Secondary metabolites produced by endophytic fungus Paecilomyces ...

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variotii Bainier with antimicrobial activity against Enterococcus ... antimicrobial activity of the crude extract obtained from Paecilomyces variotii for eight clinical ...
Secondary metabolites produced by endophytic fungus Paecilomyces variotii Bainier with antimicrobial activity against Enterococcus faecalis M. R. Oliveira Silva*,1,2, K. Xisto Sena2 and N. Buarque Gusmão2 1

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PhD Student in Biology of Fungi, Department of Mycology, Federal University of Pernambuco, Av. Prof. Morais Rego, 1235, 50670-901, Recife, Pernambuco, Brazil 2 Laboratory of Antimicrobial Assays, Department of Antibiotics, Federal University of Pernambuco, Av. Prof. Morais Rego, 1235, 50670-901, Recife, Pernambuco, Brazil *

Corresponding author: e-mail: [email protected], Phone: +55 81 21268346

Enterococcus faecalis is a Gram-positive bacteria considered an important emergent nosocomial pathogen, mainly due to its resistance to most antibiotics and responsible for a variety of human infections. In order to discover new bioactive secondary metabolites from microbial sources, the objective of this work was to evaluate the antimicrobial activity of the crude extract obtained from Paecilomyces variotii for eight clinical isolates of E. faecalis by the disk diffusion method. The results showed that all clinical isolates were inhibited by fungal extract, with inhibition zones ranging from 25 to 35.25 mm. In addition, the results also revealed resistance, in the microorganisms test, to antibiotics such as ciprofloxacin, cotrimoxazole, erythromycin, gentamicin, oxacillin and tetracycline. Keywords Antimicrobial Activity; Paecilomyces variotii; Endophytic Fungi; Enterococcus faecalis

1. Introduction Enterococci are usual inhabitants in the human intestinal microbiota, mainly in the large intestine and genitalurinary tract, and generally they are not regarded as pathogenic organisms. However, in literature it has been cited that enterococci are emerging as prominent nosocomial pathogens, being responsible for a variety of human ailments [1, 2]. Enterococcus faecalis is a member of the genus Enterococcus considered the major opportunistic pathogen isolated from hospital infections, with incidence of 80-90% [3]. The survival of this microorganism in the hospital environment is due to the elevated level of acquired resistance to conventional antibiotics, such as aminoglycosides and glycopeptides, but its resistance to ampicillin is still rare [4]. The increased in drug-resistance by bacteria is considered a serious problem for public health, constituting a challenge for researchers worldwide to search for new antimicrobials agents. Nowadays, research groups have focused interest on microorganisms obtained from different ecosystems, probably due to their metabolic pathway diversity in consequence of the selective environmental conditions. Endophytic fungi are recognized as useful sources of secondary metabolites because depending on the biotype, ecological niche and host plants, these microorganisms are able to produce appropriate bioactive compounds [5, 6]. There are several reports on antimicrobial compounds produced by this specific group of fungi [7-10]. In an attempt to discover a novel substance of medicinal importance, the present work aims to evaluate the antimicrobial activity of the secondary metabolites obtained from Paecilomyces variotii, an endophytic fungus isolated from Laguncularia racemosa mangrove plant.

2. Material and Methods 2.1

Endophytic fungus

Paecilomyces variotii strain was isolated from the leaves of Laguncularia racemosa (L.) Gaertn. (White mangrove, Combretaceae) collected from estuary of the Paripe River, Ilha de Itamaracá, Pernambuco, Brazil. The fungal identification was performed by observing macroscopic and microscopic characteristics such as colour, colony morphology and conidial size. The fungus was maintained on potato dextrose agar (PDA) slants at ± 4°C until further use.

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2.2

Fermentation

In order to obtain secondary metabolites, the fungal strain was grown in PDA culture medium at 30°C for 3 days. After that, a pre-inoculum was prepared by introducing small fragments (1 cm2) of the growth culture into 250 mL Erlenmeyer flasks containing 50 mL of Malt broth (1.5% Malt Extract, pH 6.9 ± 0.1) and cultivated on a rotary shaker at 200 rpm, ± 28°C (room temperature) for 3 days. Subsequently, the pre-inoculum was transferred to Fernbach flasks containing 450 mL of the same culture medium and cultivated as described above for 7 days.

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2.3 Obtention of crude extract At the end of fermentation, the culture broth was separated from the mycelium by filtration and the filtrate was extracted with ethyl acetate (1:1, v/v) under constant shaking for 2 hours. The organic phase was concentrated under reduced pressure using a rotary evaporator at ± 45°C and, finally, the concentrated extract was stored in a vacuum desiccator. 2.4

Microorganisms test

For the antimicrobial activity test, eight clinical isolates of Enterococcus faecalis were used: 21675, 21752, 21944, 24614, 24622, 24708, 24962 and 25392 (Table 1). All clinical isolates were provided by Laboratorial Unit of the University Hospital, Federal University of Pernambuco, Recife, Brazil. The confirmation of the genus Enterococcus was determined by plating in bile-esculin and brain heart infusion plus 6.5% NaCl agars culture media. Sequentially, the bacterial cultures were maintained on growth medium (1% Peptone, 0.3% Beef Extract, 0.5% NaCl, 1% Yeast Extract, 1% Glucose, 1.5% Agar, pH 7.0 ± 0.1) at ± 4°C. Table 1

Enterococcus faecalis isolated from different sources.

Clinical Isolates 21675 21752 21944 24614 24622 24708 24962 25392

Source Ulcer Tracheal secretion Blood Vaginal secretion Tracheal secretion Secretion* Tissue fragment Bubble fluid

* Not Specified

2.5 Antimicrobial assay Antimicrobial activity test was carried out by disk diffusion method [11]. The crude extract was dissolved in dimethylsulfoxide (DMSO) until a final concentration of 50 mg/mL. Sterile paper disks were impregnated with 20 µL of the extract and placed on the Petri dish surfaces containing Mueller-Hinton agar medium previously spread with bacteria suspensions adjusted according to McFarland standard solution 0.5. Then, the Petri dishes were incubated at 35°C and the diameter of the inhibition zones (in mm) was measured after 48 hours. All data were analyzed using Microsoft Excel for Windows. 2.6

Reference antibiotics

The susceptibility profile of each clinical isolate was determined using disks of the following reference antibiotics: ampicillin (10 μg), ciprofloxacin (5 μg), cotrimoxazole (25 μg), erythromycin (15 μg), gentamicin (10 μg), oxacillin (1 μg), penicillin G (10 UI), tetracycline (30 μg) and vancomycin (30 μg).

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3. Results and Discussion

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Fig. 1 shows that all E. faecalis clinical isolates were susceptible to the crude extract at 100 µg/disk final concentration, with inhibition diameters ranging from 25 to 35.25 mm. The isolate number 24962 was the most susceptible and presented an expressive result, with inhibition diameter of 35.25 mm, followed by the isolates 24622 and 24614 with inhibition diameters of 29.25 mm and 27.25 mm, respectively. Furthermore, these microorganisms were resistant to the antibiotics erythromycin, gentamicin, oxacillin, tetracycline and cotrimoxazole and the isolate 24614 was even resistant to ciprofloxacin as can be observed in Table 2.

Fig. 1 Antimicrobial activity test using the crude extract (100 µg/disk) obtained from P. variotii for E. faecalis clinical isolates. Table 2 

Susceptibility test using reference antibiotics.

Clinical Isolates

Antimicrobial Agents Amp Cipro Cotri

Ery

Gent Oxa

Pen G

Tet Vanc

21675

S

S

R

S

R

R

S

S

S

21752

S

S

R

S

R

R

R

S

S

21944

S

R

R

R

R

R

S

R

S

24614

S

R

R

R

R

R

S

R

S

24622

S

S

R

R

R

R

S

R

S

24708

S

S

R

R

R

R

S

R

S

24962

S

I

R

R

R

R

S

R

S

25392

S

R

R

R

R

R

S

R

S

Amp- Ampicillin, Cipro- Ciprofloxacin, Cotri- Cotrimoxazole, Ery- Erythromycin, Gent- Gentamicin, Oxa- Oxacillin, Pen G- Penicillin G, Tet- Tetracycline and Vanc- Vancomycin. R- Resistant, I- Intermediate, S- Susceptible

The efficacy of ampicillin and vancomycin was 100% for E. faecalis, indicating the success of these antibiotics in infections caused for this pathogen. The elevated incidence of E. faecalis isolated from brazilian hospitals is basically due its intrinsic resistance to several antibiotics and also acquired resistance by mutation or/and transferring of genetic material such as plasmids and transposons [12]. Previous studies have demonstrated the potential of producing substances with biological activity by endophytic fungi: Penicillium janthinellum from Melia azedarach (Meliaceae) [13]; Penicillium janczewskii from Prumnopitys andina (Podocarpaceae) [14]; Aspergillus fumigatus from Cynodon dactylon (Poaceae) [15]; Curvularia sp. from Ocotea corymbosa (Fabaceae) [16]; Phomopsis sp. from Erythrina crista-galli (Fabaceae), Garcinia species, Aspidosperma tomentosum (Apocynaceae) and Spondias mobin (Anacardiaceae) [17-19]; Guignardia sp. from Spondias mobin (Anacardiaceae) [20].

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The genus Paecilomyces presents several species that are able to produce a wide variety of bioactive secondary metabolites with cytotoxic, immunostimulating and antimicrobial activities [21]. Brefeldin A produced by Paecilomyces sp. isolated from Taxus mairei and Torreya grandis has been associated with cytotoxic activity [22]. In another work, Paecilomyces sp., an endophytic fungus isolated from medicinal plants, produces secondary metabolites that show promising antitumor and antifungal activities [23]. Recently, research on the secondary metabolites of endophytic fungi from estuarine environment has led to the isolation of many new compounds with different bioactivities. Xyloketal D, G, H are substances isolated from mangrove fungus Xylaria sp. [24-26]. Two unidentified fungi isolated from Kandelia candel (Rhizophoraceae) and Avicennia marina (Avicenniaceae) are able to produce metabolites with antitumor and cytotoxic activities, respectively [27, 28]. Avicennin A and B are compounds obtained from the mangrove fungus of Avicennia marina (Avicenniaceae) and Penicillium thomi isolated from Bruguiera gymnorrhiza (Rhizophoraceae) which produces metabolites recognized for antitumor activity [29, 30]. In addition, paecilin A and B are two new chromone derivates from mangrove endophytic fungus Paecilomyces sp. [31]. The antimicrobial potential of the mangrove endophytic fungi is also very well documented in literature [32].

4. Conclusions Our results suggest that mangrove endophytic fungi are gaining importance because of their enormous potential to produce novel bioactive compounds of medicinal importance. Further purification and characterization of the fungal extract reported in this study may lead to the discovery of novel antimicrobial compounds of pharmaceutical importance, which could then be produced on a large scale. Acknowledgements The authors are grateful to Professor Thomas Henry Greenhalgh for english review and CAPES for financial support.

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