|Year : 2015 | Volume
| Issue : 2 | Page : 277-281
Detection of mutations in mtrR gene in quinolone resistant strains of N.gonorrhoeae isolated from India
SV Kulkarni1, M Bala2, J Bhattacharya3, A Risbud1
1 Department of Microbiology and Clinical Pathology, National AIDS Research Institute, Bhosari, Pune, Maharastra, India
2 Regional STD Teaching Training and Research Centre, Vardhman Mahavir Medical College and Safdarjang Hospital, New Delhi, India
3 Department of Molecular Virology, National AIDS Research Institute, Bhosari, Pune, Maharashtra, India
|Date of Submission||20-Jan-2014|
|Date of Acceptance||28-Oct-2014|
|Date of Web Publication||10-Apr-2015|
S V Kulkarni
Department of Microbiology and Clinical Pathology, National AIDS Research Institute, Bhosari, Pune, Maharastra
Source of Support: None, Conflict of Interest: None
Background and Objectives: Emergence of multi-drug resistant Neisseria gonorrhoeae resulting from new genetic mutation is a serious threat in controlling gonorrhea. This study was undertaken to identify and characterise mutations in the mtrR genes in N.gonorrhoeae isolates resistant to six different antibiotics in the quinolone group. Materials and Methods: The Minimum inhibitory concentrations (MIC) of five quinolones for 64 N.gonorrhoeae isolates isolated during Jan 2007-Jun 2009 were determined by E-test method. Mutations in MtrR loci were examined by deoxyribonucleic acid (DNA) sequencing. Results: The proportion of N.gonorrhoeae strains resistant to anti-microbials was 98.4% for norfloxacin and ofloxacin, 96.8% for enoxacin and ciprofloxacin, 95.3% for lomefloxacin. Thirty-one (48.4%) strains showed mutation (single/multiple) in mtrR gene. Ten different mutations were observed and Gly-45 → Asp, Tyr-105 → His being the most common observed mutation. Conclusion: This is the first report from India on quinolone resistance mutations in MtrRCDE efflux system in N.gonorrhoeae. In conclusion, the high level of resistance to quinolone and single or multiple mutations in mtrR gene could limit the drug choices for gonorrhoea.
Keywords: mtrR mutations, N.gonorrhoeae, quinolone resistance
|How to cite this article:|
Kulkarni S V, Bala M, Bhattacharya J, Risbud A. Detection of mutations in mtrR gene in quinolone resistant strains of N.gonorrhoeae isolated from India. Indian J Med Microbiol 2015;33:277-81
|How to cite this URL:|
Kulkarni S V, Bala M, Bhattacharya J, Risbud A. Detection of mutations in mtrR gene in quinolone resistant strains of N.gonorrhoeae isolated from India. Indian J Med Microbiol [serial online] 2015 [cited 2019 Mar 26];33:277-81. Available from: http://www.ijmm.org/text.asp?2015/33/2/277/154878
| ~ Introduction|| |
Neisseria More Details gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhea, is a Gram-negative diplococcus and is strictly a human pathogen. Clinical isolates of N.gonorrhoeae frequently exhibit high levels of antimicrobial resistance by multiple mechanisms that include porin (uptake), cellular targets of the antibiotics, and active efflux of anti-microbials by efflux pumps. ,,, The efflux-mediated resistance to fluoroquinolones which is well-recognised in a number of Gram-negative bacteria has also been reported in N.gonorrhoeae.,
Mutations in MtrRCDE efflux system (Mtr; multiple-transferable resistance) cause the enhanced expression of efflux pump. , Earlier studies have shown that the mutation in the mtrR gene was due to the alteration in Ala-39 → Thr, Gly-45 → Asp, and Tyr-105 → His region. , This study was performed to determine mutations in the mtrR gene in quinolone-resistant strains of N.gonorrhoeae strains recently isolated from India. This study was a part of a PhD thesis on Quinolone resistance mutations of Neisseria gonorrhoeae More Details in India.
| ~ Materials and Methods|| |
Sixty-four Neisseria gonorrhoeae strains were isolated from patients attending sexually transmitted disease (STD) clinics of National AIDS Research Institute (NARI), Pune (n = 16), Safdarjang hospital, New Delhi (n = 35) and 13 strains were collected from institutional sexually transmitted infection (STI)-Operational research project (Mumbai-6, Hyderabad-6, and Nagpur-1) from Jan-2007 to Jun-2009. The study was approved by the institutional ethics committee. These strains were isolated from male (n = 49) and female (n = 15) patients presenting with acute gonococcal urethritis and endocervicitis. Urethral or cervical specimens were inoculated directly on to Modified Thayer Martin selective agar, saponin lysed blood agar with Vancomycin Colistin, Nystatin and Trimethorpim inhibitors, and chocolate agar with Columbia agar base. The plates were incubated at 37 o C in a 5% CO2 and 45% moisture. N.gonorrhoeae was identified by standard methods and were stored in cryoprotective medium (Nutrient broth containing 20% glycerol) at − 70 o C until further testing. 
Susceptibility to ciprofloxacin, ofloxacin, and norfloxacin (AB BioDisk, Solna, Sweden) lomefloxacin (Hi media laboratories, Mumbai, India) was determined by E test according to the manufacturer's instructions. The MIC of all the isolates for enoxacin (Sigma chemical) was determined by agar dilution method using GC agar base and vitamin growth supplement (Hi media laboratories, Mumbai, India). World Health Organisation (WHO) reference strains A, G, L received under WHO Gonococcal Antimicrobial Surveillance Programme from WHO GASP South East Asia Region Reference Laboratory, New Delhi, ATCC 49226 and 8 sensitive strains were included as quality controls. The strains were defined as susceptible (S), intermediate susceptible (I) and resistant (R) according to the breakpoints proposed by the Clinical and Laboratory Standards Institute (CLSI). 
Polymerase chain reaction (PCR) and direct deoxyribonucleic acid (DNA) sequencing were performed to identify mutations in mtrR genes of the gonococcal isolates. The mtrR genes were amplified using PCR primers as reported by Dewi et al. 2004. 
The PCR amplification was performed in 25 ul of a reaction mixture which contained 2.5 ul of 10× Taq polymerase buffer (500 mM KCl, 100 mM Tris-HCl [pH 8.3], 15 mM MgCl2, 0.1% gelatin), 1 ul of each of the primers (25 pmol/¼ r ), 2.5 ul r of each of the deoxynucleoside triphosphates (10 mM), 0.2 ul of Taq DNA polymerase (5 U/¼ r ) (Bangalore genei, India), and 10 ul r of template DNA. Thirty-five cycles were performed for each reaction. Each cycle consisted of 30 s at 94°C, 1 min at 60°C, and 1 min at 72°C and final extension at 72°C at 5 mins.
Amplicons were separated by 1.5% agarose gel stained with ethidium bromide for visualisation. DNA was purified using Qiagen DNA purification kit (Minielute ® PCR purification kit, Qiagen GmbH, Hilden, Germany). The purified DNA was sequenced using a Big Dye Terminator Cycle Sequencing kit (Applied Biosystems, Foster, USA) and an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) following the manufacturer's instructions. Mutations were identified by comparing translated amino acid sequences to reference sequence GenBank accession no. Z 25796. World Health Organisation (WHO) reference strains A, G, L received under WHO Gonococcal Antimicrobial Surveillance Programme from WHO GASP South East Asia Region Reference Laboratory, New Delhi were included as quality controls.
The data was analysed using Statistical Package for the Social Sciences (SPSS) 15.0 for windows (SPSS Inc. 1989-2006). Association of mutation patterns with quinolone resistance were analysed using the Pearson's Chi-square test and Fisher's exact tests using SPSS 10.0 (SPSS Inc. Chicago, II, United states). A P value of < 0.05 was considered to be significant.
| ~ Results|| |
Antibiotic susceptibility testing of all the strains revealed that 98.4% of the strains were resistant to norfloxacin and ofloxacin, 96.9% to enoxacin and ciprofloxacin, 95.3% to lomefloxacin. The strains resistant to all the antibiotics showed high MIC value (≥8 ug/ml).
Thirty-one (48.4%) of the N.gonorrhoeae strains showed mutations in the mtrR gene. Of these, nine (14%) of N.gonorrhoeae strains showed ≥ 3 mutations in mtrR gene, twenty-one (32.8%) strains showed double mutations, and one (3.1%) strain showed single mutation. Asp-79 → Asn, Arg-98 → Gln, Tyr-105 → His, Asn-110 → Ser (9.4%) was the common mutation found in the strains (n = 9) showing ≥ 3 mutations in mtrR gene followed by Ala-39 → Thr, Leu-47 → Pro, Tyr-105 → His (3.1%), Ala-77 → Thr, Ala-78 → Glu, Thr-86 → Ala in (1.6%). All these strains showed resistant to all the five antibiotics (MIC ≥ 8 ug/ml). Gly-45 → Asp, Tyr-105 → His (25%) was common mutation, followed by Ala-39 → Thr, Tyr-105 → His (3.1%), Ala-78 → Glu, Thr-86 → Ala (1.6%), Asp-79 → Asn, and Tyr-105 → His (1.6%) in strains showing double mutations and single mutation.
Thirty-three strains did not show any mutations in mtrR gene. Of these thirty-three strains, five were resistant to ≤ 4 antibiotics with MICs value of ≤ 0.032 ug/ml [Table 1] and [Figure 1]. The MICs level of these isolates were significantly associated (P < 0.001) with the number of mutations in the mtrR gene.
|Figure 1: Agarose gel electrophoresis of PCR products for mtr gene: Lane 1-Sample 1, Lane 2-Sample 2, Lane 3-WHOA, Lane 4-Negative control, Lane 5-Marker 100bp|
Click here to view
|Table 1: Minimum inhibitory concentrations (MICs) for 64 strains of N.gonorrhoeae with mutation pattern in mtrR gene with geographic source |
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WHO A strain, which is sensitive to all antibiotics (MIC of ≤ 0.016 ug/ml) had no mutations in the mtrR gene while WHO L and WHO G (MIC >=4 ug/ml) had single mutation at codon 45 (Gly-45 → Asp).
| ~ Discussion|| |
The emergence and dissemination of resistance to antimicrobial agents among N. gonorrhoeae isolates have limited the variety of drugs that can be used for the treatment of gonococcal infections. Quinolones became popular therapy during the 1980s and were widely used as effective oral therapy against penicillin-resistant N gonorrhoeae. Ciprofloxacin, one of the quinolone has been one of the recommended first-line therapies for these infections worldwide, including in India. However, a number of studies have revealed an increasing frequency of ciprofloxacin-resistant N. gonorrhoeae isolates in different geographic regions. , Due to increase in quinolone resistance, cephalosporins were the only recommended class of drugs for treatment of gonococcal infections worldwide. The current regimens for treatment of gonorrhea in all STI clinics in India is syndromic management (cefixime 400 mg + azithromycin 1 gm) as per National AIDS Control Organisation (NACO) guidelines. 
The mutations in the promoter and the encoding region of the mtrR gene may contribute to high level of quinolone resistance in N. gonorrhoeae due to increased expression of the proteins of the MtrCDE system.  Here, we studied the mutations in the mtrR gene in quinolone resistance strains of N.gonorrhoeae and the results are being presented.
Since the discovery of the mtrR repressor and the realisation that it controls mtrRCDE expression in N. gonorrhoeae, the studies carried out in the 1990s examined the impact of mtrR mutations on gene expression and antibiotic resistance endowed by the MtrC-MrD-MtrE efflux pump.  Recognising its importance, recent investigations included this locus along with others to study the resistance determinants. It was found that certain mtr mutations, A39T or G45D in the mtrR DNA-binding domain or H105Y and the E202G that impact the C-terminal region of mtrR are repeatedly detected in the quinolone resistant isolates. ,
Sequence analysis of the mtrR coding region of our isolate showed the presence of Gly-45 → Asp, Ala-39 → Thr, Tyr-105 → His mutations which were also described by others. ,, In addition, we observed Leu-47 → Pro, Thr-86 → Ala, Arg-98 → Gln mutation which has not been reported so far. All the strains (n = 31) showed multiple mutations except one strains which showed single mutation at codon 45. The strains resistant to all antibiotics were significantly associated with presence of multiple mutations in the mtrR genes (P < 0.01). All these patients had symptoms of acute gonococcal urethritis and endocervicitis and were treated according to NACO guidelines i.e. with cefixime 400 mg + azithromycin 1 gm. None of the patient reported failure to the treatment.
To our knowledge, this is the first study from India that has detected mtrR mutations in N.gonorrhoeae strains resistant to various quinolones. In conclusion, single or multiple mutations in mtrR gene may have resulted in increased resistance to quinolone and thus may limit the drug choice for gonorrhea. It is important to stress continued research on antibiotic resistance to further understand the mechanism responsible for antibiotic resistance in N.gonorrhoeae.
| ~ Acknowledgement|| |
Authors thank all the NARI Clinic staff and Mrs. Leelamma Peter, Safdarjang STD Lab for providing support for collection of samples, Mr. Sudhanshu Pandey and Ms. Suvarna Sane for providing support in data analysis. The authors also thank the Director of NARI for extending support for this study.
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