Antimicrobial Activities of Methanol, Ethanol and Supercritical CO2 Extracts of Philippine Piper betle L. on Clinical Isolates of Gram Positive and Gram Negative Bacteria with Transferable Multiple Drug Resistance.

Demetrio L Valle,Esperanza C Cabrera,Juliana Janet M Puzon,Windell L Rivera,Asad U Khan

doi:10.1371/journal.pone.0146349

Demetrio L Valle, Esperanza C Cabrera + Show 3 more

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https://doi.org/10.1371/journal.pone.0146349

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Abstract

Piper betle L. has traditionally been used in alternative medicine in different countries for various therapeutic purposes, including as an anti-infective agent. However, studies reported in the literature are mainly on its activities on drug susceptible bacterial strains. This study determined the antimicrobial activities of its ethanol, methanol, and supercritical CO2 extracts on clinical isolates of multiple drug resistant bacteria which have been identified by the Infectious Disease Society of America as among the currently more challenging strains in clinical management. Assay methods included the standard disc diffusion method and the broth microdilution method for the determination of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentrations (MBC) of the extracts for the test microorganisms. This study revealed the bactericidal activities of all the P. betle leaf crude extracts on methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), extended spectrum β-lactamase-producing Enterobacteriaceae, carbapenem-resistant Enterobacteriaceae, and metallo-β-lactamase-producing Pseudomonas aeruginosa and Acinetobacter baumannii, with minimum bactericidal concentrations that ranged from 19μg/ml to 1250 μg/ml. The extracts proved to be more potent against the Gram positive MRSA and VRE than for the Gram negative test bacteria. VRE isolates were more susceptible to all the extracts than the MRSA isolates. Generally, the ethanol extracts proved to be more potent than the methanol extracts and supercritical CO2 extracts as shown by their lower MICs for both the Gram positive and Gram negative MDRs. MTT cytotoxicity assay showed that the highest concentration (100 μg/ml) of P. betle ethanol extract tested was not toxic to normal human dermal fibroblasts (HDFn). Data from the study firmly established P. betle as an alternative source of anti-infectives against multiple drug resistant bacteria.

Highlights

Piper betle L. is a tropical vine that belongs to the family Piperaceae
Subashkumar et al demonstrated the presence of antibacterial activities of the P. betle ethanol extracts on clinical isolates of Acinetobacter spp., Escherichia coli, Klebsiella spp., Proteus spp., Pseudomonas spp., V. cholera, S. aureus and Streptococcus fecalis using the disc diffusion and well diffusion methods [12], while the same was shown by Agarwal et al [13] on pathogenic P. aeruginosa, S. aureus and E. coli, and by Chakraborty and Shah [14] on Streptococcus pyogenes, S. aureus, E. coli and Proteus vulgaris, using the well diffusion method
All these aforementioned studies did not determine the antibacterial activities of the P. betle extracts on the more novel multiple drug resistant (MDR) bacterial strains identified by the Infectious Disease Society of America (IDSA) as especially difficult to treat, and which the present study addressed

Summary

Introduction

Piper betle L. is a tropical vine that belongs to the family Piperaceae. The plant, known as “ikmo” in the vernacular is extensively cultivated throughout the Philippines [1], and in other Southeast Asian countries such as China, India, Sri Lanka, Malaysia, Nepal, Pakistan, Thailand and Indonesia [2]. Nouri et al [15] showed the inhibitory activities of P. betle on isolates of S. aureus, S. epidermidis, Bacillus cereus, Bacillus subtilis, Listeria monocytogenes, E. coli, Salmonella typhimurium, Salmonella enteritidis, Klebsiella pneumoniae, and P. aeruginosa. All these aforementioned studies did not determine the antibacterial activities of the P. betle extracts on the more novel multiple drug resistant (MDR) bacterial strains identified by the Infectious Disease Society of America (IDSA) as especially difficult to treat, and which the present study addressed. Extensive and careful review of scientific literature shows that studies on the effect of P. betle extracts on these MDR strains are notably lacking

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