Whole-genome sequencing of two clinical strains of Mycobacterium tuberculosis with phenotypic susceptibility to rifampicin but predicted resistance by Xpert MTB/RIF

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Introduction

Multidrug-resistant (MDR) tuberculosis develops in patients treated with rifampicin (RIF) and isoniazid, the most effective anti-tuberculosis drugs, also called first-line drugs. Globally, more than 40% of Mycobacterium tuberculosis (MBT) strains become MDR or at least RIF resistant¹. RIF binds close to the active site in the β subunit (rpoB gene) of the bacterial RNA polymerase enzyme [1] in the RIF resistance determining region (RRDR). The binding of RIF to RRDR sterically impedes the elongation of newly synthesized RNA, which ultimately blocks protein synthesis by the microbial cell. MBT has no known mechanism of horizontal gene transfer; RIF resistance mainly arises from chromosomal mutations within the RRDR [2]. The impact of RIF resistance is significant and is often reflected in MBT in terms of a reduced growth rate and the decreased competitiveness of RIF-resistant mutants compared to ancestral susceptible forms [3]. However, it has been observed that MBT forms with low adaptability can partially or completely restore phenotypic properties over time, in particular, increase growth rate due to the appearance of so-called compensatory mutations [4]. The identification of compensatory mutations is very difficult and depends on the methodology used.

Molecular epidemiologic studies of Beijing B0/W148 genomes belonging to the L2 genetic lineage [5, 6] indicate that more than 95% of clinical strains of this genotype contain mutations in RRDR and is one of the key factors in the epidemic spread of primary drug-resistant tuberculosis with RIF in Russia [6, 7]. Almost 90% of these strains carry the most common amino acid substitution S450L (nucleotide substitution C→T in 761155 position of the genome) [6], and it was noted that the genetic cost of this substitution in the RRDR of the rpoB gene for mutants is the lowest [2].

The aim of the study: to search for the most probable compensatory mutations in rpoA, rpoB and rpoC genes encoding α-, β- and β′-subunits of RNA polymerase of MBT, causing the phenomenon of phenotypic sensitivity to RIF.

Materials and methods

A retrospective cross-analysis of Xpert Ultra MTB/RIF and phenotypic bacteriological results for 2022 obtained in the laboratory department of the Irkutsk Regional Tuberculosis Hospital was performed. The study was conducted with the informed consent of the patients. The research protocol was approved by the Ethics Committee of the Scientific Centre for Family Health and Human Reproduction Problems (protocol No. 2, February 18, 2020).

2298 samples were examined, of which 529 were sensitive to RIF, while 363 were resistant; sensitivity to RIF using Xpert Ultra MTB/RIF was not determined in 90 samples. The main reason for the lack of positive PCR results is the low concentration of the target substance when Xpert Ultra MTB/RIF was performed. In 8 cases, resistance determined by Xpert Ultra test in sputum was not confirmed by bacteriologic methods. All cases were chronic MDR or extensively drug-resistant TB patients who had been discontinued from RIF due to the presence or acquisition of resistance to the drug in a previously isolated strain.

Two strains were obtained for repeat Xpert Ultra MTB/RIF, genotype testing, Sanger sequencing, and whole genome sequencing (WGS) (Table 1). DNA isolation, library preparation, WGS and bioinformatics, phylogenetic and statistical analyses were performed as described previously [6]. Primary nucleotide sequences were deposited in the NCBI bioproject PRJNA1215569. Resistance to anti-tuberculosis drugs was determined on a BD Bactec bacteriological analyzer (Becton Dickinson) and on Löwenstein–Jensen medium according to the Order of the Ministry of Health of Russia from 21.03.2003 No. 109 (ed. 05.06.2017). Genetic heteroresistance in individual genome positions was determined by the number of alternative short reads during WGS as described previously [8].

 

Table 1. Characterization of M. tuberculosis isolates

No.	Patient record group	HIV	Drug resistance	Genotype
Irk1	Ineffective course of tuberculosis treatment	+	To isoniazid, RIF*, capreomycin, pyrazinamide, prothianamide, bedaquiline, linezolid	Beijing B0/W148
Irk2	Tuberculosis relapse	_	To isoniazid, RIF*, ethambutol, capreomycin, pyrazinamide, levofloxacin, bedaquiline, linezolid	Beijing B0/W148

Note. *Based on the Xpert Ultra MTB/RIF results, but not the microbiological test.

 

Amino acid substitution probability in detected mutations was investigated using two approaches: PAM matrices (Point Accepted Mutation matrices) — PAM30 and PAM250 [9] and SIFT (Sorting Intolerant From Tolerant) algorithm for predicting amino acid substitutions affecting protein function [10].

Results

Repeated Xpert Ultra MTB/RIF test, Sanger sequencing and WGS revealed the presence of a single S450L mutation in the rpoB gene with phenotypic sensitivity in both strains. Phylogenetic analysis revealed that both genomes belonged to the Beijing B0/W148 genotype. The strains were characterized by a higher growth rate than the other isolates. After elucidation of the genotypic affiliation of the studied strains to the Beijing B0/W148 genotype, 513 complete B0/W148 genomes from the Short Read Archive (NCBI) online service published between 1995 and 2020 for strains from Northern Eurasia were used as reference genomes.

A total of 34 missense mutations in the rpoB gene were detected for this genome set [11]. Level 1 missense mutations were in 9 variants; level 2 — in 2; and level 3 — in 20 (Table 2). Furthermore, 3 mutations were found in the rpoB gene that were absent in the WHO catalog description: E82G, I90M, R219G. 45 missense mutations were detected in the rpoC gene, all of them belonged to mutations of level 2 significance (Table 1). Only 5 missense mutations were detected in the rpoA gene, also belonging to mutations of the 2^nd level of significance (Table 1).

 

Тable 2. Presence of mutations of the 1^st, 2^nd and 3^rd levels of significance [11]

Gene	Mutations of level 1 significance	Mutations of level 2 significance	Mutation of level 3 significance
rpoB	L430P; Q432P; D435V; D435Y; H445D; H445L; S450L; L452P; H723D	T427A; S431R	P45S; G79S; V305I; G376V; T400A; P454S; I491M; V496A; L554P; Y564H; S672Y; L731P; V800A; R827C; R827L; H835P; G836S; K891E; Q980K; R1008C
rpoC	None	E187G; G311R; G332S; G433C; P434A; P434L; K445R; L449R; F452C; V483G; D485N; E488Q; I491V; I491T; L507V; L516P; V517L; G519S; A521D; Q523E; H525N; L527V; L558L; Y586H; Q693H; N698H; N698S; N698K; E702K; D735N; D735E; D747A; H748P; E757A; R770H; T812I; S838C; D943N; D943G; M983I; P1040S; P1040R; I1046M; V1147A; K1152N	None
rpoA	None	G31C; R153R; T187P; V183A; R182Q	None

 

The following combinations of mutations were detected in the 2 strains studied: in the Irk1 strain, rpoB — S450L; rpoC — H748P; in the Irk2 strain, rpoB — S450L; rpoC — V483G. Interestingly, a similar case of drug sensitivity in the presence of a combination of rpoB — S450L; rpoC — V483G mutations was described in 2024 in the strain of Euro-American lineage (4.2.2.2.2.1) [12]. However, the authors suggested that the result obtained was a laboratory error due to the use of inflated concentrations of RIF during testing. The H748P mutation in the rpoC gene is not considered compensatory in the final version of the article, although it was described as such in the original manuscript by the same authors² [12].

The Irk1 and Irk2 genomes tested occupy the highest positions in terms of heteroresistance values (214 and 212) at position 761155 (rpoB nucleotide substitution — S450L; Figure). The highest heteroresistance (235), expressed as the presence of alternative short reads at WGS at the position under study, was observed in only one genome from Yakutia isolated in 2013. We investigated the probability of occurrence of detected amino acid substitutions in the rpoC gene using two approaches: PAM (Point Accepted Mutation) matrices — PAM30 and PAM250 [9] and the SIFT (Sorting Intolerant From Tolerant) algorithm [10]. To predict amino acid substitutions affecting protein function, PAM matrices were used to estimate the probability of amino acid substitutions during evolution [9]. The SIFT algorithm was used to determine whether amino acid substitutions affect protein function using evolutionary information and homologous sequence alignments [10].

 

Heteroresistance assessment of a sample of 515 genomes at position 761155 (rpoB nucleotide substitution — S450L).

 

A SIFT value of 0.00 was obtained for V483G (IRK2) indicating low tolerance, which may indicate a significant effect of this substitution on the function of the β'-subunit of RNA polymerase. However, the moderate values of PAM10 (0) and PAM250 (–1) suggest that this mutation does not lead to a complete loss of function and may stabilize the RNA polymerase complex, compensating for the destabilization caused by S450L. In turn, the H748P (IRK1) mutation with SIFT 0.05 and PAM250 (-3) shows moderate tolerance, indicating a slightly negative effect on the protein. It can be hypothesized that the above mutations affect the conformation of the β'-subunit, restoring the transcription efficiency caused by the major S450L mutation, where V483G may play a more pronounced compensatory role.

Discussion

The phenomenon of the emerging sensitivity in strains has been observed earlier in two international projects [8] when the minimum inhibitory concentration of MBT strains from the same patient was determined sequentially. It has been repeatedly observed (data not published) that withdrawal of certain anti-tuberculosis drugs, including RIF, leads to a decrease in the minimum inhibitory concentration down to the values of the borderline sensitivity defined by the manufacturer of the Sensititre MYCOTB kits (TREK Diagnostics). The main hypothesis that could explain this phenomenon was the assumption that antibiotic-sensitive clones begin to multiply more actively in the pathogen population after anti-tuberculosis drug withdrawal from the persister pool [8]. The Beijing B0/W148 genotype carries resistance to RIF in more than 95% of cases upon primary infection, i.e. it has already acquired all the compensatory mutations necessary for survival outside the organism in the process of evolution. The key question of the study is what fitness mutations lead to the emergence of the sensitivity phenomenon in the presence of the major rpoB mutation — S450L. The detected missense mutations V483G and H748P in the groC gene in the absence of other significant changes in the rpoA and rpoB genes may indicate that the withdrawal of certain anti-tuberculosis drugs may lead to the emergence of compensatory fitness mutations, manifested as sensitivity to anti-tuberculosis drugs in the presence of the major substitution, identified in PCR test. It can also be assumed that against the background of high heterogeneity on the rpoB gene in the pathogen population, the described combination of S450L mutations together with V483G/H748P undergoes stabilizing selection only during RIF treatment. The abolition of RIF leads to a gradual return of the pathogen population to a more stable model in which clones containing V483G/H748P and other fitness mutations are eliminated. The source of this is persisters harboring S450L but lacking mutations in the rpoC gene.

Conclusion

It can be assumed that the phenomenon of discrepancy between results of bacteriological and molecular genetic tests is associated with the acquisition of compensatory mutations in the groC gene during RIF treatment of Beijing B0/W148 strains. The identified mutations affect the conformation of the β'-subunit, restoring the transcription efficiency caused by the major S450L mutation. Further studies of the phenomenon of decreased resistance to anti-tuberculosis drugs in the tuberculosis pathogen after its withdrawal are necessary.

 

¹ WHO. Global tuberculosis report 2024. Geneva: World Health Organization; 2024. URL: https://www.who.int/teams/global-tuberculosis-programme/tb-reports/global-tuberculosis-report-2024

² URL: https://www.biorxiv.org/content/10.1101/2022.02.22.481565v1.full.pdf

About the authors

Oleg B. Ogarkov

Scientific Centre for Family Health and Human Reproduction Problems

Author for correspondence.
Email: obogarkov@mail.ru
ORCID iD: 0000-0002-3168-1983

Dr. Sci. (Med.), Director, Institute of Epidemiology and Microbiology

Россия, Irkutsk

Viacheslav V. Sinkov

Scientific Centre for Family Health and Human Reproduction Problems

Email: vsinkov@gmail.com
ORCID iD: 0000-0003-3396-9590

Cand. Sci. (Med.), senior researcher, Laboratory of epidemiologically and socially significant infections

Россия, Irkutsk

Tatyana A. Kuhtina

Irkutsk Regional Clinical Tuberculosis Hospital

Email: tuyanatsta@mail.ru

bacteriologist, Head, Bacteriological laboratory

Россия, Irkutsk

Svetlana N. Zhdanova

Scientific Centre for Family Health and Human Reproduction Problems

Email: svetnii@mail.ru
ORCID iD: 0000-0001-7160-9700

Dr. Sci. (Med.), leading researcher, Laboratory of epidemiologically and socially significant infections, Institute of Epidemiology and Microbiology

Россия, Irkutsk

Ilya G. Kondratov

Scientific Centre for Family Health and Human Reproduction Problems

Email: kondratovig@mail.ru
ORCID iD: 0000-0002-2631-4724

Cand. Sci. (Biol.), researcher, Laboratory of epidemiologically and socially significant infections, Institute of Epidemiology and Microbiology

Россия, Irkutsk

References

Supplementary files