rpoB Mutations are Associated with Variable Levels of Rifampin and Rifabutin Resistance in Mycobacterium tuberculosis

Objective To assess the relationship between the variant rpoB mutations and the degree of rifampin (RIF)/rifabutin (RFB) resistance in Mycobacterium tuberculosis (M. tuberculosis). Methods We analyzed the whole rpoB gene in 177 M. tuberculosis clinical isolates and quantified their minimum inhibitory concentrations (MICs) using microplate-based assays. Results The results revealed that of the 177 isolates, 116 were resistant to both RIF and RFB. There were 38 mutated patterns within the sequenced whole rpoB gene of the 120 isolates. Statistical analysis indicated that mutations, S450L, H445D, H445Y, and H445R, were associated with RIF and RFB resistance. Of these mutations, S450L, H445D, and H445Y were associated with high-level RIF and RFB MIC. H445R was associated with high-level RIF MIC, but not high-level RFB MIC. D435V and L452P were associated with only RIF, but not RFB resistance. Q432K and Q432L were associated with high-level RFB MIC. Several single mutations without statistical association with rifamycin resistance, such as V170F, occurred exclusively in low-level RIF but high-level RFB resistant isolates. Additionally, although cross-resistance to RIF and RFB is common, 21 RIF-resistant/RFB-susceptible isolates were identified. Conclusion This study highlighted the complexity of rifamycin resistance. Identification of the rpoB polymorphism will be helpful to diagnose the RIF-resistant tuberculosis that has the potential to benefit from a treatment regimen including RFB.


Introduction
Tuberculosis (TB) continues to be a major global public health threat causing 6.4 million new cases and 1.6 million deaths in 2021. 1 The alarming increase in drug-resistant, especially rifampicin-resistant TB (RR-TB), represents the major challenge for TB control. RR-TB requires prolonged use of a combination of drugs that are less efficient and more toxic. In the last year, there was a worldwide estimate of 450,000 incident cases of RR-TB. 1 Rifampin (RIF) is one of the principal drugs in TB treatment and serves as a hallmark for the detection of multidrugresistant TB (MDR-TB). 2 Rifabutin (RFB) is a semisynthetic derivative of rifamycin S, which together with RIF, belongs to the rifamycin family. These two drugs target the DNA-dependent RNA polymerase β-subunit, encoded by the rpoB gene. RFB is recommended for TB treatment in HIV-coinfected patients because it is less prone to drug-drug interactions than RIF in patients receiving antiretroviral therapy. 3,4 However, reports on the successful treatment of MDR-TB patients with RFB, even in patients with isolates susceptible in vitro to RFB, are very limited. 5,6 One possible reason is due to the fact that the clinical efficacy of RFB for the treatment of MDR-TB has not been well established because of general concerns about potential cross-resistance among the rifamycins. 7,8 Certain mutations in the RIF resistance-determining region (RRDR) in rpoB gene, located between codons 426 and 452 in M. tuberculosis, are associated with cross-resistance to both RIF and RFB. 2,[9][10][11] The mutations, S450L, H445Y, and H445D are most frequently observed in RIF-and RFB-resistant strains. [11][12][13] Although cross-resistance to RIF and RFB is common, some RIF-resistant strains carrying certain rpoB mutations may remain susceptibility in vitro to RFB. 14,15 Thus, RFB has the potential to be clinically effective against RIF-resistant strains.
Some reports have shown RFB susceptibility in RIF-resistant strains with specific rpoB mutations. [16][17][18] These studies also pointed out the possible uses for RFB in these cases with rpoB polymorphisms and their association with phenotypic drug susceptibility data. 16,17 However, most studies focused on the analysis of mutations in the RRDR region of rpoB. 11,17,18 Furthermore, several rpoB mutations were also detected in rifamycin-susceptible phenotypes. Thus, a detailed investigation of the different rpoB mutations and their association with different rifamycin resistance levels may be helpful to clinicians treating RIF-resistant TB, even multi-drug resistant TB (MDR-TB). To this end, we systematically performed quantitative RIF and RFP resistance phenotyping with MIC measurements for 177 M. tuberculosis clinical isolates from China and compared the effect of mutation within the whole rpoB gene on MIC changes for these two drugs.

DNA Extraction, Amplification, and Sequencing
All isolates on the L-J slants were collected and inactivated by heating at 95°C for 20 min. Supernatants containing genomic DNA were collected by centrifugation and stored at −20°C for further use.
The entire rpoB gene of each isolate was amplified using the primers and reaction conditions as previously described. 19 All amplified products were purified, dried, and loaded onto an ABI 3730XL DNA Analyzer (Applied Biosystems, Foster City, CA). The sequences generated were compared with the H37Rv reference genome (GenBank accession number NC_000962) using BioEdit v7.05.3.

Data Analysis
The effects of rpoB mutations on rifamycin drugs were evaluated by the regression multivariate model and P value less than 0.05 was considered statistically significant. All statistical analyses were performed using SAS version 9.3 (SAS Institute, Cary, USA).

Correlation of rpoB Mutations with Drug Resistance
Most of the rpoB mutations (single or double) occurred in RIF-and RFB-resistant isolates. Only one RIF-susceptible isolate (MIC = 0.5 µg/mL) and 19 RFB-susceptible isolates (MIC ≤ 0.5 µg/mL) bearing the rpoB mutation were detected ( Table 1).
As 20.0% of isolates with rpoB mutations harbored more than one mutation, the correlation between mutations and drug resistance was estimated through multivariate regression (  (Table 2). Notably, mutations D435V (OR = 37.39) and L452P (OR = 11.59) were associated with resistance to only RIF (P<0.05), but not RFB, while mutations Q432K (OR = 13.75), Q432L (OR = 13.75), and L430P (OR = 15.99) were associated with resistance to only RFB (P<0.05), but not RIF. Furthermore, other mutations, except for those locating in the RRDR, were not associated with drug resistance according to multivariate analyses. In addition, there were still 8 RIF-resistant isolates and 5 RFB-resistant isolates that harbored no mutation in the whole rpoB.

Discussion
Rapid and accurate determination of drug resistance in M. tuberculosis is essential for early initiation of appropriate anti-TB treatment. Although several molecular diagnostic techniques, such as GeneXpert MTB/RIF (Cepheid Inc., Sunnyvale, CA, USA) and GenoType MTBDRplus (HAIN Lifescience GmbH, Nehren, Germany), provide the rapid identification of M. tuberculosis drug resistance directly from specimens, 22-24 these molecular methods may miss isolates with mutations outside the target region, and cannot determine the level of resistance. Furthermore, previous reports indicated that some isolates with rpoB mutations are RIF-susceptible according to phenotypic drug susceptibility testing. 10,11,25 Similarly, RFB-susceptible isolates with rpoB mutations have also been reported, 11,14,15,18,26 highlighting the importance of the potential inclusion of RFB in the treatment regimen. For these reasons, it is crucial to understand the association of rpoB mutations with different RIF and RFB resistance levels.
Although cross-resistance to RIF and RFB is common, we still observed discordance between RIF and RFB in 21 isolates. Previous studies showed a proportion of RIF-resistant isolates with RFB-susceptibility ranging from 13% to 28%. 11,17 In our study, the proportion of RIF-resistant/RFB-susceptible isolates was 16.5% (21/127 isolates). The replacement of RIF by RFB might positively affect the treatment outcome in the cases carrying these isolates.
It is reported that the RNA polymerase β-subunit encoded by rpoB is a target for rifamycin drugs, and some amino acid changes in this protein can lead to rifamycin resistance. [27][28][29][30] Our study showed that 93.7% (119/127 isolates) of RIFresistant isolates and 95.3% (101/106 isolates) of RFB-resistant isolates harbored at least one mutation in rpoB. Overall, 19 distinct codons in rpoB had mutations contributing to amino acid replacements, of which 11 codons are located in the RRDR and 8 are located outside the RRDR. Interestingly, almost all mutations outside the RRDR were accompanied by mutations in the RRDR. These results show the potential for using DNA sequencing of RRDR to rapidly predict RIF and RFB resistance. 2,10 However, a single mutation V170F outside the RRDR was also observed in one resistant isolate, similar to some reports. 20,31 This highlights the complexity of RIF and RFB resistance and emphasizes the requirement to investigate beyond the RRDR, especially when patient treatment effects are not as expected.
Our study also showed the mutations associated with RIF and RFB. Statistical analysis indicated that, of the 38 different rpoB polymorphisms, S450L, H445D, H445Y, and H445R were significantly correlated with RIF and RFB resistance. Miotto et al 32

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Mutations D435V and L452P were significantly associated with only RIF resistance, but not RFB resistance, consistent with the prior data. 17,32 Cases carrying the isolates with these two mutations may be RFB-susceptible and benefit from the RFB-containing treatment regimen. In addition, our results indicated that Q432K, Q432L and L430P were significant with only RFB-resistance. Although mutation L430P was classified as RFB-susceptible by Whitfield et al, 17 this mutation was observed in four RFB-resistant isolates. Of them, three isolates were accompanied by another rpoB mutation (Table 1). Interestingly, one isolate harboring double mutations (L430P/H445N) classified as RFB-susceptible by Whitfield, 17 had a MIC of ≥16 µg/mL. Thus, the actual effects of these double rpoB mutations on the RFB resistance deserve further investigation. We also found that mutations 450L, H445D, and H445Y were strongly associated with high-level RIF/RFB MIC. This observation is similar to those of previous reports. [11][12][13]33 Moreover, mutation H445R was well correlated with highlevel RIF MIC, while mutations Q432K and Q432L were associated with high-level RFB MIC. Our results, therefore, add important data on the association between mutations and rifamycin-resistant levels in TB. Notably, V170F mutation occurred exclusively in two high-level RFB MIC but low-level RIF MIC isolates. The function of this mutation on the interaction between RpoB and RFB is unknown and requires further exploration.
This study used the WHO current recommended clinical breakpoints (0.5 µg/mL) for susceptibility testing of RIF. 20 However, we still identified one RIF-susceptible isolate (MIC = 0.5 µg/mL) with the D435Y mutation, which has also been reported in some RIF-susceptible isolates. 14,15,31 It was notable that 19 RFB-susceptible isolates also harbored rpoB mutations, with the use of CLSI recommended clinical breakpoints (0.5 µg/mL) for susceptibility testing of RFB. 21 Of them, 9 had MICs of 0.5 µg/mL. Although most mutations presenting in these isolates were classified as RFBsusceptible, some reports also suggested that the critical testing concentration, currently recommended at 0.5 µg/mL, may be too high. 15,16 Moreover, some RIF/RFB resistant isolates lacking rpoB mutations were identified, indicating that resistance in these isolates may be attributed to other mechanisms, such as enhanced efflux pump activity or lowered cell wall permeability. 11,34 This study also suffers some limitations. In total, large amounts of clinical isolates (n = 177) were recruited in this study. However, the number of isolates carrying mutations outside RRDR, such as T400I, G759S, and Q1056K, was very small, leading to insufficient data for further statistical analysis. Also, we only analyzed mutations in the whole rpoB gene, and changes in other genes or other mechanisms such as efflux pumps could not be identified in the present study.
In summary, we determined comprehensive profiles of rpoB mutations and their associations with RIF and RFB resistance levels in a variety of M. tuberculosis clinical isolates. Revealing the SNP-specific differential rifamycin phenotypic resistance will have the potential for using RFB to optimize the treatment of some RIF-resistant patients. However, clinical investigations are required to verify whether RFB can be applied effectively in these patients to promote outcomes.

Ethical Approval
This study was approved by the Ethics Committee of the National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention. All patients participating in the study had written consent themselves.

Funding
This study was supported by the projects from the National Key Program of Mega Infectious Diseases (Grant No. 2018ZX10302302-001). The funder had no role in the study design, data collection and analysis, decision to publish, or manuscript preparation.