Academic literature on the topic 'Mycobacterium smegmatis - Moxifloxacin'

ABSTRACTAntibiotic therapy of infections caused by the emerging pathogenMycobacterium abscessusis challenging due to the organism's natural resistance toward most clinically available antimicrobials. We investigated the bactericidal activity of antibiotics commonly administered inM. abscessusinfections in order to better understand the poor therapeutic outcome. Time-kill curves were generated for clinicalM. abscessusisolates,Mycobacterium smegmatis, andEscherichia coliby using antibiotics commonly categorized as bactericidal (amikacin and moxifloxacin) or bacteriostatic (tigecycline and linezolid). In addition, the impact of aminoglycoside-modifying enzymes on the mode of action of substrate and nonsubstrate aminoglycosides was studied by usingM. smegmatisas a model organism. While amikacin and moxifloxacin were bactericidal againstE. coli, none of the tested compounds showed bactericidal activity againstM. abscessus. Further mechanistic investigations of the mode of action of aminoglycosides inM. smegmatisrevealed that the bactericidal activity of tobramycin and gentamicin was restored by disruption of the chromosomalaac(2′) gene in the mycobacterial genome. The lack of bactericidal antibiotics in currently recommended treatment regimens provides a reasonable explanation for the poor therapeutic outcome inM. abscessusinfection. Our findings suggest that chromosomally encoded drug-modifying enzymes play an important role in the lack of aminoglycoside bactericidal activity against rapidly growing mycobacteria.

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 Quinolones were examined for rapid lethal activity against Mycobacterium smegmatis in the presence and absence of chloramphenicol, an inhibitor of protein synthesis. C-8 methoxy, C-6 fluorine, and particular C-7 ring substituents enhanced rapid killing. With the surprising exception of moxifloxacin, higher quinolone concentrations were required for lethal activity in the presence of chloramphenicol than in its absence. Moxifloxacin was also unusual in lacking the time lag characteristic of fluoroquinolone lethality. Several fluoroquinolone dimers, which represent quinolones with large C-7 substituents, showed modest bacteriostatic activity. Unlike other quinolones, the dimers failed to display lethal activity. The insensitivity of moxifloxacin to chloramphenicol has not been observed with other bacteria and may therefore reflect unique aspects of mycobacterial gyrase.

ABSTRACTThe absence of the Holliday-junction Ruv resolvase ofMycobacterium smegmatisincreased the bacteriostatic and bactericidal activities of the fluoroquinolone moxifloxacin, an important antituberculosis agent. The treatment ofruvAB-deficient cells with thiourea and 2,2′-bipyridyl lowered moxifloxacin lethality to wild-type levels, indicating that the absence ofruvABstimulates a lethal pathway involving reactive oxygen species. A hexapeptide that traps the Holliday junction substrate of RuvAB potentiated moxifloxacin-mediated lethality, supporting the development of small-molecule enhancers for moxifloxacin activity against mycobacteria.

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.

ABSTRACTAn agar plate assay was developed for detecting the induction of drug-resistant mycobacterial mutants during exposure to inhibitors of DNA gyrase. WhenMycobacterium smegmatison drug-containing agar, resistant colonies arose over a period of 2 weeks. ArecAdeficiency reduced mutant recovery, consistent with involvement of the SOS response in mutant induction. The C-8-methoxy compounds gatifloxacin and moxifloxacin allowed the recovery of fewer resistant mutants than either ciprofloxacin or levofloxacin when present at the same multiple of the MIC; a quinolone-like 8-methoxy-quinazoline-2,4-dione was more effective at restricting the emergence of resistant mutants than its cognate fluoroquinolone. Thus, the structure of fluoroquinolone-like compounds affects mutant recovery. A spontaneous mutator mutant ofM. smegmatis, obtained by growth in medium containing both isoniazid and rifampin, increased mutant induction during exposure to ciprofloxacin. Moreover, the mutator increased the size of spontaneous resistant mutant subpopulations, as detected by population analysis. Induction of ciprofloxacin resistance was also observed withMycobacterium tuberculosisH37Rv. When measured with clinical isolates, no difference in mutant recovery was observed between multidrug-resistant (MDR) and pansusceptible isolates. This finding is consistent with at least some MDR isolates ofM. tuberculosislacking mutators detectable by the agar plate assay. Collectively, the data indicate that the use of fluoroquinolones against tuberculosis may induce resistance and that the choice of quinolone may be important for restricting the recovery of induced mutants.

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.