6.0 ANTIBACTERIAL ACTIVITY OF CAROTENOID FROM HALOMONAS SPECIES AGAINST CHOSEN HUMAN BACTERIAL PATHOGENS

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1 6.0 ANTIBACTERIAL ACTIVITY OF CAROTENOID FROM HALOMONAS SPECIES AGAINST CHOSEN HUMAN BACTERIAL PATHOGENS 6.1 INTRODUCTION Microorganisms that cause infectious disease are called pathogenic microbes. Although the vast majority of bacteria are harmless or beneficial, quite a few bacteria are pathogenic. The most common bacterial disease is tuberculosis, caused by the bacterium Mycobacterium tuberculosis, which kills about 2 million people a year, mostly in sub-saharan Africa. Pathogenic bacteria contribute to other globally important diseases, such as pneumonia, which can be caused by bacteria such as Streptococcus and Pseudomonas, and foodborne illnesses, which can be caused by bacteria such as Shigella, Campylobacter and Salmonella. Pathogenic bacteria also cause infections such as tetanus, typhoid fever, diphtheria, syphilis and leprosy. Making antibacterial drug therapy effective, safe and affordable has been the focus of interest during recent years. Antibacterial therapy, since its dawn in 1940s, is changing constantly and in the last fifteen years has undergone a tremendous change due to evolvement of new concepts in the pharmacokinetics of antibacterial agents and their pharmacodynamics in microbes (Leggett et al., 1989). Previously antibacterial agents were selected on the basis of their high degree of in vitro activity but currently antibiotic use is based on its effectiveness and cost in a given situation (Cunha, 1995). The important new concepts, which determine antibiotic use, revolve around the word effectiveness and lack of side effect. According to World Health Organization, natural products are the best source to obtain a variety of newer drugs 98

2 without side effects. The use of natural products with known antimicrobial properties can be of great significance in therapeutic treatments. In the last few years, a number of studies have been conducted in different countries to prove such efficiency. Studies concerned with the antibacterial property of carotenoids from halobacteria are still lacking. Keeping this in mind, the present study has taken effort to find out the antibacterial potential of carotenoids against the chosen antibiotic resistant human bacterial pathogens and poultry bacterial pathogens MATERIALS AND METHODS Extraction of carotenoids The carotenoids from the Halomonas strains KP2, KT2 and KT3 were extracted from the cultures of pigmented strains. The carotenoids were extracted with methanol (3-4 times) until a colourless residue was obtained. The combined methanol extracts were separated into two layers by adding hexane and distilled water. The pigment was recovered in the hexane layer, which was then washed several times with distilled water and dried over anhydrous sodium sulfate and the polar lipids were removed using acetone precipitation. The pigment solution, free of polar lipids, was dried under nitrogen and the resulting pigmented residue was weighed and redissolved in hexane. The extracted pigments were subjected to phase partition between light petroleum ether and 90% aqueous methanol. Each fraction was re extracted, washed, dried and weighed. 99

3 Antibiotic Resistant Pathogens Five antibiotic resistant human pathogens viz., Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus epidermis, Pseudomonas aeruginosa and Streptococcus pneumoniae were obtained from the Institute for Microbial Technology (IMTECH) culture collections from Chandigarh, India. All the bacterial strains were suspended in nutrient broth and incubated at 37 C for 48 hrs Opthalmic Pathogens Five antibiotic resistant human pathogens viz., Staphylococcus aureus, E. coli, Streptococcus pyogens, Proteus sp and Acinitobacter sp were isolated and identified from the infected broiler in the Namakkal district by following standard methodologies. All the bacterial strains were suspended in nutrient broth and incubated at 37 C for 48 hrs Antibacterial sensitivity assay Agar well diffusion assay was performed to find out the antibacterial potential of carotenoids from halomonas species isolated from the salt pan of Kanyakumari district. Muller Hinton agar (Hi-Media, Mumbai) plates were prepared by using the sterilized molten agar medium. After solidification, well was made by using metal well cutter. To the each well, different concentrations (10, and 40 µg/well) of carotenoids diluted with DMSO were poured in to the agar well which were previously seeded with the chosen bacterial pathogens. Control plate was maintained without the addition of carotenoids but with DMSO alone. All the plates were incubated overnight at 37 C under static conditions. After 24 hrs, the zone of 100

4 inhibition appearing around the discs were measured and recorded in millimeter in diameter. Triplicate samples were maintained for each bacterial strain Determination of Minimum Inhibitory Concentration (MIC) (Hammond and Lambert, 1978) Minimum inhibitory concentration is defined as the lowest concentration of bioactive compounds that inhibit the growth of particular microorganism by broth dilution method. 0.1 ml of 24 hrs microbial broth of antibiotic resistant pathogens and ophthalmic bacterial pathogens were added to the tubes containing 0.5 ml of varying concentrations of the carotenoids (8, 16, 32, 64, 128, 256, 512 µg.ml -1 ) obtained from potential pigmented Halomonas sp KT2 and further the total volume is made upto 1 ml by adding 0.4 ml of sterilized nutrient broth. Nutrient broth alone served as negative control. Whole setup in triplicate was incubated at 37 C for 48 hrs in thermostat shaker. After incubation, the tubes were then examined for microbial growth by turbidity observations Determination of Minimum Bactericidal Concentration (MBC) To determine the minimum bactericidal concentration, a loopful inoculum of broth was collected from MIC tubes and streaked onto a nutrient agar in triplicates and then incubated at 37 C for 24 hours. After incubation, the concentration at which no visible growth was seen was noted as the minimum bactericidal concentration RESULTS The present study also made an attempt to find out the antibacterial potential of carotenoids extracted from the three pigmented Halomonas sp. It is observed from the present study that, among the three species, the KT2 showed sensitivity against 3 101

5 antibiotic resistant bacterial pathogens viz., Klebsiella sps. Staphylococcus aureus and Pseudomonas aeruginosa. However, the maximum zone of inhibition was noticed with the carotenoid concentration of 40µg against Klebsiella sp. The carotenoids from Halomonas sp. KT2 did not show sensitivity against Streptococcus pneumonia and Streptococcus epidermis (Table 1). Table 1. Antimicrobial sensitivity of different concentrations of carotenoids from Halomonas sp. (KT2) against antibiotic resistant bacteria pathogens Zone of Inhibition (mm dia.) Pathogens 10µg 20 µg 30 µg 40 µg Klebsiella sp Staphylococcus aureus Pseudomonas aeruginosa Streptococcus pneumoniae Streptococcus epidermis indicates no inhibition Moreover, the KT2 showed sensitivity against 3 opthalmic bacterial pathogens viz., E. coli, Staphylococcus aureus and Streptococcus pyogens. However, the maximum zone of inhibition was noticed with the carotenoid concentration of 40µg against Streptococcus pyogens and E. coli. The carotenoids from Halomonas sp. KT2 did not showed sensitivity against Proteus sp. and Acinitobacter sp. (Table 2). 102

6 Table 2. Antimicrobial sensitivity of different concentrations of carotenoids from Halomonas sp. (KT2) against ophthalmic bacterial pathogens Pathogens Zone of Inhibition (mm dia.) 10µg 20 µg 30 µg 40 µg E.coli Staphylococcus aureus Streptococcus pyogens Proteus sp Acinitobacter sp indicates no inhibition Moreover, the carotenoid from Halomonas sp KT3 and KP 2 did not showed any sensitivity against any one of chosen antibiotic resistant and ophthalmic bacteria pathogens (Tables 3-6). Table 3. Antimicrobial sensitivity of different concentrations of carotenoids from Halomonas sp. (KT3) against antibiotic resistant bacterial pathogens Zone of inhibition (mm dia.) Pathogens 10µg 20µg 30µg 40µg Klebsiella sp Staphylococcus aureus Pseudomonas aeuroginosa Streptococcus pneumoniae Streptococcus sp indicates no inhibition 103

7 Table 4. Antimicrobial sensitivity of different concentrations of carotenoids from Halomonas sp. (KT3) against ophthalmic bacterial pathogens Pathogen Zone of Inhibition (mm dia.) 10µg 20 µg 30 µg 40 µg E.coli Staphylococcus aureus Streptococcus pyogens Proteus sp Streptococcus sp indicates no inhibition Table 5. Antimicrobial sensitivity of different concentrations of carotenoids from Halomonas sp. (KP2) against antibiotic resistant bacterial pathogens Pathogen Zone of Inhibition (mm dia.) 10µg 20 µg 30 µg 40 µg Klebsiella sp Staphylococcus aureus Pseudomonas aeruginosa Streptococcus pneumoniae Streptococcus sp indicates no inhibition 104

8 Table 6. Antimicrobial sensitivity of different concentrations of carotenoids from Halomonas sp. (KP2) against ophthalmic bacteria pathogens Pathogen Zone of Inhibition (mm dia.) 10µg 20 µg 30 µg 40 µg E.coli Staphylococcus aureus Streptococcus pyogens Proteus sp Streptococcus sp indicates no inhibition The present study is also made with an attempt to find out the broad spectrum of sensitivity of the most promising Halomonas sp KT2 which showed maximum sensitivity against 3 pathogens by well diffusion test and the results are represented in Tables 7 and 8. It clearly reveals that, KT2 showed varied MIC values ranged from µg.ml -1. The MIC value 32 µg.ml -1 of KT 2 strain was recorded against the antibiotic resistant bacteria Klebsiella sp, Pseudomonas aeruginosa and in case of ophthalmic pathogens Streptococcus pyogens the MIC of 128 µg.ml -1 was recorded against Staphylococcus aureus. Table. 7. Minimum inhibitory concentration of carotenoid obtained from Halomonas sp. KT2 against antibiotic resistant pathogens Name of the pathogen Concentration of Extract (µg.m -1 ) Staphylococcus aureus Klebsiella pneumoniae Streptococcus epidermis Pseudomonas aeuroginosa Streptococcus pneumoniae indicates intensity of sensitivity - indicates no sensitivity 105

9 Table. 8. Minimum inhibitory concentration of carotenoid obtained from Halomonas sp. KT2 against ophthalmic pathogens Name of the pathogens Concentration of extract (µg.ml -1 ) E.coli Staphylococcus aureus Streptococcus pyogens Proteus sp Acinitobacter sp indicates intensity of sensitivity - indicates no sensitivity The minimum bactericidal concentration (MBC) test was also carried out by the present study where it shows that the, most promising strains of Halomonas sp. (KT2) which confirm the minimum bactericidal concentration against 5 tested pathogens and the results are represented in Tables 9 and 10. It clearly reveals that, KT2 showed varied MBC values ranging from µg.ml -1. The MBC value is 16 µg.ml -1 against the antibiotic resistant pathogens Klebsiella pneumoniae and Pseudomonas aeruginosa and ophthalmic pathogens Staphylococcus aureus (Plate 2). Table. 9. Minimum bactericidal concentration of carotenoid obtained from Halomonas sp. KT2 against antibiotic resistant bacteria pathogens Name of the pathogen Concentration of extract (µg.m -1 ) Staphylococcus aureus Klebsiella pneumoniae Streptococcus epidermis Pseudomonas aeuroginosa Streptococcus pneumoniae indicates intensity of sensitivity - indicates no sensitivity 106

10 Table. 10. Minimum bactericidal concentration of carotenoid obtained from Halomonas sp. against ophthalmic bacteria pathogens Name of the pathogens Concentration of extract (µg.ml -1 ) E.coli Staphylococcus aureus Streptococcus pyogens Proteus sp Streptococcus sp indicates intensity of sensitivity - indicates no sensitivity 107