Antibacterial resistance pattern among Escherichia coli strains isolated from Mansoura hospitals in Egypt with a special reference to quinolones

Extensive use of fluoroquinolone antibacterial in clinical practice has been associated with increasing frequency of quinolone-resistant Escherichia coli strains. In the current study, a total of 80 E. coli clinical isolates from Mansoura hospitals patients in Egypt were studied for antibacterial susceptibility pattern against 15 different antibacterials. These strains were tested for quinolones resistance by minimum inhibitory concentration (MIC) determination using broth micro-dilution method. The resistance rate of ciprofloxacin and levofloxacin for E. coli isolates was found to be 60%. PCR was performed for detection of plasmid-mediated quinolone resistance genes including qnrA, qnrB and qnrS. 30 and 61.3% of E. coli isolates were positive for qnrA and qnrB, respectively, whereas qnrS was identified in only 15% of isolates. Quinolone resistance-determining region (QRDR) of gyrA and ParC genes was characterized for 17 ciprofloxacin and levofloxacin resistant E. coli isolates (MIC 12.5-200 μg mL). Two mutation sites in gyrA were detected in 17 tested E. coli isolates. However, two mutation sites in parC were detected in four E. coli isolates. The amino acid change at Ser-83 and aspartic-87 in GyrA were the most common mutation sites identified in the isolates. These results indicated that multiple mechanisms of quinolone-resistance are commonly found in E. coli isolated from Mansoura hospitals.


INTRODUCTION
Quinolones are powerful broad-spectrum antibacterial agents commonly used in both human and veterinary medicine for the treatment of a wide variety of infections.In the last decade, fluoroquinolones have become first and second-line antibacterials of choice for acute respiretory, enteric and urinary tract infections as well as serious systemic infections such as bacteremia (Jamison, 2006).Their extensive use has been associated with raising level of quinolone resistance in different microorganisms (Robicsek et al., 2006).Fluroquinolones act by increasing levels of enzyme-mediated DNA cleavage affecting DNA gyrase enzyme which catalyzes the negative supercoiling of DNA and topoisomerase IV enzyme which decatenates or removes the interlinking of daughter chromosomes at the completion of a round of DNA replication allowing their segregation into daughter cell (Ambrozic et al., 2007;Wang et al., 2009).Quinolone resistance has traditionally been attributed mainly to chromosomal mutations in the gyrA and gyrB genes of DNA gyrase and in parC and parE genes of topoisomerase IV or due to decreased intracellular concentration as a result of decreased permea-bility of the membrane or over expression of efflux pump systems (Poirel et al., 2006;Oktem et al., 2008;Allou et al., 2009).
Alteration in quinolone resistance determining region (QRDR) is considered as the most important mechanism of quinolone resistance.Mutations in QRDRs of gyrA and parC are most commonly documented, however resistance is also conferred by mutations in parE (Hopkins et al., 2005).These mutations block the action of quinolones resulting in increased level of resistance to fluoroquinolones (Ruiz, 2003).In Escherichia coli and related Gram negative bacteria, DNA gyrase is the first target for fluoroquinolones.Alterations in gyrA are reported much more often than alterations in gyrB (Frank et al., 2011).
Plasmid-mediated quinolone resistance has been also previously described.The study of Martinez-Martinez et al. (1998) was the initial report of this transferable mode of resistance associated with qnr gene (now named qnrA).This gene was identified for the first time in Klebsiella pneumoniae in the United States.Several studies reported a world wide distribution of qnr determinants among bacterial isolates (Cheung et al., 2005;Cerquetti et al., 2009).The qnr gene encodes a 218amino-acid protein which protects DNA gyrase and topoisomerase IV activity from the action of quinolones (Tran and Jacoby, 2002;Strahilevitz et al., 2009).The plasmid-mediated quinolone resistance determinants are widely distributed in clinical Enterobacteriaceae isolates around the world.These genes are usually located on mobile elements, including integrons, insertion sequences and transposons (Martinez-Martinez et al., 1998;Robiesek et al., 2006;Yamane et al., 2008).Furthermore, the qnr gene carrying plasmids, which are classified as Class I integron-carrying plasmids, usually carry in addition to qnr gene multiple resistance determinants providing multidrug resistance to different antimicrobials including: aminoglycosides, B-lactams and sulfonamides (Martinez-Martinez et al., 1998;Tran et al., 2005).
The prevalence and distribution of qnr genes were different in various geographical areas.qnrA genes have been identified worldwide in a variety of Enterobacterial species.Six variants have been identified (qnrA1 to qnrA6).These genes can increase the MIC of fluoroquinolones up to 32-fold in E. coli isolates (Poirel et al., 2006;Allou et al., 2009).In addition, qnrA gene enhances the selection of chromosomal encoded quinolone resistance determinants which confer additional resistance to fluoroquinolones.Other plasmid-mediated quinolone resistance determinants qnrB (qnrB1 to qnrB6) and qnrS (qnrS1 and qnrS2) have been also identified in enterobacterial species, sharing 41 and 60% amino acid identity with qnrA, respectively (Nordmann and Poirel, 2005;Shin et al., 2009).Another mechanism of quinolone resistance relies on upregulation of efflux pump which exports El-Sokkary and Abdelmegeed 663 quinolones and other antimicrobials out of the bactereial cell.Although multiple mechanisms of quinolone resistance have been reported from many continents, there are few data from Africa on the molecular basis for quinolone resistance.In the current study, we focused on the prevalence of E. coli resistance to quinolones and the frequency of qnrA, qnrB and qnrS among clinical isolates of E. coli in Mansoura Hospitals.Also, this study was undertaken to determine the mechanism of quinolone resistance among E. coli isolates from Mansoura Hospitals.

Bacterial strains
Eighty clinical E. coli isolates were collected from Mansoura university hospitals in Dakahlia governorate, Egypt during March 2011 to February 2013.The isolates were collected from urine, wound and sputum samples.These isolates were identified using standard biotyping methods (Crichton, 1996).

Determination of MIC (Minimal inhibitory concentration) for the isolated strains
The minimal inhibitory concentrations of the isolates for ciprofloxacin and levofloxacin were determined using broth microdilution method following the Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) guidelines (CLSI, 2007).In this method, 96-well microtitre plates were filled with small volumes (200 µl) of serial two-fold dilutions of each tested antibacterial.The final concentrations of each antibacterial in the wells ranged from 1.56 to 200 µg mL -1 .The turbidity of overnight culture is adjusted to obtain visually comparable turbidity to that of the 0.5 McFarland turbidity standards, then an aliquot of 20 µl was added to each wells of the microtitre plate.The plates were incubated for 24h at 30°C before determining the results.The MICs were read visually and were defined as the lowest concentration where no viability was observed in the wells of the microplates after incubation.The MICs values indicate resistance > 1 and > 2 µgmL -1 for ciprofloxacin and levofloxacin, respectively

Screening for the qnr genes in clinical strains
Screening was carried out by polymerase chain reaction (PCR) amplification of qnrA, qnrB and qnrS using the primed sets listed in Table 1.DNA templates were prepared by transferreing bacteria isolates to distilled water in Eppendorf tubes and then boiling for 10 min then prepared DNA templates were directly used in the PCR

Mutational analysis of the quinolone-resistance determining regions of gyrA and parC
The QRDR of the gyrA and parC genes were amplified in seventeen quinolone resistant E. coli strains by PCR using the primer pairs listed in Table 1.PCR reactions began with 10 min primary denaturation at 94°C followed by 40 cycles of 94°C for 30s, annealing temperature for 30 s and 72°C for 30 s. gyrA and parC amplification primers were annealed at 55 and 62°C, respectively.PCR amplified gene fragments were purified using the PCR Purification Kit (MEGA quick-spin fragment DNA purification INtRON biotechnology, Korea) for subsequent sequencing.Purified PCR products were used as a template in sequencing reactions carried out with the ABI PRISM ® BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, USA).The reaction mixtures were analysed on an ABI 3730 DNA analyser (Applied Biosystems, Foster City, USA).
Amplicons were sequenced on both strands and predicted peptide sequences were analysed by the online BLAST of the NCBI website software (http://www.ncbi.nlm.nih.gov/BLAST/).Then point mutations were identified by comparing the identified sequences to the corresponding genes by pair-wise FASTA alignments.

Antibacterial susceptibility testing
All E. coli isolates were resistant to ampicillin.On the other hand, they were all sensitive to imipenem.In addition, the highest resistance rate was recorded against ceftazidime (72.5%), ceftriaxone and cefoperazone (71.3%), ceftizoxime and cefoxitin (70%), norfloxacin, ciprofloxacin and azithromycin (67.5%), cefotaxime (65%), tobramycin and levofloxacin (62.5%) and gentamicin (51.3%).In contrast, resistance to amikacin was less common but was seen in only 23.75% of the isolates.Quinolone resistance was always seen in multiple-resistant E. coli, as all quinolone resistant E. coli were resistant to at least one other antimicrobial (Figure 1).

Determination of minimum inhibitory concentrations (MICs) of ciprofloxacin and levofloxacin against E. coli isolates
Ciprofloxacin MICs values for 80 E. coli isolates are shown in Figure 2. The susceptibility of isolates to ciprofloxacin showed that 48 (60%) of 80 E. coli were resistant while 22 (27.5%) of E. coli isolates were sensitive to ciprofloxacin in addition to 10 isolates (12.5%) exhibiting intermediate susceptibility to ciprofloxacin.The distribution of levofloxacin MICs values are shown in Figure 3. 48 isolates exhibited high-level levofloxacin resistance producing MICs of ≥ 12.5 µg mL -1 .However, 25 isolates exhibited susceptibility to levoflo-xacin.Only 7 isolates showed intermediate susceptibility.

Characterization of quinolone resistance mechanisms in E. coli
Multiple horizontally-transmitted quinolone resistance genes were detected among E. coli strains isolated from  of the tested isolates.However, 9 of 59 qnr positive strains showed low level of resistance to ciprofloxacin and levofloxacin (MIC of 3.125-1.56µg mL -1 ).In contrast, only 4 isolates of 16 qnr negative isolates had higher level of resistance to ciprofloxacin and levofloxacin (200-12.5 µg mL -1 ).
Seventeen highly resistant E. coli isolates were selected in this study to perform gyrA and parC sequence.Mutations in gyrA and parC subunits are summarised in Table 2. Quinolone-resistant isolates had at least two non-synonymous substitutions in the QRDR of gyrA and some of these isolates also had one additional mutation in parC.Sixteen from the tested isolates having two mutations in GyrA had a serine to leucine substitution at position 83 and aspartic acid to asparagine substitution at position 87, one of the most commonly documented resistance conferring mutations.All of these isolates had MIC values of at least 12.5 and 25µg mL -1 levofloxacin and ciprofloxacin, respectively.Only one isolate had S83L and additional GyrA substitution, Aspartic acid with Tyrosine (D87Y) had MIC values of 50 and 200 µg mL -1 levofloxacin and ciprofloxacin, respectively.Four of E. coli isolates also harboured the frequently documented non-synonymous mutations in the QRDR of parC.These ParC substitutions were identified as glutamic acid substituted with glycine E84G, alanine with valine A108V and glutamic acid with valine E84V.

DISCUSSION
The emergence of fluoroquinolone-resistant E. coli is increasing in many parts of the world (Levermore, 2009).In African countries with a high infectious disease burden, formal and informal health systems depend heavily on broad spectrum orally-administrable antibacterials.In this study, most of E. coli isolates from Mansoura hospitals in Egypt were resistant to ceftazidime, ceftriaxone, cefoperazone, ceftizoxime, cefoxitin, cefotaxime, azithromycin, tobramycin and gentamicin.Fluoroquinolone antibacterials have been recently introduced as an effective alternative to antibacterials that have been compromised by resistance.
Our results indicated high level of quinolone resistance with elevated MIC levels identified among the isolated E. coli in this study.However, lower resistance rates were markedly identified previously for this class of drugs (Sreela et al., 2011).
In our study, 60% of E. coli isolates exhibited high level of levofloxacin and ciprofloxacin resistance.The rate of E. coli resistance to fluoroquinolones is increasing worldwide.In USA, the rate of resistance in E. coli isolates was 3.5 and 1.9 to 2.5% for nalidixic acid and fluoroquinolones, respectively (Karlowsky et al., 2003).Quinolones target the bacterial enzymes DNA gyrase and topoisomerase IV, which are essential for cell growth and proliferation.DNA gyrase and topoisomerase IV are both tetrameric enzymes comprising two subunits gyrA and gyrB in DNA gyrase and two subunits parC and parE in topoisomerase IV.
The association between mutations of DNA gyrase and topoisomerase IV with fluoroquinolone resistance has been previously established for both Gram-negative and Gram-positive organisms (Frank et al., 2011).Accumulation of alterations in gyrA and the simultaneous presence alterations in parC play fundamental role in developing high level of resistance to ciprofloxacin in clinical isolates.In Gram negative bacteria, the primary target of quinolones is the gyrA subunit of DNA gyrase, and point mutations A.
Our E. coli isolates exhibited MIC ranging from 12.5-200 and 50-200 μg/ml of levofloxacin and ciprofloxacin, respectively.Four of these isolates also harbored point mutations in the topoisomerase IV subunit genes parC which is previously identified in Gram negative bacteria but at a significantly lower frequency than gyrA mutations (Ling et al., 2003).These ParC substitutions were detected in Glutamic acid-84 and Alanine-108 amino acid positions in the protein sequence of ParC.The QRDR polymorphisms most commonly detected in this study are those most frequently reported in the literature in E. coli (Namboodiri et al., 2011).It is generally believed that parC gene mutations arise after gyrA gene mutations, as DNA gyrase (rather than topoisomerase IV) is the preferred target of quinolones in Gram negative bacteria (Ling et al., 2003).
This study suggests that in clinical isolates of E. coli, DNA gyrase is a primary target of quinolones.In addition, two amino acid changes at Ser-83 and Asp-87 in GyrA were identified in all of high-level quinolone resistant E. coli with decreased susceptibility to both ciprofloxacin and levofloxacin.Moreover, the simultaneous presence of the ParC alterations play additional role in developing high-level resistance to quinolones which is supported by lower frequency ParC substitutions in highly quinolone resistant strains.
qnr gene, a naturally occurring gene encoding a pentapeptide repeat protein that confers reduced susceptibility to nalidixic acid or fluoroquinolone can be easily transferred between bacterial isolates due to its presence on mobile genetic elements (Jacoby et al., 2008;Strahilevitz et al., 2009).The first qnr gene, now known as qnrA, was found to protect E. coli DNA gyrase from inactivation by ciprofloxacin (Tran et al., 2005).Other qnr genes have been isolated, including qnrS1 from Shigella flexneri (Tran et al., 2005) and qnrB (Jacoby et al., 2006).
In this study, the presence of qnr genes was investigated in some isolates.qnr positive isolates identified at higher level were: qnrB (49 isolates) as compared to qnrS and qnrA (14 isolates and 24 isolates, respectively).It is important to note that plasmid-encoded qnr genes do not confer quinolone resistance by themselves, but facilitate the selection of bacteria bearing higher-level resistance, thereby augmenting the effect of other resistance mutations.The presence of horizontallyacquired genes accounted in part for elevated nalidixic acid MICs in strains that harboured these genes, but not completely.It is therefore possible that other resistance mechanisms, such as ParE polymorphisms, other horizontally acquired resistance genes, over-active efflux, or even novel mechanisms are present in some of the isolates (Martínez et al., 1998).

Conclusion
Fluoroquinolones, largely ciprofloxacin and levofloxacin, are considered as antimicrobials of high use in Mansoura hospitals in Egypt.As expected, this study demonstrates that resistance to these drugs is common, present at high numbers in E. coli isolates and occurs through multiple mechanisms.The main mechanism is associated with gyrA alterations in QRDR.Additionally, horizontallyacquired resistance to the quinolones was also identified at high frequency in this study this is supported by the hypothesis that these genes are present on mobile elements that could be transmitted to different pathogens.Finally, resistance to other antimicrobials except imipenem was also documented in this study which may limit the use of other antimicrobials as alternatives.

Figure 1 .
Figure 1.Proportion of E. coli isolates resistant to each of the fifteen antimicrobial agents.

Table 1 .
Primers used in this study.
assay.A reaction mixture containing 0.5 μM of each primer, 1.5 mM MgCl 2 , 0.2 mM dNTPs, 1 U Taq polymerase (Thermoscientific Dream Taq Green DNA polymerase), 5 μl of template DNA and nuclease free water was added for a total volume of 25 μl per reaction.PCR reactions were carried out by using Techne progene thermocycler under the following conditions: initial denaturation at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 46°C for qnrA or 54°C for qnrB or 48°C for qnrS for 30 s, and extension at 72°C for 1 min; final extension at 72°C for 5 min.PCR products aliquots were analyzed by agarose gel electrophoresis on 1% agarose gel and visualized by ethidium bromide staining

Table 2 .
Correlation between susceptibilities of levofloxacin and ciprofloxacin and alterations in GyrA and ParC in17 E. coli isolates.