Abstract: In this study, anti-bacterial activity of aqueous extract of P. igniarius against selected gram positive and gram negative bacteria using agar diffusion, broth macrodilution and agar dilution methods were investigated. The agar diffusion method revealed that P. igniarius aqueous extract showed inhibition zone against only S. aureus ATCC 24923. The susceptibility test against MSSA and MRSA revealed that P. igniarius aqueous extract showed inhibition zone against all of MSSA strains but cannot inhibit growth of MRSA. The broth macro dilution and agar dilution methods reveal MIC and MBC of P. igniarius against S. aureus ATCC 25923 was 1.25 and 2.5 g L-, respectively while MICs and MBCs against MSSA were 1.25-2.5 g L- and 0.25-0.5 g L-, respectively. The aqueous extract of P. igniarius showed inhibitory effect on growth of MSSA but not MRSA.
INTRODUCTION
Phellinus igniarius or Phellinus igiarius is the wooden fungus belong to family Hymenochaetaceae. It has been reported as the Eskimos folklore (Blanchette et al., 2002). The fungus in Phellinus group has been intense study about it immunological and anticancer activity. P. igniarius has been reported antiproliferative, antimetastatic (Tuzz-Ying Lin et al., 2008) and anticacinogenic effect (Shon and Kyungo-Soo, 2001).
Furthermore, it also has been reported vasorelaxation (Kang et al., 2006), antioxidant and cytotoxic (Wang et al., 2005a, b), inhibition of cytochrome P450 isozyme and ornithine decarboxylase activity (Shon and Kyungo-Soo, 2004). However, P. igniarius never been reported it antibacterial activity. This is the first reported of P. igniarius antibacterial activity.
MATERIALS AND METHODS
Fungi sample and extraction: The P. igniarius was identified by Natural medicinal mushroom museum, Mahasarakham University, Thailand. The fungus was collected on July 2008 from forest in Cambodia. The 10 g of fungi were grinded and boiled in 1 L of water for 15 min and filtrated. The filtrates were spray dried. The yield of spray dried extract was 1-2.5% of dried weight of fungi. In this study used one batch of extraction throughout the studies.
Tested microorganism: All microorganisms were obtained from Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Thailand. Eight bacteria, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Micrococcus luteus ATCC 9341, Bacillus subtillis ATCC 6633, Lactobacillus plantarum ATCC 14917 were used as gram positive bacteria Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028 and Klebsiella pneumonia ATCC 10031 were used as gram negative bacteria.
Twelve clinical isolates of S. aureus were included in this study. The tested isolates were divided into 2 groups, 6 of each, according to susceptibility to methicillin where, MRSA referred to methicillin-resistant strains and MSSA referred to methicillin-susceptible strains.
Antimicrobial assay
Agar diffusion susceptibility test: Susceptibility determinations
were made as described in the standard guideline technique (Lorian, 1996). All
test bacteria were cultured overnight on Tryptic Soy Agar (TSA) slant at 37
°C. Bacteria were washed from surface agar slant with sterile normal saline
solution (0.9% NaCl) then adjusted to match turbidity of standard Mcfarland
No. 0.5 before used as starter solution. Twenty milliliter of Mueller Hinton
Agar (MHA) was putted in cultivation plates and swabbed starter solution on
agar surface by using swab cotton.
Phellinus igniarius extract was dissolved in sterile water and put in sterile stainless steel cylinders (6 mm internal diameter and 10 mm height) were placed on the inoculated agar surface. The various concentrations of P. igniarius extract solution were filled in the cylinders (300 μL cylinder-). After pre-diffusion at room temperature for 1 h, the plates were incubated at 37°C for 19 h. The normal saline solution filled in the cylinder was used as control and 10 mg L- gentamicin sulphate solution or 1 μg mL- oxacillin were used as standard in same cultivation plate.
MICs and MBCs determination using agar dilution and broth macro dilution methods: MICs of crude water extract of P. igniarius solution were determined by agar dilution method (Merck) (Lorian, 1996) while MBCs were determined by broth macro-dilution method were (Lorian, 1996) and reference antibiotics oxacillin (Sigma Chemical Co., St. Louis, USA). Inoculates were prepared in the same medium at density adjusted to 0.5 McFarland turbidity standard (108 colony-forming units (Cfu mL-) and two fold dilution for the broth macro-dilution procedure. The inoculated tube were incubated at 37°C and the MICs were recorded after 24 h of incubation. The MIC was defined as the lowest concentration of P. igniarius solution or oxacillin at which the microorganism tested did not showed visible growth while MBC was defined as the minimum bactericidal concentration with negative subcultures on agar medium. Values were means of triplicate.
RESULTS AND DISCUSSION
Phellinus sp. were reputed as oriental and Russian folklore especially P. igniarius has been record to use as remedy in Russian (Blanchette et al., 2002). Among of Phellinus sp., Phellinus linteus is the most studied of it biological activities. P. linteus has been investigated it anti-Staphylococcus aureus (Hur et al., 2004), the aqueous extract of its show anti-methicillin resistant S. aureus. However P. igniarius has never been investigated it antibacterial activity.
The used of P. igniarius has been recorded in Russian and Alaskans or Eskimos (Branchette et al., 2002). Until nowadays Alaskans still used P. igniarius as the remedy in elder to 5 years old person even in pregnancy. In oriental, P. igniarius has been interesting for treatment as immuno modulator or inhibition of cancer cells. It has been reported antiproliferative and antimetastatic effects of human hepatocacinoma (Tuzz-Ying Lin et al., 2008) and antimutagenicity in bacterial model (Salmonella typhimurium) (Shon and Kyung-Soo, 2001). It is also showed vasorelaxation effect (Kang et al., 2006) and inhibitory effect on cytochrome P450 isozymes and ornithine decarboxylase activities (Shon and Kyung-Soo, 2004).
In this study antibacterial activity of aqueous extract of P. igniarius
against selected gram positive and gram negative bacteria using agar diffusion,
broth macrodilution and agar dilution methods were investigated. The agar diffusion
method revealed that P. igniarius aqueous extract showed inhibition zone
against only S. aureus ATCC 24923 (Table 1). Thus it
was test inhibitory effect against 12 clinical isolates S. aureus. The
1 μg oxicillin disc was tested against clinical isolates of S. aureus.
The S. aureus which showed no inhibition zone with 1 μg oxacillin
were grouped as MRSA (methicillin resistant S. aureus) while S. aureus
which give inhibition zone diameter more than 9 mm, were grouped as MSSA (methicillin
sensitive S. aureus). The susceptibility test against MSSA and MRSA revealed
that P. igniarius aqueous extract showed inhibition zone against all
of MSSA strains but cannot inhibit growth of MRSA (Table 2).
The broth macro dilution and agar dilution methods reveal MIC and MBC of
P. igniarius against S. aureus ATCC 25923 was 1.25 and 2.5 g L-,
respectively while MIC and MBC against MSSA were 1.25-2.5 g L- and
0.25-0.5 g L-, respectively (Table 3). This is
the first reported antimicrobial activity of P. igniarius.
| Table 1: | Inhibition zone diameters of P. igniarius aqueous extract solution against various bacteria |
| Data are Mean±SD (n = 3); nz = No inhibition zone | |
| Table 2: | Inhibition zone diameters of P. igniarius aqueous extract solution against MSSA and MRSA |
| Data are Mean±SD (n = 3); nz = No inhibition zone | |
| Table 3: | The MICs and MBCs of P. igniarius aqueous extract against MSSA and MRSA |
In conclusion the P. igniarius aqueous extract may consist of antimicrobial active compound that can inhibit growth of S. aureus which is involving skin infection. However, P. igniarius cannot inhibit growth of MRSA which causing severe infection diseases. This study provides additional information the use of P. igniarius as remedy.
ACKNOWLEDGMENTS
Authors would like to show their appreciations to the staff of Natural Medicinal Mushroom Museum, Mahasarakham, Thailand especially Mr. Winai Klinhom (M.Sc.) Faculty of Science, Mahasarakhaum University the expertise of ethno mushroom who identified the P. igniarius and gives the mushroom sample.