Antimicrobial activity of culturable microorganisms associated with Caribbean black corals (Antipatharia) Daniela Valentina Peña Moreno*

* Departamento de Ciencias Biológicas, Universidad de los Andes, [email protected]

Abstract This study aims to examine microorganisms associated with Caribbean black corals from Colombia, their diversity and especially their possible antimicrobial activity. In recent years, it has been recognized that corals and other marine invertebrates are a great source of new biological active compounds, which may have different functions, such as providing defense against pathogens. This is one of the first studies reporting the biodiversity of microorganisms and their antimicrobial activity, associated with black corals (Antipatharia) from the Caribbean Sea in Colombia. Bacteria were identified based on the bacterial 16S rRNA sequences and for the only fungi found, using the fungal internal transcribed spacer sequences (ITS). Each microorganism was tested against two bacteria with high medical importance in human health: Staphylococcus aureus and Pseudomonas aeruginosa. Fourteen percent coral associated bacteria out of 28 isolations showed any kind of antimicrobial activity from medium to high, against S. aureus but, no bacteria displayed any kind of antimicrobial activity against P. aeruginosa, probably due to its high resistance and capacity to acquire resistance to multiple antibiotics. It is relevant to highlight that 75% of the bacteria that showed any kind of antimicrobial activity, came from deep waters (50 m), which points out the importance of studying mesophotic coral ecosystems and organisms such as as black corals and their potential as sources of new natural compounds.

Introduction Coral reefs are one of the largest structures made by living creatures, and though, they contain an enormous diversity of organisms (Rosenberg et al., 2007). A big part of these organisms are marine invertebrates, which have long been recognized as potential sources of structurally unique natural products, to mention some: sponges, corals, tunicates, algae and bryozoans (Wiese et al., 2008). Many natural products from marine invertebrates have high structural similarities to metabolites of their associated microorganisms including bacteria; this suggests that microorganisms are at least involved in the biosynthesis of these metabolites or are in fact the true sources (Proksch et al., 2002). If bacteria are indeed the producers of bioactive compounds it might provide an alternative strategy to produce the compound in large scales, which can be done by transferring gene clusters responsible for the biosynthesis of the respective natural products to a vector suitable for large-scale fermentation (Proksch et al., 2002). The discovery of natural new products is certainly an area receiving much attention now a days, including those that come from marine organisms. It is well recognized that microorganisms associated with marine invertebrates, in particular sponges and hard corals, are an excellent source of new natural products (Pham et al., 2016), that is why is necessary to study a greater variety of microorganisms and their potential bioactive compounds.

Corals in general provide three habitats for bacteria: the coral tissue, the calcium carbonate skeleton and the surface mucus layer, each having distinct bacterial populations (Rosenberg et al., 2007). Studies have shown that, for example, the mucus layer supports a diverse and abundant beneficial bacterial community (Rosenberg et al., 2007), which shows that this bacteria may have a specific role on the biology of corals. Most of the knowledge related with microbes associated with corals, comes from stony and soft corals, yet the microbial diversity of black corals (order: Antipatharia) is still poorly understood (Zhang et al., 2012), particularly there is little information on microorganisms associated with black corals of the Caribbean sea, much less in Colombia.

Black corals comprise an interesting taxon to investigate due to their global distribution in shallow and deep waters (Brugler et al., 2007). Black corals are certainly a group needed to be investigated, since there is paucity on information regarding their life styles and especially little information on the microorganisms associated with them, and their possible bioactive compounds. Unlike most cnidarians, antipatharian tissues have no structural protection against abrasive forces, and muscular systems are poorly developed, so the tentacles can only contract slightly but not retract into a groove like other anthozoans (Lesser, 2012). This suggests that black corals might have others mechanisms of protection against external forces and pathogens, such as their associated bacteria. Some studies have shown that a high proportion (51.6%) of microbial isolates coming from black corals, displayed distinct antibacterial and antifungal activities, against marine pathogens (Zhang et al., 2012), showing their potential function as protectors of corals against pathogens. It is certain that coral associated microorganisms have an important role in the immunity of corals, but these microorganisms may have important bioactive compounds, useful for humans or to understand and find new ways to produce natural beneficial products. Many studies have reported that marine bioactive peptides can be used as antihypertensive, antioxidative, anticoagulant, and antimicrobial components in functional foods or nutraceuticals and pharmaceuticals due to their therapeutic potential in the treatment or prevention of diseases (Kim et al., 2010). Currently, the search of these bioactive compounds is important, because many bacteria causing diseases is becoming resistant to antibiotics and new alternatives are urgently needed. Possible ways to decrease the incidence of nosocomial S.aureus infections include developing vaccines, and new or improved antimicrobials. Specially knowing that this pathogen is the most common cause of infection in hospitalized patients (Archer, 1998). This is one of the first studies analyzing the diversity of bacteria associated with in the Colombian Caribbean and their antimicrobial activity using the radial diffusion assay and 16S rRNA and ITS gene analysis to identify the isolates.

Methodology

Sample collection and microbial isolation Samples were collected from several visually healthy black corals in the Caribbean sea coast of Santa Marta, Colombia (El Morro). A total of 15 samples were collected at a 15-50 m of depth, most of them come from the specie Stichopathes spp and one individual from each of the following : pennacea, Antipathes furcata and Antipathes atlantica. For the bacterial growth, a piece of each organism was placed over a nutritive agar plate and PDA medium; this was done for 6 of the 15 samples. The rest of the samples were submerged in three buffers, with different salinity concentrations, then diluted, filtrated and inoculated in nutritive agar plates, for their posterior isolation, depending on their macroscopic characteristics. The resulting plates were incubated at 25ºC, based on the temperature in which they lived in the sea for 24 hours, the fungi was kept at 37ºC for five days.

Identification Bacterial and fungi DNA extraction was made following the protocol established by Coffroth et al (1992), Shaghai-Maroof et al (1984) and Murray & Thompson (1980), with some modifications. The 16S rRNA gene was amplified by the polymerase chain reaction (PCR) using primers 27F (5′-AGAGTTTGATYMTGGCTCAG-3′) and 338R (5′- GCTGCCTCCCGTAGGAGT-3′). For the fungi isolated the ITS nuclear gene was amplified by PCR, using the primers ITS1F (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4R (5′-TCCTCCGCTTATTGATATGC-3′). DNA sequencing of the selected bacteria and only fungi was carried out by Macrogen, Korea. Sequences in GeneBank were found using BLAST searches and then, when the top three matching BLAST hits were from the same genera and were 98% or more similar to the query sequence, the name was assigned. Determination of antimicrobial activity A suspension of each of the bacteria was made following the CDC manual (Procedure manual and quality control). In order to adjust the turbidity of bacterial suspensions, a Mc farland standard was made using numbers: 0.5, 1 and 2, but for the trials only the 0.5 was used as established by the CLSI (Clinical and laboratory standards institute) for the radial diffusion assays. Two bacteria with medical importance were used for the radial diffusion assays; Staphylococcus aureus and Pseudomonas aeruginosa. S. aureus is a gram positive bacteria, with high relevance due to the multiple infections it causes and P. aeruginosa a gram negative bacteria, tightly related with respiratory diseases. After having the Mc farland standard, each bacteria with medical importance was inoculated in a Petri dish with Mueller Hinton medium. The coral associated bacteria were inoculated in paper discs (previously sterilized) and placed on the petri dishes containing the bacteria with medical importance. For each coral associated bacterial, three replicates where made and for each bacteria, one antibiotic was used as a positive control. The positive controls where: for S.aureus, oxiciline and for P. aeruginosa, gentamicin, following the CLSI parameters. In each petri dish, 4 to 5 paper disks inoculated with coral associated bacteria were placed and at least one positive control for all the replicates. Following the CLSI protocol, the resulting petri dishes were incubated for 24 hours at 37°C and afterwards using a ruler, three distinct measures of the diameter of the inhibition halo were made. Following what Zhang et al (2012) established: a weak activity is a halo less than 10mm in diameter, moderated activity; a halo between 10 and 15 mm and finally a high activity is a halo higher than 15mm. Using this parameters, the determination of the antimicrobial activity (high, moderate, weak) of each isolation was made, and the posterior analysis of the data.

Fig 1. Radial diffusion assay of several microbial isolates against S. aureus. Central disk, positive control.

Results

Out of the 28 microbial isolations based on their morphological differences (visible growth characteristics), the bacterial community of the black corals was represented by various genera including: Bacillus, Pseudomonas, Kocuria, Pontibacter, Vibrio, Exiguobacterium, Stenotrophomonas and the phylum Proteobacteria. The majority of isolations came from the coral specie Stichopathes spp, but others species were also used. The bacterial and the only fungal isolation, were identify by the bacterial 16S rRNA gene sequence and fungal ITS sequence, respectively. Using comparisons with sequences in GeneBank, the sequences of the isolations shared 97-100% similarity, however many of the sequences where too noisy to identify or resulted in more than one genera in the BLAST. Unfortunately, in the majority of isolations, bacteria couldn’t be identified as well as the only fungi found. Probably, because some bacteria live in symbiosis with other organisms, which makes them difficult to isolate. Other possible explanation is that these bacteria might belong to new species, not well identified or described. Some bacteria resulted in the same percentage of similarity between several species, so only the genus was used in the final results. The most dominant isolations belonged to the genus Bacillus and bacteria with no identification.

All the bacterial isolations were tested against the two bacteria with medical importance (S. aureus and P. aeruginosa), in order to examine the antimicrobial activity. Four isolates (14%) showed any kind of antimicrobial activity against S. aureus, but no isolation showed any antimicrobial activity against P. aeruginosa. Half of the bacteria that displayed antimicrobial activity showed moderate activity and the other half showed high, which demonstrates the potential of this associated bacteria. It’s relevant to illustrate that the zone of inhibition in the different bacteria with antimicrobial activity were clear but not as strong as the controls (Fig 1.). The bacteria Bacillus velezensis and Bacillus amyloliquefaciens, showed the highest antimicrobial activity against S. aureus, however several studies have stated that this two bacteria might be the same specie (Wang et al., 2008), implying that this bacteria has definitely an important potential. The other two microbial isolations, having moderate activity couldn’t be identify, though they might represent other bacteria than B. amyloliquefaciens, because they have a lower inhibition zone and distinct morphology. The general results of diversity and the radial diffusion assay are show in Table 1. Finally an interesting result found in this study is that the majority (75%) of the bacteria that displayed any kind of antimicrobial activity, came from the corals collected deeper (Table 2).

Table 2. Coral specie, depth and associated bacteria that showed antimicrobial activity.

Coral specie Depth Bacteria Plumapathes 50 m Bacillus velezensis pennacea Antipathes furcata 50 m Bacillus amyloliquefaciens Antipathes atlantica 50 m No ID Stichopathes spp. 15 m No ID

Table 1. Antimicrobial activity of culturable microbial representative isolates from the Colombian Caribbean sea black corals

S.aureus Microbial Antimicrobial activity isolates Genus/Phylum Specie (zone of inhibition mm) Type of activity C2 Bacillus Bacillus cerus 0 Null Pseudomonas C24 Pseudomonas veronii 0 Null Bacillus C1 Bacillus amyloliquefaciens ±21,1 High C3.1 Bacillus Bacillus aryabhattai 0 Null C13 Bacillus Bacillus velezensis ±24 High C8 Kocuria Kocuris flava 0 Null C4 Bacillus sp 0 Null C5 Vibrio 0 Null C9 Bacillus 0 Null C10.1 Pontibacter 0 Null Bacillus Bacillus C11 oceanisediminis 0 Null C14 Exiguobacterium 0 Null C15.1 Pontibacter 0 Null C16 No ID 0 Null C20 No ID 0 Null C21 No ID 0 Null C23 No ID 0 Null C25 No ID 0 Null C27 Bacillus 0 Null C28 Stenotrophomonas 0 Null Stenotrophomonas C29 Stenotrophomonas maltophilia 0 Null C32 No ID 0 Null C33 No ID 0 Null Stenotrophomonas C34 Stenotrophomonas maltophilia 0 Null C35 No ID 0 Null C37 Proteobacteria 0 Null C24 Pseudomonas 0 Null C30 No ID ±12,1 Moderate C32.2 No ID ±13,2 Moderate Positive control Gentamicin 16,6 Positive control Oxiciline 21,3

Discussion The present study showed the diversity of the microorganisms found in black coral species of the Colombian Caribbean Sea. The 28 microbial isolations and the only fungi isolation where found on black corals of the species: Plumapathes pennacea, Antipathes furcata, Antipathes atlántica and Stichopathes spp. Most of the bacteria isolated came from the species: Stichopathes spp which was the most abundant in shallow waters. The dominant microbial isolations were principally of bacteria with no identification and members of the genera Bacillus. This result may indicate some bacteria were difficult to isolate or separate, because they may live in association with others and though difficult to identify separately. Certainly a well-designed isolation protocol with multiple isolation media is essential for isolating diverse and abundant fungi (Zhang et al., 2012), which may have been a factor that decreased the probability of isolating all the fungi and bacteria in this study.

In the present study, not all the bacteria genera were found in all the specimens. However, as stated before, most isolations came from the species Stichopathes spp, a specie which has few studies regarding its associated microorganisms. This demonstrates the huge microbial diversity that might be found in all the species of black corals that could be found in the Colombian Caribbean Sea. In contrast with other studies, the representatives of the genera Bacillus and Exiguobacterium were found in soft corals of the specie Alcyonium digitatum, and were the most dominant (Pham et al., 2016). On the other hand, the genus Bacillus was the most abundant in the isolates from the black coral Antipathes dichotoma from South China Sea (Zhang et al., 2012), as well as the present study.

The genus Bacillus is an important antibiotic source, over 800 antibiotic metabolites, including the important group of peptide antibiotics are produced by different species of this genus (Zhang et al., 2012), which shows that black corals, may become an important source in the search of antibiotic metabolites, being the source of a high abundance of Bacillus sp. Other genus like Stenotrophomonas, have been isolated from marine ecosystems (Malhotra et al., 2013) and Pontibacter coming from soils, which where also found in black corals in this study. These two genus, are not commonly found in diversity studies associated with corals, which may represent new genera associated with this organisms. The genus Stenotrophomonas was also found in several of the microbial isolations, and is normally found in aquatic environments, the species S. maltophilia is an opportunistic pathogen, found in several black corals. This bacteria is known to have a limited pathogenicity and may be found in contaminated waters (Martínez et al., 2007), which might explain the presence in multiple of the isolations. Other bacteria commonly found in corals include: Gammaproteobacteria such as Pseudoalteromonas spp., Vibrio spp., and Shewanella spp. (Gray et al. 2011; Lampert et al. 2006), which includes the genera Vibrio, highly associated with corals and also found in this study. In stony and soft coral the phylum Gammaproteobacteria is relatively common and abundant (Harder et al., 2003; Nithyanand & Pandian, 2009; Gray et al., 2011), however not commonly found in black corals, probably due to the different morphological structures of the black corals and stony and soft coral species or simply this bacteria phylum are not trapped in tissues of black corals (Zhang et al., 2012). The phylum Gammaproteobacteria such as species of Vibrio and Pseudoalteromonas have exhibit antimicrobial activity against gram–positive and gram- negative strains (Shnit-Orland et al. 2012; Shnit- Orland and Kushmaro 2009), which could not be evidenced in the only bacteria found from this phylum. In general, the great diversity of microorganisms was confirmed in black coral of the Colombian Caribbean, in this study.

In the antimicrobial tests, interestingly, the corals collected in deep waters (50m): Plumapathes pennacea, Antipathes furcata and Antipathes atlántica, all showed antimicrobial activity against S. aureus. Specifically the bacteria: Bacillus velezensis also known as Bacillus amyloliquefaciens, showing the highest antimicrobial activity and a second bacteria that could not be identified, that showed moderate antimicrobial activity. No isolation showed any kind of antimicrobial activity against P. aeruginosa, which seems no surprise due to its multiple mechanisms of resistance. This bacteria has an inherent resistance to many drug classes, ability to acquire resistance, via mutations, to all relevant treatments, it also has a high and increasing rate of resistance locally and plays a frequent role in serious infections (Livermore, 2002). The specie with the highest antimicrobial activity, Bacillus amyloliquefaciens, is a bacteria known to produce volatile compounds, which inhibits the growth and spore germination of Fusarium oxysporum (Yuan et al., 2012), which shows the potential of this bacteria as antifungal or antibacterial. Yoshida et al (2001), also proved the potential of this bacteria and found antimicrobial and antifungal activities, but in plant species. As established before, this genus is an important antibiotic resource which makes B. amyloliquefaciens a promising candidate for further studies on antibiotic active compounds. Although in this study, a certain amount of bacteria showed antimicrobial activity, others studies like Zhang et al (2012), found a higher percentage of bacteria with antimicrobial properties. Antibiotic profiles and the production of antibiotic substances apparently are strain specific properties and in many isolates depend on the media applied for the production of metabolites (Pham et al., 2016). Certainly, more studies using a variety of mediums, species of corals and microorganisms is needed to understand the actual role of these organisms in black corals and their antimicrobial compounds. Other studies on soft corals have shown that a large proportion of bacteria have antimicrobial activity and the broad range of activity over several phyla suggests that bacteria and their bioactive compounds, play a significant role in the life of corals (Pham et al., 2016), which supports the need to investigate black coral associated microorganisms, to provide a wider range of diversity and their bioactive compounds. Conclusions From a few number of microbial isolations, an insight of the diversity of microorganisms associated with black coral of the Colombian Caribbean Sea was made. A big part of the isolations failed to be identified, needing more precise methods, but the rest showed a wide range of genus and probably some new genus not found before in black corals. The dominant genus, Bacillus, a well-known bacteria for its important antibiotic source, is certainly a group needed to be investigated for bioactive compounds. A certain part of all the bacteria isolated, showed any kind of antimicrobial activity against the most common human pathogen in hospitalized patients, S. aureus, which might bring new insights in the developing of vaccines and new and improved antimicrobials. Out of the group of bacteria with antimicrobial properties, the majority came from deep waters, supporting the need to investigate black coral associated microorganisms, particularly those coming from deep waters.

Acknowledgments

Special thanks to Lorena Toquica and Adriana Celis for all their knowledge and help throughout this project. Thanks to Juan Armando Sánchez and all the BIOMMAR lab team for their help and support, to my family and nearest friends.

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