Antibiotic resistance in mycobacterium tuberculosis : the role of genetic mutations in resistance conferring genes and efflux transporters.

Abstract

Two decades after the World Health Organisation (WHO) declaration of tuberculosis (TB) as a global emergency, the disease remains a public health crisis of epic proportions. The emergence of drug resistant strains of Mycobacterium tuberculosis, the etiologic agent of TB, and the convergent human immunodeficiency virus (HIV) epidemic places a devastating burden on an already weakened public health care system in South Africa. Rapid and accurate detection of drug resistance to first and second line drugs to guide effective treatment of TB is central to control of the disease and in preventing further dissemination of drug resistant strains. Knowledge of the underlying resistance mechanisms driving drug resistance in M.tuberculosis is pivotal in the design of rapid molecular based assays and will impact of the development of novel drugs and regimens for the disease. The manuscript in chapter 2 of this thesis, entitled Dynamics of antimicrobial resistance in Multi-Drug and Extensively Drug resistant strains of Mycobacterium tuberculosis in KwaZulu-Natal, South Africa, demonstrated the diversity of the resistance mechanisms amongst the multidrug resistant (MDR) TB strains currently circulating in the KwaZulu-Natal province of South Africa by the analysis of the rpoB, katG, inhA, pncA and embB genes associated with resistance to key drugs used in the treatment of TB. Multiple drug resistance mechanisms in the MDR-TB isolates suggests that the strains emerged separately and acquired resistance mutations independently. The findings of this study also confirms the clonality of the XDR-TB epidemic demonstrated by the predominance of the F15/LAM4/KZN strain family and reveals that MDR-TB strains are evolving and spreading via transmission. The manuscript in chapter 3 of this thesis, entitled Streptomycin resistance in the F15/LAM4/KZN strain of Mycobacterium tuberculosis is mediated by lineage-specific

alteration of the gidB gene, demonstrated that streptomycin (STR) resistance in the F15/LAM 4/KZN MDR and XDR-TB strains was mediated by a rare, 130bp deletion within the gidB gene of M.tuberculosis leading to a complete disruption of the gene. Classical mutations in the rpsL gene mediated STR resistance in the remaining strain families. Widespread STR resistance has resulted in the exclusion of the drug from current treatment regimens. The findings of this study support the decision of policymakers and cautions the application of the drug in the absence of drug susceptibility testing. The manuscript in chapter 4 of this thesis, entitled Moxifloxacin resistance in the F15/LAM4/KZN extensively drug-resistant strain of Mycobacterium tuberculosis, demonstrated that the F15/LAM4/KZN XDR strain harboured the A90V gyrA mutation associated with high level ciprofloxacin (CPX) and ofloxacin (OFX) resistance and correlated with increased minimum inhibitory concentrations (MIC) for moxifloxacin (MXF). The results of this study cautions the utilization of MXF as part of empiric treatment protocols in the absence of moxifloxacin MIC data of the circulating XDR strains in an area. It also raises concerns regarding the regarding the use of moxifloxacin in KwaZulu-Natal. Furthermore, the current breakpoint defining resistance to MXF is of concern and requires revision. The manuscript in chapter 5 of this thesis, entitled Evaluation of Capreomycin in the treatment of the F15/LAM4/KZN extensively drug-resistant strain of Mycobacterium tuberculosis demonstrated that the A1401G rrs mutation was the main mechanism mediating resistance to the aminoglycosides, kanamycin (KAN) and amikacin (AMIK); and to capreomycin (CAP). CAP was reintroduced into TB treatment protocols without prior drug susceptibility testing. This results of this study demonstrates high level resistance to CAP and urges careful consideration in the application of CAP the KwaZulu-Natal province. Furthermore, concerns regarding the high breakpoint value that defines CAP resistance as

compared to wild-type MICs for the drug results in misdiagnosis of resistance that results inadequate patient treatment and amplifies resistance. The manuscript in chapter 6 of this thesis, entitled KZN Multidrug and Extensively drug resistant strains of Mycobacterium tuberculosis remain susceptible to Linezolid and para-Amino salicylic Acid, demonstrated that the mechanisms most commonly associated with resistance to the linezolid (LIN) and para-amino salicylic acid (PAS) were absent in the MDR and XDR-TB strains in this study. Mutations detected in the drug targets were lineage specific markers rather than resistance mechanisms. This study also highlights the poor understanding of resistance to these drugs and the need for further study to allow for resistance detection to be incorporated into diagnostic assays, thus prolonging the utility of these drugs. The manuscript in chapter 7 of this thesis, entitled Efflux mediated drug resistance in clinical isolates of Mycobacterium tuberculosis in KwaZulu-Natal, South Africa, demonstrated the role of efflux pumps in mediating low level resistance. The results of this study supports the hypothesis that efflux activity leads to decreased intracellular antibiotic concentrations, thereby allowing the survival of a sub-population of bacteria under the sub-inhibitory level of the antibiotic, from which resistant mutants emerge, leading to clinically significant levels of resistance. The results of this study strongly supports the application of efflux pump inhibitors as adjunctive to the current treatment protocols. The results emanating from this thesis has contributed to the body of knowledge of drug resistance in M.tuberculosis, especially in the KwaZulu-Natal province of South Africa. Furthermore, the results can be used to guide treatment protocols and contributes to the future development of molecular based assays aimed at detecting resistance.