Open Access Original Research Article

Bioprospecting of Cellulase Producing Extremophilic Bacterial Isolates from India

Siddarthan Venkatachalam, Manivannan Sivaprakash, Vasudevan Gowdaman, Solai Ramatchandirane Prabagaran

Microbiology Research Journal International, Page 142-154
DOI: 10.9734/BMRJ/2014/5395

Aim: To isolate and identify the potential extremophilic cellulase producing strain viz., psychrophiles, halophiles, thermophiles and to compare the Cellulase activity from samples collected from different geographical regions of India.
Place and Duration of Study: Bharathiar University, Department of Biotechnology, Molecular Microbiology Lab, Coimbatore, Tamilnadu, India, between January to April 2011.
Methodology: Cellulase-producing extremophilic bacteria viz., psychrophiles, halophiles, and thermophiles have been isolated from soil samples. According to morphology and pigmentation, 138 distinct bacteria were isolated and screened for cellulase activity by Gram’s iodine–carboxymethylcellulose plate (CMC) assay. On the basis of the cellulase activity, six potent cellulase-producing isolates from each cluster viz., P14, P36, H6, H13, T2 and T3 were selected for 16S rRNA gene based identification. The strains were optimized for maximum cellulase activity at various temperature and pH range.
Results: The phylogenetic relationship revealed that P14 and P36 psychrophilic isolates possessed maximum identity with Bacillus simplex (100%) and Arthrobactercitreus (99%), with a cellulase activity of 14.10± 1.73 and 18.27± 0.71 UmL-1 respectively. Likewise, among halophiles, H6 and H13 were identified as Bacillus subtilis and Bacillus endophyticus (99%), with a cellulase activity of 14.87 ± 0.55 and 16.83 ± 0.44 U mL-1, correspondingly. In thermophiles, T2 and T3 showed close proximity with Bacillus amyloliquefaciens and Bacillus megaterium (99%), with a cellulase activity of 21.53 ± 1.30 and 19.93 ± 0.38 U mL-1 respectively.
Conclusion: In the present study, the thermophilic isolates showed promising Cellulase activity compared to psychrophiles and halophiles.

Open Access Original Research Article

Isolation of Polysaccharides and Terpenoids from Some Basidiomycota and Their Antibacterial Activities

Swapan Kumar Ghosh

Microbiology Research Journal International, Page 155-166
DOI: 10.9734/BMRJ/2014/4615

Aims: Antibacterial chemicals were isolated from fruit bodies of three basidiomycota [Coltricia perennis (L) Murrill, Onnia tomentosa (Fr.) P. Karst., and Polyporus mori (Pollini) Fr. ] fungi and their antibacterial potential were screened against five bacteria.
Study Design: All experiments were performed thrice in completely randomized design (CRD) each, with five replications per treatment (antibacterial activity). The data was subjected to ANOVA. Means of three observations were compared with Duncan’s Multiple Range Test (DMRT).
Place and Duration of Study: Molecular Mycopathology Laboratory, Department of Botany, Ramakrishna Mission Vivekananda Centenary College, Rahara, Kolkata, between January 2012 and February 2013.
Methodology: During the rainy season in the year of 2012, a survey for mushroom collection in the forest beds, infected logs in the plain of west Bengal was conducted .The fruit bodies of some basidiomycota were collected in sterile biodegradable polythene begs and brought to laboratory. The morphology, anatomy of fruit bodies and measurement of reproductive organs were recorded. The spore prints of all collected basidiocarps were taken.The collected basidiomycota were identified. The polysaccharides from the basidiocarps of the test fungi were isolated employing the methods of Mizuno et al. [17] and Wang et al.[12].Terpeniods were isolated according to the method followed by Anke and Werte [24] and Chairul et al. [25].
Their antibacteral activities were assayed against five bacteria [three Gram positive bacteria (Staphylococcus aureus, Micrococcus roseus and Bacillus brevis ) and two Gram negative bacteria (Ralstonia solanacearum and Escherichia coli )] following the agar plates cup diffusion techniques.
Results: Terpenoid isolated from Coltricia perennis was most active in inhibiting the growth of all five bacteria. This terpenoid inhibited maximum (25 ±2.4mm) growth against Staphylococcus aureus and minimum against Micrococcus roseus (17±1.1mm). The polysaccharides isolated from these three mushrooms were less active against the test five bacteria. The terpenoids isolated from Onnia tomentosa and Polyporus mori also inhibited the growth of the test bacteria.
Conclusion: These three basidiomycetous mushrooms have antibacterial activity. After further research, their activity can be employed in medical science.

Open Access Original Research Article

Salmonella Yeerongpilly in a Chinese Owl (Columba livia domestica) in Jamaica

Suzette Curtello, Angel Alberto Justiz Vaillant, Helen Asemota, Monica P. Smikle, Patrick Eberechi Akpaka

Microbiology Research Journal International, Page 167-172
DOI: 10.9734/BMRJ/2014/5942

Salmonella infection in bird species in Jamaica was studied. This revealed that very low prevalence of salmonellosis was found (0.32%). Salmonella Yeerongpilly (newly reported in the country) was isolated from a bird collected at a bird aviary. This study showed that there was the presence of this Salmonella serovar in a Chinese owl (Columba livia domestica) in Jamaica. There were not published reports from Caribbean Islands of the presence of this serovar. Salmonella Yeerongpilly belongs to serogroup E1 and by molecular serotyping random amplification of polymorphic DNA (RAPD) fingerprinting belongs to A20, B17 and C21. This strain was isolated in Queensland Australia in the 1960s before the successful Salmonella eradication campaign. This study suggests that a larger investigation in pet birds as Salmonella carriers should be carried out in Jamaica. Mandatory screening or quarantine of birds entering the country should be institutionalized.

Open Access Original Research Article

Purification and Partial Characterization of a Non-specific Acid Phosphatase Degrading NAD from Aspergillus oryzae NRRL447

Thanaa H. Ali, Bakry M. Haroun, Amir E. Tantawy

Microbiology Research Journal International, Page 173-184
DOI: 10.9734/BMRJ/2014/5953

A non specific acid phosphatase from Aspergillus oryzae NRRL447 catalyzes the phosphate hydrolysis from nicotinamide adenine dinucleotide forming nicotinamide riboside, adenosine and Pi as the final products of the reaction. The enzyme was purified to homogeneity by a sequential treatment of acetone fractionation, DEAE-cellulose chromatography and gel filtration chromatography. The enzyme was purified 400-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of MW 52 kDa. The enzyme displayed maximum activity at pH 5.0 and 40 °C with NAD as substrate. The enzyme activity appeared to be stable over pH 2.0–5.0 and up to 40 °C. The enzyme activity was enhanced slightly by Mg2+, Ca2+ whereas inhibited strongly by F, Mo04, Cu2+ and Fe2+. The enzyme hydrolyzes several phosphate esters, suggesting a probable non-specific nature. The substrate concentration-activity relationship is the hyperbolic type and the apparent Km for NAD+ was 6.25 x 10-4 M.

Open Access Original Research Article

Antibiotic Resistance Patterns and Plasmid Profiles of Methicillin Resistant Staphylococcus aureus Isolates from Human Samples

Chibuike Ibe, Reginald Azu Onyeagba, Solomon Charles Ugochukwu

Microbiology Research Journal International, Page 185-194
DOI: 10.9734/BMRJ/2014/5533

Antibiograms and plasmid profiles are commonly used to characterizemethicillin-resistant Staphylococcus aureus (MRSA) inepidemiologic studies. However, antibiograms are frequently inadequate to accomplish the differentiation. Plasmid profile being more informative has been reported to be useful in tracing the epidemiology of antibiotic resistance. Antibiotic resistance patterns and plasmid profiles of methicillin-resistant Staphylococcus aureus (MRSA) isolates from human specimens were investigated to determine the discriminatory power of plasmid profile analysis in conjunction with antibiotic susceptibility pattern. Specimens were analyzed using disc diffusion assay and restriction enzymes analysis of plasmid DNA procedure. The 51 methicillin-resistant Staphylococcus aureus (MRSA) isolates were grouped into 18 groups using their resistogram. Twenty four (47.1%) strains out the 51 methicillin-resistant Staphylococcus aureus (MRSA) isolates harbored plasmids. Single plasmid isolates were 14(27.5%), double plasmid isolates were 6(11.8%) while tripple plasmid isolates were 4(7.8%). The 24 isolates containing plasmids were categorized into 14 groups based on their resistogram. Plasmid profile showed greater similarity between isolates (10 profiles) than antibiotic resistance pattern which showed a higher disparity (14 patterns). However, resistance to various antimicrobial agents was not consistent with the presence of plasmids. No particular molecular size plasmid could be associated with any particular antimicrobial resistance patterns. Resistance was observed in isolates with various molecular size plasmids as well as in those that had no plasmids. Nonetheless, 2 pairs of isolates with the same plasmid profile also had similar (almost the same) resistance pattern. Plasmid profile analysis in conjunction with the antibiotic resistance typing is valuable in the epidemiological investigation of methicillin-resistant Staphylococcus aureus (MRSA).

Open Access Original Research Article

Isolation and Identification of Glucose Oxidase Hyper Producing Strain of Aspergillus niger

Ikram Ul Haq, Ali Nawaz, Hamid Mukhtar, Waseem Ahmed

Microbiology Research Journal International, Page 195-205
DOI: 10.9734/BMRJ/2014/3082

Aims: Glucose oxidase is an enzyme with large scale applications in various industries. It is also used in several diagnostic kits which makes it medically important as well. Our aim was to isolate indigenous glucose oxidase hyper producing strain of Aspergillus niger from different soil samples of Punjab, Pakistan.
Study Design: An experimental study.
Place and Duration of Study: Institute of Industrial Biotechnology, GC University, Lahore from March 2011 to July 2012.
Methodology: Two hundred and seventy nine fungal strains were isolated from soil of different localities of Punjab. Isolates were screened for glucose oxidase production using submerged fermentation. Glucose oxidase hyper producer isolate was identified using morphological and molecular techniques i.e. 18S rDNA. DNA was isolated and amplified using PCR. Gene sequencing was done and homology analysis was studied. Rate of glucose oxidase production was also analysed.
Results: Glucose oxidase hyper producing isolate was identified as A. niger A247 strain. This strain gave best reproducible results (145.22 ±0.034 U/g of cell mass) after 72 hrs of fermentation at 30ºC and at a medium pH of 7.2.
Conclusion: Our results indicate the natural ability of A. niger to produce Glucose oxidase in large quantity instead of using genetic manipulation techniques.

Open Access Original Research Article

Ethnopharmacological Properties of Ceasalpinia benthamiana - A Mini Review

A. Osho

Microbiology Research Journal International, Page 206-213
DOI: 10.9734/BMRJ/2014/5560

Caesalpinia benthamiana (Baill.) Herend. and Zarucchi (synonym. Mezoneuron benthamianum Baill.) belongs to the family Fabaceae, it is a climbing or a straggling shrub and is well known in some West African countries for its medicinal properties where it is used to cure general malaise, wound, urethral discharge, ulcer, pile, skin infection and believed to have aphrodisiac property. Phytochemical studies have revealed the leaf to contain essential oils, Gallic acid derivatives, tannins, saponins, flavonoids, phenols, anthraquinones and reducing sugars while the aqueous fractions of the root contain Gallic acid, resveratrol and tannins. Pharmacological assays have established the plant to be anti-inflammatory, anti-diarrheal, anti-bacterial, anti-candida, and to have vasorelaxation and aphrodisiac properties. This review presents information on the morphology, ecology, ethnopharmacology, phytochemistry, biological activities and toxicological properties of C. benthamiana and aims at providing an up-to-date detail that should constitute baseline information for future research on the plant.

Open Access Original Research Article

A Prevalence Study of Typhoid Fever in Taiz and Al-Hodiedahcities in Yemen

Gamal Al-ameri, Nada Saif

Microbiology Research Journal International, Page 214-223
DOI: 10.9734/BMRJ/2014/6768

Aims: Typhoid fever is an acute illness associated with fever that is most often caused by the Salmonella typhi bacteria. This study was carried out to determine the prevalenece of typhoid fever and distribution among different groups in Al-hodiedah and Taiz hospitals, and to determine the relation between the two governorates.
Study Design: Seroprevalence survey.
Place and Duration of Study: This study was carried out in Taiz hospitals and Al-Hodiedah hospitals in Yemen for about 1500 cases during September to December 2012.
Methodology: A total of 1500 cases were randomly collected and examined by Widal test and blood samples for WBC to detect the typhoid fever. Also, the questionnaire data was used for determine the correlation between typhoid fever and other factors such as age, sex, and clinical symptoms, then the data analyzed by spss program.
Results: This study found that 151 cases of typhoid fever are positive for widal test from total 1500 specimens was collected from Al-hodiedah hospitals and Taiz hospitals. Also found from 151 positive cases 57 cases for male and 94 cases for female.
There were 55 cases the main complain was fever follow by diarrhea 42 cases then abdominal pain 31 cases.
Conclusion: The results of the study indicate that there is no significant different in the prevalence of typhoid fever between cases collected from Taizhospitals and Al-Hodeida hospitals. Also, no significant different between sex or age and the positive cases. The positive cases were come with different manifestations such as fever, abdominal pain and diarrhea.

Open Access Original Research Article

Sourcing Starter Cultures for Parkia biglobosa Fermentation Part II: Potential of Bacillus subtilis Strains

E. Y. Aderibigbe, W. Visessanguan, B. Somphop, K. Yutthana, D. Jureeporn

Microbiology Research Journal International, Page 224-234
DOI: 10.9734/BMRJ/2014/6471

Aim: To select good strains of Bacillus subtilis for use as starter culture in the fermentation of Parkia biglobosa.
Study Design: Fifteen (15) strains of Bacillus subtilis group obtained from commercial samples were used in starter-culture fermentation of Parkia biglobosa seeds to produce ‘iru’.
Place and Duration of Study: Food Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), Pahumthani, Thailand, between March to May 2010.
Methodology: The quality of the starter culture-fermented products were compared on the bases of sensory evaluation, degree of hydrolysis (DH), level of ammonia nitrogen (NH3-N), pH and enzymatic activities. The 15 strains were also screened for haemolytic activity.
Results: On the basis of the sensory scores of 5 parameters (color, odor, consistency, texture and over-all liking), particularly the over-all liking, 5 strains were rated the best (in descending order): BC4333 > 8B > 2B > 7A > 5A, amongst the 15 tested. There were good correlations between pH and DH (r= 0.926), DH and NH3-N (r=0.962) and between pH and NH3-N (r=0.945). The strain BC4333 produced the very soft variant of ‘iru’ (‘iru-pete’), without the addition of ‘kuuru’ (local potash). The quantity of extracellular enzymes (protease, amylase, pectinase, phytase and lipase) produced during fermentation varied significantly. None of the 5 strains was haemolytic on sheep blood agar.
Conclusion: The 5 strains of Bacillus subtilis (BC4333, 8B, 2B, 7A, 5A) that showed potentials of being used as starter cultures for industrial production of ‘iru’, were non-hemolytic on blood agar.

Open Access Original Research Article

In vitro Bioluminescence Used as a Method for Real-Time Inhibition Zone Testing for Antibiotic-Releasing Composites

Noora M. Mannisto, Niina Ahola, Matti T. Karp, Minna Veiranto, Minna Kellomaki

Microbiology Research Journal International, Page 235-254
DOI: 10.9734/BMRJ/2014/6661

Aims: This study describes the potential of real-time bioluminescence imaging in evaluating the antibiotic efficiency of two cylinder-shaped bioabsorbable antibiotic-releasing composites by in vitro inhibition zone tests. The bacterial infections of bone tissue can cause extensive hard and soft tissue damage and decrease the efficiency of oral antibiotic therapy due to the poor blood circulation in the infected area. To overcome this problem, new, locally antibiotic-releasing biodegradable composites have been developed.
Study Design & Methodology: The two composites evaluated in this study were composed of poly(L-lactide-co-ε-caprolactone) matrix, β-tricalcium phosphate ceramic and either ciprofloxacin or rifampicin antibiotic. The composites were tested with genetically modified model pathogens of osteomyelitis (Pseudomonas aeruginosa and Staphylococcus epidermidis) in vitro in inhibition zone tests using a method of real-time bioluminescence.
Results: The first signs of the effect of the released ciprofloxacin or rifampicin became visible after four hours of incubation and were seen as changed bioluminescence around the composite pellet on a culture dish. Both of the composite types showed excellent effects against the sensor bacteria within the diffusion area. Bioluminescence measurements suggested that no survivor bacteria capable of evolving resistant strains were left inside the inhibition zones. The S. epidermidis bacterial strain was an inhibition sensor and P. aeruginosa was a stress sensor.
Conclusion: These results highlight the potential of the composite materials against the pathogens of osteomyelitis. The approach allows continuous visual inspection of the efficacy of the antibiotics against the bacteria.