In vitro antimicrobial screening of Lycoperdon lividum and determination of the ethanol extract composition by gas chromatography/mass spectrometry

The aim of this study was to investigate the antimicrobial activity of Lycoperdon lividum against 17 bacterial and 1 fungal strains and analyse the composition of ethanol extracts by GC/MS. The in vitro antimicrobial activity of L. lividum extracts having 15 mg/mL concentration was assessed against a wide range of strains by disk diffusion method. The ethanol extract of L. lividum had antimicrobial activity against several microorganism tested, but it was active especially against S. carnosus . The results obtained herein indicate that L. lividum contains several active metabolites. In vitro antimi- crobial screening of Lycoperdon and of the extract composition by gas chroma-tography/mass spectrometry.


Introduction
Although there is tremendous progress in human medicine; bacterial, viral and fungal issues are still threaten the public health especially in the developing countries (Cos et al., 2006). Relative unavailability of medicines in these countries and in addition to this the extensive antibiotic resistance has a large impact on human health (Okeke et al., 2005). Therefore, further researches about investigation of new antimicrobial substances should be conducted (Altuner et al, 2010).
Mushrooms have a potential of using both as nutritive and medicinal food stuff (Imtiaj and Lee, 2007;Bonatti et al., 2004;Cheung and Cheung, 2005;Agrahar-Murugkar and Subbulakshmi, 2005;Altuner et al., 2012). Mushrooms are not only sources of nutrients but also could be used to prevent diseases such as hypertension, hypercholesterolemia and cancer (Bobek and Galbavy, 1999).
Lycoperdon lividum Pers. grows on soil and dunes, solitary to gregarious, in forests, outside of forests, in meadows and cow pastures (Breitenbach and Kränzlin, 1986).
The aim of this study was to investigate the antimicrobial activity of L. lividum against 17 bacterial and 1 fungal strains and analyse the composition of ethanol extracts by GC/MS.

Extraction procedure
L. lividum samples were air dried and samples were ground by a mortar and a pestle. In order to extract active substances, ground samples were shaken in ethanol (Merck, Germany) at 100 rpm for 3 days at room temperature. Then all the extracts were filtered through Whatman No. 1 filter paper into evaporation flasks. The filtrate was evaporated by a rotary evaporator (Heidolph Hei-Vap Value HL/HB-G1) at 30°C and lyophilized. After lyophilization the residues were collected and used to prepare 15 mg.mL -1 extracts.

Microorganisms
A wide range of bacteria (Gram positive and Gram negative) and yeast were selected to test the antimicrobial activity of L. lividium. The strains were chosen from standard strains as much as possible. Other strains which are not standard were all isolated from food and identified at the Department of Biology, Faculty of Science, Ankara University.

Prepartion of inocula
All bacterial strains were incubated at 37°C for 24 hours. But since the requirements for C. albicans is different. C. albicans was incubated at 27°C for 48 hours. Inocula were prepared by transferring morphologically similar colonies of each organism into 0.9% sterile saline solution until the visible turbidity was equal to 0.5 McFarland standard having approximately 10 8 cfu/mL for bacteria and 10 7 cfu/L for C. albicans (Canlı et al., 2015).

Disk diffusion method
Disk diffusion test was performed as described previously by Andrews (2003). The culture medium was poured into 120 mm sterile petri dish to give a mean depth of 4.0 ± 0.5 mm. 40, 60 and 100 µL aliquots of each extract was applied on sterile disks of 6 mm diameter end up with 550, 917 and 1375 µg sample on each disk. To get rid of any residual solvent which might interfere with the results, disks were left to dry overnight at 30°C in sterile conditions (Altuner et al., 2010). The surface of the plates was inoculated using previously prepared inocula containing saline suspension of microorganisms. Inoculated plates were then left to dry at room temperature before applying the disks. Disks were firmly applied to the surface of the plate which had an even contact with the agar (Savaroglu et al., 2011). Plates were incubated and inhibition zone diameters were expressed in millimetres.

Gas chromatography-mass spectrophotometry method (GC-MS)
The GC-MS analysis of etanol extract of LC were quantitatively performed by GC-MS (Agilent 19091S-433, 30 x 0.25 x 0.25 HPS MS) based on the method defined previously by Hossain and Rahman (2011) with some modifications. Split less injection was performed with a purge time of 1.0 min. The carrier gas was helium at a flow rate of 1 mL/min. The column temperature was maintained at 60°C for 2 min, then programmed at 10°C/min to 300°C and waited for 10 min. The inlet temperature was 250°C, AUX was 290°C and the solvent delay was 4 min. The identification of the peaks was based on computer matching of the mass spectra with the National Institute of Standards and Technology (NIST Mass Spectrometry DATA CENTER, 2005) library and by direct comparison with published data (Canlı et al., 2014).

Controls
Empty sterile disks and extraction solvent (ethanol) loaded on sterile disks which were dried at sterile conditions to remove solvent as done in the study were used as negative controls.

Statistics
The statistical analysis was performed using a nonparametric method Kruskal-Wallis one-way analysis of variance. A value of p<0.05 was considered statistically significant.

Discussion
Results clearly show that L. lividiumis active against several microorganisms but its antimicrobial activity is notable especially against S. carnosus. Among the microorganisms affected by L. lividum extracts are E. coli, S. carnosus, S. Epidermidis, S. Agalactiae, E. aerogenes, S. kentucky and K. Pneumonia.
It is well-known that Gram negative bacteria are in general more resistant to a large number of antibiotics and chemotherapeutic agents than Gram positive bacteria (Nikaido, 1998).
We have observed that L. lividum had a low antimicrobial activity against E. aerogenes, E. coli, K. pneumoniae, S. kentucky, S. epidermidis and S. agalactiae, when it is compared to S. carnosus for the concentrations used in the study.
Unfortunately there have been no reports neither about the antimicrobial activity of L. lividium, nor the chemical composition of its ethanol extract in order to compare with our results, as far as the current literature is concerned. Results presented in this study are the very first data both about the antimicrobial activity and the chemical composition of the ethanol extract of L. lividium.
E. aerogenes was identified as one of the "ICU bugs" which could cause significant mortality and morbidity. In addition the infection management is complicated due to its resistance to multiple antibiotics (Hidron et al., 2008). Although a very low activity was observed against E. aerogenes, the results can be accepted as noteworthy. Probably increasing amount of extracts loaded on the empty sterile antibiotic disks may increase the activity.
There are several studies reported by using differentE. coli strains. For example, Dulger et al. (2005) showed that the methanolic extract of Hypnum cupressiforme (30 mg/mL) presented 12.2 mm of inhibition zone against E. coli. When the results reported by Dulger et al. (2005) were compared with our results, which were maximum 7 mm inhibition zone, it can be concluded that L. lividum presented very low antimicrobial activity. But since the E. coli strain used in this study is different than the study conducted by Dulger et al. (2005), this difference is not surprising.
In addition it was also pointed out that Gram negative bacteria are the dominant killers among bacterial pathogens in the Intensive Care Units (ICU) (Villegas and Quinn, 2004). Klebsiella is one of these Gram  (Villegas and Quinn, 2004).
From this point of view, having antibacterial activity against K. pneumoniae may very important. Quereshi et al. (2010) identified that Ganoderma lucidum (40 μg.mL -1 ) caused 11.30 mm of inhibition zone against K. pneumoniae. In our study we observed 7 mm of inhibition zone against L. lividum extract. By comparing these two studies it can be concluded that ethanolic extracts of L. lividum has lower antimicrobial activity against K. pneumoniae when compared to G. lucidum.
It was previously uncommon but in the last decade an increase was observed in S. kentucky cases especially in Northeast Africa and Turkey. This strain displays highlevel resistance to ciprofloxacin, one of the drugs used against Salmonella infections. In addition, secondarily acquired resistances to extended-spectrum cephalosporin and trimethoprim + sulfamethoxazole was also observed (Collard et al., 2007) S. epidermidis is not usually pathogenic. But they often develop risk for infection for patients with a compromised immune system. These infections can be both nosocomial and community acquired, but they pose a greater threat to hospital patients. S. epidermidis is also a major concern for people with catheters because it is known to cause biofilms that grow on these devices (Queck and Otto, 2008;Salyers et al., 2002).
Several researchs conducted on the antibacterial activity of several higher plants against S. epidermidis. Mahida and Mohan (2007) tested 10 mg of methanolic extracts of 23 plant extracts, but the highest zone was found to be 20 mm for Mangifera indica. In another study ethanol extracts of 23 plants were tested against S. epidermidis and the highest inhibition zone diameter was given as 18 mm for Stachys leptoclada (Sarac and Ugur, 2007).
The antimicrobial nature of L. lividum extract can be explained by looking at its GC/MS analysis. The GC/ MS analysis clearly puts forward the composition of L. lividum extract, which contains several compounds that have antimicrobial activities.
For example, tetradecanoic acid, which is also called myristic acid, is a saturated fatty acid having molecular formula CH3(CH2)12COOH. It is previously presented that myristic acid is an antibacterial and antifungal agent (McGaw, 2002;Seidel and Taylor, 2004;Agoramoorthy et al., 2007). Skrivanova (2006) showed that myristic acid has antimicrobial activity against Clostridium perfringens. Batovska et al. (2009) found that myristic acid is active against L. monocytogenes with having 125 µg/mL MIC value. Narasimhan et al. (2006) tested the antimicrobial activiy of myristic acid derivatives and presented that myristic acid derivatives are more effective against Gram positive bacteria rather than Gram negative bacteria. It is also concluded that S.
Hexadecanoic acid, which is also known as palmitic acid, is the most common fatty acid found in animals, plants and microorganisms (Gunstone et al., 2007). It is mainly used to produce soaps and cosmetics.
Stearic acid is a saturated fatty acid having molecular formula C18H36O2. Stearic acid is mainly used in the production of detergents and cosmetics, but an antimicrobial activity potential against diverse pathogenic microorganism is also proposed in several studies (Zheng et al., 2005;Nalina and Rahim;Chakraborty and Shah, 2011).
On the other hand, there are several studies that show pantoyl lactone has antimicrobial activity, especially against S. equi (Bashir, 2013) and some pyrrolidinone derivatives present antimicrobial properties (Daferner et al., 2002).

Conclusion
We observed antimicrobial activity against E. aerogenes, E. coli, K. pneumoniae, S. kentucky, S. carnosus, S. epidermidis and S. agalactiae. The ethanol extract of L. lividum contains several active metabolites and it is active against several microorganisms. Its antimicrobial activity is notable especially against S. carnosus.