Open Access Short communication

Prevalence of Staphylococcus aureus subsp. anaerobius in Sub-Clinical Abscess Cases of Sheep

Nasreen O. Musa, Abdulkhalig Babiker, Kamal Eltom, Khalid Rodwan, Sulieman M. El Sanousi

Microbiology Research Journal International, Page 131-136
DOI: 10.9734/BMRJ/2012/1409

Aim: To investigate the prevalence of Staphylococcus aureus subsp. anaerobius (Saan) in sub-clinical lymph node abscesses (SLNA) in sheep.
Place and Duration of Study: Department of Microbiology (Faculty of Veterinary Medicine) and Unit Microbiology and Molecular Biology (Institute for Studies and Promotion of Animal Exports), between May 2003 and Dec. 2005.
Methodology: Enlarged superficial lymph nodes (n=137) were taken from sheep carcasses at meat inspection and were subjected for bacteriological examination.
Results: Pure cultures of Saan were obtained from 44% of the pus samples, Corynebacterium spp. from 33% and S. aureus subsp. anaerobius from 7%. The rest of the pus samples yielded mixed cultures of Saan with either Macrococcus caseolyticus (10%) or Corynebacterium spp. (6%).
Conclusion: Although these results show S. aureus subsp. anaerobius as the prime cause of SLNA, they also show the importance of inclusion of Corynebacterium pseudotuberculosis in vaccine developments or vaccination protocols against abscess diseases of sheep, especially those intended for export.

Open Access Original Research Article

Role of Arbuscular Mycorrhizae (AM) Fungi and Multi Bioinoculants in Cotton Plant Growth

Tasleem Sultana, Pavan Kumar Pindi

Microbiology Research Journal International, Page 123-130
DOI: 10.9734/BMRJ/2012/1315

Aim: The present study reflects the effect of Arbuscular Mycorrhizae (AM) fungi and other bioinoculants on the growth of cotton seedlings.
Study Design: Screening of efficient biofertilizers from undisturbed forest soils to improve the crop yield of cotton in barren lands of Mahabubnagar District.
Place and Duration of Study: Department of Microbiology, Palamuru University, Mahabubnagar, Andhra Pradesh, India, between May 2011 and February 2012.
Methodology: Mahyco hybrid variety, the most widely cultivated variety in Mahabubnagar District, Andhra Pradesh, India was selected among the sixteen varieties of cotton seeds for this study. Soil samples were analyzed for physicochemical characteristics. Seven different isolates of AM fungi were maintained as pure cultures in laboratory, which were isolated from different Agro-forestry tree rhizosphere soils. Among these pure cultures, R1-R2 has shown maximum colonization with Mahyco variety and this isolate was identified as Glomus mosseae. Mahyco hybrid variety was also tested with three different bioinoculants (Rhizobium sp., Azospirillum sp., Bacillus sp.) along with the combination of AM pure culture of R1-R2. These three potential bioinoculants were identified based on 16S rRNA gene sequence. Preliminarily Mahyco hybrid variety was investigated with individual pure cultures of AM fungi and with other bioinoculants. R1-R2 were taken in single, dual, triple and multicombinations with other three bioinoculants.
Results: In single combination M+R1-R2 showed the best growth by M+Rhizobium, followed by M+Bacillus and in dual combination M+R1-R2+Rhizobium and in triple combination M+R1-R2+Rhizobium+Azospirillum and in multicombination i.e. M+R1-R2+Rhizobium+Azospirillum+Bacillus showed the best growth among all the combinations.
Conclusion: The multicombination mediates increased the cotton growth characteristics. The effect of multicombination was not significantly different in treatment affected by various varieties. Inoculation of multicombination along with AM pure culture resulted significant increase in shoot and root length of cotton plant. So multicombination was proved to be superior.

Open Access Original Research Article

Evaluation of Antigen Preparation Methods for Polyclonal Antibody Production against Streptomyces spp.

Kusavadee Sangdee, Petcharat Thummabenjapone, Aphidech Sangdee

Microbiology Research Journal International, Page 137-145
DOI: 10.9734/BMRJ/2012/1605

Aim: To determine the optimum antigen preparation method for producing specific polyclonal antibody specific for Streptomyces species.
Study Design: Experimental study.
Place and Duration of Study: Faculty of Science, Mahasarakham University, Mahasarakham Province, Thailand, between May 2011 and December 2011.
Methodology: Two Streptomyces isolates were used for antisera production. The sonication method was chosen for antigen preparation. Antigen suspensions were emulsified with incomplete Freund’s adjuvant and 1 ml was injected into rabbit thigh muscle for the first, second and third immunization. The fourth and fifth immunizations were injected intravenously. Antibody titer, detection limit and specificity were measured using indirect-ELISA. Streptomyces antigen mixed with soil was investigated.
Results: The 5 min sonication method gave a higher protein than other test methods, so this protocol was chosen for all subsequent work. The sonicated Streptomyces antigen was used for polyclonal antibody production. At week 7, the rabbit anti-Streptomyces sp. isolate STPR78 antibody and anti-Streptomyces sp. isolate STPR84 antibody gave maximum titers at 1:64000 and 1:32000, respectively. The detection limit of the anti-Streptomyces antibodies was 125 ng and 250 ng, respectively. Both anti-Streptomyces antibodies were also found to have good specificity. Minimal cross-reactivity was detected with antigens from other test bacteria. Regrettably however, neither antibody was capable of detecting Streptomyces spp. STPR78 or STPR84 in inoculated soil.
Conclusion: A specific and high titer of polyclonal antibody was produced using sonicated antigen.

Open Access Original Research Article

Production of Halophilic α-Amylase by Immobilized Cells of Moderately Halophilic Bacillus sp. Strain TSCVKK

Kanthi K. Kiran, Podili Koteswaraiah, T. S. Chandra

Microbiology Research Journal International, Page 146-157
DOI: 10.9734/BMRJ/2012/1484

Aims: To investigate the effect of cell immobilization on amylase production by the moderately halophilic bacterium, Bacillus sp. strain TSCVKK and to compare the properties of the amylase produced under immobilized conditions with the enzyme produced by the free cells.
Study Design: Cell immobilization.
Place and Duration of Study: Department of Chemistry, Biochemistry Lab, Indian Institute of Technology (IIT Madras), Chennai, Tamil Nadu, between Jan 2009 and March 2009.
Methodology: Bacillus sp. strain TSCVKK was immobilized in alginate, agar, polyacrylamide and gelatin. Production of amylase was determined using 3, 5-dinitrosalicylic acid (DNS). Effect of NaCl, pH, temperature on the activity of amylase was determined and compared with the amylase produced by the free cells.
Results: Maximum production of 832 mU/ml was achieved with an initial cell load of 1.2% (w/v; wet weight) of 24 h grown cells immobilized in 2% agar of 4 mm3 block size using GSL-2 medium containing 10% NaCl and 1.5% dextrin at pH 8.0 at 30ºC after 36 h of growth. Amylase production was lower when the cells were immobilized in alginate (211 mU/ml) or with the free cells of same biomass concentration as used for immobilization (333 mU/ml). Amylase was not produced when gelatin or polyacrylamide was used as the immobilization matrix. The immobilized cells in 2% agar could be used up to 5 cycles without much reduction in amylase production. Amylase produced through cell immobilization retained all the properties that were shown by amylase produced under submerged fermentation.
Conclusion: Agar was the suitable matrix to immobilize Bacillus sp. strain TSCVKK for amylase production. Amylase produced under immobilization conditions retained its temperature, salt and pH requirements. Immobilized cells were used for 5 cycles without much decrease in production.

Open Access Original Research Article

Purification, Characterization and Antitumor Activity of L-asparaginase from Penicillium brevicompactum NRC 829

Ali Mohamed Elshafei, Mohamed Mohamed Hassan, Mohamed Abd-Elmontasr Abouzeid, Dalia Ali Mahmoud, Dina Helmy Elghonemy

Microbiology Research Journal International, Page 158-174
DOI: 10.9734/BMRJ/2012/1735

Aim: The aims of this study were to attempt to extract, purify and characterize of L-asparaginase, an antitumor agent, from Penicillium brevicompactum NRC 829.
Study Design: Testing of antitumor activity of L-asparaginase against four different tumor human cell lines.
Place and Duration of Study: Department of Microbial Chemistry, Genetic Engineering and Biotechnology Division, National Research Centre (NRC), Cairo, Egypt, between June 2010 and November 2011.
Methodology: Penicillium brevicompactum NRC 829, a local isolated strain from Culture Collection of the National Research Centre of Egypt, was grown and maintained on modified Czapek Dox medium. The fresh fungal biomass was thoroughly ground with washed cold sand. The cell contents were extracted with cold 0.1M Tris-HCl pH 8.0, thereafter, the slurry obtained was centrifuged at 5500 rpm for 15 min and the supernatant was directly used as the source of enzyme. The purification of L-asparaginase from crude-enzyme extracts of P. brevicompactum was achieved by a sequential multi-steps process starting by heat treatment for 20 min at 50ºC, followed by gel filtration on Sephadex G-100 column, and the most active fractions of L-asparaginase were dialyzed out, lyophilized and then loaded on a Sephadex G-200 column.
Results: An intracellular glutaminase-free-L-asparaginase from Penicillium brevicompactum NRC 829 was purified to homogeneity with an apparent molecular mass (Mr) of 94 kDa. The purified enzyme was 151.12 fold with a final specific activity of 574.24 IU/mg protein and about 40% yield recovery. The purified L-asparaginase showed its maximal activity against L-asparagine when incubated at pH 8.0 at 37ºC for 30 min. The enzyme was more stable at alkaline pH than the acidic one and thermally stable up to 60 min at 50-60ºC. L-asparaginase was highly specific for its natural substrate, L-asparagine with a Km value of 1.05 mM. The activity of L-asparaginase is activated by mono cations and various effectors including K+, Na+, 2-mercaptoethanol (2-ME), and reduced glutathione (r-GSH), whereas it is moderately inhibited by various divalent ions including Hg2+, Cu2+, and Ag+. Results indicated the involvement of sulfhydryl group(s) in the enzyme active site(s). The purified enzyme inhibited the growth of human cell line hepatocellular carcinoma (Hep-G2), with IC50 value of 43.3μg/ml.
Conclusion: L-asparaginase purified from Penicillium brevicompactum NRC 829 is a potential candidate for medical applications.

Open Access Original Research Article

Contributions of Escherichia coli to Diarrhoea among HIV/AIDS Patients at a Hospital in Tropical West Africa

Esther D. Tyodugh, Ubleni U. Emanghe, Godwin T. Jombo, Ella B. Abraham

Microbiology Research Journal International, Page 175-186
DOI: 10.9734/BMRJ/2012/1098

Aims: To ascertain the contributions of Escherichia coli to diarrhea among patients with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) in a HIV endemic community.
Study Design: Hospital based cross-sectional study.
Place and Duration of Study: Mkar Christian hospital, Mkar, Gboko local government area of Benue state, Nigeria between January and June, 2009.
Methodology: Close ended questionnaires were administered and relevant information such as age, sex, marital status, educational background, occupation and regular intake of antiretroviral drugs were obtained from HIV/AIDS patients with diarrhea and non-HIV/AIDS patients which served as control. Stool samples were collected, stored, transported and processed using standard procedures of microscopy, culture and sensitivity.
Results: Bacteria were found to contribute 59.9% (209) of the diarrhoea among HIV/AIDS patients at Mkar of which E. coli accounted for 111 (43.4%) of the cases and was significantly higher among HIV/AIDS patients on irregular or absent anti-retroviral treatment (ART) than those on regular ART, 64.0% (87) versus 36.9% (24) respectively (P< 0.05). Other bacteria recovered were Salmonella typhi (30.6%) and Shigella dysentheriae/flexneri (26.0%).
Conclusion: E. coli should be accommodated in symptomatic management of diarrhoea among HIV/AIDS patients at Mkar while efforts should be made at provision of adequate antiretroviral drugs for the people while their strict intake and adherence enforced.

Open Access Original Research Article

Culture-Independent Analysis of Bacterial Community Composition during Bioremediation of Crude Oil-Polluted Soil

Chioma Blaise Chikere

Microbiology Research Journal International, Page 187-211
DOI: 10.9734/BMRJ/2012/1565

Aim: To use cultivation-independent techniques based on DGGE of PCR-amplified 16S rRNA gene and to evaluate bacterial community composition during bioremediation of crude oil-polluted soil.
Study Design: Molecular fingerprints of bacterial populations involved in the active phase of crude oil biodegradation were generated with DGGE after 16S rRNA gene amplification.
Place and Duration of Study: Department of Microbiology and Plant Pathology, University of Pretoria, South Africa, between March and August 2008.
Methodology: Crude oil-degrading bacteria in soil microcosms contaminated with 4% crude oil and then biostimulated with nitrogen-phosphorus-potassium inorganic fertilizer (NPK: designated PN soil), calcium ammonium nitrate (designated PU soil) and poultry droppings (designated PP soil) respectively were characterized with PCR of the gene for the small subunit (SSU) of the bacterial ribosome. Total culturable heterotrophic and hydrocarbon utilizing bacteria were enumerated using plate count and Bushnell Haas media. Total organic carbon content was measured throughout the study period to indirectly determine the effect of microbial activity on carbon content in biostimulated treatments as against controls. Gas chromatography was used to monitor hydrocarbon degradation with time while electron microscopy examined community richness during hydrocarbon degradation. Reamplified dominant DGGE bands (550bp) were cleaned up and sequenced using an ABI 3130XL genetic analyzer. Electropherograms were inspected with Chromas Lite 2.01. Sequence identification was performed using BLAST.
Results: Dendogram of the DGGE bands constructed using Jaccard coefficient algorithm revealed that communities from PU and PP-amended soils each formed distinct clades whereas PN treated soil showed less association when compared with PU and PP respectively. Fifty distinct bands were excised, reamplified by PCR and sequenced. Sequence analysis revealed the presence of phylogenetically distinct known hydrocarbon degrading bacteria like Corynebacterium spp., Dietzia spp., Janibacter sp. low G+C Gram positive bacterial clones Nocardioides spp., Rhodococcus erythropolis and uncultured bacterial clones. Forty successful sequence data obtained from the excised DGGE bands were submitted to GenBank database under accession numbers GU451069 to GU451108. Chromatograms of the residual hydrocarbons in test treatments and controls showed that biodegradation occurred markedly in treated soils in this order PN>PU>PP while no significant loss was observed in the oil-contaminated control on days zero and 42 respectively. Bacterial counts increased significantly in PN, PU and PP treatments and not in controls PC and OC. Total organic carbon increased appreciably in PN, PU and PP respectively from day zero to day 28. Electron micrographs of microbial consortia in the nutrient-amended soils revealed presence of active populations induced by biostimulation as against the sparsely populated controls.
Conclusion: The results suggest that nutrient amendment stimulates and selects indigenous soil bacteria that are able to degrade petroleum hydrocarbons.