Academic literature on the topic 'M. tuberculosis - Rifampicin'

Background: The present study determined the incidence of rifampicin resistance M. tuberculosis among outpatients at the General Hospital Yauri, Kebbi State, Nigeria. Materials and Methods: The study is a cross-sectional study conducted from February 2018 to October 2019. Sociodemographic data were collected from hospital registration books. Rifampicin resistance M. tuberculosis was detected using GeneXpert Model GX-IV following manufacturers' instruction. Descriptive statistics and logistic regression were computed using SPSS version 20. The results were presented as odds ratios with associated 95% confidence intervals, and P-value at 0.05. Result: Of the 837 samples, 65.8% (551/837) were males, and 34.2% (286/837) females, 11.4% (95/837) HIV-seropositive. M. tuberculosis was detected in 15.5% (130/837), of which 116/130 (89.23%) were males and 14/130 (10.77%) females. M. tuberculosis¬-HIV coinfection was detected in 9.47% (9/95) of HIV positive. Rifampicin resistance was observed in 1.3% (11/837), 7.7% (10/130) in M. tuberculosis patients and 1.05% (1/94) in HIV seropositive. In logistic regression, the odds ratio for having a rifampicin-resistant M. tuberculosis was 0.49 (0.15-1.54) for > 30 years; taking <30 years as the reference value, 1.02 (1.00-1.03) for male; taking female as the reference value, and 0.78 (0.09-6.15) for HIV positive, taking negative as the reference value. Conclusion: This study reported the current incidence rate of rifampicin-resistant M. tuberculosis at the General Hospital Yelwa Yauri, Kebbi State, Nigeria, among presumptive TB patients. Patients diagnosed with rifampicin-resistant M. tuberculosis were predominantly male adults. Thus, frequent screening is vital for surveillance and reduces the risk of transmission and spread of M. tuberculosis infections.

ABSTRACT Resistance to rifampin (rifampicin), isoniazid, and streptomycin of 69 Mycobacterium tuberculosis isolates was analyzed by an in-house method based on mycobacteriophage D29 and a colorimetric micromethod. Both methods showed sensitivity and specificity values ranging from 93% to 100%. These simple methods offer an option for drug resistance assessment of M. tuberculosis.

Лекарственно-устойчивый туберкулез остается актуальной проблемой в здравоохранении Казахстана. Казахстан одна из стран в мире с высоким количеством мультирезистентного туберкулеза, характеризующегося устойчивостью к основным антибиотикам первого ряда – рифампицину и изониазиду. Цель данной работы: определить биологическое разнообразие и охарактеризовать мутации в rpoB гене рифампицин-устойчивых клинических изолятов M. tuberculosis из Казахстана. В данной работе от больных с легочным туберкулезом было собрано 115 клинических изолятов M. tuberculosis, устойчивых к противотуберкулезному препарату первого ряда - рифампицину. Генотипирование всех 115 рифампицин-резистентных изолятов было проведено методом MIRU-VNTRс использованием 15 локусов. ДНК-секвенирование RRDR региона rpoB гена всех 115 рифампицин-устойчивых образцов M. tuberculosis было выполнено на секвенаторе ABI 3730 DNA Analyzer (Applied Biosystems). Согласно результатам MIRU-VNTR генотипирования, 86,1% образцов среди рифампицин-устойчивых изолятов были отнесены к семейству Beijing M. tuberculosis. Анализ нуклеотидных последовательностей всех устойчивых к рифампицину изолятов показал преобладание мутации в 531 кодоне rpoB гена с аминокислотной заменой серина на лейцин Ser531Leu в 82,6% случаев. В данной работе семейство Beijing M. tuberculosis было ассоциировано с мутацией Ser531Leu в 531 кодоне гена (p<0.0001), а генотип LAM M. tuberculosis был связан с мутацией His526Leu в 526 кодоне гена (p<0.0001). Дәріге-төзімді туберкулез Қазақстанның денсаулық сақтау саласында өзекті мәселе болып қалуда. Қазақстан бірінші қатардағы негізгі антибиотиктерге – рифампицин мен изониазидке төзімділікпен сипатталатын мультирезистентті туберкулез көрсеткіші жоғары мемлекеттердің бірі. Зерттеу жұмысының мақсаты: Қазақстанда таралған рифампицинге-төзімді M. tuberculosis клиникалық изоляттарының rpoB генінде мутацияларды сипаттау және олардың биотүрлігін анықтау. Аталған зерттеу жұмысында өкпе туберкулезі науқастарынан туберкулезге қарсы бірінші қатардағы препарат – рифампицинге төзімді 115 M. tuberculosis клиникалық изоляттары жиналды. Барлық 115 рифампицинге резистентті изоляттар MIRU-VNTR әдісімен 15 локус бойынша генотиптелді. 115 рифампицинге-төзімді M. tuberculosisүлгілерінің rpoB генінің RRDR аймағын ДНҚ-секвенирлеу ABI 3730 DNA Analyzer (Applied Biosystems) секвенаторында өткізілді. MIRU-VNTR генотиптеу нәтижелеріне сәйкес, рифампицин-төзімді изоляттардың арасында 86,1% жағдайында үлгілер Beijing M. tuberculosisтұқымдасына жатты. Рифампицинге төзімділігі бар барлық изоляттардың нуклеотидтік тізбектерінің анализі 82,6% жағдайында rpoB генінің 531 кодонында сериннің лейцинге аминқышқылдық алмасуы бар Ser531Leu мутациясының басымдылығын көрсетті. Біздің жұмысымызда Beijing M. tuberculosis тұқымдасы геннің 531 кодонындағы Ser531Leu мутациясымен ассоциацияланды (p<0.0001), ал LAM M. tuberculosis тұқымдасы геннің 526 кодонындағы His526Leu мутациясымен байланысты (p<0.0001) болды. Drug-resistant tuberculosis remains one of major health problems in Healthcare system of Kazakhstan. Kazakhstan is one of countries with high quantity of multidrug-resistant tuberculosis chracterized by resistance to the main antibiotics used to cure tuberculosis – rifampicin and izoniazid. Aim of this study is to detect biodiversity and characterize mutations in rpoB gene of rifampicin-resistant M. tuberculosis clinical isolates from Kazakhstan. In this work, 115 M. tuberculosis clinical isolates resistant to the first line antibiotic – rifampicin were gathered from patients with pulmonary tuberculosis. Genotyping of 115 rifampicin-resistant isolates was performed by MIRU-VNTR method using 15 loci. DNA sequencing of RRDR region of rpoB gene of 115 rifampicin-resistant isolates of M. tuberculosis was done on ABI 3730 DNA Analyzer (Applied Biosystems). According to the MIRU-VNTR genotyping results, 86,1% isolates among the resistant samples were isolates of Beijing M. tuberculosis family. Analysis of nucleotide sequences of all resistant to rifampicin isolates showed prevalence of mutations at 531 codon of rpoB gene with the amino acid substitution of serine to leucine Ser531Leu in 82.6% of cases. In our study Beijing M. tuberculosis family was associated with the Ser531Leu mutation at 531 codon of the gene (p<0.0001), LAM M. tuberculosis family revealed association with the His526Leu mutation at 526 codon of the gene (p<0.0001)

Background: Drug resistant tuberculosis threatens global TB control and is a major public health problem in several countries and India has the highest tuberculosis in the world. The rifampicin resistance is a good predictor of multidrug resistant tuberculosis. The aim of this study was to determine the prevalence of rifampicin resistance M. tuberculosis and associated factor among presumptive tuberculosis patients in eastern Uttar Pradesh.Methods: A cross-sectional study was conducted from October 2016 to September 2017. Detection of M. tuberculosis and resistance to rifampicin was performed using Gene Xpert MTB/RIF assay. Data was collected using pre-structured questionnaire by face to face interview. The chi-square test was used to assess the statistical significance of each ratio, p<0.05 was considered significant.Results: Out of 510 patients, Mycobacterium tuberculosis was detected in 168 (32.9%). Out of these 168 patients, the prevalence of rifampicin resistance tuberculosis was 44 (26.1%). It was higher among male 38 (30.6%) than female 6 (13.6%). Regarding age distribution, maximum numbers of rifampicin resistance patients were in the age group of 20-40 years 36.7%. The prevalence of rifampicin resistance was 36 (27.6%) and 8 (21.0%) in pulmonary and extra-pulmonary respectively. Out of 44 rifampicin resistant cases, 39 (37.8%) were previously treated and 5 (7.6%) cases were treatment naïve patients. In this study, among presumptive DRTB cases, new 2 (11.7%), relapse 13 (39.3%), failure 23 (46.0%), loss to follow-up 1 (10.0%) and MDR contact 1 (20.0%) respectively were rifampicin resistant and one HIV seropositive patient was found to be rifampicin resistant.Conclusions: Previously treated cases were significantly associated with rifampicin resistance tuberculosis. The Gene Xpert is a good equipment for rapid detection and management of drug resistant tuberculosis for both pulmonary as well as extra-pulmonary tuberculosis.

The formulation of the antituberculosis drug rifampicin embedded into 20-30 nm nanoparticles from soy phosphatidylcholine and sodium oleate, is characterized by greater bioavailability as compared with free drug substance. In this study higher antituberculosis activity of this formulation was shown. Rifampicin in nanoparticles demonstrated more effective inhibition of M. tuberculosis H37Rv growth: minimal inhibiting concentration (MIC) was twice smaller than for free rifampicin. Administration of this preparation to mice with tuberculosis induced by M. tuberculosis Erdman revealed that after 6 weeks of oral administration the CUF value in lung was 22 times smaller for rifampicin in nanoparticles than for free drug (1.7 un. vs. 37.4 un.). The LD50 value in mice was two fold higher for rifampicin in nanoformulation.

Tuberculosis is a chronic infectious disease which is found in the developing as well as the developed country. This disease is oneof the community health problems which become the priority programs in the national as well as international health. In the lasttwo decades, they can be found in the emergency tuberculosis problems that is related with the Multi Drug Resistance (MDR) Strain.The detection of rifampicin resistance in M. tuberculosis infection can help clinical laboratory to find the MDR strain. Related to thisproblem the proportional culture method is still the gold standard for rifampicin resistance detection for M. tuberculosis infection. Butthis method needs 4−6 weeks to obtain the result, while its sensitivity is not very high. The development of the molecular detection forM. tuberculosis rifampicin resistance in a direct clinical specimen such as sputum, cerebrospinalfLuid, etc. will give an improvement inthe diagnosis, because it has an accurate, fast, sensitive and a specific result. Isolates from twenty six of M. tuberculosis derived fromthe sputum of tuberculosis patients that have failed the tuberculosis treatment, were examined with the proportional culture method.In this study PCR-SSCP were used for the molecular detection of rifampicin resistancy using direct sputum samples. The proportionalculture method was used as a gold standard for the rifampicin resistance detection. A set of primers was directed to conserve the regionof rpoB gene of M. tubercuLosis. This RNA polymerase gene was encondes?, which is bound on rifampicin. A 157-bp fragment wasamplified by PCR and analyzed by SSCP technique. The sensitivity of PCR-SSCP is 80% (high), its specificity is 95.2% (very high), thepositive predictive value is 80% and the negative predictive value is 95.2%. Statistically there were no significant difference between theresult of PCR-SSCP and the proportional culture method. Based on the study result, the molecular detection technique for rifampicinresistance on M. tuberculosis infection can be used as the screening device /means for Multi Drug Resistance Tuberculosis (MDR-TB),while the clinician waits the culure result.

First-line drugs (Isoniazid and Rifampicin) for the treatment of tuberculosis are known to have experienced resistance to Mycobacterium tuberculosis. The aim of this study was to determine the minimum inhibitory concentration (MIC) of maltodextrin encapsulated rosella calyx extract and its ability to provide a synergistic effect with Isoniazid (INH) and Rifampicin (RIF) on M. tuberculosis R37rv. Rosella calyxs were macerated using 50% ethanol and encapsulated using maltodextrin. Antibacterial activity was carried out by determining MIC value using Microscopic Observation and Direct Susceptibility (MODS) method. The synergistic effect was carried out by calculating the Fractional Inhibition Concentration Index (FICI). The results showed that this extract was able to inhibit M. tuberculosis H37rv with MIC of 10 mg/ ml. The FICI value of the combination of extract with INH and rifampicin was obtained 1.25. This showed that rosella calyx extract is not synergistic with INH and rifampicin, might be due to rosella calyx extract has an antibacterial effect with a different mechanism with INH and Rifampicin.

First-line drugs (Isoniazid and Rifampicin) for the treatment of tuberculosis are known to have experienced resistance to Mycobacterium tuberculosis. The aim of this study was to determine the minimum inhibitory concentration (MIC) of maltodextrin encapsulated rosella calyx extract and its ability to provide a synergistic effect with Isoniazid (INH) and Rifampicin (RIF) on M. tuberculosis R37rv. Rosella calyxs were macerated using 50% ethanol and encapsulated using maltodextrin. Antibacterial activity was carried out by determining MIC value using Microscopic Observation and Direct Susceptibility (MODS) method. The synergistic effect was carried out by calculating the Fractional Inhibition Concentration Index (FICI). The results showed that this extract was able to inhibit M. tuberculosis H37rv with MIC of 10 mg/ ml. The FICI value of the combination of extract with INH and rifampicin was obtained 1.25. This showed that rosella calyx extract is not synergistic with INH and rifampicin, might be due to rosella calyx extract has an antibacterial effect with a different mechanism with INH and Rifampicin.

The present study was undertaken to determine the drug resistance pattern of M. tuberculosis isolated from 225 pulmonary and 45 extrapulmonary tuberculosis cases. The samples were cultured on Lowenstein Jensen (L-J) media for isolation of M. tuberculosis. Drug resistance to first line anti tubercular drugsnamely isoniazid (INH), rifampicin (RIF), Ethambutol (ETH) and streptomycin (SM) were determined by indirect proportion method. The overall drug resistance of M. tuberculosis was 53.6% to any of the first line anti tubercular drugs. Rate of multi drug resistant tuberculosis (MDR-TB) among the untreated cases was 4.2%, while it was 36.0% in previously treated cases. It was found that 83.3% rifampicin resistant M. tuberculosis was cross resistant to one or more of other first line anti-tubercular drugs, while cross resistance of INH, ETH and SM resistant isolates was much low. The present study revealed that high level of drug resistance exists to individual anti tubercular drugs and MDR-TB is an emerging problem, particularly in treated cases. Rifampicin resistance could be used as a surrogate marker for drug resistance to other first line anti tubercular drugs.Ibrahim Med. Coll. J. 2014; 8(2): 41-46

Despite efforts to limit the morbidity and mortality from tuberculosis (TB), it continues to be an important cause of death. There is an urgent need for a diagnostic test that accurately and quickly diagnoses TB, especially if it is also a near-point-of-care test. The GeneXpert polymerase chain reaction test (known in India as CBNAAT [cartridge-based nucleic acid amplification test] and is capable of diagnosing TB and rifampicin resistance within 2 h) is a promising tool. The duration of our study was two years and was carried out in the DOTS centre of a tertiary care hospital in India. A total of 5449 samples were processed using CBNAAT. Of the total samples tested, 2068 were extra-pulmonary. The following information was collected: number of extra-pulmonary samples processed; number of Mycobacterium tuberculosis ( M. tuberculosis)-positive samples; patterns of rifampicin sensitivity; number of people living with HIV (PLHIV); and number of children. Of the samples, 62.1% were from suspected pulmonary TB patients. Out of the total samples tested using CBNAAT, 21.8% were positive for M. tuberculosis. Rifampicin resistance was seen in 9.2%, 8.5% and 10.3% of the total, pulmonary and extra-pulmonary samples, respectively, in M. tuberculosis-positive samples. Overall, 36.9% samples were from the paediatric population and 5.7% belonged to PLHIV. Rifampicin resistance was seen in 8.8% and 8.3% of the M. tuberculosis-positive paediatric and PLHIV samples, respectively.

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Tuberculosis is a serious disease, but curable in practically 100% of new cases, since complied the principles of modern chemotherapy. Isoniazid (ISN), Rifampicin (RIF), Pyrazinamide (PYR) and Chloride Ethambutol (ETA) are considered first line drugs in the treatment of tuberculosis, by combining the highest level of efficiency with acceptable degree of toxicity. Concerning USP 33 - NF28 (2010) the chromatography analysis to 3 of 4 drugs (ISN, PYR and RIF) last in average 15 minutes and 10 minutes more to obtain the 4th drug (ETA) using a column and mobile phase mixture different, becoming its industrial application unfavorable. Thus, many studies have being carried out to minimize this problem. An alternative would use the UFLC, which is based with the same principles of HPLC, however it uses stationary phases with particles smaller than 2 ?m. Therefore, this study goals to develop and validate new analytical methods to determine simultaneously the drugs by HPLC/DAD and UFLC/DAD. For this, a analytical screening was carried out, which verified that is necessary a gradient of mobile phase system A (acetate buffer:methanol 94:6 v/v) and B (acetate buffer:acetonitrile 55:45 v/v). Furthermore, to the development and optimization of the method in HPLC and UFLC, with achievement of the values of system suitability into the criteria limits required for both techniques, the validations have began. Standard solutions and tablets test solutions were prepared and injected into HPLC and UFLC, containing 0.008 mg/mL ISN, 0.043 mg/mL PYR, 0.030 mg.mL-1 ETA and 0.016 mg/mL RIF. The validation of analytical methods for HPLC and UFLC was carried out with the determination of specificity/selectivity, analytical curve, linearity, precision, limits of detection and quantification, accuracy and robustness. The methods were adequate for determination of 4 drugs separately without interfered with the others. Precise, due to the fact of the methods demonstrated since with the days variation, besides the repeatability, the values were into the level required by the regular agency. Linear (R> 0,99), once the methods were capable to demonstrate results directly proportional to the concentration of the analyte sample, within of specified range. Accurate, once the methods were capable to present values of variation coefficient and recovery percentage into the required limits (98 to 102%). The methods showed LOD and LOQ very low showing the high sensitivity of the methods for the four drugs. The robustness of the methods were evaluate, facing the temperature and flow changes, where they showed robustness just with the preview conditions established of temperature and flow, abrupt changes may influence with the results of methods
A tuberculose ? uma doen?a grave, por?m cur?vel em praticamente 100% dos casos novos, desde que obedecidos os princ?pios da moderna quimioterapia. S?o considerados f?rmacos de 1? linha no tratamento ? tuberculose: isoniazida, pirazinamida, etambutol e rifampicina. De acordo com USP 33 - NF28 (2010) as an?lises cromatogr?ficas para 3 dos 4 f?rmacos (isoniazida, pirazinamida e rifampicina) duram em m?dia 15 minutos e mais 10 minutos para a obten??o do 4? f?rmaco (etambutol) utilizando outra coluna, com outra mistura de fase m?vel, tornando esta aplica??o na pr?tica industrial desfavor?vel. Uma das alternativas ? utilizar o CLUE, o qual baseia-se nos mesmos princ?pios da CLAE, por?m utiliza fases estacion?rias com part?culas menores que 2 ?m. Dessa forma pretende-se com o presente estudo desenvolver e validar novos m?todos anal?ticos para determina??o simult?nea de tuberculost?ticos por CLAE/DAD e CLUE/DAD. Para isto, foi realizado um screening anal?tico, o qual verificou que ? necess?rio um gradiente de sistema de fase m?vel A (tamp?o acetato:metanol 94:6 v/v) e B (tamp?o acetato:acetonitrila 55:45 v/v). Posteriormente, ao desenvolvimento e otimiza??o do m?todo em CLAE e CLUE com a obten??o dos valores de adequabilidade do sistema dentro dos limites de aceita??es vigente para ambos as t?cnicas, as valida??es deram-se in?cio. Solu??es padr?es e solu??es testes dos comprimidos foram preparadas e injetadas no CLAE e CLUE, contendo isoniazida, pirazinamida, etambutol e rifampicina nas concentra??es de 0,008, 0,043, 0,030 e 0,016 mg.mL-1, respectivamente. A valida??o dos m?todos anal?ticos foram realizadas para: especificidade / seletividade, intervalos da curva anal?tica, linearidade, limite de detec??o, limite de quantifica??o, exatid?o, precis?o (repetibilidade, precis?o intermedi?ria) e robustez. Os m?todos foram adequados para determina??o dos 4 f?rmacos separadamente sem interfer?ncia nos demais. Precisos, devido ao fato de que os m?todos demonstraram que mesmo com varia??o de dias, al?m da repetibilidade, os valores ficaram dentro da faixa preconizada na legisla??o vigente. Lineares (R > 0,99), ou seja, os m?todos foram capazes de demonstrar que os resultados obtidos eram diretamente proporcionais ? concentra??o do analito na amostra, dentro de um intervalo especificado. Exatos, uma vez que os m?todos foram capazes de apresentar valores de coeficiente de varia??o e porcentagem de recupera??o dentro dos limites exigidos (98 a 102%). Os m?todos mostraram LD e LQ com n?veis baixos demonstrando que os m?todos possuem elevada sensibilidade aos quarto f?rmacos. A robustez foi avaliada frente ?s altera??es de temperatura e fluxo, onde os m?todos demonstraram-se robustos apenas nas condi??es previamente estabelecidas de temperatura e fluxo, altera??es bruscas podem acarretar influ?ncia nos resultados dos m?todos

The World Health Organization (WHO) estimated that eight million new cases of tuberculosis (TB) occur every year and that one-third of the world’s population is infected with Mycobacterium tuberculosis (M. tuberculosis). With the increase in HIV/AIDS in the 1980’s, an increase in transmission of TB led to an increase in TB incidence. A study showed that South African adults (ages 15 to 49) will suffer 278 154 deaths between 2008 and 2017 if current control measures are continued. A M. tuberculosis strain that is resistant to isoniazid (INH) and rifampicin (RIF) used in the treatment of TB is known as a multi-drug resistant (MDR-TB) strain. In extensively drugresistant tuberculosis (XDR-TB) the M. tuberculosis strains are not only resistant to INH, RIF and any one of the fluoroquinolones but to at least one of the three injectable second-line drugs such as amikacin or kanamycin. Unfortunately, many people with XDR-TB will die because it is virtually impossible to formulate an effective treatment before the resistance pattern of the M. tuberculosis strain has been identified. Bacteriological culture is considered the diagnostic gold standard and can identify mycobacteria in over 80% of TB cases, with a specificity of over 98%. However, culturing the mycobacteria takes 4 to 6 weeks and makes diagnosis and treatment a prolonged process. In this study 60 patients suspected of TB disease, from the Anti-retroviral (ARV) clinic at the Tshwane District Hospital (TDH) were collected from October 2008 to April 2009. This study evaluated the use of the QuantiFERON-TB GOLD ELISA assay in a high burden setting. Tshwane District Hospital, South Africa. The sensitivity and specificity of the QFT assay in the clinic were 30% (9/30) and 63% (19/30) respectively when compared to the gold standard culture results. Analysis suggested that the sensitivity of the QuantiFERON assay is determined by a limiting patient CD4 value of between 150 and 200. Real-time PCR assays were used for rapid identification of Mycobacterium spp and to determine the presence of isoniazid and rifampicin resistant genes of M. tuberculosis strains. The real-time PCR assay identified 28% (17/60) M. tuberculosis, 2% (1/60) M. kansasii and 70% (42/60) of the isolates Mycobacterium spp negative. No M. avium were detected. The 17 M. tuberculosis positive specimens were further analysed for the presence of INH and RIF resistance genes. All 17 specimens had either no mutation or one or more mutations at the specific gene targets (rpo1, rpo2, katG and inhA). This study showed several possibilities for the use of both an immunological assay as well as molecular methods for the diagnosis of TB. This study suggested that in terms of routine diagnosis of TB in high HIV prevalence settings the QFT test should be used with caution. Realtime PCR for both detection and identification showed useful results and can be used together with culture results to improve turnaround times for TB diagnosis. Copyright
Dissertation (MSc)--University of Pretoria, 2010.
Medical Microbiology
unrestricted

Tee PhD thesis presents the study of the response of Mycobacterium tuberculosis, the causative agent of tuberculosis, upon prolonged exposure to lethal concentrations of the first line anti-tuberculosis drug, rifampicin. The study shows that prolonged exposure to lethal concentration of rifampicin causes cell death initially by several log orders of cells, followed by a persistence phase, from where rifampicin resisters emerge carrying mutation in the RRDR locus of the rpoB gene. This phenomenon was found to occur even when the drug concentration is well above MBC levels. Luria-Delbruck experiment, in a modified format, showed that the resisters emerged as fresh mutants, and not due to the growth of pre-existing natural resisters to rifampicin. The per sister cells, which showed high levels of hydroxyl radical generation, were found to have thickened outer layer, unlike the mid-log phase cells, which restricts the permeability of a fluorescent-conjugate of rifampicin, 5-FAM-rifampicin, 10-fold less in per sister cells, as compared to mid-log phase cells. The thickened outer layer has high negative surface charge and is hydrophilic in nature. It is proposed that the hydrophilic-natured thickened outer layer might have restricted the permeability of lethal concentrations of hydrophobic-natured rifampicin. This, in turn, might have ensured the presence of sub-lethal concentration rifampicin inside the per sisters, which in turn might have generated the hydroxyl radical that caused mutagenesis to generate rifampicin resisters. The Chapter 1 is the Introduction to the thesis presented in 4 parts – Part 1.1, 1.2, 1.3 and 1.4, introducing briefly the history of antibiotics, antibiotic resistance, antibiotic persistence and a brief history of tuberculosis respectively. It is concluded with a rationale behind the present study. The Chapter 2 presents the entire Materials and Methods used in the experiments described in the thesis. The Chapter 3 presents the data on the response of M. tuberculosis to rifampicin upon extended exposure. Rifampicin exposure of susceptible M. tuberculosis H37Ra cells showed a decrease in their CFU/ml followed by a persistence phase wherein the CFU/ml remained constant. However, prolonged exposure of rifampicin even at higher concentrations showed regrowth in the culture, which was found to be due to the emergence of rifampicin-resistant bacteria. Screening of rifampicin-resistant mutants showed point mutations in the rifampicin resistance determining region (RRDR) of the rpoB gene in all the mutants. In parallel, using trans formants of M. tuberculosis expressing unstable GFP under the respective native ribosomal RNA promoter, the metabolic status of the per sister cells was determined. When actively growing highly fluorescing cells were exposed to lethal concentration of rifampicin, their metabolism diminished, as illustrated by the decrease in their fluorescence during persistence phase, followed by the emergence of a sub-population of bacteria which were again metabolically active. In order to verify whether the rifampicin resisters are freshly formed mutants or have come from the naturally existing resisters, Luria-Delbruck fluctuation test was performed in a modified manner. The number of rifampicin resisters that emerged from the persistent phase was found to vary amongst different cultures from different days and different times of exposure, showing fluctuation. However, the addition of theorem before persistence phase almost abolished the generation of the rifampicin-resistant bacilli, indicating the role of hydroxyl radical in the emergence of rifampicin resisters. The generation of hydroxyl radical in mycobacteria exposed to rifampicin was confirmed using electron para-magnetic resonance spectrometry (EPR), with the spin trap agent, 5,5- Dimethyl-1-pyrroline N-oxide (DMPO) specific for hydroxyl radical. An increase in the formation DMPO-OH adduct in the persistence phase cells was observed, in comparison to mid-log phase cells. Exposure to theorem significantly diminished the adduct formation. The persistence phase cells also showed significantly high levels of signal specific to the hydroxyl-specific fluorescent dye, hydroxyphenyl fluorescein (HPF), as compared to the mid-log phase cells. In addition we have determined the oxidative stress in the bacilli upon rifampicin exposure using a redox biosensor (Mrx1-roGFP) which also showed high oxidative stress in the persistent phase. These observations confirmed the presence of high levels of oxidative stress and hydroxyl radical in the rifampicin persistent cells, in comparison with mid-log phase cells. Whole genome sequencing of the four independently isolated rifampicin resistant M. tuberculosis showed genome wide mutations having less common mutations with respect to the wild type genome, indicative of the occurrence of random mutagenesis. In addition mutation frequencies were comparable between the samples with respect to the wild type sample. About 69% of the mutations were A-C or T-G, followed by A-T or T-A, which is known to be due to oxidative stress in the cells. Variations in the colony morphology were also observed on the persistent phase cells, indicating the occurrence of mutagenesis in the bacterial genome during rifampicin treatment. The Chapter 4 is on the morphological and ultrastructural studies on rifampicin-exposed M. tuberculosis cells. Transmission electron micrographs of rifampicin per sisters showed significant thickening of the outermost capsular layer (OL), as compared to the mid-log phase cells. This observation was verified by staining the cells with ruthenium red, which specifically stains anionic polysaccharide of the OL. Zeta potential (ZP) measurement of the surface charge of the persistent cells showed high negative ZP, as compared to the mid-log phase cells. The negative ZP value was found to gradually increase during the course of rifampicin treatment and to decrease during the regrowth phase. Hexadecane assay showed larger proportion of per sister cells being retained in the aqueous phase, as compared to the mid-log phase cells. This indicated the higher hydrophilicity of the per sister cells, which was in agreement with the higher surface negative charge of the cells. The permeability of rifampicin per sister cells to 5-FAM-rifampicin (rifampicin conjugated to 5-carboxy fluorescein, which is as hydrophobic as rifampicin) was found to be 10-fold less than that of the mid-log phase cells. However, removal of the thick OL by bead beating (BB) with 4 mm glass beads significantly improved the permeability of per sister cells to 5-FAM-rifampicin. On the contrary, no difference in the 5-FAM-rifampicin uptake was observed in mid-log phase cells, with or without BB. These observations implied that the thick hydrophilic-natured OL with high negative surface charge may be playing a significant role in limiting the permeability of hydrophobic-natured rifampicin entry into the persisted cells. This in turn may ensure the presence of sub-lethal concentration of rifampicin inside the persisted cells that are exposed to lethal concentration of the antibiotic. Exposure of bacteria to sub-lethal concentration of antibiotics has been reported to generate oxidative stress in the bacteria, leading to mutagenesis. A model has been proposed based on these observations in which the persistent mycobacteria are protected from lethal concentrations of the rifampicin by the thick OL which in turn ensures sub-lethal intracellular antibiotic concentration, leading to the generation of hydroxyl radical mediated mutagenesis and thereby emergence of rifampicin resisters. This thesis is concluded with the list of salient findings, publications and references.

Tee PhD thesis presents the study of the response of Mycobacterium tuberculosis, the causative agent of tuberculosis, upon prolonged exposure to lethal concentrations of the first line anti-tuberculosis drug, rifampicin. The study shows that prolonged exposure to lethal concentration of rifampicin causes cell death initially by several log orders of cells, followed by a persistence phase, from where rifampicin resisters emerge carrying mutation in the RRDR locus of the rpoB gene. This phenomenon was found to occur even when the drug concentration is well above MBC levels. Luria-Delbruck experiment, in a modified format, showed that the resisters emerged as fresh mutants, and not due to the growth of pre-existing natural resisters to rifampicin. The per sister cells, which showed high levels of hydroxyl radical generation, were found to have thickened outer layer, unlike the mid-log phase cells, which restricts the permeability of a fluorescent-conjugate of rifampicin, 5-FAM-rifampicin, 10-fold less in per sister cells, as compared to mid-log phase cells. The thickened outer layer has high negative surface charge and is hydrophilic in nature. It is proposed that the hydrophilic-natured thickened outer layer might have restricted the permeability of lethal concentrations of hydrophobic-natured rifampicin. This, in turn, might have ensured the presence of sub-lethal concentration rifampicin inside the per sisters, which in turn might have generated the hydroxyl radical that caused mutagenesis to generate rifampicin resisters. The Chapter 1 is the Introduction to the thesis presented in 4 parts – Part 1.1, 1.2, 1.3 and 1.4, introducing briefly the history of antibiotics, antibiotic resistance, antibiotic persistence and a brief history of tuberculosis respectively. It is concluded with a rationale behind the present study. The Chapter 2 presents the entire Materials and Methods used in the experiments described in the thesis. The Chapter 3 presents the data on the response of M. tuberculosis to rifampicin upon extended exposure. Rifampicin exposure of susceptible M. tuberculosis H37Ra cells showed a decrease in their CFU/ml followed by a persistence phase wherein the CFU/ml remained constant. However, prolonged exposure of rifampicin even at higher concentrations showed regrowth in the culture, which was found to be due to the emergence of rifampicin-resistant bacteria. Screening of rifampicin-resistant mutants showed point mutations in the rifampicin resistance determining region (RRDR) of the rpoB gene in all the mutants. In parallel, using trans formants of M. tuberculosis expressing unstable GFP under the respective native ribosomal RNA promoter, the metabolic status of the per sister cells was determined. When actively growing highly fluorescing cells were exposed to lethal concentration of rifampicin, their metabolism diminished, as illustrated by the decrease in their fluorescence during persistence phase, followed by the emergence of a sub-population of bacteria which were again metabolically active. In order to verify whether the rifampicin resisters are freshly formed mutants or have come from the naturally existing resisters, Luria-Delbruck fluctuation test was performed in a modified manner. The number of rifampicin resisters that emerged from the persistent phase was found to vary amongst different cultures from different days and different times of exposure, showing fluctuation. However, the addition of theorem before persistence phase almost abolished the generation of the rifampicin-resistant bacilli, indicating the role of hydroxyl radical in the emergence of rifampicin resisters. The generation of hydroxyl radical in mycobacteria exposed to rifampicin was confirmed using electron para-magnetic resonance spectrometry (EPR), with the spin trap agent, 5,5- Dimethyl-1-pyrroline N-oxide (DMPO) specific for hydroxyl radical. An increase in the formation DMPO-OH adduct in the persistence phase cells was observed, in comparison to mid-log phase cells. Exposure to theorem significantly diminished the adduct formation. The persistence phase cells also showed significantly high levels of signal specific to the hydroxyl-specific fluorescent dye, hydroxyphenyl fluorescein (HPF), as compared to the mid-log phase cells. In addition we have determined the oxidative stress in the bacilli upon rifampicin exposure using a redox biosensor (Mrx1-roGFP) which also showed high oxidative stress in the persistent phase. These observations confirmed the presence of high levels of oxidative stress and hydroxyl radical in the rifampicin persistent cells, in comparison with mid-log phase cells. Whole genome sequencing of the four independently isolated rifampicin resistant M. tuberculosis showed genome wide mutations having less common mutations with respect to the wild type genome, indicative of the occurrence of random mutagenesis. In addition mutation frequencies were comparable between the samples with respect to the wild type sample. About 69% of the mutations were A-C or T-G, followed by A-T or T-A, which is known to be due to oxidative stress in the cells. Variations in the colony morphology were also observed on the persistent phase cells, indicating the occurrence of mutagenesis in the bacterial genome during rifampicin treatment. The Chapter 4 is on the morphological and ultrastructural studies on rifampicin-exposed M. tuberculosis cells. Transmission electron micrographs of rifampicin per sisters showed significant thickening of the outermost capsular layer (OL), as compared to the mid-log phase cells. This observation was verified by staining the cells with ruthenium red, which specifically stains anionic polysaccharide of the OL. Zeta potential (ZP) measurement of the surface charge of the persistent cells showed high negative ZP, as compared to the mid-log phase cells. The negative ZP value was found to gradually increase during the course of rifampicin treatment and to decrease during the regrowth phase. Hexadecane assay showed larger proportion of per sister cells being retained in the aqueous phase, as compared to the mid-log phase cells. This indicated the higher hydrophilicity of the per sister cells, which was in agreement with the higher surface negative charge of the cells. The permeability of rifampicin per sister cells to 5-FAM-rifampicin (rifampicin conjugated to 5-carboxy fluorescein, which is as hydrophobic as rifampicin) was found to be 10-fold less than that of the mid-log phase cells. However, removal of the thick OL by bead beating (BB) with 4 mm glass beads significantly improved the permeability of per sister cells to 5-FAM-rifampicin. On the contrary, no difference in the 5-FAM-rifampicin uptake was observed in mid-log phase cells, with or without BB. These observations implied that the thick hydrophilic-natured OL with high negative surface charge may be playing a significant role in limiting the permeability of hydrophobic-natured rifampicin entry into the persisted cells. This in turn may ensure the presence of sub-lethal concentration of rifampicin inside the persisted cells that are exposed to lethal concentration of the antibiotic. Exposure of bacteria to sub-lethal concentration of antibiotics has been reported to generate oxidative stress in the bacteria, leading to mutagenesis. A model has been proposed based on these observations in which the persistent mycobacteria are protected from lethal concentrations of the rifampicin by the thick OL which in turn ensures sub-lethal intracellular antibiotic concentration, leading to the generation of hydroxyl radical mediated mutagenesis and thereby emergence of rifampicin resisters. This thesis is concluded with the list of salient findings, publications and references.

Bacterial persisters are a subpopulation of bacteria that can tolerate lethal concentrations of antibiotics. These are phenotypic variants that can give rise to drug‐susceptible population upon withdrawal of the antibiotic. Persistent bacteria play a crucial role in prolonging antibiotic treatment and are responsible for the recalcitrance of many chronic bacterial diseases, including tuberculosis. Several mechanisms have been proposed for the formation of persisters, which include expression of toxin‐antitoxin systems, generation of reactive oxygen species (ROS), and stochastic changes in gene expression and so on. Recent report from our laboratory has demonstrated that continuous prolonged exposure of Mycobacterium tuberculosis cells to lethal concentrations of antibiotics generates antibiotic persistence phase cells from which genetically resistant mutants emerge de novo either to the same antibiotic to which it was exposed to or to another antibiotic used for selection Sebastian et al., Antimicrobial Agents Chemotherapy 61(2), e01343‐16, 2016). The persistence phase cells showed high levels of oxidative stress that inflicted genome‐wide mutations in addition to the mutations for which the resistant mutants were selected against rifampicin and moxifloxacin. Thus, it was we demonstrated that the antibiotic persistence phase M. tuberculosis cells is a reservoir for the de novo emergence of antibiotic resistant mutants. In the present study, the response of Mycobacterium smegmatis upon prolonged exposure to rifampicin was examined since the bacilli has two mechanisms to inactivate or neutralise the action of rifampicin. These mechanisms include: (i). ADP‐ribosylation of rifampicin by the product of the gene ADP‐ribosyltransferase (arr); (ii). Rescue of rifampicin‐ mediated transcription inhibition by MsRbpA. The question asked was whether genetically resistant mutants against rifampicin would emerge from rifampicin persister phase cells, like in the case of M. tuberculosis cells and if they do, what are the mechanisms by which the rifampicin‐resistant mutants emerge from the persistence phase cells. For comparison and contrast purpose, and as a control sample, the response of M. smegmatis cells to moxifloxacin, against which the bacilli do not have any inherent inactivation or neutralisation mechanism, was studied. The Chapter 1, which forms the Introduction to the thesis, gives an extensive literature survey on all the different aspects of the research performed on the response of mycobacterial cells to antibiotics. The Chapter 2 presents in detail all the materials and methods used to perform the experiments. A large number of cell biological and molecular biological methods, such as fluorescence microscopy and fluorescence measurements, flow cytometry, cloning and expression, real time RT‐PCR and whole genome sequencing, and biophysical methods such as electron paramagnetic resonance spectrometry, and others were used to perform the experiments. The data Chapter 3 presents the data on the response of M. smegmatis cells to rifampicin. The data shows that exposure to MBC levels of rifampicin results in the killing of the cells to a 5‐log10 reduction in the cfu of M. smegmatis cells but the remaining cells persist and from these cells emerge rifampicin‐resistant mutants. The persistence phase cells were found to generate elevated levels of hydroxyl radical, which inflicted genome‐wide mutations, and the mutants harbouring nucleotide changes at the rifampicin resistance determining region (RRDR) could regrow back. Interestingly, the killing phase and the regrowth phase showed very low levels of hydroxyl radical unlike the persistence phase cells. The mutations, which are identical to those in the rifampicin‐resistant mutants have been reported in the M. tuberculosis cells isolated from in vitro cultures and from the TB patients. The data Chapter 4 presents the response of the arr knockout mutant to rifampicin. The persistence phase population of the arr knockout mutant showed significantly higher levels of hydroxyl radical generation than the equivalent persistence phase population of the wild type cells. While the wild type cells showed emergence of rifampicin‐resistant mutants from the persistence phase, the arr knockout mutant showed the emergence of rifampicin‐ resistant mutants from the very exposure of the cells to rifampicin. In other words, the natural mutation frequency of the arr knockout mutant was significantly higher than that of the wild type. This indicated that the arr gene might have a natural role in keeping the oxidative stress at lower levels in the cells, which needs further investigation. The data Chapter 5 presents the response of M. smegmatis cells to moxifloxacin. Here also, the bacilli exposed to lethal concentrations of moxifloxacin showed a killing phase, followed by a persistence phase and a regrowth phase. The moxifloxacin‐resistant mutants were found to emerge from the moxifloxacin persistence phase cells. The cells from the regrowth phase of moxifloxacin‐exposed cells showed mutations in the quinolone resistance determining region (QRDR) in the gyrase gene, which is the target of moxifloxacin. The mutations, which are identical to those in the rifampicin‐resistant mutants have been reported in the M. tuberculosis cells isolated from in vitro cultures and from the TB patients. The thesis is concluded with discussion of the findings presented in the three chapters by projecting the comparison and contrast of the response of M. smegmatis and M. tuberculosis cells to rifampicin. The thesis contains an extensive bibliography.

碩士
嘉南藥理科技大學
生物科技系暨研究所
98
Abstract Mycobacterium avium complex (MAC) belongs to a group of slow growing mycobacteria. Many studies showed that chronic pulmonary disease is most commonly manifestated by a non-tuberculous mycobacteria (NTM) infection. MAC, M. kansasii, and M. abscessus, in order of frequency, are the most common pathogens of NTM lung disease. We found the patients with NTM lung disease were more than those with pulmonary tuberculosis between July, 2007 and December, 2008 and MAC was the most common pathogen of NTM lung disease. A case-control was conducted study and 30 patients with a positive sputum culture for MAC as the case group and 30 patients with a positive sputum culture for M. tuberculosis as the control group were enrolled. The two groups were matched with sex, residential areas, and patient services (outpatients or inpatients). Several variables as predictors of risk factors were used to evaluate acquiring NTM lung disease, including age, height, weight﹐BMI﹐DM, smoking, upper lung fields﹐lower lung fields, and pulmonary cavities. We also compared the gene sequence by sequencing to identify the Rifampicin resistance gene of MAC. Involvement of lower lung fields were more common in MAC lung disease (McNemar test, p &lt;0.05), while the pulmonary cavities were more common in pulmonary tuberculosis (Logistic regression, p &lt;0.05). Finally, EMBOSS were used for the database of M.avium AF060366 and sequence analysis. Three strains with a point mutation (Glycine→Aspartic acid) in the nucleotide sequence 433 was noted. Twenty-six strains (87%) presented the phenotype of Rifampicin resistance, while 4 strains were sensitive to Rifampicin (13%). However, genotypes of Rifampicin resistance were noted for only 11% of the strains. It implies that a new criteria for clinical susceptibility test should be established to provide a more convenient, cheap and accurate method for the in-vitro susceptibility test.

AbstractBuruli ulcer (BU), one of the skin-related neglected tropical diseases (skin NTDs), is a necrotizing and disabling cutaneous disease caused by subcutaneous infection with Mycobacterium ulcerans. Leading on from the World Health Organization’s (WHO) establishment of a global BU initiative in 1998, >67,000 cases of BU have been reported from over 32 countries, mostly from West Africa and Australia. While treatment is currently in the transition period from rifampicin plus streptomycin (injection) to an all-oral regimen, it cannot hope to eradicate this opportunistic environmental pathogen. M. ulcerans is genetically very similar to related pathogenic organisms M. marinum, M. leprae and M. tuberculosis. However, M. ulcerans carries a unique megaplasmid, pMUM001, encoding the biosynthetic machinery responsible for production of a lipid-like exotoxin virulence factor, mycolactone. This diffusible compound causes the substantial divergence in BU’s pathogenic aetiology from other mycobacterial infections. Hence, mycolactone is cytotoxic and immunosuppressive and causes vascular dysfunction in infected skin. A major recent advance in our understanding of BU pathogenesis has been agreement on the mycolactone’s mechanism of action in host cells, targeting the Sec61 translocon during a major step in secretory and membrane protein biogenesis. While vaccine development for all mycobacteria has been challenging, mycolactone production likely presents a particular challenge in the development of a BU vaccine. The live-attenuated vaccine BCG is known to provide only partial and transient protection in humans but provides a convenient baseline in mouse preclinical studies where it can delay, but not prevent, disease progression. No experimental vaccine strategy has yet conferred greater protection than BCG. However, there is now the prospect of developing a vaccine against mycolactone itself, which may provide hope for the future.

Tuberculosis, caused by Mycobacterium tuberculosis (M.tb), remains the biggest infection burden in the word. Rifampin (RIF) and Isoniazid (INH) are the most effective antibiotics for killing M.tb. However, the resistance rate of rifampin and INH are high and lead to almost 35% treatment failure. Metformin enhanced anti tuberculosis efficacy in killing M. tuberculosis through several mechanism, firstly through autophagia mechanism and secondly by activating superoxide dismutase (SOD). Metformin activated mTOR and AMPK then induced more effective autophagy against M.tb. Superoxide Dismutase (SOD) is an enzyme produced in the host’s antioxidant defense system. SOD neutralizes reactive oxygen species (ROS) that excessively produced during phagocytosis process against M.tb. Excessive production of ROS associated with Th1 overactivation and leads into macrophage activity inhibition and excessive tissue damage. Metformin has ability in improving SOD level during inflammation.

Introduction: Lymphadenitis, also previously called “scrofula,” is the most common cause of manifestation of extrapulmonary tuberculosis (TB), an extremely prevalent disease in underdeveloped regions, causing millions of deaths around the world. This is why it must be recognized and treated as early as possible. Objective: This review aims to summarize the main topics of tuberculous lymphadenitis (TL), covering epidemiology, clinical, and recent treatments. Methods: This article consists of a review of publications on the subject. The research was carried out through SciELO, PubMed, and LILACS databases, as well as virtual scientific libraries such as DynaMed and UpToDate. Results: TL is the infection of lymph nodes caused by Mycobacterium tuberculosis, and it is the most common type of extrapulmonary TB, mainly in endemic areas. Worldwide, there is an increase in the incidence of TB in developed and underdeveloped countries, resulting in millions of deaths per year. Its relationship with HIV and the consequent development of extrapulmonary forms has been increasingly common, representing about 21% of TB cases in the United States. The main extrapulmonary TB sites are as follows: lymph nodes, pleura, meninges, bones, miliary, and disseminated. In HIV patients, atypical presentations are not uncommon. The clinical picture consists of slow lymph node growth, generally affecting the cervical region and may affect other sites; signs and symptoms of the primary TB may also be present. The diagnosis of TL is made by culture or molecular identification of M. tuberculosis in the tissue of the affected lymph node, which can be approached by excision or by fine-needle biopsy. The anatomopathological findings are giant epithelioid cells, granulomas, and caseous necrosis. Treatment should be started empirically according to the clinic, awaiting laboratory confirmation, and its first line consists of the first 2 months with RHZE (Rifampicin, Isoniazid, Pyrazinamide, and Ethambutol), followed by 4 months of Isoniazid and Rifampicin. Paradoxical worsening after starting the therapy is one of the complications, usually occurring 8 weeks after starting the treatment. Management should be monitored on an outpatient basis, with cure occurring in up to 94% of the cases. Conclusion: TL is one of the main manifestations of extrapulmonary TB, closely related to coinfection with HIV. It should be promptly investigated in patients with a compatible clinical presentation and present in endemic areas. Its treatment, despite long duration, cures the vast majority of cases and reduces the overall morbidity and mortality of properly treated patients.