Abstract: Marine bacteria are producers of secondary metabolites in the harsh ocean. In the present study, marine bacteria were isolated from marine sponges in the Gulf of Mannar. The potential isolates were selected for 16S rRNA gene sequencing. The results of phylogenetic analyses revealed that isolate No. 3 was Stenotrophomonas sp., strain SMAKK001; isolate No. 6 was Bacillus strain SMAKK002 and isolate No. 8 was identified as Bacillus sp. strain SMAKK003. Antimicrobial assays were performed against various microorganisms with bacterial metabolites. Out of the 10 marine bacterial filtrates only four filtrates exhibited antimicrobial activity. Among them, the filtrate of Stenotrophomonas sp. strain SMAKK001, Bacillus subtilis strain SMAKK002 exhibited strong activity against gram positive and negative bacterial strains. Secondary metabolite of marine bacterial isolates SMAKK001, SMAKK002 and SMAKK003 showed considerable inhibitory activity against pathogenic bacteria and fungi. Inhibitory activity was observed against important pathogenic species such as MDR Staphylococcus sp., MDR Pseudomonas sp. and MDR Klebsiella sp. which opens up interesting avenues in the search for novel compounds against multidrug-resistant pathogenic bacteria. The present study revealed that marine bacterial metabolites used against potential new anti-MDR strains.
INTRODUCTION
The ocean is the mother of life and it is believed that the most primitive forms of life originated here. It harbors a great variety of marine organisms which are diverse in their physiology and adaptations. Since the 1920s when the first antibiotic Penicillin was discovered, it proved microorganisms are the largest source of novel drugs. In spite of the long and successful history of antibiotics of terrestrial origin, the search for marine microbial metabolites is even today a nearly untouched subject. First marine antibiotic, pentabromo pseudilin was described (Burkholder et al., 1966) that discover believed that marine microorganisms as a novel source for active compounds.
Microorganisms produce secondary metabolites which may have some potential bioactivity, either against microorganism or against physiological function of the diseased body. These microbial metabolites are used as antibiotics which effectively against infectious diseases such as HIV-1 and bacterial infections. Some drugs have also been found to be useful against carcinomas (bleomycin, dactinomycin, doxorubicin and staurosporin), coronary heart disease, or may act as immune-suppressants (cyclosporin) to aid in organ transplantation (Cragg and Newman, 2001); thus making the microbial secondary metabolites an enormous source of pharmaceutical importance. Recently, the first report on antimicrobial activity of Stenotrophomonas strains isolated from deep sea invertebrates which produced remarkable anti fungal substances (Romanenko et al., 2008). Likewise, Bacillus species are usually isolated from sediments but they are sometimes associated with benthos in the marine environment (Hentschel et al., 2001).
Marine bacteria are producers of such secondary metabolites in the harsh oceanic climates. Brominated biphenyl compound was isolated from P. phenolica that inhibit the methicillin resistant S. aureus strains (Isnansetyo and Kamei, 2003). Franks et al. (2005) reported that tambjamine like alkaloid of Pseudoalteromonas tunicata has anti-fungal activity. These tambjamines have been isolated from marine invertebrates which was possessed antimicrobial, immunosuppressive and anti-proliferative activities (Lindquist and Fenical, 1991). Evidence indicates that the colonizing bacterium at the surface of higher organisms is the source of these compounds (Konig et al., 2006). This has been indicated by Burke and colleagues by elucidation of YP1 biosynthetic pathway in P. tunicate (Burke et al., 2007).
It is widely accepted that culture-based techniques are inadequate for studying bacterial diversity from environmental samples, as many bacteria cannot be cultured and/or adequately identified using current and traditional techniques (Amann et al., 1995). The use of molecular approaches to describe microbial diversity has greatly enhanced the knowledge about natural microbial communities (Friedrich et al., 2001; Webster et al., 2001; Hentschel et al., 2002). However, microbial cultivation is essential to search for new bioactive compound producing strains.
MATERIALS AND METHODS
In the present study, marine sponges from the Gulf of Mannar southeast coast of India were collected in sterile polythene bags at a depth of 5-10 m by SCUBA diving; after collection, specimens were transferred to the laboratory in the ice box and processed under aseptic conditions as follows: Other macro-organisms were removed from the fresh material. Small pieces of sponges were inoculated on ZoBell marine agar medium. The plates were incubated at 28°C until bacterial colonies developed. Bacterial colonies were then removed and transferred to marine agar slants. Each isolate was purified by streak plate method and then Gram staining was done. The potential isolates that exhibited moderate activity were selected for 16S rRNA gene sequencing. The selected pure bacterial isolates were cultured in marine broths at 25°C and the total genomic DNA of each isolate was extracted using the DNA purification Kit as per the manufacturer’s protocol (Cat. No. 69506 Qiagen Inc., CA, USA).
The 16S rRNA gene amplification was done using PCR with 16S rRNA Forward primer 5'-AGAGTRTGATCMTYGCTWAC-3' and reverse primer 5'-CGYTAMCTTWTTACGRCT-3' for isolate 3 and 16Sr RNA Forward primer 5'-AGAGTTTGATCCTGGCTCAG-3' and reverse primer 5'-ACGGCTACCTTGTTACGACTT-3' for isolate 6 and 8, using the following PCR programs: Initial denaturation was for 5 min at 94°C, Denaturation 30 sec at 94°C, Annealing: 30 sec at 55°C, Extension: 2 min at 72°C, Final extension: 15 min at 72°C, Number of Cycles: 35 and held at 4°C.
The purified PCR products were commercially sequenced (ABI 3130 Genetic Analyzer) using 16S rRNA specific primer. Phylogenetic analysis Sequences were analyzed using the Basic Local Alignment Search Tool (Altschul et al., 1997) for preliminary species identification. A phylogenetic tree was constructed by a bootstrap test, using MEGA4 program. The sequenced bacterial strains were also deposited at NCBI gene bank.
Screening of antimicrobial activity: Each pure culture isolate was inoculated on marine broth medium and incubated at 28°C for 14 days with 120 rpm agitation separately. After 14 days, the broth cultures were harvested by filtration through 0.44 μm Millipore filters and centrifuged at 10,000 rpm for 5 min. Supernatant was assessed against the bacterial and fungal cultures. This was carried out by disk diffusion assay (Manikandan et al., 2001a) using 6 mm diameter paper discs which were impregnated with 100 μL volumes of the filtrate. Then the discs were placed on Mueller-Hinton agar plates seeded with microorganisms and incubated at 37°C for 24 h. After incubation, presence of a clear circular zone around the sample impregnated disc was used as an indicator of activity. All the assays were carried out in triplicate.
RESULTS
In the present study, marine bacteria were isolated based on the morphological structure of colonies on marine agar plate. The Gram staining of sponge associated bacteria analyzed in this study showed three as Gram positive and seven as Gram negative. Out of the ten bacterial isolates, only four marine bacterial isolates were able to produce secondary metabolites and were detected by the formation of clear zones around the disc in Mueller-Hinton agar plates at 37°C after 24 h. The isolates 3, 6 and 8 were moderate antimicrobial secondary metabolite producers and were selected for the 16S rRNA sequence analysis. 16S rRNA sequence analysis for Isolate No. 3, 6 and 8 showed 778, 730 and 633 bp, respectively. These 16S rRNA sequence of bacterial isolates showed more than 99% similarity with the available database sequence in the National Center for Biotechnology Information (NCBI) (Fig. 1-3). The results of phylogenetic analyses revealed that isolate No. 3 was Stenotrophomonas sp., of Xanthomonadaceae family in the gram negative bacterium; isolate No. 6 was Bacillus subtilis of Bacillaceae family in the Gram positive bacterium and isolate No.8 was identified as Bacillus sp. of Bacillaceae family in the Gram positive bacterium.
| Fig. 1: | Phylogenetic tree of Stenotrophomonas sp. strain SMAKK001 |
| Fig. 2: | Phylogenetic tree of Bacillus subtilis strain SMAKK002 |
| Fig. 3: | Phylogenetic tree of Bacillus sp. strain SMAKK003 |
Preliminary antimicrobial assays were performed against various microorganisms with filtrate (secondary metabolites) obtained from broth culture of 10 marine bacterial isolates. Out of the 10 marine bacterial filtrates only four filtrates exhibited antimicrobial activity. Among them, the filtrate of Isolate 3G¯ (Stenotrophomonas sp. strain SMAKK001) exhibited strong activity against gram positive bacterial strains of the Staphylococcus aureus whereas it exhibited moderate activity against Streptococcus mutans, E. coli, V. cholerae, C. albicans and A. niger. The filtrate of isolate 6G+ (Bacillus subtilis strain SMAKK002) exhibited strong activity against the gram positive bacterial strains of the Staphylococcus aureus and moderate activity against the gram positive bacterial strains of the Streptococcus mutans and the gram negative bacterial strains of the Vibrio cholerae whereas, it exhibited weak activity against the gram negative bacterial strains of Pseudomonas aeruginosa and fungal strains of Candida albicans and Aspergillus niger (Table 3). The filtrate of isolate 8G+ (Bacillus spp. strain SMAKK003) exhibited moderate activity against Streptococcus mutans, Staphylococcus aureus, Vibrio cholerae and C. albicans, whereas, it exhibited weak activity against fungal strains and Aspergillus niger only (Table 1).
| Table 1: | Antimicrobial activity of methanol extract of sponges against gram positive bacterial strains |
| -: No activity; +: Weak activity (<5 mm zone of inhibition); ++: Moderate activity (>5 mm zone); +++: Strong activity (>10 mm zone) | |
| Table 2: | Antimicrobial activity of methanol extracts of sponges against gram negative bacterial strains |
| -: No activity; +: Weak activity (<5 mm zone of inhibition); ++: Moderate activity (>5 mm zone); +++: Strong activity (>10 mm zone) | |
| Table 3: | Antimicrobial activity of methanol extracts of sponges against fungal strains |
| -: No activity; +: Weak activity (<5 mm zone of inhibition), ++: Moderate activity (>5 mm zone), +++: Strong activity (>10 mm zone) | |
Table 2 shows the antimicrobial activity of the filtrate of strain SMAKK001 exhibited moderate activity against all the MDR strains tested. The filtrate of strain SMAKK002 exhibited moderate activity against MDR Pseudomonas sp. and MDR Staphylococcus sp., whereas, it exhibited weak activity against the MDR Klebsiella sp. The filtrate of strain SMAKK003 exhibited moderate activity against only MDR Staphylococcus sp., whereas, it exhibited weak activity against the MDR Pseudomonas sp. and MDR Klebsiella sp (Table 4).
| Table 4: | Antibacterial activity of filtrate of marine bacterial isolate against MDR strains |
| -: No activity; +: Weak activity (<5 mm zone of inhibition), ++: Moderate activity (>5 mm zone), +++: Strong activity (>10 mm zone); G¯: Gram negative; G+: Gram positive | |
DISCUSSION
The discovery of 3 new strains of bacteria viz., Stenotrophomonas sp., strain SMAKK001, Bacillus subtilis strain SMAKK002 and Bacillus sp., strain SMAKK003 which exhibited good antimicrobial activity and their 16S rRNA gene seguences were submitted to NCBI gene bank (Gene Bank accession numbers: JF895518 for Stenotrophomonas sp., strain SMAKK001, JF965493 for Bacillus subtilis strain SMAKK002 and JN007072 for Bacillus sp., strain SMAKK003). The discovery of new classes of antibiotics has become necessary due to the increased incidence of multiple drug resistance among pathogenic microorganisms to drugs that are currently in clinical use (Manikandan et al., 2011b, c). The sponge associated bacteria analyzed in this study by Gram staining revealed three of them as Gram positive and seven of them as Gram negative. Similarly, many of the previous studies have indicated that majority of marine bacteria are Gram negative (Taylor et al., 2007). Studies on marine microbial communities have also evidence that most bacteria from this environment appear to be Gram positive (Gontang et al., 2007). Most of the Gram positive bacteria are produced spores in the unfavorable conditions which ensure their survival nature in the marine environment (Ettoumi et al., 2009). The Gram classification can be an interesting tool if the differences between the structure and chemical composition of the two main groups of bacteria are considered.
Out of the 10 bacterial isolates from sponges, only 4 isolates were found to produce potential secondary metabolites. Among these, two isolates were Gram-positive and two were Gram-negative and bioactive producers. Bernan et al. (1997) has reported the predominance of Gram-negative bacteria as bioactive producers in the marine environment, whereas, Ivanova et al. (1999) reported the occurrence of Gram-negative bacteria as bioactive producers in the marine environment. Various studies have reported the predominance of Gram-negative bacteria as bioactive producers in the marine environment (Hentschel et al., 2001; Taylor et al., 2007). Some bacterial strains from the marine sponge showed no antibacterial activity against the same bacteria. These results indicate that certain bacterial species have selective mechanisms (Matz et al., 2008).
Out of the 4 potential bacterial isolates, only 3 isolates were found to be identified with 16S rRNA gene sequencing and phylogenetically these are located within the phyla Proteobacteria and Firmicutes. All sequences were submitted to gen bank and the accession numbers for all producer strains were sequenced. The strain SMAKK001 identified as Stenotrophomonas sp. was found exclusively on Halichondria species. Romanenko et al. (2008) have also reported that Stenotrophomonas maltophilia strain isolated from sponges collected in the Pacific Ocean displayed remarkable fungal inhibitory activity. Bacterial strain of the genus Bacillus subtilis (SMAKK002) was found associated with the sponge Tethya repens, A. cornuta and Haliclona sp. The strain SMAKK003 was identified as Bacillus sp. and it was predominantly found in all sponges. Similarly, Pabel et al. (2003) also reported marine Bacillus species in the sponges.
The present study revealed that Bacillus species was found in all sponges whereas, the bacterium Stenotrophomonas sp., was found particularly in Halichondria sp. Sponges of the genus Halichondria provide good examples for the importance of microbial association (Wang, 2006). These data support that in several cases symbioses between host sponges and their bacteria are mutual, i.e., not only beneficial to the bacteria (using the sponge as a habitat) but also to the sponge. Lemos et al. (1985) demonstrated that the production of compounds by microbes that are found on the surfaces of organisms would be extremely advantageous. They also remarked that bioactive compounds may be produced when spatial competitors or predators challenge the sponges. However, Thacker et al. (1998) found no evidence that the production of secondary metabolites was induced by the presence of a spatial competitor; rather they suggested that the constant threat of predators might maintain high concentrations of the compound. The diversity of antibiotic producing marine bacteria isolated in the present study suggests that sponges are rich sources of novel bacteria. The bacterial diversity observed in the present study may be only a fraction of the total diversity of associated bacteria.
Antibacterial activity among the marine bacteria is well-known and has been demonstrated in a number of studies (Uzair et al., 2006). Preliminary results are encouraging that the isolated strains exhibit antibacterial activity and the present study also ascertained that Stenotrophomonas sp. and Bacillus sp. are widely recognized as rich sources of antimicrobial agents as suggested by Minkwitz and Berg (2001) and Gebhardt et al. (2002).
The strain SMAKK001 exhibited antimicrobial activity against both Gram-negative and Gram-positive bacteria. The high frequency of activity against Gram-positive bacteria was expected, since Gram-negative bacteria are generally less susceptible to antimicrobials than Gram-positive bacteria because of the presence of an outer membrane and Lipopolysaccharide (LPS) which together act as an efficient barrier against hydrophobic and lipophilic molecules (Snyder and McIntosh, 2000). The strains SMAKK002 and SMAKK003 were effective against fungi. Particularly, product of the isolate SMAKK003 inhibited activity against Candida albicans which causes human genital infections, e.g., vaginitis and oral infection of infants and AIDS patients (Egusa et al., 2008).
Secondary metabolite of marine bacterial isolates SMAKK001, SMAKK002 and SMAKK003 showed considerable inhibitory activity against pathogenic bacteria and fungi. Inhibitory activity was observed against several bacteria, including important pathogenic species such as MDR Staphylococcus sp., MDR Pseudomonas sp. and MDR Klebsiella sp. which opens up interesting avenues in the search for novel compounds against multidrug-resistant pathogenic bacteria. The present study revealed that marine bacterial metabolites may be used against potential new anti-MDR strains.
CONCLUSION
Very little information is available on the production of antimicrobial substances and phylogenetic identification of sponge associated bacteria found off the coastline of Gulf of Mannar, a place known to have rich biodiversity that remains to be studied extensively. This is the first study that reports Gulf of Mannar sponge-associated bacterial strains with high potential for producing antimicrobial substances which are active against multidrug-resistant bacteria. Therefore, the isolates which showed activity against those tested bacteria are also valuable for further study. The results of the present investigation clearly revealed that the marine bacteria from the sponges are potential sources of novel antibiotics.
ACKNOWLEDGMENT
This investigation was supported by the ISRO/SAC, MoES and Department of Marine and Coastal Studies, Madurai Kamaraj University, Madurai, Tamil Nadu, India.