Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Diversity and antibacterial activity of bacteria cultured from Mediterranean Axinella spp. sponges M. Haber and M. Ilan Department of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel

Keywords antibacterial activity, microbial antagonism, porifera, secondary metabolites, symbionts. Correspondence Micha Ilan, Department of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. E-mail: [email protected] 2013/1805: received 4 September 2013, revised 23 October 2013 and accepted 15 November 2013 doi:10.1111/jam.12401

Abstract Aims: Evaluation of the diversity and antibacterial activity of bacteria cultivated from Mediterranean Axinella sponges and investigating the influence of culture conditions on antibacterial activity profiles of sponge bacteria. Methods and Results: Based on 16S rRNA gene sequence analysis, the 259 bacteria isolated from the three Mediterranean Axinella sponges A. cannabina, A. verrucosa and A. polypoides belonged to 41 genera from the four phyla Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria and included five potential newly cultured genera. In antagonistic streak assays, 87 isolates (34%) from 13 genera showed antibacterial activity towards at least one of the 10 environmental and laboratory test bacteria. The extracts and filtrates of 22 isolates grown under three different culture conditions were less often active as the isolates in the corresponding antagonistic streak assays. Changes in antibacterial activity profiles were isolate- and culture condition-specific. Conclusions: Axinella sponges are a good source to cultivate phylogenetic diverse and hitherto novel bacteria, many of which with antibacterial activity. Analysis of induced antibacterial activities might enhance the role of sponge bacteria in efforts to isolate new antibiotics in the future. Significance and Impact of the Study: This study was the first to investigate the diversity and antibacterial activity of bacteria isolated from A. cannabina and A. verrucosa. It highlights the potential importance of induced activity and the need for employing multiple culture conditions in antibacterial screening assays of sponge-associated bacteria.

Introduction Sessile marine invertebrates, such as sponges (phylum Porifera), frequently use secondary metabolites to protect themselves from competitors for space (Engel and Pawlik 2000; Pawlik et al. 2007), predators (Pawlik et al. 1995; Burns et al. 2003; Sokolover and Ilan 2007), pathogens (Kelman et al. 2001; Mayer and Hamann 2005) and the settlement of fouling organisms (Kubanek et al. 2002; Tsoukatou et al. 2002; Haber et al. 2011). In addition to their ecological importance, these compounds can have promising pharmaceutical and biotechnological activities. Marine sponges are a major source for such structural diverse novel natural products (see reviews by Mayer and Hamann 2005; Blunt et al. 2011 and references therein). However, as is the case for many natural products from

marine invertebrates, the promising sponge metabolites are often present in minute quantities in the organisms thus preventing harvest from nature without harming the survival of the sponge populations (Proksch et al. 2002; Imhoff et al. 2011). The structural similarity of many marine natural products to those of known microbial origin has lead to an increased interest in the microbial symbionts of marine invertebrates, as they might be the true producers of compounds isolated from their hosts, and therefore might be a solution to the supply problem in later stages of the pharmaceutical pipeline (Salomon et al. 2004; Waters et al. 2010). Marine sponges are hosts to a diverse microbial community including Archaea (Margot et al. 2002), diatoms (Webster et al. 2004), dinoflagellates (Garson et al. 1998), fungi (Paz et al. 2010) and bacteria (Schmitt et al. 2012).

Journal of Applied Microbiology 116, 519--532 © 2013 The Society for Applied Microbiology

519

Axinella bacteria: diversity and activity

M. Haber and M. Ilan

The potential bacterial production of natural products previously identified as sponge metabolites has been investigated by several techniques such as cell disassociation followed by sorting and chemical extraction of different cell fractions (Unson et al. 1994), antibody staining (Gillor et al. 2000) and MALDI-Tof imaging (Simmons et al. 2008; Yarnold et al. 2012). However, metabolites might be produced by one cell type and accumulated in another, especially if the target molecules are small and can be easily transported between cells (Simmons et al. 2008). Unequivocal evidence of the origin of a compound is therefore only obtained by either localizing the biosynthetic gene cluster or by isolating the compound of interest from pure cell cultures (Salomon et al. 2004; Taylor et al. 2007; Simmons et al. 2008). A bacterial origin of some sponge metabolites has successfully been confirmed by both approaches, for example the complete gene cluster for psymberin was isolated from the metagenome of the sponge Psammocinia aff. bulbosa and supported a bacterial origin based on the genetic features (Fisch et al. 2009), and manzamine A originally found in the sponge Acanthostrongylopora sp. has later been isolated from pure cultures of its symbiotic actinobacterium Micromonospora sp. strain M42 (Hill et al. 2005; Peraud 2006). Sponge bacterial communities differ from those of the surrounding seawater (Hentschel et al. 2002). Several phylogenetic clades specific to sponges have been reported, mainly by culture-independent methods (Taylor et al. 2007; Simister et al. 2012). Overall, sponge-associated bacteria are affiliated with 17 formally described bacterial phyla and several more, yet uncultured, candidate phyla (Simister et al. 2012). Despite their close phylogenetic relatedness, there is still a high diversity between sponge bacterial communities as there is only little overlap between communities from different sponge species on a bacterial species, genus and family level and most taxa are sponge species-specific (Schmitt et al. 2012; Giles et al. 2013). From a pharmacological viewpoint, unique bacteria are likely the source of hitherto unknown natural products, as they need to adapt to unique microhabitats (Jensen and Fenical 1996). Actinobacteria are among the dominant bacterial groups in sponges (Montalvo et al. 2005; Taylor et al. 2007; Vicente et al. 2013). They are known to produce a variety of interesting bioactive compounds, especially antibiotics. New antibiotics are continually needed because of the development and natural existence of resistant pathogens, the evolution of new diseases and the toxicity of some of the current compounds (Demain 1999). In this study, we investigated the culturable bacterial community of three sympatric Mediterranean sponges: Axinella cannabina, A. polypoides and A. verrucosa, and 520

evaluated their antibacterial activity. Axinella sponges are well known for their specific associations with Archaea (Margot et al. 2002), but a recent deep sequencing study also indicates the presence of a diverse bacterial community (White et al. 2012). Recent phylogenetic studies suggest that the family Axinellidae and its name-giving genus Axinella are polyphyletic (Alvarez et al. 2000; Gazave et al. 2010). The three species studied here are grouped in distinct clades with different chemical composition (Gazave et al. 2010). Isocyanids have been isolated from A. cannabina (Cimino et al. 1975), while pyrroles are present in A. verrucosa (Aiello et al. 2006; Haber et al. 2010). Both compound classes are absent in A. polypoides (Cimino et al. 1975). Thus, these three sponges represent different microhabitats, which we hypothesized should select for different adaptations in their bacterial symbionts including the production of a variety of secondary metabolites. The antibacterial activity of the isolated bacteria was screened in an antagonistic assay against environmental and laboratory test bacteria. To assess the potential for biotechnological exploitation, 22 of the more active isolates were then grown under three different conditions, and their antibacterial activity was re-examined. Material and methods Sponge sample collection and bacterial isolation Samples were taken from three specimens of each of the two sponge species A. polypoides and A. verrucosa and from a single A. cannabina specimen. The samples were collected by Scuba diving at Sdot Yam, Israel (32°29.77′ N; 34°53.23′E), at 30 m depth in November, 2006, stored underwater in ziplock bags and brought to the laboratory within 3 h after collection. Samples were cleaned from macro-epibionts (such as barnacles and hydrozoans) and rinsed with sterile artificial seawater (40 g l 1 Instant Oceanâ; United Pet Group, Blacksburg, VA, USA) (ASW). The next steps were performed under sterile conditions in a laminar flow hood. A representative piece of about 1 cm3 from each sample was cut into small pieces and thoroughly ground with sterile mortar and pestle in 9-ml sterile ASW. The liquid part of each of the obtained suspensions gave the initial 100 dilutions and was further diluted to 10 1, 10 2 and 10 3. For each sample, 100 ll of the latter three dilutions was plated on marine agar plates (37
4 g l 1 marine broth 2216, 18 g l 1 bactoagar, pH 7
2) with three replicates per dilution for A. verrucosa and A. polypoides and two replicates for A. cannabina. 100 ll of the 100 and 10 1 dilutions was plated out on SCA [10 g l 1 soluble starch, 1 g l 1 casein (dissolved in 10 ml 1 mol l 1 NaOH), 0
5 g K2HPO4, 20 g l 1 NaCl,

Journal of Applied Microbiology 116, 519--532 © 2013 The Society for Applied Microbiology

Axinella bacteria: diversity and activity

M. Haber and M. Ilan

20 g l 1 bactoagar, pH 7
2], modified ISP2 (4 g l 1 yeast extract, 10 g l 1 malt extract 4 g l 1 dextrose, 20 g l 1 NaCl, 20 g l 1 bactoagar, pH 7
2) and M1 media (10 g l 1 soluble starch, 4 g l 1 yeast extract, 2 g l 1 peptone, 20 g l 1 NaCl, 18 g l 1 bactoagar, pH 7
2). Again three replicates per dilution were prepared for A. verrucosa and A. polypoides and two for A. cannabina. M1 was not used for A. cannabina. Plating was always done until the agar surface was dry. SCA and modified ISP2 agar plates were supplemented with 10 lg ml 1 nalidixic acid (against Gram-negative bacteria), 25 lg ml 1 nystatin and 10 lg ml 1 cyclohexamide (both against fungi), while M1 was supplemented with 5 lg ml 1 rifampin (against Gramnegative bacteria) and 100 lg ml 1 cyclohexamide. Antibiotic stocks were 0
22 lm filter-sterilized before use, with the exception of nystatin, which does not completely dissolve in water, and were added to the autoclaved medium at a temperature of around 45°C under the laminar flow hood. Antibiotics were used to select for slow growing Gram-positive bacteria such as Actinobacteria, a group known for producing a wide array of bioactive compounds (B
erdy 2005; Fenical and Jensen 2006). After inoculation, plates were incubated at room temperature (20–24°C). Plates for Gram-positive bacteria were kept in the dark to avoid light degradation of the antibiotics. Plates were sealed with Parafilmâ (Brand, Wertheim, Germany) after 1 day to keep them humid and avoid contamination. Strains were selected from marine agar plates after 7 days, and from SCA, modified ISP2 and M1 plates after 19 and 39 days. The selection was based on diversity of colony morphology. Colonies were transferred to fresh plates with an inoculation loop by gently touching the surface and spread applying isolations streaks to allow the growth of single colonies. All bacteria from SCA, modified ISP2 and M1 plates were transferred to modified ISP2 plates without antibiotics to decrease the number of different media used. If these bacteria did not grow on modified ISP2 plates, they were picked again from the original plate and transferred to plates with their original growth medium without antibiotics. Colonies were restreaked on fresh plates until pure cultures were obtained. Stocks were prepared in 25% glycerol in liquid medium and stored at 80°C. Molecular identification of isolated bacteria For identification, the bacteria were grouped based on morphology and medium. After PCR amplification of the 16S rRNA gene, application of restriction fragment length polymorphism (RFLP) analysis enabled grouping of isolates, which were then compared based on 16S rRNA gene sequencing of a representative member of each RFLP pattern and its molecular identification.

DNA for PCR amplification was obtained by transferring bacterial cells of a single colony with a sterile toothpick into a 1
7-ml Eppendorf tube containing 30 ll of TE buffer (10 mmol l 1 Tris-HCl, 1 mmol l 1 EDTA) followed by three cycles of heating to 100°C for 5 min and freezing 1 h at 80°C) to lyse the cells. From the resulting suspension, 1 ll was added to the PCR mix consisted of 2
5 ll 109 PCR Buffer, 2
0 ll 2
5 mmol l 1 dNTPs, 0
25 ll of each 100 mmol l 1 primer (63f and 1387r) (Marchesi et al. 1998), 0
2 ll Dream Taq polymerase (Fisher Scientific GmbH, Schwerte, Germany) (5 units ll 1) and 19
8 ll molecular water. PCRs were run in a Bioer XP cycler (Bioer Technology, Tokyo, Japan) with the following temperature profile: 3 min 94°C, 30 cycles of 1 min 94°C, 1 : 20 min 54°C, 2 min 72°C and a final extension of 5 min 72°C. The RFLP analysis followed the approach by Bergman et al. (2011). Three micro litre of the PCR products was added to a reaction mix consisting of 1 ll 109 Buffer R+, 5
5 ll molecular water and 0
5 ll of the restriction enzyme BsuRI (=HaeIII) (10 units ll 1) (Fermentas) and incubated for 3 h at 37°C. The restriction patterns of the members within a group were compared with each other following electrophoresis at 60V for 2 h on a 2% (w/v) agarose TBE (89 mmol l 1 Tris-Base, 89 mmol l 1 boric acid, 2 mmol l 1 EDTA) gel spiked with 2 ll ethidium bromide. Gels were observed under UV, a digital photo was taken for documentation and used to determine the RFLP groups. The PCR product from a representative of each banding pattern present in a morphological group was sent to MCLAB (San Francisco, CA, USA) for sequencing with the forward primer 63f. Obtained raw sequence data were visualized and manually edited using the 4PEAKS software, version 1.7.2 (A. Griekspoor and Tom Groothuis, available at mekentosj.com). The edited sequences were compared with the closest validly described bacterial species using the EzTaxon-e server (Kim et al. 2012). Isolates with less than 97% 16S rRNA gene similarity were considered as undescribed species and as new genera if the similarity was below 95%. All bacterial 16S rRNA gene sequences obtained in this study were submitted to GenBank, and the accession numbers are JN699088-158 and JN699062 for A. polypoides, JN699163-228 and JN699063-64 for A. verrucosa and JN699065-87 for A. cannabina, respectively. To confirm the taxonomic assignment, a phylogenetic analysis was performed. For each sponge-associated isolate, the 16S rRNA gene sequence of the closest validly described species was downloaded from the EMBL database, as well as all available sequences of isolates previously cultured from the three studied sponge species. Thermotoga maritima (Phylum: Thermotogae) was used as an outgroup. Sequences were aligned using INFERNAL

Journal of Applied Microbiology 116, 519--532 © 2013 The Society for Applied Microbiology

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Axinella bacteria: diversity and activity

M. Haber and M. Ilan

1.0 (Nawrocki et al. 2009) as implemented in the ribosomal database project, version 10 (Cole et al. 2009). The obtained alignment was manually improved and ambiguously aligned positions as detected by GBLOCKS, version 0.91b, (with default settings, but allowing gaps in the final alignment if present in 10 mm

Test bacteria

Sponge bacterium

St

Sh

6–10 mm

(c) Sh

St

Test bacteria

Figure 1 (a) Result of a streak assay after incubation showing various degrees of inhibition against four test bacteria (two marked with white arrows). (b, c) Liquid cultures of two sponge bacteria after incubation under shaking (Sh) and standing (St) conditions for the analysis of culture condition on production of antibacterial compounds.

Actinobacteria genera: Curtobacterium (one isolate), Microbacterium (1) and Streptomyces (7); the Firmicutes genera: Bacillus (4) and Terribacillus (1); the Proteobacteria genera: Pseudovibrio (5) from its Alpha-class as well as Pseudoalteromonas (2) and Microbulbifer (1) from its Gamma-class. These bacteria were grown under three conditions: (i) on agar plates, (ii) in liquid medium with shaking at 150 rpm and (iii) in liquid medium without shaking (Fig. 1b,c). The culture medium for each bacterium was the same as used previously in the antibacterial screening. Liquid media were prepared the same way as solid media without the addition of agar. All cultures were grown in the dark at 30°C for 5 days, with the exception of the four Streptomyces isolates MVI.12, MVI.20, MVII.16 and MVII.23. These were incubated for 10 days to allow spore formation, which has been linked to the production of antibiotics in Streptomyces bacteria. Agar plate cultures were extracted after incubation by cutting them into 0
5 cm3 pieces and submerging them for 24 h in 30 ml methanol/dichloromethane 1 : 1 under gentle shaking, followed by a second extraction with 20 ml of the same solvent mixture for 4 h. Extracts were filtered over a Whatman filter no. 595 1/2 into the same bulb and evaporated under reduced pressure and gentle heating in the water bath (