MATERIALS AND METHODS

Plant collection and extraction: Schefflera leucantha viguier was collected and on May 2008 from Prachinburi Province, Thailand. The plant was identified by Department of Biology, Faculty of Science, Mahasarakham University, Thailand. Ten gram of dried aerial part of plant were boiled in 500 mL water and the residues of extraction were done the repeat of extraction 3 times. The yield of extraction was 0.7-1.3% of dried weight of dried plant ’ s powder. In this study used pooled of batches of the same condition of extraction throughout the studies. The extraction and antimicrobial susceptibility tests were conducted on year 2008 at Department of Science, Mahasarakham University, Thailand.

Microorganisms: All of bacteria used in this study are American Type Culture Collection (ATCC). The gram positive (Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Micrococcus luteus ATCC 9341, Bacillus subtillis ATCC 6633, Lactobacillus plantarum ATCC 14917 and gram negative (Escherichia coli ATCC25922, Salmonella typhimurium ATCC 14028, Klebsiella pneumonia ATCC 10031, Proteus vulgaris ATCC 13315 and Pseudomonas aeruginosa ATCC 9721 ) bacteria, were obtained from the Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Thailand.

Antimicrobial sensitive test
Agar diffusion susceptibility test: The antibacterial activity of plant extract were screened using agar diffusion method as described in the standard guideline (Lorian, 1996). Briefly, bacteria cultures were grown in Tryptic soy agar at 37 ° C for 19 h and wash with sterile normal saline solution (NSS) (0.9% NaCl) to prepared standard McFarland No. 0.5 (10⁸ colony-forming units (cfu)/mL. Twenty milliliter of Muller Hinton Agar (MHA) was poured in Petri disc and the bacteria suspension solution were swab in to the surface of culture medium by using sterile swabbing cotton. The sterile stainless steel cylinders (6 mm internal diameter and 10 mm height) were place on the agar medium surface.

Different concentrations (1, 2 and 4 g L ^-1) of plant solution were prepared using sterile water as vehicle and filled in to the stainless steel cylinders. After pre-diffusion at room temperature for 1 h, the plates were incubated at 37 ° C for 19 h. The 10 mg L ^-1 of gentamicin sulphate (Sigma Chemical Co., St. Louis, USA) solution was used as reference antibiotic.

MICs and MBCs determination using agar dilution and broth macro dilution: The MICs (Minimal Inhibitory Concentration) of plant extract was determined by using agar dilution method (Lorian, 1996), while MBCs (Minimal Bactericidal Concentration) were determined by using the broth macro-dilution method (Lorian,1996). Both methods were used gentamicin sulphate as reference antibiotic (Sigma Chemical Co., St. Louis, USA). The bacteria suspensions were prepared in normal saline solution at density adjusted to No. 0.5 McFarland turbidity. The inoculated bacterial cultures were incubated at 37 ° C. After 24 h of incubation the MICs were recorded by observed no spot of bacteria growth in agar dilution method, while no turbidity of broth culture tube in broth-macro dilution method. The MBCs was determined by no bacterial growth observed in sub culture of clear tube from broth-macro dilution test.

RESULTS

In this study, S. leucantha was extracted with aqueous system with of 0.7-1.3% of dried weight of dried plant ’ s powder. The antimicrobial activity of the plant was assayed in vitro by agar diffusion, agar dilution and broth macro-dilution methods against 10 bacteria species.

The agar diffusion test revealed the inhibitory effect of the plant against L. plantarum ATCC 14917, E. coli ATCC   25922,   K.   pneumoniae ATCC 10031 and P. vulgaris ATCC 13315. The clear zone diameter are in the range of 12-16 mm at plant extract concentration 4 g L ^-1 (Table 1). Among tested bacteria L. plantarum is normal flora while E.coli, K. pneumoniae and P. vulgaris are nosocomial infection bacteria. The aqueous extract of the plant showed inhibitory effect against growth of E. coli and K. pneumoniae causing urinary tract infection while P. vulgaris is an opportunistic causing both of urinary tract infections and respiratory tract infections.

The Minimal Inhibitory Concentrations (MICs) of the plant was determined by using agar dilution method and confirmed by broth macro-dilution method while the Minimal Bactericidal Concentrations (MBCs) were determined using broth macro-dilution method. The MICs of S. leucantha are in the rage of 8-16 g L ^-1 while MBCs are in the rage of 16-32 g L ^-1 (Table 2). The most sensitive bacteria to the plant extract were gram negative bacteria than gram positive bacteria. This may because of the difference cell wall structure of the bacteria.

In conclusion, aqueous extract of S. leucantha showed inhibitory effect against respiratory tract and urinary tract infection bacteria at low concentrations. The results supported used of S. leucantha as nosocomial infection treatment. In addition, the toxicity test of this plant was need to indicated the safety doses of used this plant as remedy.

Table 1:	Inhibition zone diameters of S. leucantha aqueous extract against some nosocomial infection bacteria and normal flora bacteria
nz: No inhibition zone; Data are expressed as Mean ±SD

Table 2:	The MICs and MBCs of S. leucantha aqueous extract against some nosocomial infection bacteria and normal flora bacteria
nd: Not determine

DISCUSSION

Recently, nosocomial infection bacteria decrease their susceptibility to antibiotic medicine and some developed to be multidrug resistant bacteria (Martinez and Baquero, 2002). Patients with none infection diseases who have to stay in hospital for long period such as heart disease, cancer disease and other chronic diseases have high risk to get nosocomial infection (Nicholls et al., 1975; Asefzadeh, 2005; Saonuam et al., 2008). Furthermore, in nurse and medical career person who need to contact with patient often as well. The difference structure or targets of attacking bacteria medicines were being used. However, treatment of multi-drug resistant bacteria consider complicated. It has been reported that some of bacteria were decreased its susceptibility rates to Tigecycline for 3 years follow-up study in USA (Dowzicky and Park, 2008).

The one candidate for antibacterial treatment is plant extract. The folk medicine treatment has been applied in many countries before antibiotics were existed. However, the scientific studies such biological activity and toxicology or even clinical studies of antibacterial activity of plant extracts still in few amounts. Therefore, the evaluation of antimicrobial activity of plant extract may give useful information.

In this study, antimicrobial activity of S. leucantha has been evaluated. The screening of antimicrobial activity of plant extract was conducted by agar diffusion method. The plant aqueous extract showed inhibition zone against L. plantarum ATCC 14917, E. coli ATCC 25922, K. pneumoniae ATCC 10031 and P. vulgaris ATCC 13315 (Table 1.) The L. plantarum is normal flora while E. coli, K. pneumoniae and P. vulgaris are nosocomial infection bacteria. In general, E. coli and K. pneumoniae causing urinary tract infection while P. vulgaris is an opportunistic causing both of urinary tract infections and respiratory tract infections.

The MIC of S. leucantha aqueous extract was determined using agar dilution method (and confirm with broth macro dilution method), while MBC was determined using broth macro dilution (0.5 -512 g L ^-1). The MICs of plant aqueous extract were in the range of 8-16 g L ^-1 while the MBCs were in the range of 16-32 g L ^-1 (Table 2).

Previously, antibacterial of S. leucantha methanol extract has been reported by Potduang et al. (2007). The study reported antimicrobial activity of S. leucantha methanol extract against Bacteroides sp., Enterococcus faecalis, Lactobacillus sp., Peptococcus sp., Streptococcus mutans., Klebsilla pneumoniae and Propionibacterium acnes. However, the antimicrobial activity of plant extract can be difference due to the difference solvent extraction. The using of water considers less toxicity compare with organic solvent and good as medicine vehicle as well. In this study, S. leucantha aqueous extract show good antibacterial activity in low concentration. This may supporting the used of S. leucantha as a traditional medicine for nosocomial infection especially for respiratory tract and urinary tract infections.

In conclusion, the result showed good inhibitory effect of S. leucantha against respiratory tract and urinary tract infection bacteria at low concentration. It may interesting for further investigation in clinical isolation of the bacteria to evaluate the possible use of this plant as the remedy to treat respiratory tract and urinary tract infection because the bacteria that was isolated from patient may have difference susceptibility to the plant extract and also to the medicine as well. Recently, the infection diseases treatments with medicine have to deal with multi-drug resistant and adverse effect of the drug. The plant extract seem to be one way of treatment of infection disease. This study may supply some information of antimicrobial activity of S. leucantha aqueous extract against respiratory and urinary tract infection bacteria.