Introduction

During the past two decades research on marine bacteria has highlighted the tremendous potential of these microorganisms as a source of new bioactive secondary metabolites (Jenson and Fenical, 2000 ; Ahmed et al., 2000) and there is a growing awareness of the need for development of new antimicrobial agents for the treatment of human animal and plant diseases. Marine bacteria could represent a new source of antibiotics, which are currently needed to combat emergent antibiotic resistant pathogen. Many marine heterotrophic bacteria are known to produce antibacterial substances, which inhibit or kill other bacteria. These antibacterial compounds are not only inhibitory to terrestrial bacteria but also to indigenous bacterial strains, which is of considerable ecological significance (Saz et al., 1963). It is clear that new classes of antibiotics are urgently needed. Many marine free living and sediment inhabiting marine bacteria have been shown to produce secondary metabolites that display antibacterial properties (Burgess et al., 1991 and Long and Azam, 2001) have also reported that particle attached marine bacteria are more likely to produce inhibitory compounds than free living counterparts. It has been reported that antibiotic producing marine Pseudomonas that produce antibiotic metabolite which are inhibitory for both gram negative and gram positive bacteria. (Nair and Simidu, 1987)

Antibiotics have been defined as substances produced by microorganisms that in high dilution are antagonistic to the growth or life of other microorganisms (O'Grady et al., 1997). Antibiotics may be intermediates or the end product of metabolism or may be waste products or other compounds, which have antibiotic properties. There is no doubt that the discovery of antibiotics has revolutionized the world of medicine, however sixty years later humans are far from the winning battle against infectious diseases because the pathogenic strains have emerged that are virtually unresponsive to antibiotics, such multi drug resistance, arising mainly through antibiotic misuse.

The more the bacteria come in contact with antibiotics, the greater are the chances of becoming resistant. Bacteria have proved to be amazing when it comes to circumventing the action of microbial agents particular strains which are causing problems at the oment are the Vancomycin-resistant Enterococci (VRE) (Frieden et al 1993; Leclerc, 1997) and methicillin resistant Staphylococcus aureus (MRSA). Several strains of human pathogens have been found to contain up to ten different genes coding for resistance (Henry, 2000). As Bacteria are continuously overcoming the tools with which humans have to fight there is a need for search for new antibiotics that affect the target.

Previous studies have shown that a high percentage of marine bacteria produce antimicrobial metabolites compared with the number of planktonic isolates that produce such metabolites, however it is also observed in laboratory most isolates stop producing microbial compounds when they are continuously cultivated in shake flasks. This paper describes isolation and characterization of CMG1030 Pseudomonas aeruginosa producing compounds that inhibit the growth of multi resistant clinical isolates of Staph, Salmonella, Shigella etc. The antimicrobial activity has been studied using various parameters.

Materials and Methods

Bacterial strains used

Screening of Isolates for Inhibitory Activity

Effect of time on the production of antibacterial metabolite

Thermostability of the antibacterial activity

Effect of enzymes on antibacterial metabolite

Plate assay to establish the bacteriostatic or bactericidal activity of the antibacterial metabolite

Determination of siderophore

Isolation of Antibacterial Compound

Analytical chromatography

Bioassay of crude extracts and its fractions

Determination of minimal inhibitory concentration

Phase contrast microscopy

Results and Discussion

Marine bacteria have been recognized as an important and untapped resource for novel bioactive compounds. A greater development of marine biotechnology will produce novel compounds that may contribute significantly towards drug development over the next decade(Kasanah & Hamann, 2004). Multidrug resistant strains such as methicillin resistant staphylococcus aureus (MRSA), Vancomycin resistant Enterococcus cause serious public health problem throughout the world. To isolate new type of antimicrobial compounds active against resistant organism's marine bacteria were isolated from Baluchistan coast (Crustaceans, Fish, and Water). 100 bacteria were isolated and tested for the production of antibacterial metabolite. A remarkable fraction 15% of the strains exhibited antagonistic properties against antibiotic resistant clinical isolates and against indigenous marine bacteria. CMG1030 identified as Pseudomonas aeruginosa showed good antimicrobial activity and was selected for further investigation. The production of inhibitory antibiotic compound by fluorescent Pseudomonas is well documented (Fravel, 1988 and Fenton, et al., 1992). CMG1030 showed excellent antibacterial activity. This strain is aerobic and failed to grow under anaerobic and acetic conditions it did not require organic growth factors, grow on acetate as the sole carbon source, was oxidase, catalase and citrate positive but indol negative and found to be resistant to several antibiotics (ampicillin, kanamycin, erythromycin, and tetracycline) and sensitive to chloramphenicol. A bioassay based on clearing zone on agar was used to determine the antibiotic activity of the metabolite produced by CMG1030 against clinical isolates and indigenous bacteria (Table 2). The antimicrobial metabolite has bactericidal activity against MRSA These results are similar to Isnansetyo et al., 2001 reported that Pseudomonas.sp isolated from marine red -alga produce anti MRSA compound having bactericidal nature.

Figure 1

Table 1: Morphological and Biochemical Characters of CMG1030

Figure 2

Table 2: Zones of inhibition produced by whole cell, culture filtrate and crude extract of CMG1030 against clinical isolates (By agar well diffusion method)

CMG 1030 showed varied spectra of activity inhibiting indigenous marine isolates (Vibrio algiolyticus, Shewanella putrifacians, E.coli, Bacillus subtilis, Staphylococus.aureus, Staph epidermidis) and antibiotic resistant clinical isolates (MRSA, Staph.aureus, Staph.epidermidis, E.coli. This may be due to the fact that same strain might produce different antibacterial compounds, depending on culture medium. To determine whether siderophore or antibiotics are responsible for the antagonistic properties of strain CMG1030 we tested for antibacterial activity in presence of FeCl3 because this Pseudomonas strain inhibited the growth of test strains in ferric ion rich medium, siderophore may not be involved in the antagonistic response, effect of time on production of antibacterial compound was also studied by growing it on King B medium and it was observed that degree of production of antibacterial compound increased with increasing culture age maximum zone size observed after 72 hrs of growth (Fig2).

Figure 3

Figure 1: Phase contrast images of live cells of treated with antibacterial compound, A control cells without treatment, B cells treated with Crude extract of Pseudomonas aeruginosa

Figure 4

Figure 2: Effect of time on production of antibacterial metabolite by CMG1030

The different thermal treatments conducted with supernatants demonstrate the thermo resistance of the inhibitory metabolite. Growth inhibition zones produced were the same for all supernatants independent of the thermal treatments (Table 3) to which they were subjected these results are in agreement with the reported thermo resistant of antibiotic produced by Pseudomonas. Cepacia (Jayaswal 1990) and to check the effect of enzymes the supernatant was treated with different enzymes (Pepsin, Protease P, Protease K, RNase) enzymes treated supernatant was found to be resistant to enzymes indicating antibiotic metabolite is not protein in nature.

Figure 5

Table 3: Effect of different thermal treatments on the antibiotic Activity of supernatant of CMG1030

Next we investigated the possibility of the production of antibiotic substances by this strain crude extract from a 5 day old culture of CMG1030 grow on King B medium were prepared by portioning with 80% acetone followed by ethylacetate these solvents were same ones used by (Jayaswal 1990) for the extraction of antifungal compounds from the strain of Pseudomonas, as( Bonsall 1997) reported these types of solvents allow extraction of metabolites such as phenazine, pyoluteorin, 2-4 diacetylphloroglucinol produces by numerous strains of Pseudomonas, extracts . The crude product appeared as dark brown oil and yield was between 100-150mg per 1000mL broth. Crude extract of CMG1030 tested positive in bioassay against all tested strains. These results indicated that the antagonistic activity was due to the production of antibacterial compound and that the compound could be extracted from growth medium by portioning with organic solvents. A thin layer chromatography of crude extract was performed and 13 bands were observed under UV transilluminater. (Table4). Bioassay of elutes from compounds separated by TLC showed one compound with activity against test strains. The MIC of isolated compound for S.aureus S. epidermidis were 75 µg/ml for Enterobacter faecalis 100 µg/ml. Log phase cells of S.aureus were treated with MIC concentration of isolated compound and incubated at 30°C for 30 min and slides were prepared and observed under phase contrast microscope, microscopic examination of crude extract treated cells of S.aureus suggest lysis of cells of S.aureus and some big and ghost cells were also observed (Fig2).

Acknowledgment