rpoB Mutations Research Articles

The relative transmission fitness of multidrug-resistant Mycobacterium tuberculosis in a drug resistance hotspot

Multidrug-resistant tuberculosis (MDR-TB) is among the most frequent causes of death due to antimicrobial resistance. Although only 3% of global TB cases are MDR, geographical hotspots with up to 40% of MDR-TB have been observed in countries of the former Soviet Union. While the quality of TB control and patient-related factors are known contributors to such hotspots, the role of the pathogen remains unclear. Here we show that in the country of Georgia, a known hotspot of MDR-TB, MDR Mycobacterium tuberculosis strains of lineage 4 (L4) transmit less than their drug-susceptible counterparts, whereas most MDR strains of L2 suffer no such defect. Our findings further indicate that the high transmission fitness of these L2 strains results from epistatic interactions between the rifampicin resistance-conferring mutation RpoB S450L, compensatory mutations in the RNA polymerase, and other pre-existing genetic features of L2/Beijing clones that circulate in Georgia. We conclude that the transmission fitness of MDR M. tuberculosis strains is heterogeneous, but can be as high as drug-susceptible forms, and that such highly drug-resistant and transmissible strains contribute to the emergence and maintenance of hotspots of MDR-TB. As these strains successfully overcome the metabolic burden of drug resistance, and given the ongoing rollout of new treatment regimens against MDR-TB, proper surveillance should be implemented to prevent these strains from acquiring resistance to the additional drugs.

Molecular Epidemiology of Rifampicin-Resistant Mycobacterium tuberculosis by GeneXpert MTB/RIF Assay in Renal Transplant Recipients: A Single-Center Experience.

There are scarce data on the incidence and resistance pattern of rifampicin-resistant Mycobacterium tuberculosis among kidney transplant recipients. This is a retrospective, single- center study of kidney transplantrecipients suspected of M. tuberculosis infection. The GeneXpert assay we used detected mutations in the rpoB gene that confer rifampicin resistance using 5 overlapping probes (A, B, C, D, and E). The probes can detect mutations in the codons 507 to 511 (probe A), 511 to 518 (probe B), 518 to 523 (probe C), 523 to 529 (probe D), and 529 to 533 (probe E).We also detailed the treatment protocol and outcomes of kidney transplantrecipients infected with rifampicin-resistant M. tuberculosis. In total, 2700 samples were processed during the period from October 2018 to February 2022 with successful results in 2640 samples (97.04%). One hundred and ninety (7.19%) samples were positive for M.tuberculosis, and rifampicin resistance was detected in 12 (0.45%) cases (11 pulmonary, 1 genitourinary). The most common rpoB mutation was located in the region of probe E (75.0%), followed by probe A (16.6%) and in 1 combination probe DE (8.33%). The rpoB mutations were not observed in probe B and probe C. Six patients received bedaquiline-based treatmentfor a short course of 11 months, whereas the other 6 patients required a long course of 18 to 20 months. Three patients died, 2 were lost to follow-up, and 7 were cured. During treatment, 4 patients experienced acute rejection, and 1 graft loss was reported. We report for the first time the incidence and pattern of rifampicin resistance among kidney transplant recipients with tuberculosis infection. Further investigations are required for exploring the molecular and clinical phenotypes.

Spatial Distribution of Drug-Resistant Mycobacterium tuberculosis Infections in Rural Eastern Cape Province of South Africa.

Tuberculosis (TB), an infectious airborne disease caused by Mycobacterium tuberculosis (Mtb), is a serious public health threat reported as the leading cause of morbidity and mortality worldwide. South Africa is a high-TB-burden country with TB being the highest infectious disease killer. This study investigated the distribution of Mtb mutations and spoligotypes in rural Eastern Cape Province. The Mtb isolates included were 1157 from DR-TB patients and analysed by LPA followed by spoligotyping of 441 isolates. The distribution of mutations and spoligotypes was done by spatial analysis. The rpoB gene had the highest number of mutations. The distribution of rpoB and katG mutations was more prevalent in four healthcare facilities, inhA mutations were more prevalent in three healthcare facilities, and heteroresistant isolates were more prevalent in five healthcare facilities. The Mtb was genetically diverse with Beijing more prevalent and largely distributed. Spatial analysis and mapping of gene mutations and spoligotypes revealed a better picture of distribution.

Consequences of rpoB mutations missed by the GenoType MTBDRplus assay in a programmatic setting in South Africa.

Rifampicin resistance missed by commercial rapid molecular assays but detected by phenotypic assays may lead to discordant susceptibility results and affect patient management. This study was conducted to evaluate the causes of rifampicin resistance missed by the GenoType MTBDRplus and its impact on the programmatic management of tuberculosis in KwaZulu-Natal, South Africa. We analysed routine tuberculosis programme data from January 2014 to December 2014 on isolates showing rifampicin susceptibility on the GenoType MTBDRplus assay but resistance on the phenotypic agar proportion method. Whole-genome sequencing was performed on a subset of these isolates. Out of 505 patients with isoniazid mono-resistant tuberculosis on the MTBDRplus, 145 (28.7%) isolates showed both isoniazid and rifampicin resistance on the phenotypic assay. The mean time from MTBDRplus results to initiation of drug-resistant tuberculosis therapy was 93.7 days. 65.7% of the patients had received previous tuberculosis treatment. The most common mutations detected in the 36 sequenced isolates were I491F (16; 44.4%) and L452P (12; 33.3%). Among the 36 isolates, resistance to other anti-tuberculosis drugs was 69.4% for pyrazinamide, 83.3% for ethambutol, 69.4% for streptomycin, and 50% for ethionamide. Missed rifampicin resistance was mostly due to the I491F mutation located outside the MTBDRplus detection area and the L452P mutation, which was not included in the initial version 2 of the MTBDRplus. This led to substantial delays in the initiation of appropriate therapy. The previous tuberculosis treatment history and the high level of resistance to other anti-tuberculosis drugs suggest an accumulation of resistance.

Rifampicin resistance conferring mutations among Mycobacterium tuberculosis strains in Rwanda.

The World Health Organization-endorsed phenotypic and genotypic drug-susceptibility testing (gDST/pDST) assays for the detection of rifampicin-resistant (RR) tuberculosis (TB), may miss some clinically relevant rpoB mutants, including borderline mutations and mutations outside the gDST-targeted hotspot region. Sequencing of the full rpoB gene is considered the reference standard for rifampicin DST but is rarely available in RR-TB endemic settings and when done indirectly on cultured isolates may not represent the full spectrum of mutations. Hence, in most such settings, the diversity and trends of rpoB mutations remain largely unknown. This retrospective study included rpoB sequence data from a longitudinal collection of RR-TB isolates in Rwanda across 30 years (1991-2021). Of 540 successfully sequenced isolates initially reported as RR-TB, 419 (77.6%) had a confirmed RR conferring mutation. The Ser450 Leu mutation was predominant throughout the study period. The Val170Phe mutation, not covered by rapid gDST assays, was observed in only four patients, three of whom were diagnosed by pDST. Along with the transition from pDST to rapid gDST, borderline RR-associated mutations, particularly Asp435Tyr, were detected more frequently. Borderline mutants were not associated with HIV status but presented lower odds of having rpoA-C compensatory mutations than other resistance-conferring mutations. Our analysis showed changes in the diversity of RR-TB conferring mutations throughout the study period that coincided with the switch of diagnostic tools to rapid gDST. The study highlights the importance of rapid molecular diagnostics reducing phenotypic bias in the detection of borderline rpoB mutations while vigilance for non-rifampicin resistance determinant region mutations is justified in any setting.

Molecular Characterization of Mutations in Isoniazid- and Rifampicin-Resistant Mycobacterium tuberculosis Isolated in Thailand.

The control of drug-resistant tuberculosis (TB) is a major challenge. The frequency and mutation characteristics indicate the efficiency of molecular tests for the rapid detection of TB drug resistance. This study examined the existence of katG and inhA mutations for isoniazid (INH) resistance and rpoB mutations for rifampicin (RFP) resistance. In total, 178 drug-resistant Mycobacterium tuberculosis (MTB) isolates were analyzed. Mutations in katG encoding and inhA regulatory regions were detected in 136/168 (81.0%) and 29/168 (17.3%), respectively, with the most prominent mutation of Ser315Thr substitution in katG in 126/168 (75.0%), and -15 C to T substitution in the regulatory region of the inhA (26/168; 15.5%). Two distinct katG mutations (Tyr337Cys, 1003InsG) were identified. Of 125 RFP-resistant isolates, 118 (94.4%) carried mutations affecting the 81-bp RFP resistance-determining region, with the most commonly affected codons 450, 445, and 435 identified in 74 (59.2%), 26 (20.8%), and 12 (9.6%) isolates, respectively. Genetic mutations were highly associated with phenotypic INH and RFP resistance, and the majority shared similarities with those reported in previous studies in Thailand and other Asian countries. These data are useful for guiding the use and improvement of molecular tests for TB drug resistance.

Multidrug-resistant toxigenic Corynebacterium diphtheriae sublineage 453 with two novel resistance genomic islands.

Antimicrobial therapy is important for case management of diphtheria, but knowledge on the emergence of multidrug-resistance in Corynebacterium diphtheriae is scarce. We report on the genomic features of two multidrug-resistant toxigenic isolates sampled from wounds in France 3 years apart. Both isolates were resistant to spiramycin, clindamycin, tetracycline, kanamycin and trimethoprim-sulfamethoxazole. Genes ermX, cmx, aph(3')-Ib, aph(6)-Id, aph(3')-Ic, aadA1, dfrA15, sul1, cmlA, cmlR and tet(33) were clustered in two genomic islands, one consisting of two transposons and one integron, the other being flanked by two IS6100 insertion sequences. One isolate additionally presented mutations in gyrA and rpoB and was resistant to ciprofloxacin and rifampicin. Both isolates belonged to sublineage 453 (SL453), together with 25 isolates from 11 other countries (https://bigsdb.pasteur.fr/diphtheria/). SL453 is a cosmopolitan toxigenic sublineage of C. diphtheriae, a subset of which acquired multidrug resistance. Even though penicillin, amoxicillin and erythromycin, recommended as the first line in the treatment of diphtheria, remain active, surveillance of diphtheria should consider the risk of dissemination of multidrug-resistant strains and their genetic elements.

The mutation rate of rpoB gene showed an upward trend with the increase of MIRU10, MIRU39 and QUB4156 repetitive number

BackgroundMycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) is a frequently used typing method for identifying the Beijing genotype of Mycobacterium tuberculosis (Mtb), which is easily transformed into rifampicin (RIF) resistance. The RIF resistance of Mtb is considered to be highly related with the mutation of rpoB gene. Therefore, this study aimed to analyze the relationship between the repetitive number of MIRU loci and the mutation of rpoB gene.MethodsAn open-source whole-genome sequencing data of Mtb was used to detect the mutation of rpoB gene and the repetitive number of MIRU loci by bioinformatics methods. Cochran-Armitage analysis was performed to analyze the trend of the rpoB gene mutation rate and the repetitive number of MIRU loci.ResultsAmong 357 rifampicin-resistant tuberculosis (RR-TB), 304 strains with mutated rpoB genes were detected, and 6 of 67 rifampicin susceptible strains were detected mutations. The rpoB gene mutational rate showed an upward trend with the increase of MIRU10, MIRU39, QUB4156 and MIRU16 repetitive number, but only the repetitive number of MIRU10, MRIU39 and QUB4156 were risk factors for rpoB gene mutation. The Hunter-Gaston discriminatory index (HGDI) of MIRU10 (0.65) and QUB4156 (0.62) was high in the overall sample, while MIRU39 (0.39) and MIRU16 (0.43) showed a moderate discriminatory Power.ConclusionThe mutation rate of rpoB gene increases with the addition of repetitive numbers of MIRU10, QUB4156 and MIRU39 loci.

Biofilms on Indwelling Artificial Urinary Sphincter Devices Harbor Complex Microbe-Metabolite Interaction Networks and Reconstitute Differentially In Vitro by Material Type.

The artificial urinary sphincter (AUS) is an effective treatment option for incontinence due to intrinsic sphincteric deficiency in the context of neurogenic lower urinary tract dysfunction, or stress urinary incontinence following radical prostatectomy. A subset of AUS devices develops infection and requires explant. We sought to characterize biofilm composition of the AUS device to inform prevention and treatment strategies. Indwelling AUS devices were swabbed for biofilm at surgical removal or revision. Samples and controls were subjected to next-generation sequencing and metabolomics. Biofilm formation of microbial strains isolated from AUS devices was reconstituted in a bioreactor mimicking subcutaneous tissue with a medical device present. Mean patient age was 73 (SD 10.2). All eighteen artificial urinary sphincter devices harbored microbial biofilms. Central genera in the overall microbe-metabolite interaction network were Staphylococcus (2620 metabolites), Escherichia/Shigella (2101), and Methylobacterium-Methylorubrum (674). An rpoB mutation associated with rifampin resistance was detected in 8 of 15 (53%) biofilms. Staphylococcus warneri formed greater biofilm on polyurethane than on any other material type (p < 0.01). The results of this investigation, wherein we comprehensively characterized the composition of AUS device biofilms, provide the framework for future identification and rational development of inhibitors and preventive strategies against device-associated infection.

Large-scale genomic analysis of Mycobacterium tuberculosis reveals extent of target and compensatory mutations linked to multi-drug resistant tuberculosis

Resistance to isoniazid (INH) and rifampicin (RIF) first-line drugs in Mycobacterium tuberculosis (Mtb), together called multi-drug resistance, threatens tuberculosis control. Resistance mutations in katG (for INH) and rpoB (RIF) genes often come with fitness costs. To overcome these costs, Mtb compensatory mutations have arisen in rpoC/rpoA (RIF) and ahpC (INH) loci. By leveraging the presence of known compensatory mutations, we aimed to detect novel resistance mutations occurring in INH and RIF target genes. Across ~ 32 k Mtb isolates with whole genome sequencing (WGS) data, there were 6262 (35.7%) with INH and 5435 (30.7%) with RIF phenotypic resistance. Known mutations in katG and rpoB explained ~ 99% of resistance. However, 188 (0.6%) isolates had ahpC compensatory mutations with no known resistance mutations in katG, leading to the identification of 31 putative resistance mutations in katG, each observed in at least 3 isolates. These putative katG mutations can co-occur with other INH variants (e.g., katG-Ser315Thr, fabG1 mutations). For RIF, there were no isolates with rpoC/rpoA compensatory mutations and unknown resistance mutations. Overall, using WGS data we identified putative resistance markers for INH that could be used for genotypic drug-resistance profiling. Establishing the complete repertoire of Mtb resistance mutations will assist the clinical management of tuberculosis.

Performance Evaluation of the BACTEC MGIT 960 System for Rifampin Drug-Susceptibility Testing of Mycobacterium tuberculosis Using the Current WHO Critical Concentration.

The World Health Organization recently lowered the rifampin (RIF) critical concentration (CC) for drug-susceptibility testing (DST) of Mycobacterium tuberculosis complex (MTBC) using the mycobacterial growth indicator tube (MGIT) 960 system. Here, we evaluated the diagnostic performance of the MGIT system with the revised CC for determining MTBC RIF resistance with 303 clinical MTBC isolates, including 122 isolates with rpoB mutations, of which 32 had single borderline-resistance mutations, and 181 wild-type rpoB isolates. The phenotypic RIF resistance was determined via the absolute concentration method (AC) and via MGIT using both previous (1 mg/L) and revised (0.5 mg/L) CCs for the latter method. The diagnostic accuracy of each phenotypic DST (pDST) was assessed based on rpoB genotyping as the reference standard. The overall sensitivity of the AC was 95.1% (95% confidence interval [CI], 89.6 to 98.2%), while the MGIT results with previous and revised CCs were 82.0% (95% CI 74.0 to 88.3%) and 83.6% (95% CI 75.8 to 89.7%), respectively. The 32 MTBC isolates with single borderline-resistance mutations showed a wide range of MICs, and sensitivity was not significantly increased by reducing the MGIT CC. All 181 wild-type rpoB isolates were RIF-susceptible in the AC and with MGIT using the previous CC, whereas 1 isolate was misclassified as RIF-resistant with the revised CC. Our results demonstrate that the overall diagnostic performances of the MGIT DST with the revised RIF CC and previous CC were comparable. A further large-scale study is required to demonstrate the optimal RIF CC for MGIT.

ГЕНОТИПІЧНА ТА ФЕНОТИПІЧНА ХАРАКТЕРИСТИКА СТІЙКОСТІ ДО АНТИМІКОБАКТЕРІАЛЬНИХ ПРЕПАРАТІВ 8 ШТАМІВ M. TUBERCULOSIS, ВИДІЛЕНИХ ВІД ХВОРИХ З ВПЕРШЕ ДІАГНОСТОВАНИМ ТУБЕРКУЛЬОЗОМ ЛЕГЕНЬ

The article presents the experience of whole-genome sequencing of M. tuberculosis (MBT) strains. Materials and methods. 8 MBT isolates were tested. The strains were obtained from patients with newly detected pulmonary tuberculosis (NDPTB). Critical concentrations of anti-mycobacterial drugs (AMBD): H; R; E; Z; Lfx; Mfx; Am; cm; Lzd; Bdq; Dlm and Cfz were determined using the BACTEC MGIT system. Sequencing of MBT strains was performed on Illumina MiSeq equipment at the laboratory of microbiology and biochemistry of the National Institute of phthisiology and pulmonology (NIPP). The obtained whole-genome sequences of MBT strains were deposited in fastq files and analyzed using the PhyResSE program (https://bioinf.fz-borstel.de/mchips/phyresse/) with the bioanalytical solution package. The panel of this package includes gene targets involved in the development of MBT to AMBP. Phylogenetic information and a certain spoligotype make it possible to obtain neutral single nucleotide polymorphisms. The genetic profile of the drug was performed according to the WHO mutation catalog (https://www.who.int/publications/i/item/9789240028173). The results. The dominant in Ukraine Beijing genotype was represented by 7 strains. The non-beijing group included 1 strain: Ural. 7 MBT strains belonged to the Beijing family, Lineage 2 phylogenetic line with the same spoligotype indices. The analysis of mutations according to the “Catalogue of mutations of the M. tuberculosis complex and their association with drug resistance”, WHO, 2021, allowed all detected mutations to be divided into categories - associated with resistance, associated with resistance - intermediate, not associated with resistance, with uncertain significance. Comparison of the resistance profile with the corresponding genotype showed that isolates, belonging to the Beijing genotype, usually carried MDR/pre-XDR mutations. Comparison of the resistance profile with the corresponding genotype showed that isolates belonging to the Beijing genotype usually carried MDR/pre-XDR mutations. All MBT strains with resistance to R had mutations found in the rpoB gene. The canonical amino acid substitution of rpoB S450L, associated with a high level of resistance to R with a non-significant decrease in the viability of the strains, was identified in 6 isolates. Another clinically significant mutation in rpoB (S435Y) was found in 2 cases and only in representatives of the Beijing family. Mutations of the categories: not associated with resistance (rpoC (E1092D)) and of uncertain significance: rpoC (I491V), rpoB (H674N), rpoB (V496A), were detected only in combination with “strictly associated” positions in phenotypic-resistant isolates of the genotype Beijing. The canonical single amino acid substitution katG S315T was detected in all isolates selected for sequencing. All 8 strains resistant to H showed mutations in genes associated with resistance to the drug. Mutations in the key Bdq resistance genes - atpE and mmpR - were not detected in any genome. In 1 strain of MBT, a C154R mutation associated with Lzd resistance was detected in the rplC gene. Similarly, in 1 strain of MBT, a mutation in ddn (D30S0) was detected with an as yet undetermined significance of association with resistance to Dlm. Conclusions. The study provided information on the genetic diversity of modern MBT isolates in TB patients. The predominance of the Beijing genotype testified to the stable population structure of the TB pathogen and the preservation of trends of high levels of the spread of TB forms of infection on the territory of the country. The combination of phenotype and molecular genetic data made it possible to give a more complete assessment of DR isolates. A large share of MDR was caused by mutations that did not have a negative effect on the viability and transmissibility of the pathogen, indicating a large epidemic reservoir of MBT strains with MDR/pre-XDR. The highly transmissible strains of Beijing carried mostly single uncombined R and H mutations, which were associated with a high level of resistance. Key words: genetic diagnostics, sequencing, pulmonary tuberculosis.

The development of variation-based rifampicin resistance in Staphylococcus aureus deciphered through genomic and transcriptomic study.

Rifampicin (RIF) resistance imposes a challenge on the antimicrobial treatment of pathogen infections. Figuring out the development mechanism of RIF resistance is critical to improving antimicrobial therapy strategy in clinics and biological treatment strategy of RIF polluted sewage in environmental engineering. The RIF resistance development of Staphylococcus aureus (S. aureus) with exposure to RIF at sub-inhibitory concentrations was comprehensively investigated via genomic and transcriptomic approaches in this study. RIF minimal inhibitory concentration (MIC) for S. aureus rapidly increased from 0.032 to 256mg/L. Membrane permeability decrease, biofilm formation enhancement, and ROS production increase associated with RIF resistance were observed in RIF-induced strains. Through comparative genomic analysis, mutations in rpoB and rpoC were considered to be associated with RIF resistance in S. aureus mutants. Pan-genome-wide single-nucleotide variant analysis indicated that mutations at rpoB-1412, rpoB-1451, and rpoB-1457 were prevalent in 13849 public genomes of S. aureus, while mutations at rpoB-2256, and rpoC-3092 were first discovered in this study. The panorama of adaptative alteration of cellular physiological processes was observed via transcriptomic analysis. The oxidation pressure responses, metabolism, transporters, virulence factors, and multiple steps of DNA and RNA machinery were found to be perturbed by RIF in S. aureus.

Rifampicin exerts anti-mucoviscous activity against hypervirulent Klebsiella pneumoniae via binding to the RNA polymerase β subunit

In hypervirulent Klebsiella pneumoniae (hvKP), the hypermucoviscous capsule is known to be a major virulence determinant. We previously discovered that rifampicin (RFP), a bactericidal drug that binds to and inhibits the β subunit of RNA polymerase (RpoB), elicits anti-mucoviscous activity against hvKP by suppressing rmpA, a regulator of capsule production. Here, we aimed to determine whether RFP exerts this effect at sub-growth-inhibitory concentrations via its binding to RpoB. Five spontaneous RFP-resistant mutants (R1-R5) were prepared from an hvKP clinical isolate and subjected to whole genome sequencing and mucoviscosity analyses. Subsequently, a two-step allelic exchange procedure was used to create a rpoB mutant R6 and revertants with wild-type rpoB from R1-R5 (named R1'-R5'). Transcription levels of rmpA and the capsular polysaccharide polymerase gene magA and capsule thickness of R1-R5 and R1'-R5' grown without or with RFP were evaluated by quantitative reverse transcription polymerase chain reaction and microscopic observation using India ink staining. R1-R5 all had non-synonymous point mutations in rpoB and were highly resistant to the bactericidal effects and anti-mucoviscous activity of RFP. While the properties of R6 were similar to those of R1-R5, the responses of R1'-R5' to RFP were identical to those of the wild type. rmpA and magA transcription levels and capsule thickness correlated well with the mucoviscosity levels. RFP exerts anti-mucoviscous activity by binding to RpoB. The mechanism of how this causes rmpA suppression remains to be explored.

Strain diversity and gene mutations associated with presumptive multidrug-resistant Mycobacterium tuberculosis complex isolates in Northwest Ethiopia

In this study, we assessed the genetic diversity and gene mutations that confer resistance to rifampicin (RIF), isoniazid (INH), fluoroquinolone (FQ), and second-line injectable (SLI) drugs in RIF-resistant (RR)/multidrug-resistant tuberculosis (MDR-TB) isolates in Northwest Ethiopia. Spoligotyping was used to assign isolates to TB lineages (Ls), and Hain line probe assays were used to detect resistance to RIF, INH, and FQs, and SLIs. Among 130 analyzed strains, 68.5% were RR, and four major Mycobacterium tuberculosis complex lineages (L1, L3, L4, and L7) were identified with a predominance of the Euro-American L4 (72, 54.7%), while L7 genotypes were less common (3, 2.3%). Overall, the L4-T3-ETH (41, 32.0%), L3-CAS1-Delhi (29, 22.7%), and L3-CAS1-Killi (19, 14.8%) families were most common. Line probe analysis showed that among rpoB mutants, 65.2% were S450L, while 87.8% of katG mutants were S315T. Only three isolates showed mutation (c-15t) at the inhA gene, and no double mutation with katG and inhA genes was found. Six strains, two each of L1, L3, and L4, were resistant to FQs, having gyrA mutations (D94G, S91P), of which three isolates had additional resistance to SLI (rrs A1401G or C1402T mutations) including one isolate with low-level kanamycin (KAN) resistance. This study showed a predominance of L4-T3-ETH, L3-CAS1-Delhi, and L3-CAS1-Killi families, with a high rate of rpoB_S450L and katG_S315T mutations and a low proportion of gyrA and rrs mutations. L7 was less frequently observed in this study. Further investigations are, therefore, needed to understand L7 and other lineages with undefined mutations.

Genotypic Distribution and the Epidemiology of Multidrug Resistant Tuberculosis in Upper Northern Thailand.

The epidemiology and genotypes of multidrug-resistant tuberculosis (MDR-TB), a global public health threat, remain limited. The genotypic distribution and factors associated with MDR-TB in upper northern Thailand between 2015 and 2019 were investigated. The DNA sequencing of rpoB, katG, and inhA promoter of 51 multidrug-resistant Mycobacterium tuberculosis isolates revealed nine patterns of the rpoB gene mutation distributed in seven provinces. The S531L mutation was the most common mutation in all provinces. The rpoB mutation in Chiang Rai, Chiang Mai, and Lampang was highly diverse compared to other areas. Here, the mutation profiles that have yet to be reported in northern Thailand (H526P, Q513P, and H526C) were detected in Chiang Rai province. The S315T katG mutation was the most common genotype associated with INH resistance, especially in Chiang Mai and Lampang. Further analysis of data from 110 TB patients (42 MDR-TB and 68 drug-susceptible TB) revealed that <60 years of age was a significant factor associated with MDR-TB (OR = 0.316, 95% CI 0.128-0.784, p = 0.011) and ≥60 years of age was a significant factor associated with the S315T katG-mutation (OR = 8.867, 95% CI 0.981-80.177, p = 0.047). This study highlighted the necessity for continuous surveillance and risk factor monitoring for effective control of MDR-TB.

RpoB Gene Mutation Characteristics of Rifampicin-resistant Tuberculosis in Anqing, China

Background: Rifampicin resistant tuberculosis is a serious problem faced by tuberculosis control in China, and rapid detection of rifampicin resistance is urgently needed. Objectives: This study aimed to describe the molecular characteristics and frequency of RNA polymerase β subunit (rpoB) gene mutations in rifampicin-resistant tuberculosis (RR-TB) in the Anqing area. Methods: The rpoB gene fragment was amplified by polymerase chain reaction (PCR), and all isolates were sequenced for mutations in the rpoB gene. The mutations were obtained by comparing the sequencing results with the MUBII database. In addition, logistic regression was used to analyze the relationship between rpoB mutations and rifampicin (RIF) resistance. Results: There were 152 males and 42 females in this study, and the mean age was 56.60 ± 17.91 years. Mutations in the rpoB gene were a risk factor for rifampicin resistance (β = 5.271, P < 0.001 OR = 195.192). Among the 19 RR-TB strains, 16 (84.21%) had mutations in the ropB gene, and three (1.71%) of 175 rifampicin-sensitive strains were mutated. The mutation sites of five strains (31.58%) were at the codon 526 and five strains (31.58%) at the codon 531. However, there were two strains at the codon 513 and two strains at the codon 533 (15.79%), and two strains (10.53%) were double mutations. Conclusions: The mutation characteristics of the rpoB gene in the Anqing area are complex, and rpoB mutation detection can be used as an indicator to screen drug resistance of RIF.

Detection and factors associated with tuberculosis and rifampicin resistance among presumptive patients at the Thailand-Myanmar border.

Tuberculosis (TB) continues to be a major public health problem in Thailand and many countries. Endemic TB and outbreaks of TB drug resistance in the borderlands are particularly important. The Thailand-Myanmar border has extensive cross-border travel that may accelerate TB's spread. This cross-sectional study aimed to determine the frequency and factors associated with TB, and rifampicinresistant TB (RR-TB) among presumptive tuberculosis patients in Mae Sot Hospital. Sputum was processed by microscopic examination and Xpert MTB/RIF assay. Laboratory results and socio-demographic characteristics were collected and analyzed. Univariate and multivariate analyses were performed to assess the association of the risk factors with TB and RR-TB. The significant variables at p-values < 0.05 in univariate analysis were selected for multivariate analysis. Of 365 presumptive patients enrolled, 244 (66.85%) were males and 199 (54.52%) were Burmese. Of these, 314 (86.03%) were registered as new cases and 183 (50.14%) worked as laborers. Sputum microscopy was positive in 132 (36.16%) cases. Based on Xpert MTB/RIF, the frequency of TB was 136 (37.26%) and RR-TB was 15 (11.03%). TB was more common in males than females. The majority of the cases belonged to the 26-50-year-old age group and migrant workers. In RR-TB detection, the rpoB mutations covered by probe E were the most frequently observed. Sequencing showed that the most highly mutated codon was codon 531 and Ser531Thr was the most common mutation. For risk factor analysis, working as laborers was significantly (p-value < 0.05) associated with TB (aOR 2.83; 95% CI 1.43-5.63) and previously treated cases were significantly associated with RR-TB (aOR 12.33; 95% CI 2.29-66.49). The high frequency of TB and RR-TB in migrants highlights the problem and factors associated with TB at the border and the need for efforts in TB control programs in this setting.

Analysis of molecular mechanisms of drug resistance of Mycobacterium tuberculosis in patients with pulmonary tuberculosis and its pharmacoeconomics

Purpose: To investigate the molecular mechanisms of drug resistance of Mycobacterium tuberculosis in patients with pulmonary tuberculosis and its pharmacoeconomics. &#x0D; Methods: Data pertaining to patients with primary tuberculosis treated in the First Affiliated Hospital of Zhaoqing Medical College, Zhaoqing, China from January 2020 to June 2021 were retrospectively analyzed. Sputum specimens were collected from all eligible patients, and 151 uncontaminated specimens with good bacteriophage activity were screened. &#x0D; Results: A total of 107 Mycobacterium tuberculosis strains were isolated from the 151 specimens, 31 of which strains were resistant to varying degrees to rifampicin, isoniazid, streptomycin, and ethambutol with an overall resistance of 28.97 %. There were 16 strains with rpoB mutation, 22 strains with katG mutation, and 8 strains with inhA mutation. The difference in the sputum negative rate, lesion absorption rate, and tuberculosis cavity closure rate, and total medical cost between the two group were not statistically significant (p &gt; 0.05). The incidence of adverse reactions in the FDC group was significantly lower than that in the blister pack group (p &lt; 0.05). &#x0D; Conclusion: The total resistance of Mycobacterium tuberculosis in primary tuberculosis patients remains at a high level, and the development of resistance is associated with mutations in rpoB, katG, and inhA genes. FDC regimen provides more pharmacoeconomic and therapeutic benefits than blister pack regimen.

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

To assess the relationship between the variant rpoB mutations and the degree of rifampin (RIF)/rifabutin (RFB) resistance in Mycobacterium tuberculosis (M. tuberculosis). We analyzed the whole rpoB gene in 177 M. tuberculosis clinical isolates and quantified their minimum inhibitory concentrations (MICs) using microplate-based assays. 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. 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.