Molecular characteristics of Mycobacterium tuberculosis drug-resistant isolates from HIV- and HIV+ tuberculosis patients in Russia

Background High burden of drug-resistant (DR) tuberculosis (TB) is a significant threat to national TB control programs all over the world and in the Russian Federation. Different Mycobacterium tuberculosis (MTB) genotypes are hypothesized to have specific characteristics affecting TB control programs. For example, Beijing strains are supposed to have higher mutation rates compared to strains of other genotypes and subsequently higher capability to develop drug-resistance. Results Clinical MTB isolates from HIV- and HIV+ patients from four regions of Russia were analyzed for genotypes and mutations conferring resistance to Isoniazid, Rifampicin, Ethambutol, aminoglycosides, and fluoroquinolones. Analysis of genotypes and polymorphism of genomic loci according to the HIV status of the patients – sources of MTB isolates were performed. Studied MTB isolates from HIV- TB patients belonged to 15 genotypes and from HIV + TB patients – to 6 genotypes. Beijing clinical isolates dominated in HIV- (64,7%) and HIV+ (74,4%) groups. Other isolates were of LAM (including LAM1 and LAM9), Ural, and 4 minor groups of genotypes (including 5 subclones T). The spectrum of genotypes in the HIV- group was broader than in the HIV+ group. PR of B0/W148 Beijing was significantly lower than of other Beijing genotypes in susceptible and MDR-XDR isolates. Rates of isolates belonging to non-Beijing genotypes were higher than Beijing in susceptible isolates from HIV- patients. Conclusions Beijing genotype isolates prevailed in clinical isolates of all drug susceptibility profiles both from HIV- and HIV+ patients, although B0/W148 Beijing genotype did not dominate in this study. Genome loci and mutations polymorphisms were more pronounced in clinical isolates from HIV- patients, than from HIV+.


Background
High burden of drug resistant (DR) tuberculosis (TB) is a significant threat to national TB control programs all over the world [1,2]. The Russian Federation (RF) is in the group of countries with high level emergence of multidrug resistant (MDR) TB, with estimated proportion of MDR TB cases in 2019 amounting 35% of new Open Access *Correspondence: m_shulgina@mail.ru 1 National Medical Research Center of Phthisiopulmonology and Infectious Diseases, Ministry of Public Heath of the Russian Federation (NMRC PhID), Moscow, Russian Federation Full list of author information is available at the end of the article cases and 71% of previously treated cases. Rates of HIV positive (HIV+) patients with TB are also high [1].
Geographic distribution of Mycobacterium tuberculosis (Mtb) genotypes is of unrelenting interest of the researchers in many countries. Data on genotyping as specific characteristics of MTB strains gives a new impulse to developing molecular epidemiology, and induced discussions on molecular peculiarities of Mtb, causing disease in a particular region of the world. Results of numerous researches on Mtb genotyping using spoligotyping, MIRU-VNTR typing and whole genome sequencing are accumulate in electronic databases, for example in Pasteur Institute database [3]. Most of these investigations are aimed at phylogeny and evolution of MTB.
Studies of Mtb genotypes circulating at the territory of the Russian Federation have been going on for decades. The prevailing genotype is Beijing. Beijing genotype belongs to East-Asian lineage (lineage 2) and widely spreads all over the world [4,5]. Beijing strains are abundant in East Asia and in the former Soviet Union republics. They are frequently isolated from TB patients in Russia and among immigrants from the former Soviet Union. Strains belonging to Beijing subclone BO/W148 are isolated from TB patients all over the territory of the Russian Federation [5,6]. Other Mtb genotypes isolated at the territory of the Russian Federation less frequently than Beijing strains are genotypes of Euro-American family, such as LAM, Ural, Haarlem, S, T and X-types and others [7,8].
Different MTB genotypes are hypothesized to have specific characteristics effecting TB control programs. Beijing strains are supposed to have higher mutation rates compared to strains of other genotypes and subsequently higher capability to develop drug-resistance [5,[9][10][11]. Beijing genotype is also associated with unfavorable outcomes of TB treatment [12]. Other researchers have demonstrated that Beijing strains had not developed resistance to rifampin in elevated rate and there were no significant differences in occurrence of mutations between Beijing and non-Beijing clinical isolates (ClIs) [13][14][15].
Currently the best method for revealing Mtb genotyping and molecular DR profile is whole genome sequencing [16,17]. However, the use of this method is limited by high level technology laboratories. Use of commercial genotyping test-systems made it possible for regional clinical laboratories to be included in the pool of centers performing molecular epidemiological surveys and to introduce molecular characteristics of an etiological agent to routine clinical practice. These researches provide more data on the mechanisms of DR and mutations conferring DR phenotype of Mtb to develop and update tests for DR TB diagnosis and personalized chemotherapy regimens.
In 2018-2019 the National Medical Research Center of Phthisiopulmonology and Infectious Diseases (NMRC PhID) and its' branch the Ural Research Institute of Phthisiopulmonology (URIPh) had collected Mtb ClIs from different regions of Russia for their drug sensitivity testing (DST) reanalysis as a part of the regional External Quality Control program. Subsequently molecular characteristics of the ClIs, including their genotyping and testing for mutations associated with Mtb DR were studied. ClIs were attributed according to HIV status of TB patients. In this research we had evaluated rates of different genotypes according to clinical Mtb ClIs' DR profiles of loci and types of mutations conferring DR. The aim of this study was to analyze polymorphisms of different DNA loci associated with Mtb DR according to the ClIs' genotypes and HIV status of patients, who provided material for ClIs.
PRs of Beijing and Ural ClIs from HIV+ patients were significantly higher than from HIV-patients (p = 0, 04 for both). PR for ClIs of LAM genotype was significantly higher in the group from HIV-patients, than in the group of ClIs from HIV+ patients (p = 0,008).

Resistance to INH
Total number of INH resistant ClIs was 233 from HIVand 79 from HIV+ patients. All ClIs with mutations in katG and inhA genes, both from HIV-and HIV-patients were resistant to INH according to DST. Mutations in both genes were detected in 21 (PR = 9,0%, CI95% = 3,7%) ClIs from HIV-patients. All ClIs with double mutations were of Beijing genotype (other Beijing), four were monoresistant to INH, 12 -MDR and five -XDR. In all cases mutation were katG315Ser315Thr, inhA15Th-r15Ala. No ClIs with double mutations were detected in the group from HIV+ patients. In analysis presented below each of these mutations were considered as mutations in separate ClIs with total number of evaluated ClIs from HIV-patients -254, and from HIV+ patients -79 (Table 3).
In one isolate a mutation often associated with INH resistance -ahpC12 (Cys12Thr) as a sole mutation was detected, however the isolate was susceptible to INH in DST.

Resistance to RIF
Total number of ClIs from HIV-patients bearing mutations associated with resistance to RIF was 215, and of ClIs from HIV+ patients -62. Mutations in rpoB gene were only detected ( Table 4). All ClIs with rpoB mutations were resistant to RIF according to phenotypic tests.
No mutations were revealed in loci rpoB507 and rpoB513, neither in ClIs from HIV-patients, nor in ClIs from HIV+ patients (Table 4).

Resistance to EMB
Total number of ClIs from HIV-patients with embB mutations was 198, and of ClIs from HIV+ patients -66. Mutations in embB gene were only detected in EMB resistant ClIs (Table 5). However, we identified 42 ClIs from HIV-patients and 5 ClIs from HIV+ patients as resistant to EMB by DST but did not detect mutations in their embB genes.
Beijing genotypes ClIs prevailed among EMB resistant ClIs with mutations in EMB (EMB resistant further on): 165 ClIs from HIV-patients of 198 tested  Mutations in ClIs from HIV-patients were detected in seven loci of embB, total number of types of mutations was 12. In locus embB306 two mutations substituting Methionine by Leucine and Valine, in locus embB319two types of mutations substituting Tyrosine by Cysteine, and Serine, embB406 -Glycine by Alanine, Aspartic acid and Serine, embB497 -Glutamine by Lysine and Arginine. In three other loci there were only one type of mutations detected in each: at embB296 -substitution of Asparagine by Histidine, at embB309 -Valine by Phenylalanine and at embB354 -Asparagine by Alanine (Table 5).

Resistance to fluoroquinolones
In this study resistance to Ofl was chosen as a marker of resistance to FQ, although some of ClIs resistant to Ofl in the studied groups of clinical ClIs retained susceptibility to Levofloxacin and/or Moxifloxacin. The choice of Ofl resistance gave the possibility to enlarge the group of FQ resistant ClIs. In this group all ClIs bear mutations in gyrA and gyrB genes. We were not aimed to analyze associations of particular mutations with resistance to different FQ.
In the HIV+ patients' group of ClIs gyrA mutations were detected in 24 ClIs, four in monoresistant, 13-in preXDR and 10 in XDR ClIs. No gyrB mutations or ClIs with double mutations in gyrA and gyrB gene were detected in this group.
No dominating loci or type of mutations in gyrB gene were observed (Table 6).

Resistance to aminoglycosides
Although the latest WHO clinical guidelines and recommendations of 2021 exclude injectable drugs Kn and Cap from the chemotherapy regimens and DST [18,19], phenotypic DST for these drugs were included in this study as it was done mainly in 2018-2019. However, we found different mutations spectra conferring to Kn, Ami, Cap, like in other researches [20,21]. Total number of ClIs resistant to Kn or Ami from HIV-patients was 97, and of ClIs from HIV+ patients -24. Mutations in regulatory regions of eis and rrs genes were detected ( Table 6). All ClIs with mutations in eis and rrs genes were resistant to Kn according to DST, five were also resistant to Ami and Cap. All mutants in rrs gene both in HIV-and HIV+ groups of ClIs were resistant to Ami and Cap.
Prevailing mutation in promoter of rrs gene was Ala1401Gly, both in ClIs from HIV-and HIV+ patients. One isolate from HIV-patients with mutation rrs Cys1402Thr and one from HIV+ patients rrs Gly1484Thr were revealed.
No ClIs with mutations in locus eis13, were found neither from HIV-, nor from HIV+ patients.

Discussion
MTB genotypes and mutations' landscapes in many regions of the world had been described during the last 30 years [22,23]. Genotypes typical for the Russian Federation revealed with spoligotyping, MIRU-VNTR analysis and whole genome sequencing were also described in many publications [8,22,23]. Extended research is in progress today to reveal MICs of anti-TB drugs' associations with particular mutations, directed to development Table 6 Loci and types of mutations conferring FQ resistance in MTB clinical isolates belonging to different genotypes 0 :-loci and types of mutations that were tested but not revealed of reliable DST and molecular tests highly correlated with efficiency of chemotherapy with a particular drug [24]. In our study we used commercial test-system, designed to reveal most frequent in the Russian Federation genotypes and mutations, associated with DR in MTB, and conventional DST methods. Despite certain limitations of the methods used we had revealed certain regularities in genotypes, types of mutations conferring DR of MTB and HIV status of patients -sample sources.

Genotypes and DR profiles
In our research Beijing genotype (including 82 B0/W148 Beijing) ClIs prevailed in the group from HIV-patients (64,7%) and from HIV+ patients (74,4%) -the difference is statistically significant. Beijing genotype was the most frequent genotype in different Russian regions according to other publications [22,23]. Beijing subclone B0/W148 was assigned as one of the most widely distributed clusters in the Russian Federation and a "successful" clone in other studies [25][26][27][28]. However, it was not the most frequent genotype in the groups of ClIs we studied: PRs of B0/W148 were 19,0% in ClIs from HIV-patients and 20,2% in ClIs from HIV+ patients. Other Beijing ClIs comprised 45,7% and 54,2%, correspondingly.
Beijing genotype ClIs were revealed in ClIs of all DR profiles, both in the groups isolated from HIV-and HIV+ patients. PRs of Beijing genotypes in MDR-XDR ClIs were higher than in susceptible ClIs, both in ClIs from HIV-and HIV+ patients: 86,6% and 88,5% in drug resistant ClIs and 36,6% and 63,7%, correspondingly. Differences in PRs of Beijing phenotypes in susceptible and MDR-XDR ClIs were statistically significant (for ClIs from HIV-patients p < < 0, 0001 and for ClIs from HIV+ patients p = 0,0002, correspondingly).
B0/W148 subclone of Beijing genotype was identified in rare compared to other Beijing subclones in ClIs with majority of mutations conferring resistance to Table 7 Loci and types of mutations conferring aminoglycosides and capreomycin resistance in MTB clinical isolates belonging to different genotypes 0 :-loci and types of mutations that were tested but not revealed  were for katG -30,3% and  53,6%, inhA -7,0% and 65,1%, rpoB 30,2% and 57,2%,  gyrA 31,3% and 53,7%, eis -21,4% and 74, 4%, rrs 33,3% and 63,05 in B0/W148 subclone and other Beijing subclones ClIs from HIV-patients, correspondingly. There were no differences in PRs in ClIs from HIV+ patients for rpoB, gyrB, and in ClIS from both HIV+ and HIVpatients for embB. Prevalence of Beijing phenotypes in MDR-XDR profiles agrees with the hypothesis of higher mutation rates in these types of strains [9,15,23,[31][32][33]. However, in our study frequencies of Beijing genotype in ClIs susceptible to all drugs both in groups from HIV-and HIV+ patients were also high: 37,7% and 63,7%, correspondingly. It implies that Beijing genotype may not be the only factor of DR development, but rather cooperate with other factors, such as, for example, individual characteristics of the host immunity. B0/W148 genotype was not detected in susceptible ClIs neither from HIV-nor HIV+ patients. In MDR-XDR ClIs from HIV-patients its PR was significantly lower than of other Beijing genotypes (29,1% and 55,3%, correspondingly, p = 0,0002), no significant difference was revealed in these two groups of ClIs from HIV+ patients.

Number of MTB isolates with eis and rrs mutations from HIV+ patients
PRs of non-Beijing genotypes ClIs (LAM, LAM1, LAM9, URAL, and minor genotypes) were significantly higher in susceptible ClIs from HIV-and HIV+ patients, than in MDR-XDR groups.

Mutations conferring resistance to INH
In this study mutations conferring INH resistance were detected in katG, inhA and ahpC genes. In 9,0% of all INH resistant ClIs from HIV-patients' mutations in both katG and inhA genes were detected. All double mutants were of Beijing genotype (other than B0/W148 Beijing genotypes). No double mutants were detected in the HIV+ group. All ClIs with mutations, conferring resistance to INH (except one isolate with sole ahpC C12T mutation) were resistant to corresponding drugs in DST.
Only one isolate from HIV-group with mono ahpC C12T mutation was identified, and this isolate was susceptible to all drugs. Gene ahpC is coding for Alkylhydroperoxide reductase C and is a part of ahpC-OxiR regulon, coding for enzymes responsible for the detoxification of reactive oxygen. The system is normally inactive in MTB cells and is activated by mutations inducing its expression when inactivation of catalase-peroxidase by katG mutation occur, ahpC mutations are compensatory mutations for mutations in katG gene [24,34,35]. As compensatory mutations ahpC mutations are often detected in katG mutants and so are associated with INH resistance [24,[35][36][37][38].
Analysis of prevalence INH resistance conferring mutations revealed patients Beijing ClIs.
In 254 ClIs from HIV-patients 83,1% harbored mutations in katG gene and 16,9% in inhA gene. All katG mutations in ClIs from HIV-patients were in four loci: three types of mutations at katG315 (substitution of Serine by Threonine, Arginine and Glycine), at kat328substitution of Tryptophan by Cysteine and at katG335substitution of Isoleucine by Valine. Variability of types of mutations in katG gene was lower in HIV+ group of ClIs: only two types of mutations were detected, both in katG315 loci. Most frequent mutation in HIV-and HIV+ group of ClIs was Ser315Thr (PR = 94,8% and 98,7%, correspondingly) associated with non-B0/W148 Beijing genotypes.
All detected mutations in inhA loci were in the inhA promotor region [30,35]. The most frequent mutation type in ClIs from HIV-and HIV+ patients was T15A (PR = 81,4% in HIV-group of ClIs and 100% in HIV+ ClIs). This type of mutations was associated with other than B0/W148 Beijing genotypes in ClIs from HIVpatients (PR = 65,7%). Number of HIV+ ClIs with inhA mutations were only five, no significant differences were revealed. Other investigators published similar results on prevalence of katG and inhA mutations in INH resistant MBT ClIs, and locus katG235 as the most frequent locus of mutations [35,[38][39][40]. Studies of mutations associated with INH resistance in MTB in Kyrgyz Republic also revealed prevailing mutations katG Ser315Thr (88.6%) and the only one mutation in inhA region was T15A [40].
Polymorphism in katG gene was higher in Beijing ClIs in HIV-group compared to other genotypes, and in HIVgroup compared to HIV+ group.

Mutations conferring resistance to RIF
Mutations in rpoB gene were the only detected, according to the design of test system used.
Most frequent type of mutation in ClIs from HIVand HIV+ patients was Ser531Leu -(PR = 87,4% and PR = 82,3%, correspondingly). Similar results were reported in other publications [14,39]. No significant differences in rates of this type of mutation in Beijing and other genotype groups of ClIs were observed. Mutations Leu511Pro (PR = 4,7%) and Asp516Ala (PR = 2,3%) were detected in ClIs of Beijing genotype only.
According to the test system facilities all mutations detected were in the region of rpoB511-rpoB533 (23bp long), which is a part of the rpoB 81-bp core region [41] -the RIF resistance-determining region (RRDR). Ser531Leu mutation dominated in RIF resistant mutants in the group of ClIs we had studied, as in ClIs from Kirgizia [40]. This mutation was associated with high MICs for RIF and Rifabutin [24,42]. Some of other mutations revealed in our investigations (Leu511Pro, PR = 4,3% of RIF resistant ClIs from HIV-patients and PR = 3,2% of ClIs from HIV-patients) and rare Asp516Val (4 ClIs from HIV-patients) and Ser522Leu (1 isolate from HIVpatients) were resistant to RIF in our study and were found susceptible in another [42]. However, according to WHO recommendations any mutation of RRDR, except for synonymous mutations, should be assumed to confer RIF resistance [24,43].

Mutations conferring resistance to EMB
Variability of loci and mutations conferring resistance to EMB was higher in ClIs from HIV-than from HIV+ patients: loci with mutations detected were seven and five, correspondingly, number of mutation's types were 12 and eight, correspondingly.

Mutations conferring resistance to fluoroquinolones
Rate of gyrA mutants among ClIs from HIV-patients of B0/W148 genotype was significantly lower than of other Beijing genotypes (31,3% and 53,7%, correspondingly, p = 0,014). There was no significant difference between HIV+ groups of ClIs with gyrA mutations of B0/ W148 and other Beijing genotype. Non-Beijing ClIs from HIVgroup with gyrA mutations were LAM, Ural and X1, in HIV+ group of ClIs one isolate was of Ural genotype only.

Mutations conferring resistance to aminoglycosides and Capreomycin
We were detecting mutations in eis and rrs genes. All ClIs with mutations in these genes were resistant to Kn according to DST. All rrs mutants both from HIV-and HIV+ groups of ClIs were also resistant to Ami and Cap, only five of eis mutants from HIV-group were additionally resistant to Ami and Cap. These findings agreed with other studies' data rrs mutants had high levels of MICs for Kn and cross-resistance to Ami and Cap, eis -moderate level of resistance to Kn and in some cases to Ami and Cap [20,21,24]. In the group of ClIs from HIV-patients eis mutations were detected in 70 ClIs (PR = 72,2%) and rrs mutations in 27 ClIs (PR = 27,8%). In the group of ClIs from HIV+ patients eis mutations were detected in 15 ClIs (PR = 62,5%) and rrs in 9 ClIs (PR = 37,5%).
Prevailing mutation in promoter of rrs gene was rrs Ala1401Gly, both in ClIs from HIV-and HIV+ patients. One isolate from HIV-patients with mutation rrs Cys1402Thr and one from HIV+ patients rrs Gly1484Thr were revealed. These findings agreed with other studies' data: rrs mutations and Ala1401Gly were associated with MICs high levels for Kn [20,21].
Mutations in eis and rrs genes detected in our study were described in other publications as conferring different levels of resistance to Kn, Ami and Cap [20,21,24].
Prevailing mutations conferring resistance to INH were katG Ser315Thr and T15A and conferring RIF resistance -rpoB Ser531Leu, both in ClIs from HIV-and HIV+ patients.
In ClIs resistant to Ofl most frequent were gyrA mutations in loci gyrA 90-91 and gyrA 94-95 both in ClIs from HIV-and HIV+ patients.
The dominating mutation in rrs gene was Ala1401Gly, both in ClIs from HIV-and HIV+ patients. Variability of mutations in eis gene was higher: mutations Gly10Ala, Cys12Thr, Cys14Thr and Gly37Thr were frequently detected both in ClIs from HIV-and HIV+ patients.
Spectra of genotypes and types of mutations detected in ClIs from HIV-patients were wider than in ClIs from HIV+ patients.

Design
Mtb ClIs were received in the NMRC PhID and URIPh laboratories from four different regions of the Russian Federation. Mtb were isolated from pulmonary specimens (mainly sputum) from newly diagnosed TB patients in 2018-2019 using liquid culturing on Bactec MGIT 960 system in laboratories of regional centers. In this study only one isolate collected from a patient before the beginning of chemotherapy were included.
Sampling of ClIs in all regions was for the purpose of external quality control, was random and targeted to adequate representation of susceptible, drug resistant (including MDR and XDR) isolates. ClIs in the samples were to be both from HIV-and HV+ patients and isolated from biological materials collected before beginning of chemotherapy, one isolate from a patient. Although these rules were followed in most of cases and considering that the only information on patients, we can operate was their HIV status (no personal data were used in this study) we cannot totally exclude clustering of isolates in exceptional cases. None of the rates presented in this manuscript could be considered as an epidemiological indicator.
Phenotypic drug susceptibility retesting, detection of mutations conferring drug resistance to specific drugs and genotyping of Mtb isolates had been carried out in NMRC PhID and URIPh with similar technologies.
Isolates were characterized according to their DR profiles according to WHO recommendations [46] as: • Susceptible-susceptible to all drugs tested. • Mono-resistant -resistant to only one of the tested drugs. • Poly-resistant -resistant to 2 and more drugs tested, except resistance to both INH and RIF. • Multidrug resistance (MDR) -resistance at least to INH and RIF, • pre-XDR -resistance at least to INH and RIF and at least to one of fluoroquinolones (FQ) or at least one of the injectable drugs (Kn, Ami, Cap), • XDR -resistance at least to INH and RIF (MDR) and at least to one of the FQ and at least one of the injectable drugs (Kn, Amk,Cap).
Studies had been held in the period of 2018-2019, before new WHO recommendations on Mtb DST (including recommendations of the use of critical concentrations for RIF as 0,5 ml/l and exclusion of Kn and Cap from the list of antituberculosis drugs to be tested) and new definitions were issued [18,19].
Molecular-genetic analysis of ClIs was performed with commercially available microarray test-systems (TB-TEST,BIOCHIP-IMB, Russia) which were developed by the Institute of molecular biology named after V.A. Engelhard of the Russian Academy of Science. Assay procedures include decontamination of biological specimens, DNA extraction, two consecutive multiplex PCR and PCR-products (amplicons) hybridization on a biologic microchip [47,48]. Detection of hybridization results, registration, and interpretation were performed with Chipdetector portable fluorescence analyzer. ClIs belonging to Mtb complex were identified by repetitive DNA IS6110, no species identification within the complex was possible, although members of the complex other than M. tuberculosis are extremely rare in Russia.

DNA extraction
Mbt DNA was extracted from Mtb culture using TB-TEST extraction system (BIOCHIP IMB Ltd., Moscow, Russian Federation) following the manufacturer's protocol.
Genotyping was carried out by microarray technology using TB-TEST (IMB-BIOCHIP, Russia). This method allowed to identify Mtb genotypes endemic for the Russian Federation (Beijing, Beijing B0/W148, LAM, Haarlem, Ural and others) by detection of genotype-specific single nucleotide polymorphism according the algorithm as in [7,49].
Genotyping tests were performed according to the manufacturer's instructions.
All loci and mutations detectable by the test-system used are listed in columns "Loci and types of mutations" of the Tables 3, 4, 5, 6 and 7.

Statistical analysis
95% confidence intervals (CI 95%) were calculated for prevalence rates (PR) -the rate (%) of genotypes, mutation loci and types. Fisher's exact test in analysis of two by two tables were used to assess differences between groups of data. Accepted Fisher's statistical significance level p was 0,05. Statistical analysis was done with Statistica software version 12.