Mycobacterium tuberculosis Lineage Distribution Using Whole-Genome Sequencing and Bedaquiline, Clofazimine, and Linezolid Phenotypic Profiles among Rifampicin-Resistant Isolates from West Java, Indonesia

Tuberculosis (TB) is caused by Mycobacterium tuberculosis infection. Indonesia is ranked second in the world for TB cases. New anti-TB drugs from groups A and B, such as bedaquiline, clofazimine, and linezolid, have been shown to be effective in curing drug resistance in TB patients, and Indonesia is already using these drugs to treat patients. However, studies comparing the TB strain types with anti-TB resistance profiles are still relevant to understanding the prevalent strains in the country and their phenotypic characteristics. This study aimed to determine the association between the TB lineage distribution using whole-genome sequencing and bedaquiline, clofazimine, and linezolid phenotypic profile resistance among M. tuberculosisrifampicin-resistant isolates from West Java. M. tuberculosis isolates stock of the Department of Microbiology, Faculty of Medicine, Universitas Indonesia, was tested against bedaquiline, clofazimine, and linezolid using a mycobacteria growth indicator tube liquid culture. All isolates were tested for M. tuberculosis and rifampicin resistance using Xpert MTB/RIF. The DNA genome of M. tuberculosis was freshly extracted from a Löwenstein–Jensen medium culture and then sequenced. The isolates showed phenotypically resistance to bedaquiline, clofazimine, and linezolid at 5%, 0%, and 0%, respectively. We identified gene mutations on phenotypically bedaquiline-resistant strains (2/3), and other mutations also found in phenotypically drug-sensitive strains. Mykrobe analysis showed that most (88.33%) of the isolates could be classified as rifampicin-resistant TB. Using Mykrobe and TB-Profiler to determine the lineage distribution, the isolates were found to belong to lineage 4 (Euro-American; 48.33%), lineage 2 (East Asian/Beijing; 46.67%), and lineage 1 (Indo-Oceanic; 5%). This work underlines the requirement to increase the representation of genotype-phenotype TB data while also highlighting the importance and efficacy of WGS in predicting medication resistance and inferring disease transmission.


Introduction
Tuberculosis (TB) is one of the leading causes of death worldwide after HIV/AIDS in infectious diseases, and Indonesia is ranked second globally in terms of TB cases [1].TB is caused by Mycobacterium tuberculosis infection, especially in the lungs [1,2].About one-third of the world's population is infected with M. tuberculosis, and infection with this bacterium contributes to the deaths of about two million people each year, which makes TB the eighth most common cause of death and the second leading cause of death from infectious agents.Tis number will increase with the emergence of drug-resistant strains of M. tuberculosis [3].Indonesia is one of 10 countries attempting to prevent the incidence of rifampicin-resistant (RR)/multidrugresistant TB (MDR-TB).Given the signifcant achievements in global treatment coverage, Indonesia needs to make special eforts to improve the screening and diagnosis of drug-resistant TB as well as access to treatment [1].
Rifampicin resistance has become a signifcant problem in TB control during long-term treatment with rifampicin.Resistance to this drug can lead to severe consequences, such as TB treatment failure, the prolongation of treatment, and increased retreatment rates.As very few mono-resistant strains are resistant to rifampicin, and most are also resistant to isoniazid, it has become a marker for MDR-TB [1,4].According to World Health Organization (WHO) guidelines, the anti-TB drugs used to treat RR/MDR-TB can be classifed into groups A, B, and C. Tis new classifcation is based on the drug class and the certainty of the evidence about the drugs' efcacy and safety.Te drugs in these groups play a therapeutic role in the MDR-TB regimen [5].Te efcacy of bedaquiline (group A) in patients with pulmonary MDR-TB was frst demonstrated in 2014 when the addition of bedaquiline to the regimen showed a faster and increased number (78%) of culture conversions after 120 weeks compared with placebo (58%) [6].MDR-TB patients treated with bedaquiline also had TB-negative sputum within six months [7,8].Bedaquiline is strongly recommended for the long-term treatment of patients with RR/MDR-TB [9].Currently, bedaquiline resistance mechanisms include mutations in the atpE and cross-resistance to clofazimine in the Rv0678, Rv1979c, and pepQ genes, with linezolid resistance encoded by the rrl and rplC genes.
Bedaquiline inhibits adenosine triphosphate (ATP) synthesis and several resistance-associated mutations encoded by the atpE gene as an additional resistance mechanism [10,11].Mutations in the atpE are also associated with resistance both in vitro and in vivo [6,10,12,13], and a high mutation frequency of Rv0678 contributes to low resistance to bedaquiline in vitro and clinically.In 2016, the mutations in Rv1979c and pepQ were found to be associated with bedaquiline and clofazimine resistance.Rv0678, Rv1979c, and pepQ also have cross-resistance between bedaquiline and clofazimine [13][14][15][16][17].
Te distribution of M. tuberculosis strains in various regions shows variations in the level of virulence in the process of human adaptation, with epidemiological diferences dominating.Currently, M. tuberculosis has nine lineage strains distributed across many parts of the world [18].Te lineages include Indo-Oceanic (L1), East Asian/Beijing (L2), East African-Indian (L3), European-American (L4), West African 1 (L5), West African (L6), Aethiops vetus (L7) [19], and East African (L8 [20] and L9 [18,21]).Several molecular identifcation methods have been developed from these strains of M. tuberculosis, including IS6110-RFLP, MIRU-VNTR, and spoligotyping.Tese methods have demonstrated high resolution and good performance in clinical trials, tracing, and reinfection detection.However, the great diversity or, in some cases, excessive homogeneity makes the application of this method unsuitable for the phylogenetic analysis of strains of M. tuberculosis [22,23].Whole-genome sequencing has facilitated advances in the study of M. tuberculosis resistance, transmission kinetics, and phylogenetic analysis, as well as developments in sequencing technology [23].In this study, we aimed to determine the relationship between lineage and bedaquiline, clofazimine, and linezolid resistance based on the phenotypic profles of M. tuberculosis RR isolates from West Java, Indonesia, using whole-genome sequencing technology.

Materials and Methods
2.1.Isolates Collection and Identifcation.Tis study was conducted at the Laboratory of the Department of Microbiology, Faculty of Medicine, Universitas Indonesia.A total of 60 isolates from the stock of the Department of Microbiology, Faculty of Medicine, Universitas Indonesia, were subcultured on a Löwenstein-Jensen (LJ) medium and identifed as M. tuberculosis bacteria using the SD MPT64 Rapid test (SD Bioline) to ensure all the bacteria are M. tuberculosis and not contaminated by other bacteria.Previously before bacterial stock was made, we tested the phenotype of bacteria to rifampicin using the GeneXpert method and to bedaquiline, clofazimine, and linezolid using the mycobacteria growth indicator tube (MGIT) liquid culture method.

Genomic DNA Isolation and Whole-Genome Sequencing.
Genomic DNA extraction from the M. tuberculosis isolates was conducted using the modifed cetyl trimethyl ammonium bromide (CTAB) method.Briefy, 3-4 M. tuberculosis colonies from the LJ medium were scrapped and transferred into a screw cap tube containing 100 μL Tween 80 and 6-8 sterile beads and then vortexed.Tereafter, 1 mL of nuclease-free water was added to each tube and vortexed until a bacterial suspension was obtained.A 300 μL bacterial suspension was transferred to a microcentrifuge tube, heated in a water bath for 30 min at 95 °C, and cooled to room temperature.Subsequently, 50 μL lysozyme (10 mg/mL) was added to each tube, which was then vortexed and incubated for 2 hours at 37 °C.Following incubation, a mixture of 70 μL 10% SDS and 5 μL proteinase K was added to each tube, which was then vortexed and incubated for 10 minutes at 65 °C.Ten, 100 μL 5M NaCl and 100 μL of CTAB-NaCl solution preheated to 65 °C were added.Te mixture was vortexed until it turned milky and then incubated for 10 min at 65 °C.A 750 μL of chloroform/ isoamyl alcohol (24 :1) solution was added, vortexed for 1 minute, and centrifuged at 12,500 rpm for 8 min at room temperature.Next, 200 μL supernatant was transferred to a tube, and 300 μL of cold isopropanol was added.Tis was mixed with the tube inverted 2-3 times and then incubated for 2 hours at −20 °C.DNA pellets were obtained by centrifugation at 12,500 rpm for 15 min at 4 °C.Te supernatant was removed and added 1 mL cold ethanol 70%, and then inverted 2-3 times.DNA pellets were obtained by centrifugation at 12,500 rpm for 5 min at 4 °C.Te centrifugation was repeated at 12,500 rpm for 2 min at 4 °C, and the remaining ethanol was removed with a micropipette and dried.Finally, 37 μL of nuclease-free water was added.Te quality and quantity of the genomic DNA were measured using a NanoDrop A260/280, Qubit Fluorometer, and agarose gel electrophoresis.Te DNA samples were sent to Omics Drive Pte Ltd Co. for sequencing 2 International Journal of Microbiology using a NovaSeq 6000 (Illumina).Library preparation used Illumina DNA Prep Kit and was undertaken according to the guidelines, and the library pools were subjected to single-end sequencing (150 bp).

Bioinformatics and Data Analysis. Omics Drive Pte Ltd
Co. performed the following bioinformatics analysis.FastQC v0.11.9 was used to check the quality of the raw sequences.Trimmometric v0.39 was used to eliminate any poor-quality reads.Te high-quality reads were mapped using BWA v0.7.17.Te SAM format data were converted to BAM format, and the BAM fles were sorted and indexed using SAMtools v1. 15

M. tuberculosis Phenotypic DST Profle Characteristics.
Te geographical origins of the studied strains from West Java are shown in Figure 1.In total, 397,377 cases of TB were recorded in 2021 with 1.5% of them are rifampicin-resistant (RR)/MDR-TB; West Java province reported the most signifcant number of cases at 91,368 with a detection rate of 90.6%, and the data thus accurately depicted the distribution of M. tuberculosis strains in this population.Many people still have pulmonary TB, and the number of new cases being identifed each year is increasing due to a lack of public awareness of the available TB treatments [28,29].Sixty M. tuberculosis RR isolates from Cisarua, West Java, were available for drug susceptibility testing with bedaquiline, clofazimine, and linezolid.We found that while 5% of the isolates were resistant to bedaquiline, 100% were phenotypically sensitive to linezolid and clofazimine (Table 1).Bedaquiline is a novel regimen that is benefcial for treating TB, as the administration of this drug can convert TB-positive sputum to negative within six months [7,8].Bedaquiline is used to treat MDR-TB patients via the BPal (bedaquiline, pretomanid, and linezolid) regime in some provinces in Indonesia.Te data from our study indicate that resistance to bedaquiline has emerged.Although minimal, it serves as a warning, and a strategy to prevent bedaquiline resistance from increasing must be prepared.
In 2005, the U.S. Centers for Disease Control and Prevention introduced the concept of TB infected by M. tuberculosis extremely drug-resistant (XDR) as a diferent entity.Tese bacteria cannot be killed by fuoroquinolone therapy, such as levofoxacin and moxifoxacin, or other second-line drugs, such as amikacin and kanamycin, and are resistant to frst-line drugs such as isoniazid and rifampicin.XDR M. tuberculosis can occur due to the inappropriate handling of MDR-TB patients [20,30].On the other hand, phenotypic studies of bedaquiline, clofazimine, and linezolid to RR background (RR, MDR, and XDR) M. tuberculosis isolates were low.However, the data indicated that coresistance to bedaquiline, linezolid, and clofazimine was still very low in the tested population [31].Recently, a series of clinical trials evaluating new and alternative oral anti-TB drugs (bedaquiline, clofazimine, and linezolid) have demonstrated their potential in treating resistant TB in a shorter time with lower toxicity and higher efcacy [32].It was previously believed that phenotypic drug susceptibility testing (DST) employing automated methods like the MGIT 960 system could accurately detect M. tuberculosis susceptibility or resistance to frst-line anti-TB medications [33].However, these techniques require considerable time and efort and produce results in weeks rather than days [33,34].Methods that address these limitations are thus needed as alternatives or companions to culture-based methods.Whole-genome sequencing has emerged as an alternative to further understand resistance profles and lineage dissemination within a country [23,33].Tis method has been implemented in many countries, primarily to detect the resistance profles of frst-line anti-TB drugs and to conduct epidemiological studies [4,18].We undertook whole-genome sequencing using genes possibly associated with M. tuberculosis resistance to bedaquiline and identifed mutations linked to these phenotypic traits.Tese fndings demonstrate that the wholegenome sequencing method can serve as a suitable alternative alongside culture methods or potentially replace them.

Whole-Genome Sequencing Results of M. tuberculosis
Isolates.Our tools for genome mutation analysis, Mykrobe and TB-Profller, have long been widely used.Tese two tools promise to make it easy for users to analyze antituberculosis drug resistance profles and determine lineage quickly [35,36].
Te genes encoding bedaquiline resistance, specifcally atpE, Rv0678, Rv1979c, and pepQ, was then looked for in the data collected from the WGS study.Figure 2 displays the distribution of bedaquiline-encoding gene variants discovered using WGS.Out of the 60 isolates, three isolates were found to be phenotypically resistant to bedaquiline; however, only two isolates were found to have gene mutations, and ten isolates were found to be phenotypically sensitive but harbored mutations in the bedaquiline resistance gene.Te Rv1979c T⟶C (c.A-129G) gene, located upstream of the Rv1979c gene, was determined to be the most often detected mutation in all isolates according to the fndings of this investigation.One of the 2/60 phenotypically and genotypically resistant organisms has the Rv1979c gene mutation, which is linked to bedaquiline resistance in Asp286Gly and Leu393His.Val426Ile, Asp286Gly, Gly31Gly, and Leu139Leu were among the other variants in the Rv1979c gene that were discovered, but they were shown to be International Journal of Microbiology phenotypically sensitive.In the upstream region of the atpE gene, 3/60 mutations (C⟶T) were discovered, but in the upstream region of the Rv0678 gene, only 1/60 isolates (T⟶−) were discovered.Te pepQ G⟶A (Pro69Leu) gene mutation was discovered in the genotypically but phenotypically sensitive strains (3/60).

International Journal of Microbiology
Numerous studies have reported various mutations discovered as a result of WGS analysis.Rv1979c and pepQ gene alterations were reported by Ismail et al. [15].According to the research by Ismail et al. [15], the Rv1979c gene missense mutation Asp286Gly and the synonymous mutations Leu139Leu and Gly31Gly were discovered in M. tuberculosis isolates that had drug susceptibility test fndings that showed they were sensitive to bedaquiline phenotypically.Te WHO database of resistance coding gene variants has confrmed the mutation often discovered in this study, which is the mutation at codon position −129 (A⟶G) in the upstream region of the Rv1979c gene, and it does not correlate with bedaquiline resistance [37].
Both of those genes, the efux pump repressor mmpR (Rv0678) and the atpE gene (Rv1305), specifcally the F0 domain of the ATP synthase enzyme, exhibit alterations in bedaquiline-producing mutants that are resistant to the drug [38,39].Te uncharacterized transporter Rv1979c and pepQ, which are both encoded as genes involved in cross-resistance between bedaquiline and clofazimine, have statistically signifcant co-occurrences with Rv0678 [40,41].However, the connection between bedaquiline and clofazimine resistance in these two genes (Rv1979c and pepQ) has to be explored further.Mutations in the M. tuberculosis genome can alter drug targets or metabolic pathways involved in drug resistance.Tese mutations can have a signifcant impact on the efectiveness of antituberculosis drugs [42].Drug resistance in M. tuberculosis is a complicated phenomenon with numerous interrelated genetic components.Drug resistance can result from changes in a single gene or a combination of abnormalities in several genes connected to the resistance pathway [43].

M. tuberculosis Resistance Type and Lineage Distribution.
We found agreement between the drug-resistant type of RR/ MDR M. tuberculosis from the genotypic results using Mykrobe analysis and the phenotypic DST results for bedaquiline (Table 2).Te studied strains from the genotypic results showed 88.33% (53/60) were RR M. tuberculosis, and the remainder had an MDR 11.67% (7/60) background (Table 2).Table 2 demonstrates the association between anti-TB drug resistance to rifampicin (RR or MDR) and bedaquiline.Our results revealed that one bedaquiline-resistant isolate had an RR background and two had an MDR background.Tis discovery is crucial as it demonstrates the occurrence of bedaquiline-resistant cases within the context of RR/MDR M. tuberculosis.Te WHO recommends bedaquiline as one of the three essential medications that must be used to treat RR-TB in every regimen.
Notwithstanding, bedaquiline resistance was already noted shortly following its introduction.Te establishment of bedaquiline resistance and difculties in measuring resistance to bedaquiline make it difcult to employ bedaquiline efectively [44].MDR-TB and RR-TB are both conditions that can be treated with bedaquiline [45].According to a recent cost-efectiveness study, adding bedaquiline to a baseline MDR-TB regimen will enhance health outcomes and lower costs in high TB environments [46].With success rates as high as 78% and fewer fatalities compared to normal regimens, bedaquiline has been proven to be successful in treating MDR-TB [46,47].
Several factors can afect discrepancies between phenotypic and genotypic data.According to numerous studies, quiet or disputed mutations and heteroresistance are the most frequent reasons for discrepancies between phenotypic and genotypic techniques when testing drug resistance in TB.Silent mutations in M. tuberculosis DNA afect the nucleotide but not the amino acid.Some disputed variants in the gene are found genotypically but produce drugresistance results in phenotypic testing, particularly in liquid media (MGIT 960).Tese mutations afect the "ftness" of M. tuberculosis, causing it to evolve slowly in the presence of the drugs during phenotypic DST, which prevents it from being detected [48].
In this study, 48.33% of the isolates belonged to the Euro-American lineage (L4), with most of them being of the Haarlem clade, followed by the East Asia/Beijing lineage (L2) at 46.67% and the Indo-Oceanic lineage (L1) at 5% (Table 3).Lineage 4 has also been found in Upper Myanmar, which could be explained by cross-border transmission with Tailand.Information about the future distribution of these M. tuberculosis strains can be linked to information across borders to assess transboundary transmission [49,50].In a recent TB study from Tailand [51], L2 was implicated in the increased incidence of MDR-TB, as it is associated with drug resistance and increased transmissibility.Another possibility could be that these isolates were part of an outbreak, but as this is an archived collection with no epidemiological data, we could not investigate further.In Cambodia, the majority of the lineages are also L1 (Indo-Oceanic), L2 (East Asian), and L4 (Euro-American) [52].Te high prevalence of the L1 M. tuberculosis strain in Cambodia is perhaps not surprising as it is a common strain in the Southeast Asian countries of India and Bangladesh [51].Te distribution between lineage and drug resistance type is shown in Figure 3.
In this study, we aimed to establish knowledge regarding the type distribution of M. tuberculosis RR isolates in Indonesia, especially in West Java province.Such a knowledge base is essential for epidemiological research aimed at infection control and in helping to provide efective antibiotic therapy.Furthermore, detecting drug-resistant TB in Indonesia is reliant on the use of Xpert M. tuberculosis/rifampicin and line probe assays (MTBDRplus and MTBDRsl), which are limited to detecting resistance in only frst-and second-line drugs and not the new anti-TB drugs used to treat MDR-TB [24].Next-generation sequencing (NGS) technology, especially for TB, provides the most comprehensive approach to molecular-based DST.Wholegenome sequencing is a method of determining the exact nucleotide sequence of a particular genome, which is the entire genetic material of an organism [53,54].NGS can sequence millions to billions of reads in a single process within a shorter time and with more accurate results, which makes it a cost-efective and powerful sequencing technology with high-throughput data [55,56].Te results concordance data between Xpert MTB/RIF, a line probe International Journal of Microbiology assay, and whole-genome sequencing with phenotypic DST to predict resistance to 15 anti-TB drugs were reported as 40%, 63%, and 93%, respectively [57].
Te results of this study are fascinating but also limited to the conditions of the origin of the samples.In addition, this study only focuses on in vitro phenotypic profles and does not involve the clinical status of patients.To fully understand the role of specifc mutations in conferring resistance to TB drugs, further data on WGS and phenotypic testing are required, especially for isolates that are resistant to bedaquiline, clofazimine, and linezolid.

Conclusions
Tis study demonstrates the applicability and value of WGS in identifying drug resistance and estimating disease transmission in West Java.Some mutations were found to support phenotypically resistant.However, this study revealed an uncorrelation between specifc lineage and phenotype profles where the bedaquiline resistance was found in all lineages.All the mutation data will be a helpful database to determine antituberculosis drug choice if the WGS is used for a laboratory tool examination in Indonesia.6 International Journal of Microbiology Our study further showed that the M. tuberculosis isolates from West Java, Indonesia, used in this study predominantly belonged to the Euro-American lineage, followed by the East Asian and Indo-Oceanic lineages.

Table 1 :Figure 2 :
Figure 2: Te distribution of mutations encoding bedaquiline resistance genes was discovered using a whole-genome sequencing study of 60 M. tuberculosis isolates.

Figure 3 :
Figure 3: MTB lineage distribution and drug resistance type.
[24][25][26][27]3 was used for the de novo assembly.GATK-HaplotypeCaller was used for the variant calling and SnpEf v4.3 for the variant annotations.Te analysis information is then compiled into a fle.Mykrobe v0.10 and TB-Profler v4.4.1 were used to predict the lineage classifcation, and only Mykrobe was used to predict the type of drug resistance among the isolates[24][25][26][27].Descriptive statistics were used to describe the results of this study.
2.4.Ethics Approval of Research.Te Health Research EthicsCommittee of the Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia, approved the study protocol (protocol no.22-12-1429) and issued it on December 5th, 2022.

Table 2 :
MTB isolates characteristics based on Mykrobe analysis.

Table 3 :
Lineage distribution based on TB-Profler analysis.