Plasmid-Mediated Quinolone Resistance Genes in Escherichia coli Urinary Isolates from Two Teaching Hospitals in Turkey : Coexistence of TEM , SHV , CTX-M and VEB-1 Type β-lactamases

Purpose: To evaluate the occurrence of plasmid-mediated quinolone resistance (PMQR) genes and the prevalence of extended spectrum β-lactamase (ESBL) types in Escherichia coli clinical isolates. Methods: Sixty-one ESBL-producing urinary E. coli isolates were studied. An antibiotic susceptibility test was performed using the disc diffusion method. ESBL production was determined using a double-disc synergy test for all isolates; E-test and Vitek 2 were used for plasmid-mediated quinolone resistance (PMQR)-positive isolates and their transconjugants. The presence of PMQR and β-lactamase genes was determined by polymerase chain reaction (PCR). Results: The strains displayed high rates of resistance to norfloxacin (80 %). The most frequent PMQR gene was aac(6’)-Ib-cr (45.9 %). In all, one qnrA1 (1.6 %), one qnrS1 (1.6 %), and two qepA1-positive isolates (5.7 %) were identified. The genes, qnrS1+aac(6’)-Ib-cr and qepA1, were co-expressed with blaCTX-M-15 gene, while qnrA1 occurred with blaTEM-1, blaSHV, and blaVEB-1 genes. The most frequent β-lactamase type was cefotaximase (CTX-M), which generally hydrolyzes cefotaxime (92 %) more than it does ceftazidime; followed by temoneira (TEM, 39 %); sulfhydryl variable (SHV, 5 %), and Vietnamese extended-spectrum beta– lactamase (VEB, 1.6 %). Conclusion: A high prevalence of aac(6’)-Ib-cr and CTX-M type β-lactamase was detected in ESBLproducing E. coli strains. This study also identified the co-expression of qnrA1 and blaVEB-1 genes and of qnrS1+aac(6’)-Ib-cr in E. coli isolates. The co-existence of PMQR genes with ESBLs may lead to a serious public health problem.


INTRODUCTION
Although bacterial resistance to quinolones is usually due to chromosomally encoded mechanisms, it can also originate from plasmid-mediated genes.
After the identification of qnr determinants, which protect target enzymes against quinolone inhibition, two other mechanisms have now been described: the qepA gene encodes an efflux pump, which confers reduced susceptibility on hydrophobic fluoroquinolones such as norfloxacin and ciprofloxacin; and the aac(6')-Ib-cr gene, which encodes modified aminoglycosideacetylating enzymes and can inactivate both aminoglycosides and fluoroquinolones [1][2][3].
Over the last decade, plasmid-mediated quinolone resistance (PMQR), particularly among the various species of Enterobacteriacae, has been increasingly reported from many regions of the world.Plasmids carrying genes may contribute to the development of higher levels of fluoroquinolone resistance and may pose a threat by allowing the rapid spread of resistance among organisms.Although these PMQR genes have been associated with low levels of quinolone resistance, it could cause high-level quinolone resistance by facilitating the selection of chromosomal mutations.Several studies have demonstrated that most qnr-positive enterobacterial isolates are associated with extended spectrum βlactamases (ESBLs), including TEM, SHV, VEB, and CTX-M types, which are generally located on plasmids that are highly transferable and may harbor resistance genes to several different groups of antibiotics [4].Today, many antibiotics, such as β-lactams and fluoroquinolones, which are widely prescribed by clinicians for the treatment of urinary tract E. coli infection, are in limited use.
The aim of this study was to determine the prevalence of PMQR genes and ESBL types in clinical urinary isolates of E. coli collected from two large teaching hospitals located in the European and Asian parts of Istanbul in Turkey.

Antimicrobial susceptibility and synergy testing
Individual strains were tested based on the recommendations of the Clinical and Laboratory Standards Institute (CLSI), using the Kirby-Bauer disc diffusion method for susceptibility [12].The double disc synergy test with cefotaxime and ceftazidime was used for screening ESBL production.

Enterobacterial repetitive consensus PCR (ERIC-PCR)
The Enterobacterial Repetitive Intergenic Concensus (ERIC)-PCR with ERIC1 and ERIC2 primers was used to analyze the epidemiological relationship between PMQRpositive E. coli isolates.Cycling conditions were as follows: 5 min at 94 °C; 40 cycles of 1 min at 94 °C, 1 min at 36 °C, 2 min at 72 °C; and final extension of 10 min at 72 °C.The PCR products were separated by electrophoresis in 1.5 % agarose gel and visualized on a UV transilluminator, and fingerprints were compared [13].

Transferability of PMQR genes and plasmid analysis
Conjugation experiments with an azideresistant E. coli J53 (AzR) as the recipient were performed in liquid culture media, as described previously [14].Transconjugants were selected on trypticase soy agar plates containing sodium azide (100 µg/ml) for counter selection and amoxicillin (100 µg/ml), cefotaxime (8 µg/ml), ceftazidime (8 µg/ml), nalidixic acid (16 µg/ml).The High Pure Plasmid Isolation Plasmid DNA Kit (Roche, Mannheim, Germany) was used for the extraction of plasmid DNA.E. coli V517 cells harboring plasmids of 54.4, 7.1, 5.6, 5.2, 3.0, 2.7, and 2.1 kb were used as the size marker for the plasmids.The presence of transferred PMQR genes and related ESBLs were confirmed by PCR.

Characterization of ESBL and PMQR genes and sequencing
DNA extraction was performed, as described previously.Briefly, bacterial colonies were suspended in 2 ml centrifuge tubes and then centrifuged at 12,000 g.The pellets were washed in 750 µl TE buffer (10 mM Tris HCl, pH 8.0, 1 mM EDTA) and then boiled for 10 min in 500 µl TE buffer and centrifuged.The supernatants were stored at -20 °C prior to subsequent DNA amplification [9].
The bla TEM, bla SHV, bla CTX-M, and bla VEB genes were investigated by PCR, as previously described [15,16].A multiplex PCR was performed to detect qnrA, qnrB, and qnrS, as previously described by Cattoir et al [17].PCR amplification of qnrC, qnrD, qepA, and aac (6')-Ib was carried out with specific primers and conditions [2,3,18].The DNA for control for each specific gene region was included with each group of tested strains.After PCR amplifications, the products of aac(6')-Ib positives were further analysed by digestion with BtsCI for detection of the -cr variant (New England Biolabs, Ipswich, MA, USA).
The amplification products of PMQR and related β-lactamases were sequenced with an Applied Biosystems sequencer (ABI PRISM 310 Genetic Analyzer; Applied Biosystems, Foster City, CA, USA).The nucleotide and amino acid sequences were analyzed and compared by BLAST search (www.ncbi.nlm.nih.gov).

Statistical analysis
Statistical analysis was performed using SPSS for Windows, version 11.5 (SPSS, Inc, Chicago, IL, USA).Rates of resistance were compared using the Chi-square test.A pvalue of < 0.05 was considered to be statistically significant.

Antibiotic susceptibilities and the prevalence of PMQR determinants/βlactamase genes
Table 1 shows the prevalence and susceptibility data.The strains displayed highest resistance to norfloxacin (80 %).The most active antibiotics were imipenem (100 %), fosfomycin (100 %) and nitrofurantoin (93.4 %).The rate of resistance to amoxicillin-clavulanic acid for the isolates of IMF was higher than that of the isolates of GMMA (p = 0.022); the opposite was found for the resistance to norfloxacin (p = 0.001).Co-resistance was identified in 96 % of the strains.The highest co-resistance was determined for norfloxacin and the most common two co-resistance phenotypes were amoxicillin-clavulanic acid/norfloxacin/cotrimoxazole (24.5 %) and amoxicillinclavulanic acid/gentamicin/norfloxacin/cotrimoxazole (15 %).
The prevalence of PMQR genes for aac(6')-Ib-cr, qepA, qnrA, and qnrS were 45.9, 5.7, 1.6, and 1.6%, respectively.qnrA1 and qepA1 were detected alone in strains, but qnrS1 was co-expressed with aac(6')-Ib-cr.All PMQR-positive isolates were resistant to norfloxacin, except the qnrA1positive strain.In addition, norfloxacin resistance in aac(6')-Ib-cr-positive isolates (all were resistant) was significantly higher than in the aac(6')-Ib-crnegative ones (p = 0.001).No isolates carrying the qnrB, qnrC, or qnrD genes were detected in this study (Table 1).The most prevalent ESBL type was CTX-M (92 %) (mostly CTX-M group 1 (66 %)), followed by TEM and SHV.Only one isolate harbored the VEB-1 type β-lactamase.Ciprofloxacinresistant E. coli 4 and E. coli 6 were isolated from patients with nephrolithiasis, who were operated in the same division of GMMA during the same period, and these were qepA1-positive.E. coli 4 was isolated 36 days after the operation and the patient was treated with fosfomycin.E. coli 6 was isolated from a patient who was admitted with high fever 11 days after the operation and treated with a imipenem-gentamicin combination.Ciprofloxacin-resistant E. coli34 (from a kidney transplant patient treated with piperacillin-tazobactam) and ciprofloxacinsusceptible E. coli 210 (from a patient who was born with premature rupture of membranes treated with an ampicillingentamicin combination) were isolated in different divisions of ITF.E. coli 24 harbored both qnrS1 and aac(6')-Ib-cr, while qnrA1 was detected in E. coli 210.

RAPD-PCR typing
E. coli 4 and E. coli 6 have similar antibiotic patterns; i.e., they are resistant to amoxicillinclavulanic acid, cefriaxone, amikacin, gentamicin, norfloxacin, levofloxacin, and cotrimoxazole.RAPD-PCR typing was carried out on the four PMQR-positive isolates.The results showed that the qepA1-positive isolates were clonally related (data not shown).

Characteristics of PMQR-positive isolates, transconjugants, and plasmid analysis
PCR assays were used to detect βlactamase, and identified the bla CTX-M-15 gene in E. coli 4, E. coli 6, and E. coli 34, while bla TEM-1 , bla SHV , and bla VEB-1 genes were detected in E. coli 210.Despite three separate attempts, conjugative assays failed with the E. coli 4 and E. coli 6 isolates.However, plasmid analysis demonstrated that both strains harbored multiple plasmids changing 1-7 kb (Figure 1).The MICs of ciprofloxacin and other antibiotics for E. coli 34 and E. coli 210 parenteral isolates and their transconjugants are presented in Table 2.The MICs for ciprofloxacin were increased 63 and 8 times in the transconjugants of E. coli 34 and E. coli 210, respectively.

DISCUSSION
It is more difficult to treat ESBL-producing E. coli because most β-lactams are no longer therapeutic options.In particular, CTX-M type enzymes have emerged worldwide and have rapidly increased in E. coli isolated from both community and nosocomial settings [19].The associated co-resistance of ESBL producers to different groups of antimicrobials, such as quinolones, sulfonamides, and aminoglycosides, is another issue of concern.Limited data have been reported on the epidemiology of E. coli that produce CTX-M type enzymes in Turkey.Two recent reports from Turkey have shown that the CTX-M enzyme is common among ESBL positive isolates (86.8 %) at our hospital (IMF) in Istanbul [20] and from patients with urinary tract infections (76.5%) [21].The latter finding of the predominance of CTX-M type enzymes according to TEM and SHV types is reflected in our study, as well.These reports suggest that CTX-M type enzymes are more prevalent than other ESBLs in Turkey.Consistently, a high prevalence of CTX-M type enzymes has been reported in several studies from other countries.In addition, the present study demonstrates that the prevalence of VEB type β-lactamase is low (1.6 %).
Over the past 10 years, PMQR has emerged as an important issue.Different rates of PMQR have been reported depending on the country of origin of the isolates [22].E. coli carrying qnrA, qnrB, qnrS, and aac(6')-Ib-cr MICs (µg/ml) against PMQR-positive isolates and their transconjugants  [16].In addition, a qnrA-positive-Citrobacter freundii isolate that produces bla VEB-1 and bla OXA-48 has been reported in Turkey [8].Here, in addition to the TEM and SHV type, a VEB type β-lactamase was detected, but distinct from that of a qnrApositive E. coli isolate from the same hospital.This finding showed that VEB-1 type βlactamase persists in microorganisms in Turkey.The present study also demonstrated the co-expression of the PMQR genes, similar to previous reports from France, UK, China, and Turkey [11,[24][25][26].

Antibiotics
The strain, qepA, was first identified in 2007 in two E. coli clinical isolates from Japan and Belgium [27], while a new variant (qepA2) has already been detected in France [28].However, recently, a qepA producing E. coli strain possessing qnrB2 and aac(6')-Ib-cr gene has been reported in Turkey [11].In the present study, in addition to qnrA1, qnrS1 and aac(6')-Ib-cr, qepA1-positive E. coli isolates that produce CTX-M-15 type βlactamase were indentified.No qepA was found in the isolates screened in ITF, in contrast to other hospitals located in the Asian part of Istanbul.Although these PMQR mechanisms are rare, the association of qepA1 with multi-drug resistant CTX-M-15 producing E. coli can be a cause for concern.There may be a rapid spread of E. coli, especially in hospital settings where various antimicrobials are largely used and thus may support the dissemination of these microorganisms.

CONCLUSION
This study documents the high prevalence aac(6')-Ib-cr and CTX-M type enzymes in Turkey.In addition to aac(6')-Ib-cr and CTX-M type enzymes, the qepA1 and VEB-1 type enzymes are alarming for Turkey.Our study confirms that CTX-M producing E. coli isolates from urine specimens are highly resistant/co-resistant to norfloxacin, cotrimoxazole, amoxicillin-clavulanic acid, and gentamicin.Empiric therapy with these antibiotics may not be adequately effective.However, fosfomycin and nitrofurantoin resistance rates seem low and they may be alternatives for therapy.The emergence of the combination of PMQR and ESBL compromise the usage of valuable antibiotics worldwide.Antibiotic resistance is a public health problem, which requires continuous surveillance, monitoring, and revision of the policy of antibiotic use.

Fig 1 :Table 2 :
Fig 1: Plasmid DNAs from E. coli 4 and E. coli 6. Line M, E. coli V 517 (used as standard for plasmid size)