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Efflux pumps as a possible mechanism of resistance to 2nd line drugs: phenotypic and genotypic evaluation in resistant clinical isolates of Mycobacterium tuberculosis

Grant number: 18/12135-1
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): September 01, 2018
Effective date (End): February 29, 2020
Field of knowledge:Biological Sciences - Microbiology - Applied Microbiology
Principal researcher:Fernando Rogério Pavan
Grantee:Júlia Araújo Grecco
Home Institution: Faculdade de Ciências Farmacêuticas (FCFAR). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil

Abstract

On the planet, the Tuberculosis (TB) mortality rate decreased by 47% since 1990 to 2015, however, an estimated 1.3 million TB deaths by 2016. This means that, even as the mortality rate decreases over the years, it is still considered one of the 10 leading causes of death worldwide, and an important contributing factor is the emergence of multidrug resistant strains (MDR- TB) and / or extensively resistant (XDR-TB). Among resistance mechanisms of Mycobacterium tuberculosis, the main causative agent of the disease, mutations in drug target genes or in metabolizing enzymes account for most, but not all, cases of antibiotic resistance. Efflux pumps (EP) are membrane-carrying proteins capable of expelling several metabolic substances from the mycobacteria, as well as antimicrobial agents, and are also a mechanism of resistance. It is known that when M. tuberculosis is exposed to subinhibitory concentrations of drugs, it may increase the expression and / or activity of its EP in order to resist the presence of these antimicrobials. In view of these considerations, this work has the objective of searching for the possible mechanism of resistance of 21 clinical isolates, being 12 resistant to aminoglycosides and 9 resistant to fluoroquinolones (FLQ), which did not show mutations, previously studied, in rrs and gyrA genes, the main related to aminoglycoside resistance and FLQ, respectively. To evaluate the effect of the antimicrobial presence on the activity and the expression of the EP, a pre-treatment of the clinical isolates with 24 and 48h exposure at subinhibitory concentrations will be done and also withdrawing this exposure for an equal period to then use the accumulation methodologies of ethidium bromide and RT-PCR. By analyzing EP as a mechanism of resistance from the phenotypic perspective, using the combination with BE inhibitors, and from the genotypic perspective by analyzing the expression of some resistance genes already related to EP. It is expected to better understand the EP behavior of resistant bacteria in the presence of antimicrobials and, if possible, to elucidate for this library of clinical isolates the resistance mechanism. In addition, to our knowledge, is this the first work that seeks to answer whether the increase in the expression of BE after exposure to antimicrobials is a constitutive or inductive factor?

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