Bangladesh J Pharmacol. 2015; 10: 321-325

Available Online: 11 April 2015; DOI: 10.3329/bjp.v10i2.22463

Antimicrobial screening of Mnium stellare

¹Dokuz Eylül University, Faculty of Science, Department of Biology, Buca, İzmir, Turkey; ²Kastamonu University, Faculty of Science and Arts, Department of Biology, Kuzeykent, TR 37 100, Kastamonu, Turkey; ³Ankara University, Faculty of Science, Department of Biology, Tandogan, Ankara,Turkey.

Abstract

Many plants contain active substances that are known to be effective in both enhancing the wound healing process and lowering the incidence of wound infections. Previous studies have shown that bryophytes produce a variety of secondary metabolites that present pharmaceutical activities including antimicrobial activity against various pathogenic bacteria and fungi. The aim of this study was to investigate the antimicrobial activity of Mnium stellare against 17 bacterial and 1 fungal strains. Our present study has shown that the ethanol extract of M. stellare has antimicrobial activity against several Gram positive and Gram negative microorganism tested, but its antimicrobial activity is notable especially against B. subtilis, S. typhimirium, S. aureus, S. carnosus, and S. epidermidis. These results are the very first report of the antimicrobial activity of M. stellare.

Introduction

It has been known for years that in Traditional Chinese Medicine Bryophytes were used to treat several diseases such as cardiovascular diseases, tonsillitis, bronchitis, cystitis and skin infections especially. In addition, previous studies presented that some of the secondary metabolites extracted from Bryophytes are effective in wound healing process and have anti-infective effect on some microorganisms (Altuner et al., 2010).

Especially in last decades the anti-infective activities of plant-derived products come up focus of interest. Antibacterial and antifungal resistance rates of microorganisms accelerated the research on new antimicrobial agents due to increasing morbidity and mortality rates of bacterial and fungal infections (Basile et al., 1998; Ilhan et al., 2006; Agoramoorthy et al., 2007; Veljic et al., 2008; Altuner, 2008; Altuner and Çetin, 2009; Altuner et al., 2010a, 2010b, 2010c, 2011a; Savaroğlu et al., 2011a, 2011b; Onbaşılı et al., 2011, 2013; Oztopcu-Vatan et al., 2011; Savaroglu et al., 2011a, 2011b; Altuner and Canlı, 2012; Altuner et al., 2014).

The secondary metabolites synthesized by plants have several advantages for plants themselves such as acting as a defence mechanism against microorganisms, insects and herbivores (Samidurai and Saravanakumar, 2009, Altuner et al., 2012c). These secondary metabolites have some applications such as being used as antimicrobial agents. The antimicrobial activity of plants has many applications, including raw and processed food preservation, pharmaceuticals, alternative medicine and natural therapies (Lis-Balchin and Deans, 1997; Reynolds, 1996; Canlı et al, 2014).

In this study, antimicrobial activity of Mnium stellare was determined against 17 bacterial and 1 fungal strains.

Materials and Methods

Moss samples: Bryophytes are plants which define about 14,500 species (Veljic et al., 2008). In contrast to the extensive utilisation of higher plants as a source of antimicrobial substances, Bryophytes have rarely been considered for this purpose (Basile et al., 1998). M. stellare Hedw. (Family: Mniaceae Schwagr.) samples used in this study were collected from Akdağ Mountain (N 40° 44′ - E 035° 59′), Amasya, which is located between Central Anatolia and the Middle Black Sea region. M. stellare is a well-known species, easily recognised by its lighter green color and unbordored leaves. (Smith, 2004). Voucher specimens were deposited for further reference.

Extraction procedure: All M. stellare samples were dried after collection and the samples were ground by a mor-tar and a pestle. In order to extract active substances, ethanol (Merck, Germany) was chosen as an extraction solvent. Ground samples were shaken in ethanol at 100 rpm for 3 days at room temperature. All the extracts were filtered through Whatman No. 1 filter paper into evaporation flasks (Altuner et al., 2011b). The filtrate was evaporated by a rotary evaporator (Heidolph Hei-Vap Value HL/HB-G1) at 30°C. After evaporation the residues were collected and used to prepare 9 mg/mL extracts.

Microorganisms: A wide range of Gram positive and Gram negative bacteria and yeast were selected to test the antimicrobial effect of M. stellare. The strains were chosen from standard strains as much as possible. Other strains which are not standard were all isolated from food and identified in Ankara University, Faculty of Science, Department of Biology.

Bacillus subtilis ATCC 6633, Candida albicans ATCC 10231, Enterobacter aerogenes ATCC13048, Enterococcus durans, Enterococcus faecalis ATCC 29212, Enterococcus faecium, Escherichia coli ATCC 25922, Escherichia coli CFAI, Klebsiella pneumoniae, Listeria monocytogenes ATCC 7644, Salmonella enteritidis ATCC 13075, Salmonella infantis, Salmonella kentucky, Salmonella typhimurium SL 1344, Staphylococcus aureus ATCC 25923, Staphylococcus carnosus MC1.B, Staphylococcus epidermidis DSMZ 20044 and Streptococcus agalactiae DSMZ 6784 were used in the study.

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 inoculated 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, thus standard inocula is adjusted to contain approximately 10⁸ cfu/mL for bacteria and 10⁷ cfu/mL for C. albicans (Hammer et al, 1999; Altuner, 2011).

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 mm ± 0.5 mm (Altuner and Çetin, 2009; Altuner and Akata, 2010). 60 µL, 100 µL and 150 µL aliquots of each extract was applied on sterile paper disks of 6 mm diameter end up with 440 µg/µL, 917 µg/µL and 1375 µg/µL sample on each disk (Mahasneh and El-Oqlah, 1999; Silici and Koc, 2006). 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 (Silici and Koc, 2006; Altuner et al., 2012a, 2012b). The surface of the plates was inoculated using previously prepared inocula containing saline suspension of microorganisms. Inoculated plates were then left to dry for 5 min 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. Plates were incubated and inhibition zone diameters were expressed in millimetres.

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.

Results

The diameter of the inhibition zones recorded as the diameter of the zones in millimetres for the samples are given in Table I. No activity was observed for the negative controls; solvents and empty sterile disks. Table I clearly shows that ethanol extracts of M. stellare were presented antimicrobial activity against B. subtilis, L. monocytogenes, S. enteritidis, S. infantis, S. kentucky, S. typhimurium, S. aureus, S. carnosus and S. epidermidis.

Discussion

Results given in Table I clearly show that M. stellare are active against several microorganisms but its antimicrobial activity is notable especially against B. subtilis, S. typhimirium, S. aureus, S. carnosus and S. epidermidis. There have been no reports about the antimicrobial activity of M. stellare as far as the current literature is concerned. These results are the very first data about the antimicrobial activity of M. stellare.

Among the microorganisms which were affected by M. stellare extracts B. subtilis, L. monocytogenes, S. aureus, S. carnosus and S. epidermidis are Gram positive where S. enteritidis, S. infantis, S. kentucky and S. typhimurium are gram negative strains.

It is a well known fact that Gram negative bacteria are in general more resistant to a large number of antibiotics and chemotherapeutic agents than Gram positive bacteria (Nikaido, 1998). In addition, it was also pointed out previously that Gram negative bacteria are the dominant killers among bacterial pathogens in the Intensive Care Units (ICU) (Villegas and Quinn, 2004).

It was reported that although serovar Typhimurium of Salmonella has a less alarming public image than serovar Typhi, it is a bigger health problem and it is thought by researchers to be at least 30-fold underreported. There are probably hundreds of millions of cases every year in the world in which serovar Typhimurium kill twice as many people as serovar Typhi which were mostly infants and the elderly people (McClelland et al., 2001). According to results, 100 µL of M. stellare extract showed low antibacterial activity against S. typhimurium. Since the inhibition zone is quite low, increasing the active substance loaded on the empty sterile antibiotic disks may also increase the activity.

On the other hand ethanol extracts of M. stellare are active against several gram positive strains, as stated previously. The results of the disk diffusion tests applied on the gram positive strains are more remarkable than the results of gram negative strains. The activity against especially on B. subtilis, S. carnosus and S. epidermidis are noteworthy.

The pathogenic potential of B. subtilis is generally described as low or absent (De Boer and Diderichsen, 1991). B. subtilis is only known to cause disease in severely immunocompromised patients (Galieni and Bigazzi, 1998). Several researchers study antimicrobial activity of some plant extracts on B. subtilis ATCC 6633. Khalid et al. (2011) compared four different methanolic plant extracts, namely Pistacia integerrim, Polygonum bistorta, Swertia chirata and Zingiber officinale. In this study maximum 30 mg of extracts were loaded on sterile antibiotic disks and inhibition zones were found to be 12 mm for P. integerrim, 11 mm for P. bistorta, 12 mm for S. chirata and 17 mm for Z. officinale. In our study we observed 14 mm zone for 1.375 mg of M. stellare extract which is about 22 times lower than the amount used for study conducted by Khalid et al. (2011). Comparing these results clearly puts forward how M. stellare is active against B. subtilis when compared to some other higher plants.

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 patients hospitalized. S. epidermidis is also a major concern for people with catheters or other surgical implants because it is known to cause biofilms that grow on these devices (Queck and Otto, 2008; Salyers et al., 2002).

Several studies conducted on the antimicrobial 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).

In our study we observed a 24 mm inhibition zone for 1.375 mg of M. stellare extract against S. epidermidis which is relatively high when compared to other previous studies.

Scientists defined S. kentucky as a “superbug” since it can develop resistance to some antibiotics, which means it is difficult to treat. It was previously uncommon but after 2006 an increase was observed in S. kentucky cases especially in Northeast Africa and Turkey. This strain display high-level resistance to ciprofloxacin, one of the drugs used against Salmonella diseases. In addition, secondarily acquired resistances to extended-spectrum cephalosporin and trimethoprim + sulfamethoxazole was also observed (Collard et al., 2007).

As a result, the ethanol extract of M. stellare has antimicrobial activity especially against B. subtilis, S. typhimirium, S. aureus, S. carnosus, and S. epidermidis. But further researches, especially cytotoxicity and genotoxicity tests are needed to be conducted to conclude whether M. stellare extracts can be used safely in terms of their antimicrobial activity.

References

Agoramoorthy G, Chandrasekaran M, Venkatesalu V, Hsu MJ. Antibacterial and antifungal activities of fatty acid methyl esters of the blind-your-eye mangrove from India. Braz J Microbiol. 2007; 38: 739-42.

Altuner EM. Bazı karayosunu türlerinin antimikrobiyal aktivitesinin belirlenmesi. Ankara, TURKEY, 2008, p 1-300. (PhD dissertation, Ankara Üniversitesi, Fen Bilimleri Enstitüsü)

Altuner EM. Investigation of antimicrobial activity of Punica granatum L. fruit peel ash used for protective against skin infections as folk remedies especially after male circumcision. Afr J Microbiol Res. 2011; 5: 3339-42.

Altuner EM, Akata I. Antimicrobial activity of some macro-fungi extracts. SAÜ Fen Bil Der. 2010; 14: 45-49.

Altuner EM, Akata I, Canlı K. In vitro antimicrobial screening of Bovista nigrescens (Pers.). Kastamonu U J For Fac. 2012a; 12: 90-96.

Altuner EM, Akata I, Canlı K. In vitro antimicrobial screening of Cerena unicolor (Bull.) Murrill (Polyporaceae Fr. Ex Corda). Fresen Environ Bullet. 2012b; 21: 3704-10.

Altuner EM, Canlı K. In vitro antimicrobial screening of Hypnum andoi A.J.E. Sm. Kastamonu U J For Fac. 2012; 12: 97-101.

Altuner EM, Canlı K, Akata I. Antimicrobial screening of Calliergonella cuspidata, Dicranum polysetum and Hypnum cupressiforme. J Pure Appl Microbiol. 2014; 8: 539-45.

Altuner EM, Çeter T, Bayar E, Aydın S, Arıcı F, Süleymanoğlu G, Edis A. Investigation on antimicrobial effects of some moss species collected From Kastamonu Region. Commun Fac Sci Univ Ank Series C. 2011a; 23: 33-43.

Altuner EM, Çeter T, Demirkapı D, Özkay K, Hayal U, Eser G. Investigation on antimicrobial effects of some lichen species collected from Kastamonu region. Commun Fac Sci Univ Ank Series C. 2011b; 23: 21-31.

Altuner EM, Çeter T, İşlek C. Investigation of antifungal activity of Ononis spinosa L. ash used for the therapy of skin infections as folk remedies. Mikrobiyol Bul. 2010; 44: 633-39.

Altuner EM, Çetin B. Antimicrobial activity of Thuidium delicatulum (Bryopsida) extracts. Kafkas U Fen Bil Enst Der. 2009; 2: 85-92.

Altuner EM, Çetin B, Çökmüş C. Antimicrobial screening of some Mosses collected from Anatolia. Pharmacogn Mag. 2010a; 6: 56.

Altuner EM, Çetin B, Çökmüş C. Isotechium alopecuroides Türünün antimikrobiyal aktivitesi Üzerine Lokasyon Parametresinin Etkisi. Ecology 2010 Symposium Proceedings. 2010b, p 96.

Altuner EM, Çetin B, Çökmüş C. Antimicrobial activity of Tortella tortulosa (Hedw.) Limpr. extracts. Kastamonu U J For Fac. 2010c; 10: 111-16.

Altuner EM, İşlek C, Çeter T, Alpas H. High hydrostatic pressure extraction of phenolic compounds from Maclura pomifera fruits. Afr J Biotechnol. 2012c; 11: 930-37.

Andrews JM. BSAC standardized disk susceptibility testing method (version 6). J Antimicrob Chemother. 2003; 60: 20-41.

Basile A, Vuotto ML, Ielpo TL, Moscatiello V, Ricciardi L, Giordano S, Cobianchi RC. Antibacterial activity in Rhynchostegium riparoides (Hedw.) Card. Extract (Bryophyta). Phytother Res. 1998; 12: 146-48.

Canlı K, Çetin B, Altuner EM, Türkmen Y, Üzek U, Dursun H. In vitro antimicrobial screening of Hedwigia ciliata var. leucophaea and determination of the ethanol extract composition by gas chromatography/mass spectrometry (GC/MS). J Pure Appl Microbiol. 2014; 8: 2987-98.

Collard JM, Place S, Denis O, Rodriguez-Villalobos H, Vrints M, Weill FX, Baucheron S, Cloeckaert A, Struelens M, Bertrand S. Travel-acquired salmonellosis due to Salmonella Kentucky resistant to ciprofloxacin, ceftriaxone and co-trimoxazole and associated with treatment failure. J Antimicrob Chemother. 2007; 60: 190-92.

De Boer AS, Diderichsen B. On the safety of Bacillus subtilis and B. amyloliquefaciens: A review. Appl Microbiol Biotechnol. 1991; 36: 1-4.

Galieni P, Bigazzi C. Recurrent septicemia in an immunocompromised patient due to probiotic strains of Bacillus subtilis. J Clin Microbiol. 1998; 36, 325-26.

Ilhan S, Savaroglu F, Colak F, Filik Iscen C, Erdemgil FZ. Antimicrobial activity of Palustriella commutata (Hedw.) Ochyra extracts (Bryophyta). Turk J Biol. 2006; 30: 149-52.

Khalid A, Waseem A, Saadullah M, Rehman UU, Khiljee S, Sethi A, Asad MHHB, Waqas MK, Murtaza G. Antibacterial activity analysis of extracts of various plants against Gram-positive and -negative bacteria. Afr J Pharm Pharmacol. 2011; 5: 887-93

Kumar PP, Kumaravel S, Lalitha C. Screening of antioxidant activity, total phenolics and GC-MS study of Vitex negundo. Afr J Biochem Res. 2010; 4: 191-95.

Lis-Balchin M, Deans SG. Bioactivity of selected plant essential oils against Listeria monocytogenes. J Appl Bacteriol. 1997; 82: 759-62.

Mahida Y, Mohan JSS. Screening of plants for their potential antibacterial activity against Staphylococcus and Salmonella spp. Indian J Nat Prod Resour. 2007; 6: 301-05.

Mahasneh AM, El-Oqlah AA. Antimicrobial activity of extracts of herbal plants used in the traditional medicine of Jordan. J Ethnopharmacol. 1999; 64: 271-76.

McClelland M, Sanderson KE, Spieth J, Clifton SW, Latreille P, Courtney L, Porwollik S, Ali J, Dante M, Du F, Hou S, Layman D, Leonard S, Nguyen C, Scott K, Holmes A, Grewal N, Mulvaney E, Ryan E, Sun H, Florea L, Miller W, Stoneking T, Nhan M, Waterston R, Wilson RK. Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature 2001; 413: 852-56.

Nikaido H. Antibiotic resistance caused by gram-negative multidrug efflux pumps. Clin Infect Dis. 1998; 27: 32-41.

Onbaşılı D, Altuner EM, Çelik GY. Mnium marginatum Özütlerinin Antimikrobiyal Aktivitesi. Kastamonu U J For Fac. 2011; 11: 205-08.

Onbaşlı D, Yuvalı Çelik G, Altuner EM, Altınsoy B, Aslım B. In vitro antimicrobial, antioxidant, and antibiofilm activities of Bryum capillare, a bryophyte sample. Curr Opin Biotechnol. 2013; 24 (Suppl 1): 113.

Oztopcu-Vatan P, Savaroglu F, Filik Iscen C, Kabadere S, Ilhan S, Uyar R. Antimicrobial and antiproliferative activities of Homalothecium sericeum (Hedw.) Schimp. Extracts. Fresen Environ Bull. 2011; 20: 461-66.

Queck SY, Otto M. Staphylococcus epidermidis and other coagulase-negative Staphylococci. Staphylococcus: Molecular genetics. Caister Academic Press, 2008.

Raman BV, Samuel LA, Pardha Saradhi M, Narashimha Rao B, Naga Vamsi Krishna A, Sudhakar M, Radhakrishnan TM. Antibacterial, antioxidant activity and GC-MS analysis of Eupatorium odoratum. Asian J Pharm Clin Res. 2012; 5(Suppl.): 99-106.

Reynolds JEF. Martindale: The extra pharmacopoeia. 31^st ed. London, Royal Pharmaceutical Society of Great Britain, 1996.

Salyers A, Whitt D. Bacterial pathogenesis: A molecular approach. 2^nd ed. Washington DC, ASM Press, 2002.

Samidurai K, Saravanakumar A. Antibacterial activity of Pemphis acidula Forst. Global J Pharmacol. 2009; 3: 113-15.

Sarac N, Ugur A. Antimicrobial activities and usage in folkloric medicine of some Lamiaceae species growing in Mugla, Turkey. Eur Asian J Bio Sci. 2007; 4: 28-37.

Savaroglu F, Filik-Iscen C, Oztopcu-Vatan P, Kabadere S, Ilhan S, Uyar R. Determination of antimicrobial and antiproliferative activities of the aquatic moss Fontinalis antipyretica Hedw. Turk J Biol. 2011a; 35: 361-69.

Savaroglu F, Filik İscen C, İlhan S. An evaluation of the antimicrobial activity of some Turkish mosses. J Med Plants Res. 2011b; 5: 3286-92.

Silici S, Koc AN. Comparative study of in vitro methods to analyse the antifungal activity of propolis against yeasts isolated from patients with superficial mycoses. Lett Appl Microbiol. 2006; 43: 318-24.

Smith AJE. The moss flora of Britain and Ireland. 2^nd ed. London, Cambridge University Press, 2004.

Veljic M, Tarbuk M, Marin PD, Ciric A, Sokovic M, Marin M. Antimicrobial activity of methanol extracts of mosses from Serbia. Pharm Biol. 2008; 46: 871-75.

Villegas MV, Quinn JP. An update on antibiotic-resistant gram-negative bacteria. Infect Med. 2004; 21: 595-99.