Open Access Original Research Article

Production and Partial Purification of Cellulase from a New Isolate, Penicillium verruculosum BS3

Sreedharan Sajith, Sasidharan Sreedevi, Prakasan Priji, Kizhakkepowathial Nair Unni, Sailas Benjamin

Microbiology Research Journal International, Page 1-12
DOI: 10.9734/BMRJ/2015/17865

Aims: This study describes the production of cellulases by a newly isolated fungus, Penicillium verruculosum BS3 on lignocellulosic biomass with special emphasis on endoglucanase.
Study Design: Isolation, screening and characterization of cellulolytic fungus. Production of cellulase by submerged and solid state fermentation. Partial purification of cellulase by ammonium sulphate precipitation, dialysis and spin column. Characterization by Native PAGE and SDS-PAGE.
Place and Duration of Study: Enzyme Technology Laboratory, School of Biological Sciences, University of Calicut, Kerala - 673635, India, between October 2013 and November 2014.
Methodology: The lignocellulolytic fungus was characterized and identified by lacto-phenol cotton blue staining and 28S rRNA gene sequencing. Preliminary confirmation of cellulase production was done by iodine plate assay method. Production of cellulase was carried out on synthetic and natural raw substrates employing submerged and solid state fermentation strategies. The produced cellulase was purified by ammonium sulphate precipitation, dialysis and spin column followed by characterization using native-PAGE and SDS-PAGE.
Results: Primary screening on carboxymethyl cellulose (CMC) agar plates showed cellulase production potential of P. verruculosum BS3. Production of cellulases was quantitatively estimated on basal salt medium (BSM) supplemented with 1% CMC by submerged fermentation (SmF). Among cellulases, endoglucanase showed the highest yield (930.9 U/ml), which was focused on the subsequent studies. The CMC in BSM was replaced by the natural flour (1%) of banana, tapioca, potato or banana peel, of which tapioca flour supported the maximum production of endoglucanase (598.3 U/ml). In solid state fermentation (SSF), P. verruculosum BS3 was grown on sawdust supplemented with 10% banana flour as a solid support, moistened with BSM and the maximum production of endoglucanase (3214.1 U/gds) was observed on day 9 of incubation at 28ºC. The partially purified (9.1 folds purification with 2.1% yield) cellulase was then characterized by native PAGE and SDS-PAGE. Upon staining with Congo-red (1%), on the CMC (0.25%) impregnated native gel, the zymogram showed a major cellulase fraction as yellow opaque zone. From the SDS-PAGE profile, the approximate MW of cellulase was estimated as 17 kDa.
Conclusion: This study signifies the industrial importance of the cellulase produced by P. verruculosum BS3 on cheap agro-products, and that the low MW cellulase has added importance as it offers potentials for protein engineering.

Open Access Original Research Article

Plasmid Profile of Antibiotics Heteroresistant Escherichia coli Isolates from Diarrhoeic Children Attending Ahmadu Bello University Teaching Hospital, Shika, Zaria, Nigeria

A. Sani, P. M. Djauro, J. A. Onaolapo, Y. K. E. Ibrahim, H. W. Idris

Microbiology Research Journal International, Page 1-10
DOI: 10.9734/BMRJ/2015/17640

In this study, 100 Escherichia coli (E. coli) isolates were obtained from the stool of 222 diarrhoeic children attending Ahmadu Bello University Teaching Hospital, Shika, Zaria. Out of the 100 isolates of E. coli obtained, five (5) were found to be heteroresistant to different antibiotics. The isolation and biochemical identification were performed using standard microbiological procedures. The antimicrobial susceptibility testing and the determination of the heteroresistance were performed using modified Bauer-Kirby sensitivity testing technique. The isolation of plasmid DNA was performed using the protocol described in the plasmid extraction kit (Thermo Scientific, UK). Multiplex PCR assay was performed to identify the different diarrhoeagenic groups of the antibiotics heteroresistant E. coli isolates. The results of antimicrobial susceptibility testing shows that 5 (100%) of the isolates are resistant to amoxicillin, 4 (80%) to trimethoprim/sulfamethoxazole, 2 (40%) to ciprofloxacin and chloramphenicol, 1(20%) to gentamicin and none was resistant to amoxicillin-clavulanic acid and ceftriaxone. Multiple antibiotic resistance (MAR) indexing of the E. coli isolates in this study showed that 80% have MAR index above 0.2, at such a MAR index, there is a significant level of misuse of these antibiotics within a particular environment. Among the five (5) heteroresistant E. coli isolates, 2 (40%) were found to be β-lactamase positive while 3 (60%) were found to be β-lactamase negative. The plasmid analysis showed that, four (4) of the five (5) E. coli isolates were found to harbor plasmids. However, there was no correlation between β-lactamase production and possession of plasmid by the antibiotics heteroresistant E. coli isolates. Following the Multiplex PCR assay, none of the antibiotics heteroresistant E. coli isolates harbor the virulence gene for any of the diarrhoeagenic E. coli.

Open Access Original Research Article

Effect of Probiotics (Lactobacillus acidophilus and Bifidobacterium bifidum) as Immune Stimulant on Hybrid Catfish Heteroclarias

T. A. Yisa, O. A. Ibrahim, S. M. Tsadu, U. P. Yakubu

Microbiology Research Journal International, Page 1-6
DOI: 10.9734/BMRJ/2015/17703

Study on the effect of probiotics (Lactobacillus acidophilus and Bifidobacterium bifidum) as feed supplement and immune stimulant on hybrid Heteroclarias was carried out with 240 hybrid Heteroclariasfingerlings with mean weight 1.90 ±0.40 g fed probiotic supplement (GNC Ultra 25) for fifty six (56) days in 12 number 30 x 60 x 30 cm rectangular aquarium tanks. There were four treatments and each replicated three times. The inclusion level of probiotic fed supplement was at 0 g/kg, 1 g/kg, 2 g/kg and 3 g/kg. The experimental fish were fed at 3%,5%,7%, and 9% body weight at two weeks interval, The result showed that there was significant difference p(<0.05) between control and other treatments in terms of specific growth rate and percentage survival. The microbial analysis showed that there was presence of Gram positive bacterial in the gut of the hybrid Heteroclarias. The treatments fed with probiotics have the higher plate count as compared to the control, the haematological analysis showed that blood parameters of control differed significantly p(<0.05) from other treatments. The inclusion levels of the probiotic supplement use in this study enhanced the growth and immune response of hybrid Heteroclarias, however inclusion level at 1 g/kg is sufficient to enhance the proliferation of the required bacteria.

Open Access Original Research Article

Phenotypic Detection of Extended Spectrum Beta-Lactamase and Carbapenemases Produced by Klebsiella spp Isolated from Three Referrals Hospitals in Yaounde, Cameroon

Anicette Chafa Betbeu, Hortense Gonsu Kamga, Michel Toukam, Calixte Didier Mbakop, Emilia Enjema Lyonga, Serge Bilong, Sinata Koulla- Shiro

Microbiology Research Journal International, Page 1-9
DOI: 10.9734/BMRJ/2015/18250

Aims: The main objective of this study was to determine the resistance phenotype of β-lactamines by Klebsiella in three hospitals in Yaoundé.
Study Design: A cross-sectional descriptive study.
Place and Duration of Study: Bacteriology laboratories of three referrals hospitals in Yaounde (University Teaching Hospital, General Hospital and Gynaeco-Obstetric and Pediatric Hospital) between May and November 2013.
Methodology: A cross-sectional descriptive study was carried out over a 6 month-period from May to November 2013 in the bacteriology laboratories of three referrals hospitals in Yaounde. 99 strains ofKlebsiella spp. were collected for the study. Antimicrobial susceptibility testing was done using the disc diffusion method. The antibiotics tested were the β-lactams, other inhibitors like clavulanic acid, tazobactam, Ethylene-Diamine-Tetra-Acetic Acid (EDTA), cloxacillin and 3-aminophenyl boronic acid hydrochloride. The minimum inhibitory concentration (MIC) was also determined to enable the classification of the different resistance phenotypes.
Results: Ninety-nine Klebsiella spp. were identified from urine (52.5%), blood (21.2%), pus (15.2%) and others sites (11.1%). The distribution of the Klebsiella spp. was: Klebsiella pneumoniae pneumoniae (78.7%), Klebsiella oxytoca (12.12%), Klebsiella pneumoniae ozaenae (5.05%), and Klebsiella pneumoniae rhinoscleromatis (4.04%). The isolates were most resistant to piperacillin (76%) and cephalothin, (85%). The most active antibiotics were imipenem (99%), and ertapenem (77%). The phenotypic tests revealed the following resistance phenotypes: extended-spectrum beta-lactamase (30.30%), wild (27.27%), penicillinase resistant to inhibitors (16.16%), carbapenemase (11.11%). Out of these 99 Klebsiella spp., 5 were carbapenemases producers of class C and 6 of class D. The MIC were variable with different antibiotics tested but the MIC of imipenem were always lower than 1 µg/ml.
Conclusion: The interpretation of the antimicrobial susceptibility testing has enabled the establishment of a high prevalence of expanded spectrum β-lactamase and consequently leading to an increase in the presence of carbapenemase producing Klebsiella spp. This could lead to therapeutic failure in case of treatment with beta-lactamines antibiotics. Therefore this trend needs to be monitored.

Open Access Original Research Article

Phytochemical Study and Antimicrobial Activity of Bark Extracts of Ceiba pentandra (L.) Gaertn. (Bombacaceae) from Côte d’Ivoire on Antibiotic Resistant Staphylococcus aureus and Pseudomonas aeruginosa

Golly Koffi Julien, Siaka Sorho, Soro Yaya, Guessennd Nathalie, Dosso Mireille, Djaman Allico Joseph

Microbiology Research Journal International, Page 1-7
DOI: 10.9734/BMRJ/2015/18103

One of the health problems in recent decades is the emergence of multi-antibiotic resistant bacteria. Among the solutions to this concern, the secondary metabolites of medicinal plants seem to be privileged. In this context, our work is devoted to the phytochemical study and evaluation of antibacterial activity of bark extracts of Ceiba pentandra from Ivorian Pharmacopoeia on methicillin resistant S. aureus and on imipenem and ceftazidime resistant P. aeruginosa. The phytochemical study shows that bark of Ceiba pentandra harvested in the center of Côte d’Ivoire contains alkaloids, flavonoids, saponins, tannins, steroids, terpenoids and cardiac glycosides. Microbiological testing showed that in liquid medium, ethyl acetate extract is active on all studied bacteria. Minimum Inhibitory Concentration (MIC) range from 0.78 to 6.25 mg/mL and Minimum Bactericidal Concentration (MBC) from 1.04 to 8.33 mg/mL. Purification of ethyl acetate extract had no influence on its activity against the studied bacteria. In fact, the most active fraction has MIC ranging from 0.52 to 6.25 mg/mL and MBC from 1.04 to 10.42 mg /mL. According to the ratio MBC /MIC, which was nearly 2, ethyl acetate extract and his active fraction are bactericidal.

Open Access Original Research Article

Diversity of Rotavirus VP7 and VP4 Genotypes Associated with Severe Childhood Diarrhea in North West Cameroon: Detection of Unusual Strains G1P[6], G2P[6], G2P[8] and G3P[6]

Florence A. Mbuh, Susan Damanka, Gorge E. Armah, Sunday A. Omilabu, Aliyu A. Ahmad, Jarlath U. Umoh

Microbiology Research Journal International, Page 1-11
DOI: 10.9734/BMRJ/2015/18071

Aims: To determine rotavirus genotypes and distribution among children 0 - 5 years old with severe acute diarrhea in the North West Region (NWR) of Cameroon.
Study Design: Cross sectional.
Place and Duration of Study: Hospitals and health centers in the NWR served as sample collection centers between January and December 2004.
Methodology: Fifty-six Enzyme Immunoassay (EIA) and Polyacrylamide gel electrophoresis (PAGE) - positive rotavirus stool specimens were analyzed for VP7 and VP4 genotypes by reverse transcriptase polymerase chain reaction (RT-PCR).
Results: A total of 51 (91.1%) samples genotyped as G and P types while the remaining five samples (8.9%) were partially characterized. Four VP7 genotypes (G1, G2, G3 and G9) and two VP4 genotypes, P[6] and P[8] were detected. The predominant G and P types were; G3 (34%) and P[8] (41.1%). Genotypes G1-G3 accounted for 85.8% of isolates while G9 represented 3.6% of isolates. Genotype P[6] represented 30.4% of all VP4 genotypes and was the most widespread strain occurring in all age groups. Twelve single G and P-type combinations were identified. Genotype G1P[8] (19.6%) predominated, followed by G3P[6] (17.9%). Unusual strains detected were G1P[6], G2P[6], G2P[8] and G3P[6] accounting for 32% of cases. Mixed infections were detected from 15 (27%) isolates comprising G1/G3P[6], G2/G9P[6], G2P[6]/P[8] and G1/G3/G8 P[6]/P[8]. Twenty isolates (35.7%) had unusual genotype / electropherotypes combinations. All G9 strains, mixed G genotypes and strains with atypical electrpherotypes occurred in children hospitalized with severe gastroenteritis.
Conclusion: There is high incidence of unusual rotavirus strains circulating in the NWR of Cameroon that could have an impact on rotavirus vaccine performance. Future studies will investigate post vaccine prevalence and characterization of non-typeable strains by other methods.