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Transmission of Mycobacterium tuberculosis bacilli from one individual to another is the basis of the disease process. While considerable emphasis has been placed on the role of host mechanisms of resistance in establishing or preventing new infection, far less has been expended on understanding possible factors operative at the bacterial level. In this study we established a panel of clinical isolates of M. tuberculosis strains obtained from the Western Cape region of South Africa, each of which had been carefully tracked in terms of their degree of transmission in the community. Each of the panel were used to infect guinea pigs with 15–20 bacilli by aerosol exposure and the course of the infection then determined. Strains with different degrees of transmission could not be distinguished in terms of their capacity to grow in the main target organs of infected animals. However, rather surprisingly, while strains with no evidence of transmission [NOT] in general caused moderate to severe lung damage, this parameter in animals infected with highly transmitted [HT] strains was mostly mild. In terms of TH1 immunity these signals were strongest in these latter animals, as was IL-17 gene expression, whereas minimal signals for regulatory molecules including IL-10 and FoxP3 were seen across the entire panel. In terms of T cell numbers, responses of both CD4 and CD8 were both far faster and far higher in animals infected with the HT strains. At the gene expression level we observed a major three-fold difference [both up and down] between NOT and HT strains, but in terms of proteins of key interest only a few [including PD-L1 and HIF-3] showed major differences between the two groups. Overall, it was apparent that NOT strains were far more inflammatory that HT strains, and appeared to trigger a much larger number of genes, possibly explaining the observed damage to the lungs and progressive pathology. In contrast, the HT strains, while equally virulent, were more immunogenic and developed much stronger T cell responses, while keeping lung damage to a minimum. Hence, in terms of trying to explain the capacity of these strains to cause transmission, these results are clearly paradoxical. [ABSTRACT FROM AUTHOR]

A series of molecules containing bulky lipophilic scaffolds was screened for activity against Mycobacterium tuberculosis and a number of compounds with antimycobacterial activity were identified. The most active compound, (2 E)- N -(adamantan-1-yl)-3-phenylprop-2-enamide (C1), has a low micromolar minimum inhibitory concentration, low cytotoxicity (therapeutic index = 32.26), low mutation frequency and is active against intracellular Mycobacterium tuberculosis. Whole genome sequencing of mutants resistant to C1 showed a mutation in mmpL 3 which may point to the involvement of MmpL3 in the antimycobacterial activity of the compound. In silico mutagenesis and molecular modelling studies were performed to better understand the binding of C1 within MmpL3 and the role that the specific mutation may play in the interaction at protein level. These analyses revealed that the mutation increases the energy required for binding of C1 within the protein translocation channel of MmpL3. The mutation also decreases the solvation energy of the protein, suggesting that the mutant protein might be more solvent-accessible, thereby restricting its interaction with other molecules. The results reported here describe a new molecule that may interact with the MmpL3 protein, providing insights into the effect of mutations on protein-ligand interactions and enhancing our understanding of this essential protein as a priority drug target. • A new MmpL3 inhibitor with intracellular antimycobacterial activity was identified. • The compound has a good selectivity index and low mutation frequency. • In vitro mutagenesis identified resistance-associated mutations. • Molecular modelling suggested how an observed mutation causes drug resistance. • MmpL3 inhibitors that remain effective despite resistance mutations can be designed. [ABSTRACT FROM AUTHOR]

Summary This study was part of a larger cross-sectional survey that was evaluating tuberculosis (TB) infection in humans, livestock and wildlife in the Serengeti ecosystem in Tanzania. The study aimed at evaluating the genetic diversity of Mycobacterium tuberculosis isolates from TB patients attending health facilities in the Serengeti ecosystem. DNA was extracted from 214 sputum cultures obtained from consecutively enrolled newly diagnosed untreated TB patients aged ≥18 years. Spacer oligonucleotide typing (spoligotyping) and Mycobacterium Interspersed Repetitive Units and Variable Number Tandem Repeat (MIRU-VNTR) were used to genotype M. tuberculosis to establish the circulating lineages. Of the214 M. tuberculosis isolates genotyped, 55 (25.7%) belonged to the Central Asian (CAS) family, 52 (24.3%) were T family (an ill-defined family), 38 (17.8%) belonged to the Latin American Mediterranean (LAM) family, 25 (11.7%) to the East-African Indian (EAI) family, 25 (11.7%) comprised of different unassigned (‘Serengeti’) strain families, while 8 (3.7%) belonged to the Beijing family. A minority group that included Haarlem, X, U and S altogether accounted for 11 (5.2%) of all genotypes. MIRU-VNTR typing produced diverse patterns within and between families indicative of unlinked transmission chains. We conclude that, in the Serengeti ecosystem only a few successful families predominate namely CAS, T, LAM and EAI families. Other types found in lower prevalence are Beijing, Haarlem, X, S and MANU. The Haarlem, EAI_Somalia, LAM3 and S/convergent and X2 subfamilies found in this study were not reported in previous studies in Tanzania. [ABSTRACT FROM AUTHOR]

Abstract: Drug resistant tuberculosis (TB) has reached alarming proportions in South Africa, draining valuable resources that are needed to fight drug susceptible TB. It is currently estimated that 9.6% of all TB cases have multi-drug resistant (MDR)-TB, thereby ranking South Africa as one of the highest MDR-TB burden countries in the world. Molecular epidemiological studies have demonstrated the complexity of the epidemic and have clearly shown that the epidemic is driven by transmission as a consequence of low cases detection and diagnostic delay. The latter has in turn fueled the amplification of drug resistance, ultimately leading to the emergence of extensively drug resistant (XDR)-TB. Despite the introduction of new drugs to combat this scourge, culture conversion rates for XDR-TB remain below 20%. Failure to achieve cure may be explained from DNA sequencing results which have demonstrated mutations in 7 genes encoding resistance to at least 8 anti-TB drugs. This review shows how molecular epidemiology has provided novel insights into the MDR-TB epidemic in South Africa and thereby has highlighted the challenges that need to be addressed regarding the diagnosis and treatment of MDR-TB. An important step towards for curbing this epidemic will be collaboration between clinicians, laboratories and researchers to establish scientific knowledge and medical expertise to more efficiently guide public health policy. [Copyright &y& Elsevier]

Summary Pyrazinamide resistance is largely unknown in the spectrum of drug resistant phenotypes. We summarize data on PZA resistance in clinical isolates from South Africa. PZA DST should be performed when considering its inclusion in treatment of patients with rifampicin-resistant TB or MDR-TB. [ABSTRACT FROM AUTHOR]

Whole genome sequencing (WGS) can investigate the entire Mycobacterium tuberculosis (Mtb) genome but currently requires large amounts of mycobacterial DNA, necessitating culture. Culture-free Mtb WGS could revolutionize the clinical use of WGS but is hampered by the high viscosity, low mycobacterial load, and high contamination with bacterial and human DNA in sputum samples. To improve the sputum liquefaction and decontamination step prior to DNA extraction, we assessed the efficiency of Myco-TB, MycoPrep, and Sputolysin with/without TiKa-Kic in liquefying and decontaminating sputum and aimed to evaluate the effect of these approaches on mycobacterial viability, and Mtb DNA quality and quantity. Experiments using spiked sputum samples showed that Myco-TB and BD MycoPrep with standard (15 min) or increased (30 min) incubation time, but not reduced (7,5 min) incubation time performed well in liquefying and decontaminating sputum. No difference in DNA quality or quantity, contamination, or the amount of human DNA present was observed. In comparison, Sputolysin with/without TiKa-Kic was less effective for liquefaction and decontamination of sputum. PCR amplification of the human GAPDH gene after sputum treatment, showed the presence of human DNA in all samples, regardless of sputum treatment. Focused efforts are needed to deplete contaminating DNA for culture-free Mtb WGS. • Myco-TB and BD MycoPrep have similar liquefaction and decontamination efficiency. • Sputolysin ± TiKa-Kic is not effective for sputum liquefaction and decontamination. • DNase treatment of sediments did not completely deplete human DNA. [ABSTRACT FROM AUTHOR]

Summary: We report a case of extensive necrogranulomatous pneumonia due to infection with the dassie bacillus (Mycobacterium tuberculosis complex sp.) in a free-living pregnant adult female dassie (rock hyrax—Procavia capensis). A juvenile female dassie from the same colony also showed a focal lesion in the lungs suggestive of mycobacterial pneumonia. Our findings indicate the widespread occurrence of the dassie bacillus in free-living dassies and suggest very high infection rates in some populations. The introduction of South African dassies into novel environments should be considered in this light. [Copyright &y& Elsevier]

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a major cause of death worldwide. Diverse genotypes have been demonstrated to drive the epidemiology of drug resistant (DR-) TB globally. Currently, there is limited knowledge on the genotypes and transmission dynamics of M. tuberculosis in Zambia. This study aimed to describe the genotypes of DR-TB from the Copperbelt and Northern regions of Zambia. Molecular typing tools of insertion sequence 6110- restriction fragment length polymorphism (IS 6110- RFLP) and spacer oligonucleotide typing (spoligotyping) were applied. We demonstrate that diverse genotypes are associated with DR-TB in Zambia. The predominant genotype was lineage 4; other strains belonged to lineage 2 and 3. Genotypes previously identified as driving the epidemiology of drug susceptible TB have been identified as drivers of DR-TB. Genotyping analysis showed clustering of strains among patients from different regions of the country; suggesting that DR-TB is widespread. Molecular findings combined with phenotypic and epidemiologic findings play a critical role in identifying circulating genotypes and possible transmission chains. Clustering of drug resistant strains was demonstrated to be 48% and 86% according to IS 6110 -RFLP and spoligotyping, respectively. However, gaps in clinical and demographic data skew the interpretation, and call for data collection policy improvements. [ABSTRACT FROM AUTHOR]