Gotowa bibliografia na temat „M. smegmatis - Moxifloxacin”

ABSTRACT The Mycobacterium tuberculosis Rv2686c-Rv2687c-Rv2688c operon, encoding an ABC transporter, conferred resistance to ciprofloxacin and, to a lesser extent, norfloxacin, moxifloxacin, and sparfloxacin to Mycobacterium smegmatis. The resistance level decreased in the presence of the efflux pump inhibitors reserpine, carbonyl cyanide m-chlorophenylhydrazone, and verapamil. Energy-dependent efflux of ciprofloxacin from M. smegmatis cells containing the Rv2686c-Rv2687c-Rv2688c operon was observed.

ABSTRACT The outer membrane of mycobacteria presents an effective permeability barrier for many antibiotics. Transport pathways across this membrane are unknown for most drugs. Here, we examined which antibiotics utilize the porin pathway across the outer membrane of the model organism Mycobacterium smegmatis. Deletion of the porins MspA and MspC drastically increased the resistance of M. smegmatis ML10 to β-lactam antibiotics, while its β-lactamase activity remained unchanged. These results are consistent with the ninefold-reduced outer membrane permeability of the M. smegmatis porin mutants for cephaloridine and strongly indicate that β-lactam antibiotics rely on the porin pathway. The porin mutant ML10 accumulated less chloramphenicol and norfloxacin and was less susceptible to these antibiotics than wild-type M. smegmatis. These results demonstrated that small and hydrophilic antibiotics use the Msp porins for entering the cell. In contrast to norfloxacin, the hydrophobic moxifloxacin was 32-fold more effective in inhibiting the growth of M. smegmatis, presumably because it was able to diffuse through the lipid membrane. Structural models indicated that erythromycin, kanamycin, and vancomycin are too large to move through the MspA channel. This study presents the first experimental evidence that hydrophilic fluoroquinolones and chloramphenicol diffuse through porins in mycobacteria. Thus, mutations resulting in less efficient porins or lower porin expression levels are likely to represent a mechanism for the opportunistic pathogens M. avium, M. chelonae, and M. fortuitum, which have Msp-like porins, to acquire resistance to fluoroquinolones.

Active efflux of drugs across the membrane is a major survival strategy of bacteria against many drugs. In this work, we characterize an efflux pump EfpA, from the major facilitator superfamily, that is highly conserved among both slow growing and fast-growing mycobacterium species and has been found to be upregulated in many clinical isolates of Mycobacterium tuberculosis . The gene encoding EfpA from Mycobacterium smegmatis was over-expressed under both constitutive and an inducible promoter. Expression of efpA gene under both the promoters resulted in greater than 32-fold increased drug tolerance of M. smegmatis cells to many first-line (rifampicin, isoniazid and streptomycin) and second-line (amikacin) anti-tuberculosis drugs. Notably, drug tolerance of M. smegmatis cells to moxifloxacin increased by more than 180-fold when efpA was over-expressed. The increase in minimum inhibitory concentration (MIC) correlated with the decreased uptake of drugs including norfloxacin, moxifloxacin and ethidium bromide and the high MIC could be reversed in the presence of an efflux pump inhibitor. A correlation was observed between the MIC of drugs and the efflux pump expression level, suggesting that the latter could be modulated by varying the expression level of the efflux pump. The expression of high levels of efpA did not impact the fitness of the cells when supplemented with glucose.The efpA gene is conserved across both pathogenic and non-pathogenic mycobacteria. The efpA gene from the Mycobacterium bovis BCG/ M. tuberculosis , which is 80% identical to efpA from M. smegmatis , also led to decreased antimicrobial efficacy to many drugs, although the fold-change was lower. When over-expressed in M. bovis BCG, an 8-fold higher drug tolerance to moxifloxacin was observed . This is the first report of an efflux pump from mycobacterium species that leads to higher drug tolerance to moxifloxacin, a promising new drug for the treatment of tuberculosis.

We had earlier reported the de novo emergence of genetic resisters of Mycobacterium tuberculosis and Mycobacterium smegmatis to rifampicin and moxifloxacin from the antibiotic-surviving population containing elevated levels of the non-DNA-specific mutagenic reactive oxygen species (ROS) hydroxyl radical. Since hydroxyl radical is generated by Fenton reaction between Fe(II) and H 2 O 2 , which is produced by superoxide dismutation, we here report significantly elevated levels of these three ROS and Fe(II) in the M. smegmatis rifampicin-surviving population.

ABSTRACT The bacterial populations surviving in the presence of antibiotics contain cells that have gained genetic resistance, phenotypic resistance and tolerance to antibiotics. Isolation of live bacterial population, surviving against antibiotics, from the milieu of high proportions of dead/damaged cells will facilitate the study of the cellular/molecular processes used by them for survival. Here we present a Percoll gradient centrifugation based method for the isolation of enriched population of Mycobacterium smegmatis surviving in the presence of bactericidal concentrations of rifampicin and moxifloxacin. From the time of harvest, throughout the enrichment and isolation processes, and up to the lysis of the cells for total RNA preparation, we maintained the cells in the presence of the antibiotic to avoid changes in their metabolic status. The total RNA extracted from the enriched population of live antibiotic-surviving population showed structural integrity and purity. We analysed the transcriptome profile of the antibiotic-surviving population and compared it with the orthologue genes of Mycobacterium tuberculosis that conferred antibiotic tolerance on tubercle bacilli isolated from the tuberculosis patients under treatment with four antitubercular antibiotics. Statistically significant comparability between the gene expression profiles of the antibiotic tolerance associated genes of M. smegmatis and M. tuberculosis validated the reliability/utility of the method.

ABSTRACT The emergence of antibiotic genetic resisters of pathogenic bacteria poses a major public health challenge. The mechanism by which bacterial antibiotic genetic resister clones formed de novo multiply and establish a resister population remained unknown. Here, we delineated the unique mode of cell division of the antibiotic genetic resisters of Mycobacterium smegmatis and Mycobacterium tuberculosis formed de novo from the population surviving in the presence of bactericidal concentrations of rifampicin or moxifloxacin. The cells in the rifampicin/moxifloxacin-surviving population generated elevated levels of hydroxyl radical-inflicting mutations. The genetic mutants selected against rifampicin/moxifloxacin became multinucleated and multiseptated and developed multiple constrictions. These cells stochastically divided multiple times, producing sister-daughter cells phenomenally higher in number than what could be expected from their generation time. This caused an abrupt, unexpectedly high increase in the rifampicin/moxifloxacin resister colonies. This unique cell division behavior was not shown by the rifampicin resisters formed naturally in the actively growing cultures. We could detect such abrupt increases in the antibiotic resisters in others’ and our earlier data on the antibiotic-exposed laboratory/clinical M. tuberculosis strains, M. smegmatis and other bacteria in in vitro cultures, infected macrophages/animals, and tuberculosis patients. However, it went unnoticed/unreported in all those studies. This phenomenon occurring in diverse bacteria surviving against different antibiotics revealed the broad significance of the present study. We speculate that the antibiotic-resistant bacillary clones, which emerge in patients with diverse bacterial infections, might be using the same mechanism to establish an antibiotic resister population quickly in the continued presence of antibiotics. IMPORTANCE The bacterial pathogens that are tolerant to antibiotics and survive in the continued presence of antibiotics have the chance to acquire genetically resistant mutations against the antibiotics and emerge de novo as antibiotic resisters. Once the antibiotic resister clone has emerged, often with compromise on growth characteristics, for the protection of the species, it is important to establish an antibiotic-resistant population quickly in the continued presence of the antibiotic. In this regard, the present study has unraveled multinucleation and multiseptation followed by multiple constrictions as the cellular processes used by the bacteria for quick multiplication to establish antibiotic-resistant populations. The study also points out the same phenomenon occurring in other bacterial systems investigated in our laboratory and others’ laboratories. Identification of these specific cellular events involved in quick multiplication offers additional cellular processes that can be targeted in combination with the existing antibiotics’ targets to preempt the emergence of antibiotic-resistant bacterial strains.

Bacterial persisters are a subpopulation of bacteria that can tolerate lethal concentrations of antibiotics. These are phenotypic variants that can give rise to drug‐susceptible population upon withdrawal of the antibiotic. Persistent bacteria play a crucial role in prolonging antibiotic treatment and are responsible for the recalcitrance of many chronic bacterial diseases, including tuberculosis. Several mechanisms have been proposed for the formation of persisters, which include expression of toxin‐antitoxin systems, generation of reactive oxygen species (ROS), and stochastic changes in gene expression and so on. Recent report from our laboratory has demonstrated that continuous prolonged exposure of Mycobacterium tuberculosis cells to lethal concentrations of antibiotics generates antibiotic persistence phase cells from which genetically resistant mutants emerge de novo either to the same antibiotic to which it was exposed to or to another antibiotic used for selection Sebastian et al., Antimicrobial Agents Chemotherapy 61(2), e01343‐16, 2016). The persistence phase cells showed high levels of oxidative stress that inflicted genome‐wide mutations in addition to the mutations for which the resistant mutants were selected against rifampicin and moxifloxacin. Thus, it was we demonstrated that the antibiotic persistence phase M. tuberculosis cells is a reservoir for the de novo emergence of antibiotic resistant mutants. In the present study, the response of Mycobacterium smegmatis upon prolonged exposure to rifampicin was examined since the bacilli has two mechanisms to inactivate or neutralise the action of rifampicin. These mechanisms include: (i). ADP‐ribosylation of rifampicin by the product of the gene ADP‐ribosyltransferase (arr); (ii). Rescue of rifampicin‐ mediated transcription inhibition by MsRbpA. The question asked was whether genetically resistant mutants against rifampicin would emerge from rifampicin persister phase cells, like in the case of M. tuberculosis cells and if they do, what are the mechanisms by which the rifampicin‐resistant mutants emerge from the persistence phase cells. For comparison and contrast purpose, and as a control sample, the response of M. smegmatis cells to moxifloxacin, against which the bacilli do not have any inherent inactivation or neutralisation mechanism, was studied. The Chapter 1, which forms the Introduction to the thesis, gives an extensive literature survey on all the different aspects of the research performed on the response of mycobacterial cells to antibiotics. The Chapter 2 presents in detail all the materials and methods used to perform the experiments. A large number of cell biological and molecular biological methods, such as fluorescence microscopy and fluorescence measurements, flow cytometry, cloning and expression, real time RT‐PCR and whole genome sequencing, and biophysical methods such as electron paramagnetic resonance spectrometry, and others were used to perform the experiments. The data Chapter 3 presents the data on the response of M. smegmatis cells to rifampicin. The data shows that exposure to MBC levels of rifampicin results in the killing of the cells to a 5‐log10 reduction in the cfu of M. smegmatis cells but the remaining cells persist and from these cells emerge rifampicin‐resistant mutants. The persistence phase cells were found to generate elevated levels of hydroxyl radical, which inflicted genome‐wide mutations, and the mutants harbouring nucleotide changes at the rifampicin resistance determining region (RRDR) could regrow back. Interestingly, the killing phase and the regrowth phase showed very low levels of hydroxyl radical unlike the persistence phase cells. The mutations, which are identical to those in the rifampicin‐resistant mutants have been reported in the M. tuberculosis cells isolated from in vitro cultures and from the TB patients. The data Chapter 4 presents the response of the arr knockout mutant to rifampicin. The persistence phase population of the arr knockout mutant showed significantly higher levels of hydroxyl radical generation than the equivalent persistence phase population of the wild type cells. While the wild type cells showed emergence of rifampicin‐resistant mutants from the persistence phase, the arr knockout mutant showed the emergence of rifampicin‐ resistant mutants from the very exposure of the cells to rifampicin. In other words, the natural mutation frequency of the arr knockout mutant was significantly higher than that of the wild type. This indicated that the arr gene might have a natural role in keeping the oxidative stress at lower levels in the cells, which needs further investigation. The data Chapter 5 presents the response of M. smegmatis cells to moxifloxacin. Here also, the bacilli exposed to lethal concentrations of moxifloxacin showed a killing phase, followed by a persistence phase and a regrowth phase. The moxifloxacin‐resistant mutants were found to emerge from the moxifloxacin persistence phase cells. The cells from the regrowth phase of moxifloxacin‐exposed cells showed mutations in the quinolone resistance determining region (QRDR) in the gyrase gene, which is the target of moxifloxacin. The mutations, which are identical to those in the rifampicin‐resistant mutants have been reported in the M. tuberculosis cells isolated from in vitro cultures and from the TB patients. The thesis is concluded with discussion of the findings presented in the three chapters by projecting the comparison and contrast of the response of M. smegmatis and M. tuberculosis cells to rifampicin. The thesis contains an extensive bibliography.