Isolation and Characterization of Pseudomonas Strain That Inhibit Growth of Indigenous and Clinical Isolate
B Uzair, N Ahmed, F Kousar, D Edwards
antibacterial pseudomonas, marine bacteria, thin layer chromatography, zone of inhibition
B Uzair, N Ahmed, F Kousar, D Edwards. Isolation and Characterization of Pseudomonas Strain That Inhibit Growth of Indigenous and Clinical Isolate. The Internet Journal of Microbiology. 2005 Volume 2 Number 2.
The use of microorganisms for biological purpose has become an effective alternative to control pathogens. Bacteria from marine environment are an underutilized source of novel antibiotics. A total of 100 bacteria were isolated from marine samples (Fish, Water and Crustaceans) of Baluchistan coast and screened for antagonistic interaction. Out of these 15 strains showed antibacterial activity. One strain designated as CMG1030 identified as
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
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
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
Materials and Methods
Bacterial strains used
CMG1030 was used as producer strain and (MRSA)
Screening of Isolates for Inhibitory Activity
A total of 100 strains isolated from samples of Sindh and Baluchistan Coast were screened for production of antibacterial substance by Burk holder agar diffusion assay of Burkholder. A lawn of sensitive strain was prepared by mixing 25 ml of molten 44°C nutrient agar with 30µL of a suspension of overnight culture of sensitive strains and it was vortexed and poured on nutrient agar plate, 10µL of potential producer were spotted on the lawn. The plates were incubated face up for 24 at 30°C and examined for zones of inhibition (area where target isolate failed to grow). The strain (CMG1030) showing promising activities against clinical isolates were selected for further studies.
Effect of time on the production of antibacterial metabolite
CMG1030 was grown in king B medium and incubated at 30°C for 72 hrs and samples were pulled out after every 6 hrs. All the samples were centrifuged and filtered using millipore filters (GyroDisc CA 0.2µm) and activity of the filtrates were checked by agar well diffusion method.
Thermostability of the antibacterial activity
1.5 ml of filtrate of CMG1030 was dispensed in sterile eppendorf tubes. The tubes were incubated as follows.
a) Room temperature for 30 minutes
b) At 80°C for 30 minutes
c) Autoclaved at 120°C for 20 minutes
d) Freezer -20°C for 24 h
From each set of tubes, fractions of 100µL were tested against MRSA for inhibitory activity.
Effect of enzymes on antibacterial metabolite
To find out chemical nature of the antibacterial metabolite. Filtrate of CMG1030 was treated with different enzymes (Pepsin10µg/mL, ProteaseP10µg/mL, ProteaseK1µg/mL, RNase10µg/mL) and treated samples were tested by agar well diffusion method.
Plate assay to establish the bacteriostatic or bactericidal activity of the antibacterial metabolite
From inhibitory assay conducted samples with sterile cotton tips were taken from the surface of the plate where no growth of tested strain was observed the tips were surface streaked to nutrient agar plates and incubated at 30°C for 48 hrs, the plates were observed for growth or no growth.
Determination of siderophore
To determine whether siderophores are responsible for the antagonistic properties of CMG1030.CMG1030 was grown in nutrient agar plate supplemented with 1%ferric chloride and tested for antagonistic activity by agar well diffusion method.
Isolation of Antibacterial Compound
An antibacterial compound was isolated from CMG1030 grown on king B agar plates incubated at 30°C for 5 days. Plates after incubation was cut into 1-cm square and the first extraction was carried out with 3 volume of 80% acetone with water for 2 hrs by using a rotary shaker the combined extracts were filtered through cheesecloth to remove pieces of agar other particulate matters were removed by centrifugation (9000xg for 20 minutes at 4°C the supernatant fraction was evaporated at room temperature to ensure complete solvent removal. Portioning with ethylacetate carried out a second extraction. The extracts were combined evaporated by dryness. The resulting residue was dissolved in acetone and stored at –20°C until further purified.
Thin layer chromatography was carried out with the crude extract on silica gel (TLC Aluminum sheets silica gel 60 F254) with ethylacetate and hexane as a mobile phase (1:1). The crude extract 30µL was spotted on TLC plate and the solvent front was allowed to run for approximately18 cm. The relative mobility of each spot was determined considering the retarding factor of each solute (RF) using an ultraviolet-light (312 - 365nm) transilluminater.
Bioassay of crude extracts and its fractions
Crude extract and TLC elutes were tested for antibacterial activity. Test samples (100uL) were pipetted into 5mm diameter well bored into nutrient agar plates swabbed with test strain. Control plates consisted of crude extracts from un inoculated king B medium. The presence of inhibition zones around the well was recorded after incubation for 24 hrs at 30°C. Fractions from TLC plates were cut into portions. These portions were scrapped into eppendorf tubes and extracted with 100% acetone. The silica residues were removed by centrifugation and supernatant was transferred into second set of eppendorf tubes. Each fraction was concentrated by evaporating off acetone and tested for antibacterial activity by using disc diffusion method.
Incubated for 15 min at 30°C.
Determination of minimal inhibitory concentration
The minimum inhibitory concentration (MICs) of crude extract was determined by the standard microdilution method described in the national committee for clinical laboratory standards using Muller Hinton broth medium (Oxoid) incubated at 37°C for 24 h.
Phase contrast microscopy
An exponential phase culture of
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
CMG 1030 showed varied spectra of activity inhibiting indigenous marine isolates (
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
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
We thank to Dr Bashir Siddiq Director Spectrum Fisheries Ltd for providing samples for isolation of bacteria and we are also thankful to Miss Noureen Saeed for providing clinical isolates. Thanks to HEC for scholarship to Bushra Uzair.