Screening Bactericidal Action of Cytoplasm Extract from Kumazasa Bamboo (Sasa veitchii) Leaf against Antibiotics-

We aimed to develop a new treatment against antibiotic-resistant bacteria by focusing on cytoplasm of Kumazasa bamboo leaf. The cytoplasmic extract displays potent bactericidal action against Gram-positive bacteria such as Staphylococcus aureus, Enterococci and Streptococcus pneumoniae and also multi-antibiotic resistant MRSA (methicillin-resistant Staphylococcus aureus) and VRE (vancomycin-resistant Enterococci) strains. It induced bacteriolysis by firmly aggregating MSSA (methicillin-susceptible Staphylococcus aureus) and MRSA, and by lysis of VSE (vancomycin-susceptible Enterococci) and VRE. Clinical isolates of MRSA (30 strains in total) were susceptible to this extract, which gave MICs of Kumazasa-cytoplasmic extract ranging from 1.6 to 6.3%. The antibacterial action was bactericidal on scanning electron microscopy (SEM) analysis. A synergistic effect of this extract with ampicillin (ABPC) was obviously observed in all MRSA strains. The synergistic effect with vancomycin (VCM) was observed against VRE. The combination with clarithromycin (CAM) or tetracycline (TC) additively enhanced the antibacterial activity of CAM against CAM-susceptible Enterococci strains, and also that of TC against TC-resistant VRE strains. The present results should encourage research on antibacterial–hydrophilic ingredients derived from Kumazasa for use against antibiotic-resistant bacteria. The combination of Kumazasa-cytoplasmic extract with a cell wall synthesis inhibitor (ABPC or VCM) will be a highly efficient treatment for infections caused by multidrug-resistant MRSA and VRE strains. *Corresponding author: Shoichi Shirotake, Department of Pharmaco-Therapeutics, Graduate School of Medicine, Yokohama City University, Fuku’ura3-9, Knazawa-ku, Yokohama, 236-004, Japan, E-mail: shirophd@med.yokohama-cu.ac.jp Received September 17, 2009; Accepted October 08, 2009; Published October 09, 2009 Citation: Shirotake S, Nakamura J, Kaneko A, Anabuki E, Shimizu N (2009) Screening Bactericidal Action of Cytoplasm Extract from Kumazasa Bamboo (Sasa veitchii) Leaf against Antibiotics-Resistant Pathogens such as MRSA and VRE Strains. J Bioequiv Availab 1: 080085. doi:10.4172/jbb.1000012 Copyright: © 2009 Shirotake S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Introduction
More than 50 years of widespread use of antibiotics has resulted in the gradual appearance of antibiotic-resistant bacteria (Noble et al., 1992;Bozdogan et al., 2003). Methicillin-resistant Staphylococcus aureus (MRSA, detection rate; ca 80%) (Uehara et al., 2000) have acquired resistance due to the mecA gene, which encodes an altered penicillin-binding protein with reduced affinity for methicillin. In recent years, vancomycinresistant Enterococci (VRE) and vancomycin-resistant Staphy-lococcus aureus (VRSA) have appeared (detection rate for VRE; 20% or more of Enterococci in intensive care units in Europe and the U.S.A) (Frieden et al., 1993). One of the biggest therapeutic issues at the moment is the emergence of multiple antibiotic-resistant pathogenic bacteria which is very dangerous.
For thousands of years, plants have proved to be a rich source of biologically active compounds. Indeed throughout human history, plant extracts have been used to cure various chronic diseases. In recent years, numerous therapeutically useful drugs have been extracted from plants, (Kumarasamy et al., 2002;) such as Aspirin, Kinin, Artemisinin, Taxol and Camptothecin. Plants have developed some unique antibacterial mechanisms against infection. Traditional medicines, such as mixtures of oriental herbs ) and tea (Cho et al., 2008) were used as folk remedies to alleviate bacterial infections. Subsequent analysis has shown that these crude plant extracts contain biologically active components, such as essential oils (Duan and Zhao, 2009;Roller et al., 2009), flavonoids (Xu and Lee, 2001; Falcão-Silva et al., 2009), polyphenols (Cho et al., 2008) and alkaloids (Zuo et al., 2008), that display antibacterial activities (Pesewu et al., 2008;Shimizu et al., 2001). We have endeavored to identify and utilize hydrophilic antibacterial products from plant extracts for clinical applications.
In Japan, Kumazasa bamboo (Sasa veitchii) leaf have been used as SUSHI sheets or to package rice balls (known as CHIMAKI) to protect food from rotting due to the growth of bacteria. The traditional medicine from Kumazasa has a wide range of applications such as treatment of halitosis and body odor, stomatitis, gingivitis, bedsores, burns, hemorrhoids, cuts and purulent wounds. Organic solvent extracts (e.g., using diethyl ether) from leaves of Kumazasa (Sasa albo-marginata) were reported to possess antimicrobial activity against bacteria, fungi,  (Chuyen et al., 1982). To date, no studies have been conducted to investigate the antibacterial activity in the organic extracts against antibiotic-resistant bacterial strains. Herein, we examine the cytoplasmic extract from Kumazasa leaf in terms of its antibacterial activity against multidrug-resistant pathogens.

Cytoplasmic Extraction from Kumazasa Bamboo Leaf
The cytoplasm hydrophilic extraction was obtained by removing resin and lignin from green leaves of striped Kumazasa (Sasa veitchii), as following. Fresh Kumazasa leaves were crushed and homogenized by a mixer, then steeped in a 3%NaOH solution at room temperature overnight (the cell membrane was destroyed by alkaline hydrolysis). The pH value of the solution was adjusted to 4, by dropping H 2 SO 4 solution, and the residue was collected by centrifugation (x15000rpm), then the residue was dissolved at concentration of 40mg/ml in warm distilledwater as a green-colloidal solution, then the pH value was adjusted to 7.4, by dropping NaOH solution (cytoplasmic crude extraction).

Determination of Antibacterial Activity and Combination Effect with Antibiotic
The minimum inhibitory concentrations (MICs) of Kumazasa medicine were determined by the microbroth dilution methods (National Committee for Clinical Laboratory Standards Institute, 1997). A dilute suspension of bacteria was inoculated into each well of a 96-well microplate, and the range of Kumazasacytoplasmic extract was 1.25% to 0.2% in the MH broth (Difco.). The final inoculum of bactera was approximately 7.5X10 4 cells/ well by MacFarland. MBCs (Peterson and Shanholzer, 1997) were defined as the lowest concentration of antimicrobial agent where was no bacterial growth on LB agar plates (Difco.).
The antibacterial effects of a combination of antibiotics and Kumazasa was assessed by the checkerboard method (White et al., 1996).

Morphological Analysis of MRSA and VRE
MRSA and VRE were incubated in MH broth with or without Kumazasa medicine and/or antibiotics for 24hr. After incubation, culture suspension was filtered using Nuclepore Track-Etch membrane of pore size 0.1 um (Whatman Inc, Clifton, NJ USA). Morphological changes of MRSA and VRE were assessed by scanning electron microscopy (type:S-800, Hitachi Corp., Tokyo, Japan).

Results and Discussion
Plants have developed unique antibacterial mechanisms for protection against infection. In this study, we focused on anti-     Table 1). The Kumazasa-cytoplasmic extract (Kumazasa-CE) displayed a markedly potent antibacterial activity against Gram-positive bacteria such as Staphylococcus aureus, Enterococci and Streptococcus pneumoniae (MICs; 0.8-6.3 (v/v) %, 1%Kumazasa-CE contained 0.4mg of the dry extract). The antibacterial activity against Gram-positive bacteria is much stronger than that against Gram-negative bacteria such as E.coli or P.aeruginosa (MICs of Kumazasa medicine >25% in both cases) (in Table 1), because of the hydrophilic property of this extract. The Kumazasa-CE displayed strong antimicrobial activity (MICs; 0.8-6.3 (v/v) %) against both antibiotic-susceptible and -resistant Staphylococcus aureus and Enterococci strains, such as MSSA and MRSA, or VSE and VRE, respectively. Furthermore, this extract is also effective against multi-antibiotic resistant MRSA and VRE, although MRSA and VRE were simultaneously resistant to many antibiotics (in Table 1). Clinical isolates of MRSA (30 strains in total) were also tested, which gave MICs of Kumazasa-CE ranging from 1.6 to 6.3%. The MICs were compared with 16 to 128 times dilution of Kumazasa-CE, and the MIC value was corresponded to the Minimum bactericidal concentration value (MBC (Peterson and Shanholzer, 1997)). Scanning electron microscopy (SEM) images clearly showed that this extract induces firm aggregation of MRSA (in Fig.1A). This firm aggregation is a characteristic action to Staphylococcus aureus. Using VRE (NCTC12201), bacteriolysis was clearly observed after addition of this extract alone, but the firm aggregation was not evident from the SEM images (in Fig.1B). SEM analysis unmistakably showed the bacteriolytic action of this extract against VRE (in Fig.1B). Such antibacterial action was clear against also each antibiotic-susceptible strain on SEM image(data not shown). The cytoplasmic extract from Kumazasa displays surely bactericidal action against Gram-positive strains and also the multiple antibiotic-resistant bacteria as MRSA and VRE strains.
The synergistic effect was examined by checkerboard methods (White et al., 1996) in combination between this cytoplasmic extract and antibiotics (ABPC, VCM, CAM, and TC). ABPC and VCM inhibit a synthesis of bacterial cell-wall on binding to the target site on the cell-wall, while CAM and TC inhibit a protein synthesis on binding target enzyme inside of cell. The combination effect with antibiotic was evaluated by fractional inhibitory concentration (FIC) index in the method. FIC index < 0.5 were judged to be a synergistic effect; 0.5<FIC indexi < 1.0 for an additive effect; 1.0<FIC index < 2.0 no effect, as shown in Table 2.         autoclave had no effect on the observed antibacterial activity and the synergistic effect (data not shown). Therefore, the active components are heat stable. Kumazasa leaf containes many ingredients, as minerals, vitamins, polysaccharides, flavonoids, and other macromolecules (Chuyen et al., 1982). The present results should encourage research on antibacterial-hydrophilic ingredients derived from Kumazasa for use against antibioticresistant pathogen.
We conclude that the cytoplasmic extract from Kumazasa leaf displayed the unique bactericidal action against multi-antibiotic resistant MRSA and VRE strains, and that the combination with a cell wall synthesis inhibitor (ABPC or VCM) will be a highly efficient treatment for infections caused by multidrug-resistant MRSA and VRE strains.
The combination with ABPC strengthened the antibacterial activity of ABPC against MRSA, since MICs of ABPC diminished in a dose-dependent manner (Fig. 2). The FIC index values were under 0.5 indicating the synergistic effect on antibacterial activity of ABPC. Although the synergistic effect of the components in tea, such as catechins, flavonoids and polyphenols, with β-lactam antibiotic against MRSA strains has been reported, (Cho et al., 2008;Xu and Lee, 2001;) the precise-synergistic mechanism remained unclear. As observed from the present SEM images (in Fig.1A), the combination with ABPC resulted in firm aggregation of MRSA, which then triggered bacteriolysis from mycelia caking change. Our SEM analysis indicated that the synergistic effect is derived from a combination of both bacterial aggregation, mediated by this extract, and morphological transformation, mediated by ABPC. A combination of this extract with VCM synergistically enhanced the antimicrobial activity of VCM against VRE, but did not affect against susceptible Enterococci (Fig.2 and Table2). Moreover, the observed bacteriolytic activity against VRE (NCTC122201) by co-administration of VCM and this extract was greater than anticipated from the sum of the antibacterial activities of VCM or the Kumazasa-CE alone (i.e., a non additive effect). This synergistic effect with VCM is a novel finding. The combination effect was not observed against the strains susceptible to ABPC or VCM (in Table 2). Interestingly, the efficacy of this extract is closely related to an inhibitor of bacterial cell-wall synthesis on multi-antibiotic resistant MRSA and VRE strains.
The synergistic effect on CAM could not be measured because of the strong resistance of MRSA to CAM (MIC >128 µg/ml). The antibacterial activity of CAM was increased by half upon using 0.8% of the Kumazasa-CE against MSSA susceptible to CAM, showing an additive effect. However, the extracts at concentrations 0-0.4% did affect the MIC values of CAM. Similarly, the antimicrobial activity of CAM was enhanced by a combination of this extract against VSE and VRE(GTC0200), which are susceptible to CAM. However, such combination effect could not be detected against VRE(12201), which is strongly resistant to CAM. This extract additively enhanced the antibacterial activity of TC against MRSA (JCM8703, and clinical isolate 2005) and VRE (GTC02000 and NCTC12201) in checkerboard method, however it was not obtained against TC-highly resistant and susceptible Staphylococcus aureus strains (in Tables  1 and 2). The target sites for both CAM and TC are intracellular, and the resistant mechanism consists of a modulation on target sites and an acceleration of efflux pump. Herbal extracts were reported to reinforces synergistically the antibacterial activity by inhibition of the efflux pump for antibiotics (Rosato et al., 2007;Lee et al., 2008;. No such synergistic effect between this extract and CAM and/or TC was observed from the SEM images or from the fractional inhibitory concentrations determined using the checkerboard method. The absence of synergistic action in this extract is presumably because the target sites for both CAM and TC are intracellular. Although there is no question concerning the observed synergistic effect between the Kumazasa-cytoplasmic extract and ABPC or VCM against antibiotic-resistant bacteria (e.g., MRSA or VRE), the precise mechanism of action needs further study. Furthermore, heat treatment of this Kumazasa extract using an » CrossRef » Pubmed » Google Scholar » CrossRef » Pubmed » Google Scholar » CrossRef » Google Scholar » CrossRef » Pubmed » Google Scholar » CrossRef » Pubmed » Google Scholar