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HM Adnan Hameed1– 4*, Cuiting Fang1– 4*, Zhiyong Liu,1– 3 Yanan Ju,1– 3 Xingli Han,1– 4 Yamin Gao,1– 4 Shuai Wang,1– 3,5 Gift Chiwala,1– 4 Yaoju Tan,6 Ping Guan,6 Jinxing Hu,6 Xiaoli Xiong,1– 3 Jiacong Peng,3,7 Yongping Lin,3,7 Muzammal Hussain,4 Nanshan Zhong,3,7,8 Dmitry A Maslov,9 Gregory M Cook,10,11 Jianxiong Liu,6 Tianyu Zhang1– 4 1State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health (GIBH), Chinese Academy of Sciences (CAS), Guangzhou, People’s Republic of China; 2China-New Zealand Joint Laboratory of Biomedicine and Health, Guangzhou Institutes of Biomedicine and Health (GIBH), Chinese Academy of Sciences (CAS), Guangzhou, People’s Republic of China; 3Guangdong-Hong Kong-Macau Joint Laboratory of Respiratory Infectious Diseases, Guangzhou, People’s Republic of China; 4University of Chinese Academy of Sciences (UCAS), Beijing, People’s Republic of China; 5National Clinical Research Center for Infectious Diseases, Guangdong Provincial Clinical Research Center for Tuberculosis, Shenzhen Third People’s Hospital, Shenzhen, People’s Republic of China; 6State Key Laboratory of Respiratory Disease, Guangzhou Chest Hospital, Guangzhou, People’s Republic of China; 7State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, People’s Republic of China; 8Guangzhou National Laboratory, Guangzhou, People’s Republic of China; 9Laboratory of Bacterial Genetics, Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia; 10Department of Microbiology and Immunology, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand; 11Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Auckland, New Zealand*These authors contributed equally to this workCorrespondence: Tianyu Zhang, Guangzhou Institutes of Biomedicine and Health (GIBH), Chinese Academy of Sciences (CAS), Room A207, 190 Kaiyuan Ave, Science Park, Huangpu District, Guangzhou, 510530, People’s Republic of China, Tel +86-2032015270, Email zhang_tianyu@gibh.ac.cn Jianxiong Liu, Guangzhou Chest Hospital, 62 Hengzhigang Road, Yuexiu District, Guangzhou, People’s Republic of China, Tel +86-2083595977, Email ljxer64@qq.comObjective: Rifampicin (RIF)-resistance, a surrogate marker for multidrug-resistant tuberculosis (TB), is mediated by mutations in the rpoB gene. We aimed to investigate the prevalence of mutations pattern in the entire rpoB gene of Mycobacterium tuberculosis clinical isolates and their association with resistance level to RIF.Methods: Among 465 clinical isolates collected from the Guangzhou Chest Hospital, drug-susceptibility of 175 confirmed Mtb strains was performed via the proportion method and Bactec MGIT 960 system. GeneXpert MTB/RIF and sanger sequencing facilitated in genetic characterization, whereas the MICs of RIF were determined by Alamar blue assay.Results: We found 150/175 (85.71%) RIF-resistant strains (MIC: 4 to > 64 μg/mL) of which 57 were MDR and 81 pre-XDR TB. Genetic analysis identified 17 types of mutations 146/150 (97.33%) within RRDR (codons 426– 452) of rpoB, mainly at L430 (P), D435 (V, E, G, N), H445 (N, D, Y, R, L), S450 (L, F) and L452 (P). D435V 12/146 (8.2%), H445N 16/146 (10.9%), and S450L 70/146 (47.94%) were the most frequently encountered mutations. Mutations Q432K, M434V, and N437D are rarely identified in RRDR. Deletions at (1284– 1289 CCAGCT), (1295– 1303 AATTCATGG), and insertion at (1300– 1302 TTC) were detected within RRDR of three RIFR strains for the first time. We detected 47 types of mutations and insertions/deletions (indels) outside the RRDR. Four RIFR strains were detected with only novel mutations/indels outside the RRDR. Two of the four had (K274Q + C897 del + I491M) and (A286V + L494P), respectively. The other two had (G1687del + P454L) and (TT1835-6 ins + I491L) individually. Compared with phenotypic characterization, diagnostic sensitivities of GeneXpert MTB/RIF and sequencing analysis were 95.33% (143/150), and 100% (150/150) respectively.Conclusion: Our findings underscore the key role of RRDR mutations and the contribution of non-RRDR mutations in rapid molecular diagnosis of RIFR clinical isolates. Such insights will support early detection of disease and recommend the appropriate anti-TB regimens in high-burden settings.Keywords: rifampicin-resistance, Mycobacterium tuberculosis, rpoB, GeneXpert MTB/RIF, resistance-determining region

Background & objectives: Drug-resistant tuberculosis (TB) jeopardizes the treatment process with poor outcomes. Efflux pumps (EPs) belonging to the ABC transporter family in Mycobacterium tuberculosis confer resistance to rifampicin (RMP) besides genetic mutations thus serving as a target for a potential adjunct therapeutic inhibitory molecule. Rv1218c is one such pump that was previously reported to be active in multidrug-resistant TB clinical isolates. Methods: In this study, the inhibition potential of Rv1218c-EP was tested on 8 molecules that were shortlisted by in silico methods. These molecules were subjected to the minimum inhibitory concentration (MIC) determination, checkerboard drug combination assay, ethidium bromide-DNA binding assay, and in vitro and ex vivo cytotoxicity assay. Results: Based on the outcome of the study, two molecules dodecanoic acid (DA) and palmitic acid (PA) were found to be potential enough to decrease the MIC of RMP by 8 to 1000 folds against multidrugresistant clinical isolates and Rv1218c expressing recombinant Mycobacterium smegmatis. Interpretation & conclusions: These molecules were also found to reduce the time taken by RMP to kill these drug-resistant Mycobacteria to 48 h, unlike control isolates that survived more than 240 h of RMP exposure. The functional concentration of both molecules was non-toxic to the epithelial and blood mononuclear cells. With further comprehensive scientific validation, PA and DA could be recommended as adjunct therapeutic molecules with first-line anti-TB drugs to treat drug-resistant TB. [ABSTRACT FROM AUTHOR]

Jidong Guo,1,* Rongmei Liu,1,* Jin Shi,1,* Fengmin Huo,2 Yuanyuan Shang,3 Fen Wang,2 Mengqiu Gao,1 Shanshan Li3 1Department of Tuberculosis, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis & Thoracic Tumor Research Institute, Beijing, 101149, People’s Republic of China; 2National Clinical Laboratory on Tuberculosis, Beijing Key laboratory on Drug-resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis & Thoracic Tumor Research Institute, Beijing, 101149, People’s Republic of China; 3Department of Bacteriology and Immunology, Beijing Key Laboratory on Drug-Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis & Thoracic Tumor Research Institute, Beijing, 101149, People’s Republic of China*These authors contributed equally to this workCorrespondence: Shanshan LiDepartment of Bacteriology and Immunology, Beijing Key Laboratory on Drug-Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis & Thoracic Tumor Research Institute, No. 9, Beiguan Street, Tongzhou District, Beijing, 101149, People’s Republic of ChinaTel +86-10-8950 9368Fax +86-10-8950 9366Email Lss9011@126.comMengqiu GaoDepartment of Tuberculosis, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis & Thoracic Tumor Research Institute, No. 9, Beiguan Street, Tongzhou District, Beijing, 101149, People’s Republic of ChinaTel +86-10-8950 9322Fax +86-10-8950 9322Email gaomqwdm@aliyun.comBackground: Early and accurate diagnosis of rifampicin (RIF)-resistant Mycobacterium tuberculosis (MTB) is essential for controlling community spread of drug-resistant tuberculosis (TB). In order to discover mutations residing outside the rifampicin resistance-determining region (RRDR) of the MTB rpoB gene, we conducted this retrospective study.Methods: We retrospectively screened patient records to obtain Xpert MTB/RIF assay results for patients who received care at the Beijing Chest Hospital from 2016 to 2019 in order to identify subjects who met study selection criteria. Stored frozen patient isolates were cultured, harvested, and then subjected to drug susceptibility testing. Concurrently, entire rpoB gene DNA of each isolate was amplified and then sequenced to reveal rpoB mutations.Results: Overall, 104 RIF-susceptible tuberculosis patients who were tested using the Xpert MTB/RIF assay also had poor first-line regimen treatment responses. Isolates obtained from these cases included 101 MTB isolates that possessed wild-type rpoB allelic profiles, as demonstrated using Sanger sequencing. However, sequences from the other three isolates confirmed that rpoB of one isolate harbored a mutation encoding the amino acid substitution Ile491Phe and that rpoB genes of two isolates contained a mutation encoding the amino acid substitution Ser450Leu.Conclusion: Our data demonstrated that mutations found outside the RRDR of MTB rpoB are rare in Beijing, China, indicating that World Health Organization-approved molecular diagnostics are generally suitable for diagnosing RIF resistance.Keywords: tuberculosis, rifampicin-resistance, rpoB, diagnosis

Mohammed Hasen Bedaso,1 Falaho Sani Kalil2 1Public Health Emergency Management, East Bale Zonal Health Department, Gindhir, Ethiopia; 2Negelle Borena Health Science College, Negelle Borena, Oromia Regional State, EthiopiaCorrespondence: Mohammed Hasen BedasoPublic Health Emergency Management, East Bale Zonal Health Department, P.O. Box: 61, Gindhir, EthiopiaTel +251 923728803Email direhasen@gmail.comPurpose: Multidrug-resistant tuberculosis threatens global tuberculosis care and prevention and remains a major public health concern in many countries. In 2016, there were an estimated 490,000 cases of MDR and 110,000 more cases resistant to rifampicin (RR TB). Ethiopia is among the highest MDR TB burden countries according to the WHO. This study aims to describe the magnitude, trends, and geographical distribution of the drug-resistant TB in Bale Zone during study period.Materials and Methods: A descriptive study was conducted. We reviewed secondary data of MDR and RR TB cases from July 2014 to June 2018. Data were extracted from the Bale zone health management information system database, checked for completeness, and then analyzed for trends over time.Results: A total of 43 cases (67.4% female) of drug-resistant TB were reviewed, with 30.2% MDR and 69.8% RR TB. The prevalence of drug-resistant tuberculosis cases declined from 0.81% to 0.62% (trend χ2=2.18; P=0.14) during study period. Among drug-resistant TB cases, RR TB increased from 52.6% to 81% (trend χ2=6.5; P=0.01).Conclusion: Drug-resistant TB decreased over the period studied, although the trend did not reach statistical significance. These trends may reflect the efficacy of TB control programs to reduce drug-resistant TB transmission, as well as improved RR TB detection due to increased use of molecular diagnostic platforms like GeneXpert MTB/RIF.Keywords: tuberculosis, multidrug-resistance, rifampicin-resistance

Abstract Background Drug-resistant tuberculosis (DR-TB), including rifampicin-resistant tuberculosis (RR-TB) and multidrug-resistant tuberculosis (MDR-TB, or RR-TB with additional isoniazid resistance), presents challenges to TB control. In Uganda, the GeneXpert test provides point-of-care testing for TB and rifampicin resistance. Patients identified with RR-TB receive culture-based drug susceptibility testing (DST) to identify additional resistance, if any. There are few data on the epidemiological profiles of current DR-TB patients in Uganda. We described patients with RR-TB in Uganda and assessed the trends of RR-TB to inform TB control interventions. Methods We identified patients with RR-TB whose samples were referred for culture and DST during 2014–2018 from routinely-generated laboratory surveillance data at the Uganda National Tuberculosis Reference Laboratory. Data on patient demographics and drug sensitivity profile of Mycobacterium tuberculosis isolates were abstracted. Population data were obtained from the Uganda Bureau of Statistics to calculate incidence. Descriptive epidemiology was performed, and logistic regression used to assess trends. Results We identified 1474 patients whose mean age was 36 ± 17 years. Overall incidence was 3.8/100,000 population. Males were more affected by RR-TB than females (4.9 vs. 2.7/100,000, p ≤ 0.01). Geographically, Northern Uganda was the most affected region (IR = 6.9/100,000) followed by the Central region (IR = 5.01/100,000). The overall population incidence of RR-TB increased by 20% over the evaluation period (OR = 1.2; 95% CI 1.15–1.23); RR-TB in new TB cases increased by 35% (OR = 1.35; 95% CI 1.3–1.4) and by 7% in previously-treated cases (OR = 1.07; 95% CI 1.0–1.1). Of the 1474 patients with RR-TB, 923 (63%) were culture-positive of whom 670 (72%) had full DST available. Based on the DST results, 522/670 (78%) had MDR-TB. Conclusion Between 2014 and 2018, the incidence of RR-TB increased especially among newly-diagnosed TB patients. We recommend intensified efforts and screening for early diagnosis especially among previously treated patients. Mechanisms should be in put to ensure that all patients with RR-TB obtain DST.

Abstract Background Mortality due to tuberculosis (TB) has increased due to the development of drug resistance, the mechanisms of which have not been fully elucidated. Our research group identified a low expression of lipF gene in Mycobacterium tuberculosis clinical isolates with drug resistance. The aim of this work was to evaluate the effect of lipase F (LipF) expression on mycobacterial drug resistance. Results The effects of expressing lipF from Mycobacterium tuberculosis in Mycobacterium smegmatis on resistance to antituberculosis drugs were determined with resazurin microtiter assay plate and growth kinetics. Functionality of ectopic LipF was confirmed. LipF expression reduced the rifampicin (RIF) and streptomycin (STR) minimum inhibitory concentration (MIC) from 3.12 μg/mL to 1.6 μg/mL and 0.25 μg/mL to 0.06 μg/mL respectively, moreover a reduced M. smegmatis growth in presence of RIF and STR compared with that of a control strain without LipF expression (p

Abstract Background Xpert® MTB/RIF assay is currently used in Ethiopia for the rapid diagnosis of Mycobacterium tuberculosis (MTB) and mutations that confer Rifampicin resistance. Rifampicin resistance is determined based on any mutation in the 81 bp of rpoB gene using five overlapping probes represented as Probe A (codons 507–511), Probe B (codons 512–518), Probe C (codons 518–523), Probe D (codons 523–529) and Probe E (codons 529–533). In this review, we assessed the frequency of missed probe types for Rifampicin Resistance results. Methods Data were reviewed from specimens received and tested using Xpert® MTB/RIF assay at Ethiopian National Tuberculosis Reference Laboratory, in Addis Ababa from 15 July 2016 to 31 December 2018 retrospectively. All archived data were reviewed carefully to describe missed probe types and the quantity of DNA in the sample. Results A total of 100 specimens were reported as MTB Detected Rifampicin Resistance Detected by Xpert® MTB/RIF assay. More than half (55%) of these results were reported from male patients. The median age was 28.0 years (5 months to 88 years). Majorities (62%) of the cases were detected from sputum. Among the total of 38 extrapulmonary samples, lymph node aspirates were accounted for 50% (19/38). The most common mutations (81.0%) were found in the Probe E region followed by Probe D (10.0%), and Probe B (3.0%). Mutations in Probe A and Probe C regions were not observed. However, six (6.0%) Rifampicin resistance cases were found without any missed probe type. The delta Ct max is ≥4.3. No specimen yielded Rifampicin resistance associated with more than one probe failure or mutation combinations. Conclusion Mutations associated with Probe E (codons 529–533) region were identified as the commonest rpoB gene mutations. The Rifampicin resistance results found without any identified missing probe needs further study. The lower DNA amount was observed in extrapulmonary specimens compared with sputum.

Background: Drug-resistant tuberculosis (DR-TB), including rifampicin-resistant tuberculosis (RR-TB) and multidrug-resistant tuberculosis (MDR-TB, or RR-TB with additional isoniazid resistance), presents challenges to TB control. In Uganda, the GeneXpert test provides point-of-care testing for TB and rifampicin resistance. Patients identified with RR-TB receive culture-based drug susceptibility testing (DST) to identify additional resistance, if any. There are few data on the epidemiological profiles of current DR-TB patients in Uganda. We described patients with RR-TB in Uganda and assessed the trends of RR-TB to inform TB control interventions. Methods: We identified patients with RR-TB whose samples were referred for culture and DST during 2014–2018 from routinely-generated laboratory surveillance data at the Uganda National Tuberculosis Reference Laboratory. Data on patient demographics and drug sensitivity profile of Mycobacterium tuberculosis isolates were abstracted. Population data were obtained from the Uganda Bureau of Statistics to calculate incidence. Descriptive epidemiology was performed, and logistic regression used to assess trends. Results: We identified 1474 patients whose mean age was 36 ± 17 years. Overall incidence was 3.8/100,000 population. Males were more affected by RR-TB than females (4.9 vs. 2.7/100,000, p ≤ 0.01). Geographically, Northern Uganda was the most affected region (IR = 6.9/100,000) followed by the Central region (IR = 5.01/100,000). The overall population incidence of RR-TB increased by 20% over the evaluation period (OR = 1.2; 95% CI 1.15–1.23); RR-TB in new TB cases increased by 35% (OR = 1.35; 95% CI 1.3–1.4) and by 7% in previously-treated cases (OR = 1.07; 95% CI 1.0–1.1). Of the 1474 patients with RR-TB, 923 (63%) were culture-positive of whom 670 (72%) had full DST available. Based on the DST results, 522/670 (78%) had MDR-TB. Conclusion: Between 2014 and 2018, the incidence of RR-TB increased especially among newly-diagnosed TB patients. We recommend intensified efforts and screening for early diagnosis especially among previously treated patients. Mechanisms should be in put to ensure that all patients with RR-TB obtain DST. [ABSTRACT FROM AUTHOR]

Background: Mortality due to tuberculosis (TB) has increased due to the development of drug resistance, the mechanisms of which have not been fully elucidated. Our research group identified a low expression of lipF gene in Mycobacterium tuberculosis clinical isolates with drug resistance. The aim of this work was to evaluate the effect of lipase F (LipF) expression on mycobacterial drug resistance. Results: The effects of expressing lipF from Mycobacterium tuberculosis in Mycobacterium smegmatis on resistance to antituberculosis drugs were determined with resazurin microtiter assay plate and growth kinetics. Functionality of ectopic LipF was confirmed. LipF expression reduced the rifampicin (RIF) and streptomycin (STR) minimum inhibitory concentration (MIC) from 3.12 μg/mL to 1.6 μg/mL and 0.25 μg/mL to 0.06 μg/mL respectively, moreover a reduced M. smegmatis growth in presence of RIF and STR compared with that of a control strain without LipF expression (p < 0.05 and p < 0.01) was shown. Conclusions: LipF expression was associated with increased RIF and STR sensitivity in mycobacteria. Reduced LipF expression may contribute to the development of RIF and STR resistance in Mycobacterium species. Our findings provide information pertinent to understanding mycobacterial drug resistance mechanisms. [ABSTRACT FROM AUTHOR]

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Background: Xpert® MTB/RIF assay is currently used in Ethiopia for the rapid diagnosis of Mycobacterium tuberculosis (MTB) and mutations that confer Rifampicin resistance. Rifampicin resistance is determined based on any mutation in the 81 bp of rpoB gene using five overlapping probes represented as Probe A (codons 507-511), Probe B (codons 512-518), Probe C (codons 518-523), Probe D (codons 523-529) and Probe E (codons 529-533). In this review, we assessed the frequency of missed probe types for Rifampicin Resistance results.Methods: Data were reviewed from specimens received and tested using Xpert® MTB/RIF assay at Ethiopian National Tuberculosis Reference Laboratory, in Addis Ababa from 15 July 2016 to 31 December 2018 retrospectively. All archived data were reviewed carefully to describe missed probe types and the quantity of DNA in the sample.Results: A total of 100 specimens were reported as MTB Detected Rifampicin Resistance Detected by Xpert® MTB/RIF assay. More than half (55%) of these results were reported from male patients. The median age was 28.0 years (5 months to 88 years). Majorities (62%) of the cases were detected from sputum. Among the total of 38 extrapulmonary samples, lymph node aspirates were accounted for 50% (19/38). The most common mutations (81.0%) were found in the Probe E region followed by Probe D (10.0%), and Probe B (3.0%). Mutations in Probe A and Probe C regions were not observed. However, six (6.0%) Rifampicin resistance cases were found without any missed probe type. The delta Ct max is ≥4.3. No specimen yielded Rifampicin resistance associated with more than one probe failure or mutation combinations.Conclusion: Mutations associated with Probe E (codons 529-533) region were identified as the commonest rpoB gene mutations. The Rifampicin resistance results found without any identified missing probe needs further study. The lower DNA amount was observed in extrapulmonary specimens compared with sputum. [ABSTRACT FROM AUTHOR]

Following its introduction in 1967, rifampicin has become a mainstay of therapy in the treatment of tuberculosis, leprosy and many other widespread diseases. Its potent antibacterial activity is due to specific inhibition of bacterial RNA polymerase. However, resistance to rifampicin was reported shortly after its introduction in the medical practice. Studies in the model organism Escherichia coli helped to define the molecular mechanism of rifampicin-resistance demonstrating that resistance is mostly due to chromosomal mutations in rpoB gene encoding the RNA polymerase β chain. These studies also revealed the amazing potential of the molecular genetics to elucidate the structure–function relationships in bacterial RNA polymerase. The scope of this paper is to illustrate how rifampicin-resistance has been recently exploited to better understand the regulatory mechanisms that control bacterial cell physiology and virulence, and how this information has been used to maneuver, on a global scale, gene expression in bacteria of industrial interest. In particular, we reviewed recent literature regarding: (i) the effects of rpoB mutations conferring rifampicin-resistance on transcription dynamics, bacterial fitness, physiology, metabolism and virulence; (ii) the occurrence in nature of “mutant-type” or duplicated rifampicin-resistant RNA polymerases; and (iii) the RNA polymerase genetic engineering method for strain improvement and drug discovery. [ABSTRACT FROM AUTHOR]

Genotyping and molecular characterization of drug resistance mechanisms in Mycobacterium leprae enables disease transmission and drug resistance trends to be monitored. In the present study, we performed genome-wide analysis of Airaku-3, a multidrug-resistant strain with an unknown mechanism of resistance to rifampicin. We identified 12 unique non-synonymous single-nucleotide polymorphisms ( SNPs) including two in the transporter-encoding ctpC and ctpI genes. In addition, two SNPs were found that improve the resolution of SNP-based genotyping, particularly for Venezuelan and South East Asian strains of M. leprae. [ABSTRACT FROM AUTHOR]

Summary: Tuberculosis (TB) is still a major life-threatening infectious disease, within which especially the rise of multidrug resistant TB (MDR-TB) is currently worrying. This study focuses on mechanisms of development of rifampicin resistance, since rifampicin seems to play an important role in the development of MDR-TB. To provide further insight in rifampicin resistance, we performed a genome-wide transcriptional profile analysis for Mycobacterium tuberculosis (M. tuberculosis) using microarray technology and qRT-PCR analysis. We exposed a rifampicin-susceptible H37Rv wild type (H37Rv-WT) and a rifampicin-resistant progeny H37Rv strain with a H526Y mutation in the rpoB gene (H37Rv-H526Y) to several concentrations of rifampicin, to define the effect of rifampicin on the transcription profile. Our study showed that there are resistance-dependant differences in response between both M. tuberculosis strains. Gene clusters associated with efflux, transport and virulence were altered in the rifampicin-resistant H37Rv mutant compared to the rifampicin-susceptible H37Rv-WT strain after exposure to rifampicin. We conclude that the small gene cluster Rv0559c–Rv0560c in the H37Rv-H526Y strain was remarkably up-regulated in the microarray analysis and qRT-PCR results and appeared to be dependent on rifampicin concentration and time of exposure. Therefore this study suggests that Rv0559c and Rv0560c play a pivotal role in rifampicin resistance of M. tuberculosis. Further investigation of Rv0559c and Rv0560c is needed to reveal function and mechanism of both genes that were triggered upon rifampicin exposure. [ABSTRACT FROM AUTHOR]

Abstract: Sinefungin, a nucleoside antibiotic with potent antifungal, antiviral, and anti-trypanosome activities, has been a target for production enhancement in the past decades through medium optimization and strain improvement. For the purpose of introducing a more rational approach, we induced rpoB mutation in the producer strain, Streptomyces incarnatus NRRL 8089, by optimized UV-irradiation, and a resulting rifampicin-resistant strain rif-400 increased the sinefungin production by 7-fold. The growth and melanin production were obviously accelerated in the rifampicin-resistant high-producer mutant, while the morphological differentiation such as aerial mycelia and spiked-spore formation was retained. Molecular cloning and DNA sequencing identified a single mutation A1340G in the rpoB gene, which encodes the β-subunit of RNA polymerase, and the resulting amino acid substitution Asp447Gly corresponded to one of mutations that reportedly allowed the transcriptional up-regulation of actinorhodin production in S. coelicolor A3(2). Sinefungin production was further enhanced by resting cell system using the rpoB mutant strain in the presence of 10 mM l-Arg. d-Arg or l-ornithine did not enhance the sinefungin production, and >50 mM urea strongly suppressed the nucleoside antibiotic production, supporting the proposed biosynthetic mechanism by which urea is liberated from the guanidino-group-bearing intermediate that is produced by enzymatic condensation of l-Arg and ATP. [Copyright &y& Elsevier]

Mutations of the 81-bp-core region of rpoB gene associated with rifampicin resistance were studied in 169 clinical strains of Mycobacterium tuberculosis isolated from Spain between 1996 and 2001. The analysis identified 16 single base changes, one deletion, two insertions and five multiple mutations (with two or three codons implicated). Eight strains had no mutations, although they were resistant by the proportion method. The five single nucleotide changes with two or three codons implicated and one insertion have not been previously reported. The analysis of the RFLP-IS6110 pattern showed a great heterogeneity (159 individual patterns and four patterns with two or three strains) that suggests no geographical or temporal relations between the studied strains. The concordance rate of InnoLipa Rif-Tb probe with sequencing and phenotypic methods was 94.6%. The results indicate that the InnoLipa probe assay may be useful for rapid screening of the most frequent mutations found in rifampicin-resistant M. tuberculosis strains. [Copyright &y& Elsevier]

Mycobactenum marinum is a rare non-tuberculous mycobacterium known commonly to cause fish tank granuloma. It is found in aquatic surroundings and is common in healthy individuals. We report a case and review the literature in an immunocompromised HIV-1 individual with a first reported case of a rifampicin-resistant M. marinum organism in the literature. [ABSTRACT FROM AUTHOR]

Introduction: Tuberculosis (TB) is one of the leading causes of morbidity and mortality with a global mortality rate at two million deaths per year while one third of the world's population is infected with the TB bacillus., Objective: To determine the efficacy of the crude extracts of Erythrina abyssinica root bark on rifampicin-resistant TB., Methods: Crude extracts of root bark of Erythrina abyssinica, were screened against three strains of Mycobacterium tuberculosis including rifampicin-resistant TMC-331. Susceptibility tests used the disc diffusion method and were done on solid Middle brook 7H10, while the Minimum Inhibitory concentration (MICs) and minimum bactericidal concentration (MBCs) were determined by the Microtitre plate method using Middle brook 7H9 broth., Results: The total crude methanol extract showed activity against all the three strains of mycobacterium used, at 50mg/ml and diameters of zones of inhibition of up to 26 mm. Erythrina abyssinica total crude methanol extract showed the highest activity on the pan sensitive strain H37Rv (0.39±0.0 mg/ml) and the rifampicin resistant strain (TMC-331) (2.35±1.11 mg/ ml) and was also active on Mycobacterium avium (0.39±0.mg/ml. The values for isoniazid were 0.25µg/ml and 9.38µg/ml for H37Rv and TMC-331 respectively, while for rifampicin the MIC value was 0.25µg/ml for H37Rv but it was not active on TMC-331. Acute toxicity test gave an LD50 of 776.2mg/kg body weight while the phytochemical analysis showed the presence of alkaloids, tannins and flavones., Conclusion: The conclusion from the study was that Erythrina abbyssinica has antimycobacterial activity and reasonable safety that merits further research.