- Email: [email protected]
Genotypic detection of fluoroquinolone resistance in drug-resistant Mycobacterium tuberculosis at a tertiary care centre in south Coastal Karnataka, India
Accepted Manuscript Title: Genotypic Detection of fluoroquinolone resistance in drug resistant Mycobacterium tuberculosis at a tertiary care centre in south coastal Karnataka Authors: Kiran Chawla, Ajay Kumar, Vishnu Prasad Shenoy, Sanjiban Chakrabarty, Kapaettu Satyamoorthy PII: DOI: Reference:
S2213-7165(18)30025-0 https://doi.org/10.1016/j.jgar.2018.01.023 JGAR 592
To appear in: Received date: Revised date: Accepted date:
1-9-2017 6-1-2018 29-1-2018
Please cite this article as: Kiran Chawla, Ajay Kumar, Vishnu Prasad Shenoy, Sanjiban Chakrabarty, Kapaettu Satyamoorthy, Genotypic Detection of fluoroquinolone resistance in drug resistant Mycobacterium tuberculosis at a tertiary care centre in south coastal Karnataka (2010), https://doi.org/10.1016/j.jgar.2018.01.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title page Genotypic Detection of fluoroquinolone resistance in drug resistant Mycobacterium tuberculosis at a tertiary care centre in south coastal Karnataka
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Short title: Fluoroquinolone resistance in MTB in south coastal Karnataka.
Kiran Chawla1*; Ajay Kumar 1; Vishnu Prasad Shenoy1; Sanjiban Chakrabarty2;
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Kapaettu Satyamoorthy2
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1. Department of Microbiology, Kasturba Medical College Manipal. MAHE. India
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2. School of Life Sciences, MAHE Manipal
KC
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Affiliations:
Professor & Head, Department of Microbiology,
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Kasturba Medical College Manipal. MAHE. India [email protected] Research Fellow, Department of Microbiology,
PT
AK
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Kasturba Medical College Manipal. MAHE. India
VPS
[email protected]
Associate Professor, Department of Microbiology,
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Kasturba Medical College Manipal. MAHE. India [email protected]
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Assistant Professor, School of Life Sciences MAHE. India [email protected]
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Professor & Director, School of Life Sciences MAHE. India [email protected]
*Corresponding Author: Dr Kiran Chawla, Prof & Head of Microbiology,
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Kasturba Medical College, Manipal. MAHE. Karnataka. 576104 Email: [email protected] (KC)
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Mob: 9980220484
Emergence of fluoroquinolones resistant strains of MTB is a major threat for Global
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Highlights:
Present study highlights major mutations conferring fluoroquinolone resistance in MTB
M
A
Tuberculosis control program.
isolates in south coastal Karnataka, India gyrA mutation at codon 94, 91, 90 and gyrB mutation G1498A (D500N) were the most
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common mutations associated with fluoroquinolone resistant in this region. Inclusion of fluoroquinolone testing in first line DST would be beneficial for better
PT
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treatment outcomes.
Abstract
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Objectives:
The study was aimed to characterize mutations associated with fluoroquinolone resistance in
drug resistant isolates of M. tuberculosis at a tertiary care centre in south coastal Karnataka. Methods:
Fifty drug resistant isolates were sub cultured on Lowenstein-Jensen media. Purified amplicons of gyrA and gyrB were sequenced using ABI Big Dye Terminator cycle sequencing kit. Extended sequencing PCR products were analysed using ABI 3130 Genetic Analyzer. Mutations analysis of gyrA and gyrB gene was performed using MUBII-TB-DB database. Statistical analysis was achieved using SPSS version 22. Data were compared using Chi square
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test. P-value <0.05 was considered statistically significant. Results:
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Mutations conferring resistance to fluoroquinolones were observed in 9 (18%) clinical isolates. gyrA A281G (D94G) mutation was observed in 3(6%) clinical isolates, whereas mutations
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G280T (D94Y), and A281C (D94A) were observed in 1(2%) isolate each. Mutation G1498A
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(D500N) in gyrB alone was observed in 2 (8%) clinical isolates. Two isolates (4%) had
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mutations in both gyrA and gyrB; gyrA mutation T271C (S91P) was observed in one isolate
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whereas the other had gyrA C269G (A90G) mutation but both these isolates had common G1498A (D500N) gyrB mutation. G284C mutation conferring S95Tpolymorphism was
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observed in 39(78%) isolates. Conclusion:
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gyrA mutations at codon 94, 91, 90 and gyrB mutation G1498A (D500N) were found to be the most common mutations associated with fluoroquinolone resistant in clinical isolates in the
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present study. Future studies including larger number of samples are desirable to fully explore
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the true extent of fluoroquinolone resistance and mutations associated with them.
Keywords: Fluoroquinolone, Genes, Mycobacterium Resistance, Sequencing, Karnataka, India.
INTRODUCTION
Despite extensive worldwide efforts to combat tuberculosis (TB), it remains a major global health care concern accounting for 1.67 million lives in 2016 globally [1]. One of the major challenges threatening global tuberculosis control program is emergence of drug resistant TB. Over last decade, incidence of multidrug resistant TB (MDR-TB) showing resistance to both isoniazid and rifampicin, has globally increased affecting significantly treatment outcomes and
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increasing mortality. Fluoroquinolones (moxifloxacin, gatifloxacin, levofloxacin, and
ofloxacin) are the most effective drugs against MDR-TB and are used as second line anti-
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tubercular drugs [2]. Fluoroquinolones inhibits DNA replication of Mycobacterium tuberculosis (M. tuberculosis) by inhibiting DNA gyrase- an enzyme that consist of two
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subunits encoded by genes gyrA and gyrB [3]. Being broad-spectrum antibiotics
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fluoroquinolones are also prescribed in many other bacterial infections and in many countries
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these are readily available as over the counter medication. It has led to injudicious use of these
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drugs contributing to emergence of fluoroquinolones resistant strains of M. tuberculosis in those countries [4]. Traditionally culture based method is considered as gold standard for drug
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susceptibility testing (DST) of M. tuberculosis, but standardization of DST for second line drugs is difficult and time consuming. Delayed DST results increases the risk of transmission
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of resistant strains in the community by deferring the initiation of suitable treatment [5]. Molecular diagnostic test provides rapid detection of fluoroquinolone resistance in M.
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tuberculosis by determining mutations in two small regions known as “quinolone resistance determining region (QRDR)” of gyrA and gyrB [6]. Previous studies have suggested that
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analysis of QRDR by molecular method determines resistance in vast majority of Fluoroquinolone resistant isolates [7]. Despite being one of the highest burden countries with MDR-TB, very few studies have been carried out in India to determine mutations associated with fluoroquinolone resistance in gyrA and gyrB genes in clinical isolates of MTB [8]. As per Revised National Tuberculosis Control Program annual status report in 2016, Karnataka
accounted for 68,462 TB cases, 1338 among them were MDR and 48 were XDR [9]. To our knowledge, there is almost no information regarding fluoroquinolone resistance in clinical isolates of M. tuberculosis in Udupi district of south coastal Karnataka. Thus in the present study, we aimed to look for fluoroquinolone resistance genotypically in drug resistant clinical isolates of M. tuberculosis and seeking the common mutations conferring such resistance in
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this geographical area. MATERIALS AND METHODS
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Study settings:
A cross sectional study was carried out in department of Microbiology, Kasturba Medical
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College, Manipal, in collaboration with department of Cell and Molecular Biology, School of
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Life Sciences, Manipal Academy of Higher Education (MAHE) from January 2017 to July
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Sample Processing & DNA Extraction
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2017 after obtaining the approval of Institutional Research and Ethical Committee.
For the present study, we used 50 stock isolates of drug resistant MTB strains collected from
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our previous study [10] and routine diagnostics. The stock cultures of drug resistant isolates were sub cultured on Lowenstein-Jensen (LJ) media (HiMedia, Mumbai, India), and were
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incubated for 4 to 8 weeks at 370C. After obtaining the growth on LJ media, DNA extraction was performed using commercially available kit from Qiagen (Hilden, Germany) following
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manufacturer’s instructions.
Amplification of gyrA and gyrB For amplification previously described primers for gyrA (forward: 5’-CAG CTA CAT CGACTA TGC GA-3’ and reverse:5’-GGG CTT CGG TGT ACC TCA T-3’) and gyrB (forward:5’-CGT AAG GCA CGA GAG TTG GT-3’ and reverse 5’-ATC TTG TGG TAG
CGC AGC TT-3’) were used [11].The amplification was performed in basic 50µL amplification reaction mixture containing 2 µL DNA, 25 µL 2X PCR master mix (Qiagen, Hilden, Germany), 2mM of each primer (IDT,USA) and 21 µL distilled water. Amplification was performed in Master cycler gradient 5331 (Eppendorf, Germany) following one denaturation cycle at 94 °C for 10 min, followed by 30 cycles of 45 s at 94 °C, 30 s at 56 °C
base pairs in size and were used as template for sequencing reaction.
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Sequencing
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and 50 s at 72 °C, followed by elongation at 72 °C for 10 min. Both PCR products were 320
The purified PCR amplicons of gyrA and gyrB were sequenced using the ABI Big Dye
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Terminator cycle sequencing kit (Applied Biosystems, Foster City, CA). Cycling conditions
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used were: 25 cycles of denaturation at 96°C for 10 sec, annealing at 50°C for 5sec and extension at 60°C for 4 min. Extended sequencing PCR products were purified using
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HighPrep™ PCR clean-up reagent (MagBio Genomics, Gaithersburg, USA) and analysed using ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA).
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Analysis of mutations from sequencing data
Mutational analysis of gyrA and gyrB gene was performed by aligning the sequences of
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resistant isolates to reference sequences of gyrA (AL123456; region 7302 to 9818) and gyrB (AL123456, region 5123 to 7267) using MUBII-TB-DB database which contains mutation
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associated with antibiotic resistance in M. tuberculosis [12].
Statistical analysis All statistical analysis was performed using SPSS version 22 (SPSS Inc., USA). Data were compared using Chi square test. For all analyses, a p value <0.05 was considered statistically significant.
RESULTS DNA isolated from 50 drug resistant clinical isolates were sequenced and analysed for detection of mutations conferring resistant to fluoroquinolones. Forty-seven isolates were procured from sputum of pulmonary TB patients whereas 3 isolates were procured from pus
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from extrapulmonary sites. The mean age of the patients from whom the isolates were isolated was 39.9 ± 14.27 (Mean ± SD, range: 14-72 years), thirty-five among them were male and 15
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were females. Twenty-three were previously treated cases, whereas 27 were newly diagnosed. Thirty-two isolates were Multidrug resistant, 8 were poly drug resistant other than MDR, 5
isolates were mono-resistant to rifampicin, and two isolates each were mono-resistant to
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streptomycin and isoniazid whereas one isolate was resistant to prazinamide.
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On analyzing the sequencing data, mutations conferring resistance to fluoroquinolones were
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observed in 9 (18%) clinical isolates. Mutations in gyrA alone were observed in 5 (10%) clinical
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isolates, comprising A281G (D94G) mutation in 3(6%) clinical isolates, whereas mutations
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G280T (D94Y), and A281C (D94A) observed in 1(2%) isolate each. Mutation G1498A (D500N) in gyrB alone was observed in 2 (8%) clinical isolates. Two isolates (4%) had
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mutations in both gyrA and gyrB - gyrA mutation T271C (S91P) was observed in one isolate whereas the other had gyrA C269G (A90G) mutation, but both these isolates had common
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G1498A (D500N) gyrB mutation. G284C mutation conferring S95Tpolymorphism was observed in 39(78%) isolates.
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While analyzing the drug sensitivity profile of fluoroquinolone resistant isolates we observed that 5 (55.56%) of these isolates were MDR, 2 (22.22%) isolates were mono-resistant to rifampicin. There was no significant difference between MDR isolates and Rifampicin monoresistant isolates (p 0.32). One (11.11%) isolate was resistant to isoniazid along with ethambutol whereas 1 (11.11%) isolate was mono-resistant to streptomycin. Six fluoroquinolone resistant isolates were from newly diagnosed cases whereas 3 isolates were
from previously treated cases. No statistically significant difference was seen when fluoroquinolone resistance was compared in previously treated cases and newly diagnosed cases (p 0.48). A detailed description of mutations in gyrA and gyrB, demographic details and first line DST of these isolates is shown in table I.
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DISCUSSION
Studies in the past from various parts of India have reported 3% to 35% fluoroquinolone
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resistant isolates, In the present study 18% (9/50) isolates harboured mutations conferring resistant to fluoroquinolones [7]. Majority of (70-90%) fluoroquinolone resistant isolates have
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been reported to harbour gyrA mutation. A small number of isolates show resistance either due
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to gyr B mutation, or because of efflux pumps, decreased cell-wall permeability to drugs and
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drug inactivation [3]. A recent study also emphasized the need of combined gyrA and gyrB
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sequencing in investigation of fluoroquinolone susceptibility in M. tuberculosis isolates and reported a sensitivity of >93 % for detecting fluoroquinolone resistance [13]. Thus, we
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preferred gyrA and gyrB sequencing method for detecting fluoroquinolone resistance in the present study. Though the gold standard method for detection of drug resistance in
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M.tuberculosis is the phenotypic method, but due to lack of facilities to do second line drug resistance in our settings and constraint of funds we could do only genotypic detection of
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quinolones resistance.
Resistance to fluoroquinolone is predominantly associated with mutations at codons 90, 91 and
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94 of quinolone resistance determining region (QRDR) of gyrA [12]. In the present study 55.56% (5/9) fluoroquinolone resistant isolates were observed to have mutation in codon 94 of gyrA which is associated with high level fluoroquinolone resistance [14]. Out of these, three isolates (33.33%) were observed to have A281G (D94G) mutation, which is the most common mutation associated with fluoroquinolone resistance [15]. G280T(D94Y) and A281C (D94A)
mutations observed in 1 (11.11%) isolate each in the present study have been previously reported by other researchers in 0-17.6% strains [9]. Mutations in codon 91 in gyrA are associated with lower level resistance to fluoroquinolones [16] that was noted in 1 isolate in the present study with T271C (S91P) mutation. All four isolates with gyrB mutation in present study had mutation in gyrB G1498A (D500N),
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which is reported to confer resistance only to levofloxacin and ofloxacin [17]. Further two isolates with gyrB mutation also had mutation T271C (S91P) and C269G (A90G) in gyrA. M.
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tuberculosis isolates bearing gyrA A90G mutation were reported to be hyper-susceptible to the
action of many quinolones though the isolate in present study also conferred mutation in gyrB
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[18].
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In the present study gyrA G284C(S95T) polymorphism was observed in 78% (39/50) isolates,
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which is reported as a lineage specific polymorphism as codon 95 encodes either serine (in M.
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tuberculosis H37Rv and H37Ra) or threonine (in M. tuberculosis Erdman, Mycobacterium bovis BCG and Mycobacterium africanum) [19]. Further S95T polymorphism has been
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observed in fluoroquinolone susceptible isolates by many authors and is not considered as a fluoroquinolone resistant conferring mutation [20].
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Although 66.67% (6/9) of fluoroquinolone resistant isolates in the present study were from newly diagnosed cases and 55.56% (5/9) were multidrug resistant, any significant association
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of fluoroquinolone resistance with newly diagnosed cases and multidrug resistances was not observed due to limited sample size, which is one of the limitations of this study.
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To conclude gyrA mutation at codon 94, 91, 90 and gyrB mutation G1498A (D500N) were found to be the most common mutations associated with fluoroquinolone resistance in clinical isolates in Udupi district of south coastal Karnataka. Though present study was a preliminary insight; future studies including larger number of samples are desirable to fully explore the true extent of fluoroquinolone resistance and mutations associated with them. In past few years, a
gradual increase in XDR cases has been observed worldwide thus fluoroquinolone testing should be included as a part of the first line DST.
Acknowledgement
Conflict of interest: All the authors declare no conflicts of interest.
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Declarations
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We thank all the technical staff for their various roles and contributions to this project.
Funding: No funding
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Competing Interests: No conflict of interest
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Ethical Approval: The ethical approval was obtained from Institutional Research and Ethical
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Committee, Manipal University
References 1. World Health Organization (WHO). Global Tuberculosis report 2017. Available from: http://apps.who.int/iris/bitstream/10665/259366/1/9789241565516-eng.pdf?ua=1 2. World Health Organization, 2014. Companion handbook to the WHO guidelines for the programmatic
management
of
drug-resistant
tuberculosis.
Available
from:
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https://www.ncbi.nlm.ih.gov/books/NBK247420/pdf/Bookshelf_NBK247420.pdf
3. Hillemann D, Rüsch-Gerdes S, Richter E. Feasibility of the Geno- Type MTBDRsl assay
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for fluoroquinolone, amikacin-capreomycin, and ethambutol resistance testing of
Mycobacterium tuberculosis strains and clinical specimens. J Clin Microbiol 2009;
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47:1767–1772.
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4. Dooley KE, Golub J, Goes FS, Merz WG, Sterling TR. Empiric treatment of community
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acquired pneumonia with fluoroquinolones and delays in treatment of tuberculosis. Clin
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Infect Dis 2002; 34:1607–1612.
5. Jabeen K, Shakoor S, Hasan R. Fluoroquinolone-resistant tuberculosis: implications in
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settings with weak healthcare systems. Int J Infect Dis 2015; 32:118–123. 6. Sun Z, J Zhang, X Zhang, S Wang, Y Zhang, and C. Li. Comparison of gyrA gene
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mutations between laboratory-selected ofloxacin-resistant Mycobacterium tuberculosis strains and clinical isolates. Int. J. Antimicrob Agents 2008; 31:115–121.
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7. Chang KC, Yew WW, Chan RCY. Rapid assays for fluoroquinolone resistance in Mycobacterium tuberculosis: a systematic review and meta analysis. J Antimicrob
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Chemother 2010; 65:1551-1561.
8. Singhal R, Reynolds PR, Marola JL, Epperson LE, Arora J, Sarin R, et al. Sequence Analysis of Fluoroquinolone Resistance-Associated Genes gyrA and gyrB in Clinical Mycobacterium tuberculosis Isolates from Patients Suspected of Having MultidrugResistant Tuberculosis in New Delhi, India. J. Clin. Microbiol 2016; 54:2298-2305.
9. TB India 2017, Revised National Tuberculosis Control Program Annual status report. Central TB Division. Available from: https://tbcindia.gov.in. 10. Rao P, Chawla K, Shenoy VP, Mukhopadhyay C, Brahmavar V, Kamath A, et al. Study of drug resistance in pulmonary tuberculosis cases in south coastal Karnataka. J of Epidem and Glob Healt 2015; 5: 275– 281.
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11. Li J, Gao X, Luo T, Wu J, Sun G, Liu Q, et al. Association of gyrA/B mutations and
resistance levels to fluoroquinolones in clinical isolates of Mycobacterium tuberculosis.
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Emerg Microbes Infect 2014; 3(3):e19.
12. Flandrois JP, Lina G, Dumitrescu O. MUBII-TB-DB: a database of mutations associated
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with antibiotic resistance in Mycobacterium tuberculosis. BMC bioinformatics 2014; 14;
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15:107.
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13. Bernard C, Veziris N, Brossier F, Sougakoff W, Jarlier V, Robert J, Aubry A. Molecular
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diagnosis of fluoroquinolone resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 2015;59(3):1519-1524.
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14. Maruri F, Sterling TR, Kaiga AW, Blackman A, van der Heijden YF, Mayer C et al. A systematic review of gyrase mutations associated with fluoroquinolone-resistant
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Mycobacterium tuberculosis and a proposed gyrase numbering system. J Antimicrob Chemother 2012; 67:819–831.
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15. Avalos E, Catanzaro D, Catanzaro A, Ganiats T, Brodine S, Alcaraz J et al. Frequency and geographic distribution of gyrA and gyrB mutations associated with fluoroquinolone
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resistance in clinical Mycobacterium tuberculosis isolates: a systematic review. PLoS One 2015; 10(3) :e0120470. https://doi.org/10.1371/journal.pone.0120470
16. Sirgel FA, Warren RM, Streicher EM, Victor TC, van Helden PD, Böttger EC. gyrA mutations and phenotypic susceptibility levels to ofloxacin and moxifloxacin in clinical
isolates of Mycobacterium tuberculosis. J Antimicrob Chemother . 2012 Feb 22; 67:10881093. 17. Malik S, Willby M, Sikes D, Tsodikov OV, Posey JE New Insights into Fluoroquinolone Resistance in Mycobacterium tuberculosis: Functional Genetic Analysis of gyrA and gyrB Mutations. PLoS ONE 2012; 7(6): e39754. doi:10.1371/journal.pone.0039754
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18. Aubry A, Veziris N, Cambau E, Truffot-Pernot C, Jarlier V, Fisher LM. Novel gyrase
mutations in quinolone-resistant and-hypersusceptible clinical isolates of Mycobacterium
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tuberculosis: functional analysis of mutant enzymes. Antimicrob Agents Chemother 2006; 50:104-112.
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19. Takiff HE, Salazar L, Guerrero C, Philipp W, Huang WM, Kreiswirth B, et al. Cloning
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and nucleotide sequence of Mycobacterium tuberculosis gyrA and gyrB genes and
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detection of quinolone resistance mutations. Antimicrob Agents Chemother 1994; 38:773–
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780.
20. Bravo LT, Tuohy MJ, Ang C. Pyrosequencing for rapid detection of Mycobacterium
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tuberculosis resistance to rifampin, isoniazid, and fluoroquinolones. J Clin Microbiol .
A
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2009; 47:3985–3990.
Table 1: Description of Fluoroquinolone resistant isolates. Isolate
Age
ID
(year)
Gyr_5
44
Gyr_12
55
Gender
Type
of
Mutation in gyrA
Mutation in gyrB
Case Male
Male
New case
New case
First line DST SM
INH
RMP
EMB
PZA
R
S
S
S
S
T271C
G1498A
(S91P)
(D500N)
G280T (D94Y),
-
R
R
R
R
S
-
S
S
R
S
S
-
R
R
Gyr_13
36
Male
New case
A281G (D94G)
Gyr_14
Gyr_18
36
23
Female
Male
Previously
A281G (D94G),
treated
G284C (S95T)
New case
A281C (D94A),
-
G284C (S95T) 58
Male
Previously
-
G1498A
Gyr_29
26
Female
Previously
(D500N)
Female
New case
S
S
R
S
R
S
S
S
R
S
S
G1498A
S
R
R
S
S
A281G (D94A),
S
R
R
S
S
G1498A
S
S
R
S
S
(D500N) -
A
29
R
C269G (A90G)
treated Gyr_35
R
N
treated
U
Gyr_26
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G284C (S95T)
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G284C (S95T)
Gyr_50
56
Male
New case
M
G284C (S95T) G284C (S95T)
(D500N)
A
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Sensitive, R: Resistance
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SM: Streptomycin, INH: Isoniazid, RMP: Rifampicin, EMB: Ethambutol, PZA: Pyrazinamide, S:
S2213-7165(18)30025-0 https://doi.org/10.1016/j.jgar.2018.01.023 JGAR 592
To appear in: Received date: Revised date: Accepted date:
1-9-2017 6-1-2018 29-1-2018
Please cite this article as: Kiran Chawla, Ajay Kumar, Vishnu Prasad Shenoy, Sanjiban Chakrabarty, Kapaettu Satyamoorthy, Genotypic Detection of fluoroquinolone resistance in drug resistant Mycobacterium tuberculosis at a tertiary care centre in south coastal Karnataka (2010), https://doi.org/10.1016/j.jgar.2018.01.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title page Genotypic Detection of fluoroquinolone resistance in drug resistant Mycobacterium tuberculosis at a tertiary care centre in south coastal Karnataka
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Short title: Fluoroquinolone resistance in MTB in south coastal Karnataka.
Kiran Chawla1*; Ajay Kumar 1; Vishnu Prasad Shenoy1; Sanjiban Chakrabarty2;
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Kapaettu Satyamoorthy2
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1. Department of Microbiology, Kasturba Medical College Manipal. MAHE. India
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2. School of Life Sciences, MAHE Manipal
KC
M
Affiliations:
Professor & Head, Department of Microbiology,
ED
Kasturba Medical College Manipal. MAHE. India [email protected] Research Fellow, Department of Microbiology,
PT
AK
CC E
Kasturba Medical College Manipal. MAHE. India
VPS
[email protected]
Associate Professor, Department of Microbiology,
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Kasturba Medical College Manipal. MAHE. India [email protected]
SC
Assistant Professor, School of Life Sciences MAHE. India [email protected]
KS
Professor & Director, School of Life Sciences MAHE. India [email protected]
*Corresponding Author: Dr Kiran Chawla, Prof & Head of Microbiology,
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Kasturba Medical College, Manipal. MAHE. Karnataka. 576104 Email: [email protected] (KC)
SC R
Mob: 9980220484
Emergence of fluoroquinolones resistant strains of MTB is a major threat for Global
N
U
Highlights:
Present study highlights major mutations conferring fluoroquinolone resistance in MTB
M
A
Tuberculosis control program.
isolates in south coastal Karnataka, India gyrA mutation at codon 94, 91, 90 and gyrB mutation G1498A (D500N) were the most
ED
common mutations associated with fluoroquinolone resistant in this region. Inclusion of fluoroquinolone testing in first line DST would be beneficial for better
PT
CC E
treatment outcomes.
Abstract
A
Objectives:
The study was aimed to characterize mutations associated with fluoroquinolone resistance in
drug resistant isolates of M. tuberculosis at a tertiary care centre in south coastal Karnataka. Methods:
Fifty drug resistant isolates were sub cultured on Lowenstein-Jensen media. Purified amplicons of gyrA and gyrB were sequenced using ABI Big Dye Terminator cycle sequencing kit. Extended sequencing PCR products were analysed using ABI 3130 Genetic Analyzer. Mutations analysis of gyrA and gyrB gene was performed using MUBII-TB-DB database. Statistical analysis was achieved using SPSS version 22. Data were compared using Chi square
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test. P-value <0.05 was considered statistically significant. Results:
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Mutations conferring resistance to fluoroquinolones were observed in 9 (18%) clinical isolates. gyrA A281G (D94G) mutation was observed in 3(6%) clinical isolates, whereas mutations
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G280T (D94Y), and A281C (D94A) were observed in 1(2%) isolate each. Mutation G1498A
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(D500N) in gyrB alone was observed in 2 (8%) clinical isolates. Two isolates (4%) had
A
mutations in both gyrA and gyrB; gyrA mutation T271C (S91P) was observed in one isolate
M
whereas the other had gyrA C269G (A90G) mutation but both these isolates had common G1498A (D500N) gyrB mutation. G284C mutation conferring S95Tpolymorphism was
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observed in 39(78%) isolates. Conclusion:
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gyrA mutations at codon 94, 91, 90 and gyrB mutation G1498A (D500N) were found to be the most common mutations associated with fluoroquinolone resistant in clinical isolates in the
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present study. Future studies including larger number of samples are desirable to fully explore
A
the true extent of fluoroquinolone resistance and mutations associated with them.
Keywords: Fluoroquinolone, Genes, Mycobacterium Resistance, Sequencing, Karnataka, India.
INTRODUCTION
Despite extensive worldwide efforts to combat tuberculosis (TB), it remains a major global health care concern accounting for 1.67 million lives in 2016 globally [1]. One of the major challenges threatening global tuberculosis control program is emergence of drug resistant TB. Over last decade, incidence of multidrug resistant TB (MDR-TB) showing resistance to both isoniazid and rifampicin, has globally increased affecting significantly treatment outcomes and
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increasing mortality. Fluoroquinolones (moxifloxacin, gatifloxacin, levofloxacin, and
ofloxacin) are the most effective drugs against MDR-TB and are used as second line anti-
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tubercular drugs [2]. Fluoroquinolones inhibits DNA replication of Mycobacterium tuberculosis (M. tuberculosis) by inhibiting DNA gyrase- an enzyme that consist of two
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subunits encoded by genes gyrA and gyrB [3]. Being broad-spectrum antibiotics
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fluoroquinolones are also prescribed in many other bacterial infections and in many countries
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these are readily available as over the counter medication. It has led to injudicious use of these
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drugs contributing to emergence of fluoroquinolones resistant strains of M. tuberculosis in those countries [4]. Traditionally culture based method is considered as gold standard for drug
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susceptibility testing (DST) of M. tuberculosis, but standardization of DST for second line drugs is difficult and time consuming. Delayed DST results increases the risk of transmission
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of resistant strains in the community by deferring the initiation of suitable treatment [5]. Molecular diagnostic test provides rapid detection of fluoroquinolone resistance in M.
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tuberculosis by determining mutations in two small regions known as “quinolone resistance determining region (QRDR)” of gyrA and gyrB [6]. Previous studies have suggested that
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analysis of QRDR by molecular method determines resistance in vast majority of Fluoroquinolone resistant isolates [7]. Despite being one of the highest burden countries with MDR-TB, very few studies have been carried out in India to determine mutations associated with fluoroquinolone resistance in gyrA and gyrB genes in clinical isolates of MTB [8]. As per Revised National Tuberculosis Control Program annual status report in 2016, Karnataka
accounted for 68,462 TB cases, 1338 among them were MDR and 48 were XDR [9]. To our knowledge, there is almost no information regarding fluoroquinolone resistance in clinical isolates of M. tuberculosis in Udupi district of south coastal Karnataka. Thus in the present study, we aimed to look for fluoroquinolone resistance genotypically in drug resistant clinical isolates of M. tuberculosis and seeking the common mutations conferring such resistance in
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this geographical area. MATERIALS AND METHODS
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Study settings:
A cross sectional study was carried out in department of Microbiology, Kasturba Medical
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College, Manipal, in collaboration with department of Cell and Molecular Biology, School of
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Life Sciences, Manipal Academy of Higher Education (MAHE) from January 2017 to July
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Sample Processing & DNA Extraction
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2017 after obtaining the approval of Institutional Research and Ethical Committee.
For the present study, we used 50 stock isolates of drug resistant MTB strains collected from
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our previous study [10] and routine diagnostics. The stock cultures of drug resistant isolates were sub cultured on Lowenstein-Jensen (LJ) media (HiMedia, Mumbai, India), and were
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incubated for 4 to 8 weeks at 370C. After obtaining the growth on LJ media, DNA extraction was performed using commercially available kit from Qiagen (Hilden, Germany) following
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manufacturer’s instructions.
Amplification of gyrA and gyrB For amplification previously described primers for gyrA (forward: 5’-CAG CTA CAT CGACTA TGC GA-3’ and reverse:5’-GGG CTT CGG TGT ACC TCA T-3’) and gyrB (forward:5’-CGT AAG GCA CGA GAG TTG GT-3’ and reverse 5’-ATC TTG TGG TAG
CGC AGC TT-3’) were used [11].The amplification was performed in basic 50µL amplification reaction mixture containing 2 µL DNA, 25 µL 2X PCR master mix (Qiagen, Hilden, Germany), 2mM of each primer (IDT,USA) and 21 µL distilled water. Amplification was performed in Master cycler gradient 5331 (Eppendorf, Germany) following one denaturation cycle at 94 °C for 10 min, followed by 30 cycles of 45 s at 94 °C, 30 s at 56 °C
base pairs in size and were used as template for sequencing reaction.
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Sequencing
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and 50 s at 72 °C, followed by elongation at 72 °C for 10 min. Both PCR products were 320
The purified PCR amplicons of gyrA and gyrB were sequenced using the ABI Big Dye
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Terminator cycle sequencing kit (Applied Biosystems, Foster City, CA). Cycling conditions
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used were: 25 cycles of denaturation at 96°C for 10 sec, annealing at 50°C for 5sec and extension at 60°C for 4 min. Extended sequencing PCR products were purified using
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HighPrep™ PCR clean-up reagent (MagBio Genomics, Gaithersburg, USA) and analysed using ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA).
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Analysis of mutations from sequencing data
Mutational analysis of gyrA and gyrB gene was performed by aligning the sequences of
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resistant isolates to reference sequences of gyrA (AL123456; region 7302 to 9818) and gyrB (AL123456, region 5123 to 7267) using MUBII-TB-DB database which contains mutation
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associated with antibiotic resistance in M. tuberculosis [12].
Statistical analysis All statistical analysis was performed using SPSS version 22 (SPSS Inc., USA). Data were compared using Chi square test. For all analyses, a p value <0.05 was considered statistically significant.
RESULTS DNA isolated from 50 drug resistant clinical isolates were sequenced and analysed for detection of mutations conferring resistant to fluoroquinolones. Forty-seven isolates were procured from sputum of pulmonary TB patients whereas 3 isolates were procured from pus
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from extrapulmonary sites. The mean age of the patients from whom the isolates were isolated was 39.9 ± 14.27 (Mean ± SD, range: 14-72 years), thirty-five among them were male and 15
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were females. Twenty-three were previously treated cases, whereas 27 were newly diagnosed. Thirty-two isolates were Multidrug resistant, 8 were poly drug resistant other than MDR, 5
isolates were mono-resistant to rifampicin, and two isolates each were mono-resistant to
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streptomycin and isoniazid whereas one isolate was resistant to prazinamide.
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On analyzing the sequencing data, mutations conferring resistance to fluoroquinolones were
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observed in 9 (18%) clinical isolates. Mutations in gyrA alone were observed in 5 (10%) clinical
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isolates, comprising A281G (D94G) mutation in 3(6%) clinical isolates, whereas mutations
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G280T (D94Y), and A281C (D94A) observed in 1(2%) isolate each. Mutation G1498A (D500N) in gyrB alone was observed in 2 (8%) clinical isolates. Two isolates (4%) had
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mutations in both gyrA and gyrB - gyrA mutation T271C (S91P) was observed in one isolate whereas the other had gyrA C269G (A90G) mutation, but both these isolates had common
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G1498A (D500N) gyrB mutation. G284C mutation conferring S95Tpolymorphism was observed in 39(78%) isolates.
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While analyzing the drug sensitivity profile of fluoroquinolone resistant isolates we observed that 5 (55.56%) of these isolates were MDR, 2 (22.22%) isolates were mono-resistant to rifampicin. There was no significant difference between MDR isolates and Rifampicin monoresistant isolates (p 0.32). One (11.11%) isolate was resistant to isoniazid along with ethambutol whereas 1 (11.11%) isolate was mono-resistant to streptomycin. Six fluoroquinolone resistant isolates were from newly diagnosed cases whereas 3 isolates were
from previously treated cases. No statistically significant difference was seen when fluoroquinolone resistance was compared in previously treated cases and newly diagnosed cases (p 0.48). A detailed description of mutations in gyrA and gyrB, demographic details and first line DST of these isolates is shown in table I.
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DISCUSSION
Studies in the past from various parts of India have reported 3% to 35% fluoroquinolone
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resistant isolates, In the present study 18% (9/50) isolates harboured mutations conferring resistant to fluoroquinolones [7]. Majority of (70-90%) fluoroquinolone resistant isolates have
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been reported to harbour gyrA mutation. A small number of isolates show resistance either due
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to gyr B mutation, or because of efflux pumps, decreased cell-wall permeability to drugs and
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drug inactivation [3]. A recent study also emphasized the need of combined gyrA and gyrB
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sequencing in investigation of fluoroquinolone susceptibility in M. tuberculosis isolates and reported a sensitivity of >93 % for detecting fluoroquinolone resistance [13]. Thus, we
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preferred gyrA and gyrB sequencing method for detecting fluoroquinolone resistance in the present study. Though the gold standard method for detection of drug resistance in
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M.tuberculosis is the phenotypic method, but due to lack of facilities to do second line drug resistance in our settings and constraint of funds we could do only genotypic detection of
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quinolones resistance.
Resistance to fluoroquinolone is predominantly associated with mutations at codons 90, 91 and
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94 of quinolone resistance determining region (QRDR) of gyrA [12]. In the present study 55.56% (5/9) fluoroquinolone resistant isolates were observed to have mutation in codon 94 of gyrA which is associated with high level fluoroquinolone resistance [14]. Out of these, three isolates (33.33%) were observed to have A281G (D94G) mutation, which is the most common mutation associated with fluoroquinolone resistance [15]. G280T(D94Y) and A281C (D94A)
mutations observed in 1 (11.11%) isolate each in the present study have been previously reported by other researchers in 0-17.6% strains [9]. Mutations in codon 91 in gyrA are associated with lower level resistance to fluoroquinolones [16] that was noted in 1 isolate in the present study with T271C (S91P) mutation. All four isolates with gyrB mutation in present study had mutation in gyrB G1498A (D500N),
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which is reported to confer resistance only to levofloxacin and ofloxacin [17]. Further two isolates with gyrB mutation also had mutation T271C (S91P) and C269G (A90G) in gyrA. M.
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tuberculosis isolates bearing gyrA A90G mutation were reported to be hyper-susceptible to the
action of many quinolones though the isolate in present study also conferred mutation in gyrB
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[18].
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In the present study gyrA G284C(S95T) polymorphism was observed in 78% (39/50) isolates,
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which is reported as a lineage specific polymorphism as codon 95 encodes either serine (in M.
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tuberculosis H37Rv and H37Ra) or threonine (in M. tuberculosis Erdman, Mycobacterium bovis BCG and Mycobacterium africanum) [19]. Further S95T polymorphism has been
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observed in fluoroquinolone susceptible isolates by many authors and is not considered as a fluoroquinolone resistant conferring mutation [20].
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Although 66.67% (6/9) of fluoroquinolone resistant isolates in the present study were from newly diagnosed cases and 55.56% (5/9) were multidrug resistant, any significant association
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of fluoroquinolone resistance with newly diagnosed cases and multidrug resistances was not observed due to limited sample size, which is one of the limitations of this study.
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To conclude gyrA mutation at codon 94, 91, 90 and gyrB mutation G1498A (D500N) were found to be the most common mutations associated with fluoroquinolone resistance in clinical isolates in Udupi district of south coastal Karnataka. Though present study was a preliminary insight; future studies including larger number of samples are desirable to fully explore the true extent of fluoroquinolone resistance and mutations associated with them. In past few years, a
gradual increase in XDR cases has been observed worldwide thus fluoroquinolone testing should be included as a part of the first line DST.
Acknowledgement
Conflict of interest: All the authors declare no conflicts of interest.
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Declarations
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We thank all the technical staff for their various roles and contributions to this project.
Funding: No funding
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Competing Interests: No conflict of interest
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Ethical Approval: The ethical approval was obtained from Institutional Research and Ethical
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Committee, Manipal University
References 1. World Health Organization (WHO). Global Tuberculosis report 2017. Available from: http://apps.who.int/iris/bitstream/10665/259366/1/9789241565516-eng.pdf?ua=1 2. World Health Organization, 2014. Companion handbook to the WHO guidelines for the programmatic
management
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Table 1: Description of Fluoroquinolone resistant isolates. Isolate
Age
ID
(year)
Gyr_5
44
Gyr_12
55
Gender
Type
of
Mutation in gyrA
Mutation in gyrB
Case Male
Male
New case
New case
First line DST SM
INH
RMP
EMB
PZA
R
S
S
S
S
T271C
G1498A
(S91P)
(D500N)
G280T (D94Y),
-
R
R
R
R
S
-
S
S
R
S
S
-
R
R
Gyr_13
36
Male
New case
A281G (D94G)
Gyr_14
Gyr_18
36
23
Female
Male
Previously
A281G (D94G),
treated
G284C (S95T)
New case
A281C (D94A),
-
G284C (S95T) 58
Male
Previously
-
G1498A
Gyr_29
26
Female
Previously
(D500N)
Female
New case
S
S
R
S
R
S
S
S
R
S
S
G1498A
S
R
R
S
S
A281G (D94A),
S
R
R
S
S
G1498A
S
S
R
S
S
(D500N) -
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29
R
C269G (A90G)
treated Gyr_35
R
N
treated
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Gyr_26
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G284C (S95T)
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G284C (S95T)
Gyr_50
56
Male
New case
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G284C (S95T) G284C (S95T)
(D500N)
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Sensitive, R: Resistance
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SM: Streptomycin, INH: Isoniazid, RMP: Rifampicin, EMB: Ethambutol, PZA: Pyrazinamide, S: