Introduction

Drug resistance problems are increasing worldwide. There is a need to find novel sources of antimicrobial drugs. At present, natural products still remain the most important resources for discovering new drugs. Endophytic fungi have been shown to produce a wide range of biologically active metabolites (Aly et al. 2010). Natural products from mangrove fungal endophytes with biological activity have been reported (Chen et al. 2007; Huang et al. 2008). However antimicrobial activity from endophytic fungi isolated from mangroves in Thailand has been rarely studied. Recently, (Chareprasert et al. 2010) reported that an endophytic fungus Xylaria sp.1 isolated from Acanthus ilicifolius from Thailand had antibacterial properties against both Gram-positive and Gram-negative bacteria. However, only the agar diffusion test method was used in their study. This study aimed to screen for fungal endophytes from mangrove plants in the south of Thailand that produced antimicrobial metabolites against potential human pathogens. Minimal inhibitory concentrations (MIC), minimal bactericidal concentrations (MBC) or minimal fungicidal concentrations (MFC) of crude extracts from their culture media and hyphae were determined.

Materials and methods

Sampling of mangrove plants

Leaves and branches from 54 healthy plants of 12 mangrove species were collected from mangrove areas in the south of Thailand in Satun, Songkhla, Surat Thani and Trang provinces. The twelve mangrove species were Aegiceras corniculatum (n = 1), Avicennia alba (n = 4), Avicennia officinalis (n = 5), Bruguiera gymnorrhiza (n = 1), Bruguiera parviflora (n = 5), Lumnitzera littorea (n = 2), Rhizophora apiculata (n = 12), Rhizophora mucronata (n = 9), Sonneratia caseolaris (n = 4), Scyphiphora hydrophyllacea (n = 3), Xylocarpus granatum (n = 4), and Xylocarpus moluccensis (n = 4). Plant specimens were compared with the voucher specimens at Prince of Songkla University Herbarium.

Isolation and identification of endophytic fungi

The isolation methods for fungi were as previously described (Phongpaichit et al. 2006). Briefly, leaves and branches were cut into small segments and surface-sterilized by sequential washes in 95% ethanol (30 s), 5% sodium hypochlorite (5 min), 95% ethanol (30 s) and rinsed with sterile water. The sample segments were then placed on corn meal agar medium supplemented with antibiotics (penicillin plus streptomycin sulphate 50 mg l−1) to restrict bacterial growth. Plates were incubated at 25°C for 1 week. Fungal growth was observed every day. Pure cultures were obtained by hyphal tip isolation and stored in 15% glycerol at −80°C. A total of 619 fungal isolates were obtained and 150 were selected for antimicrobial assays based on their morphotypes and host plant source.

Endophytic fungi that showed good antimicrobial activity were identified based on morphology and the analyses of the DNA sequences of the large subunit (LSU) and the internal transcribed spacer (ITS1-5.8S-ITS2), ITS regions of their ribosomal RNA gene using fungal universal primers (White et al. 1990; Bunyard et al. 1994; Landvik 1996). The LSU and ITS sequences of active endophytic fungi have been submitted to GenBank for retrieving their accession numbers.

Fermentation and extraction of endophytic fungi

Endophytic fungal cultures were grown on potato dextrose agar and incubated at 25°C for 3–5 days. Six mycelial plugs (1 × 1 cm2) were inoculated into 500 ml Erlenmeyer flasks containing 300 ml potato dextrose broth and incubated for 3 weeks at 25°C under a stationary condition (Phongpaichit et al. 2006). The culture broth was filtered to separate the filtrate and mycelium. The filtrate was extracted with ethyl acetate (EtOAc) and this was evaporated to dryness under reduced pressure at 45°C using a rotary vacuum evaporator to give the BE extract. The fungal mycelia were extracted with methanol (MeOH) for 2 days. The aqueous MeOH layer was concentrated under reduced pressure. Distilled water (50 ml) was added to the extract and the mixture was then mixed with hexane (100 ml). The aqueous layer was then extracted with an equal volume of EtOAc. The hexane extract and the combined EtOAc extracts were evaporated to dryness under reduced pressure at 45°C using a rotary vacuum evaporator to give CH and CE extracts respectively.

Antibacterial assay

The dried fungal extracts were dissolved in dimethyl sulfoxide and stored at −4°C until used. They were tested against Staphylococcus aureus ATCC25923, a clinical isolate of methicillin-resistant S. aureus (MRSA) SK1, Escherichia coli ATCC25922, and Pseudomonas aeruginosa ATCC27853 by a microdilution method according to a modification of Clinical and Laboratory Standards Institute (CLSI) M7-A4 (CLSI 2000). The lowest concentration of extract that inhibited visible growth was recorded as the MIC. Concentrations of crude extract less dilute than the MIC and the MIC were streaked onto a nutrient agar plate and incubated under appropriate conditions. The lowest concentration of extract that showed no growth was recorded as the MBC. Vancomycin and gentamicin were used as standard antibacterial agents for positive inhibitory controls.

Antifungal assay

The MICs of fungal crude extracts were determined by a modification of the microbroth dilution CLSI M27-A2 (CLSI 2002a) against yeasts (Candida albicans ATCC90028, Cryptococcus neoformans ATCC90112) and a modification of the microbroth dilution CLSI M38-A (CLSI 2002b) against a clinical isolate of Microsporum gypseum MU-SH4. Microtiter plates were incubated at 35°C for 24 h for C. albicans, 48 h at room temperature for C. neoformans, and 7 days at room temperature for M. gypseum. The MFCs of the active extracts were determined by the streaking method on Sabouraud’s dextrose agar. Amphotericin B was used as a positive inhibitory control for yeasts and miconazole for M. gypseum.

Results and discussion

Antimicrobial activity

A total of 385 extracts from 150 fungal endophytes were evaluated in antimicrobial screening tests. The results showed that 47% of extracts from 92 isolates (61.3%) inhibited at least one test microorganism. Of these, the endophytic fungi that produced secondary metabolites had most activity against Gram-positive bacteria (S. aureus ATCC25923 and MRSA-SK1) with 65 and 70 isolates, followed by M. gypseum (59 isolates), C. neoformans (35 isolates) and C. albicans (21 isolates). Only two endophytic fungal extracts inhibited P. aeruginosa and none inhibited E. coli (Table 1). It is interesting to note that both strains of S. aureus tested gave very similar results. S. aureus ATCC25923 is a susceptible strain whereas MRSA is resistant to methicillin. MRSA has the mecA gene encoding for an altered penicillin binding protein gene (PBP2a) which has low affinity to beta-lactam antibiotics (Barber 1963). It is well known that MRSA are multi-drug resistant strains (Kwong et al. 2008). This may indicate that different resistance mechanisms are involved in the resistance of MRSA to different classes of antibiotics. Active fungal crude extracts in this study may act on different or new targets.

Table 1 Selected endophytic fungi from various mangrove species and their antimicrobial activity against pathogenic microorganisms
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Among the active crude extracts, hexane extracts from fungal mycelia had the highest inhibitory activity in the screening tests with 51%, followed by ethyl acetate extracts from the mycelia (46.5%) and ethyl acetate extracts from the fermentation broths (44.8%). This result indicated that most of the active materials were present in the fungal mycelia. Active extracts may contain cell-bound components and low polarity substances.

A total of 181 active extracts inhibited 1–5 strains of the test microorganisms (Fig. 1). Most of them (35.4%) inhibited 2 strains. Only 7.2% had a broad antimicrobial spectrum that inhibited 5 strains of test microorganisms that included S. aureus, MRSA-SK1, C. albicans, C. neoformans and M. gypseum. In this study, we found six endophytic fungi that produced extracts with low MIC values against test microorganisms (Table S1, see Electronic Supplementary Material). These isolates were MA12, MA34, MA96, MA148, MA156 and MA194. The MIC values of their extracts were comparable to the MIC values of standard drugs. MA194 isolated from Rhizophora apiculata showed the broadest antimicrobial spectrum against Gram-positive bacteria, yeasts and M. gypseum (MIC 8–32 μg ml−1).

Fig. 1
figure 1

Number of test strains inhibited by mangrove endophytic fungal crude extracts

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Distribution and identification of endophytic fungi with antimicrobial activity

Among the antimicrobial active isolates listed in Table S1 only two isolates (MA34 and MA99) can be identified based on their morphology as Xylaria cubensis and Pestalotiopsis sp., respectively. The rest did not sporulate in culture and were identified as sterilia mycelia. Based on their morphology and LSU and ITS sequence analyses, our active mangrove endophytic fungal isolates can be classified into six genera, Acremonium, Diaporthe, Hypoxylon, Pestalotiopsis, Phomopsis, and Xylaria. The genera Diaporthe, Phomopsis, Pestalotiopsis and Xylaria have been reported to be the most common endophytes isolated from mangrove plants (Cheng et al. 2008; Huang et al. 2008; Pang et al. 2008; Chareprasert et al. 2010). Many mangrove endophytes have been reported to produce antimicrobial substances such as Colletotrichum sp., Xylaria sp., Pestalotiopsis sp., Paecilomyces sp., Phomopsis sp. and Phoma sp. (Pang et al. 2008; Schulz et al. 2008). Moreover, Hypoxylon spp. found in this study have never been previously reported as mangrove fungal endophytes. (Chareprasert et al. 2010) reported that Cladosporium sp. from Thespesia populneoides and Xylaria sp. 1 from Acanthus ilicifolius caused considerable inhibition to Gram-positive and Gram-negative bacteria. They collected mangrove plants from the west coast area in Chanthaburi Province and the upper southern part of Thailand, Prachuap Khiri Khan and Ranong Provinces whereas our collecting sites were from the lower southern part. Results from these studies have indicated that endophytic fungi from mangrove plants from Thailand could be a good source of antimicrobial natural products.