Kateete David Patrick, Namakula Shakira, Kigozi Edgar, Katabazi Fred A, Kasule George William, Musisi Kenneth, Wampande Edward, Lukoye Deus, Joloba Moses L
Department of Medical Microbiology, School of Biomedical Sciences, Makerere University College of Health Sciences, Kampala, Uganda.
Department of Immunology and Molecular Biology, School of Biomedical Sciences, Makerere University College of Health Sciences, Kampala, Uganda.
bioRxiv. 2025 Jul 11:2025.07.11.664293. doi: 10.1101/2025.07.11.664293.
Mutations in , a gene that encodes the bacterial RNA polymerase (RNAP) beta-subunit, can cause high-level resistance to rifampicin. Approximately 95% of rifampicin-resistant clinical isolates possess mutations in an 81-base pair region referred to as the rifampicin-resistance determining region (/RRDR). Also, rifampicin-resistant clinical isolates carry multiple mutations in RNAP genes (i.e., , ), particularly and , which encode the alpha- (α) and beta'- (β') subunits, respectively. Such secondary mutations offset the fitness cost associated with rifampicin-resistance mutations in , resulting in resistant strains that are as fit as the wildtype drug-susceptible strains. To analyse the patterns of compensatory mutations in RNAP encoding genes of rifampicin-resistant clinical isolates in Uganda, whole genome sequencing and Sanger DNA sequencing were performed on 52 clinical isolates - 20 drug-susceptible and 32 multidrug resistant (MDR). A total of 24 (75%) MDR-TB isolates had high-level rifampicin-resistance conferring mutations in /RRDR i.e., Ser531Leu (31%); His526Asp (6%); His526Leu (3%); His526Tyr (3%); His526Arg (3%); His526Gly (3%); Asp516Tyr (13%); Asp516Val (6%); Glu513Lys (3%); Leu511Pro (3%); Leu492Leu (3%); Gln490Arg (3%). Further, two putative compensatory mutations (Gln490Arg & Lys1025Glu) outside the RRDR and not resistance conferring were found in . Altogether, 16 (50%) MDR-TB isolates with /RRDR resistance conferring mutations had non-synonymous mutations in of the following patterns Leu39Phe (3%); Tyr61His (3%); Asp271Gly (3%); Ser377Ala (3%); Pro481Thr (3%); Val483Ala (6%); Leu516Pro (3%); Ala521Asp (3%); Gly594Glu (13%); Asn698Ser (3%); Leu823Pro (3%). In conclusion, putative compensatory mutations are prevalent in rifampicin-resistant clinical isolates in Uganda, with /Gly594Glu and /Val483Ala as the most frequent. Further studies will determine their association with strain genetic background, fitness and transmission in an endemic setting with a high burden of HIV-TB coinfection.
rpoB基因编码细菌RNA聚合酶（RNAP）的β亚基，该基因的突变可导致对利福平的高水平耐药。约95%的耐利福平临床分离株在一个被称为利福平耐药决定区（rpoB/RRDR）的81个碱基对区域存在突变。此外，耐利福平临床分离株在RNAP基因（即rpoA、rpoC）中携带多个突变，特别是rpoA和rpoC，它们分别编码α（α）和β'（β'）亚基。这些继发突变抵消了与rpoB中利福平耐药突变相关的适应性代价，从而产生了与野生型药物敏感株适应性相当的耐药菌株。为了分析乌干达耐利福平临床分离株的RNAP编码基因中的补偿性突变模式，对52株临床分离株——20株药物敏感株和32株耐多药（MDR）株进行了全基因组测序和桑格DNA测序。共有24株（75%）耐多药结核病分离株在rpoB/RRDR中存在赋予高水平利福平耐药性的突变，即Ser531Leu（31%）；His（526）Asp（6%）；His526Leu（3%）；His526Tyr（3%）；His526Arg（3%）；His526Gly（3%）；Asp516Tyr（13%）；Asp516Val（6%）；Glu513Lys（3%）；Leu511Pro（3%）；Leu492Leu（3%）；Gln490Arg（3%）。此外，在rpoC中发现了RRDR之外的两个假定补偿性突变（Gln490Arg和Lys1025Glu），它们不赋予耐药性。总共16株（50%）具有rpoB/RRDR耐药性赋予突变的耐多药结核病分离株在rpoA中存在以下模式的非同义突变：Leu39Phe（3%）；Tyr61His（3%）；Asp271Gly（3%）；Ser377Ala（3%）；Pro481Thr（3%）；Val483Ala（6%）；Leu516Pro（3%）；Ala521Asp（3%）；Gly594Glu（13%）；Asn698Ser（3%）；Leu823Pro（3%）。总之，假定补偿性突变在乌干达耐利福平临床分离株中普遍存在，其中rpoA/Gly594Glu和rpoC/Val483Ala最为常见。进一步的研究将确定它们在艾滋病毒-结核病合并感染负担高的地方流行环境中与菌株遗传背景、适应性和传播的关联。
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