|Year : 2012 | Volume
| Issue : 4 | Page : 442-447
Plasmid mediated quinolone resistance determinants qnr, aac(6′)-Ib-cr, and qep in ESBL-producing Escherichia coli clinical isolates from Egypt
WM Hassan1, A Hashim2, RAA Domany1
1 Department of Microbiology and Immunology, Faculty of Pharmacy, Helwan University, Egypt
2 Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Egypt
|Date of Submission||24-Apr-2012|
|Date of Acceptance||06-May-2012|
|Date of Web Publication||24-Nov-2012|
Department of Microbiology and Immunology, Faculty of Pharmacy, Helwan University
Purpose: To characterize the prevalence of plasmid-mediated quinolone resistance determinants qnr, aac(6′)-Ib-cr and qep in extended-spectrum β-lactamase (ESBL) -producing E. coli and to determine the association of these determinants with CTX-M group in Cairo, Egypt. Materials and Methods: MICs of 15 antimicrobial agents against 70 E. coli clinical isolates were determined using agar dilution technique according to CLSI. Screening for the qnrA, qnrB, qnrS, aac(6′)-Ib, qep and CTX-M genes was carried out by PCR amplification and DNA sequencing. Curing was used to confirm whether qnr, aac(6′)-Ib, qep or ESBL-encoding genes were located on plasmids. Results: Out of 70 E. coli clinical isolates, 61 were resistant to at least one antibiotic, 16 (22.8%) were multidrug resistant and 30 (42%) were ESBL producers. Out of 30 ESBL producers E. coli isolates, 8 (26.6%) were positive for qnr genes, and the qnrA1-, qnrB1-and qnrS1-type genes were detected alone or in combination in 5 (16.6%), 7 (23.3%) and 5 (16.6%) isolates, respectively. Seven (23.3%) isolates were positive for aac(6′)-Ib-cr and only two (6.6%) isolates were positive for qepA4. Loss of all plasmids upon curing suggested that qnr, aac(6′)-Ib-cr , qep A4 and ESBL-encoding genes were always plasmid mediated. Out of 8 Qnr positive isolates 5 were associated with both CTX-M-1 and CTX-M-9 while 2 from 6 aac(6′)-Ib-cr positive isolates were associated with both CTX-M-1 and CTX-M-9. Conclusions: This study highlights the prevalence of quinolone resistance determinants qnr, aac(6′)-Ib-cr , qep A4 associated with CTX-M positive E. coli isolates from Egypt. This is the first report of the plasmid mediated fluoroquinolone efflux pump, Qep A from Egypt.
Keywords: E. coli, quinolones, plasmid, extended-spectrum β-lactamase, Egypt
|How to cite this article:|
Hassan W M, Hashim A, Domany R. Plasmid mediated quinolone resistance determinants qnr, aac(6′)-Ib-cr, and qep in ESBL-producing Escherichia coli clinical isolates from Egypt. Indian J Med Microbiol 2012;30:442-7
|How to cite this URL:|
Hassan W M, Hashim A, Domany R. Plasmid mediated quinolone resistance determinants qnr, aac(6′)-Ib-cr, and qep in ESBL-producing Escherichia coli clinical isolates from Egypt. Indian J Med Microbiol [serial online] 2012 [cited 2013 May 21];30:442-7. Available from: http://www.ijmm.org/text.asp?2012/30/4/442/103766
| ~ Introduction|| |
Plasmid-mediated quinolone resistance associated with qnrA, qnrB and qnrS has been reported among enterobacterial species in Asia, the USA, South America and several countries in Europe.  Moreover, it has been frequently reported that the qnr genes have been detected among isolates producing extended spectrum β-lactamases (ESBLs).
The second mechanism, an aac(6′)-Ib-cr aminoglycoside acetyltransferase variant, with two specific amino acid substitutions, enables the acetylation of the piperazinyl substituent of ciprofloxacin and norfloxacin, reducing their activity.  Plasmid-mediated quinolone resistance determinants, including Qnr peptides and aac(6′)-Ib-cr, are increasingly identified worldwide among various clinical isolates of Enterobacteriacea. 
The last PMQR determinant, qepA, was first identified in an Escherichia More Details coli clinical isolate from Japan  and later found also in an E. coli isolate in Belgium.  This protein confers resistance to hydrophilic quinolones, i.e., norfloxacin, ciprofloxacin and enrofloxacin by efflux pump system which showed a considerable similarity to the major facilitator superfamily-type efflux pumps. The aim of this study was to evaluate the presence of quinolone resistance genes among ESBL producing E. coli clinical isolates and their association with CTX-M genes.
| ~ Materials and Methods|| |
Ninety non duplicate clinically relevant Enterobacteriaceae isolates were collected from inpatients and outpatients attending different hospitals in Cairo, Egypt during the period from January 2007 to September 2007. The samples collected from the patients were urine and stool.
Antimicrobial susceptibility testing
Susceptibility and MICs were determined by disc diffusion method and agar dilution technique, following the CLSI guidelines (MDR defined as bacteria resistant to β-lactams, quinolones and aminoglycosides antibiotics.
Antibiotics were Ampicillin, Ampicillin/Sulbactam, Amoxacillin/Clavulinic acid, Cefotaxime, Cefoperazone, Cefaclor, Cephradine, Imipenem, Amikacin, Gentamicin, Ciprofloxacin, Ofloxacin, Chloramphenicol, Tetracycline and Trimethoprim/Sulphamethoxazole.
Detection of ESBLs
Suggestive evidence of ESBL production was defined as synergy between Amoxicillin/ Clavulanate and at least one of the following antibiotics: Cefotaxime, Ceftazidime, Aztreonam, Cefotriaxone  and confirmed by the combined disc method according to CLSI.  E. coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603 were used as a negative and positive control for ESBL test, respectively.
Plasmid extraction and curing
The Sigma-Aldrich Kit was used to extract plasmids from E. coli clinical isolates according to the manufacturer's instructions.
Three plasmid curing agents were used including norfloxacin disc, sodium dodecyl sulfate and ethidium bromide at elevated temperature (42 o C). 
PCR amplification and sequencing
The qnrA, qnrB, qnrS, qep, aac(6′)-Ib-cr , blaCTX-M-1 , blaCTX-M-9 genes were detected by PCR in clinical isolates using the following primers: for qnrA, qnrA_up (5′-AGAGGATTTCTCACGCCAGG-3′) and qnrA_dw (5′-TGCCAGGCACAGATC-TTGAC-3′) to give a 580 bp product; for qnrB, qnrB_up (5′-GGMATHGAAATTCGCCACTG-3′) and qnrB_dw (5′-TTTGCYGYYCGCCAGT-CGAA-3′) to give a 264 bp product; and for qnrS, qnrS_up (5′-GCAAGTTCATTGAACAGGGT-3′), qnrS_dw (5′-TCTAAACCGTCGAGTTCG-GCG-3′) to give a 428 bp product. 
Primers for aac(6′)-Ib-cr were aac(6′)-Ib-cr _up (5'-TTGCGATGCTCTATGAGTGGCTA-3') and aac(6′)-Ib-cr _dw (5'-CTCGAATGC-CTGGCGTGTTT-3'), generating a 482 bp fragment.  Primers for qep were qep_up (5′-GCAGGTCCAGCAGCGGGTAG-3′) and qep_dw (5′-CTTCCTGCCCGAGTATCGTG-3′), to give a 199 bp fragment. Amplification and identification of ESBL-encoding genes were performed with blaCTX-M-1 -group, CTX-M1_up (5′-GACGATGTCACTGGCTGAGC-3′), CTX-M-1_dw (5′-AGCCGCG-GACGCTAATACA-3′) to give a 499 bp product and blaCTX-M-9 -group, CTX-M9_up (5′-GCTGGAGAAAAGCAGCGGAG-3′), CTX-M-9_dw (5′-GTAAGCTGACG-CAACGTCTG-3′) to give a 474 bp product. 
Both strands of amplicons were sequenced twice with an automatic sequencer (model 3730 xl; Applied Biosystems, Weiterstadt, Germany). The genes sequences were subjected to BLAST to perform sequence similarity searches. The program selection was optimized for highly similar sequences (Megablast). Sequences were initially aligned using the Bioedit built-in clustal W program. Resulting alignments were compared and the final alignments were improved manually and prepared in FASTA, MEGA and NEXUS formats. Amino acid translations of partial nucleotide genes sequences were obtained and analyzed by MEGA 3.1 software and confirmed using ExPaSy translate tool available online at http://www.expasy.ch/tools/dna.html and compared to the available protein sequence in Genbank.
| ~ Results|| |
Seventy out of 90 Enterobacteriaceae clinical isolates were E. coli. Out of 70 E. coli clinical isolates 61 were resistant to at least one antibiotic, 16(22.8%) were multi-drug resistant and 30 were ESBL producers. Among ESBL producers 60% and 57% were ciprofloxacin and gentamycin resistant respectively while one isolate was amikacin resistant.
Plasmid profile and curing analysis
All ESBL-producing E. coli were harboring plasmids. The MDR isolates showed different numbers of plasmids ranging from one (isolate14) to six plasmids (isolates 6 and 24). All plasmids were lost upon curing and resistance to different antibiotics was also lost; thereby confirming their location on plasmid.
Prevalence of qnr-group, aac(6′)-Ib-cr and qep genes
PCR and sequence analysis indicated that eight of 30 ESBL producing isolates were positive for qnr genes, with qnrA-, qnrB-and qnrS-type alleles detected in 5 (16.6%), 7 (23.3%) and 5 (16.6%) E. coli isolates, respectively. Seven (23.3%) isolates were positive for aac(6′)-Ib-cr and only two (15%) isolates contained qepA genes.
Detection and prevalence of CTX-M group in Qnr positive E. coli isolates
PCR and sequence analysis indicated the presence of blaCTX-M-14 and/or blaCTX-M-15 in qnr positive E. coli isolates.
Out of 8 Qnr positive isolates 6 were associated with CTX-M-1, 7 were associated with CTX-M-9 and 5 were associated with both CTX-M-1 and CTX-M-9. The 2 Qep positive isolate were associated with either CTX-M-1 or CTX-M-9 while 2 from 6 aac(6′)-Ib-cr positive isolates were associated with both CTX-M-1 and CTX-M-9 [Table 1].
|Table 1: Genotypes and antibiotic resistance phenotypes of PMQR E. coli clinical isolates with CTX-M1 and CTX-M9|
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DNA sequencing results
Nucleotide composition analysis showed that the aac (6′) -Ib detected was aac(6′)-Ib-cr gene with GC value of 54.6. Among the studied 484 nucleotide bases compromising for aac(6′)-Ib-cr gene, 478 bases were conserved while only 6 sites were variable. Surprisingly, 4 out of six base substitutions were transversional changes, from T→A, A→T and G→T. Only two base substitutions were transitional changes [Figure 1]. Among the studied 172 amino acid residues, 168 amino acid residues were conserved and only 4 amino acids were variable. Amino acid changed from Try→Arg, Leu→Ser, Asp→Tyr, Asp→Val at 74 th , 89 th , 151 th 170 th residues, respectively.
|Figure 1: Multiple DNA sequence alignment of aac(6?)-Ib-cr gene isolated from E. coli tested isolate and retrieved sequences from Genbank. Hyphen indicates alignment gaps or missing data|
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Nucleotide composition analysis showed that the detected qepA1 gene was with a high GC value of 71.1%. The detailed composition of this gene was: T (10.1), C (37.6), A (18.3) and G (33.9) and the bases were conserved among different genes from different sources and there is no base substitution observed.
Nucleotide composition analysis showed that our qnrB1 gene has GC value of 52.1. Among the studied 263 nucleotide bases compromising for qnrB1 from different bacterial species, including our gene, 243 bases were conserved and 20 sites were variable among the 263 nucleotide bases.
It is worth mentioning that, out of 20 base substitutions 5 were transversional changes, from C→A (2), T→A (1) and G→T (2). Fifteen base substitutions were transitional changes [Figure 2]. Among these base substitutions 16 were parsimony informative changes. Among the studied 87 amino acid residues, 82 amino acid residues were conserved and only 5 amino acids were variable. Amino acid change from Ile→Met, Ala→Thr, Ser→Gly, Ser→Thr and from Phe→Leu at 74 th, 60 th , 62 nd, 70 th , and 82 nd respectively.
|Figure 2: Multiple DNA sequence alignment of qnrB1 and its variants genes isolated from E. coli tested isolate and retrieved sequences from Genbank|
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Nucleotide composition analysis showed that the detected qnrS gene was qnrS1 with low GC value of 43.7 and the detailed composition was: T (29.7), C (19.2), A (26.6) and G (24.2). Among the studied 428 nucleotide bases compromising for qnrS1 from different bacterial species, including our gene, all bases were conserved.
| ~ Discussion|| |
ESBL prevalence varies in different countries. E. coli producing ESBL has been reported at a prevalence rate of 67%, 42% and 43% in Iran and India respectively. , While less than 1% of E. coli isolates produce ESBL in the Scandinavian countries.  In the United States, occurrence of ESBL production in Enterobacteriaceae ranges from 0 to 25%, depending on the institution, with the national average being around 3% (CDC National Nosocomial Infections Surveillance). In this study the combined disc method confirmed ESBL production in 30 (42%) E. coli isolates, which is in accordance with the results from developing countries.
The prevalence of the qnr genes in ESBL producing E. coli clinical isolates collected from hospitals in this study was 26.6%. This is not consistent with the study conducted in Denmark that showed only 1.63% (2/122) of nalidixic acid-resistant E. coli isolates as qnr-positive.  In France, the prevalence of qnr genes was 1.6% (2/125) among ESBL producing E. coli and Klebsiella spp. Isolates. , While in Canada only about 1% (5/550) of ciprofloxacin and/or tobramycin resistant E. coli and Klebsiella spp. isolates were qnr-positive.  Nevertheless, high prevalence has also been detected in other parts of the world such as Spain (5%) ,  and China (8%). 
qnr-positive isolates were resistant to antimicrobials of three different classes; β-lactams, aminoglycosides and quinolones and were classified as multi-drug resistant isolates. The prevalence of such multi-drug resistant clinical isolates could be due to either the spread of a successful single or few clonal groups or the presence of transferable R-plasmids in these isolates.  In this study, plasmids were detected in all of the qnr-positive isolates suggesting that plasmids were responsible for the spread.
qnrS1 and qnrA1 were detected in five isolates (16.6%), while qnrB was found in seven isolates (23.3%). The dominance of qnrS and qnrB in our isolates collections is similar to other studies from Europe. 
The prevalence of aac (6′) -Ib-cr among the selected E. coli clinical isolates was 23.3 % (7/30). This finding was higher than what has been found in other studies; the prevalence of aac(6′)-Ib-cr was 11.3% (62/549) among ciprofloxacin-and/or tobramycin-resistant E. coli and Klebsiella spp. clinical isolates from Canada  and 9.9% (36/365) among ESBL-producing E. coli and K. pneumoniae isolates from six provinces in China. 
In our study aac(6′)-Ib-cr positive clinical isolates showed reduced susceptibility or resistance to gentamicin except isolate 17 which is the sensitive to gentamicin. The gene aac(6′)-Ib-cr was geographically, widespread, most commonly detected in E. coli, and equally prevalent in both ciprofloxacin-susceptible and resistant strains.  The considerable association between aac(6′ )-Ib and gentamicin resistance was an unexpected finding since aac(6′)-Ib-cr does not confer resistance to gentamicin. The same unanticipated finding was reported from USA.  The explanation could simply be that the isolates harbor other resistance determinants affecting gentamicin. In addition, the aac(6′)-Ib-cr positive clinical isolates were in addition resistant to trimethoprim-sulfmethoxazole. This could be due to the fact that aac(6′)-Ib-cr has mostly been found in complex integrons  and that the 3′-CS region of such integrons contains the sul1 gene encoding resistance to sulphonamides.
The prevalence of qepA1 was low (0.3%) in E. coli clinical isolates collected from 140 Japanese hospitals between 2002 and 2006.  Our study revealed a higher prevalence of qepA1 in E. coli clinical isolates (2/30, 6.6%). This efflux pump, first described in 2007 in two E. coli clinical isolates from Japan and Belgium, , has already been detected in France with a new variant QepA2. 
CTX-M type ESBLs have been extensively reported for the past 10 years in both community and nosocomial settings and a strong linkage between their production and quinolone resistance has been reported in Enterobacteriaceae. , In the present study all qnr positive E. coli isolates were associated with CTX-M-1, CTX-M-9 or both. Our findings indicated high prevalence of quinolone resistance determinants qnr, aac(6′)-Ib-cr and qep in ESBL producing E. coli from Egypt. Moreover, 63.3% and 53.3% of ESBL producing E. coli showed co-resistance to ciprofloxacin and gentamicin respectively. This association could be clinically significant since the therapeutic options for treatment of the increasingly encountered quinolones, β-lactam, and gentamicin-resistant E. coli are limited.
| ~ Acknowledgments|| |
We thank Dr. Amr T. M. Saeb. Department of Bioinformatics and Biotechnology, Strategic Centre of Diabetes Research, King Saud University KSA for interpretation of DNA sequencing results regarding bioinformatics and We also thank Dr. Noha Gamal Khalaf, Lecturer at the Department of Microbiology and Immunology, Faculty of Pharmacy, El Asher University for reviewing the manuscript.
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[Figure 1], [Figure 2]