The diverse pattern of resistance by methicillin-resistant Staphylococcus aureus (MRSA) is the major obstacle in the treatment of its infections. The key reason of resistance is the poor membrane permeability of drug molecules. Over the last decade, cell-penetrating peptides (CPPs) have emerged as efficient drug delivery vehicles and have been exploited to improve the intracellular delivery of numerous therapeutic molecules in preclinical studies. Therefore, to overcome the drug resistance, we have investigated for the first time the effects of two CPPs (P3 and P8) in combination with four antibiotics (viz. oxacillin, erythromycin, norfloxacin, and vancomycin) against MRSA strains. We found that both CPPs internalized into the MRSA efficiently at very low concentration (<10 μM) which was non-toxic to bacteria as well as mammalian cells and showed no significant hemolytic activity. However, the combinations of CPPs (≤10 μM) and antibiotics showed high toxicity against MRSA as compared to antibiotics alone. The significant finding is that P3 and P8 could lower the MICs against oxacillin, norfloxacin, and vancomycin to susceptible levels (generally <1 μg/mL) for almost all five clinical isolates. Further, the bacterial cell death was confirmed by scanning electron microscopy as well as propidium iodide uptake assay. Simultaneously, time-kill kinetics revealed the increased uptake of antibiotics. In summary, CPPs assist to restore the effectiveness of antibiotics at much lower concentration, eliminate the antibiotic toxicity, and represent the CPP-antibiotic combination therapy as a potential novel weapon to combat MRSA infections.

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium responsible for several infections in humans. The infections caused by this bacterial strain are difficult to treat due to the resistance of MRSA to clinically used antibiotics. Several medicinal plants extracts and their phytoconstituents have been reported to possess modulation and efflux pump inhibitory (EPI) activity against MRSA strains. Alpinia calcarata rhizomes have been reported to be used in Ayurveda for several ailments including fungal infections. Based on this information and in continuation with our efforts to discover EPIs from Indian medicinal plants, we describe EPI activity of flavonoids isolated from A. calcarata. Galangin and kaempferol showed ≥ 32-fold modulation in minimum inhibitory concentration (MIC) of ethidium bromide (EtBr) as well as norfloxacin in NorA-overexpressed S. aureus (SA-1199B) strain. Pinocembrin showed 32-fold modulation of EtBr MIC in SA-1199 strain, but not in SA-1199B and K1758 strains. A significant difference was not observed in the modulation of norfloxacin MIC by galangin in SA-1199 and SA-1199B strains, which may be due to non-specific nature of galangin as modulator or EPI. However, kaempferol modulated the MIC of EtBr as well as norfloxacin 64-fold and 32-fold, respectively. Also, the best modulatory effect of kaempferol was observed only in SA-1199B strain compared to two other strains. The EPI activity of kaempferol and galangin were found to be competitive with respect to verapamil. In dose-response assay, kaempferol at 31.25 µg/mL concentration was found to be better EPI by inhibiting NorA pump in SA-1199B strain and also demonstrated further confocal microscopy.

Nosocomial infections caused by antibiotic-resistant Gram-negative pathogens are of grave concern today. Polymyxins are considered as the last resorts of therapy to treat these multi-drug resistant (MDR) bacteria. But their associated nephrotoxicity and neurotoxicity calls for the development of safer polymyxin therapy until novel and less toxic antibiotics are discovered. No other polymyxin molecule except polymyxin B and E (colistin) is explored thoroughly in literature to demonstrate its clinical relevance. In the present study, we have isolated two antimicrobial compounds named P1 and P2 from the soil isolate Paenibacillus dendritiformis strain PV3-16, which we later identified as polymyxin A2 and A1 respectively. We tested their minimum inhibitory concentrations (MICs) against MDR clinical isolates, performed membrane permeabilization assays and determined their interaction with lipopolysaccharide (LPS). Finally, we studied their toxicity against human Leukemic monocyte cell line (THP-1) and embryonic kidney cell line (HEK 293). Both compounds displayed equal efficacy when compared with standard polymyxins. P1 was 2–4 fold more active in most of the clinical strains tested. Moreover, P1 showed higher affinity toward LPS. In cytotoxicity studies, P1 had IC50 value (>1000 μg/ml) similar to colistin against HEK cells but immune cells, i.e., THP-1 cell lines were more sensitive to polymyxins. P1 showed less toxicity in THP-1 cell line than all other polymyxins checked. To sum up, P1 (polymyxin A2) possessed better efficacy than polymyxin B and E and had least toxicity to immune cells. Since polymyxin A was not investigated thoroughly, we performed the comprehensive in vitro assessment of this molecule. Moreover, this is the first report of isolation and characterization of polymyxin A from P. dendritiformis. This compound should be further investigated for its in vivo efficacy and toxicity to develop it as a drug candidate.

Endothelial dysfunction and vascular remodeling are the hallmarks of pulmonary arterial hypertension (PAH). For PAH treatment, there is a rising demand of Stem cell therapy. Interestingly, research reveals that stem/progenitor cells may have an impact in disease progression and therapy in PAH patients. Clinical trials for stem cell therapy in cardiac cell regeneration for heart repair in PAH patients are now underway. The clinical potential of stem/progenitor cell treatment that offers to PAH patients helps in lesion formation which occurs through regaining of vascular cell activities. Majorly the stem cells which are specifically derived from bone marrow such as mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs) and induced pluripotent cells (iPSCs), adipose-derived stem cells (ADSCs), and cardiac stromal cells (CSCs) are among the subtypes that are proved to play a pivotal role in the repair of the heart. But with only MSCs and EPCs, have shown positive outcomes and act as therapeutically efficient in regaining cure for PAH in clinical trials. This chapter also seeks to explain the potential limitations and challenges with most recent achievements in stem/progenitor cell research in PAH.