Antifungal activity of Arctic andAntarctic bacteria isolates

Polar Biol (2011) 34:139–143DOI 10.1007/s00300-010-0854-4SHORT NOTEAntifungal activity of Arctic and Antarctic bacteria isolates Raeesh M. Shekh · P. Singh · S. M. Singh · Utpal RoyReceived: 1 April 2010 / Revised: 8 June 2010 / Accepted: 15 June 2010 / Published online: 6 July 2010© Springer-Verlag 2010Abstract Various psychrotolerant bacterial strains with broad-spectrum antimicrobial potential were isolated from a number of Antarctic and Arctic samples (lake sediments, water, as well as faeces, feathers and soils collected in pen-guin rookeries). Seven isolates from all types of samples exhibited clear antifungal activities against the multidrug-resistant pathogenic yeast strain Candida albicans NCIM 3471. One isolate from the penguin rookery, identi W ed by means of 16S rDNA sequencing as a strain of Enterococcus faecium, showed very strong antimycotic activity against a total of six C. albicans strains. The antibiotic activity was found after ammonium sulphate precipitation and dialysis but was sensitive to proteolytic enzymes.Keywords16S rDNA · Antimicrobial activity · Antarctica · Candidiasis · Inhibition zoneIntroductionPsychrophilic microorganisms are widely prevalent on earth, as there is a vast area where temperatures constantly remain below 10°C, such as in deep-sea waters, mountains and polar regions. The constraints on and sustainability of life in frozen environments are of considerable importance in a number of contexts, from polar microbial ecology and astrobiology to cryopreservation and other industrial appli-cations (Rothschild and Mancinelli 2001; Singh and Elster 2007). Psychrophilic bacterial strains, and particularly their enzymes, that are able to perform catalysis e Y ciently at low temperatures have been proposed for use in a number of biotechnology applications (Priscu et al. 1999; Groudieva et al. 2004). In view of the severe environmental conditions prevailing in Antarctica and the Arctic, it has been argued that the production of extracellular antimicrobial com-pounds would be a particular advantage in reducing inter-species competition (O’Brien et al. 2004). Therefore, it was suggested that polar regions may be viewed as a vast untapped reservoir of microorganisms with manifold anti-biotic potential.The aim of this study was to screen and isolate antimi-crobial polar wild-type bacterial isolates from lake sedi-ment cores of the Schirmacher Oasis, soil and faecal samples of Antarctica and water samples of the Arctic. Our W rst objective was to screen and isolate polar psychrotrophs that could produce cold-active substances capable of inhib-iting multidrug-resistant strains of Candida albicans. The second aim was to preliminarily characterize the biochemi-cal nature of the antimycotic substance produced by the most promising isolate.Materials and methodsSampling, enrichment and isolationSediment cores from di V erent sites at Lake Priyadarshini, Schirmacher Oasis (70°45 47.3 SЈ, 11°44 22.8EЈ), East Antarctica were sampled by helicopter method (Singh and Tiwari 2004) using a Hydro-Bios gravity core sampler.R. M. Shekh · P. Singh · U. Roy (&)Biological Sciences Group,Birla Institute of Technology & Science (BITS), Pilani K K BIRLA Goa Campus, Goa 403 726, India e-mail: ray_utp@yahoo.co.inS. M. SinghPolar Biology Laboratory,National Centre for Antarctic and Ocean Research, Sada-Headland, Vasco-da-Gama, Goa 403 804, IndiaCores collected from a depth of 6.5m were found to be 80cm in length. After dredging, the cores were cut into 2-cm slices starting from the top, labelled and stored in sterile polythene bags.Soil, faeces and feather samples were collected from penguin rookeries of Larsemann Hills, East Antarctica (69°21.68ЈS, 76º07.76ЈE; 69°21.68ЈS, 76º07.70ЈE and 69º22.433ЈS, 76°08.940ЈE). In Arctic, Ny Alesund, water samples were collected from a glacier mouth (78°53.703ЈN, 12°02.475ЈE), glacier stream (78°53.75ЈN, 12°02.502ЈE), sea convergence (78°56.106ЈN, 11°51.854ЈE) and perma-frost soil samples (78°55.165ЈN, 11°52.660ЈE). The col-lected water and soil samples were stored at 4°C until processed.A total of 100 l of each sample was spread on 1/10 Zobell marine agar (ZMA) and 1/10 malt-glucose-yeast-peptone extract (MGYP, pH 6.4–6.8) agar plates. In a separate experiment, samples were W ltered using 0.4- m millipore membranes. The W lter membranes were placed upon 1/10 ZMA, 1/10 nutrient agar media and incubated at 4 and 15°C for 1–2weeks. Appropriately diluted penguin rookery samples were also plated on two types of media mentioned earlier in order to recover most of the representative bacte-ria at two di V erent temperatures suitable for psychrophilic and psychrotolerant species (5 and 15°C, respectively). After the appropriate incubation period, all plates were screened for colonies. Distinct and isolated colonies were counted, picked and transferred to MGYP broth with 1.9% NaCl and further incubated at 5 and 15°C until su Y cient growth was observed.Tests for antifungal activityThe wild-type isolates were screened by examining the pro-duction of antimicrobial/antimycotic substances against various multidrug-resistant yeast strains (namely, Candida albicans NCIM 3471, NCIM 3557, MTCC 3958, MTCC183, MTCC 227, MTCC 7315 and C. krusei NCIM 3129). Initially, the distinct, isolated colonies from all MGYP or nutrient agar plates were randomly picked, trans-ferred onto modi W ed trypticase soy broth (mTSB) and incu-bated for 48h at 15°C. Cell-free supernatants (CFS) were prepared by centrifuging at 10,000rpm for 30min at 4°C. CFS was stored at ¡20°C until further use. This was done to prevent any undesirable enzymatic degradation of the extracellular inhibitory substances produced by the isolates.The 8-mm wells were made using a sterile well borer on solidi W ed MGYP agar (pH 6.6–6.8) plates. A total of 100 l of CFS was added to each well, and the plates were incu-bated at 37°C for 12h for di V usion. An aliquot of 100 l of appropriately diluted (1:1 dilution), freshly grown indicator organism (Candida albicans) was spread uniformly with a sterile, bent glass rod. Plates were incubated at 27°C for 2–3days and then were inspected for clear zones of inhibi-tion around the wells, and the zone diameter was measured (Roy et al. 2009). For the bacterial strains used as test organisms, instead of MGYP, brain heart infusion (BHI) medium (pH 7.40§0.2) was used.Species identi W cation16S rDNA gene sequence typing was performed to identify the selected polar isolates to the species level. Genomic DNA was extracted from bacterial isolates using a KT 83B kit (cat no. 105604, GeNeI, Bangalore, India). The 1.5-kb 16S rDNA gene was ampli W ed by PCR using universal primers and high-W delity PCR polymerase. The length of primers was 20 mers (forward primer 5Ј-AGAGTRTG ATCMTYGCTWAC-3Ј, reverse primer 5Ј-CGYTAMCTT WTTACGRCT-3Ј), and the amplicon generated was 1,500bp long. The PCR conditions were 94°C for 5min, 94°C for 30s, 55°C for 30s, 72°C for 2min and W nally 72°C for 5min. The number of cycles was 35. The PCR product was checked on a 1% agarose gel and puri W ed using a Qiagen puri W cation kit (Hilden, Germany). The puri W ed product was cloned into a TA vector (KT 63A, cat no. 107416, GeNeI, (Bangalore, India) and bidirectionally sequenced with vector-speci W c forward, reverse and inter-nal primers.The sequence data obtained for 16S rDNA genes were entered into the GenBank database and assigned accession number (HM481246). The nearest published homologues for each isolate were determined by comparison with the database using the BLAST search tool (Altschul et al. 1990).E V ect of enzymes on dialysed concentrateThe APR 210 isolate was cultured in 100ml of modi W ed TSB (pH 7.4) for 48h at 15°C. Cell-free supernatant was prepared (12,000rpm for 30min, 4°C; Eppendorf Centri-fuge, Hamburg, Germany) and stored at ¡80°C until use. The thawed CFS was saturated in up to 75% (w/v) pulver-ized ammonium sulphate (E. Merck, Darmstadt, Germany). The sample was subjected to continuous stirring for 3h at 4°C on a magnetic stir plate. The sample was dialysed using a dialysis membrane–60 (HiMedia), with a 12,000-Da cut-o V against one-litre chilled, sterile distilled water with four changes after every 4h at 4°C. The dialysed sample was concentrated using a lyophilizer (Martin-Christ, Osterode am Harz, Germany), and the biological activity was checked by cut-well agar assay. To test for enzyme sensi-tivity, the dialysed concentrate was treated with trypsin (0.5M Tris HCl bu V er, pH 8.0; SD-Fine, India), pronase E (10mM sodium phosphate bu V er, pH 7.0; Calbiochem) and proteinase K (50mM Tris HCl, pH 7.5; Sigma) at a W nalconcentration of 1.0mg/ml. Bu V ers were used as controls.Samples were incubated at 37°C for 90min. The residual activity was determined by cut-well agar assay.E V ect of temperature and pHThe crude cell-free supernatants were stored at ¡80, ¡20and 4°C for a period of 4weeks to 3months. Furthermore,the cell-free Enterococcus sp . APR 210 supernatants were used to test for heat sensitivity or heat stability. Aliquots of the cell-free supernatants were exposed to heat treatments of 60°C for 90min, 90°C for 20min, 100°C for 20 and 30min or autoclaved, and after each heat treatment, the sample was brought to room temperature. The remaining activity of the aliquot was determined using a cut-well agar assay against C. albicans MTCC 7315, an indicator strain.All experiments were performed in triplicate. The pH of CFS was neutralized. The pH values of the CFS aliquots were also adjusted to pH 8.0 and 10.0 with 5 (N) NaOH and to 2.0 and 4.0 with 6 (N) HCl, respectively. All the aliquots were incubated at 37°C for 1h. The resulting CFS solutions were subjected to an agar-well di V usion assay to record the loss or retention of biological activity.Results and discussionScreening and identi W cation of bacterial isolates with antifungal activityA total of 218 isolates were screened against the multi-drug-resistant fungal pathogenic yeast strain Candida albi-cans NCIM 3471. As a result of screening, seven bacterial isolates were recovered all of which inhibited C. albicans NCIM 3471 (Table 1). These isolates were further tested against several other strains of C. albicans (Table 1). The three isolates, AGM 111, AGM 108-5,and APR 210, repro-ducibly exhibited strong antimycotic activity. CFS of APR 210 also inhibited some bacteria such as Bacillus sp., Pseu-domonas aeruginosa , P. putida and Klebsiella pneumoniae (data not shown).The 16S rDNA sequence of isolate APR 210 showed closest homology (99.6%) to the Enterococcus faecium strain DSM:20477. Fatty acid methyl ester (FAME) analy-sis suggested, however, that APR 210 most closely related to the E. faecalis strain ATCC 828.Our results indicate that the Arctic water bacterial com-munity is comprised of mostly psychrotrophic, halotolerant species, whereas the bacteria from the penguin rookery and sediment cores were psychrotrophic, including Psychrob-acter immobilis , Yersinia aldovae and Carnobacterium maltaromatiucum , and a few were producers of antimicro-bial compounds (unpublished data). Few reports have beenT a b l e 1O r i g i n , t a x o n o m i c a l a Y l i a t i o n a n d a n t i f u n g a l a c t i v i t i e s o f s e l e c t e d b a c t e r i a l i s o l a t e sS o u r c e : G M W S C G l a c i a l m e l t w a t e r a n d s e a c o n v e r g e n c e . S t r a i n c o d e : A P R A n t a r c t i c p e n g u i n r o o k e r y ; A G M A r c t i c g l a c i a l m e l t . A n t i b a c t e r i a l a c t i v i t i e s w e r e i n d i c a t e d a s t h e d i a m e t e r (m m ) o f t h e c l e a r i n h i b i t i o n z o n e s o b t a i n e d i n a g a r d i V u s i o n a s s a y s o f v a r i o u s m u l t i d r u g -r e s i s t a n t s t r a i n s o f C a n d i d a a l b i c a n s (N C I M 3471, M T C C 183, M T C C 7315 a n d C k r u s e i N C I M 3129)a h a z y i n h i b i t i o n z o n eA r e aS o u r c eS t r a i n c o d e S p e c i e sN e a r e s t p h y l o g e n e t i c n e i g h b o u rD i a m e t e r o f a n t i m y c o t i c a c t i v i t y z o n e s (m m )C a n d i d a a l b i c a n s s t r a i n sC . k r u s e i347173151833129A n t a r c t i cL a r s e m a n n H i l l sP e n g u i n r o o k e r yA P R 12P s y c h r o b a c t e r i m m o b i l i sM o r a x e l l a17––10L a r s e m a n n H i l l sP e n g u i n r o o k e r yA P R 79P . i m m o b i l i sM o r a x e l l a15––10L a r s e m a n n H i l l sP e n g u i n r o o k e r yA P R 210E n t e r o c o c c u s s p .E n t e r o c o c c u s f a e c a l i s20232310aS c h i r m a c h e r O a s i sL a k e s e d i m e n t4AB a c i l l u s m e g a t e r i u m B . m e g a t e r i u m2213––S c h i r m a c h e r O a s i sL a k e s e d i m e n t21CB . m y c o i d e s B . p s e u d o m y c o i d e s12–––A r c t i cN y A l e s u n d G M W S C A G M 108-5Y e r s i n i a a l d o v a eE . h o r m a e c h i i 20151512a N y A l e s u n d G M W S C A G M 111C a r n o b a c t e r i u m m a l t a r o m a t i c u mC . m a l t a r o m a t i c u m18151811apublished so far about polar isolates inhibiting multi-drug-resistant organisms e.g., the Antarctic Arthrobacter sp.HPH17 and Planococcus sp. CHF8 that have been demon-strated to produce antimicrobial peptides inhibiting patho-gens like Listeria spp. and non-pathogenic strain of Brochothrix thermosphacta (O’Brien et al. 2004), a major food spoiler.This study revealed the presence of Enterococcus sp .(isolate APR 210 from penguin rookery), which corrobo-rates earlier W ndings regarding the presence of E. faecalis from penguins (Soucek and Mushin 1970). This isolate was shown to have a particularly pronounced activity, strongly inhibiting as many as seven Candida strains.Characterization of the antimycotic substanceThe antimicrobial activity of the supernatant of the strain E .faecium APR210 withstood heating (i.e., 60min at 60°C). However, biological activity was completely lost at 100 and 121°C (Table 2). The complete loss of heat stabil-ity of the anti-Candida substance reportedly di V ers from another anti-Candida substance, pentocin TV 35b (Okkers et al. 1999). The antimicrobial activity was not lost by repeated freezing and thawing or by long-term storage (i.e.,3months) at ¡20 and ¡80°C. The activity was completely lost at pH values 2, 4 and 10. However, no loss of activity at pH values 6, 6.9 and 8 was observed.Given that the molecular weight cut-o V (MWCO) of the dialysing membrane was 12kDa, the molecular weight of the inhibitory substance seemed to be greater than 12kDa.The upper part of the Nanosep membrane of MWCO of 30kDa showed antifungal activity (Fig.1a). The dialysed concentrate (10X) of APR 210 was able to produce a zone diameter of 47 to 55mm against MTCC 7315 and 183,respectively (Fig.1b), and the clarity of zones indicated that the antimicrobial principle was candidacidal. The dia-lysed substance was assessed for its sensitivity to three pro-teolytic enzymes. The antimycotic substance in the dialysed concentrate was sensitive to proteinase K, which caused a complete loss of antimicrobial activity (Table 2) and to pronase E, which led to partial loss of biological activity.Treatment with trypsin had no e V ect on the biological activ-ity. Bu V er and enzyme preparations added to the wells alone had no e V ect on the indicator lawns of C. albicans MTCC183.These results suggest that the inhibitory principle in CFS produced by the Enterococcus isolate APR 210 might belong to a protein of class II bacteriocins (Cotter et al.2006; Pascual et al. 2008) or bacteriocin-like inhibitory substances (BLIS).Table 2E V ects of enzymes, heat and pH on the antifungal activity of cell-free supernatants (CFS) of Enterococcus sp a++: no loss of activity; +: partial loss; –: complete lossTreatmentCFS Activity aEnzymes (1.0mg/ml)Trypsin ++Pronase E +Proteinase K¡Control (not treated with enzymes)++Heat90min at 37°C ++90min at 60°C ++20min at 90°C ++30min at 100°C ¡20min at 100°C ¡15min at 121°C ¡Control (at 4°C)++pH2.0, 4.0 and 10.0¡6.0, 7.0 and 8.0++Fig.1a The strong antimycotic activity produced by the dialy-sate (using a 12kDa cut-o V ) and Nanosep (MWCO 30kDa, Pall science, Germany) membrane separated dialysed concentrate of APR 210 against NCIM 7315. b Antimycotic activity of the dialysate concentrate of CFS of E. faecium APR 210 against Candida albicans NCIM 7315 and MTCC 183Acknowledgments This work was W nancially supported by the Council of Scienti W c and Industrial Research (CSIR), New Delhi, India. The samples were provided by the Polar Biology Laboratory NCAOR, Goa, India. Thanks are also due to the Sanmar Specialties for ribotyping. We are thankful to the directors of the Birla Institute of Technology & Science, Pilani KK Birla Goa Campus and National Centre for Antarctic & Ocean Research Goa for providing research facilities. We are particularly grateful to Dr. T. Hoshino (Sapporo) and two anonymous reviewers for their valuable advice that helped a lot in improving the manuscript.ReferencesAltschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410Cotter PD, Hill C, Ross RP (2006) What’s in a name? Class distinction for bacteriocins. Nature Rev Microbiol 4(2). doi:10.1038/nrmicro 1273-c1Groudieva T, Kambourova M, Yusef H, Royter M, Grote R, Trinks H, Antranikian G (2004) Diversity and cold-active hydrolytic enzymes of culturable bacteria associated with Arctic sea ice, Spitsbergen. 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