Tuberculosis (TB) is a worldwide pandemic, which claims approximately 2 million lives annually. Though treatable through chemotherapy, TB still causes 8-10 million new infections annually. The problem is further complicated by the emergence of multi-drug resistant strains of Mycobacterium tuberculosis (Mtb) the causative agent of TB. The unabated spread of this disease and associated high levels of mortality has prompted the search for new drugs with novel modes of action. The peptidoglycan (PG) component of the cell wall of Mtb is an incredibly complex structure and has been the focus of antimicrobial development in other organisms. In this study, we characterize PG remodelling N-acetylmuramyl-L-alanine amidases (cell wall amidases) in Mycobacterium smegmatis (Msm), a model organism for TB research by gene knockout/knockdown. Cell wall amidases cleave the bond between the stem peptide and the glycan backbone in PG and have been shown to play an essential role in cell growth, cell signalling and antibiotic resistance in other organisms. Our bioinformatics analysis revealed that M. smegmatis encodes three possible amidase homologues designated ami1, ami2 and ami3. Deletion mutagenesis in Msm resulted in successful deletion of ami1 whilst repeated attempts to delete ami2 did not yield a knockout mutant, suggesting that ami2 is essential for growth. Deletion of ami1 results in the formation of long filaments consisting of 3 to 8 cells attached to each other due to incomplete resolution of the septum. In these filaments, lateral growth at both the internal septation sites and extreme poles resulted in irregular cell width, strongly implicating Ami1 in bacterial cell division, the maintenance of bacterial cell shape and possibly balancing the growth at the cell pole. Since deletion of ami2 was not possible, a knockdown strain allowing for anhydrotetracycline (ATC)-regulated conditional gene expression of ami2 was generated. In this system, withdrawal of ATC results in repression of expression. Expression analysis of ami2 in this strain revealed that whilst significant gene knockdown was achieved, ami2 expression was not completely abolished in the absence of the inducer ATC, suggesting the presence of basal level ami2 expression. The repression of ami2 expression results in retarded growth, diminished motility, unusual colony morphology consisting of miniature colonies lacking any form of serpentine cording and the formation of miniature cells consisting of globular bulges. These data implicate Ami2 in bacterial growth and maintenance of bacterial cell shape. Collectively, our data comprise the first demonstration of an important role for peptidoglycan degrading amidases in mycobacterial growth and cell division. Furthermore, the phenotypic defects in colony formation due to deletion or depletion of amidases suggest that these enzymes play an important role in cell-cell communication during colony formation. These data validate this class of enzymes as an untapped, legitimate source of novel targets for anti-tubercular drug discovery.

Mycobacterium tuberculosis is a facultative pathogen that causes tuberculosis and it accounted for 1.4 million deaths worldwide in 2011. During infection reactive species are released by macrophages as part of the hosts‟ immune response, causing damage to the pathogen‟s DNA. Since mycobacterial genomes are G+C rich the guanine base is more susceptible to oxidative damage which results in the formation of 7,8-dihydro-8-oxoguanine (8-oxoG) lesions which are subsequently repaired by the Fpg/Nei family of DNA glycosylases of the base excision repair (BER) pathway. Mycobacteria possess four copies of the Fpg/Nei glycosylases with the Fpg homologue displaying an overlapping role with MutM in the GO system. Loss of the Fpg/Nei DNA glycosylases leads to mispaired bases during replication which are subsequently repaired by the MutY DNA glycosylase. Previously, in our laboratory we showed that a deficiency of two Fpg/Nei glycosylases displayed no differences in survival of M. smegmatis mutants under oxidative stress and a 2-3 log difference was observed only when three or all four of the DNA glycosylases were inactivated. Surprisingly, there was no corresponding increase in mutator phenotype for all the combinatorial Fpg/Nei deficient mutants compared to the parental strain. A recent study further showed that a deficiency in M. smegmatis MutY glycosylase also displayed no notable susceptibility to oxidative stress or increase in mutagenesis. Since in E.coli a double mutM and mutY mutant displayed an increased mutator phenotype compared to the individual mutants, it was plausible to investigate the role of MutY in combination with the Fpg/Nei family of DNA glycosylases in mycobacteria to understand whether these DNA glycosylases display overlapping and/or compensatory functions in dealing with oxidative damage. Using homologous recombination the mutY gene was deleted in the parental and in the Fpg/Nei deficient mutant strains. Deletion of mutY together with the Fpg/Nei family of DNA glycosylasesdisplayed similar in vitro growth kinetics as the parental strain under normal culture conditions. However, under in vitro oxidative stress conditions the mutY deficiency especially in the absence of all four Fpg/Nei DNA glycosylases results in a greater reduction in survival of the mutants with a general increase in mutation rates. Consistent with these data was the significant increase in C → A and A → C mutations with the progressive loss of the DNA glycosylases as assessed by the spectral analysis of rifampicin resistant mutants. Taken together these data indicate that the mycobacterial MutY DNA glycosylase has antimutator properties and possibly has a more significant role in mycobacterial genome maintenance compared to the Fpg/Nei family of DNA glycosylases.

Berlin, Humboldt-University, Diss., 2004.

Th. 3e cycle--Chim. org.--Toulouse 3, 1980. N°: 2319.

Made available in DSpace on 2015-07-22T20:50:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-11-25 / Micobacterias de crescimento rapido (MCR) tem sido frequentemente descritas em surtos de infeccoes em seres humanos. Mycobacterium abscessus subsp. massiliense e uma MCR que foi descrita pela primeira vez em 2004, e desde entao, 10 trabalhos identificando essa especie em surtos e casos clinicos esporadicos sao os relatos presentes na literatura. Assim, a interacao M. abscessus subsp. massiliense-hospedeiro foi o objetivo principal deste trabalho. Para isso, variantes de colonia lisa e rugosa de um isolado do surto que ocorreu em Belem, entre 2004 e 2005 (B7 L e R), e de 2 isolados nao relacionados a surtos (B67 (L) e MG3-6 (R)) foram utilizadas em infeccoes intraperitoneal e endovenosa de camundongos e ex vivo em macrofagos peritoneais e celulas epitelioides-like. No que tange a resposta imunologica apos a infeccao intraperitoneal, o nivel de citocinas foi semelhante para os 4 isolados. Entretanto, apos 14 dias da infeccao os bacos dos animais infectados pelos isolados B7 R e MG3-6 apresentavam um aumento significativo de peso em relacao aos demais grupos. Apos 90 dias, esta diferenca se manteve apenas para o grupo infectado com B7 R. A histologia dos bacos destes animais mostrou que a resposta proliferativa nos foliculos linfoides foi mais pronunciada para a infeccao por B7 R, levando a necrose central dos mesmos apos 14 dias. Nestes, bacilos alcool-acido resistentes foram encontrados penetrando a zona perifolicular, e tambem em vasos de maior calibre, sugerindo disseminacao via corrente sanguinea. A infeccao endovenosa corroborou os resultados obtidos para a infeccao intraperitoneal. Apos 2 dias de infeccao, a recuperacao de unidades formadoras de colonia (UFCs) do baco foi significativamente maior para os animais infectados por B7 R. Entretanto, a infeccao foi igualmente controlada em todos os grupos, semelhantemente a infeccao intraperitoneal. A infeccao de culturas de macrofagos e CEs-like corrobou os achados in vivo, pois B7 R foi capaz de se proliferar no interior de macrofagos e, apenas para este isolado, houve o aumento na porcentagem de CEs-like infectadas. Adicionalmente, o isolado B7 R estimulou uma producao menor de TGF-ƒÒ nas culturas quando comparado ao isolado de fenotipo liso. CEs-like nao foram capazes de conter a infeccao causada pelo isolado B7 R, talvez pela interacao micobacteria-CEs-like resultar em menor producao de TGF-£].Tomados em conjunto, os resultados apresentados sugerem que micobacterias formadoras de colonias rugosas associadas a surtos sao mais patogenicas que as de fenotipo liso e que aquelas presentes no ambiente. Os mecanismos subjacentes responsaveis por estes achados estao sendo estudados. / Rapidly growing mycobacteria (RGMs) have been frequently identified in outbreaks of human infections. Mycobacterium abscessus subsp. massiliense is an RGM which was first described in 2004, and since then 10 publications identifying this species in outbreaks and sporadic clinical cases have been reported. The interaction between M. abscessus subsp. massiliense and host was the main objective of this work. Therefore, variants of smooth and rough colonies (B7 S and B7 R), isolated from an outbreak which took place in Belém, from 2004 to 2005, and two non-related isolates [B67 (S) and MG3-6 (R)] were used to produce intraperitoneal and intravenous infections in mice, and ex vivo infection in peritoneal macrophages and epithelioid-like cells. In terms of immune response following intraperitoneal infection cytokine levels were similar for the four isolates. However, after 14 days, the spleens of animals infected with B7 R and MG3-6 showed a significant increase in comparison to the spleens of animals infected with the other two isolates. After 90 days, this difference was maintained only for the group infected with B7 R. The histological analysis of the spleens showed that the proliferative response in lymphoid follicles was more evident in animals infected with B7 R, leading to central necrosis after 14 days. Acid-fast bacilli were found penetrating the perifollicular zone of these spleens, and also large blood vessels, suggesting blood dissemination. The intravenous infection corroborated the results obtained for intraperitoneal infections. After two days of infection, the recovery of colony forming units (CFUs) from spleen was significantly larger in animals infected with B7 R. Nevertheless, the infection was likewise controlled by all groups, similarly to the intraperitoneal infection. In the intravenous infection model, animals infected with B7 L, B67 and MG3-6 showed low production of TGF- in early time points. The infection of macrophages and epithelioid-like cells corroborated the in vivo findings, and B7 R was capable of proliferating inside macrophages and only for this isolate there was an increase in the percentage of infection in epithelioid-like cells. Furthermore, B7 R and B7 S stimulated a modest production of TGF- in these cultures when compared to the nonrelated isolates. Contrary to what was observed previously for M. avium, epithelioid-like cells were not able to restrain the infection caused by B7 R. Taken together, the results suggest that rough colony forming mycobacteria associated to outbreaks are more pathogenic than bacteria of smooth phenotype and those present in the environment, maybe because of the lower levels of TGF- upon mycobacteria-infected cells interaction. The underlying mechanisms are under investigation. / TEDE / BV UNIFESP: Teses e dissertações

The global burden of tuberculosis (TB) has reached an alarming status with numerous countries reporting an incidence of greater than 300 cases per 100 000 population per capita. By far, the most concerning statistic regarding TB is that estimates indicate 2 billion people worldwide are infected with Mycobacterium tuberculosis, the causative agent of TB. Only a small proportion (10%) of these individuals progress to active disease whilst the majority control the infection in an asymptomatic state that is termed latent TB infection (LTBI). These individuals carry a lifetime risk, which is significantly enhanced in the presence of HIV co-infection, of progressing to active disease. It has been hypothesized that during LTBI, the infecting bacterial population adopts a non-growing, dormant state and that progression to active disease is a result of reactivation of these organisms from dormancy. M. tuberculosis encodes for 5 homologues for the resuscitation promoting factor (Rpf), designated rpfA-E which have been shown to be important for reactivation from dormancy and critical for virulence during TB infection. As such, these factors may play an important role in bacterial reactivation during LTBI. In this study, we further characterize Rpf function in Mycobacterium smegmatis, a non-pathogenic relative of M. tuberculosis. M. smegmatis encodes four rpf–like homologues and using sequential deletion mutagenesis, we constructed a panel of deletion mutants that were defective in one, two, three or all four rpf-like genes in M. smegmatis. The successful construction of a quadruple mutant lacking all four rpf-like genes demonstrated that, as observed in M. tuberculosis, the rpf-like genes are collectively dispensable for growth of M. smegmatis. A select group of rpf deficient mutant strains were then characterized further. Our analysis indicates that the double ΔrpfA ΔrpfB, triple ΔrpfA ΔrpfB ΔrpfC, and quadruple ΔrpfA ΔrpfB ΔrpfC ΔrpfE, mutants displayed no growth defects in vitro but were impaired for biofilm formation and formed abnormal colonies that lacked the surface cording which is characteristic of mycobacterial colonies. Genetic complementation reversed these defects. Furthermore, the triple ΔrpfA ΔrpfB ΔrpfC, and quadruple ΔrpfA ΔrpfB ΔrpfC ΔrpfE, mutants displayed increased susceptibility to vancomycin, erythromycin and cephalosporins, suggesting some change in peptidoglycan structure or composition. Microscopic analysis of rpf deletion mutants revealed that the double ΔrpfA ΔrpfB, triple ΔrpfA ΔrpfB ΔrpfC, and quadruple ΔrpfA ΔrpfB ΔrpfC ΔrpfE, mutants displayed cells that were smaller than the wild type. In an interesting development, analysis of growth and survival in an in vitro model of mycobacterial dormancy indicated that the triple ΔrpfA ΔrpfB ΔrpfC, and quadruple ΔrpfA ΔrpfB ΔrpfC ΔrpfE mutants were unable to enter into a viable but non-culturable state. These results point to a novel role for Rpfs in the establishment of the dormant state in mycobacteria. Transmission Electron Microscopy on cells isolated from the dormancy model confirmed the progressive accumulation of inclusion bodies in the double ΔrpfA ΔrpfB, triple ΔrpfA ΔrpfB ΔrpfC, and quadruple ΔrpfA ΔrpfB ΔrpfC ΔrpfE mutants as these stains were passaged through our model of dormancy. Further analysis with Nile Red staining identified these inclusion bodies as lipid bodies. Collectively, our data reveal an important role for Rpfs in regulating essential growth processes and reveal a new role for Rpfs in the ordered shutdown of bacterial growth and establishment of dormancy in M. smegmatis.

Thesis (M.S.)--Oregon State University, 2006. / Printout. Includes bibliographical references (leaves 53-57). Also available on the World Wide Web.

Abstract not available

Thesis (MSc. (Medical Sciences)) -- University of Limpopo, 2016. / The reference phenotypic methods for Mycobacterium tuberculosis drug susceptibility testing are qualitative and based on drug critical concentrations. Limitations include lack of standardization and variations in laboratory preparation of drug stock solutions. The recommended critical concentrations are determined by consensus and experience rather than scientific data. Consequently incorrect and inadequate susceptibility breakpoints are used and patients receive ineffective antimicrobial therapy. The determination of wild-type minimal inhibitory concentration distribution is an important tool used by European Committee for antimicrobial susceptibility Testing (EUCAST) to establish clinical breakpoints in Europe. This could be applicable in South Africa. Aim To determine wild-type minimal inhibitory concentration distributions of first and secondline drugs against Mycobacterium tuberculosis complex clinical isolates and compare these with the recommended critical concentration in Limpopo province. Methods A sample of 101 Mycobacterium tuberculosis complex positive cultures were collected from National Health Laboratory Services in Polokwane (Limpopo province) and subcultured on BACTEC MGIT 960 system. The isolates were inoculated on MYCOTB MIC plates to determine the wild-type MIC distributions of first and second-line drugs. The data were compared with currently recommended critical concentrations. DNA was extracted and amplified by PCR. Genotypic drug susceptibility testing was performed using GenoType MTBDRplus version 2.0 and GenoType MTBDRsl version 2.0 for the first- and second-line drugs, respectively. Genotyping of clinical isolates was performed to determine M. tuberculosis strain families using spoligotyping. vi Results Wild-type MIC distributions range reported in this study are as follows rifampin (≤ 0.12 - 0.5 μg/μg/ml), isoniazid (≤ 0.3 - 2.00 μg/ml), rifabutin (≤ 0.12 - 0.25 μg/ml), ethionamide (≤ 0.12 - 5 μg/ml), ethambutol (≤ 0.5 - 2 μg/ml), streptomycin (≤ 0.25 - 0.5 μg/ml), paraaminosalicylic (≤ 0.5 - 4.0 μg/ml), cycloserine (≤ 2 -16 μg/ml), amikacin (≤ 0.12 - 0.5 μg/ml), kanamycin (≤ 0.6 -2.5 μg/ml), moxifloxacin (≤ 0.6 - 0.5 μg/ml), ofloxacin (≤ 0.25 - 1 μg/ml). GenoType MTBDRplus detected (n= 68, 67%) rifampin resistance (MUT 3=26, MUT 2=18, MUT 2B=8) on the rpoB gene. Isoniazid resistant (n=20, 19.8%) was detected katG MUT (n=20, 19.8%) on katG gene (S315T1). Genotypic resistance to second-line drugs determined by GenoType MTBRsl detected no mutations in (n= 98, 97%) isolates on gyrA, gyrB rrs and eis gene and (n=3, 2.9%) isolates non mycobacterium tuberculosis complex were detected. The frequency and percentage of Mycobacterium tuberculosis family strain were identified in (n= 81, 80%) of the clinical isolates which matched 18 pre-existing shared types. The results showed high genotype diversity with the Beijing strain (n= 30, 29.7%) and T family (n= 19, 18.8%) dominating. Twenty isolates (19.8%) had no shared types thus reported as orphan. Conclusion The findings obtained in this study suggest wild-type Minimal Inhibitory Concentration distributions may be considered when setting clinical breakpoints. Discordant results were observed between phenotypic and genotypic DST for rifampin, isoniazid, streptomycin, rifabutin and ethambutol, suggesting that breakpoint concentrations for some drugs are set too high while others are too low. The Mycobacterium tuberculosis clinical isolates displayed diverse family strain with Beijing and T strain predominate breakpoints for first-line and second-line drugs used in Mycobacterium tuberculosis treatments. Poster Presentations Poster presented at faculty of Health science first annual research day on Second-line drug susceptibility breakpoints for Mycobacterium tuberculosis using MYCOTB MIC plate. University of Limpopo Tiro hall 16th to 17th September 2014. Poster presented at National Health Laboratory Service Pathology Research and Development Congress (PathReD) on Determination of families strains of Mycobacterium tuberculosis circulating in Limpopo Province, South Africa. Emperors Palace 14th April-16th April 2015.

South Africa has a heavy burden of tuberculosis (TB) which is exacerbated by the concurrent epidemic of HIV. Molecular techniques have been used in most developed countries to investigate the dynamics of the TB epidemic, but despite the high prevalence of TB in sub-Saharan Africa, little data on strain types are available outside of the Western Cape. This study aims to provide information on the genotypic characteristics of multidrug-resistant (MDR) Mycobacterium tuberculosis strains in Johannesburg. Patient data obtained from the National Health Laboratory Service (NHLS) referral TB diagnostic laboratory and from Sizwe hospital, a MDR-TB referral hospital, were used to determine the risk factors for treatment outcomes in patients with MDR tuberculosis. Multidrug-resistant M. tuberculosis isolates from over 100 clinics and hospitals in Johannesburg were stored for the study. Spoligotyping and MIRU-VNTR were used to genotype the strains. Drug susceptibility profiles showed that 238 (55%) of the 434 M. tuberculosis isolates tested were resistant to streptomycin and ethambutol, in addition to being resistant to rifampicin and isoniazid. A comparison of spoligotyping results with the international spoligotyping database (SITVIT2) showed a total of 50 shared international types (SITs). Forty-five shared types, containing 417 isolates (96%) matched a pre-existing shared type whereas 5 shared types (containing 11 isolates) were newly created. Diverse strain types were noted, with Beijing, LAM, EAI, T, S, H and X families being dominant. Spoligotype defined families were split into sub-clusters by MIRU typing, resulting in 76 MIRU international types (MITs), containing 389 isolates and 45 orphan isolates. Spoligotyping showed lower discrimination (Hunter-Gaston discriminative index (HGDI) of 0.917) compared with MIRU typing (HGDI = 0.957) but there was no remarkable difference in the discriminatory power of combined spoligotyping and MIRU (HGDI = 0.962) compared with MIRU typing used alone. Twenty-four loci MIRU-VNTR typing was performed on strains from Beijing and CAS, EAI and H families to identify loci with high discriminatory power in our region. The proposed 15 MIRU-VNTR locus combination, together with MIRU 39, was found to be sufficient as a secondary typing method for the routine epidemiological investigation of the Beijing family isolates. Non-Beijing families could be sufficiently differentiated by the 15 MIRU locus combination. This study also describes the treatment outcomes of 351 MDR-TB patients at Sizwe hospital, who started treatment between 2004 and 2007, and investigates possible risk factors associated with poor outcomes. Final treatment outcome was available for 324 (92%) of the patients. Treatment success (completion and cure) was recorded in 158 (48.8%) of patients, while 73 (22.5%) had poor outcomes and 93 (28.7%) defaulted. Eleven (3.1%) patients were transferred out to another health facility and 16 (4.6%) had no recorded final outcome. The proportion of successful treatment increased significantly over time. Univariable and multivariable analysis (P = 0.05) identified the year of MDR-TB diagnosis and spoligotype-defined families as factors associated with treatment outcome. No associations were found between treatment outcome and HIV status, previous TB and additional MDR resistance to either streptomycin or ethambutol. The patient isolates were also characterised molecularly, complementing the study of isolates from Johannesburg alone, and providing information for the Gauteng Province. A sub-study illustrating genotypic diversity of the families constituting extensively drug-resistant TB (XDR-TB) strains in South Africa was conducted subsequent to the nosocomial outbreak in KwaZulu Natal (KZN). The results show that multiple, parallel development of resistance, rather than transmission alone, also plays an important role in the incidence of this extended form of resistance.