Sequence analysis of fluoroquinolone resistance associated ... · 4 61 INTRODUCTION 62 Treatment of multidrug-resistant tuberculosis (MDR-TB) patients, with strains resistant to 63

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Sequence analysis of fluoroquinolone resistance associated genes gyrA and gyrB in clinical 1

Mycobacterium tuberculosis isolates from suspected multidrug-resistant tuberculosis 2

patients in New Delhi, India 3

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Authors: Ritu Singhala, b, Paul R. Reynoldsc, d, Jamie Marolaa, L. Elaine Eppersone, Jyoti Arorab, 5

Rohit Sarinf, Vithal Prasad Myneedu b, #, Michael Stronge, g, Max Salfinger a, h, # 6

a Mycobacteriology and Pharmacokinetics Laboratories, National Jewish Health, Denver, CO, 7

USA; b National Reference Laboratory and Centre of Excellence (WHO), Department of 8

Microbiology, National Institute of Tuberculosis and Respiratory Diseases, New Delhi, India; c 9

Department of Academic Affairs, National Jewish Health; d Department of Pediatrics, National 10

Jewish Health; e Center for Genes, Environment, and Health, National Jewish Health; f 11

Department of Tuberculosis and Chest Diseases, National Institute of Tuberculosis and 12

Respiratory Diseases, New Delhi, India; g Computational Bioscience Program, University of 13

Colorado, Denver, CO, USA; h Department of Medicine, National Jewish Health 14

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Running Title: Sequence analysis of gyrA and gyrB genes 16

#Correspondence to: 17

Max Salfinger ([email protected]) and 18

Vithal Prasad Myneedu ([email protected]) 19

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JCM Accepted Manuscript Posted Online 22 June 2016J. Clin. Microbiol. doi:10.1128/JCM.00670-16Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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Address: 23

Max Salfinger, MD 24

Mycobacteriology & Pharmacokinetics Laboratories, 25

National Jewish Health, K412a 26

1400 Jackson Street, 27

Denver, CO 80206, USA 28

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Dr. Vithal Prasad Myneedu 30

Head of Laboratory, National Reference Laboratory and Centre of Excellence (World Health 31

Organization, WHO), 32

Department of Microbiology, National Institute of Tuberculosis and Respiratory Diseases, 33

Sri Aurobindo Marg, 34

New Delhi, 110030, India 35

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ABSTRACT 39

Fluoroquinolones (FQ) are broad-spectrum antibiotics recommended for treatment of 40

multidrug-resistant tuberculosis (MDR-TB) patients. FQ resistance, caused by mutations in the 41

gyrA and gyrB genes of Mycobacterium tuberculosis (MTB), is increasingly reported world-42

wide; however, information on mutations occurring in strains from the Indian sub-continent is 43

scarce. Hence, in this study we aimed to characterize mutations in the gyrA and gyrB genes of 44

acid-fast bacilli (AFB) smear positive sediments or MTB isolates from AFB smear negative TB 45

suspects from India. A total of 152 samples from suspected MDR-TB patients were included in 46

the study. Of these, 146 strains detected were characterized by sequencing of the gyrA and 47

gyrB genes. The extracted DNA was subjected to successive amplification using a nested PCR 48

protocol, followed by sequencing. 49

A total of 27 mutations were observed in 25 strains in the gyrA gene, while no mutations 50

were observed in the gyrB gene. The most common mutation occurred at amino-acid position 51

94 (13/27; 48.1%) of which the D94G mutation was the most prevalent. The gyrA mutations 52

were significantly associated with rifampin (RIF) resistant-TB patients in comparison with non-53

RIF resistant patients. Heterozygosity was seen in 4/27 (14.8%) mutations, suggesting the 54

occurrence of mixed populations with different antimicrobial susceptibilities. 55

A high rate of FQ-resistant mutations (17.1%), were obtained among the isolates of TB 56

patients suspected of MDR-TB. These observations emphasize the need for accurate and 57

rapid molecular tests for the detection of FQ-resistant mutations at the time of MDR-TB 58

diagnosis. 59

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INTRODUCTION 61

Treatment of multidrug-resistant tuberculosis (MDR-TB) patients, with strains resistant to 62

rifampin (RIF) and isoniazid (INH), is further complicated with the presence of fluoroquinolone 63

(FQ) resistance, due to prolonged, limited, and expensive treatment options (1,2). A recent 64

meta-analysis on response to treatment of 6,724 MDR-TB patients from 26 centers revealed 65

that treatment success of patients with MDR-TB was 64%, while success of patients with 66

MDR-TB plus FQ resistance was only 48% (3). This clearly indicates the need for routine 67

molecular screening for FQ resistance-associated molecular markers so that treatment of 68

such patients can be better optimized and without delay. Many of such cases develop 69

extensively drug-resistant tuberculosis (XDR-TB), defined as MDR-TB plus resistance to any 70

one FQ and any of the aminoglycosides/cyclic peptides (A-CP) (4). This poses an even more 71

serious threat in TB management since only 40% of XDR-TB patients have successful 72

treatment outcomes (2). Previous studies suggest that 5.4% (95% CI: 3.4–7.5) of MDR-TB 73

cases may actually be XDR-TB (2). 74

FQ are oral, antibacterial agents that have activity against Mycobacterium tuberculosis (MTB) 75

(5, 6). Hence, FQs are recommended for the treatment of MDR-TB patients and patients with 76

intolerance to RIF (2). FQ are also being evaluated in conjunction with first-line regimens for 77

newly diagnosed drug susceptible TB (7). Since FQ are broad-spectrum antibiotics, these 78

antimicrobials are often over-prescribed by clinicians for diverse infections (8). In many 79

resource-limited countries, FQ are readily available as over-the-counter medications, 80

propagating their misuse (9, 10). Such indiscriminate use has contributed to the increasing 81

emergence of FQ-resistant MTB world-wide, as well as India (2, 11). Newer FQ-based 82


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regimens are being explored, which necessitates the knowledge of FQ resistance data from 83

high TB burden countries (12). 84

Traditionally, culture-based methods have been used for determining antimicrobial 85

susceptibility, which usually takes several weeks. Thus, a patient harboring a drug resistant 86

MTB strain may continue to spread undetected in the community (13). Molecular methods, 87

however, have decreased the time for detection of drug resistance, especially for MDR-TB 88

(14). Among these methods, reverse hybridization or line probe assays (LPA) have been 89

implemented as part of the national TB programs in high burden countries for rapid detection 90

of MDR-TB (15). These assays are directed towards the detection of the most common 91

mutations for rifampin resistance (14). In contrast to RIF resistance tests, fewer molecular 92

tests are available for diagnosing FQ resistance, and the existing methods are limited by lower 93

level of association with phenotypic drug resistance (16, 17, 18). 94

The principal target of FQ drugs in MTB is the DNA gyrase protein complex, which catalyzes 95

the ATP-dependent introduction of negative supercoils into closed circular DNA molecules. 96

The gyrA and gyrB genes encode the two subunits of DNA gyrase, which form the catalytically 97

active GyrA2 GyrB2 complex. Resistance to FQ in MTB is primarily caused by mutations in the 98

quinolone resistance-determining region (QRDR), a conserved 320bp region within the gyrA-99

gene. FQ resistance has also been shown to be associated, but less commonly attributable, to 100

mutations in a 375 bp region of the gyrB gene (5). 101

Mutations in the gyrA and gyrB genes of clinical isolates of MTB have been described in many 102

populations and studies (5, 17, 18, 19, 20, 21, 22, 23); however, the information on mutations 103

in the gyrA-gyrB genes from the Indian sub-continent is scarce (24, 25), although India has 104

one of the highest percentages of MDR-TB cases in the world (2). There is evidence that 105


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different mutations are associated with different levels of phenotypic resistance, or different 106

minimum inhibitory concentrations (MIC), to FQs; moxifloxacin (MOX) has been suggested to 107

be more active with lower MICs for all isolates, compared to ofloxacin (OFL) (23, 25, 26). Thus, 108

knowledge of the specific mutation may help reveal the level of FQ resistance, which can in 109

turn inform decisions regarding the selection of OFL or higher generation FQs like MOX or 110

levofloxacin (LVX). This information can also be used to determine whether phenotypic 111

testing should be extended from OFL to MOX and LVX. In the present study, we therefore 112

aimed to characterize mutations in gyrA and gyrB genes in MTB isolates from the Indian 113

subcontinent in MDR-TB suspected patients. 114

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MATERIALS AND METHODS 116

Sample selection. Samples from presumptive MDR-TB patients from districts within New 117

Delhi, sent to the Mycobacteriology Laboratory in New Delhi, India over a period from 118

November 1 to November 30, 2013, were analyzed in this study. The presumptive MDR-TB 119

patients were identified as per the national guidelines (Programmatic management of drug-120

resistant tuberculosis; PMDT), which includes smear positive failures on category I or category 121

II regimen, history of contact with MDR-TB, all re-treatment cases (smear positive and smear 122

negative) and HIV-TB (27). Category I regimen is prescribed for new patients (intensive phase 123

for 2 months and consists of INH, RIF, pyrazinamide (PZA) and ethambutol (EMB) given under 124

observation thrice weekly, followed by continuation phase of 4 months of INH and RIF given 125

thrice weekly) (27). Category II regimen is prescribed for TB patients who have had a history 126

of at least one month of anti-tuberculosis treatment. Relapses, treatment after default and 127

failures are treated with a category II regimen (Intensive phase for 2 months and consists of 128

INH, RIF, PZA, EMB and Streptomycin (SM) given under observation thrice weekly, followed by 129

INH, RIF, PZA and EMB given under observation thrice weekly for 1 month and then followed 130

by continuation phase of 5 months of INH, Rif and EMB given thrice weekly (27). 131

Sample testing. Testing was performed using Genotype MTBDRplus (Hain Lifescience, 132

Nehren, Germany), a line probe assay (LPA), and a WHO endorsed molecular test. This 133

diagnostic tool may be used for detection of MDR-TB (both RIF & INH) directly on smear 134

positive sputum samples or on culture isolates in case of smear negative samples. Smear 135

positive sputum samples were identified as MDR-TB directly by LPA, with a turn-around time 136

of approximately 5 days. Smear negative sputum were inoculated for culture, and if positive 137

for MTB, subjected to LPA (27) according to PMDT. The site of testing was at the 138


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Mycobacteriology Laboratory, located in the Department of Microbiology at the National 139

Institute of Tuberculosis and Respiratory Diseases (NITRD) in New Delhi, India, and is one of 140

the National Reference Laboratories (NRL) under the Revised National Tuberculosis Control 141

Program (RNTCP) in India and a WHO center of excellence. 142

Processing of samples. The samples were processed by a standard N-acetyl-L-cysteine - 143

sodium hydroxide (NALC-NaOH) method (28), with a final concentration of 1.0% NaOH, (28). 144

The samples were stained for acid-fast bacilli (AFB) by Ziehl-Neelsen (ZN) method. All 145

processed AFB smear-positive samples were considered for DNA extraction and LPA, and all 146

AFB-smear negative samples were inoculated into BACTEC MGIT (Becton Dickinson, Sparks, 147

MD, USA) tubes, and when positive for MTB were considered for LPA. The cultures were 148

incubated for 42 days before being considered negative. 149

DNA extraction. For DNA extraction, 500 μL of NALC-NaOH processed samples or 1 mL of a 150

culture from BACTEC MGIT tubes were used. The DNA extraction was performed using 151

Genolyse version 1.0 kit (Hain Lifescience, Nehren, Germany). Supernatants containing DNA 152

was transferred into a fresh tube and stored at - 20˚C. 153

Genotype MTBDRplus assay. The amplification and hybridization using GenoType MTBDRplus 154

kit 2.0 were performed as per the manufacturer’s instructions and previously described (29). 155

For quality control of each assay, M. tuberculosis H37Rv was used as a positive control and 156

molecular grade water as negative control was put in every run. 157

Primer Design for Sequencing of FQ-resistance regions of gyrA and gyrB. The molecular 158

amplification, sequencing, and polymorphism detection was carried out at National Jewish 159

Health, Denver, CO. A consensus sequence of the target regions was created using 160

alignments of approximately 10 to 15 MTB gyrA or gyrB sequences present in the National 161


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Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/). Two 162

pairs of primers each for gyrA and gyrB were designed using Primer 3 163

(http://bioinfo.ut.ee/primer3-0.4.0/primer3/). The gyrA primer sets were gyrARS-164

Forward(F)/gyrAPRR-Reverse(R):5’-CAGCTACATCGACTATGCGA-3’/5’-165

ATTTCCCTCAGCATCTCCA-3’; 358 bp amplicon. And a nested set: gyrAPRR-F/gyrARS-R: 5’-166

GACTATGCGATGAGCGTGAT-3’/5’-GGGCTTCGGTGTACCTCAT-3’; 310bp amplicon. The two 167

primer sets designed for gyrB were gyrBPRR-F/gyrBPRR-R: 5’-AACAGCTGACCCACTGGTTT-168

3’/5’-CGCTGCCACTTGAGTTTGTA-3’; 556 bp amplicon and gyrBPRS-F/gyrBPRS-R:5’-169

CGCAAGTCCGAACTGTATGTCGTAG-3’/5’-GTTGTGCCAAAAACACATGC-3’; 346 bp amplicon. 170

Annealing temperature and magnesium concentrations were optimized prior to sample 171

analysis. 172

Amplification. Initial amplification of gyrA and gyrB using 40 cycles and with a single pair of 173

primer sets for each gene gave poor amplification, likely due to low DNA quantity in the 174

Genolyse-extracted DNA. Secondary purification and concentration of DNA also did not yield 175

consistent amplification. Thus, nested amplification was performed with primer sets as 176

described above. 177

gyrA amplification. PCR was performed with the Advantage-GC Genomic LA Polymerase Mix 178

(Clontech #639153, Mountain View, CA, USA) using 400 µM dNTP and 400 nM primers. The 179

first amplification employed gyrAPRR-F/RS-R in a 10 µL reaction mix at the following 180

conditions: 1 X 2 min at 94oC, 15 X [15s at 95°C, 20s at 60°C, and 75s at 72°C] 1 X 2 min at 181

72°C, hold at 4oC. The second amplification was performed with gyrARS-F/gyrAPRR-R for 35 182

cycles at the same conditions listed above, using 2 µL of the first PCR reaction mix as 183


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template. Supplementary Figure S1 illustrates the positioning of forward and reverse primers 184

for gyrA and gyrB. 185

gyrB amplification: The first PCR reaction was performed with Amplitaq Gold (ThermoFisher 186

Scientific, Carlsbad, CA) using the gyrBPRR primer set with 2.5 mM Mg++, 200uM dNTP and 187

200 nM primers at the following conditions: 1 X 95oC 10min; 20 X [95oC 15s, 59.1oC 20s, 72oC 188

75s], 72oC 2min, 4oC hold. The second amplification used primer set gyrBRS at 2 mM Mg++, 189

200 uM dNTP, 200 nM primers as follows: 1 X 95oC 10min; 40 X [95oC 15s, 60oC 20s, 72oC 190

75s], 72oC 2min, 4oC hold. Supplementary Figure S1 illustrates the positioning of forward and 191

reverse primers for gyrA and gyrB. 192

Amplicon purification and sequencing. The amplicons were prepared for sequencing by 193

treatment with Exonuclease I and Shrimp Alkaline Phosphatase (Exo-SAP). Ten µL of 1X PCR 194

buffer/ 0.375 mM Mg++ containing 0.05 U SAP and 2 U Exo-nuclease I was added to each PCR 195

reaction. The mixture was incubated at 37°C for 30 minutes followed by 95°C for 10 minutes. 196

Sequencing was performed with Big Dye 3.1 (ABI, Applied Biosystems, Foster City, CA). Each 197

10 µL reaction mix contained 1.5 µL 5X buffer, 0.875 µL BDX64 (MCLAB, San Francisco, CA), 198

0.35 µL Big Dye 3.1, 1 µL 5 µM primer, and 2 µL Exo/SAP-treated amplicon. Cycling conditions 199

were 2 min at 96°C, 35 cycles as follows: 10s at 96°C, 5s at 52°C, and 2min at 60°C. 200

Sequencing products were purified with spin columns (EdgeBio, Gaithersburg, MD) according 201

to manufacturer’s directions, dried, and loaded onto an ABI 3730 Genome Analyzer (Applied 202

Biosystems, Foster City, CA). 203

Sequence analysis. Sequence traces were imported into a BioNumerics database (Applied 204

Maths, Austin, TX), and trimmed if necessary. Sequences were exported in FASTA format. The 205

genetic polymorphisms of gyrA and gyrB were compared to the reference sequence from M. 206


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tuberculosis strain H37Rv (GenBank accession number NC_000962/ L27512). The 207

polymorphisms obtained were annotated as per the M. tuberculosis genome numbering 208

system. 209

Structure Analysis. Gyrase A mutations were mapped onto the X-ray crystal structure of the 210

M. tuberculosis gyrase A protein and color coded using the PyMol structure visualization 211

software package (https://www.pymol.org/ ). The coordinates of the gyrase A X-ray crystal 212

structure were obtained from the Protein Data Bank 213

(http://www.rcsb.org/pdb/home/home.do ), PDB reference code 3ILW . 214

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RESULTS 217

A total of 152 samples from patients suspected of MDR-TB from the Delhi region of North 218

India were considered for the Genotype MTBDRplus assay. History of anti-tuberculosis drugs 219

under category I regimen or category II regimen or history of contact was collected for these 220

patients. Overall, 104/152 (68.4%) patients were at the start of retreatment/ category II 221

regimen due to default, incomplete treatment, treatment failures or relapse. 16/152 (10.5%) 222

patients were failures of a new TB/ category I regimen. Thirty out of 152 (19.7%) patients 223

were failures of retreatment/ category II regimen. Out of 152, 1 patient had a history of 224

contact with MDR-TB and one was HIV positive. The LPA was performed on 16 MTB isolates 225

from cultures of AFB smear-negative sputum samples, whereas remaining LPA was performed 226

directly on the AFB smear-positive sputum samples. 227

The 179 bp region between 148 to 327 bp of the gyrA gene, encoding amino-acids 49 to 109, 228

was studied for the identification of mutations. Six strains yielded poor gyrA sequences even 229

on repeat sequencing and were excluded from the analysis. A total of 27 mutations in 25 230

strains (17%) were obtained in 146 strains studied for gyrA gene, in which two strains had 231

more than one mutation. Table 1 provides the clinical details, susceptibility to INH and RIF 232

and mutations observed in gyrA. The substitution at amino acid 94 was most common 233

mutation (13/27; 48.1%). Mutations observed include A281G D94G (8/ 27; 29.6%) and C269T 234

A90V (8/ 27; 29.6%), followed by A281C D94A (2/27; 7.4%), G280T D94Y (2/27; 7.4%), C269Y 235

A90V* (2/27; 7.4%), G280A D94N (1/27;3.7%), A283G S95A (1/27;3.7%), T271C S91P 236

(1/27;3.7%), and A281R D94G* (2/27; 7.4%). Strains ID12, and ID18 had double mutations 237

A90V/D94G and A90V/D94A respectively. The location of the mutations in the codons and 238


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respective amino-acid changes in the primary sequence is depicted in Figure 1, and in the 239

gyrA protein structure in Figure 2. 240

Co-existence of wild-type and mutated base (Heterozygosity) was seen in 4/27 (14.8%) 241

mutations which included strains ID12, ID24 and ID25 of which two resistance-associated 242

mutations were seen in ID12 detailed in Table 1, Supplementary Figure S2. Polymorphism 243

G284C, which encodes a S95T polymorphism, as compared to the reference H37Rv gyrA 244

sequence, was found in 138/146 (94.5%) of the strains. This S95T polymorphism is a common 245

lineage specific polymorphism, not likely to be involved in fluoroquinolone resistance. 246

Among the patients with the isolates having gyrA mutations, 22/25 (88%) were AFB smear-247

positive and 3/25 (12%) were AFB smear-negative when identified as MDR-TB suspects. Of 248

these isolates, the majority (24/25; 96%) were previously treated pulmonary TB cases, and 249

one (1/25; 4%) case was failure of category I regimen. Four of previously treated (4/24; 250

16.7%) cases were failures of category II regimen. 251

Of 146 MDR-TB suspects, 21/146 (14.4%) were found to be MDR-TB, 10/146 (6.8%) RIF mono-252

resistant, 5/146 (3.4%) INH mono-resistant, and 110/146 (75.3%) susceptible to both 253

antibiotics based on the well characterized mutations. In the MDR-TB strains, 14/21 (66.7%) 254

had FQ resistance mutations in the gyrA gene, and of the RIF monoresistant strains 2/10 255

(20%) had FQ resistance mutations in the gyrA gene. In the non-MDR cases, FQ resistance was 256

observed in 10/115 (8.7%) strains, the difference being statistically significant (< 0.0001). 257

Similarly, the RIF resistant strains were found in 16/25 (64%) strains with gyrA mutations in 258

comparison with 15/121 (12.4%) strains without gyrA mutations. This difference was also 259

found to be statistically significant (< 0.0001). The 418 bp region between 1229 to 1647 bp of 260


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gyrB gene encoding the amino-acids 410 to 543 for 109 strains was also investigated, and no 261

mutations were found in any of the samples. 262

DISCUSSION 263

This study aimed at the detection of mutations in gyrA and gyrB genes for FQ resistance. The 264

study population included TB patients from North India suspected to be MDR-TB patients. 265

Most of the patients had a history of irregular or defaulted TB treatment using the first-line 266

anti-tuberculosis agents like RIF, INH, ethambutol, and pyrazinamide. It has been shown 267

earlier that such patients are much more likely to develop MDR-TB. The treatment regimen 268

involving such patients includes FQ or aminoglycosides (2). 269

In the present study, 17.1% of patients as described above were found to harbor FQ 270

mutations. In India, with a population of over a billion individuals, this has significant 271

implications, due to the high potential proportion of FQ resistance among TB and MDR-TB 272

suspects. This is likely due to FQ’s being over-prescribed in certain regions of the world, 273

especially in many resource-limited countries, where they are readily available over the 274

counter (8). In the Indian subcontinent, FQ resistance for MTB has increased from 3% of 275

laboratory cultures in 1996 to 35% in 2004 in Mumbai and from 17.4% in 2005 to 42.9% in 276

2009 in Pakistan (9, 10). FQ resistance is known to be associated with poor treatment 277

outcomes among MDR-TB patients (30), and the increasing prevalence of FQ resistance TB 278

isolates is of great concern. 279

Resistance to FQ in MTB is caused mainly by the mutations in the QRDR region, a highly 280

conserved region in the gyrA protein, located at the N-terminal portion extending from amino 281

acid 74 to amino acid 113. FQ resistance in MTB is less common due to mutations in the 282

second protein of the gyrase complex, gyrB, with QRDR mutations observed from codon 461 283


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to 499 of gyrB (31). In the previous review of mutations by Maruri et al, 59% and 41% of 284

mutations were in gyrA and gyrB respectively. For gyrA mutations, 81% and 19% of mutations 285

were inside and outside the QRDR respectively. Whereas for gyrB mutations, 44% of 286

mutations were inside the QRDR region, 50% were outside the QRDR, and 1% were deletions 287

(31). 288

In the present study, mutations were found only in the gyrA gene, with substitutions at codon 289

94 in gyrA being the most common at 48.1%. The substitutions in codon 94 (37%) are the 290

most common as reviewed by Maruri et al (31). In another review by Avalos et al, across 18 291

countries, mutation data of 3,846 clinical isolates with known phenotypic resistance profiles 292

to FQ was studied. The most frequent gyrA mutation was D94G which ranged from 21-32% 293

(32). The relative frequency of mutations in the QRDR region reported from different regions 294

world-wide are detailed in table 2. D94G is reported as the most common codon 94 295

substitution in most reports, as seen in studies from Pakistan, China, The Philippines, 296

Thailand, Vietnam, Uzbekistan, Russia, Germany, France and USA (33, 34, 35, 36, 37, 38, 39, 297

40, 17, 41). All mutations at codon 94 have been associated with in-vitro resistance as seen in 298

earlier studies (17, 24, 33,36, 37, 40, 41) The codon 90 mutation, A90V has also been found in 299

high frequency with studies from India, The Philippines, China, and Belgium reporting it as the 300

most common (24, 35, 42, 23). The review by Avalos et al. found 13-20% A90V mutations 301

contributing to resistance (32). In the present study, both D94G and A90V were the most 302

commonly observed mutations. Interestingly, both these mutations have been reported in all 303

gyrA mutation studies. However, other amino acid 94 substitutions, including D94A, D94Y, 304

D94N and D94H have been reported with varying frequency from none to 17.6%, with a rare 305

D94F substitution reported only in one study (35). 306


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The frequency of A90V mutations has been found to be less in some studies from Germany 307

(10.3%), and the USA (5.9%), suggesting that the frequency of mutations in a gene tend to 308

vary by geographic location (40, 41, 43). Two strains in the present study had double 309

mutations ID18; A90V/D94A and ID12; A90V/D94G. The presence of double mutations has 310

been related to high-level drug resistance, as reported previously (17, 31, 39, 40, 42). Aubrey 311

et al reported high-level drug resistance on a strain with A90V/D94G mutation, which had 312

very high MIC of >160 µg/mL (5). 313

In the present study, a novel mutation was found in gyrA in ID23; S95A; within the QRDR 314

region. Figure 2 demonstrates the X-ray crystal structure of the M. tuberculosis gyrase A 315

protein (Protein Data Bank code 3ILW) with amino acids at positions A90, S91, D94, S95 316

shown in red within the QRDR. Although the S95A mutation is in close proximity to well 317

characterized fluoroquinolone resistance mutation sites, including D94, further 318

microbiological confirmation would be necessary to assess the impact of this specific 319

mutation on fluoroquinolone resistance. 320

Hetero-resistance, defined as both wild type and a mutation at specific loci, was seen in 321

14.8% of FQ mutations identified by sequencing. Presence of hetero-resistant mutations 322

suggest, mixed susceptible and resistant bacteria in the same clinical sample. Two different 323

mechanisms for hetero-resistance have been postulated, co-existence of two genotypically 324

different strains i.e. susceptible and resistant, or a single strain evolving from susceptible and 325

resistant organisms. It has been shown earlier that infection with two different strains was 326

responsible for hetero-resistance in 71% of the patients and could serve as a quality indicator 327

for TB control programs in various countries (44). This occurrence may be present in higher 328

frequencies in hyper-endemic regions (15). The evolution of a susceptible strain into a 329


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resistant strain could be the result of poor treatment in terms of dose and/or duration. Other 330

studies have also reported hetero-resistance with a frequency varying from 4 to 37.1% (17, 331

37, 39, 40). 332

An interesting observation in the QRDR region of gyrA of MTB is the presence of G to C 333

polymorphism at position 284; S95T as compared to the reference H37Rv gene. This encodes 334

either serine (in M. tuberculosis H37Rv and H37Ra) or threonine (in M. tuberculosis Erdman, 335

M. bovis BCG, M. africanum) (19). In the present study, this polymorphism was found in 336

94.5% isolates. The S95T polymorphism has not been implicated to FQ resistance since it has 337

been found in strains with MIC values to ofloxacin at less than the critical concentration, <2 338

µg/mL (6, 33, 35, 42). An exception was an earlier Indian study, which found S95T in 88.2% of 339

ofloxacin-resistant isolates with no other mutation in QRDR gyrA or gyrB (24). 340

We looked for gyrB mutations in the QRDR region, as several worldwide studies have 341

reported mutations associated with in vitro resistance such as D472H, R485C, D495N, D500N, 342

D500H, N510D, N533T, N538D, N538I, T539N, 546M (5, 23, 36, 45). However, in our study, we 343

did not find any gyrB mutations among our samples, which agrees with other studies 344

performed in Indian and in China (24, 34). 345

Interestingly, we found a significant association of MDR-TB with the gyrA mutations, in 346

comparison with strains without the gyrA mutations. Over half of the MDR-TB strains 347

examined in this study also had mutations in gyrA (66.7%). The rate of FQ resistance among 348

MDR-TB patients also has been found to be high in other countries in the region, The 349

Philippines; 51.4% (46), Taiwan; 22.2% (47), and in China (Shanghai); 25.1% (20). The rates 350

from countries in other continents are lower, with 4.1% in the United States and Canada (48) 351


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and 4.3% in MDR-TB in Russia (49). It has been hypothesized that resistance to other anti-TB 352

drugs could potentially influence resistance to FQ (13). 353

In summary, our study revealed a high rate, 17.1%, of FQ mutations among the isolates of 354

Indian TB patients suspected of MDR-TB. The FQ mutations in sequence verified MDR strains 355

was 66.7% whereas, in non-MDR cases, it was 18.7%. All the mutations were found 356

exclusively in gyrA gene, with A90V and D94G being the most common. One novel mutation 357

S95A was also observed. The data strongly suggests for conducting expanded studies, 358

examining both phenotypic and genotypic data on a larger number of isolates from India and 359

surrounding countries. This would further help in developing, refining, and improving rapid 360

molecular diagnostic tests for determining resistance (50). This would also aid in the control 361

and identification of pre-XDR-TB and XDR-TB in a timelier and effective manner, preferably at 362

the time of detection of MDR-TB. 363

364

Acknowledgement 365

This work was supported by the NIH/FIC AIDS and TB International Training and Research 366

Program (D43TW001409) at NYU School of Medicine, Principle Investigator: Dr. Suman Laal 367

(PhD). Michael Strong acknowledges support from the Boettcher Foundation Webb-Waring 368

program. 369

370


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542


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Legends 543

544

Figure 1 545

Location of the mutations in the analyzed strains. Sequence polymorphisms likely to be 546

involved in fluoroquinolone resistance are indicated in the boxes above (nucleotide changes) 547

and below (amino acid changes) the primary sequence. A common lineage specific 548

polymorphism is indicated by the circles, and is not likely to be involved in resistance. 549

550

Figure 2 551

Location of the identified MTB gyrA mutations. Mutations identified in this study are 552

indicated in red on the gyrA protein structure, and cluster at the quinolone resistance-553

determining region. 554

555

556

557


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Table 1. Characteristics of gyrA mutations and corresponding amino-acid changes in various 558

Mycobacterium tuberculosis strains isolated from clinically suspected multidrug-resistant 559

tuberculosis cases 560

Strain Number

Age

Sex

Category of treatment

Type of patient

Stage of treatment

Acid-fast bacilli smear INH RIF

Nucleotide change(s) (Amino-acid change[s])

ID 1 21 M 2 Retreatment Entry 2+ S S C269T (A90V) ID 2 33 M 2 Retreatment Entry 1+ S S A281G (D94G) ID 3 11 M 2 Retreatment Entry Neg. R R G280A (D94N) ID 4 11 M 2 Retreatment Entry Neg. R R A281G (D94G) ID 5 67 F 2 Retreatment Entry 3+ S S G280T(D94Y) ID 6 26 F 2 Retreatment FUP 3+ S R C269T (A90V) ID 7 50 M 2 Retreatment Entry 3+ S R C269T (A90V) ID 8 13 F 2 Retreatment Entry 1+ R S A281C (D94A) ID 9 26 F 2 Retreatment Entry 3+ R R A281G (D94G) ID 10 28 F 2 Retreatment Entry 1+ R R A281G (D94G) ID 11 20 F 2 Retreatment Entry 3+ R R A281G (D94G)

ID 12 35 M 2 Retreatment Entry Neg. R R C269Y; (A90V)*, A281R (D94G)*

ID 13 30 M 2 Retreatment Entry 3+ R R C269T (A90V) ID 14 28 F 2 Retreatment Entry 3+ R R A281G (D94G) ID 15 20 M 2 Retreatment Entry 3+ S S C269T (A90V) ID 16 24 M 2 Retreatment Entry 3+ R R T271C (S91T) ID 17 51 M 2 Retreatment Entry 2+ S S A281G (D94G)

ID 18 22 M 2 Retreatment Entry 3+ R R C269T (A90V), A281C (D94A)

ID 19 17 F 2 Retreatment FUP 1+ S S C269T (A90V) ID 20 23 M 2 Retreatment Entry 3+ R R A281G (D94G) ID 21 21 F 2 Retreatment FUP 3+ R R C269T (A90V) ID 22 22 M 2 Retreatment FUP 2+ R R G280T (D94Y) ID 23 13 F 1 New case FUP Scanty S S A283G (S95A) ID 24 70 M 2 Retreatment Entry 1+ S S C269Y (A90V) * ID 25 30 M 2 Retreatment Entry 3+ R R A281R (D94G)* M: Male; F: Female FUP: Acid-fast bacilli smear positive on follow up samples MDR-TB: Multidrug-resistant tuberculosis; INH: Isoniazid; RIF: Rifampin S: Susceptible; R: Resistant Neg.: Negative * Hetero-resistant


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Table 2: Frequency of gyrA mutations in QRDR region in Mycobacterium tuberculosis isolates in different geographic regions

Mut

ated

codo

ns

Pres

ent s

tudy

, n=2

8

Indi

a, n

=8 (2

4)

Paki

stan

n=

41 (3

3)

Chen

a, C

hina

, n=5

6 (3

4)

Huan

g, C

hina

, 201

1, n

=63

(18)

Phili

ppin

es, n

=7 (3

5)

Thai

land

, n=2

1 (3

6)

Viet

nam

, n=3

0 (3

7)

Uzbe

kist

an, n

=23

(38)

Russ

ia, n

= 40

(39)

Belg

ium

, n=2

8 (2

3)

Fran

ce, 2

010,

n=

25 (1

7)

Germ

any,

200

9, n

=29

(40)

USA,

n=1

7 (4

1)

Phen

otyp

ic an

timicr

obia

l su

scep

tibili

ty re

sults

to

flour

oqui

nolo

ne (R

efer

ence

)

T80A 7.1%* 8.0% S (23), R or S (17)

M81T 5.9% R (41)

G88A 4.8% 2.5% 8.0% R (17, 36, 39)

G88C 9.5% 4.0% R (17, 18)

D89N 1.6% 3.6% R (18, 23)

A90V 28.6% 37.5% 26.8% 17.9% 18% 42.9% 23.8% 20.0% 21.7% 17.5% 39.3% 20.0% 10.3% 5.9% R (17, 18, 23,

24, 33 -41)

A90E 4.0% R (17)

A90G 3.6% 4.0% R (17, 23)

A90V* 7.1% 3.4% R (40)

S91T 3.6% 1.8% R (34)

S91P 12.5% 4.9% 7.1% 3.2% 9.5% 4.3% 3.4% 5.9%

R (18, 24, 33, 34, 36, 38, 40, 41)

D94G 28.6% 25% 39% 46.4% 47.6% 42.9% 33.3% 23.3% 43.5% 32.5% 14.3% 28.0% 44.8% 41.2% R (17, 18, 23, 33

- 41)

D94A 7.1% 25% 4.9% 5.4% 9.5 9.5% 13.3% 17.4% 7.5% 10.7% 8.0% 13.8% R (17, 18, 23,

33, 34, 36 - 40)

D94Y 7.1% 12.2% 7.1% 11.1% 3.3% 4.3% 17.5% 10.7% 11.8% R (18, 33, 34, 37

-39, 41)

D94N 3.6% 4.9% 10.7% 9.5% 9.5% 4.3% 2.5% 7.1% 8.0% 3.4% 17.6%

R (17, 18, 23, 33, 34, 36, 38 - 41)

D94H 3.6% 9.5% 4.3% 3.6% 4% R (17, 23, 34,

36, 38)

D94F 14.3% R (35)

D94G* 7.1% 16.7% 5% 4% R (17, 37, 39)

D94N* 3.3% 2.5% R (37, 39)

D94Y* 2.5% R (39)

D94A* 3.3% 3.4% R (37, 40)

S95A 3.6%

L96P 2.4% R (33)

L109P 5.9% R (41)


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D111N 4.9% R (33)

Q113L 5.9% R (41) A90V*, D94G*

10% 2.5% 3.4% R (37, 39, 40)

A90V*, D94A*

3.3% R (37)

S91P*, D94G*

3.4% R (40)

D94N*, D94G*

5% 6.9% R (39, 40)

D94A*, D94G* 3.3%

R (37) D94N*, D94Y*

3.4% R (40)

D94N*, D94G*, D94Y*

2.5% R (39)

561

n=Total number of mutations reported (Reference No) 562

*Heteroresistance 563

R=Resistant 564

S=Susceptible 565


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566 567

568 Figure 1 569 570

571 572

573 L 574 575 576 577


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Figure 2 578

579

580

581

582

583


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Documents

Sequence analysis of fluoroquinolone resistance associated ... · 4 61 INTRODUCTION 62 Treatment of multidrug-resistant tuberculosis (MDR-TB) patients, with strains resistant to 63