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

Metformin Prevents Nigrostriatal Dopamine Degeneration Independent of AMPK Activation in Dopamine Neurons

Metformin is a widely prescribed drug used to treat type-2 diabetes, although recent studies show it has wide ranging effects to treat other diseases. Animal and retrospective human studies indicate that Metformin treatment is neuroprotective in Parkinson’s Disease (PD), although the neuroprotective mechanism is unknown, numerous studies suggest the beneficial effects on glucose homeostasis may be through AMPK activation. In this study we tested whether or not AMPK activation in dopamine neurons was required for the neuroprotective effects of Metformin in PD. We generated transgenic mice in which AMPK activity in dopamine neurons was ablated by removing AMPK beta 1 and beta 2 subunits from dopamine transporter expressing neurons. These AMPK WT and KO mice were then chronically exposed to Metformin in the drinking water then exposed to MPTP, the mouse model of PD. Chronic Metformin treatment significantly attenuated the MPTP-induced loss of Tyrosine Hydroxylase (TH) neuronal number and volume and TH protein concentration in the nigrostriatal pathway. Additionally, Metformin treatment prevented the MPTP-induced elevation of the DOPAC:DA ratio regardless of genotype. Metformin also prevented MPTP induced gliosis in the Substantia Nigra. These neuroprotective actions were independent of genotype and occurred in both AMPK WT and AMPK KO mice. Overall, our studies suggest that Metformin’s neuroprotective effects are not due to AMPK activation in dopaminergic neurons and that more research is required to determine how metformin acts to restrict the development of PD

Impact of caloric and dietary restriction regimens on markers of health and longevity in humans and animals: a summary of available findings

Considerable interest has been shown in the ability of caloric restriction (CR) to improve multiple parameters of health and to extend lifespan. CR is the reduction of caloric intake - typically by 20 - 40% of ad libitum consumption - while maintaining adequate nutrient intake. Several alternatives to CR exist. CR combined with exercise (CE) consists of both decreased caloric intake and increased caloric expenditure. Alternate-day fasting (ADF) consists of two interchanging days; one day, subjects may consume food ad libitum (sometimes equaling twice the normal intake); on the other day, food is reduced or withheld altogether. Dietary restriction (DR) - restriction of one or more components of intake (typically macronutrients) with minimal to no reduction in total caloric intake - is another alternative to CR. Many religions incorporate one or more forms of food restriction. The following religious fasting periods are featured in this review: 1) Islamic Ramadan; 2) the three principal fasting periods of Greek Orthodox Christianity (Nativity, Lent, and the Assumption); and 3) the Biblical-based Daniel Fast. This review provides a summary of the current state of knowledge related to CR and DR. A specific section is provided that illustrates related work pertaining to religious forms of food restriction. Where available, studies involving both humans and animals are presented. The review includes suggestions for future research pertaining to the topics of discussion

Synthesis, biological activity and in silico study of alkyl eugenol derivatives as Mycobacterium tuberculosis inhibitors

Since ancient times, tuberculosis (TB) has been a fatal infectious disease, made even more difficult to treat with the emergence of drug-resistant strains. The present work aimed to evaluate the antituberculosis activity of newly synthesized eugenol derivatives, and their iron chelation ability as well. The mycobacterium H37Rv strain was sensitive to all tested compounds. The compounds 2-methoxy-4-propenyl-1-propynyloxybenzene (1), 3,5-bis(trifluoromethyl) benzyloxy-2-methoxy-4-propenylbenzene (3) and ethyl 2-methoxy-4-propenylphenylcarbonate (5) showed the best growth inhibitory activity at a concentration of 10 µg/mL. Also, a considerable iron chelation activity for 1 and 5 translated by EC50 values of 0.94 and 0.33 µg/mL, respectively. Generally, the synthesized derivatives illustrated an encouraging in silico pharmacokinetic profile. The molecular docking study revealed that compound 3 exhibited the excellent binding affinity within the active sites of MabA and PanK targets, involving hydrogen bond interactions, fluorine interactions and hydrophobic interactions. Our results suggest that compound 3 could potentially act via the inhibition of MabA and PanK proteins. While, derivatives 1 and 5 could have a potential iron chelation mechanism against mycobacterial growth

Plasmid-based high-resolution melting analysis for accurate detection of rpoB mutations in Mycobacterium tuberculosis isolates from Moroccan patients

Abstract Background Rapid diagnosis of drug resistance in tuberculosis (TB) is pivotal for the timely initiation of effective antibiotic treatment to prevent the spread of drug-resistant strains. The development of low-cost, rapid and robust methods for drug-resistant TB detection is highly desirable for resource-limited settings. Methods We report the use of an in house plasmid-based quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis for the detection of mutations related to rifampicin-resistant Mycobacterium tuberculosis (MTB) in clinical isolates from Moroccan patients. Five recombinant plasmids containing predominant mutations (S531L, S531W, H526Y and D516V) and the wild-type sequence of the Rifampicin Resistance-Determining Region (RRDR) have been used as controls to screen 45 rifampicin-resistant and 22 rifampicin-susceptible MTB isolates. Results The sensitivity and the specificity of the qPCR-HRM analysis were 88.8% and 100% respectively as compared to rifampicin Drug Susceptibility Testing (DST). The results of qPCR-HRM and DNA sequencing had a concordance of 100%. Conclusion Our qPCR-HRM assay is a sensitive, accurate and cost-effective assay for the high-throughput screening of mutation-based drug resistance in TB reference laboratories

Whole genome sequencing-based analysis of tuberculosis (TB) in migrants: rapid tools for cross-border surveillance and to distinguish between recent transmission in the host country and new importations

14 páginas, 7 figurasBackgroundThe analysis of transmission of tuberculosis (TB) is challenging in areas with a large migrant population. Standard genotyping may fail to differentiate transmission within the host country from new importations, which is key from an epidemiological perspective.AimTo propose a new strategy to simplify and optimise cross-border surveillance of tuberculosis and to distinguish between recent transmission in the host country and new importationsMethodsWe selected 10 clusters, defined by 24-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR), from a population in Spain rich in migrants from eastern Europe, north Africa and west Africa and reanalysed 66 isolates by whole-genome sequencing (WGS). A multiplex-allele-specific PCR was designed to target strain-specific marker single nucleotide polymorphisms (SNPs), identified from WGS data, to optimise the surveillance of the most complex cluster.ResultsIn five of 10 clusters not all isolates showed the short genetic distances expected for recent transmission and revealed a higher number of SNPs, thus suggesting independent importations of prevalent strains in the country of origin. In the most complex cluster, rich in Moroccan cases, a multiplex allele-specific oligonucleotide-PCR (ASO-PCR) targeting the marker SNPs for the transmission subcluster enabled us to prospectively identify new secondary cases. The ASO-PCR-based strategy was transferred and applied in Morocco, demonstrating that the strain was prevalent in the country.ConclusionWe provide a new model for optimising the analysis of cross-border surveillance of TB transmission in the scenario of global migration.This project was funded by ISCIII: ERANET-LAC (TRANS-TB-TRANS REF AC16/00057; ELAC2015/T08-0664), FIS (13/01207; 15/01554) and cofunded by ERDF Funds from the European Commission: “A way of making Europe”. Miguel Servet grant (CP15/00075) for LPL. Ministerio de Economía y Competitividad (grant SAF2016-77346-R), ERC (638553-TB-ACCELERATE) to IC. FPU13/00913 (Ministerio de Educación y Ciencia) to ACO.Peer reviewe