Oleanolic acid: an antimycobacterial component of Syzygium aromaticum L. and inhibitor of efflux mediated drug resistance.

Oleanolic acid (OA) was isolated from Syzygium aromaticum L. buds, and structurally characterised using different spectroscopic techniques; MS, IR,1H/13C-NMR and 2D NMR experiments. The antimycobacterial activity according to a resazurin microtiter assay (REMA) showed important inhibitory effect of OA on the virulent H37Rv strain, with the lowest minimum concentration of 50 µg/mL, compared to other fractions. Molecular docking of OA with BacA drug efflux pump resulted in good binding affinity of hydrophobic interaction type. Therefore, OA could contribute to the antimycobacterial action of clove buds, and has potential as an efflux pump inhibitor. Further studies are required on its use to combat multidrug resistant strains.

Clove Buds Volatile Compounds: Inhibitory Activity on Mycobacterium Growth and Molecular Docking on Mmr Efflux Pump Drug Resistance.

Syzygium aromaticum is used in traditional and modern medicine for its various and outstanding pharmacological properties. Here, we studied the chemical composition of hexane extract and non-polar fractions (NPF) obtained from the maceration and fractionation of clove buds, in order to evaluate their in vitro antimycobacterial activity, as well as their contribution against efflux pump (EP) resistance through molecular docking experiments. The gas chromatography-mass spectrometry (GC-MS) analysis of the volatile profiles revealed the presence of eugenol, followed by eugenyl acetate, and ß-caryophyllene as common major compounds. According to Resazurin microtiter assay (REMA), Mycobacterium tuberculosis H37 Rv strain was sensitive to all volatile samples at concentration range between 10 and 100 µg/mL. The NPF of ethanol extract was the best inhibitor with a MIC=10 µg/mL. The in silico study revealed a strong binding affinity between eugenol and Mmr EP protein (-8.1 Kcal/mol), involving two binding modes of hydrogen bond and π-alkyl interactions. The non-polarity character of clove volatile constituents, and their potential additive or synergistic effects could be responsible for the antimycobacterial activity. In addition, these findings suggest the benefic effect of eugenol in the management of mycobacterium drug resistance, whether as potential inhibitor of Mmr drug EP, or modulator during combination therapy.

Genotypic diversity of multi- and pre-extremely drug-resistant Mycobacterium tuberculosis isolates from Morocco.

In Morocco, the prevalence of multidrug resistant tuberculosis (MDR-TB) continues to increase especially within previously treated cases; these MDR cases may evolve to extensively drug resistant tuberculosis (XDR-TB) raising major concern to TB control programs. From an epidemiological window, scarce informations are available about the genetic diversity of Mycobacterium tuberculosis (MTB) strains fueling these forms of resistance. The aim of this study was to assess to genetic diversity of MDR-MTB strains. Hence, this prospective study was conducted on patients diagnosed with MDR-TB at Pasteur Institute of Casablanca from 2010 to 2013. A total of 70 MDR-MTB isolates were genotyped by spoligotyping and 15-loci MIRU-VNTR methods. Spoligotyping generated four orphan patterns, five unique profiles whereas 61 strains were grouped in nine clusters (2 to 25 strains per cluster), the clustering rates being 87.1%. Subtyping by 15 loci MIRU-VNTR splitted all clusters already established by spoligotyping and generated 70 unique profiles not recognized in SITVIT2 database; clustering rate was equal to zero. HGDI analysis of 15 loci MIRU demonstrated that eight out of 15 loci were highly discriminant. Of note, all pre-XDR strains belongs to many clades, meaning that there no association between gyrA mutants and particular clade. Overall, the data generated by this study (i) describe the population structure of MDR MTBC in Morocco which is highly homogenous, (ii) confirm that TB in Morocco is almost exclusively transmitted by modern and evolutionary lineages with high level of biodiversity seen by MIRU, and (iii) validate the use of optimized 15-loci MIRU-VNTR format for future investigations in Morocco.

Molecular characterization of mutations associated with resistance to second line drugs in Mycobacterium tuberculosis patients from Casablanca, Morocco.

The emergence and spread of extensively drug-resistant tuberculosis (XDR-TB) is a serious threat to global health. Therefore, its rapid diagnosis is crucial. The present study aimed to characterize mutations conferring resistance to second line drugs (SLDs) within multidrug Mycobacterium tuberculosis (MDR-MTB) isolates and to estimate the occurrence of XDR-TB in Casablanca, Morocco. A panel of 200 MDR-TB isolates was collected at the Pasteur Institute between 2015-2018. Samples were subjected to drug susceptibility testing to Ofloxacin (OFX), Kanamycin (KAN) and Amikacin (AMK). The mutational status of gyrA, gyrB, rrs, tlyA and eis was assessed by sequencing these target genes. Drug susceptibility testing for SLDs showed that among the 200 MDR strains, 20% were resistant to OFX, 2.5% to KAN and 1.5% to AMK. Overall, 14.5% of MDR strains harbored mutations in gyrA, gyrB, rrs and tlyA genes. From the 40 OFXR isolates, 67.5% had mutations in QRDR of gyrA and gyrB genes, the most frequent one being Ala90Val in gyrA gene. Of note, none of the isolates harbored simultaneously mutations in gyrA and gyrB genes. In eight out of the 200 MDR-TB isolates resistant either to KAN or AMK, only 25% had A1401G or Lys89Glu change in rrs and tlyA genes respectively. This study is very informative and provides data on the alarming rate of fluoroquinolone resistance which warrants the need to implement appropriate drug regimens to prevent the emergence and spread of more severe forms of Mycobacterium tuberculosis drug resistance.