Screening and Preliminary Identification of Medicinal Plants Endophytic Actinomycetes Used for Inhibiting Penicillin-Resistant Staphylococcus aureus

65 strains of endophytic actinomycetes were isolated from Achyranthes bidentata, Paeonia lactiflora, Radix Platycodi and Artemisiae argyi. Active strains of inhibiting penicillin-resistant staphylococcus aureus were screened from these strains and some were identified preliminarily. The results showed that 12 strains among these 65 strains of endophytic actinomycetes were able to suppress penicillin-resistant staphylococcus aureus. Through the observation on morphology of mycelium and phylogenetic analysis of 16S rDNA, a strain belongs to the genus Glycomyces and most other as the genus Streptomyces.


Introduction
Since Fleming discovered the first antibiotic-penicillin in 1929, the discovery and application of antibiotics successfully prevent and treat various diseases infected by microbes .However, due to the long-term and extensive use of antibiotics, a large number of drug-resistant strains emerged in clinic.According to reports, the average chance of infection by drug-resistant Mycobacterium tuberculosis has reached 10.2%, and Mycobacterium tuberculosis and Staphylococcus aureus as a common pathogenic bacteria are prone to mutation and drug resistance (Monaghan, et al., 2006).The enhancement of bacterial resistance to drugs caused great difficulties to clinical anti-infective therapy, hence it was concerned widely.The people are constantly trying to find new antimicrobial substances from different organisms to resist the various germs which can threat to human life and health.
Actinomycetes is the main source of antibiotics and endophytic actinomycetes isolated from medicinal plants has considerable development potential.From the present findings, some new actinomycetes from tissue of medicinal plants have been founded constantly (Kudo, et al., 1998;Chen, et al., 2009;Qin, et al., 2008Qin, et al., , 2010)).Moreover, most endophytic actinomycetes of medicinal plants can produce important compounds and some of them are new chemical structure (Caruso, et al., 2000;Ezra, et al., 2004;Liu, et al., 2007).
The purpose of this study is to isolate endophytic actinomycetes from the medicinal plants and to screen antagonistic strains of drug-resistant Staphylococcus aureus among the isolating strains.

Pathogenic instructions bacteria
Penicillin-resistant staphylococcus aureus HBUY100301 was reserved by this laboratory.

Isolation and cultivation of endophytic actinomycetes
The root tissue of medicinal plants was washed by tap water and surface sterilized with 75% alcohol for 5~10min and 0.1% HgCl2 for 2~5min, finally washed by sterile water for 4 times before being ground and spread onto S Agar (Lechevalier, et al., 1983), Starch-Casein Agar (Ku¨ster, et al. 1964) and Flour yeast-extract sucrose casein hydrolysate agar (Coombs, et al., 2003) supplemented with (ml-1) 30μg nalidixic acid, 20μg ketoconazole and 50μg cycloheximide.After incubation at 28 for 2 ℃ -4 weeks, the strains were isolated and purified on ISP2 agar (Shirling & Gottlieb, 1966).The pure culture was maintained as a suspension in 30% glycerol at -80 and lyophilized cells for long ℃ -term preservation.

Preparation of inoculum and fermentation
Spore suspension was prepared in distilled water from culture grown on S medium at 28 for 7 days.The ℃ suspension was added to ISP-2 broth in each 500 ml Erlenmeyer flask at 10% inoculum rate in 100 ml liquid medium.Cultures were kept on a shaker at 180 r/min at 28 for 7 days.The fermentation liquid were extracted ℃ with isopyknic methyl alcohol and centrifugation at 5000 r/min for 20 min.Extracting solution was concentrated under vacuum and then preserved at -20 .℃

Antagonistic-activity assay
Two 8 mm discs of sterile paper was soaked in extracting solution above.The air-dried discs were palced on a NA plate which was inoculated penicillin-resistant staphylococcus aureus.After incubation at 28℃ for 24h, the result with or without inhibition zone was observed.Each treatment consisted of three replicates.The experiment was repeated twice.

Observation of morphological characteristics
Strain was inoculated on ISP-2 agar and coverslip was inserted into medium, cultivated at 28 for 5 days.Then ℃ the coverslip was placed under the light microscope to observe morphological characteristics of Mycelium.

Phylogenetic analysis of 16S rDNA
(1) Extration of genomic DNA Genomic DNA was extracted by the method of Fred (Fred at al., 1996).
(3) Construction of phylogenetic tree The obtained 16S rDNA sequences was undertaken homology comparison with the known sequences of Genbank database using BLAST method.Multiple alignment of the sequences obtained from GenBank was performed using CLUSTAL W (Thompson, et al., 1994).Phylogenetic trees were constructed with MEGA version 5.0 (Tamura, et al., 2011) using the neighbour-joining method (Saitou & Nei, 1987) and the reliability of the tree topology was evaluated by bootstrap analysis with 1000 resamplings (Felsenstein, 1985).

Screening of antagonistic strains
12 among 65 strains of endophytic actinomycetes were able to suppress penicillin-resistant staphylococcus aureus.The Numbers of strains is as table 1 and some photos of bacteriostasis are as (figure 1)

Morphological characteristics
The result of observation of morphological characteristics showed most active strains had typical spiral spore chain (Figure 1, A) and these feature consistented with the genus Streptomyces, in addition to some strains (Figure 1, B). 16S rDNA phylogenetic analysis of part of the representative strains were carried o further determine their taxonomic status.

Discussions
With the increasing number of antibiotic applications, bacterial resistance has become a serious problem faced by the world anti-infective therapeutic area.In the present study, 12 strains of endophytic actinomycetes which could inhibit penicillin-resistant staphylococcus aureus were successfully isolated from Achyranthes bidentata, Paeonia lactiflora, Radix Platycodi, Artemisiae argyi.The results showed endophytic actinomycetes of medicinal plants are important resource and finding some new compounds to suppress drug-resistant strains from secondary metabolites of medicinal plants endophytic actinomycetes is expected to become an effective way to resolve the above problem.
In addition, the preliminary identification of some active strains in this study showed that the most belonged to the genus Streptomyces.This result is consistent with previous report which Streptomyces is the most important strains of antibiotic production; from another perspective, the rapid growth of the Streptomyces are likely to affect the separation of other actinomycetes.Therefore, the selectivity of isolation and culture methods to the slower growth of endogenous actinomycetes should be increased.