Dataset Information

Development of an In Vitro Assay for Detection of Drug-Induced Resuscitation-Promoting-Factor-Dependent Mycobacteria.

ABSTRACT: Tuberculosis is a major infectious disease that requires prolonged chemotherapy with a combination of four drugs. Here we present data suggesting that treatment of Mycobacterium tuberculosis, the causative agent of tuberculosis, and Mycobacterium smegmatis, a model organism widely used for the screening of antituberculosis agents, with first-line drugs resulted in the generation of substantial populations that could be recovered only by the addition of a culture supernatant from growing mycobacteria. These bacilli failed to grow in standard media, resulting in significant underestimation of the numbers of viable mycobacteria in treated samples. We generated M. smegmatis strains overexpressing M. tuberculosis resuscitation-promoting factors (Rpfs) and demonstrated their application for the detection of Rpf-dependent mycobacteria generated after drug exposure. Our data offer novel opportunities for validation of the sterilizing activity of antituberculosis agents.

SUBMITTER: Loraine J

PROVIDER: S-EPMC5038329 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Development of an In Vitro Assay for Detection of Drug-Induced Resuscitation-Promoting-Factor-Dependent Mycobacteria.

Loraine Jessica J Pu Feifei F Turapov Obolbek O Mukamolova Galina V GV

Antimicrobial agents and chemotherapy 20160923 10

Tuberculosis is a major infectious disease that requires prolonged chemotherapy with a combination of four drugs. Here we present data suggesting that treatment of Mycobacterium tuberculosis, the causative agent of tuberculosis, and Mycobacterium smegmatis, a model organism widely used for the screening of antituberculosis agents, with first-line drugs resulted in the generation of substantial populations that could be recovered only by the addition of a culture supernatant from growing mycobact ...[more]

PMID: 27503641

Similar Datasets

Project description:One third of the world population carries a latent tuberculosis (TB) infection, which may reactivate leading to active disease. Although TB latency has been known for many years it remains poorly understood. In particular, substances of host origin, which may induce the resuscitation of dormant mycobacteria, have not yet been described. In vitro models of dormant ("non-culturable") cells of Mycobacterium smegmatis (mc(2)155) and Mycobacterium tuberculosis H37Rv were used. We found that the resuscitation of dormant M. smegmatis and M. tuberculosis cells in liquid medium was stimulated by adding free unsaturated fatty acids (FA), including arachidonic acid, at concentrations of 1.6-10 µM. FA addition enhanced cAMP levels in reactivating M. smegmatis cells and exogenously added cAMP (3-10 mM) or dibutyryl-cAMP (0.5-1 mM) substituted for FA, causing resuscitation of M. smegmatis and M. tuberculosis dormant cells. A M. smegmatis null-mutant lacking MSMEG_4279, which encodes a FA-activated adenylyl cyclase (AC), could not be resuscitated by FA but it was resuscitated by cAMP. M. smegmatis and M. tuberculosis cells hyper-expressing AC were unable to form non-culturable cells and a specific inhibitor of AC (8-bromo-cAMP) prevented FA-dependent resuscitation. RT-PCR analysis revealed that rpfA (coding for resuscitation promoting factor A) is up-regulated in M. smegmatis in the beginning of exponential growth following the cAMP increase in lag phase caused by FA-induced cell activation. A specific Rpf inhibitor (4-benzoyl-2-nitrophenylthiocyanate) suppressed FA-induced resuscitation. We propose a novel pathway for the resuscitation of dormant mycobacteria involving the activation of adenylyl cyclase MSMEG_4279 by FAs resulted in activation of cellular metabolism followed later by increase of RpfA activity which stimulates cell multiplication in exponential phase. The study reveals a probable role for lipids of host origin in the resuscitation of dormant mycobacteria, which may function during the reactivation of latent TB.

Project description:The anaerobic bioremediation of polychlorinated biphenyls (PCBs) is largely impeded by difficulties in massively enriching PCB dechlorinators in short periods of time. Tetrachloroethene (PCE) is often utilized as an alternative electron acceptor to preenrich PCB-dechlorinating bacteria. In this study, resuscitation promoting factor (Rpf) was used as an additive to enhance the enrichment of the microbial communities involved in PCE/PCBs dechlorination. The results indicated that Rpf accelerates PCE dechlorination 3.8 to 5.4 times faster than control cultures. In Aroclor 1260-fed cultures, the amendment of Rpf enables significantly more rapid and extensive dechlorination of PCBs. The residual high-chlorinated PCB congeners (≥5 Cl atoms) accounted for 36.7% and 59.8% in the Rpf-amended cultures and in the corresponding controls, respectively. This improvement was mainly attributed to the enhanced activity of the removal of meta-chlorines (47.7 mol % versus 14.7 mol %), which did not appear to affect dechlorination pathways. The dechlorinators, including Dehalococcoides in Chloroflexi and Desulfitobacterium in Firmicutes, were greatly enriched via Rpf amendment. The abundance of nondechlorinating populations, including Methanosarcina, Desulfovibrio, and Bacteroides, was also greatly enhanced via Rpf amendment. These results suggest that Rpf serves as an effective additive for the rapid enrichment of active dechlorinating cultures so as to provide a new approach by which to massively cultivate bioinoculants for accelerated in situ anaerobic bioremediation. IMPORTANCE The resuscitation promoting factor (Rpf) of Micrococcus luteus has been reported to resuscitate and stimulate the growth of functional microorganisms that are involved in the aerobic degradation of polychlorinated biphenyls (PCBs). However, few studies have been conducted to investigate the role of Rpf on anaerobic microbial populations. In this study, the enhancement of Rpf on the anaerobic microbial dechlorination of PCE/PCBs was discovered. Additionally, the Rpf-responsive populations underlying the enhanced dechlorination were uncovered. This report reveals the rapid enrichment of active dechlorinating cultures via Rpf amendment, and this sheds light on massively enriching PCB dechlorinators in short periods of time. The enhanced in situ anaerobic bioremediation of PCBs could be expected by supplementing Rpf.

Dataset Information

Development of an In Vitro Assay for Detection of Drug-Induced Resuscitation-Promoting-Factor-Dependent Mycobacteria.

Publications

Development of an In Vitro Assay for Detection of Drug-Induced Resuscitation-Promoting-Factor-Dependent Mycobacteria.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets