Open Access Original Research Article

Proteases Production by a Bacterial Isolate Bacillus amyloliquefaciens 35s Obtained from Soil of the Nile Delta of Egypt

Fatma Refaat Nassar, Ahmed Abdelwahab Abdelhafez, Tarek Said El-Tayeb, Samah Hashem Abu-Hussein

Microbiology Research Journal International, Page 315-330
DOI: 10.9734/BMRJ/2015/15435

Aims: The present work was designed to investigate the production of proteases from an Egyptian bacterial isolate.
Study Design: Samples were collected from soil, water and food, and screened for the isolation of proteases producing bacteria. The highest proteases producing isolate was selected for proteases production.
Place and Duration of Study: Department of Agricultural Microbiology, Faculty of Agriculture, Ain Shams University, between March 2014 and September 2014.
Methodology: Twenty samples were obtained. Primary screening of proteolytic bacteria was done by inoculating plates of skim milk agar. Secondary screening of proteolytic bacteria was done by quantitative determination of proteases concentration. One isolate from soil was chosen due its high proteolytic activity. Identification of the selected isolated was done 16S rRNA gene sequencing. Optimization of proteases production was conducted using a two-step approach. First, a quick identification of the important factors by simple screening experiments (this manuscript) including inoculum size, incubation period, carbon and nitrogen sources, temperature and pH. In a subsequent manuscript, application of complex response surface methodology for further optimization will be studied. Data generated from the above experiments were analyzed using one-way ANOVA with post hoc multiple comparison analysis performed using Tukey’s HSD.
Results: In the first optimization step, the production of extracellular proteases was favored in the presence of starch, and peptone. A 2.1 fold increase in proteases production was obtained using the design space in the optimized medium as compared with the un-optimized basal medium. Enzyme production increased significantly with optimized medium (845 U/ml) compared to un-optimized medium (405 U/ml). Optimum production conditions were composed of incubation period of 24 h at 37ºC, pH 8 and agitation speeds of 120 rpm. Optimum medium for proteases production was composed of (g/l): peptone (10), starch (10), KH2PO4, MgSO4.7H2O, and CaCl2. Interaction between the variables and optimizing these variables will be studied in a later manuscript using a Plackett-Burman design and further with the Face Centered Central Composite Design (FCCCD) of Response Surface Methodology (RSM).
Conclusion: This work succeeded to obtain a novel bacterial isolate capable of producing proteases and the production process was optimized by screening the physical and nutritional parameters influencing proteases production by employing one-variable-at-time approach.

Open Access Original Research Article

Effect of Black Cumin Seed Oil (Nigella sativa) on Enhancement of Immunity in the Climbing Perch, Anabas testudineus

Aisha Khatun, M. M. M. Hossain, M. Z. Rahman, M. E. Alam, Farzana Yasmin, M.S. Islam

Microbiology Research Journal International, Page 331-339
DOI: 10.9734/BMRJ/2015/15330

Aims: The study was conducted to examine the efficacy of dietery black cumin seed oil (Nigella sativa) on the immune response of climbing perch, Anabas testudineus against A. hydrophila.
Place and Duration: This experiment was performed in the Laboratory of Fisheries and Marine Bioscience (FMB), Jessore University of Science and Technology (JUST), on July to December 2013.
Methodology: Fish husbandry and experimental design, Culture and Aeromonas hydrophila Isolation, Diet Preparations, Serum preparation (immune response assay), Growth performance, bactericidal activity, phagocytic activity and challenge test have been performed in this study.
Results: Climbing perch (Anabas testudineus) of average weight 25±5 g were fed for 1, 2 and 4 weeks with diet supplemented 20 ml (20%), 30 ml (30%) and 40 ml (40%) 100 g-1 of N. sativa oil and with normal diet as control (0%). Immunological parameters including bactericidal activity and phagocytic activity were investigated. Treatment groups recorded enhancement in those parameters compared to the control. Treatment groups fed the dose 30% N. sativa oil showed a significant enhancement in bactericidal activity and phagocytic activity. The highest weight gain (WG) 41.7±1.5 was significantly increased with the 30% dose of N. sativa oil but specific growth rate (SGR) and feed conversion ratio (FCR) did not change significantly when compared to the control. Feeding with 30% dose diet to A. testudineus showed lowest cumulative mortality 20% compared to other dose diets and played most effective performance during challenge test.
Conclusion: This result suggests that 30% dose of N. sativa oil enriched diet significantly enhanced the immune response and disease resistance of A. testudineus against A. hydrophila.

Open Access Original Research Article

Molecular Epidemiology of Shiga Toxin-Producing Escherichia coli Isolated from School Children in Ondo State, Nigeria

A. K. Onifade, M. A. Oladoja

Microbiology Research Journal International, Page 340-347
DOI: 10.9734/mrji/2015/v6i65676

Molecular techniques were used for studying the epidemiology of Escherichia coli from five major towns in Ondo state, namely; Akure and Ondo, Owo, Okiutipupa and Ikare-Akoko. In total, 807 urine and faecal samples of apparently healthy primary school pupils were gathered between December 2012 and March 2013. Withal, 206 E. coli strains were isolated and characterized by biochemical tests. Polymerase chain reaction (PCR) assay was performed to determine the presence of the genes-encoding virulence factors. A total of 22 (12 male, 10 female) isolates out of 28 subjected to PCR amplification were found to possess only stx1 virulence gene associated with Enterohaemorrhagic E. coli strains. PCR assay was demonstrated to be a useful technique for the epidemiological work of E. coli where this organism is a major cause of infection.

Open Access Original Research Article

Isolation and Characterization of a Marine Bacterium from Sundarbans, Bangladesh

Ashish Kumar Sarker, Md. Anwarul Haque, Mohammad Sayful Islam, Md. Ajijur Rahman, Md. Anwar Ul Islam

Microbiology Research Journal International, Page 348-357
DOI: 10.9734/BMRJ/2015/15907

Aims: Marine environment is a vast but represents a largely untapped source for isolation of new microorganism having ability to produce novel bioactive compound(s). The present study was designed with an aim to discover and identify marine bacterial strain producing antibacterial metabolites from marine soil samples of Sundarbans, Bangladesh.
Study Design: We used serial dilution technique for isolation of marine bacteria, streak plate technique for screeninig of priliminery antibacterial activity, cover slip culture method for morphological characterization and the 16S rDNA sequencing approach for phylogenetic characterization.
Place and Duration of Study: Pharmaceutical Microbiology Research Laboratory, Department of Pharmacy, University of Rajshahi, Rajshahi-6205, Bangladesh between August 2010 and July 2011.
Methodology: A total of 39 bacterial colonies were isolated from the marine soils collected from different locations of Sundarbans, a mangrove forest of Bangladesh. Among them ANAM-39 was selected on the basis of morphological, cultural, physiological, biochemical and taxonomic characterization, and it exhibited the highest antibacterial activity against a series of test organisms.
Results: The 16S rDNA sequence of the strain ANAM-39 showed close similarity with Smithella propionica(95.99%), Syntrophus aciditrophicus (93.63%) and Syntrophus gentianae (93.17%). However, the cultural, morphological, physiological and biochemical characteristics of the strain and S. propionica were completely different. So, the strain ANAM-39 may not be assigned within the genus Smithella.
Conclusion: To confirm the taxonomic position of the strain further studies are needed in respect to the DNA relatedness studies, DNA-DNA hybridization, small subunit (SSU) sequences, cell wall composition and other characterization.

Open Access Original Research Article

Anaerobic Microbial Activities of a Nigerian Onshore Oil Production Facility that Uses Underground Water with Zero Sulfate Concentration for Injection

Okoro Chuma Conlette

Microbiology Research Journal International, Page 358-366
DOI: 10.9734/BMRJ/2015/13564

Aim: To evaluate the anaerobic microbial activities and souring potential of a Nigerian onshore oil production facility that uses zero sulfate underground water for injection.
Methodology: Key functional group activities such as the ability to reduce sulfate and generate sulfide by sulfate reducing bacteria (SRB), the ability to reduce nitrate to nitrite by the heterotrophic nitrate reducing bacteria (hNRB) and the ability to reduce nitrate and oxidize sulfide by sulfide oxidizing, nitrate reducing bacteria (so-NRB) were determined in samples using CSB-K medium.
Results: Lactate utilizing SRBs and hNRBs were found to be common in most oil field samples while the activities of so-NRBs were limited to very few samples. It was also observed that the underground water with zero sulfate concentration and negligible microbial activity poses no souring risk to the oil field under investigation. The produced water and oil samples from the facility though with considerable populations and activities of SRBs also recorded negligible concentration of sulfate and some organic nutrients and therefore are not likely to pose some souring risks to the oil facility under investigation.
Conclusion: From our investigation, it is evident that the zero sulfate underground water with negligible SRB populations and activities poses no souring risks to the facility under investigation but corrosion risks cannot be completely ruled out since some methanogens that are indigenous to the oil field can initiate corrosion using alternative pathways in the absence of sulfate.

Open Access Original Research Article

Prevalence of Newcastle Disease in Gombe, Northeastern Nigeria: A Ten-Year Retrospective Study (2004 – 2013)

J. R. Lawal, S. M. Jajere, M. Mustapha, A. M. Bello, Y. Wakil, Y. A. Geidam, U. I. Ibrahim, I. A. Gulani

Microbiology Research Journal International, Page 367-375
DOI: 10.9734/BMRJ/2015/15955

Aim: A ten – year retrospective study was conducted with the aim to determine the prevalence of Newcastle Disease (ND) in relation to other poultry diseases diagnosed in the State area veterinary clinics.
Study Design: Retrospective study.
Place and Duration of the Study: This study was conducted in Gombe State, Northeastern Nigeria. Case files from Tashan Dukku and Pantami area veterinary clinics in Gombe metropolis were reviewed for the study. File of cases of poultry diseases reported between January 2004 and December 2013 were studied.
Methodology: Information on ND in poultry only was extracted from the clinical records and the non – ND cases were considered together as a group. A case of ND was defined as a farm that reported an outbreak of diseases diagnosed as ND based on history, clinical signs and post – mortem findings in the study area.
Results: Of 9970 cases of poultry diseases studied, 5531 (55.50%) were cases of ND (P >0.05). The Year Specific Rate (YSR) ranged from 51.5% (OR = 1.0) in 2005 to 63.3% (OR = 1.63; 95%CI: 0.81 – 2.45) in 2006 (P>0.05). The Monthly Specific Rates (MSR) of ND was 66.5% (OR = 5.94; 95%CI: 4.57 – 7.31) in October, 64.4% (5.42; 4.29 – 6.55) in December, 64.2% (5.36; 4.17 – 6.55) in January, 61.3% (4.74; 3.61 – 5.87) in November, 27.0% (1.10; 0.15 – 2.05) in June and 25.1% in July. ND was 3.4 times more likely to occur during the Pre-dry season (October – December) and 3.0 times more likely to occur during the Dry season (January - March) as compared with the other seasons of the year.
Conclusion: The study revealed that ND is still endemic in Gombe State – Nigeria, with peak outbreaks during dry and cold seasons. Purchasing day old chicks from farms known to give booster vaccinations to their parent stock hens, vaccinating chicks with vaccines that are immunogenic, use of thermo-stable vaccines for routine vaccinations of chicks and maintenance of strict bio-security measures are recommended to reduce the prevalence of the disease in the study area.