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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Conclusion
 ~  References
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BRIEF COMMUNICATION
Year : 2018  |  Volume : 36  |  Issue : 1  |  Page : 131-135
 

Observation of a new pattern of mutations in gyrA and parC within Escherichia coli exhibiting fluroquinolone resistance


1 Department of Microbiology, Assam University, Silchar, Assam, India
2 Department of Microbiology, Silchar Medical College and Hospital, Silchar, Assam, India

Date of Web Publication2-May-2018

Correspondence Address:
Dr. Amitabha Bhattacharjee
Department of Microbiology, Assam University, Silchar - 788 011, Assam
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_17_181

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 ~ Abstract 

Therapeutic options with quinolones are severely compromised in infections caused by members of Enterobacteriaceae family. Mutations in chromosomal region are one of the major reasons for bacterial resistance towards this group of antibiotic. The aim of the study is to detect the mutations in gyr A and par C responsible for quinolone resistance among clinical isolates of Escherichia coli. A total of 96 quinolone-resistant clinical isolates of E. coli were collected from a tertiary care hospital of North-east India during March 2015 to August 2015. All the quinolone-resistant E. coli strains were investigated for mutations in the topoisomerases genes gyrA and parC by amplifying and sequencing the quinolone resistance determining regions. Among the 96 E. coli isolates, 83.3% were resistant to nalidixic acid and 80.2%, 66.6%, 23.9% and 50% to ciprofloxacin, norfloxacin, levofloxacin and ofloxacin, respectively. Several alterations were detected in gyrA and parC genes. Three new patterns of amino acid substitution are reported in E. coli isolates. The findings of this study warrant a review in quinolone-based therapy in this region of the world to stop or slow down the irrational use this drug.


Keywords: Escherichia coli, fluoroquinolones, gyrA, parC, quinolone resistance determining region


How to cite this article:
Dasgupta N, Paul D, Chanda DD, Chetri S, Chakravarty A, Bhattacharjee A. Observation of a new pattern of mutations in gyrA and parC within Escherichia coli exhibiting fluroquinolone resistance. Indian J Med Microbiol 2018;36:131-5

How to cite this URL:
Dasgupta N, Paul D, Chanda DD, Chetri S, Chakravarty A, Bhattacharjee A. Observation of a new pattern of mutations in gyrA and parC within Escherichia coli exhibiting fluroquinolone resistance. Indian J Med Microbiol [serial online] 2018 [cited 2018 Nov 17];36:131-5. Available from: http://www.ijmm.org/text.asp?2018/36/1/131/231660



 ~ Introduction Top


Fluoroquinolones (FQs) are broad-spectrum antimicrobial agents used for the treatment of a wide range of community-acquired and nosocomial infections. FQs inhibit deoxyribonucleic acid (DNA) gyrase and topoisomerase IV activities. Soon its discovery, widespread use of this agent has resulted in the emergence of FQ resistance. Mechanisms of resistance to quinolones include alterations in DNA gyrase and topoisomerase IV (quinolone resistance determining region [QRDR]) and decreased intracellular accumulation of the antimicrobial agent due to modifications of efflux pump activity. The primary target of FQs in Gram-negative bacteria is DNA gyrase, a type II topoisomerase required for DNA replication and transcription.[1] DNA gyrase, which is composed of two A subunits and two B subunits, is encoded by gyrA and gyrB genes, and topoisomerase IV is a tetrameric enzyme composed of two ParC and ParE subunits, encoded by parC and parE genes, respectively. Plasmid-mediated quinolone-resistant mechanisms such as qnr, aac (6') 1b-cr and qepA confer low levels of resistance but provide a favourable background in which selection of additional chromosomally encoded quinolone-resistant mechanisms may occur.[2] In Enterobacteriaceae, resistance to FQs has been shown to be associated most frequently with alterations in gyrA.[3] Resistance to FQs is a result of a combination of mechanisms acting either singly or in combination to produce the resistance phenotype. Point mutations within DNA gyrase (gyrA and gyrB genes) cause a reduction in the affinity of the enzyme for FQs and decrease the susceptibility of the organisms to FQs. Mutations at Ser83 and Asp87 codons of GyrA subunit and Ser80 and Glu84 codons of ParC subunit have been commonly reported in FQ-resistant Escherichia coli isolates worldwide.[4]

Thus, the current study was undertaken to investigate and detect the mutations in gyrA and parC responsible for quinolone resistance among clinical isolates of E. coli.

Permission – The work was approved by Assam University Institutional Ethical committee vide no IEC/AUS/C/2014-002dt-14/08/14.


 ~ Materials and Methods Top


Bacterial strain

A total of 96 consecutive nonduplicate clinical isolates of quinolone-resistant E. coli were collected from a tertiary care hospital of North-east India, during March 2015 to August 2015, which were resistant to at least one of the quinolone antibiotics tested. Of these cases of quinolone-resistant E. coli infections, 49 were diagnosed with urinary tract infection (UTI), 26 with gastrointestinal tract infection and 21 with wound infection. All of them were isolated from inpatients admitted to surgery, obstetrics and gynaecology and medicine wards of the hospital.

Screening of quinolone resistance

Screening was done on Mueller-Hinton agar (HiMedia, Mumbai, India) plate by Kirby–Bauer disc diffusion method and interpreted as per the Clinical Laboratory Standard Institute (CLSI) recommendations. The antibiotics tested were nalidixic acid (30 μg), norfloxacin (10 μg), ciprofloxacin (5 μg), ofloxacin (5 μg), gemifloxacin (5 μg), sparfloxacin (5 μg), levofloxacin (5 μg), gatifloxacin (5 μg) and lomefloxacin (10 μg) (HiMedia, Mumbai, India).

Minimum inhibitory concentration (MIC) was done for norfloxacin, ciprofloxacin, ofloxacin and levofloxacin by agar dilution method. The quality control strain used for was E. coli ATCC 25922. Results were interpreted as per the CLSI guidelines 2014.

Antibiotic susceptibility testing of quinolone-resistant isolates

Susceptibility was done by disc diffusion method for the following antibiotics: ampicillin (10 μg), cefotaxime (30 μg), ceftriaxone (30 μg), ceftazidime (30 μg), imipenem (10 μg), co-trimoxazole (25 μg), gentamicin (10 μg) and amikacin (30 μg) (HiMedia, Mumbai, India). The results were interpreted as per the CLSI guidelines 2014.

Detection of acquired quinolone-resistant determinants

To detect the presence of FQ-resistant determinants genes, polymerase chain reaction (PCR) assay was performed for the detection of qnr A,[5] qnr B,[5]qnr C,[5]qnr D,[6]qnr S [5] and aac (6')-Ib-cr.[7] Reactions were run under the following conditions; initial denaturation at 95°C for 2 min; 35 cycles of 95°C for 50 s, 53°C for 40 s and 72°C for 1.20 min and a final extension at 72°C for 5 min. The amplified PCR products were resolved by electrophoresis in 1% agarose gel and visualised after staining with ethidium bromide.

Typing of isolates

Typing of isolates was done by enterobacterial repetitive intergenic sequence (ERIC)-PCR to assess clonal relationships. Each single reaction mixture (25 μl) contained 1 μl (10 ng) of DNA suspension, 15 pmol of each primer, 12.5 μl of 2X GoTaq Green Master Mix (Promega, Madison, USA) and nuclease-free water is added to make a volume of 25 μl. The reaction condition and primers were used as described previously.[8]

Amplification of quinolont-resistance determining regions

PCR was performed to amplify the QRDRs of gyr A and par C for mutation detection. A 586 and a 265 base pair regions of DNA within the gyr A and par C gene were amplified for all the quinolone-resistant isolates The primers sequence used in this study are as follows; gyrA F-CGTCGCGTACTTTACGCCATGAACG, gyrA R-ATACCTTGCCGCGACCGGTACGG and par C F-TGTATGCGATGTCTGAACTG, par C R-CTCAATAGCAGCTCGGAATA.[9] The reaction conditions were as follows; initial denaturation at 95°C for 2 min; 32 cycles of 95°C for 25 s, 52°C for 1 min and 72°C for 1.20 min and a final extension at 72°C for 7 min.

Denaturing gradient gel electrophoresis

To identify the mutation within the QRDR, the amplified products of the above gene were subjected to denaturing gradient gel electrophoresis (DGGE). The DGGE analysis was performed using a DCode™ Universal Mutation Detection System (Bio-Rad Laboratories, Hercules, CA, USA). PCR-DGGE amplicons were loaded in polyacrylamide gels (8% [wt/vol] acrylamide in 0.5X TAE buffer) with 40%–65% denaturant gradient being 7 M urea and 40% deionised formamide. The gels were electrophoresed at a constant voltage of 20 V for 15 min followed by 5 h 30 min at 200 V. After electrophoresis, gels were stained with ethidium bromide for 5 min and destained in distilled water for 20 min with agitation, after which their image the gel was placed on a UV transilluminator and visualised in Gel Doc EZ imager (Bio-Rad, USA).


 ~ Results Top


Screening results for seven quinolone resistance are illustrated in [Table 1]. Among the collected isolates, 80 (83.3%) were resistant to nalidixic acid, 7 (7.2%) were intermediate and 9 (11%) were susceptible with MIC ≤ 0.05 μg/ml. Similarly, resistance rates to ciprofloxacin, norfloxacin, levofloxacin and ofloxacin were 80.2% (n = 77), 66.6% (n = 64), 23.9% (n = 23) and 50% (n = 48) with MIC 64, 256, 4 16 μg/ml, respectively [Table 1].
Table 1: Antibiogram profiling of isolates towards quinolone

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Multiplex PCR amplification with qnr and aac(6')-Ib-cr primers confirms the presence of aac(6')-Ib-cr genes in five isolates and no other gene could be found. All the resistant isolates were subjected for mutational analysis of QRDR region by DGGE. Amplification with oligonucleotide primers gyr A and par C amplified the expected 586 bp and 265 bp DNA fragments, respectively [Figure 1].
Figure 1: Lane 1–5 showing the amplification of gyr A (586 bp) and par C (265 bp). L-100 bp ladder

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The amplified products were subjected to DGGE. Analysis of the gel revealed four types (A–D) of band pattern of each gyrA and parC gene [Figure 2]. To determine the contribution of mutation in QRDR which attributes FQ resistance, sequencing of gyrA and parC patterns were done. When the DNA sequence of the gyrA was compared with gyrA subunit of EC493/89, it revealed nucleotide differences at many positions. Pattern A was found to have 9-point mutations (EGYMU1), Pattern B with 13-point mutations (EGYMU2), Pattern C with 11-point mutations (EGYMU3) and Pattern D was found to have 10-point mutations (EGYMU4). Similarly, the isolates showing the four mutation patterns of parC were named as EPRMU1, EPRMU2, EPRMU3 and EPRMU4. Two transition mutations were common in all the isolates. Three insertion mutations of a single nucleotide were found between 166th and 167th base by T (starting with position 1 at the A of the start codon of gyr A) of gyr A pattern EGYMU1. At 181th and 190th base, deletion of single nucleotide A was observed in all gyrA pattern [Figure 3]. Mutation in codons 83 and 87 in gyr A displays the most common alteration in clinical isolates. Transition mutation at codon 83 was a C-T that resulted in the substitution of leucine for serine in pattern A, B and D, but in pattern C (EGYMU3), transition mutation at codon 83 resulted in the substitution of glycine for serine. Another transition mutation was C-A at position 87, which resulted in an Asp87 Leu and Asp87 Asn. Twenty-one different types of mutation in gyr A were found amongst the isolates analysed. They were Trp56Met, Asn57Thr, Asn57His, Trp59Met, Asn60Thr, Asn60Asp, Lys61Gly, Lys61Ser, Ala62Thr, Ala62Pro, Tyr63Ser, Tyr63Ile, Lys64 Leu, Lys65Ile, Ser83 Leu, Ser83Gly, Val85Ala, Asp87Asn, Asp87 Leu, Arg91Tyr and Ser111Asn [Figure 4].
Figure 2: Denaturing gradient gel electrophoresis pattern of gyr A

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Figure 3: Sequence alignment of the four types of gyr A sequences with the sequence of Escherichia coli strain 493/89 (EC493/89)

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Figure 4: Protein sequence alignment of gyr A with the sequence of Escherichia coli strain 493/89 (EC493/89)

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Codons 74, 76, 77, 80, 82, 84, 89, 91, 105 and 107 showed alterations in the QRDR of the par C gene. The replacements were Tyr74 Leu, Pro76Phe, His77Arg, Ser80Ile, Ser80Arg, Cys82 Leu, Glu84Gly, Glu84 Lys, Met89 Leu, Gln91His, Asn105Thr and Gly107Ala. Mutations outside the QRDR were also observed during the study. A deletion of G at the 390th base and a transversion of G-T at the 393rd base in par C have resulted in the substitution of Glu130Asp and Leu131Phe, respectively [Figure 5] and [Figure 6].
Figure 5: Sequence alignment of the four types of par C sequences with the sequence of Escherichia coli strain 493/89 (EC493/89)

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Figure 6: Protein sequence alignment of par C with the sequence of Escherichia coli strain 493/89 (EC493/89)

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Based on the results of ERIC-PCR, isolates were grouped into eight different groups designated as 1–8 [Figure 7]. The largest one belonged to cluster-4 and consisted of seven strains detected exclusively in UTI patients with length of hospital stay of 10–20 days. It is possible that this clone was spread among the UTI patients through contaminated catheters or contaminated urinary devices. Cefotaxime came up with moderate activity followed by imipenem and ceftazidime [Table 2].
Figure 7: Dendrogram established by the biostatistical analysis program NTSYS-pc on the basis of enterobacterial repetitive intergenic sequence-polymerase chain reaction profile of Escherichia coli strains

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Table 2: Antibiotic susceptibility pattern of quinolone resistant isolates

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 ~ Discussion Top


Quinolones and FQs are the first-line drugs for the treatment of UTI and enteric infections for many years. For antibiotics such as FQs, chromosomal mutations play a more important role than horizontally transferable resistance genes in the development of clinically resistant pathogens. Resistance to FQs in bacteria is mainly mediated by spontaneous mutations in the QRDR of gyr A and par C genes, either gyr A or par C, or both genes, especially at the highly conserved residues (Ser-83 and Asp-87) of gyr A.[10] Here, we have determined how the abundance of chromosomal FQ-resistant mutations in the clinical E. coli isolates from hospital environment from India, a country with relatively high FQ consumption and resistance.

The widespread use of FQ antibiotics in clinical practice leads to the rapid development of resistant phenotype. One of the most remarkable findings of this study was the widespread resistance to quinolones and FQs. This study has found that ciprofloxacin showed a higher number of resistant strains than ofloxacin and levofloxacin among the clinical isolates of E. coli. Similar findings have been reported in a previous study of E. coli isolates in Brazil.[11] Somewhat similar results were recorded in another study from Saudi Arabia [12] where the resistance rate to ciprofloxacin is quite high. This study also highlighted that mutation in the QRDR conferring resistance to FQ is associated with high MIC values. Molecular analysis by a combination of DGGE and DNA sequencing revealed various gyr A and par C sequence patterns. Accumulation of Ser83 Leu and Asp87Asn mutations in the gyr A gene of E. coli was common. Mutation in the gyr A and par C gene leading to FQ resistance in Enterobacteriaceae,[13]Pseudomonas aeruginosa[14] and Gram-positive [15] had been reported by many authors. Previous studies showed that a high level of resistance to FQs is associated with double mutations in Gyr A and an additional mutation in Par C in E. coli.[16] We also found mutations in codon 83, 87 of gyr A and 80, 84 of par C along with some unique mutations.

To the best of our knowledge, this study also reported some unique mutation in codon 60, 64, 111 of GyrA; in addition, we identified silent mutations in codons 85, 86 and 91 in resistant strains. Mutations in par C were always found together with mutations in gyr A. This is due to topoisomerase IV being a secondary target for quinolones in E. coli.[17] However, mutations in par C play an important role in the formation of highly resistant strains. Except for one pattern A, amino acid change at codon 80 in the QRDR of the Par C protein was observed in all the analysed strains. Unique mutation in codon 74 of Par C was also reported in this study. Whether the aminoacid substitution at codon 60, 64, 111 of GyrA associated with FQ resistance needs to be verified. Furthermore, the novel mutation outside the QRDR of par C should be studied thoroughly to detect the effect on drug target enzyme activity. These data indicate that high-level FQ resistance in E. coli involves the acquisition of mutations at multiple loci in the QRDRs and strategies should be adopted to limit the spread of resistance.


 ~ Conclusion Top


This study highlighted the predominance of FQ-resistant E. coli in this region due to mutation in the chromosomal region. Overall, the results of our study suggest that acquisition of mutations in gyr A and par C genes plays a significant role in the development of high-level resistance to quinolones in certain cluster lineages of E. coli. This study also poses a serious threat to the dissemination of this new point mutation. Findings of this study warrant a review of quinolone-based therapy in this region of the world and restriction in their over-the-counter availability and irrational use.

Acknowledgement

We would like to acknowledge the help of HOD, Microbiology, Assam University, for providing infrastructure. We also acknowledge the help from Assam University Biotech Hub for providing laboratory facility to complete this work.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
 ~ References Top

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Shakibaie MR, Shakibaie S, Nave HH, Azizi O, Narouzi A. Analysis of amino acid substitution mutations of gyrA and parC genes in clonal lineage of Klebsiella pneumoniae conferring high-level quinolone resistance. J Med Microbiol Infec Dis 2014;2:1-9.  Back to cited text no. 10
    
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Al Johani SM, Akhter J, Balkhy H, El-Saed A, Younan M, Memish Z, et al. Prevalence of antimicrobial resistance among gram-negative isolates in an adult Intensive Care Unit at a tertiary care center in Saudi Arabia. Ann Saudi Med 2010;30:364-9.  Back to cited text no. 12
    
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
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