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ORIGINAL ARTICLE
Year : 2019  |  Volume : 149  |  Issue : 2  |  Page : 192-198

Plasmid-mediated fluoroquinolone resistance associated with extra-intestinal Escherichia coli isolates from hospital samples


1 Division of Infectious Diseases, Nitte University Centre for Science Education & Research, Mangaluru, India
2 Department of Microbiology, Madras Medical Mission, Chennai, India
3 Nitte University Centre for Science Education & Research, Mangaluru, India

Date of Submission30-Dec-2017
Date of Web Publication3-Jun-2019

Correspondence Address:
Dr Indrani Karunasagar
Nitte Centre for Science Education and Research, Kotekar-Beeri Road, Paneer Campus, Derelakatte, Mangalore 575 018, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmr.IJMR_2092_17

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   Abstract 

Background & objectives: Infection from fluoroquinolone-resistant extra-intestinal Escherichia coli is a global concern. In this study, isolation and characterization of fluoroquinolone-resistant extra-intestinal E. coli isolates obtained from hospital samples were undertaken to detect plasmid-mediated quinolone resistance (PMQR) genes.
Methods: Forty three isolates of E. coli obtained from patients with extra-intestinal infections were subjected to antibiogram to detect fluoroquinolone resistance. The mechanism of fluoroquinolone resistance was determined by the detection of PMQR genes and mutations in quinolone resistance determining region (QRDR).
Results: Of the 43 isolates, 36 were resistant to nalidixic acid (83.72%) and 28 to ciprofloxacin (65.11%). Eight E. coli isolates showed total resistance to both the antimicrobials without any minimum inhibitory concentration. The detection of PMQR genes with qnr primers showed the presence of qnrA in two, qnrB in six and qnrS in 21 isolates. The gene coding for quinolone efflux pump (qepA) was not detected in any of the isolates tested. The presence of some unexpressed PMQR genes in fluoroquinolone sensitive isolates was also observed.
Interpretation & conclusions: The detection of silent PMQR genes as observed in the present study presents a risk of the transfer of the silent resistance genes to other microorganisms if present in conjugative plasmids, thus posing a therapeutic challenge to the physicians. Hence, frequent monitoring is to be done for all resistance determinants.

Keywords: Antibiotic resistance - plasmid-mediated quinolone resistances - quinolone resistance determining regions


How to cite this article:
Shetty SS, Deekshit VK, Jazeela K, Vittal R, Rohit A, Chakraborty A, Karunasagar I. Plasmid-mediated fluoroquinolone resistance associated with extra-intestinal Escherichia coli isolates from hospital samples. Indian J Med Res 2019;149:192-8

How to cite this URL:
Shetty SS, Deekshit VK, Jazeela K, Vittal R, Rohit A, Chakraborty A, Karunasagar I. Plasmid-mediated fluoroquinolone resistance associated with extra-intestinal Escherichia coli isolates from hospital samples. Indian J Med Res [serial online] 2019 [cited 2019 Sep 20];149:192-8. Available from: http://www.ijmr.org.in/text.asp?2019/149/2/192/259589

The increasing trend of antibiotic resistance among bacterial pathogens is a cause of global concern [1].  Escherichia More Details coli, a member of the family Enterobacteriaceae, is known to cause extra-intestinal infections frequently showing resistance to fluoroquinolones [2]. Although a normal flora in the intestinal tract of human and animals, pathogenic strains of E. coli cause intestinal infections such as gastroenteritis and extra-intestinal conditions such as urinary tract infection (UTI), meningitis, septicaemia, nosocomial pneumonia, osteomyelitis and wound infections. Virulence factors, such as adhesins and exotoxins, play an important role in the pathogenesis of this microorganism [3]. Although the major reservoir of extra-intestinal pathogenic E. coli (ExPEC) causing infection remains the alimentary tract, other sources, such as contaminated food, are also incriminated [4]. ExPEC is known to harbour specialized virulence factors to cause extra-intestinal disease particularly UTI infections [5],[6]. Such isolates can pose a significant threat to public health since these can also harbour resistance determinants that can make a pathogen resistant to multiple antimicrobial classes including present generation cephalosporins and fluoroquinolones [7].

Fluoroquinolones are an effective class of drugs used by clinicians for treating the infections of Gram-negative pathogens including UTI and hospital-acquired infections. However, the indiscriminate use of these antimicrobials has increased the prevalence of quinolone and fluoroquinolone resistance in bacterial pathogens usually mediated by point mutations in topoisomerase II (gyrA and gyrB) and topoisomerase IV (parC and parE) genes, as well as by the overexpression of efflux pumps [8]. In addition, plasmid-mediated quinolone resistance (PMQR) genes (qnrA, qnrB, qnrC, qnrS, qnrD, qnrE and qnrVC), associated with a modified aminoglycoside acetyltransferase gene [aac (6′)-1b- cr] and a specific quinolone efflux pump qepA and oqxAB have also been described in Enterobacteriaceae[2],[9],[10]. The PMQR genes in bacteria are known to display reduced susceptibility to fluoroquinolones; however, these may not present mutations in quinolone resistance determining region (QRDR)[11].

Plasmid-mediated quinolone resistance mechanism is a cause of concern since PMQR genes are located on conjugative plasmids and have been shown to disseminate fluoroquinolone resistance in E. coli isolates. The pentapeptide protein complex encoded by qnr determinants are thought to bind to topoisomerase II preventing it from the action of fluoroquinolones, aac(6')-lb-cr is known to modify fluoroquinolones with piperazinyl moiety, while plasmid-mediated qep A encodes an efflux pump of major facilitator family [12].


   Material & Methods Top


Bacterial strains & determination of antimicrobial susceptibility testing: Phenotypic identification of E. coli isolates (n=43) from clinical samples obtained from Madras Medical Mission, Department of Microbiology, Mogappair and Chennai, India, over the period of two years (2015-2017) was done and these isolates were subjected to antibiotic sensitivity testing. The isolates were analyzed for quinolone/fluoroquinolone susceptibility using disc diffusion assay according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [13]. Briefly, the bacterial isolates with the density of 0.5 McFarland turbidity were swabbed onto the pre-poured and dried Mueller Hinton agar (HiMedia Laboratories Pvt. Ltd., Mumbai). The antibiotic discs of ciprofloxacin (CIP) (5 μg) and nalidixic acid (NA) (30 μg) (HiMedia) were placed on the bacterial lawn using a sterile applicator. After overnight incubation at 37°C, inhibition zone diameters were measured and interpreted as resistant, sensitive or intermediate sensitive as per CLSI guidelines [13]. E. coli (ATCC 25922) was used as a quality control strain.

Characterization of PMQR genes: All the 43 isolates were checked for genus specific gene and for the presence of PMQR genes using PCR [14],[15]. PCR was performed in 30 μl reaction volumes containing 3 μl of 10 × buffer [100 mM Tris-HCl (p H 9), 1.5 mM MgCl2, 50 mM KCl and 1% gelatine], 100 μM of four deoxyribonucleotide triphosphates each (dATP, dGTP, dCTP and dTTP), 10 pmol of each forward and reverse primers and 1.0 U of Taq DNA polymerase with 2 μl of template DNA. All the isolates were tested for the presence of PMQR genes using primers listed in the [Table 1]. The amplified PCR products were further purified using a QIAquick PCR purification Kit (Qiagen, Hilden, Germany).
Table 1: Oligonucleotide primers used for determining gyrase and topoisomerase IV target genes

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Detection of mutation in QRDR using mismatch amplification mutation-MAMA-PCR: A MAMA assay was performed on the 43 isolates to detect the point mutations in the QRDR [16]. The known point mutations at amino acid position 83 and 87 of gyr A, 80 and 84 of par C, 447 of gyr B and 416 amino acid position of parE were targeted [17]. The primers used in the study are outlined in [Table 1].

PCR was performed in 30 μl reaction volumes containing 3 μl of 10X Taq buffer, 83 μM of four deoxyribonucleotide triphosphates, 30, 20 and 10 picomoles of forward, MAMA reverse and control reverse primers, respectively and 1 U of Taq DNA polymerase with 2 μl of DNA as a template. PCR amplification was carried out in a thermal cycler (Bio-Rad, USA) with the initial denaturation at 95°C for 5 min followed by 35 cycles of denaturation at 94°C, annealing at 55°C and extension at 72°C for 40 seconds respectively, and a final extension at 72°C for 10 min. PCR products were visualized on two per cent agarose gel stained with ethidium bromide (0.5 μg/ml) in 1× tris-acetate EDTA (TAE) buffer loaded with 10 μl of the reaction mixture and observed under UV light in a Gel Documentation system (Bio-Rad, USA).

DNA sequence analysis: The purified PCR products were sequenced in an automated ABI 3100 Genetic analyser (Applied Biosystems, USA) using fluorescent label dye terminators. The nucleotide sequences were analyzed using BLAST programmes, blastn and blastp (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The amino acids deduced from the DNA sequences were obtained through a web-based programme Expasy Translate tool (https://web.expasy.org/translate/) following which the novel sequences were submitted to the GenBank.


   Results Top


Forty-three extra-intestinal isolates of E. coli obtained from biological samples from patients with UTIs (21), wound infections (6), neonatal meningitis (5), septicaemia (5), nosocomial pneumonia (4) and osteomyelitis (2) from all age groups confirmed by phenotypic tests were reconfirmed as E. coli by PCR-based genotypic test for uid Agene.

Antibiogram analysis: Of the 43 quinolone/fluoroquinolone-resistant isolates, 28 (65.11%) were found to be resistant to ciprofloxacin while 36 (83.72%) were resistant to nalidixic acid. Seven isolates were sensitive to both the antibiotics and three showed intermediate sensitivity to ciprofloxacin.

Characterization of PMQR genes: Results of plasmid-mediated quinolone resistance genes identified by PCR using PMQR primers revealed 29 of the 43 isolates (67.44%) harbouring PMQR genes. Among these, two (6.89%), six (20.68%) and 21 (72.41%) were positive for qnrA, qnrB and qnrS, respectively [Figure 1]A and [Figure 1]B. Seven (16.27%) isolates which were sensitive to both the antibiotics by phenotypic test, possessed plasmid-mediated quinolone resistance genes, one of which was (qnrB) sequenced and submitted to the GenBank.
Figure 1: Agarose gel electrophoresis image of PMQR genes (A) M1: 100 bp DNA ladder; lane 1: qnrB PCR product; lane 2: negative control; lanes 3-5: isolates positive for qnrB. (B) M2: 100 bp DNA ladder; lane 1: qnr S PCR product; lane 2: negative control; lanes 3-5: isolates positive for qnrB. PMQR, plasmid-mediated quinolone resistance.

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Detection of mutations in QRDR using MAMA-PCR: The MAMA PCR in the absence of mutations(s) in sensitive isolates generated two PCR products from the wild-type gene using universal forward/ reverse and MAMA reverse primers whereas, a single amplicon was produced in resistant isolates with QRDR mutation(s) due to the inhibition of PCR in the presence of two or more mismatches at the 3'end of the MAMA primer [Figure 2]A, [Figure 2]B, [Figure 2]C, [Figure 2]D. Nine (20.9%) of the 43 isolates resistant to both the antimicrobial agents harboured mutation only at amino acid position 83 of gyr A while one of the isolates possessed mutation only at position 87 of the same gene. Thirteen isolates (30.23%) resistant to both the antibiotics presented mutations at gyr A 83, 87 and at par C 80 regions. Two (4.65%) of the isolates which were resistant to only nalidixic acid harboured mutations at both gyr A position 83 and 87. Four (9.30%) isolates showing resistance to both the antibiotics harboured mutation at amino acid position 83 of gyr A and 80 of par C while four (9.30%) isolates had mutation at position 83 of gyr A and 84 of par C. An isolate of E. coli resistant to both the antibiotics displayed mutation at all the four important positions of QRDR (gyr A 83, 87 and par C 80, 84) [Table 2]. An isolate resistant to nalidixic acid did not harbour any mutation in the QRDR. None of the isolates showed mutation in the gyr B and par Eregions. The PCR products of gyr A and par C of one of the representative E. coli isolate were sequenced and analyzed. The partial sequences with possible QRDR mutations were submitted to the GenBank and were assigned GenBank accession numbers MF288967 for gyr A and MF2889868 for parC.
Figure 2: PCR products of duplex MAMA-PCR assays (A) MAMA gyrA 83. M1: 100 bp DNA ladder; lane 1: gyrA PCR; lane 2: negative control; lane 3: gyrA 83 without mutation; lane 4: gyrA 83 with mutation. (B) MAMA gyrA 87. M2:100 bp DNA ladder; lane 1: gyrA PCR; lane 2: negative control; lane 3: gyrA 87 without mutation; lane 4: gyrA 87 with mutation. (C) MAMA parC 80. M1:100 bp DNA ladder; lane 1: parC PCR; lane 2: negative control; lane 3: parC 80 without a mutation; lane 4: parC 80 with a mutation. (D) MAMA parC 84. M2:100 bp DNA ladder; lane 1: parC PCR; lane 2: negative control; lane 3: parC 84 without mutation; lane 4: parC 84 without mutation. MAMA-PCR, mismatch amplification mutation assays-polymerase chain reaction.

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Table 2: Mutation status of Escherichia coli isolates at topoisomerase targets

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   Discussion Top


Fluoroquinolones are a major class of antimicrobial drugs used widely for the treatment of infections caused by Gram-negative bacterial pathogens. E. coli being one of the major causes of several extra-intestinal and hospital-acquired infections is recognized as a major problem to tackle as it shows resistance to most of the quinolones and fluoroquinolones. Mechanisms underlying fluoroquinolone resistance were earlier thought to be confined to vertical inheritance due to the spontaneous occurrence of point mutations in QRDR regions [18]. However, now it is reported to be spread horizontally using plasmid-mediated qnr genes and efflux pump genes [19]. Although, in most of the cases, fluoroquinolone resistance is attributed to mutations in the QRDR regions, a reasonable percentage of isolates in this study also harboured PMQR genes and is in agreement with the reports of Kao et al[20]. Among the PMQR positive isolates the highest percentage harboured qnr S(77.77%) followed by qnr B(22.22%) and qnr A(11.11%);however, qep Awas not observed in any of the isolates. In contrast, a study from China [21] showed presence of qnr S in isolates followed by qep Awith the absence of qnr A and qnr B. Another study from China [22] showed the presence of qnr, aac(6′)-Ib-cr, qep A and oqx AB in 2.7, 24.5, 11.9 and 6.3 per cent, respectively of fluoroquinolone-resistant E. coli isolates. In a study from Korea, Yang et al[23] observed PMQR genes in 73.8 per cent of ciprofloxacin-resistant E. coli isolates. Although, in our study, the sample size was small, yet our results showed the presence of PMQR genes in 67.44 per cent of the E. coli isolates included. PMQRs, such as qep A and aac-(6′)-Ib, were found to be dominant in the aquatic environments [24]. This observation supported the notion that the aquatic environment might constitute the original source of PMQR genes [25]. Our study also demonstrated that extra-intestinal E. coli isolates might carry silent antibiotic resistance genes, since a few fluoroquinolone sensitive isolates (16.27%) of E. coli harboured PMQRs such as qnr B and qnr S. Perhaps this gene did not express in these isolates as phenotypic resistance. The reason for the silent nature of the qnr S and qnr B of the sensitive isolates needs further study. Silencing of antibiotic resistance genes may be a phenomenon that has not received much attention. Enne et al[26] reported silencing of several plasmid-borne (pVE46) antibiotic resistance genes such as blaoxa-2, aadA1, sul1 and tetA in E. coli isolated from pig following oral inoculation of organic piglets. There was no deletion of genes or promoter regions. However, the silent resistance genes were expressed again when the plasmid carrying resistance genes transferred to a new host. This suggested that the silencing phenomenon was due to the chromosomal effects of the host. Later, it was also found that the silencing was reversed at a low frequency of 10−6-10−10 in the original host [26]. Deekshit et al[27] suggested that the deletion of promoter region was the main reason for unexpressive nature of the chloramphenicol acetyltransferase (catA) gene in  Salmonella More Details Weltevreden. These reports show the future risk associated with the global emergence of plasmid-borne resistance pattern of clinical pathogens and these PMQRs can also contribute to the elevated levels of ciprofloxacin minimum inhibitory concentrations (MICs) in clinical isolates.

All the fluoroquinolone-resistant isolates harbouring PMQR genes also had QRDR mutations. A MAMA-PCR was used to detect mutations at four major QRDRs (gyr A 83, gyr A 87, par C 80 and par C 84). It has been used to detect point mutations in gyr A and par C regions in fluoroquinolone-resistant bacterial pathogens [28],[29],[30]. The point mutation S83L at gyr A 83 was the most commonly observed change [31],[32] followed by mutation at gyr A 87. In the present study, 76.44 per cent (33/43) of the isolates harboured point mutation at gyr A 83 and 39.53 per cent (17/43) at gyr A 87. Thus, it is important to analyze these regions to check the multiple mutation status of resistant pathogens.

Among the 43 isolates of E. coli, seven different patterns of QRDR mutations were observed. Although, in the present study, all the mutations at gyr A 87 were associated with mutation at gyr A 83, one nalidixic acid-resistant isolate (S5) harboured mutation only at gyr A position 87. Higher levels of quinolone/fluoroquinolone resistance with increased MICs in many bacterial pathogens are usually associated with double mutations in gyr A region [33]. Similarly, in our study, the isolates resistant to both the antibiotics harboured double mutation at gyr A and other regions. Some of the nalidixic acid-resistant isolates did not show any mutation in the QRDRs as reported by us earlier also [34]. Since PMQRs are mainly known to increase the MIC in resistant isolates, its exact relevance as a sole mechanism of fluoroquinolone resistance needs further study.

In conclusion, our study showed the occurrence of plasmid-mediated resistance as the second most encountered fluoroquinolone resistance mechanism among clinical isolates of E. coli. In addition, the detection of silent PMQR genes in sensitive isolates could pose a future risk of these isolates transforming into resistant forms upon antibiotic challenge. Hence, it is advisable to check for the presence of resistance determinants even in phenotypic sensitive isolates.

Financial support & sponsorship: Financial support received by the last author (IK) from Nitte University intramural research fund and Indian Council of Medical Research (AMR/37/2011-ECD-1) towards this study is acknowledged.

Conflicts of Interest: None.



 
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