Symbiotic marine bacteria chemically protect their host organisms from pathogenic organisms [1]. Additionally, symbiotic bacteria are known to have various roles, such as host health, nutrition, and antibiotic production for host organisms [2-6]. Over the past 85 years, about 50,000 natural products have been discovered from microorganisms. More than 10,000 of these compounds are biologically active and more than 8,000 are antibiotics [7]. Bacteria efficiently produce natural products that could prove to be useful drugs [8]. Over the past few decades, bacteria have evolved to resist well-known antibiotics [9]. As a result, hospitals have seen a dramatic rise in drug-resistant infections, many of which are lethal. To identify new antibiotics, scientists often make use of the natural chemical defenses of fungi and bacteria, altering these natural antibiotics to produce new ones. Bacteria have an ability to produce bioactive secondary metabolites, such as antimicrobials, antifungals, antitumorals, immunosuppressants, and antibiotics [10]. Biochemical and physiological pathways of bacteria may be responsible for antibiotic resistance [11]. Therefore, we have attempted to isolate marine bacteria that live together with marine organisms and explore their antibiotic resistance to help identify novel marine derived antibiotics such as Fijimycins and Marinopyrroles [12,13].

Collection of marine organisms

We collected a marine sponge (Callyspongia confoederata), brown algae (Sargassum siliquastrum (Merens ex Turner) C. Agardh), S. macrocarpum C. Agardh, and Myagropsis myagroides (Mertens ex Turner) Fensholt, green algae (Cladophora wrightiana var. minor C. Hoek & M. Chihara), and sea water by SCUBA diving from Biyangdo on Jeju Island in March 2016.

Isolation and culture of bacteria associated with marine organisms

These six samples are washed with sterile sea water as soon as they are collected, diluted 1:20 with sterile sea water and heated at 55°C for 10 min and 20 μl was inoculated onto A1SW isolation medium (10 g soluble starch, 4 g yeast extract, 2 g peptone, 16 g agar, 1 L filtered and sterilized sea water) and incubated at 25°C [14]. After 7 days, separated single colonies from the cultured media were transferred to new A1SW plates for pure isolation of marine bacteria, incubated at 25°C for 3-4 days, and then stored in 20% glycerol (v/v) suspensions at -80°C.

DNA extraction, PCR amplification, DNA sequencing, and phylogenetic analysis

Chromosomal DNAs of pure cultivated bacteria were isolated using a LaboPass^TM tissue genomic DNA isolation kit (Cosmogenetech, Daejeon, Korea). PCR was employed to amplify 16S rRNA genes using the primers 27F and 1492R [15], and the products of which were purified with a LaboPass^TM PCR purification kit (Cosmogenetech, Daejeon, Korea), according to the manufacturer’s protocol, and sequenced on a capillary electrophoresis instrument (Applied Biosystems 3730XL, CA, USA). Similarities between the 16S rRNA gene sequence of pure isolated bacteria and those of other bacteria previously described were determined by performing BLAST searches of the GenBank/EMBL/ DDBJ database [16]. Multiple sequence alignment of representative sequences was carried out using Clustal W version 2·0 [17]. Maximumlikelihood, neighbor-joining, and minimum-evolution trees were generated using MEGA 6·0·6 [18].

Disc diffusion test for Antibiotic susceptibility

Susceptibility to six antibiotics (30 μg chloramphenicol, 15 μg erythromycin, 30 μg gentamicin, 15 μg lincomycin, 30 μg tetracycline, and 30 μg vancomycin) (Liofilchem, Roseto degli Abruzzi, Italy) was determined by the disc diffusion method [19]. For all isolated strains, 100 μl (5 × 105 CFU ml-1) was spread onto A1SW medium, and 6 antibiotics were inoculated onto the center of the agar plates, which were incubated at 25°C for 48 h. After incubation, clear zones of growth inhibition were measured to the nearest millimeter. The clear zone disc is the zone of inhibition that indicates the extent of the test organism’s inability to survive in the presence of the test antibiotic.

Nucleotide sequence accession numbers

All bacteria 16S rRNA gene sequences isolated from marine organisms described in this study have been deposited in the GenBank nucleotide sequence database under the accession numbers MF461049- MF461065.

Culture, isolation and species identification of marine bacteria

We isolated 17 strains of marine bacteria from marine sponges (Callyspongia confoederata), algaes (Sargassum siliquastrum, S. macrocarpum, Cladophora wrightiana, Myagropsis myagroides), and sea water collected from Biyangdo on Jeju Island (Table 1). We found that the bacterial strains belong to six families/orders (Halomonadaceae / Oceanospirillales, Pseudomonadaceae / Pseudomonadales, Pseudoalteromonadaceae / Alteromonadales, Rhodobacteraceae / Rhodobacterales, Flavobacteriaceae / Flavobacteriales, and Bacillaceae / Bacillales) and four classes (Gammaproteobacteria, Alphaproteobacteria, Flavobacteria and Bacilli) (Table 1). Analysis of strain JJS3-4 isolated from S. siliquastrum revealed 98% similarity to the 16S rRNA gene of Formosa spongicola A2^T [20].

Table 1: Sources for isolation of marine bacteria.

Phylogenetic analysis of marine bacteria isolated from marine organisms

Analysis of the 16S rRNA sequences from the 17 isolates, along with similar type strains, revealed a significant level of diversity (Figure 1). This approach identified species that have been exclusively reported from marine habitats. These bacteria consist of four classes (Alphaproteobacteria and Gammaproteobacteria, Flavobacteria, and Bacilli) based on 16S rRNA gene sequencing and phylogenetic analysis (Figure 1).

Figure 1:Phylogenetic tree conducted using 16S rRNA gene sequences of 17 marine-derived bacteria and type strains in this study. Bootstrap values 40% (from 1000 replicates) from maximum-likelihood / neighbor-joining / minimum-evolution analyses are shown at branch points. Type strain Streptomyces felleus AS4.1677T was used as outgroup. Bar 0.05 substitutions per nucleotide position.

Antibiotic susceptibility of isolated marine bacteria

We tested the antibiotic resistance of the 17 strains against 6 antibiotics (30 μg chloramphenicol, 15 μg erythromycin, 30 μg gentamicin, 15 μg lincomycin, 30 μg tetracycline, and 30 μg vancomycin) by disc diffusion test (Table 2). In contrast to the other bacteria tested, strain JJS3-4, identified as Formosa sp. and isolated from S. siliquastrum, was resistant to the six antibiotics, indicating that the need to study the useful secondary metabolites production of this strain (Table 2). Strain JJS1-1, JJS1-5, and JJS2-3, which were identified as Pseudovibrio spp., and Stappia sp. JJS5-1 were susceptive to chloramphenicol (Table 2). In addition, Halomonas anticariensis (strains JJS2-1, 2-2, and 3-2) and Pseudomonas rhodesiae (strain JJS4-1 and JJS4-2) showed similar resistance pattern against six antibiotics (Table 2). We performed this experiment three times and then calculated the mean value except for the bounce value and are shown to Table 2 as mean values.

Table 2: The result of 17 strains for disc diffusion antibiotic susceptibility test.

Many marine bacteria are associated with marine sponges and marine algae and such isolated bacteria have useful physiological activities [21,22]. The media and culture conditions for bacteria can significantly influence the isolation and cultivability of distinct bacterial isolates [23]. In this study, we obtained 17 cultivable heterotrophic bacteria isolates from marine organisms using a culture-dependent method. Strain JJS1-1, JJS1-5 and JJS 2-3, Pseudovibrio spp. isolated from sea water and sponge were shown susceptibility against chloramphenicol. Among these bacteria, Pseudovibrio spp. Strain JJS1-1 and JJS1-5 showed high antioxidant activities and are considered to be useful marine resources as antioxidant-producing strains (Table 3). Bacterial isolates belonging to the genera Pseudovibrio are known for their ability to produce antibiotics. Pseudovibrio isolates originating from marine invertebrates such as tunicates, corals, and sponges showed antimicrobial activity [24-26]. The antioxidants generally produced by marine environment include polyphenolic metabolites such as flavonoids, cinnamic acid, furan, and phlorotannins [27,28]. So, we are doing to search bioactive compounds from these results now.

Table 3: ABTS radical scavenging activities of 17 strains of marine bacteria.

Also, strain JJS3-4 was shown 98% similarity in 16S rRNA gene sequence with Formosa spongicola A2T that was represented strong resistance against all antibiotics. In addition to, this strain is considered to be a new strain based on the 16S rRNA gene results. By Challinor and Bode, unexplored bacterial strains from unusual sources could play in the search for such novel compounds [9]. So, it will be worth exploring the potentially useful materials that these bacteria produce via large-scale culture approaches.

Based on our findings, our results show that 17 marine bacteria derived from marine resources have species-specific results for antibiotics although the isolated sources are different. Our study not only suggests the possibility of novel antibiotic materials that can be used as a source for natural products (chemically identical to the pure natural product) but also indicates that this approach can be useful in delineating the taxonomic composition of associated microbial communities.

This work was supported by a grant from the National Marine Biodiversity Institute of Korea (2018M00700).