Inhibition of penicillin-binding protein 2a (PBP2a) in methicillin resistant Staphylococcus aureus (MRSA) by combination of ampicillin and a bioactive fraction from Duabanga grandiflora

Abstract

BackgroundThe inhibition of penicillin-binding protein 2a (PBP2a) is a promising solution in overcoming resistance of methicillin resistance Staphylococcus aureus (MRSA). A potential approach in achieving this is by combining natural product with currently available antibiotics to restore the activity as well as to amplify the therapeutic ability of the drugs. We studied inhibition effects of a bioactive fraction, F-10 (isolated from the leaves of Duabanga grandiflora) alone and in combination with a beta-lactam drug, ampicillin on MRSA growth and expression of PBP2a. Additionally, phytochemical analysis was conducted on F-10 to identify the classes of phytochemicals present.MethodsFractionation of the ethyl acetate leaf extract was achieved by successive column chromatography which eventually led to isolation of an active fraction, F-10. Both extract and F-10 were analyzed for the presence of major classes of phytochemicals in addition to obtaining a high performance liquid chromatography (HPLC) profile to reveal the complexity of the fraction F-10. Broth microdilution method was employed to determine minimum inhibitory concentration (MIC) of the extract and fractions against MRSA. Evaluation of synergistic activity of the active fraction with ampicillin was determined using checkerboard methodand kinetic growth experiments. Effect of combination treatments on expression of PBP2a, a protein that confers resistance to beta-lactam antibiotics, was elucidated with the Western blot assay.ResultsMIC of F-10 against MRSA was 750 mg/L which showed an improved activity by 4-fold compared to its crude extract (MIC = 3000 mg/L). Phytochemical analysis revealed occurrence of tannins, saponin, flavonoids, sterols, and glycosides in F10 fraction. In FIC index interpretation, the most synergistic activity was achieved for combinations of 1/64 × MIC ampicillin + 1/4 × MIC F-10. The combination also evidently inhibited MRSA growth in kinetic growth curve assay. As a result of this synergistic interaction, MIC of ampicillin against MRSA was reduced to 0.78 mg/L (64-fold) from initial value of 50 mg/L. Western blot analysis suggested inhibition of PBP2a in MRSA cultures grown in synergistic combination treatment in which no PBP2a band was expressed.ConclusionsThe results demonstrated synergism between fraction F-10 of D. grandiflora with ampicillin in suppressing MRSA growth via PBP2a inhibition.

The Interactive Antimicrobial Activity of Conventional Antibiotics and Petalostigma spp. Extracts Against Bacterial Triggers of some Autoimmune Inflammatory Diseases

“…Alternatively (or in addition to MDR efflux pumps), the P. mirabilis strain used in our study may have acquired genes encoding for reduced-affinity penicillin-binding protein 2a (PBP2a) (rendering β-lactam antibiotics ineffective). 29 It is likely that as penicillin binding proteins are a group of protein enzymes, these phytochemicals may form nonspecific interaction and affect the bacterial cell biosynthesis. The P. pubescens fruit and P. triloculare leaf hexane extracts may also contain a β-lactamase inhibitor.…”

The Effect of Cu-BPDCA-Ty on Antibacterial Activity and The Expression of mecA Gene in Clinical and Standard Strains of Methicillin-Resistant Staphylococcus aureus

“…MRSA could be prevented if penicillin-binding proteins are intact (8). The emergence of resistance to MRSA has developed the need for novel strategies to replace alternatives to traditional antibiotics (9).…”

Rosa canina L. Fruit Extracts Inhibit the Growth of Bacterial Triggers of some Autoimmune Inflammatory Diseases and Potentiate the activity of Conventional Antibiotics

“…All of these antibiotics are susceptible to resistance due to efflux pumps. [22][23][24][25][26][27][28] A single pump can provide bacteria with resistance to a wide array of chemically and structurally diverse antibiotics and it is not uncommon for an organism to code for more than one efflux pump. 22,28 It is therefore imperative to identify agents that can block the efflux mechanism (efflux pump inhibitors -EPIs) or alter the process of efflux and in so doing, extend the life of existing antibacterial drugs.…”