Open Access Short Research Article

Selection of Marine Actinomycetes with Bioactive Potential Isolated from Sediments of Bay of Bengal and Characterization of Promising Isolate, ABT-103

Venigalla Sridevi, Pyla Swetha Priyadarshini, Yendamuri Ashish Gautam

Microbiology Research Journal International, Page 1-9
DOI: 10.9734/BMRJ/2015/20132

Aim: Marine actinomycetes are a potential and untapped source for the production of novel bioactive compounds. The aim of the present work is to isolate actinomycetes strains from the marine sediments of Bay of Bengal for the production of bioactive compounds and characterization of the potential actinomycetes.

Place and Duration of Study: Department of Chemical Engineering and Biotechnology, ANITS, Visakhapatnam, between March, 2014 and April­­­­­­­­­­­­­­­­­­­­­­, 2015.­

Materials and Methods: Marine sediment samples were collected along the coast of Bay of Bengal, Visakhapatnam, India. Actinomycetes were isolated on Starch Casein Agar plates by pour plate and spread plate methods. Morphologically distinct pure isolates were tested for antimicrobial activity by Cross-streak method in preliminary screening and by Cup-plate method in secondary screening. The marine isolates were also screened for enzyme activities of amylase, lipase, protease and L-asparaginase. The most potential isolate was characterized up to genus level based on morphological, chemotaxonomic, biochemical and physiological characteristics.

Results: A total of 74 bacterial strains were isolated from marine sediment samples of Bay of Bengal. Among them, 13 morphologically distinct pure isolates were screened for antimicrobial activity and also for enzyme activities. Five isolates exhibited antimicrobial activity among which ABT-205 isolate showed broad spectrum antimicrobial activity against both bacteria and fungi, and ABT-103 exhibited maximum antifungal activity. Screening for enzyme activities revealed that nine isolates exhibited enzyme activities of which, ABT-101 and ABT-201 are highly potential for the production of Protease, whereas ABT-103 and ABT-206 were best producers of Amylase and Lipase enzymes, respectively. Among all the isolates screened, ABT-103 was found to be a promising isolate as it produced red pigment, exopolysaccharide, amylase and exhibited antifungal activity. Hence, the isolate ABT-103 was characterized and identified as Streptomycesspecies.

Conclusion: The selected marine actinomycetes isolates, ABT-101, ABT-103, ABT-201, ABT-205 and ABT-206 could be useful or the production of novel antibiotics, enzymes with different physical and physiological properties, red pigment, exopolysaccharide and other compounds for various Biotechnological applications.


Open Access Original Research Article

Emphasis on Techno-functional Properties of Bacillus Strains Involved in Ivorian Cocoa Fermentation towards their Use as Potential Starter

Wilfried Yao, Honoré G. Ouattara, Ginette Doué, Bernadette G. Goualié, Gisèle A. Koua, Sébastien L. Niamké

Microbiology Research Journal International, Page 1-10
DOI: 10.9734/BMRJ/2015/18937

Aims: To investigate in Bacillus strains some functional properties potentially interesting for cocoa fermentation processing

Place and Duration of Study: Laboratory of Biotechnology, UFR Biosciences, University Félix Houphouet-Boigny (Côte d’Ivoire), between May 2014 and March 2015.

Methodology: Spontaneous heaps fermentation were conducted in three cocoa producing regions. Bacilluswere isolated from cocoa fermentation using plate agar on nutrient medium and analyzed for pectinolytic enzymes production, citric acid breakdown, acidification and thermotolerance: these properties are known as essential for cocoa fermentation.

Results: A total of 600 Bacillus strains were isolated and 42.50% of them produced pectinolytic activity with different levels of enzymes production. The production of these enzymes is influenced by sugars content and ethanol beyond 2%, while the influence of acid was sharper with 0.15% as limit of tolerance. A large proportion of Bacillus population (67.16%) exhibited acidifying capacity, this property was accompanied by gas production in some strains. The total acidity yield ranged from 30 to 300 mg/L and strains presenting gas production tend to produce more acid. Additionally, more than 75% of acidifying strains presented the ability to breakdown citric acid. Moreover, all the strains exhibited a strong thermotolerance at 45ºC with a level of bacterial growth round 80% of that reached at 35ºC, the optimal growth temperature. The distribution of these properties in the Bacillus population was not uniform in the different regions.

Conclusion: This study indicates that Bacillus strains involved in Ivorian cocoa fermentation possess some properties such as ability to produce pectinolytic enzymes, capacity to breakdown citric acid and strong thermotolerance at 45ºC, essential for a well-fermented cocoa. These strains may play a more important role in cocoa fermentation, than it is believed at date. Taken together, these results show that Bacillusstudied should be potential candidate as starter for cocoa fermentation control.


Open Access Original Research Article

Comparative Assessment of Antibiotic Susceptibility Pattern of Gram Negative Pathogens Isolated from Intensive Care Unit Patients in Pune

S. Arora, N. Munshi

Microbiology Research Journal International, Page 1-9
DOI: 10.9734/BMRJ/2015/18199

Introduction and Aim: Extended spectrum β-lactamases (ESBLs) and Metallo-β-lactamases (MBLs) production is one of the main means of the resistance developed by Gram negative bacteria against β-lactam antibiotics. The present study was carried out to evaluate the incidences of ESBL and MBL producers in gram negative bacteria isolated from Ruby Hall Clinic, Pune, Maharashtra, India and to evaluate the efficacy of drugs against these bacteria.

Methodology: 254 different samples collected from various sources were screened for the presence of bacterial pathogens. The pathogens were identified using selective media technique. The ESBL and MBL producer's screening and the antimicrobial susceptibility testing (AST) of pathogens towards a new drug; Elores (ceftriaxone + sulbactam with adjuvant ethylenediaminetetraacetic acid, EDTA) in comparison with commonly used antibiotics like meropenem, imipenem, piperacillin-tazobactam and cefoperazone-sulbactam was carried out according to CLSI guidelines. 

Results: Among 254 samples collected, 200 samples showed the presence of bacterial infections with Klebsiella sp. (39%) as the most predominant pathogens followed by, E. coli (32%) and Pseudomonas sp. (16.5%), Acinetobacter sp. (12.5%). Of the identified pathogens, 61% (122/200) were found to be ESBL producers and 4.5% (9/200) were MBL producers. Nearly, 3.5% (7/200) pathogens were both ESBL and MBL producers. However another significant number (66 isolates) of pathogens were identified as non-ESBL/ non-MBL producers. Further, our data showed that, Elores was highly susceptible (87 to 100%) followed by imipenem-cilastatin (30 to 67%), meropenem (33 to 68%), cefoperazone-sulbactam (24 to 70%) and piperacillin-tazobactam (4 to 81%) against Gram negative bacteria.

Conclusion: The results of the present study concludes, that Elores is an useful option to treat the infections caused by carbapenemase producing multi-drug resistance Gram negative bacteria.


Open Access Original Research Article

Co-production of Pectinase and Biosurfactant by the Newly Isolated Strain Bacillus subtilis BKDS1

Bijesh Kavuthodi, Steni K. Thomas, Denoj Sebastian

Microbiology Research Journal International, Page 1-12
DOI: 10.9734/BMRJ/2015/19627

Aim: To isolate pectinase producing bacterial strains from various samples and to investigate the co-production of pectinase and biosurfactant by this isolated strain.

Study Design: Isolation, screening, selection and identification of pectinolytic bacteria. Production of pectinase and analysis of exo-pectinase types. Partial purification by ammonium sulphate precipitation and dialysis. The study also analyses the capability of the selected strain for biosurfactant production.

Place and Duration of Study: Department of Life Sciences, University of Calicut, Kerala - 673635, India, between July 2013 and November 2014.

Methodology: The pectinolytic bacteria was isolated and screened by iodine plate assay method. The enzyme production was confirmed by 3,5-dinitrosalicylic acid (DNS) method. The potent enzyme producing strain was biochemically characterised and identified by 16S rRNA gene sequence analysis. Thiobarbituric acid (TBA) and DNS assay were performed to test the types of exo-pectinase produced. Partial purification of the enzyme was done by ammonium sulphate precipitation. Biosurfactant production by the strain was tested by methods like foam formation, drop collapse test, oil displacement test, microplate assay, hemolytic assay, penetration assay, emulsification activity and bacterial adhesion to hydrocarbon (BATH) assay.

Results: Thirty six pectinolytic bacterial strains were screened from the collected samples. Four isolates were selected on the basis of zone size (20 mm to 26 mm) in well plate screening method. In which, the isolate showed maximum enzyme production in DNS assay (0.707 U/mL) is the same which exhibited larger pectin depolymerization area (26 mm) in well diffusion assay. This strain was identified as Bacillus subtilisBKDS1 using 16S rDNA based molecular technique. The optimum incubation time was found to be 72 h for the maximum enzyme production (1.288 U/mL). Assay of exo-pectinase revealed that, the organism was able to produce polygalacturonase (PG), pectin lyase (PNL) and pectate lyase (PEL). In ammonium sulphate precipitation, the enzyme activity was found in 40 -100% salt saturation fractions. On further analysis, it was observed that the organism also produced biosurfactant along with pectinase in the same culture medium.

Conclusion: The present study indicates the possibility of an integrated process for obtaining pectinase enzymes and biosurfactants in the same culture media. So this could greatly increase the economic viability of the isolated strain.


Open Access Original Research Article

Rare Codons Optimizer Host Strain of E. coli Improves Expression of Clostridium septicum Alpha Toxin Gene

Reza Pilehchian Langroudi

Microbiology Research Journal International, Page 1-7
DOI: 10.9734/BMRJ/2015/18922

Aims: Clostridium septicum is an anaerobic, gram positive bacterium that is able to form resistant spores. It produces numerous toxins that cause many diseases such as gas gangrene in humans and braxy in farm animals. In the present study, an attempt was done to express an active fragment of alpha toxin gene of C. septicum vaccine strain, in E. coli strains BL21 (DE3) and Rosetta (DE3) strains.

Methodology: In the present study, the active fragment of alpha toxin gene of C. septicum vaccine strain was amplified and ligated with pJET1.2blunt cloning vector and after cloning was extracted from the pJETαsep recombinant cloning vector. After purification and evaluation, it was ligated with pET22b(+) expression vector and the pET22αsep was transformed into E. coli strains BL21(DE3) and Rosetta(DE3) strains.

Results: Results showed that a recombinant protein was expressed as a soluble protein after IPTG induction in Rosetta (DE3) rare codons optimizer host strain, but not in BL21 (DE3). Further optimization on expression conditions of the recombinant alpha toxin protein was achieved by incubating the culture of recombinant E. coli/ Rosetta (DE3[pET22αsep] cells in 37ºC and induction with 0.5, 1.0 and 1.5 mM IPTG for 3-6 h. Protein expression was evaluated by SDS-PAGE and the recombinant alpha toxin protein was purified using Ni-NTA resin and for its reconfirmation was analysed by Western blot.

Conclusions: We concluded that, E. coli strain Rosetta (DE3) is a suitable expression host for production of C. septicum alpha toxin and the obtained recombinant plasmid could be used for further research on production of recombinant vaccine against braxy disease.


Open Access Original Research Article

Antibiotic Susceptibility of Neisseria gonorrhoeae in Bacolod City, Philippines

Alain C. Juayang, Joseph Peter T. Lim, Michael Angelo D. Acosido, Dominador G. Maestral Jr, Christine T. Gallega

Microbiology Research Journal International, Page 1-6
DOI: 10.9734/BMRJ/2015/19641

Resistance of Neisseria gonorrhoeae against clinically recommended antibiotics continues to rise globally. This retrospective study aimed to describe the antimicrobial resistance and resistance trend of N. gonorrhoeae in Bacolod City from February 2010 to January 2015. A total of 99 isolates (97 males and 2 females) from ages 15 to 65 were included in the study. The highest incidence was observed between 25 - 29 age bracket. Resistance rate against the tested antibiotics were as follows: penicillin (100%), ciprofloxacin (75.3%), tetracycline (69%), spectinomycin (8%), cefixime (0%) and ceftriaxone (0%). Production of beta-lactamase was also found in all isolates; while presumed plasmid mediated tetracycline resistance was found in 34 isolates. Resistance to ciprofloxacin and tetracycline were also seen to be increasing during the five year study, but resistance to third generation cephalosporins, namely cefexime and ceftriaxone remains unchanged.