aldrich).Minimum inhibitory concentrations (MICs) for the donor, recipient, and transconjugant strains were measured by agar dilution, according to Clinical and Laboratory Standards Institute (CLSI) guidelines.
Total RNA was extracted from 1.5 mL of exponentially growing cellsusing TRIzol (Invitrogen, Carlsbad, USA),according to the manufacturer'sinstructions.Real-time quantitative PCR (qPCR) was used to study gene expression of qnrS.Using the Reverse Transcription System (Promega, Madison, USA), 200 ng of total RNA were reverse transcribed in a final volume of 20 µL.Primers for qnrS were 5'-CTCTTCCAAGTGTAGGGAGATAT-3' and 5'-ATTGGGATAGATTTCGCTTATTG-3'.Using primers as described previously [15], 16S rRNA was used as the reference gene.PCR reactions were performed using 1 µL of cDNA with RealMasterMix (Tiangen Biotech Co. Ltd., Beijing, China).
Quinolone resistance could be transferred by conjugation from nine of the eleven qnr-positive donors, and one of the five donors carried the aac(6')-Ib-cr gene alone.PCR experiments confirmed that the transconjugants harbored the same PMQR determinants as their donors, and that the qnr and aac(6')-Ib-cr genes could be cotransferred from different donors (Table 2).The 10 transconjugants showed 32-to 128-fold increases in the MICs of ciprofloxacin and 16-to 32-fold increases in the MICs of levofloxacin, relative to those of the recipient.All transconjugants showed 4-to 128-fold increases in the MICs of ceftazidime, and three showed more than a 256-fold increase in the MICs of gentamicin.Thus, ciprofloxacin resistance was cotransferred with resistance to other antimicrobial agents, such as cephalosporins, aminoglycosides, and sulfamethoxazole.
DNA sequencing of the PCR products covering the entire QRDRs of gyrA demonstrated the presence of mutations at codon 83 in the 10clinical isolates examined (Table 2).Point mutations in gyrA were found in E. coliand K. pneumoniae, and the resulting amino acid substitutions were Ser83Leu and Ser83Ile, respectively.Four E. coli isolates had an additional mutation, Asp87Asn.In parC, a Ser80Ile substitution was found in seven of ten clinical isolates.There were no mutations in the target genes among the transconjugants and the recipient.
There were fourfold and twofold differences in the MICs for ciprofloxacinand levofloxacin, respectively, among the three transconjugants that only harbored qnrS.The basal expression level of qnrS in H483T was 4.9-fold, much higher than in the transconjugants H707T and K337 (Table 3).The effect of ciprofloxacinand levofloxacin on qnrS transcription was investigated, and maximal transcription was observed at 0.4MIC with both ciprofloxacinand levofloxacin (data not shown).The qnrS transcript levels increased in all three transconjugants when ciprofloxacin was present, with 1.9-fold, 2.7-fold, and 5.4-fold increases for H707T, K337T, and H483T, respectively (Table 3).The increases over baseline observed when levofloxacin was added to the medium were 1.7-fold, 3.0-fold, and 5.1-fold for the three transconjugants, respectively (Table 3).Gene expression was measured in the presence of ciprofloxacin and levofloxacin relative to the drug-free condition.The results are expressed in terms of the mean ± standard deviation.The value obtained from non-quinolone-exposed cells of strain H707T was considered as 1.

Discussion
The results of our study demonstrated a proportion of 7.4% for the PMQR genes among clinical isolates of Enterobacteriaceae.The rate of aac(6')-Ib-cr carriage among qnr-positive isolates was much higher than that among qnr-negative isolates, which was agreement with previous studies [11,16].Conjugation experiments proved that the plasmid-mediated quinolone resistance was transferable.However, six other qnrand/or aac(6')-Ib-cr-bearing isolates failed to produce transconjugants.The result showed that qnr and aac(6')-Ib-cr genescould be cotransferred from different donors.The enzyme encoded by the aac(6')-Ib-cr gene could inactivate quinolones (ciprofloxacin and norfloxacin) by N-acetylation of the amino nitrogen on its piperazinyl group [17].Transconjugants carrying aac(6')-Ib-cr aloneshowed a 32-fold increase in ciprofloxacin with that of the recipient, suggesting that aac(6')-Ib-critself causes low-level ciprofloxacin resistance, as reported by other studies [14,16,17].Furthermore, another study reported that qnr and aac(6')-Ib-crcould act additively to generate the ciprofloxacin resistance [17].It was reported that the qnr and aac(6')-Ib-cr genes were always co-resident on the same plasmids and cotransferred to the recipient, which may have contributed to the rapid increase in resistance to quinolones among bacteria [14].Resistance to ciprofloxacinand levofloxacinwas lower in the transconjugants than in their donors.The DNA sequencing results indicated that chromosomal QRDR mutations in GyrA and ParC played an important role in mediating high-level quinolone resistance among the donor isolates.
Xu et al. reported that the differences in qnrA expression levels may account for the variations in the ciprofloxacin MICs of different transconjugants [15].In our study, qnrS was found most frequently among the isolates examined, and the expression of qnrS was investigated to understand the relationship between qnrS expression level and MICs of ciprofloxacin.Our results showed that the basal expression level of qnrS in H483T was much higher than in H707T and K337.The higher basal expression of qnrS in H483T may explain, at least in part, the higher MICs of quinolones for this transconjugant.The qnrS transcript levels increased in all three isolates when ciprofloxacin or levofloxacin was present, suggesting that the qnrSexpression was induced by quinolones, which is consistent with previous reports [9,18].It can therefore be proposed that the level of qnrS expression was associated with the quinolone resistance in the transconjugants analyzed.A higher level of qnrS transcript could provide the cell with a rapid response to the damaging effects of antimicrobial agents that inhibit DNA replication by protecting the topoisomerases;this wouldpromote bacterial survival [19].

Conclusions
In summary, PMQR genes are implicated in lowlevel quinolone resistance, and may play an important role in the generation of resistant mutants and the rapid increase in bacterial resistance.The level of qnrS expression appeared to contribute to quinolone resistance in the transconjugants analyzed.

Table 3 .
Relative expression data for qnrS in transconjugants