Abstract
Tuberculosis (TB) is a public health threat and is among the world’s most lethal infectious diseases. Globally, Cambodia ranks 15th among the 30 countries with the highest TB incidence rate. The increasing emergence of MDR-TB as well as pre-XDR and XDR-TB has been observed in this country. However, the mechanisms underlying this escalation of resistance are still poorly understood. In this context, this thesis aims to understand the emergence, spread and evolution of antibiotic resistance in Mycobacterium tuberculosis (M.tb) in Cambodia.Two collections of M.tb are included in the study: (i) 404 isolates from MDR-TB presumptive patients between 2012 and 2017, (ii) 19 isolates from bears from a wildlife rescue center and one isolate from a staff member. The isolates were analyzed by two techniques: spoligotyping, 24-MIRU-VNTR, sequencing of drug resistance genes and drug susceptibility testing for first (FLD) and second line (SLD) drugs.First, this work describes the genetic diversity of the clinical M.tb isolates and their link with FLD resistance. The two predominant families, Beijing and EAI, represent almost 90% of the sample. The analysis showed a significant association between the Beijing family and phenotypic drug resistance, in particular with MDR and quadruple resistance. The clustering suggests the existence of recent transmissions also associated with the Beijing family.Second, while focusing on resistance to SLD, the data suggest that the proportion of XDR and pre-XDR isolates remains low but is on the rise compared to previous reports. The Beijing family was predominant among these highly resistant isolates. One Beijing isolate, named XDR+, was resistant to all anti-TB drugs tested (FLD and SLD). A cluster comprising 2 pre-XDR and XDR isolates was observed, suggesting recent transmission of these strains.Third, the genetic determinants of resistance to FLD and SLD and the evolution of resistance over time were studied. The data demonstrated a diversity of drug resistant patterns from mono-resistance to XDR, an important diversity of mutation patterns in each M.tb family and each cluster and the existence of compensatory mutations in some resistant isolates. These results suggest various evolutionary trajectories towards resistance to FLD and SLD. The Beijing family was also associated with MDR and XDR and low fitness cost mutations in resistant genes. Our data suggest a cumulative effect of mutations, a role of epistasis in the acquisition of multiple resistance and the spread of highly resistant and transmissible isolates in the population.Finally, concerning the M.tb in bear population and the staff isolate, my work has confirmed a human-animal transmission of M.tb sensitive and resistant with the presence of two clusters; EAI cluster and Beijing streptomycin and isoniazid resistant cluster including the human case. The timing and exposure indicated probable transmission from bear to human and back to bear.In conclusion, this work provides knowledge on the diversity, population structure of M.tb and resistance to FLD and SLD in our sample collected from MDR-TB presumptive patients between 2012 and 2017. These data predict an evolution of resistance to a more problematic situation in the future. First, the Beijing family is associated with MDR and XDR as well as mutations associated with high level of resistance and low fitness cost and recent transmission. Second, the proportion of XDR and pre-XDR isolates remains low but appears to be increasing over time. This study strongly indicates the need for rapid interventions in terms of diagnostic and treatment to prevent the spread of the Beijing pre-XDR and XDR isolates in the population and the emergence of more resistant strains. This work also illustrates that the susceptibility of the endangered species, Helarctos malayanus sun bear, to human TB in particular to resistant TB poses problems in terms of conservation of the species but also of public health.