Tricyclic SpiroLactams Kill Mycobacteria In Vitro and In Vivo by Inhibiting Type II NADH Dehydrogenases.

It is critical that novel classes of antituberculosis drugs are developed to combat the increasing burden of infections by multidrug-resistant strains. To identify such a novel class of antibiotics, a chemical library of unique 3-D bioinspired molecules was explored revealing a promising, mycobacterium specific Tricyclic SpiroLactam (TriSLa) hit. Chemical optimization of the TriSLa scaffold delivered potent analogues with nanomolar activity against replicating and nonreplicating Mycobacterium tuberculosis. Characterization of isolated TriSLa-resistant mutants, and biochemical studies, found TriSLas to act as allosteric inhibitors of type II NADH dehydrogenases (Ndh-2 of the electron transport chain), resulting in an increase in bacterial NADH/NAD+ ratios and decreased ATP levels. TriSLas are chemically distinct from other inhibitors of Ndh-2 but share a dependence for fatty acids for activity. Finally, in vivo proof-of-concept studies showed TriSLas to protect zebrafish larvae from Mycobacterium marinum infection, suggesting a vulnerability of Ndh-2 inhibition in mycobacterial infections.

Stress responses, outer membrane permeability control and antimicrobial resistance in Enterobacteriaceae.

Bacteria have evolved several strategies to survive a myriad of harmful conditions in the environment and in hosts. In Gram-negative bacteria, responses to nutrient limitation, oxidative or nitrosative stress, envelope stress, exposure to antimicrobials and other growth-limiting stresses have been linked to the development of antimicrobial resistance. This results from the activation of protective changes to cell physiology (decreased outer membrane permeability), resistance transporters (drug efflux pumps), resistant lifestyles (biofilms, persistence) and/or resistance mutations (target mutations, production of antibiotic modification/degradation enzymes). In targeting and interfering with essential physiological mechanisms, antimicrobials themselves are considered as stresses to which protective responses have also evolved. In this review, we focus on envelope stress responses that affect the expression of outer membrane porins and their impact on antimicrobial resistance. We also discuss evidences that indicate the role of antimicrobials as signaling molecules in activating envelope stress responses.

Dual Regulation of the Small RNA MicC and the Quiescent Porin OmpN in Response to Antibiotic Stress in Escherichia coli.

Antibiotic resistant Gram-negative bacteria are a serious threat for public health. The permeation of antibiotics through their outer membrane is largely dependent on porin, changes in which cause reduced drug uptake and efficacy. Escherichia coli produces two major porins, OmpF and OmpC. MicF and MicC are small non-coding RNAs (sRNAs) that modulate the expression of OmpF and OmpC, respectively. In this work, we investigated factors that lead to increased production of MicC. micC promoter region was fused to lacZ, and the reporter plasmid was transformed into E. coli MC4100 and derivative mutants. The response of micC-lacZ to antimicrobials was measured during growth over a 6 h time period. The data showed that the expression of micC was increased in the presence of ß-lactam antibiotics and in an rpoE depleted mutant. Interestingly, the same conditions enhanced the activity of an ompN-lacZ fusion, suggesting a dual transcriptional regulation of micC and the quiescent adjacent ompN. Increased levels of OmpN in the presence of sub-inhibitory concentrations of chemicals could not be confirmed by Western blot analysis, except when analyzed in the absence of the sigma factor σE. We suggest that the MicC sRNA acts together with the σE envelope stress response pathway to control the OmpC/N levels in response to ß-lactam antibiotics.

Disruption of Yarrowia lipolytica biofilms by rhamnolipid biosurfactant.

BACKGROUND: Yarrowia lipolytica is an ascomycetous dimorphic fungus that exhibits biofilm mode of growth. Earlier work has shown that biosurfactants such as rhamnolipids are efficient dispersants of bacterial biofilms. However, their effectiveness against fungal biofilms (particularly Y. lipolytica) has not been investigated. The aim of this study was to determine the effect of rhamnolipid on a biofilm forming strain of Y. lipolytica. Two chemical surfactants, cetyl-trimethyl ammonium bromide (CTAB) and sodium dodecyl sulphate (SDS) were used as controls for comparison. RESULTS: The methylene blue dye exclusion assay indicated an increase in fungal cell permeability after rhamnolipid treatment. Microtiter plate assay showed that the surfactant coating decreased Y. lipolytica biofilm formation by 50%. Rhamnolipid treatment disrupted pre-formed biofilms in a more effective manner than the other two surfactants. Confocal laser scanning microscopic studies showed that biofilm formation onto glass surfaces was decreased by 67% after sub-minimum inhibitory concentration (sub-MIC) treatment with rhamnolipids. The disruption of biofilms after rhamnolipid treatment was significant (P<0.05) when compared to SDS and CTAB. CONCLUSION: The results indicate a potential application of the biological surfactant to disrupt Y. lipolytica biofilms.