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Sources of Mycobacterium bovis contamination remain unclear for many cases of animal and human disease. A major limitation is the lack of sufficiently informative or epidemiologically well evaluated molecular methods for typing. Here, we report an evaluation of a high-throughput method based on 29 mycobacterial interspersed repetitive unit-variable-number tandem-repeat (MIRU-VNTR) loci to genotype 127 M. bovis isolates from cattle from 77 different Belgian farms, representative of a nationwide collection obtained from 1995 to 2003. MIRU-VNTR stability was demonstrated by analyzing a series of 74 isolates in total, obtained from different animals from a single farm or from different farms with an identified epidemiological link. The genotyping results and the genotypic diversity (h) were compared with those obtained by IS6110 restriction fragment length polymorphism (RFLP) analysis and spoligotyping. Among 68 isolates with no known epidemiological link, MIRU-VNTR typing discriminated better than either RFLP analysis or spoligotyping, with isolates taken individually (32 versus 16 and 17 genotypes; h = 0.91 versus 0.73 and 0.85, respectively) or in combination (32 versus 28 genotypes; h = 0.91 versus 0.92). Maximal resolution was already achieved with a subset of 9 loci. The observed congruence of the genetic relationships based on IS6110 RFLP analysis, spoligotyping, and MIRU-VNTR markers is consistent with a clonal population structure of M. bovis. These results support MIRU-VNTR typing as a convenient and discriminatory technique for analysis of the population structure of M. bovis in much greater detail and for addressing some still unresolved issues in the epidemiology of the pathogen.

Mycobacterium tuberculosis strains of the Beijing lineage are globally distributed and are associated with the massive spread of multidrug-resistant (MDR) tuberculosis in Eurasia. Here we reconstructed the biogeographical structure and evolutionary history of this lineage by genetic analysis of 4,987 isolates from 99 countries and whole-genome sequencing of 110 representative isolates. We show that this lineage initially originated in the Far East, from where it radiated worldwide in several waves. We detected successive increases in population size for this pathogen over the last 200 years, practically coinciding with the Industrial Revolution, the First World War and HIV epidemics. Two MDR clones of this lineage started to spread throughout central Asia and Russia concomitantly with the collapse of the public health system in the former Soviet Union. Mutations identified in genes putatively under positive selection and associated with virulence might have favored the expansion of the most successful branches of the lineage.

Tuberculosis (TB) is a potentially fatal contagious disease that can affect almost any part of the body but is mainly an infection of the lungs. It is caused by micro-organisms of the Mycobacterium tuberculosis complex. It is the second greatest killer worldwide due to a single infectious agent, after the Human Immunodeficiency Virus (HIV). Without treatment, fatality is 50% in immune competent persons. TB remains the leading cause of death among HIV positive persons, causing one fifth of the deaths. The World Health Organization estimates that one third of the world population is infected by this micro-organism but only 5 to 10% develop TB disease. Nevertheless, this enormous reservoir leads to around 1.4 millions deaths annually. Standard curative treatment lasts at least 6 months and includes 4 different drugs. Toxicity of the drugs leading to (severe) adverse events and the long duration of the daily administration challenges patient’s compliance. Subinhibitory concentration of the drugs (due to poor adherence) can induce resistance of the mycobacteria to the provided drugs. Unlike most bacteria where resistance is acquired by plasmids, drug resistance of mycobacteria is obtained by genomic mutations. “Multi drug-resistant tuberculosis (MDR-TB)” is strictly defined as TB resistant to specifically isoniazid and rifampicin, the two main first line drugs. “Extensively drug resistance (XDR)” is defined as MDR-TB with additional resistance to any of the fluoroquinolones (such as ofloxacin or moxifloxacin) and to at least one of three injectable second-line drugs (amikacin, capreomycin or kanamycin). The increase of MDR-TB represents an enormous challenge to Public Health globally. This research examined different aspects of tuberculosis resistance performed in the Belgian National Reference Center, a clinical laboratory setting. First of all, a profound analysis of the MDR-TB situation in Belgium was conducted. It is the first retrospective population-based survey of MDR-TB in Belgium, covering a 15-year period (1994-2008). It comprises 174 patients representing more than 80% of the culture positive MDR-TB patients reported to the Belgian register, thus this study is considered of national relevance. It includes bacteriological and molecular data on the isolates as well as clinical aspects of the patients and treatment results. Considering only the patient’s first MDR-TB isolate, an increase over time was observed in the number of isolates resistant to a second-line drug as well as the total number of drugs each isolate was resistant to. XDR-TB was detected since 2002 and panresistant TB (resistant to every available antituberculosis drug) since 2009. Overall, a successful treatment outcome was obtained for 67.8% of the MDR-TB cases. Drug susceptibility testing (DST) of Mycobacterium tuberculosis to first line drugs (isoniazid, rifampicin, ethambutol and pyrazinamide) in liquid culture medium has a turn around time of at least two weeks, after identification of the positive culture (obtained after 2 to 4 weeks) from the patient’s clinical isolate. In order to provide the clinician with valuable information about the isolated mycobacteria leading to patient adapted therapy before bacteriological DST results are available, resistance is predicted by detection of mutations in certain genes of the mycobacteria. It is common practice for rifampicin (rpoB gene) and isoniazid (katG gene and/or inhA promoter region). In this MDR-TB collection, rifampicin resistant related mutations were found in 97.1% (168/173) of the clinical isolates and isoniazid resistant related mutations in 94.1% (160/170). The pncA, embB and gyrA genes have been sequenced to identify possible mutations because of their possible involvement with resistance to pyrazinamide, ethambutol and the fluoroquinolones respectively. However, little is known about the resistance prediction value of the mutations in these genes. The study is also the first study on the molecular epidemiology of MDR-TB in the country. DNA fingerprinting showed a large diversity of strains (67% of the patients were infected by a strain with a unique pattern) and further epidemiological examination revealed limited local transmission of MDR-TB in Belgium. The second part investigated the pncA gene and its association with pyrazinamide resistance in MDR-TB isolates from Belgium and in vitro cultured spontaneous mutants. The genetic analysis showed that 98.3% (59/60) of the Belgian clinical MDR pyrazinamide resistant (PZAR) isolates present a mutation in the pncA gene. We found 1.7% (1/60) of the PZAR MDR-isolates encoding wild type pncA and flank. A total (PZAR and PZAS) of 41 different amino acid changes, 3 protein truncations and 5 frameshifts were observed including eight novel mutations: 8Asp>Ala, 13Phe>Leu, 64Tyr>Ser, 107Glu>stop, 143Ala>Pro, 172Leu>Arg and frameshifts starting in codon 55 and 82. Analysis of all observed mutations (i.e. in clinical isolates as well as spontaneous mutants) revealed that they are not always associated with drug resistance and that they are not scattered randomly throughout the gene, but occur rather at preferential sites such as in codons with amino acids associated with either iron or substrate binding and catalytic active sites. The frequency of in vitro mutagenesis to pyrazinamide at pH 6.0 was determined and found to be relatively high at 10-5 CFU/ml. Finally, the in vitro activity of tobramycin and clarithromycin (with unclear efficacy against M. tuberculosis) was evaluated on 25 M. tuberculosis clinical isolates with various resistance profiles. The effect of the drugs administered together was examined for possible synergistic effect. The median minimum inhibitory concentration (MIC) of 8 µg/ml obtained for both drugs in this study is rather high but are beyond the concentrations obtained in lung tissues. This suggests that both drugs should be investigated further as potential adjuncts to the treatment of resistant TB when other alternatives have failed; in particularly through new drug delivery systems such as the Dry Power Inhaler which allows local drug deposition with high drug concentrations in the lungs but low toxicity due to limited systemic absorption. In addition, for 36% of the tested isolates a decrease of the MIC of clarithromycin by a single or twofold dilution was observed in the presence of a subinhibitory concentration of tobramycin and no antagonistic effect was seen for the remaining isolates. This research illustrates different (laboratory) aspects in the fight against drug resistant TB, all using the Belgian TB collection: characterisation of the Belgian MDR-TB situation on bacteriological, molecular and epidemiological level; profound analysis of genomic mutations and their possible association with drug resistance; and investigation of synergistic activity of drugs with low efficacy against M. tuberculosis., Doctorat en Sciences biomédicales et pharmaceutiques, info:eu-repo/semantics/published