https://orcid.org/0000-0001-7727-7452
Figures
Abstract
Type II toxin-antitoxin systems such as mazEF3, vapBC3, and relJK play a role in antibiotic resistance and tolerance. Among the different known TA systems, mazEF3, vapBC3, and relJK, which are type II systems, have specific roles in drug resistance. Therefore, the aim of this study was to investigate the mutations in these genes in sensitive and resistant isolates of Mycobacterium tuberculosis. Thirty-two rifampin-resistant and 121 rifampin-sensitive M. tuberculosis isolates were collected from various regions of Iran. Lineage typing was performed using the ASO-PCR method. Mutations in the rpoB gene were analyzed in all isolates by MAS-PCR. Furthermore, mutations in the mazEF3, relJK, and vapBC3 genes of the type II toxin system were assessed through PCR sequencing. These sequences were analyzed using COBALT and SnapGene 2017, and submitted to the GenBank database. Among the 153 M. tuberculosis samples, lineages 4, 3 and 2 were the most common. Lineage 2 had the highest rate of rifampin resistance. Mutations in rpoB531 were the most frequent in resistant isolates. Examination of the toxin-antitoxin system showed that rifampin-resistant isolates belonging to lineage 3 had mutations in either the toxin or antitoxin parts of all three TA systems. A mutation in nucleotide 195 (codon 65) of mazF3 leading to an amino acid change from threonine to isoleucine was detected in all rifampin-resistant isolates. M. tuberculosis isolates belonging to lineage 2 exhibited the highest rifampin resistance in our study. Identifying the mutation in mazF3 in all rifampin-resistant isolates can highlight the significance of this mutation in the development of drug resistance in M. tuberculosis. Expanding the sample size in future studies can help develop a new method for identifying resistant isolates.
Citation: Shafipour M, Mohammadzadeh A, Mahmoodi P, Dehghanpour M, Ghaemi EA (2024) Distribution of lineages and type II toxin-antitoxin systems among rifampin-resistant Mycobacterium Tuberculosis Isolates. PLoS ONE 19(10): e0309292. https://doi.org/10.1371/journal.pone.0309292
Editor: Francesca Boldrin, University of Padova, ITALY
Received: May 18, 2024; Accepted: August 7, 2024; Published: October 24, 2024
Copyright: © 2024 Shafipour et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All gene sequence files are available from the GenBank NCBI database (accession numbers: OR268987.1-OR269002.1 for mazEF3, OR269015.1- OR269030.1 for relJK, and OR269043.1-OR269058.1 for vapBC3). All necessary data are either are provided in the text of the manuscript and its Supporting Information files or deposited in NCBI with the mentioned accession numbers in the text. Additionally, all raw data set are available upon reasonable request to the corresponding author of the article. The manuscript includes methods and essential information, such as accession numbers, figure guides, primer sequences, and testing protocols.
Funding: This work was supported by Golestan University of Medical Sciences, Gorgan, Iran (grant number: IR.GOUMS.REC.1401.224).
Competing interests: The authors have declared that no competing interests exist.
Introduction
Several genetic modules are involved in antibiotic resistance. Transcriptomic analysis of antibiotic-resistant Mycobacterium tuberculosis (M. tuberculosis) isolates revealed that in pathogenic Mycobacteria, Toxin-Antitoxin (TA) systems are significantly expressed [1, 2]. To date, approximately 90 putative TA systems have been identified in M. tuberculosis. It has been proven that these systems help the survival of bacteria under stressful conditions, including starvation, oxidation, immune response, and exposure to antimicrobial agents [3–5].
The TA system consists of two parts: a protein toxin and a cognate antitoxin (protein or nonprotein). The antitoxin controls the toxin [6, 7]. In M. tuberculosis, the most abundant TA system is type II. Eighty-eight type II families have been identified in M. tuberculosis H37Rv and play important roles in bacterial survival under different adverse conditions. Type II toxins act as RNases, kinases, and acetyltransferases and are involved in bacterial virulence, biofilm formation, phage inhibition, and various types of stress management, including antibiotic tolerance and persister formation [5]. When the bacterium is exposed to stress conditions such as starvation, hypoxia, and antibiotic exposure, the antitoxin is decomposed, and the toxin affects its target. As a result, the cells in stressful conditions become persistent [5, 8]. The most abundant family within the type II TA systems is vapBC. To date, at least 50 vapBC families have been identified. Other significant families include 10 mazEFs, 3 relBE families, 3 relBE-like modules (relBE, relFG and relJK), and the tripartite type II TAC (toxin-antitoxin-chaperone) system [5, 8, 9]. Studies have shown that the expression of certain type II families, such as mazEF3, vapBC3, and relJK, is altered in antibiotic-resistant strains. The mazEF3 family consists of the mazF3 toxin and its cognate mazE3 antitoxin. The MazF toxins act as ribosome-independent mRNA endoribonucleases. The toxic effect of MazF in the cell is neutralized by the MazE antitoxin [10]. MazEF plays roles in promoting programmed cell death, inhibition of bacterial growth, persistence of infections, latent TB, and bacterial adaptability to the microenvironment [11]. Additionally, M. tuberculosis H37Rv contains three RelBE-like modules, relBE, relFG, and relJK, which are expressed in human macrophages during infection [12]. RelK and VapC3 are toxins, both of which have RNase activity, and these toxins are neutralized by RelJ and VapB3, respectively [13]. RelK and VapC3 demonstrate RNase activity, leading to the suppression of colony formation upon overexpression. RelK functions bacteriostatically by impeding bacterial growth and inducing cells to transition into the stationary phase. Interestingly, research indicates that heightened levels of RelK may contribute to a rise in rifampin-tolerant cells [11, 13]. Given that the expression of MazF3, RelK, and VapC3 toxins has been shown to increase in the presence of antibiotics in several studies, this study aims to investigate mutations in these toxins and compare their presence in both rifampin-sensitive and rifampin-resistant strains.
On the other hand, molecular epidemiological studies on M. tuberculosis infection have shown that drug resistance, tolerance, bacterial adaptation, and different responses to vaccination are related to specific genotypes and/or lineages of M. tuberculosis [14]. Additionally, recent evaluations have revealed that the type II toxin—antitoxin system varies among different lineages of M. tuberculosis, with mutations being observed in various lineages [15, 16].
Molecular techniques such as allele-specific oligonucleotide multiplex PCR (ASO-PCR) can be used to classify clinical isolates into genetic groups. Based on phylogenetic relationships, M. tuberculosis isolates are categorized into 7 main lineages: Indian Ocean (lineage 1), East Asian (lineage 2, which includes the Beijing clade), Indian and East African (lineage 3), Euro-American (lineage 4), West African-1 (lineage 5), West African-2 (lineage 6), and Aethiops vetus (lineage 7) lineages. Studies have indicated that certain lineages of M. tuberculosis, particularly lineage 2, exhibit increased virulence and antibiotic resistance [14, 17].
Studies have shown that 96% of rifampin-resistant isolates have mutations in the hotspot region of rpoB, which encodes the β-subunit of RNA polymerase [18]. Therefore, the present study aimed to investigate the relationships between rifampin resistance and the type of mutation in the rpo gene with the lineage and sequence of three important type II genes of the TA system, mazEF3, relJK, and vapBC3.
Materials and methods
Sample collection and DNA extraction
Thirty-two rifampin-resistant M. tuberculosis isolates were collected from four provinces: Tehran, Golestan, Khorasan Razavi, and Semnan. In addition, 121 rifampin-sensitive M. tuberculosis samples were collected from Golestan Province, which is one of the main centers of tuberculosis in Iran. The required permission to conduct the study was granted by the National Tuberculosis Center and the ethics committee of Golestan University of Medical Sciences. Subsequently, in collaboration with the Infectious Diseases Research Center and the Central Tuberculosis Laboratory of Golestan Province, Iran, access to the samples was authorized. A confirmatory test for rifampin resistance was conducted using the proportional method for all M. tuberculosis isolates [19]. For molecular evaluation, DNA extraction was carried out using a Cinnagen DNA Extraction Kit (Cinnagen, Co. Iran). The extracted DNA was stored at -20 °C. Subsequently, molecular confirmation of the bacteria and antibiotic resistance to rifampin was performed for all M. tuberculosis isolates.
Molecular confirmation of M. tuberculosis isolates
Molecular confirmation of M. tuberculosis species was conducted using IS6110 PCR. The PCR was carried out in a 25 μl volume, consisting of 10x PCR buffer, 1.5 mM MgCl2, 10 mM dNTPs, 0.5 μM of each primer, 1.5 U of Taq DNA polymerase, and 100 ng of extracted DNA. The Thermocycler program included initial denaturation at 94°C for 4 minutes, followed by 30 cycles denaturation at 94°C for 30 seconds, annealing at 68°C for 40 seconds, and extension at 72°C for 55 seconds, and final extension at 72°C for 8 minutes. The resulting amplicon size was 123 bp. Positive control sample included M. tuberculosis H37Rv and distilled water were used as positive and negative controls, respectively [20].
Evaluation of rpoB gene mutations
The confirmation of rifampin resistance was initially conducted using the proportional method for all 153 isolates [19]. In addition, mutations in the rpoB516, rpoB526, and rpoB531 genes were assessed for all M. tuberculosis-resistant isolates. Furthermore, resistance to isoniazid was determined by analyzing mutations in katG codon 315 and mab-inhA-15 using multiple allele-specific polymerase chain reaction (MAS-PCR) based on the method outlined by Chia et al. The reaction mixture included the following primers: rpoB516 (1 pmol), rpoB526 (5 pmol), rpoB531 (32.5 pmol), RIRm (30 pmol), katG F (1 pmol), katG R (1 pmol), inhAP-15 (6 pmol) and inhAP F (6 pmol). The other reagents used were 10X PCR buffer (2.5 ml), 50 mM MgCl2 (2 ml), 10 mM dNTP mixture (0.5 ml), 5 U/ml Taq Polymerase (0.1 ml), DNA template (20 ng), and PCR-grade water to obtain a final volume of 25 ml. The Thermocycler temperature and time protocol were as follows: 96°C for 3 minutes, 25 cycles of 95°C for 50 seconds, 68°C for 40 seconds, and 72°C for 1 minute, and a final extension at 72°C for 7 minutes. The PCR products were electrophoresed on a 2.5% agarose gel to evaluate of mutations [21]. The absence of bands at 293, 270, 218, 184, and 170 bp indicated the mutations in the katG, mabA-inhA, rpoB516, rpoB526, and rpoB531 gene, respectively.
Lineage typing of M. tuberculosis isolates
For lineage typing, the ASO-PCR method designed by Carcelén et al. was utilized, with specific primers listed in Table 1 [22]. The Thermocycler temperature and time protocol were as follows: 95°C for 10 minutes, 29 cycles of 95°C for 1 minute, 62°C for 1 minute, and 72°C for 1 minute, and a final extension at 72°C for 10 minutes. After electrophoresis of the PCR product, the lineage type was determined based on the presence or absence of the amplicon. According to Carcelén et al. study, lineage 1, lineage 2, lineage 3, lineage 4, lineage 5, and lineage 6 lack amplicons of 95 bp, 345 bp, 434 bp, 520 bp, 183 bp, and 267 bp, respectively [22].
Evaluation of the mazEF3, relJK, and vapBC3 genes of type II toxin-antitoxin complexes in M. tuberculosis isolates
Mutations in the mazEF3, relJK, and vapBC3 genes in lineage 2, 3, and 4 of M. tuberculosis (sensitive and resistant isolates) were investigated using specific primers listed in Table 1 [23]. The mazEF3, relJK, and vapBC3 genes were sequenced in 16 isolates (8 rifampin-sensitive and 8 rifampin-resistant isolates). Finally, the relationships of these mutations with protein changes and antibiotic resistance were evaluated.
Among the rifampin-sensitive and resistant M. tuberculosis isolates belonging to each lineage, certain isolates with varying mutations in the rpoB gene were chosen, and their PCR products were sent to the Momgene Company (in Iran) for sequencing. Their sequences were analyzed using the NCBI database, COBALT for protein alignment, and SnapGene 2017. Subsequently, the sequences were submitted to the NCBI website, and assigned accession numbers.
Results and discussions
Molecular confirmation of M. tuberculosis isolates and detection of rifampin resistance
The molecular approach using IS6110 PCR, confirmed the accurate detection of all M. tuberculosis isolates. All the investigated isolates showed the desired amplicon (123 bp). Among the isolates, 121 were sensitive and 32 were resistant to rifampin, as determined by the proportional method. These isolates were analyzed using the MAS‒PCR method to identify mutations in the rpoB hotspot regions. Mutations were only observed in the resistant isolates, confirming their resistance. This molecular approach successfully confirmed the sensitivity and resistance of the M. tuberculosis isolates.
Distribution of mutations in the rpoB gene
In phenotypically rifampin-sensitive isolates, no gene mutation leading to resistance to rifampin were observed. However, all phenotypically rifampin-resistant isolates had at least one mutation in the hotspot region of the rpoB gene. This indicates that mutations in these three codons of the rpoB gene directly correlate with phenotypic resistance. Mutations in the rpoB531 gene were found in 20 isolates (62.5% of the resistant isolates), with 15 samples showing individual mutations and 5 isolates showing mutations in combination with others. Mutations in the rpoB516 gene were individually observed in 12 patients and in combination with other mutations in 5 samples. Mutation in the rpoB526 gene was the least frequent and was observed in only one isolate. This mutation occurred simultaneously with other mutations. In the MAS-PCR method, mutations in the katG and mabA-InhA genes, which are responsible for isoniazid resistance, were also investigated. Four isolates (12.5%) were determined to be isoniazid resistant. These strains are also resistant to rifampin, which is known as MDR.
Lineage of M. tuberculosis isolates
Twenty-eight (18.30%), 50 (32.67%), and 75 (49.01%) MTB isolates belonged to lineages 2, 3, and 4, respectively. The frequency of rifampin resistance among these strains was 7 (25%), 9 (18%), and 16 (21.3%), respectively, showing that the resistance in lineage 2 was greater than that in the other lineages (Table 2). The Chi-square test was used to investigate the relationship between lineage type and resistance and sensitivity to rifampin. Although the amount of antibiotic resistance in lineage 2 was higher than that of lineages 3 and 4, a p-value of 0.761 was obtained, indicating the absence of a significant relationship between antibiotic resistance and lineage type. However, in various studies, this relationship has been found to be significant.
Among all the samples, 4 isolates (12.5%) were considered multidrug-resistant (MDR), and all of them belonged to lineage 2. In this lineage, none of the rifampin-resistant isolates had a single mutation in rpoB516, and 5 isolates (71.4%) had simultaneous mutations. However, simultaneous mutations were not found in members of lineages 3 and 4 (Table 3).
Toxin-antitoxin system analysis
The genes mazEF3, relJK, and vapBC3 were sequenced in 16 isolates half of which were rifampin-sensitive and the other half rifampin-resistant. The accession numbers of these samples in GenBank are provided in Table 4.
Sequence analysis in SnapGene 2017 software revealed that the mazE3 gene and its corresponding protein, MazE3 antitoxin, were similar in all investigated isolates across the three lineages. Comparison of these sequences with the standard sequence of M. tuberculosis H37Rv did not show any mutations. However, the mazF3 toxin gene in lineage 3 rifampin-resistant isolates had a mutation at nucleotide 473, changing the nucleotide G to A without altering the amino acid sequence. In all rifampin-resistant isolates, regardless of lineage, a mutation occurred at codon 65 of the toxin gene, changing the nucleotide C to T and resulting the amino acid T (threonine) changing to I (isoleucine). Figs 1 and 2 display the protein sequences of the antitoxin (MazE3) and toxin (MazF3), respectively.
S: Sensitive isolates, R: Resistant isolates. Blue: no mutation.
S: Sensitive isolates, R: Resistant isolates. White: no mutation; yellow: mutation.
Compared to the standard sequence of M. tuberculosis H37Rv, the resistant isolates in lineage 3 had a mutation at nucleotide 237 in the relJ gene where the nucleotide T was changed to G, but the amino acid sequence remained unchanged. Figs 3 and 4 shows the antitoxin and toxin, respectively. In comparison to the M. tuberculosis H37Rv sequence, the toxin sequence (relK) does not contain any mutations.
S: Sensitive isolates, R: Resistant isolates. Blue: no mutation; yellow: mutation.
S: Sensitive isolates, R: Resistant isolates. Blue: no mutation; yellow: mutation.
Sequence analysis of VapB3 (antitoxin) using SnapGene software revealed that this sequence has no mutations compared to the standard sequence of M. tuberculosis H37Rv. VapC3 consists of 137 amino acids, and the resistant isolates of lineage 3 exhibit a mutation in codon 123 (nucleotide 369), where the nucleotide C is changed to G, resulting in a changed from amino acid D (aspartic acid) to E (glutamic acid). Figs 5 and 6 depict VapB3 (antitoxin) and VapC3 (toxin), respectively.
S: Sensitive isolates, R: Resistant isolates. Blue: no mutation; yellow: mutation.
S: Sensitive isolates, R: Resistant isolates. White: No mutation; Yellow: Mutation.
In total, the evaluation of the mazEF3, relJK, and vapBC3 gene sequences of the type II toxin-antitoxin system in M. tuberculosis revealed that the toxin sequences in lineage 3 had mutations in all three families (Table 5). Specifically, in the vapC3 toxin, this mutation resulted in an amino acid change from aspartic acid to glutamic acid.
Tuberculosis remains a significant global health issue. Over the past 50 years, Iran’s health authorities have made significant progress in controlling tuberculosis. As a result, the incidence of tuberculosis has decreased from 142 cases per 100,000 people in 1964 to 12.6 cases in 2016 [24, 25]. Despite Iran’s proximity to countries such as Azerbaijan, Pakistan, Afghanistan, and Iraq, as well as high rates of immigration, the incidence of drug-resistant tuberculosis in Iran is lower than expected, ranging from 0–2.9% [26].
Genomic mutations including SNPs, small insertions, or deletions, and occasionally larger deletions or inversions, are major contributors to antibiotic resistance in M. tuberculosis. Due to absence of horizontal gene transfer or episomal resistance genes, these mutations typically occur spontaneously and are encoded in chromosome. The spread of resistant bacteria occurs through replication within the host and onward transmission between hosts. Although horizontal gene transfer is absent in M. tuberculosis, target-based mutations, activator mutations, and modulation of efflux pumps are major mechanisms for acquiring antituberculosis drug resistance [27–29].
In addition to these factors, several elements play a role in the development of antibiotic resistance, including the phylogenetic lineage of M. tuberculosis, genetic modules, metabolic adaptation to the environment, and host-specific factors. Furthermore, specific lineages of M. tuberculosis may exhibit high mutation rates and a strong ability to acquire drug resistance. Several studies have demonstrated that the drug resistance of the Beijing clade from lineage 2 (East Asian Lineage) was higher than that of other lineages [30]. This study also investigated the mutations in the mazEF3, vapBC3, and relJK families of the type II toxin-antitoxin system in both resistant and sensitive isolates, with the goal of determining the differences in the gene structure of these families between these isolates.
In this study, only three lineages (2, 3, and 4) of M. tuberculosis were found in various regions of Iran, among both drug-sensitive and drug-resistant isolates. Lineage 4 was the most prevalent lineages among rifampin-sensitive isolates, while lineage 2 was most common among rifampin-resistant isolates. Although the incidence of resistance was higher in lineage 2 (25%), in the present study, this difference is not significant in sensitive and resistant isolates of the lineages (P>0.05).
The diversity of M. tuberculosis strains in Iran, based on family/subspecies, showed that New-1 (L4) had the highest frequency, at 21.94%. CAS (L3) with 19.21%, EAI (L1) with 12.95%, and T (L4) with 12.16% were the dominant circulating M. tuberculosis genotypes, while lineages 5, 6 and 7 were not reported from Iran. Additionally, the observation of lineage 3 as one of the common subpopulations of M. tuberculosis in Iran may reflect the characteristics and pathogenic potential of this genotype. Recently, the incidence of MDR strains in lineage 3 in the world has been estimated to be 30.63% [31]. However, this amount is estimated at 21.1% in Iran [32]. In a study by Babaii Kochkaksaraei et al. in Golestan Province, which is the second center of tuberculosis in Iran, lineage 4 (36%), lineage 2 (22%) and lineage 3 (15%) had the highest frequencies. The greatest amount of antibiotic resistance was observed in lineage 2 [33].
In the study by Mansoori et al. in Golestan Province, one-third of the studied strains were found to be related to lineage 3, which had primarily migrated to this province from Sistan and Baluchistan [34]. This finding highlights the importance of conducting additional research and monitoring of this subpopulation.
In M. tuberculosis, resistance to rifampin is primarily linked to resistance to isoniazid, categorizing it as MDR. Another objective of this study was to identifying the specific mutations and their frequency in the key genes responsible for rifampin resistance (rpoB531, rpoB516 and rpoB526). All rifampin-resistant M. tuberculosis isolates exhibited at least one mutation in the rpoB hotspot region. Among these, the rpoB531 mutation was the most common in the rifampin-resistant isolates (62.5%), consistent with findings from other studies [35, 36]. In studies conducted in Iran, the prevalence of rpoB531 was reported to be 40% in Tehran and 26% at the border of Afghanistan [37]. This mutation was detected in 53% of Brazil, 59.83% of North India, 58% of Thailand, 60% of India, 53% of Greece, and 59% of Italy [38–43].
Although the prevalence of rifampin resistance in lineage 2 members is higher than in other lineages, our results showed that there is no relationship between rifampin resistance and the lineage of M. tuberculosis. One important finding in our study is that multiple mutations in the rpoB gene were only found in lineage 2.
Several studies have investigated the role of the type II toxin—antitoxin system in the emergence of antibiotic resistance in the presence of antibiotics. Keren et al. (2011) reported that drug-resistant isolates show increased expression levels of certain TA systems under antibiotic stress conditions. These systems include VapC3/VapB3, Rv2021c/Rv2022c, Rv1989c/Rv1990c, RelF antitoxin/RelG toxin, Rv0918/Rv0919, Rv3180c/Rv3181c, higB/higA, Rv3188/Rv3189, and Rv2034/Rv2035. The authors also demonstrated that the expression of higB/higA, Rv2034/Rv2035 and Rv2022c increased under starvation and hypoxia conditions [1]. Additionally, Singh et al. showed that the expression level of RelE2 significantly increased in the presence of rifampin, and the expression level of RelE3 increased with gentamicin, levofloxacin, and isoniazid [44].
In several other studies it was found that certain toxins, such as the MazF family, Rv1577c, Rv2651c, Rv0366c, and RelE, are responsible for specific antibiotic tolerance and resistance [27, 28]. Therefore, the present study examined mutations in three families (mazEF3, relJK, and vapBC3) of the type II toxin-antitoxin system in lineages 2, 3, and 4 of M. tuberculosis. No mutations were found in vapB3, mazE3, or relK in any of the sensitive or resistant isolates, regardless of their lineage, and the sequences of these genes were similar to each other and the H37RV strain.
However, lineage 3 of the M. tuberculosis-resistant isolates had mutations in nucleotide 369 of the vapC3 gene. This mutation changed the amino acid from aspartic acid to glutamic acid. Both glutamic acid and aspartic acid are classified as acidic amino acids. Additionally, a nonsense mutation was detected at nucleotide 473 of the vapC3 gene.
On the other hand, all the rifampin-resistant M. tuberculosis isolates of lineage 3 had a nonsense mutation at nucleotide 237 of the relJ gene. The role of this gene has not been determined, but it could provide a basis for future studies.
MazEF consists of an antitoxin (MazE) and a toxin (MazF), which form a complex under normal conditions. Previous studies have shown that the mazE and mazF genes are differentially expressed in drug-sensitive and drug-resistant bacteria [11, 45]. In a study conducted in 2021 by Kazemian et al., it was found that the expression of MazF3 and MazF6 was significantly higher in resistant strains compared to sensitive strains [46]. In the present study, the mazF3 (toxin component) was mutated in all rifampin-resistant M. tuberculosis isolates. The mutation occurred at codon 65, where threonine was changed to isoleucine. Threonine and isoleucine are amino acids have two stereogenic centers [47]. Additionally, another nonsense mutation at nucleotide 473 was observed in the mazF3 gene.
Therefore, although the highest level of resistance to rifampin in M. tuberculosis was observed in lineage 2, the highest level of mutation in the three gene operons encoding the toxin-antitoxin system was observed in lineage 3. In the Solano-Gutierrez study, a total of 65 mutations were observed in Lineage 4 and 28 mutations in Lineage 3. Protein MazF8, VapC49, MazE2, and VapC24 proteins had amino acid deletions, while Rv0918, Rv2653c, VapC25, VapC49, and Rv0836c showed several amino acid changes. Additionally, mazF3, vapC47, vapC6, and vapC10 were identified as new biomarkers suitable for genotyping [15]. These findings suggest the diversity of genes affecting resistance to rifampin in different parts of the world and can serve as the basis for further research on the toxin—antitoxin system and its impact on rifampin resistance.
On the other hand, a study conducted by Zaychikova et al observed that certain genes in type II TA systems have SNPs associated with specific genotypes. Consequently, a set of genes in TA systems was proposed to classify M. tuberculosis into nine main genotypes. The gene set included higA1, vapC6, vapC10, vapC38, mazF3, mazF8, and vapC47 genes [48]. Additionally, in Solano-Gutierrez’s study, the mazF3, vapC47, vapC6 and vapC10 genes were identified as suitable markers for genotyping of M. tuberculosis isolates [15]. Our results show that the mazF3 gene can be used to identify resistant isolates because, in our study, regardless of the lineage type, all resistant isolates have a mutation in codon 65. Therefore, in our study, mazF3 can be used as a new biomarker for identification of resistant isolates, and our findings are consistent with Zaychikova’s and Solano-Gutierrez’s studies. However, it is suggested that more isolates should be investigated in future studies. It is also suggested that future studies investigate the effect of this mutation on its expression and antibiotic resistance.
Conclusion
In 153 isolates investigated, Lineage 4 (49%), Lineage 3 (7.32%), and Lineage 2 (3.18%) were the most frequent. Among the 32 resistant isolates, rifampin resistance was highest in Lineage 2 (25%), followed by lineage 4 (21%). The most common mutation in rifampin-resistant isolates of M. tuberculosis was the rpoB531 gene mutation (5.62%). In lineage 2, unlike other lineages, mutations were mainly observed in multiple points in the rpoB gene. Analysis of the mazEF3, relJK, and vapBC3 genes structure showed a distinct difference between resistant and sensitive strains in mazF3, suggesting a potential link to resistance.
Since only the mutations in these genes were examined in the present study, it would be beneficial to also analyze the expression of these genes, especially mazF3, in both sensitive and resistant strains. The mazF3 gene is specifically mutated in resistant isolates of M. tuberculosis. Therefore, it is recommended to increase the sample size to conduct a more comprehensive investigation of mazF3. If successful, it could potentially be used as a biomarker for developing new methods to identify resistant isolates.
Acknowledgments
The authors are especially grateful for the kind cooperation of the Department of Pathobiology at the Faculty of Veterinary Medicine, Bu-Ali Sina University.
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