In-vitro antibacterial activity of coral reef associated bacteria and optimization of bioactive metabolite production.

This study reports the antibacterial activity of coral reef associated bacteria against bacterial pathogens and optimization of metabolite production. Twelve morphologically different bacterial were isolated from stony coral reef collected from Rameshwaram coastal area, Tamil Nadu. Cell free supernatant of all the bacterial isolates were tested against Staphylococcus aureus, Bacillus cereus, Salmonella typhi, Escherichia coli, and Klebsiella pneumoniae by agar well diffusion method. Of the 12 isolates tested, seven isolates showed antibacterial activity again at least one of the test organisms with the zone of inhibition in the range of 9-14 mm. The antibacterial compounds were extracted from the cell free supernatant using ethyl acetate and tested for antibacterial activity. In diffusion method, crude extract from strain RC12 showed 11-17 mm inhibition against all the test pathogens and the same was selected for optimization studies. Effect of culture conditions and medium components such as different carbon, nitrogen, mineral, pH, temperature on antibacterial metabolite production was studied by adopting one-factor-at-a- time method. The metabolite production was influenced by lactose, ammonium sulphate, K2HPO4 while the optimal cultivation conditions were pH 7 and 37°C. The potent strain RC12 was identified as Pseudomonas sp based on their phenotypic and biochemical characteristics. The finding of the present study concludes that coral reef bacteria found to be the best source for bioactive metabolites with broad spectrum activity.

Antibacterial and antifungal activities of Actinobacteria isolated from Rathnagiri hills.

This study was performed to isolate actinomycete colonies having antibacterial and antifungal activity from soil samples. A total of 27 actinomycete colonies were isolated in pure culture from five soil samples using Starch casein agar medium. Entire isolates were screened for their antimicrobial activity by agar plug method against five each of human pathogenic bacteria and fungi. Of this, 7 strains inhibits B. substilis, 3 strains inhibits Klebseilla sp, 6 strains inhibits B. cerus, 5 strains inhibits S. aureus and only 2 strains inhibits E. coli. Incase of fungi all the actinobateria has moderate activity with less fungal strains, only 1 strain (RA 5) inhibits entire fungus except Penicillium sp. The metabolites from potent strain was produced by fermentation, separated by centrifugation, it was tested for their antimicrobial activity against the test bacterial and fungal strains by well diffusion and disc diffusion method. In this study, the metabolites from RA5 (identified as Streptomyces sp.) have showed good antibacterial and antifungal activity. Since many isolates showed inhibitory activity against indicator bacteria, it is suggestive that Rathnagiri hill’s soil could be an interesting source to explore for antibacterial secondary metabolites.

Studies on antimicrobial activity of actinomycetes against MDR wound bacterial isolates.

A total of five different actinomycete isolates were recovered from mine soil samples collected from Salem, Tamilnadu. These were then assessed for their antibacterial activity against five multidrug resistance bacterial wound isolates. All five isolates of actinomycete exhibited antagonistic activity. The zone of inhibition ranged between 11-25 mm. Among the 5 isolates of actinomycetes A5 isolate has highest antibacterial activity against S.aureus and E.coli. Out of five bacterial isolates Pseudomonas aeruginosa was highly suppressed by actinomycetes followed by E.coli. The maximum antibacterial activity was observed on 14th day incubation. The result of primary screening reveals that most of the active actinomycetes isolates were active against gram positive bacteria (S.aureus) than gram negative bacteria. The antibiotic profile of these isolates underlined their potential as a source of novel antibiotics.

Bioactive potential of actinobacteria against drug resistant pathogens.

The present investigation was focused to study the antagonistic potential of actinobacterial strain TK2 isolated from Thirukurungudi Hills (Western Ghats, Tamil Nadu), against selected drug resistant bacterial pathogens. Of the 9 clinical bacterial strains, S. aureus was found to be resistant against methicillin and vancomycin while the remaining 8 gram negative pathogens were confirmed as extended spectrum beta lactamase (ESBL) producers. Strain TK2 showed good antagonistic activity against all the bacterial pathogens, except the three isolates of P. aeruginosa. Strain TK2 produced the antimicrobial activity on 4th day of incubation when growing on ISP2 agar medium whereas the same strain showed activity only on 8th day of incubation when it was grown on ISP2 broth. Of the various solvents tested for extraction, bioactive compound was extracted only in ethyl acetate. The crude extract showed 14-18 mm inhibition zone in disc diffusion method. The crude extract produced two spots in thin layer chromatography (TLC) in chloroform: methanol (30:60) solvent system. In bioautography, the second spot (Rf value 0.685) showed activity. The active compound was purified by preparative TLC, which showed maximum activity (15-20 mm inhibition) against test pathogens. Based on the results of chemical screening, the active compound was identified as sugar containing substance. Strain TK2 showed good growth on various growth media and culture conditions. Based on the studied phenotypic characteristics strain TK2 was identified as the species of the genus Streptomyces. Of the various growth parameters tested, ISP2 medium, glucose, pH 7, 1% NaCl and temperature 300C was influenced the antagonistic activity of strain TK2. Strain TK2 will be a potential for the isolation of bioactive compound(s) which will be a candidate for the development of antibiotic against drug resistant bacterial pathogens.

Multidrug resistant gram negative pathogens antibiotic profile and its effective control using secondary metabolites from marine actinobacteria.

Aim: To screen the spread of resistance in ESBLs producer’s particularly non lactose fermenting gram negative Acinetobacter spp. and Pseudomonas spp. and study antimicrobial activity with crude extract from novel marine actinomycetes in India. Methods: Fifty clinical isolates in a period of one year were processed and the antibiotic susceptibility was determined by double disk approximation test, the ESBLs production was screened with phenotypic confirmatory methods using disks of amikacin, meropenem, netilimicin, ciprofloxacin, gentamicin, tigecycline and piperacillin along with cephalosporin disks. Antimicrobial activity of the crude extract was determined by agar plug method. Results: The isolates collected from different samples were found resistant to third and fourth generation cephalosporins. ESBL production was detected in 56 % to 66 % of the isolates, amikacin and netilmicin showed 50% to 60% resistance they were also found resistant to carbapenems, 86% resistance was observed in Acinetobacter spp. Two strains PM21 and PM27 selected from 24 actinobacterial isolates had zone of inhibition >21mm. Conclusion: A high level of antibiotic resistance was found in Acinetobacter spp. in our study and may reflect the scenario in India. Earlier detection and reporting of ESBL producers will help in treating individual cases and also in controlling the spread of these resistant genes to other sensitive nosocomial isolates. The medical need for new agents is most acute and the future of this work aims to identify one such novel compound from marine actinobacteria.

Exploitation of bacteria from forest ecosystem for antimicrobial compounds.

This study deals with bacterial prospecting from forest soil with special reference to antimicrobial substances. Total of 25 morphologically different bacterial colonies were isolated from soil samples collected from Anaimalai forest and Parambikulam tiger reserve forest, Western Ghats, India. About 12 (48%) out of 25 isolates showed antibacterial activity in which strain AF1 showed inhibitory activity against more number of test pathogens. Bioactive substance from strain AF1 was produced by adopting submerged fermentation and extracted using ethyl acetate and chloroform. In disc diffusion method, ethyl acetate extract showed good antibacterial activity (9-17 mm zone of inhibition). Active fraction present in the ethyl acetate extract was determined by thin layer chromatography based bioautography. Findings of this work supported that the forest ecosystems investigated in this study will be potential place for bacterial bioprospecting.

Biosynthesis of gold nanoparticles by actinomycete Streptomyces viridogens strain HM10.

Biosynthesis of gold nanoparticles by Streptomycetes from Himalayan Mountain was undertaken for the first time. Out of 10 actinomycete strains tested, four strains (D10, HM10, ANS2 and MSU) showed evidence for the intracellular biosynthesis of gold nanoparticles, among which the strain HM10 showed high potency. Presence of spherical and rod shaped gold nanoparticles in mycelium of the strain HM10 was determined by transmission electron microscopy (TEM) and X-ray diffraction analysis. The average particle size ranged from 18-20 nm. UV spectral analysis indicated that the reduction of chloroauric acid (HAuCl4) occurred within 24 h of reaction period. Further, the strain HM10 showed enhanced growth at 1 and 10 mM concentration of HAuCl4. The gold nanoparticles synthesized by the strain HM10 showed good antibacterial activity against S. aureus and E. coli in well-diffusion method. The potential actinomycete HM10 strain was phenotypically characterized and identified as Streptomyces viridogens (HM10). Thus, actinomycete strain HM10 reported in this study is a newly added source for the biosynthesis of gold nanoparticles.

Studies on extended beta lactamase producing, biofilm forming clinical bacterial pathogens and its invitro inhibition by Actinobacterial extracts.

At present scenario, the extended spectrum beta lactamase (ESBL) producing bacterial pathogen causes various life threatening infections especially by the members of the family Enterobacteriaceae in hospital settings. In order to study the prevalence of ESBLs in Kanchipuram hospital, the bacterial strains were isolated from patients having Urinary tract infections (UTI), diabetic foot ulcer, pregnancy women’s, surgical wound infections, deep wounds, and genitourinary tract problems. Totally 40 bacterial isolates were recovered from 30 samples and the isolates were identified as Escherichia coli (45%), Pseudomonas sp, (25%) and Klebsiella sp (30%). The ESBL production was confirmed with third generation cephalosporins (cetixime, cephoxitin, ceftazidime, cetepime, ceftriaxone, ceftazidime/clavulanic acid) using the Kirby- bauer disc diffusion method and also by double disc diffusion method. The highest ESBL production was found among E. coli (42%), followed by Pseudomonas sp. (25%) and Klebsiella sp (20%). All the ESBL producer were tested for biofilm formation by tube method in which E.coli (43%) was found to be the good biofilm producer followed by the Klebsiella sp (31%) and Pseudomonas sp (25%). An attempt was also made to study the in-vitro inhibition of biofilm forming ESBL pathogens by actinobacterial extracts by disc diffusion method. Of the five actinobacterial extracts tested, extracts produced from the strain MA7 inhibited (8-12 mm zone of inhibition) all the biofilm forming ESBL pathogens. Further purification and characterisation of active compound from actinobacterial strain MA7 is in progress.

Metallo beta lactamase producing Pseudomonas aeruginosa and Acinetobacter baumannii.

This study aims in identifying MBLs particularly Zn requiring Molecular Class B enzymes produced by Pseudomonas aeruginosa and Acinetobacter baumannii .The resistance by these organisms are in a rise against all antibiotics including carbapenems and no prescribed CLSI guidelines is available for detecting them. Clinical isolates antibiotic susceptibility was determined by number of phenotypic tests by addition of 50mM of 10 μl zinc as cofactor for metallo beta lactamase production along with 0.5M ETDA of 5μl (930 μg per disk) plain disks. Increase in zone size of the meropenem -EDTA disk compared to the meropenem disk without EDTA was recorded positive. For Zn requiring MBLs zone towards both disks of EDTA and Zn along with meropenem is detected by DDST.