Received date: March 16, 2012; Accepted date: May 25, 2012; Published date: April 27, 2012
Citation: Malki Fatima EL, Bouraissi Meriem EL, Said B (2012) Plasmid Mediated Quinolones Resistance ESBL-Enterobactériaceae in Moroccan. Pharmaceut Anal Acta S15:006. doi: 0.4172/2153-2435.S15-006
Copyright: © 2012 Malki Fatima EL, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Multidrug Resistance (MDR) in Enterobacteriaceae including resistance to quinolones is rising worldwide. This study was conducted to assess the resistance level to antibiotics and to detect plasmid genes mediated quinolones resistance in Extended-Spectrum β-lactamase (ESBL)-producing Enterobacteriaceae collected from regional hospitals of Fes–Meknes in central Morocco. ESBL phenotype was determined according to the combination disc method recommended by the Clinical and Laboratory Standards Institute (CLSI) using double disc synergy test (DDST). The antimicrobial susceptibility patterns of isolates showed high resistance rate to most antibiotics except imipenem which showed 100% of susceptibility.
A sub-site of 27 isolates was screened for qnr genes by multiplex PCR. qnrB gene was detected in 8 ESBL isolates (2 E. coli, 4 K. pneumoniae, 01 E. aerogenes and 01 C. freundii) while no qnrA neither qnrS could be detected. aac(60)-Ib-cr gene was detected in 15 strains, 13 of them were ESBL.
Our results are in agreement with the general rule that imipenem stays the drug of choice for the treatment of infections caused by ESBL producers. Moreover, the presence of qnr determinants is closely related to ESBL phenotype while aac(60)-Ib-cr gene could be detected in isolates with or without ESBL phenotype.
Enterobacteriaceae; ESBL; Qquinolone; Plasmid genes
Enterobacteriaceae represent the major class of Gram Negative Bacteria (GNB) responsible for the majority of infectious diseases. They acquired mechanisms of resistance to various families of antibiotics .
Although quinolone resistance in Enterobacteriaceae results mostly from chromosomal mutations, it may also be mediated by plasmidencoded Qnr genes . Qnr proteins have been identified worldwide with a frequent association with clavulanate inhibited expandedspectrum β-lactamases and plasmid-mediated cephalosporinases. Qnr proteins protect DNA from the inhibitory activity of quinolones such as nalidixic acid.
The first transferable plasmid-encoded quinolone resistance gene (qnrA, qnrB and qnrS) was isolated from a clinical isolate of ciprofloxacin- resistant Klebsiella pneumonia in 1998 .
The isolated qnrA, qnrB and qnrS gene products protect DNA gyrase from inhibition by ciprofloxacin .
QnrB determinants are associated with the ESBL SHV-12 inch several isolates which may explain in part the frequent association between fluoroquinolone and expanded-spectrum cephalosporin resistance in Enterobacteriaceae [4,5].
Two other plasmid-mediated quinolone resistance mechanisms have been described. The aac (60)-Ib-cr determinant, a variant of aminoglycoside acetyltransferase capable of modifying ciprofloxacin and reducing its activity, is widely prevalent and seems to be associated to qnr genes [6-9].
The emergence of plasmid-mediated quinolone resistance determinants in Enterobacteriaceae may compromise further the efficacy of quinolones that are, together with β-lactams and aminosides, the most commonly prescribed antibiotics for treating human infections.
The aim of this study is to evaluate the efficiency of divers antibiotic classes in multidrug resistance and also to assess an eventual correlation between the presence of qnr and aac(60)-Ib-cr genes and the ESBLproduction from Enterobacteriaceae in Morocco.
We examined 148 Entrerobacteriaceae (E. coli = 96; Klebseilla spp. = 40; others = 12) collected from hospital bacteriological laboratories in Fes-Maknes region during 2010 (from 1th July to 30th September). They were isolated from urinary samples (n=97), surgical wound sepsis (n=24), blood cultures (n=15), genital tract infections (n=05), catheter (04), endotracheal tube-associated pneumonias (n=03).
Identification of species was carried out by API 20E (Bio-Mérieux SA, France).
ESBL detection methods
ESBL screening and confirmation were performed according to the Clinical and Laboratory Standards Institute (CLSI, 2008)  criteria. All isolates were tested by double disc synergy test (DDST) described by Jarlier .
ESBL producer was detected by using a disc containing ceftazidime (CAZ, 30 μg) or cefotaxime (CTX, 30 μg) in combination with and without clavulanic acid (CLA, 10 μg). The presence of an ESBL was determined by a 05 mm increase in zone diameters for CAZ/CLA and CTX/CLA compared with those for CAZ and CTX, respectively (Figure 1).
Susceptibility and synergy-testing for ESBLs were simultaneously performed on Mueller- Hinton agar. The following antibiotic disks (Bio- Rad) were tested: amoxicillin (AMX), amoxicillin-clavulanic acid (AMC), cefalotin (CF), cefotaxime (CTX), ceftazidin (CAZ), cefuroxime (CRO), imipenem (IPM), Tobramycin (TB), ofloxacin (OFX), ciprofloxacin (CIP), norfloxacin (NOR), corticomoxasol (SXT), colistin (CL).
The reference strains were E. coli ATCC 25922 and K. pneumoniae ATCC 700603.
PCR Amplification and Molecular Detection of qnr and aac(60)-Ib-cr genes
A subset of 27 non-duplicates isolates (16 E. coli, 09 Kleb. spp, 01 E. aerogenes and 01 C. freundii) was subjected to molecular analysis. The characteristics of those isolates are listed in Table 2.
DNA preparation was performed by a boiling technique including a heating step at 100C of a single colony in a total volume of 100 μL of distilled water followed by centrifugation of the cell suspension.
The amplified DNA products were examined as described previously; the expected sizes for qnrA, qnrB and qnrS were 580, 264 and 428 bp respectively.
A pair of degenerated primers was specifically choused to amplify variants of qnrB and another pair of primers was used to detect the aac(60)-Ib-cr in selected stains. The expected size was 473 bp (result none shown).
DNA amplification was carried out by using 2 μl of the extract in 500 mM, 2mM KCl, 10mM Tris HCl, 1.5 mM MgCl , containing 500 μM of each dNTP, 0.2 μM of each primer, 0.5U of Taq DNA polymerase (Roche Diagnostics, France) in a final volume of 25 μl.
PCR products were analyzed by electrophoresis in 1.2 % agarose gel in TBE buffer staining with ethidium bromide and visualized with UV light.
Phenotypic detection and antimicrobial susceptibility patterns
The antimicrobial susceptibility patterns of isolates were examined. Enterobacteriaceae showed high resistance rate to β-lactam in general with values surrounding 80% for amoxicillin, 60% for clavulanic acid, 40% for cephalosporin of first generation (C1G) and 30% for cephalosporin of third generation (C3G). 56% of isolates were resistant to sulfamethoxazole/ trimethoprim (SXT). Resistance to fluoroquinolones showed a rate of about 40%, whereas for the tested aminoglycosides (tobramycin) is only 14%. Colistin stills an efficient antibiotic against Enterobactériaceae with 96% of susceptibility while all examined isolates were susceptible to imipenem (Table 1).
|Antibiotic||E.coli n =96 (%)||Klebseilla spp n=40 (%)||Other n=12 (%)||Total N=148 (%)|
|AMX||67 (69,79)||40 (100)||10 (83,33)||117 (79,05)|
|AMC||51 (53,12)||30 (75)||10 (83,33)||91 (61,48)|
|CF||30 (31,25)||22 (55)||8 (66,66)||60 (40)|
|CTX||24 (25)||17 (42,5)||5 (41,66)||46 (31,08)|
|CAZ||24 (25)||18 (45)||5 (41,66)||47 (31,75)|
|CRO||24 (25)||18 (45)||5 (41,66)||47 (31,75)|
|TM||17 (17,7)||4 (10)||0||21 (14,18)|
|OFX||40 (41,66)||17 (42,5)||5 (41,66)||62 (41,89)|
|CIP||40 (41,66)||17 (42,5)||5 (41,66)||62 (41,89)|
|NOR||40 (41,66)||15 (37,5)||5 (41,66)||60 (40)|
|SXT||55 (57,29)||24 (60)||4 (33,33)||83 (56,08)|
|CL||4 (4,16)||0||2 (16,66)||6 (4,04)|
Table 1: Antibiotic resistance profile of the bacterial isolates.
|Isolate||Species||BLSE: 13||qnr: 8||Aac: 15||Resistance profile|
|E1||E.C||—||—||—||AMX, AMC, CF,CTX, CRO, CAZ, OFX,CIP,NOR, NET, SXT]|
|E4||E. aero||+||+||+||[AMX,AMC,CF, CRO,CAZ,CTX, OFX ,CIP,NOR,NET,SXT,CL]I=NA|
|E5||E.C||+||+||+||AMX, AMC,CF, CTX ,CRO,CAZ,OFX, CIP, NOR,SXT,NET,CL|
|E6||K.P||+||+||+||AMC, AMX, CF, CTX, CRO, CAZ, CIP,NOR, OFX, NET, SXT|
|E7||K.P||+||+||+||AMX, AMC, CF, CTX, CRO, CAZ, CIP, OFX, NOR,NET, SXT|
|E8||K.P||+||—||+||AMX, AMC ,CF, CRO, CTX, CIP, OFX,NOR,, NET,TOB, SXT|
|E9||E.C||+||—||+||AMX, AMC, CF, CTX, CRO, CAZ, CIP, NOR, OFX, NET, TOB, SXT|
|E10||E.C||—||—||—||AMX,AMC, CF, CAZ, NOR, OFX, CIP,|
|E12||K.P||+||+||+||AMX, AMC,CF, CTX, CRO, CAZ, CIP,OFX, NOR, TOB,NET, SXT|
|E13||K.O||+||—||+||AMX, AMC, CF, CTX, CRO, CAZ, CIP/ NOR/ OFX, NET, TOB, SXT|
|E14||E.C||—||—||—||AMX, AMC, CIP,NOR, OFX, SXT|
|E15||E.C||—||—||—||AMX, AMC, CIP, OFX NOR, SXT,|
|E16||CITRO||—||+||—||AMX, AMC, CIP, NA, SXT|
|E17||K.O||—||—||—||AMX, AMC, CF, CRO, CAZ, TOB, SXT|
|E18||E.C||—||—||—||AMX,AMC ,NOR,OFX,CIP,,SXT, CL|
|E19||E.C||—||—||+||AMX, AMC, CIP,OFX, NOR, SXT|
|E20||E.C||—||—||—||AMX, AMC, CIP, OFX|
|E21||K.O||—||—||—||AMX, OFX, CIP, NOR, NET, SXT|
|E22||K.P||+||—||+||AMX, AMC, CF, CTX, CRO, CAZ, TOB|
|E23||E.C||—||—||+||AMX, AMC,CF,NA,NOR,OFX,CIP, SXT|
|E24||E.C||—||—||—||AMX, AMC, CF, CIP, OFX,NOR TOB, SXT|
|E25||E.C||—||—||—||AMX, CIP,NOR, OFX, SXT|
|E26||E.C||—||—||—||AMX, CF, CTX, CRO, CAZ, SXT|
|E27||E.C||+||+||+||AMX,AMC,CF,CRO,CAZ, CTX, NOR, OFX,CIP, NET, TOB, SXT, CL|
Table 2: Resistance profile for 27 analyzed isolates.
The high ratio of co-resistance to quinolone, trimethoprim/ sulfamethoxazole and aminoglycosides among ESBL producers collected from this region has greatly limited the therapeutic role of those classes of antibiotics. However, our results are in agreement with the general rule that imipenem stays the drug of choice for the treatment of infections caused by ESBL producers.
Clavulanic acid and 3 surrounding cephalosporins CAZ, CTX and ATM;
ESBL-producer E.coli detected by DDST using CTX in combination without (b1) and with (b2) CLA showing increased zone inhibition diameter for CTX/CLA.
ESBL test revealed that 13 of 27 isolates were ESBL- producers (5 E. coli, 6 K pneumonia, 1 K. oxytoka and 1 E. aerogenes). 11 isolates presented resistance simultaneously to β-lactam, quinolones and aminosides (Table 2). This may be explaining by the association of antibiotic resistance genes between fluoroquinolone and expandedspectrum cephalosporin in Enterobacteriaceae.
Prevalence of qnr and aac(60)-Ib-cr genes
Screening of qnr genes resulted in 8 positives cases all of them carried qnrB-like gene with the expected size (2 E. coli, 4 K. pneumoniae, 01 E. aerogenes and 01 C. freundii), while no qnrA neither qnrS could be detected (result non shown. The rate of qnr carriage among K. pneumonia was higher than in E. coli. To date, qnr genes have been widely detected worldwide.
The 27 analyzed isolates showed the presence of aac(60)-Ib-cr variant gene with the expected size in 15 strains (result non shown), 13 of them were ESBL. All qnr strains showed ESBL phenotype and possessed aac (60)-Ib-cr determinant except a C. freundii which carried only QnrB gene and showed relatively a lower resistance profile (Table 2). The rate of aac (60)-Ib-cr carriage was comparable between E. coli and klebseilla.
Plasmid-mediated genes, such as qnr and aac(60)-Ib-cr, may facilitate spread and increase the prevalence of quinolone-resistant strains.
The aac (60)-Ib-cr gene was detected in non-ESBL-producing E. coli isolates which were C3G and aminoglycosides susceptible.
Total results obtained for this work is summarized in Table 2 with phenotypic and genotypic patterns. Further study is required to precise aac (60)-Ib-cr functions and relationship with divers' classes of antibiotics resistance. Furthermore, plasmid's carriage of these genes (qnr and aac(60)-Ib-cr) is assessed by conjugation test while the identification of divers variants is running by sequencing.