Frequency of Mutations Associated with Rifampicin Resistance in Mycobacterium tuberculosis Strains Isolated from Patients in West of Iran

Abstract

Background and Objective: Tuberculosis (TB) is a devastating infectious disease causing high mortality and morbidity worldwide. The most serious threat related to tuberculosis control is the recent emergence of drug-resistant tuberculosis strains. The aim of the present study was to identify various types of mutations in the rpoB region in rifampicin-resistant strains isolated from sputum of tuberculosis patients. Materials and Methods: Drug susceptibility of 125 Mycobacterium tuberculosis isolates was determined using the CDC standard conventional proportional method. Target DNA of M. tuberculosis was amplified by polymerase chain reaction, hybridized, and scanned. We used the low cost density array (LCD-array) to detect mutations within the 90-bp rpoB region. Each array is a transparent, prestructured polymer support using a nonfluorescent detection principle based on the precipitation of a clearly visible dark substrate. Results: Of the 125 M. tuberculosis isolates, 35 (28%) were found to be rifampicin-resistant and using the LCD array revealed point mutations at nine different codons as follows: S512T (AGC→ACC) (20%), D516V (GAC→GTC) (20%), H526D (CAC→GAC) (6%), H526R (CAC→CGC) (20%), H526Y (CAC→TAC) (23%), and S531W (TCG→TGG) (8%), and the most frequent site mutations were L511P (CCG→CTG) (46%), followed by S531l (TCG→TTG) (40%) and D516Y (GAC→TAC) (26%). Conclusion: Our data significantly differs from previously reported mutation frequencies for codon 526 (CAC to GAC) among Italian isolates (40.1%) and Greek isolates (17.6%). Phenotypic testing is time-consuming and requires laboratory resources. Microarray rpoB is useful in detecting rifampicin resistance-determining region-associated site mutations of rifampicin-resistant M. tuberculosis isolates.

Introduction

Tuberculosis (TB) is the most hazardous bacterial infection worldwide, caused by Mycobacterium tuberculosis and still remains a major public problem, especially in developing countries, with approximately 8 million new infections and 2.5 to 3 million deaths per year.^15,18

A more alarming aspect of tuberculosis is the emergence of multidrug-resistant strains of M. tuberculosis (MDR-TB), which is a widespread phenomenon. MDR-TB is a form of TB that is difficult and expensive to treat because it fails to respond to two important first-line drugs, rifampicin (RIF) and isoniazid (INH).⁷ The development of drug resistance may be a tragedy not only for the patients alone but also for others as they can infect other people with their drug-resistant organisms.¹⁰ Resistance to RIF is almost exclusively associated with mutations in the rpoB gene, which encodes the β subunit of RNA polymerase in M. tuberculosis.²³ More than 70 distinct rpoB mutations have been characterized from RIF-resistant isolates of M. tuberculosis worldwide.^{3,17,20,24,35}

In general, mutations of rpoB can be found in 96.1% of rifampicin-resistant M. tuberculosis isolates worldwide, and rpoB mutations usually occur in the 81-bp region corresponding to 507–533 amino acid residues called the rifampicin resistance-determining region (RRDR). Of the site mutations at the RRDR, H526D/Y/L/R, S531L, D516V, and L533P are closely correlated with rifampicin resistance.²³

There are various methods for detection of rifampicin-resistant strains, including conventional and molecular methods. The conventional methods based on culture and proportional assay of the bacterial sensitivity are time-consuming and need several weeks for bacterial growth, which leads to delay in the identification of resistant strains and jeopardizes the efforts to control the disease.²⁵ Exploring the molecular genetic basis of drug resistance will contribute to the establishment of efficient methods for the rapid identification of drug-resistant strains. In recent years, several molecular techniques have been applied to detect mutations related to anti-tuberculosis drug resistance. These include amplification and restriction fragment length polymorphism amplification and sequencing.^8,9,26

Over the last two decades, many studies have utilized molecular biological methods to detect rpoB gene mutations related to rifampicin-resistant M. tuberculosis in various cohorts of patient samples.^13,19,35 In this study, we investigated the rifampicin resistance frequency of M. tuberculosis strains isolated from sputum samples of tuberculosis patients and the dominant site mutation types in rifampicin-resistant M. tuberculosis isolates.

Materials and Methods

A total of 125 clinical M. tuberculosis strains isolated from sputum samples collected at the TB reference laboratory of Kermanshah, Iran, between September 2011 and March 2013, were tested by conventional biochemical methods. Bacterial culture was accomplished in the Lowenstein-Jensen medium and the isolates were identified by standard microbiological methods such as Ziehl-Neelsen staining, morphology of colony, pigment and niacin production, and nitrate and catalase tests.

Antimicrobial susceptibility testing (AST) was performed using the CDC standard conventional proportional method. Rifampicin (40 μg/ml) was used in slants and, in addition to the breakpoint concentration for rifampicin (2.0 μg/ml) in the Bactec system, AST was performed to identify resistance using different concentrations of rifampicin (50, 75, and 100 μg/ml) through the slant proportional method.⁴

For all experiments, M. tuberculosis H37Rv was used as the control strain. Chromosomal DNA was extracted from M. tuberculosis using the QIAamp genomic DNA Kits. Extracted bacterial DNA, free of inhibitors of the polymerase chain reaction (PCR) reaction, was used as the starting material. The prelabeled PCR primer mixes provided with the kit generated labeled fragments of the bacterial DNA. The fragment amplified from the rpoB gene (β subunit of RNA polymerase) spanned the 81-bp mutation hot spot region where point mutations could be localized in ∼90% of rifampicin-resistant isolates of M. tuberculosis. This was followed by the low cost density array (LCD-array; Myco-Resist 3.5 Kit; Chipron, Berlin, Germany) that has been developed for identification of point mutations in the rpoB gene.

The final image was captured using a transmission-light film scanning device (Chipron). Two software packages (SlideReader 1.1; Chipron and GenePix Pro 5.0; Axon Instruments, Inc.) and the online analysis module (Chipron) available at www.chipron.com were used to analyze the hybridization signals.

Results

A total of 125 M. tuberculosis strains were isolated from tuberculosis patients, 85 (68%) of which were from males and 40 (32%) from females. All isolates were from newly diagnosed, previously untreated cases in our study. The mean age of the patients was 44.2±17.4 (SD). Among these isolates, 35 (28%) were resistant to rifampicin. Analysis of rifampicin-resistant isolates showed nine different mutations in the rpoB gene.

From our microarray results, 100% of the rifampicin-resistant M. tuberculosis isolates were found to have site mutations at the RRDR of the rpoB gene. The most frequent site mutations were L511P (CCG→CTG) (n=16), followed by S531l (TCG→TTG) (n=14) and D516Y (GAC→TAC) (n=9). All the site mutation types found in this study are shown in Tables 1 and 2. Mutations in codons 526, 531 and 511, 516 were observed in 48% and 46% of rifampicin-resistant isolates, respectively. In total, 19 (54.28%) of the 35 rifampicin-resistant isolates demonstrated combinations of multiple mutations (double [n=6, 17%], triple [n=8, 23%], quadruple [n=4, 11%], and quintuple [n=1, 2.8%] mutations) in at least 2, 3, 4, and 5 codons (511, 512, 516, 526, and 531), respectively. Furthermore, an isolate had five different mutations simultaneously: D516V, H526D, S531L, H526R, and S512T. This particular isolate had a high minimum inhibitory concentration (MIC) of ≥100 μg/ml (Table 2).

Table 1.

Frequency of Amino Acid and Nucleotide Changes in Different Codons of the rpoB Gene in Rifampicin-Resistant Strains

Codon	Amino acid changes	Nucleotide mutation	Tape	No. of isolates in rifampicin-resistant strains (%)
511	Leu→Pro	CTG→CCG	Substitution	16 (46)
512	Ser→Thr	AGC→ACC	Substitution	7 (20)
516	Asp→Tyr	GAC→TAC	Substitution	9 (26)
516	Asp→Val	GAC→GTC	Substitution	7 (20)
526	His→Asp	CAC→GAC	Substitution	2 (6)
526	His→Arg	CAC→CGC	Substitution	7 (20)
526	His→Tyr	CAC→TAC	Substitution	8 (23)
531	Ser→Trp	TCG→TGG	Substitution	3 (8)
531	Ser→Leu	TCG→TTG	Substitution	14 (40)

Table 2.

Distribution of rpoB Mutations (Single, Double, Triple, Quadruple, and Quintuple) in Rifampicin-Resistant Strains

No. of mutations	Codon number	No. of isolates	MIC (μg/ml)
1 mutation	Leu511 Pro	3	≤75
	Ser512 Thr	4	∼50–75
	His526 Arg	1	≤75
	Ser531 Leu	5	≥100
	His526 Asp	1	∼50–75
	His526 Tyr	1	≤75
	Asp516Val	1	≤75
2 mutations	Leu 511 Pro-Ser531 Leu	1	∼50–75
	His526 Arg-Ser531 Leu	1	∼50–75
	Asp516Val-Ser531 Leu	1	≥100
	His526 Arg-Ser531 Trp	1	∼50–75
	Leu 511 Pro-Asp 516Val	1	≤75
	Leu 511 Pro-Asp516 Tyr	1	≤75
3 mutations	Leu 511 Pro-His 526 Tyr-Ser531 Leu	1	≥100
	Leu 511 Pro-Asp 516 Tyr-His526 Tyr	4	≥100
	Leu 511 Pro-Asp 516 Tyr-Ser531 Leu	1	∼50–75
	Leu 511 Pro-Asp 516 Val-His526 Tyr	1	≥100
	Ser 512 Thr-Asp 516 Tyr-Ser531 Leu	1	≥100
4 mutations	Ser512Thr-Asp516Val-His526Arg-Ser531Trp	1	≥100
		1	≥100
	Leu 511 Pro-516 Tyr-526 Tyr-531 Leu	1	≥100
	Leu 511 Pro-516 Val-526Arg-531Leu	1	≥100
	Leu 511 Pro-516 Tyr-526 Arg-531 Trp
5 mutations	Ser512 Thr-Asp516Val-His526 Asp-His526 Arg-Ser531-Leu	1	≥100

MIC, minimum inhibitory concentration.

Discussion

Rifampicin resistance in M. tuberculosis is widespread in many areas of the world. Rifampicin and isoniazid resistance is a surrogate marker for MDR M. tuberculosis. This study was focused on determining the frequency of specific rpoB mutations in rifampicin-resistant M. tuberculosis isolates from TB patients. Notably, all isolates in our study were from newly diagnosed cases in the region. Resistance to rifampicin is almost exclusively associated with mutations in the rpoB gene, which encodes the β subunit of RNA polymerase.³⁸ More than 95% of rpoB mutations in rifampicin-resistant clinical isolates have been found within the RRDR. The rpoB codons 531, 526, 516, and 511 are the most frequently mutated sites observed worldwide.^5,26,27,32

In this study, 28% of isolates were rifampicin-resistant, which was still lower than in many other areas in Europe and Africa (51.9%–68.9%).³⁸

This is the first report describing the genetic characteristics of rifampicin-resistant strains isolated from TB patients in the west of Iran. The common mutations observed in this region included 531 (n=17, 48%), 526 (n=17, 48%), 516 (n=16, 46%), and 511(n=16, 46%). A study by Jun et al. from China reported frequent occurrence of mutations at codon 531 (51.6%) and codon 526 (32.26%).¹⁶ Abdelaal et al. from Egypt reported frequencies of mutations as follows: codons 531 (45%), 526 (30%), and 516 (20%).¹ This indicates that mutations at 531, 526, and 516 are common, with some geographical variations in frequencies.⁶ These differences reflect the complex and crucial interactions between the drug and its target at the molecular level where the position of the affected allele seems to be variable.³³

All rifampicin-resistant isolates studied had mutations of different types in the rpoB region; however, the relationship between the combination of specific types of mutations and rifampicin resistance is unclear.

Patra from India reported the frequency of mutation in codon 531 (TCG→TTG), 526 (CAC→TAC), and 516 (GAC→GTC) to be 60%, 26.6%, and 6.6%, respectively.²⁹

In another investigation from Brazil, the codons most frequently affected were 531 (TCG→TTG), 526 (CAC→TAC), and 516 (GAC→GTC) with frequencies of 54%, 21%, and 7%, respectively.³⁹ In the other study, codon 526 (CAC→TAC) was reported to be the most frequent site of mutation in the RRDR of the rpoB gene.³⁴

However, variations in the relative frequencies of mutations in these codons have been described for isolates from different geographic locations.^5,21 In our study, three types of mutations were demonstrated in codon 526: CAC→TAC (n=8; 23%), CAC→CGC (n=7; 20%), and CAC→GAC (n=2; 6%) and two types of mutations were seen in codon 531:TCG→TGG (n=3; 8%) and TCG→TTG (n=14; 40%). Researchers elsewhere have reported additional variations of nucleotide changes in codon 531 (in India, TCG→TGG or TTG; in Russia, TCG→TGG, CAG, or TGT; in China, TCG→TTG; in Japan, TCG→TTG; in Korea, TCG→TTG; and in Taiwan, TCG→TTG) and in codon 526 (in India, CAC→CTC, TAC, GAC, CGC, or ACC; in Russia, CAC→CTC, GAC, CAA, CAG, TGC, AAC, CGC, or CCC; in China, CAC→TAC; in Japan, CAC→TAC; in Taiwan, CAC→TAC and CGC; in Korea, CAC→TAC; and in Brazil, TCG→TTG).^11,12,26,39

Our data significantly differs from previously reported mutation frequencies for codon 526 (CAC to GAC) among Italian isolates (40.1%)¹⁶ and Greek isolates (CAC to GAC; 17.6%).²² The high level of mutations conferring resistance in this unique geographic area where tuberculosis is endemic could be explained by the poor border control, population movement due to economic and tribal communications, lack of rapid identification methods for MDR M. tuberculosis, and inadequate chemotherapy.

Prevalence of two and three point mutations was high in our region at 40% (14 of 35). In a study from India,³⁷ the prevalence of double–triple point mutations was found to be 48.6% (17 of 35). Furthermore, a study from Thailand³⁰ showed 5.2% and another study from China¹² observed 12.4% prevalence of two to three point mutations, which were low in comparison to our findings.

The manufacturer's instructions warn that other mutations than those detected by the LCD-Array MYCO Resist 3.5 as well as unknown mechanisms can be the cause of resistance to rifampicin in M. tuberculosis and this assay should be run along with conventional (e.g., microbiological) methods, not to replace it. In our study, only 8.5% of strains had mutations at codon P511 (MIC ≤75), which is consistent with the results of other studies.^2,36

On the other hand, other studies have reported isolates with mutations at codon P 511L and associated this mutation with susceptibility to rifampicin.^{14,28,31,40,41} While the MIC corresponding to this mutation was ≤75 μg/ml, other mutations are most likely involved in these isolates, but not detectable with this kit (array). Explanation for differences of MIC values is as follows: Strains with the single mutation L511P show lower values (<75 μg/ml) than double or triple mutation where L511P is only one of the mutations (MIC ∼75 or >100 μg/ml). These results (MIC values) can be an indicator for the existence of other mutations outside of the 81-bp rpoB hotspot covered by our array.

In conclusion, although culture-based phenotypic susceptibility test is the gold standard for detecting drug resistance in MTB and no molecular method can yet completely substitute it, microarray provides a rapid screening tool for the majority of mutations occurring in genes related to rifampicin. However, the molecular assays only detected known mutations, which is the most important limitation in detection of drug resistance by such techniques. Based on the other studies, not all mutations related to anti-TB drugs are known yet. Since the prevalence of mutations may vary by geographic area, identification of a resistance-associated mutation can be instructive, but lack of a mutation in the target sequence must be interpreted with caution.

These results illustrate the need for further research to compare a more sensitive and specific technical assay for the detection of MDR M. tuberculosis in both rifampicin and isoniazid resistance to be used as a screening method in regions with high endemicity of M. tuberculosis infection.

Footnotes

Acknowledgment

We gratefully acknowledge Vice-Chancellor for Research and Technology, Kermanshah University of Medical Sciences, for financial support of this study resulting from MSc Medical Microbiology thesis of Hadis Sadri, Kermanshah University of Medical Sciences, Iran (Grant No. 91179).

Disclosure Statement

No competing financial interests exist.

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