ABSTRACT
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.
ABSTRACT
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.
