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The World Health Organization has categorized the Gram-negative superbugs, which are inherently impervious to many antibiotics, as critical priority pathogens due to the lack of effective treatments. The breach in our last-resort antibiotic (i.e., colistin) by extensively drug-resistant and pan-drug-resistant Enterobacteriaceae strains demands the immediate development of new therapies. In the present study, we report the discovery of tridecaptin M, a new addition to the family, and its potential against colistin-resistant Enterobacteriaceae in vitro and in vivo. Also, we performed mode-of-action studies using various fluorescent probes and studied the hemolytic activity and mammalian cytotoxicity in two cell lines. Tridecaptin M displayed strong antibacterial activity (MICs of 2 to 8 μg ml−1) against clinical strains of Klebsiella pneumoniae (which were resistant to colistin, carbapenems, third- and fourth-generation cephalosporins, fluoroquinolones, fosfomycin, and other antibiotics) and mcr-1-positive Escherichia coli strains. Unlike polymyxins, tridecaptin M did not permeabilize the outer membrane or cytoplasmic membrane. It blocked ATP synthesis in bacteria by dissipating the proton motive force. The compound exhibited negligible acquired resistance, low in vitro cytotoxicity and hemolytic activity, and no significant acute toxicity in mice. It also showed promising efficacy in a thigh infection model of colistin-resistant K. pneumoniae. Altogether, these results demonstrate the future prospects of this class of antibiotics to address the unmet medical need to circumvent colistin resistance in extensively drug-resistant Enterobacteriaceae infections. The work also emphasizes the importance of natural products in our shrunken drug discovery pipeline.

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The use of efflux pump inhibitors (EPIs) as potentiators along with the traditional antibiotics assists in the warfare against antibiotic-resistant superbugs. Efflux pumps of the resistance-nodulation-cell division (RND) family play crucial roles in multidrug resistance in Escherichia coli and Pseudomonas aeruginosa. Despite several efforts, clinically useful inhibitors are not available at present. This study describes ethyl 4-bromopyrrole-2-carboxylate (RP1) isolation, an inhibitor of RND transporters from the library of 4000 microbial exudates. RP1 acts synergistically with antibiotics by reducing their minimum inhibitory concentration in strains overexpressing archetype RND transporters (AcrAB-TolC and MexAB-OprM). It also improves the accumulation of Hoechst 33342 and inhibits its efflux (a hallmark of EPI functionality). The antibiotic-RP1 combinations prolong the postantibiotic effects and reduce the mutation prevention concentration of antibiotics. Additionally, from Biolayer Interferometry spectra, it appears that RP1 is bound to AcrB. RP1 displays low mammalian cytotoxicity, no Ca2+ channel inhibitory effects, and reduces the intracellular invasion of E. coli and P. aeruginosa in macrophages. Furthermore, the RP1-levofloxacin combination is nontoxic, well-tolerated, and notably effective in a murine lung infection model. In sum, RP1 is a potent EPI and worthy of further consideration as a potentiator to improve the effectiveness of existing antibiotics.

Efflux pumps are always at the forefront of bacterial multidrug resistance and account for the failure of antibiotics. The present study explored the potential of 2-(2-Aminophenyl) indole (RP2), an efflux pump inhibitor (EPI) isolated from the soil bacterium, to overcome the efflux-mediated resistance in Staphylococcus aureus. The RP2/antibiotic combination was tested against efflux pump over-expressed S. aureus strains. The compound was further examined for the ethidium bromide (EtBr) uptake and efflux inhibition assay (a hallmark of EPI functionality) and cytoplasmic membrane depolarization. The safety profile of RP2 was investigated using in vitro cytotoxicity assay and Ca 2+ channel inhibitory effect. The in vivo efficacy of RP2 was studied in an animal model in combination with ciprofloxacin. RP2 exhibited the synergistic activity with several antibiotics in efflux pump over-expressed strains of S. aureus. In the mechanistic experiments, RP2 increased the accumulation of EtBr, and demonstrated the inhibition of its efflux. The antibiotic-EPI combinations resulted in extended post antibiotic effects as well as a decrease in mutation prevention concentration of antibiotics. Additionally, the in silico docking studies suggested the binding of RP2 to the active site of modeled structure of NorA efflux pump. The compound displayed low mammalian cytotoxicity and had no Ca 2+ channel inhibitory effect. In ex vivo experiments, RP2 reduced the intracellular invasion of S. aureus in macrophages. Furthermore, the RP2/ciprofloxacin combination demonstrated remarkable efficacy in a murine thigh infection model. In conclusion, RP2 represents a promising candidate as bacterial EPI, which can be used in the form of a novel therapeutic regimen along with existing and upcoming antibiotics, for the eradication of S. aureus infections.

Probiotic industries strive for new, efficient and promising probiotic strains that impart a positive impact on consumer health. Challenges are persisting in isolation, screening, and selection of the new indigenous probiotic strains. In the present research, we explored the probiotic potential of 17 lactic acid bacteria isolated from Yak milk in a series of in vitro tests. We also demonstrated their health benefits, i.e., cholesterol degradation, lactose digestion, antimicrobial activity, antioxidant, and anticancer activities. Principal component analysis revealed that more than 50% of the strains fulfilled the examined criteria, e.g., survival in acidic pH, bile concentrations, and adherent property. Approximately all the strains produced antimicrobial substances against the maximum number of tested strains including clinical strains. Most strains degraded cholesterol in comparison to the reference probiotic strain whereas strain Yc showed 1.5 times higher the degradation efficiency of the control strain. Lan4 strain exhibited remarkable anticancer activity and induced the maximum apoptosis (87%) in the Hela cells and was non-toxic to the non-cancerous HEK293 cells. Around ten strains showed positive lactose digestion. Overall, this can be concluded that selected lactic acid bacteria revealed excellent probiotic properties along with desirable health benefits. These strains need to be further investigated in details for their application in the development of novel probiotic preparations for the improvement of public health.

Nosocomial infections caused by Klebsiella pneumoniae are primarily characterized by a high prevalence of extended-spectrum β-lactamases (ESBL's) and a soaring pace of carbapenemase dissemination. Availability of limited antimicrobial agents as a therapeutic option for multidrug-resistant bacteria raises an alarming concern. This study aimed at the molecular characterization of multidrug-resistant K. pneumoniae clinical isolates and studied the role of efflux pumps in β-lactam resistance. Thirty-three isolates confirmed as ESBL-positive K. pneumoniae that harbored resistance genes to major classes of antibiotics. The results showed that CTX-M15 was the preeminent β-lactamase along with carbapenemases in ESBL-positive isolates. However, the efficacy of different antibiotics varied in the presence of lactamase inhibitors and efflux pump inhibitors (EPIs). Those showing increased efficacy of antibiotics with EPI were further explored for the expression of efflux pump genes and expressed a significantly different level of efflux pumps. We found that an isolate had higher expression of kpnF (SMR family) and kdeA (MATE family) pump genes relative to RND family pump genes. No mutations were observed in the genes for porins. Together, the findings suggest that β-lactamases are not the only single factor responsible for providing resistance against the existing β-lactam drugs. Resistance may increase many folds by simultaneous expression of RND family (the most prominent family in Gram-negative bacteria) and other efflux pump family.

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