Significance of Coexisting Mutations on Determination of the Degree of Isoniazid Resistance in Mycobacterium tuberculosis Strains

Abstract

The emergence and spread of drug-resistant tuberculosis (TB) pose a threat to TB control in Sri Lanka. Isoniazid (INH) is a key element of the first-line anti-TB treatment regimen. Resistance to INH is mainly associated with point mutations in katG, inhA, and ahpC genes. The objective of this study was to determine mutations of these three genes in INH-resistant Mycobacterium tuberculosis (MTb) strains in Sri Lanka. Complete nucleotide sequence of the three genes was amplified by polymerase chain reaction and subjected to DNA sequencing. Point mutations in the katG gene were identified in 93% isolates, of which the majority (78.6%) were at codon 315. Mutations at codons 212 and 293 of the katG gene have not been reported previously. Novel mutations were recognized in the promoter region of the inhA gene (C deletion at −34), fabG1 gene (codon 27), and ahpC gene (codon 39). Single S315T mutation in the katG gene led to a high level of resistance, while a low level of resistance with high frequency (41%) was observed when katG codon 315 coexisted with the mutation at codon 463. Since most of the observed mutations of all three genes coexisted with the katG315 mutation, screening of katG315 mutations will be a useful marker for molecular detection of INH resistance of MTb in Sri Lanka.

Introduction

Tuberculosis (TB) is a preventable and curable disease, yet about 710,000 people died of TB worldwide in 2015.¹ The latest estimates state that there are 10.4 million new cases of TB with 5.9, 3.5, and 1.04 million among men, women, and children, respectively.¹ According to the World Health Organization (WHO), the Southeast Asia region bears 46.5% of the global TB burden.¹

Sri Lanka is reported to be a middle TB burden country, with around 25% of total TB cases reported from the Colombo district.¹ The estimated incidence remained stable at 66 new cases per 100,000 population over the 2000–2012 time period,² and 65 new cases per 100,000 population were reported in 2015.³ The WHO 2016 TB report has shown that the multidrug-resistant (MDR) rate in Sri Lanka is 0.54% among newly diagnosed cases and 1.7% among retreatment cases.³ Irrespective of these incidence rates, early diagnosis of drug resistance in Mycobacterium tuberculosis (MTb) isolates is very important to control the spread of the disease to reach targets of the End TB strategy.⁴

Incomplete treatment can lead to drug-resistant TB, and around 490,000 people worldwide were found to be harboring MDR TB in 2016.⁴ The emergence and spread of MDR TB pose a threat to global TB control. MDR TB is defined as resistance to at least rifampicin (RIF) and isoniazid (INH). Resistance to these two first-line drugs therefore becomes a determining factor in disease control since patients carrying MDR strains of MTb need to be treated using combinations of second-line anti-TB drugs. MDR TB greatly complicates patient management particularly within resource-poor national TB programs due to lower treatment efficacy and higher cost of treatment.⁵

INH is considered as a prodrug that is activated by the catalase peroxidase (CP) enzyme encoded by the katG gene.⁵ In the presence of CP, INH is converted to a radical form (through an oxidation reaction) and covalently attached to NAD⁺, forming an INH-NADH adduct. It binds the active site of enoyl-acyl carrier protein (ACP) reductase enzyme to inactivate it.^6,7 This inactivation leads to inhibition of the synthesis of mycolic acid, a major constituent of the mycobacterial cell wall.^6,7 ACP reductase is encoded by the inhA gene, which is a component of the fabG1-inhA operon. This operon includes another gene, fabG1 (mabA), coding for b-keto acyl reductase involved in the mycolic acid biosynthesis pathway. katG gene mutations affect the expression of CP enzyme, whereas inhA gene mutations lead to reduced affinity of INH-NADH for ACP reductase.⁷ Therefore, mutations in the katG gene and/or fabG1-inhA operon result in INH resistance. Furthermore, MTb depends on the CP enzyme to tolerate oxidative stress. When CP becomes nonfunctional due to katG mutations, the ahpC gene is frequently overexpressed to produce alkyl hydroperoxide reductase to overcome oxidative stress.⁸ Mutations in the ahpC gene serve as compensatory alterations for the upregulation of alkyl-hydroperoxide reductase to survive in the presence of toxic effects of organic peroxides. Therefore, the ahpC gene mutations indirectly contribute to INH resistance.^9–11

The main objective of this study was to determine the effect of coexisting mutations on the level of INH resistance. Since there were no significant reports on genetic characterization of INH-resistant MTb isolates in Sri Lanka, mutations in katG, inhA, and ahpC genes that are responsible for INH resistance were studied as a prerequisite for the above.

Methodology

Sample collection

Acid-fast bacillus (AFB)-positive sputum specimens (n = 541) were collected from patients suspected of having TB at the Central Chest Clinic, Colombo, from 2013 to 2016. In addition, 41 INH-resistant MTb cultures were collected from the National Tuberculosis Reference Laboratory (NTRL, Welisara, Sri Lanka).

Isolation and identification of MTb strains

The sputum specimens were processed by the modified Petrof method¹² and cultured on Lowenstein–Jensen medium for isolating MTb strains. AFB-positive culture isolates were confirmed as MTb using phenotypical characters (growth rate, color, and colony morphology) and nitrate reduction. Furthermore, suspected MTb isolates were confirmed by amplification of the IS6110 insertion element of MTb genome using pt18- 5′-GAACCGTGAGGGCATCGAGG-3′ and INS2 5′-GCGTAGGCGTCGGTGACAAA-3′ primers¹³ (Integrated DNA Technologies).

Antibiotic susceptibility testing

The agar proportion method (APM),¹⁴ considered as the gold standard, was carried out using 450 pure MTb cultures on Middlebrook 7H10 agar (Difco) plates to detect phenotypic resistance to INH according to the NCCLS guidelines.¹⁵ To classify the strains as susceptible or resistant, a critical INH concentration of 0.2 μg/mL for 7H10 agar was used as recommended by WHO.^16,17

INH (Sigma) stock solution (10 mg/mL) was prepared to obtain two final INH concentrations of 1 and 0.2 μg/mL, which were used for the antibiotic susceptibility testing. The H37Rv strain and the known INH-resistant MTb strain confirmed by NTRL were used as the quality control strains.

Determination of the minimum inhibitory concentration of INH for resistant isolates

Minimum inhibitory concentration (MIC) of INH for MTb isolates was determined for 0.2, 1, 5, 10, 15, and 20 μg/mL INH concentrations. McFarland 0.5 bacterial suspension (100 μL) was inoculated on INH incorporated 7H10 agar plates^17,18 and incubated at 37°C in a 5% CO₂ environment. The growth of isolates was observed weekly for 42 days. MIC levels were categorized as low level (0.2–5 μg/mL)¹⁹ and high level of resistance (≥10 μg/mL).¹⁸

DNA extraction and polymerase chain reaction amplification

Genomic DNA of phenotypically identified INH-resistant isolates was extracted using the phenol–chloroform method.²⁰ Extracted DNA was quantified using standard DNA markers (lambda DNA 539 ng/μL; Promega) on 1.5% agarose gel electrophoresis.

The katG, inhA, and ahpC genes were polymerase chain reaction (PCR) amplified using gene-specific primers and extracted DNA as the template (Table 1). The amplified fragments covered the full length of the katG gene, full length of the structural inhA gene, and the promoter region of fabG1 (mabA)-inhA operon and the full length of the structural ahpC gene, oxyR pseudogene, and the oxyR- ahpC intergenic region.

Table 1.

Primers and Polymerase Chain Reaction Cycle Parameters Used for Amplification of Two Overlapping Regions of katG, inhA, and ahpC Genes

Primers were designed using the IDT Oligo Analyzer 3.1 tool and one from literature.²¹ A 50-μL PCR mixture containing 5 × PCR buffer (pH = 8.3; Promega), 1.5 mM MgCl₂ (Promega), 0.5 pM each of the primer (IDT), 0.2 mM DNTP (Promega), and 1 U/μL Taq DNA polymerase (Promega) was used with 2.5 μL of genomic DNA (∼10 ng) per PCR. Thermocycling parameters used for PCR amplification are also shown in Table 1.

Sequencing of PCR-amplified products of INH-resistant isolates

PCR products were custom DNA sequenced (Macrogen) using both forward and reverse primers. The results were analyzed using BioEdit7.2.5 and Chromas LITE2.1.1 software and NCBI sequence alignment tools to identify point mutations. PCR products of reference strain H37Rv and five INH-susceptible strains were also sequenced for quality control.

Results

Of the four hundred fifty pure MTb cultures isolated from randomly collected sputum samples, 15 INH-resistant isolates were identified by the APM. The rest of the resistant cultures (41) were directly collected from the NTRL.

DNA sequencing of katG, inhA, and ahpC genes

Point mutations of DNA sequences, alterations in amino acid, and the frequency of each mutation are summarized in Table 2. Collectively, stand-alone katG mutations (n = 18) and inhA (n = 1) were identified. There were no stand-alone ahpC mutations. Five isolates carried point mutations in all 3 genes, while katG+inhA (n = 12) and katG+ahpC (n = 17) combinations were observed in other 29 isolates. Furthermore, the DNA of one MTb isolate did not PCR amplify the katG gene, but it revealed wild-type (wt) inhA and ahpC genes. Two phenotypically INH-resistant MTb isolates did not show point mutations in any of the three genes.

Table 2.

The Frequency of Point Mutations of katG, inhA, and ahpC Genes

Gene	Position	Nucleotide change	Amino acid change	Frequency %
katG gene	Codon 315	AGC to ACC	S315T	67.8 (n = 38)
		AGC to AAC	S315N	7.1 (n = 4)
		AGC to AGA	S315R	1.8 (n = 1)
		AGC to ACA	S315T	1.8 (n = 1)
	Codon 463	CGG to CTG	R463L	62.5 (n = 35)
	Codon 275	ACC to GCC	T275A	1.8 (n = 1)
	Codon 212	GGT to GAT^a	G212D	1.8 (n = 1)
	Codon 293	CTA to CTG^a	L293L	1.8 (n = 1)
	Codon 521	CTG to CTC	L521L	1.8 (n = 1)
inhA promoter region	−34	C deletion^a	—	12.5 (n = 7)
	−34	C to T	—	1.8 (n = 1)
	−15	C to T	—	8.9 (n = 5)
inhA gene	Codon 3	GGA to GGC	G3G	1.8 (n = 1)
	Codon 94	TCG to GCG	S94A	5.4 (n = 3)
	Codon 158	TAC to TAT	Y158Y	1.8 (n = 1)
fabG1 gene	Codon 27	ATC to ATT^a	I27I	1.8 (n = 1)
oxyR-ahpC intergenic region	−10	C to T	—	1.8 (n = 1)
oxyR-ahpC intergenic region	−46	G to A	—	3.6 (n = 2)
ahpC gene	Codon 39	ACC to AGC^a	T39S	1.8 (n = 1)
OxyR pseudogene	37	C to T	—	32 (n = 18)

The mutations not previously reported.

Sequence variations of the katG gene

Point mutations in katG were observed in 52 (93%) of 56 isolates, of which the majority (n = 44, 78.6%) were at codon 315. The point mutation at codon 463, reported to be a polymorphism, was observed in 35 isolates (62.5%). In addition, four single mutations at codons 212, 275, 293, and 521 were identified (Table 2). Codon 212 [GGT (Gly) to GAT (Asp)] and 293 [CTA (Leu) to CTG (Leu)] mutations have not been reported previously. Stand-alone single-point mutations at codon 315 (n = 16) and codon 463 (n = 7) were observed in 23 isolates. Dual-point mutations at codons 315 + 463 (n = 26), 315+275 (n = 1), and 315+521 (n = 1) were identified in 28 isolates, while 1 isolate carried 4 mutations (S315T+R463L+L293L+G212D).

Sequence variations of fabG1-inhA operon

Point mutations of fabG1-inhA operon were identified in 18 (32%) resistant isolates. A novel mutation at −34 (C deletion) in the promoter region of seven isolates and a single C to T transition mutation at the same position in one isolate were identified. Promoter mutation at −15, which is the globally most common inhA mutation, was found only in five (8.9%) isolates. Two silent mutations were found at codon 27 in the fabG1 gene and codon 158 in the inhA gene. Structural inhA gene mutations at codon 94 (n = 3) and codon 3 (n = 1) were identified in four isolates. All these inhA mutations were single-point mutations except one isolate, which carried double-point mutations at codon 158 and at −15 of the promoter region.

Sequence variations of oxyR-ahpC region

The majority of sequence variation was identified at position 37 (n = 18, 32%) of the oxyR pseudogene. It is reported to be a C to T polymorphism. A novel C to G transversion mutation at codon 39 was observed in one isolate, while the common mutations at −46 (n = 2) and −10 (n = 1) of the intergenic region were identified in three isolates.

Correlation between the MIC of INH and the presence of mutations

Twenty-one (37.5%) isolates showed a high level of resistance, while 35 (62.5%) isolates revealed a low level of resistance. The presence of mutations and the level of resistance of INH-resistant MTb isolates are given in Table 3.

Table 3.

Mutations Present in katG, inhA, and ahpC Genes of Isoniazid-Resistant Mycobacterium tuberculosis Isolates with the Resistance Level to Isoniazid

	Resistance genotype			Resistance level to INH (μg/mL)
No. of isolates, n (%)	katG	inhA/fabG1/promoter region (P)	ahpC/oxyR/intergenic region (I)	High ≥10 > low
4 (7.1)	S315T	wt	wt	≥10 (High)
7 (12.5)	S315T	−34 (C deletion) (P)	wt	≥10 (High)
3 (5.4)	S315T	S94A (inhA)	wt	≥10 (High)
2 (3.6)	S315T+R463L	wt	wt	≥10 (High)
1 (1.8)	S315T+T275A	wt	wt	≥10 (High)
1 (1.8)	S315T+R463L	wt	37 (C to T) (oxyR)	≥10 (High)
1 (1.8)	S315R+R463L	wt	37 (C to T) (oxyR)	≥10 (High)
2 (3.6)	R463L	wt	wt	≥10 (High)
1 (1.8)	S315T	I27I (fabG1)	wt	1 (Low)
1 (1.8)	S315T	wt	T39S (ahpC)	1 (Low)
6 (10.7)	S315T+R463L	wt	wt	1, 5 (Low)
1 (1.8)	S315N+R463L	wt	wt	0.2 (Low)
7 (12.5)	S315T+R463L	wt	37 (C to T) (oxyR)	0.2, 1 (Low)
3 (5.4)	S315N+R463L	wt	37 (C to T) (oxyR)	0.2 (Low)
1 (1.8)	S315T+R463L	wt	−10 (C to T) (I)	1 (Low)
1 (1.8)	S315T+R463L	wt	−46 (G to A) (I)	0.2 (Low)
1 (1.8)	S315T+R463L	G3G (inhA)	wt	1 (Low)
1 (1.8)	S315T+R463L	−34 (C to T) (P)	−46 (G to A) (I)	5 (Low)
1 (1.8)	S315T+R463L	−15 (C to T) (P)	37 (C to T) (oxyR)	5 (Low)
1 (1.8)	S315T+R463L+L293L+G212D	wt	37 (C to T) (oxyR)	0.2 (Low)
2 (3.6)	R463L	wt	wt	5 (Low)
2 (3.6)	R463L	−15 (C to T) (P)	37 (C to T) (oxyR)	0.2 (Low)
1 (1.8)	R463L	−15 (C to T) (P)+Y158Y (inhA)	37 (C to T) (oxyR)	0.2 (Low)
1 (1.8)	R463L+L521L	wt	37 (C to T) (oxyR)	0.2 (Low)
1 (1.8)	wt	−15 (C to T) (P)	wt	1 (Low)
1 (1.8)	Not amplified	wt	wt	1 (Low)
2 (3.6)	wt	wt	wt	0.2, 1 (Low)

INH, isoniazid; wt, wild-type.

In the high level of the INH resistance category, katG 315 codon mutation was observed in majority of isolates either with stand-alone S315T transversion mutation (n = 4) or S315T mutations coexisting with other mutations within the same gene (codons T275A and R463L: n = 3) or with the inhA gene (codon S94A and C deletion at −34: n = 10). In addition, a single isolate having a katG S315R together with R463L mutation and two isolates with stand-alone R463L mutation also revealed a high level of resistance. However, 23 of 56 (41%) isolates that had mutations at katG 315 codon (S315T/S315N) coexisting with R463L mutation evidenced a low level of resistance.

Statistical analysis was performed for isolates having katG S315T mutation with (n = 22) and without (n = 17) R463L mutation to investigate the resistance-lowering effect of R463L mutation (Table 4). Low level of resistance was revealed in a higher percentage (86.4) of isolates with R463L (p < 0.05).

Table 4.

Coexistence of katG R463L Mutation with katG S315T Mutation in Relation to Lowering of the Level of Isoniazid Resistance

	katG S315T mutation
			Total
Resistance level	With R463L mutation N (%)	Without R463L mutationN (%)	N (%)
Low	19 (86.4)	2 (11.8)	21 (53.8)
High	3 (13.6)	15 (88.2)	18 (46.2)
Total	22 (100.0)	17 (100.0)	39 (100.0)

χ² (df) = 21.474 (1), p < 0.05.

inhA promoter mutation at −15 always revealed a low level of resistance. Furthermore, the frequency of point mutations (7.2%) was low in the oxyR-ahpC region which leads to the low level of resistance.

Discussion

The present study observed the highest frequency of mutation at katG315 codon (78.6%), which is similar to the reported mutation frequency (78.4%) in Southeast Asia, while the global frequency is 64.2%.²² However, variations in frequencies are reported when different countries are considered individually, for example, Shanghai China (62.8%),²³ Bangladesh (83.9%),²⁴ Myanmar (57.3%),²⁵ and India (65.8%).²⁶

High level of resistance with stand-alone katG mutation S315T is comparable with findings in previous studies.^9,27–29 Ser-315 is located at the periphery of the INH binding pocket³⁰ of CP enzyme. Ser-315 interacts with the hydrazine part of INH.³¹ The heme binding site is considered as the active site of CP containing the energetically favorable binding site for INH. Addition of a methyl group in Thr in the mutated CP limits the accessibility of INH to the heme site by reducing dimensions of the narrowest part of the access channel; from 6 Å in the wt CP to 4.7 Å.³² The total number of hydrogen bonds binding to the heme moiety also plays an important role in INH activation and the lower number of hydrogen bonds in mutated CP than wt CP leads to INH resistance due to low INH binding affinity.²⁶

Mutation at codon 463 of the katG gene is reported as a polymorphism that is not associated with INH resistance.^33,34 However, in silico translation analysis of the mutant CP demonstrated that the Arg463Leu mutation contributes to INH resistance.³⁵ The presence of stand-alone Arg463Leu mutation in four high/low resistant isolates also agrees with Purkan's explanation. Nevertheless, the high frequency of the low level of resistant isolates (n = 23, 41.07%) with occurrence of the katG codon 315 and 463 mutation combination raises the need for further investigations to find a mechanism to explain how it reduces the level of resistance.

An interesting observation of the current research was the extremely slow growth rate (5 weeks to see the first colony) of isolates (n = 4) with the S315N mutation compared with other isolates in both control and INH-included plates. It confirms the observation of slow growth of Mtb associated with the S315N mutation.³⁶ Furthermore, an in silico analysis based on the heme binding score explained that S315N contributes to high degree of resistance,²⁶ but the level of resistance observed for katG S315N was always low in the present investigation. This incompatible observation compared with in silico analysis may be due to the association of katG S315N mutation along with the R463L mutation.

The substitution of proline for threonine at codon 275 was explained by De Vito and Morris.³⁷ They showed that codon 275 is positioned near the heterodimer active site of CP and replacement of a proline for threonine at this residue should change the physical structure of the active site, resulting in reduced substrate binding,³⁷ which favors high resistance. Furthermore, Cade et al. explained the loss of catalase activity with this mutation by the conformational stringency of proline, which considerably perturbs the H bond network.³⁸ In the current investigation, threonine at codon 275 was substituted by alanine. As Cade et al. explained, the resistance in this instance also could be due to the hydrophobic nature of the methyl group that may interfere with the H bond network.

Studies on mutations of fabG1, which encodes b-keto acyl reductase, have reported controversial views on INH resistance. Banerjee et al. report that fabG1 is not involved in INH resistance,³⁹ but recent studies have found that synonymous mutation at fabG1 (g609a) contributes to INH resistance.^40,41 It has been reported that silent mutations can slower the protein folding mechanism and lead to altered formation of protein.⁴² Furthermore, the coexistence of silent mutation with other mutations can enhance the effect of the mutated protein.⁴² However, in the present study, a novel silent mutation was observed at codon 27 of the fabG1 gene occurring along only with the katG S315T mutation, which revealed a low level of resistance. Therefore, it could be suggested that codon 27 mutation of the fabG1 gene may be responsible for lowering the otherwise high resistance due to stand-alone S315T mutation.

There were four other silent mutations: G3G and Y158Y in the inhA gene and L293L and L521L in the katG gene with a low resistance level. All these silent mutations coexisted with either S315T or R463L katG mutations or both plus inhA or ahpC mutations [G3G+S315T+R463L; Y158Y−15 (C to T)+R463L+37 (C to T); L293L+S315T+R463L+G212D+37 (C to T); and L512L+R463L+37 (C to T)]. Therefore, the effect of these silent mutations for lowering of the resistance level is not clear.

Although the point mutation at −15 of the inhA promoter region has been reported as the most common inhA mutation (19.2%) worldwide,²² it was observed with 8.9% frequency in the present study. The presence of this mutation always led to a low level of INH resistance, confirming the observations made by previous studies.²⁷

The C deletion mutation at the −34 promoter region has not been reported hitherto in Asian or in Western countries. It was the most common mutation (12.5%) identified among inhA mutations in this study. This deletion mutation occurred along with the katG 315 mutation and revealed a high level of resistance. As it is located in the promoter region, it may affect the initiation of the transcription process. Therefore, it is important to do further computer simulation protein folding studies to see whether this mutation contributes to the high level of resistance. As this deletion always occurred with the katG315 mutation, it lacks significance as an independent marker for diagnosis in INH resistance of MTb.

The globally reported ahpC mutation frequency is <1.3%.²² However, in the present study, four (7.2%) isolates had ahpC mutations. Furthermore, these mutations have little diagnostic significance since they always coexisted with the other katG 315 mutation. This was an important observation as the contribution of ahpC mutations for INH resistance of Sri Lankan MTb isolates has not been determined previously. A high frequency of polymorphism at position 37 in the oxyR pseudogene was observed, irrespective of resistance levels. However, this polymorphism is not associated with INH resistance, as reported by Sreevatsan et al.¹¹

A single resistant isolate failed to PCR amplify the katG gene, but remained wt to inhA and ahpC genes. It may be due to deletion of the katG gene of this isolate, similar to observations reported previously.^43,44

Two INH-resistant isolates did not divulge mutations in any of the three genes that were investigated. It is possible that the resistance may be due to INH mutations that occur at low frequency such as ndh, kasA, or other recently found novel mutations by whole genome sequencing.⁴⁵ Furthermore, these isolates may confer efflux-mediated INH resistance due to one or more efflux pumps working alone or in coordination with other mechanisms.⁴⁶

Conclusion

In conclusion, the katG gene mutation at codon 315 was the highest prevalent mutation among all the tested INH-resistant MTb isolates. The S315T contributed to the high resistance level, whereas the presence of the katG 463 mutation along with the katG 315 mutation revealed a tendency to reduce the resistance level. With the exception of one isolate with a −15 inhA mutation, all the other inhA and ahpC gene mutations coexisted with katG 315 mutations, indicating that there is little clinical importance of these mutations being used as diagnostic tools in molecular detection methods. Therefore, screening of katG 315 mutations is confirmed as a useful marker for molecular detection of INH resistance of MTb in Sri Lanka.

Footnotes

Acknowledgments

The authors thank Dr. Channa Senanayake and the staff of the Department of Microbiology and RPFC, Faculty of Medicine, University of Colombo, Dr. Kirthi Gunesekara, Chest Physician, Central Chest Clinic, Colombo, and the laboratory staff of NTRL for providing assistance. The study was supported by National Research Council, Sri Lanka (NRC 12–140 grant).

Ethical Approval

The study was approved by the Ethics Review Committee of the Faculty of Medicine, University of Colombo, Sri Lanka (EC/06/062).

Disclosure Statement

No competing financial interests exist.

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