Open Access Original Research Article

Fungal Flora and Ochratoxin A Associated with Coffee in Cameroon

N. D. Nganou, N. Durand, N. L. Tatsadjieu, I. Métayer, D. Montet, C. M. F. Mbofung

Microbiology Research Journal International, Page 1-17
DOI: 10.9734/BMRJ/2014/4284

Aims: 104 samples were collected from the west region and the coastal plain of Cameroon during two coffee campaigns, 2009 and 2010. Two coffee processes were evaluated (wet and dry processes) at different stages from harvesting to storage.
Study Design: Food contaminants.
Place and Duration of Study: Food Microbiology Laboratory, Department of Food Science and Nutrition (ENSAI) University of Ngaoundere; UMR 95 Qualisud, CIRAD of Montpellier, between May 2009 and September 2012.
Methodology: Fungi profile was evaluated by direct plating techniques and identified using morphological and molecular tools. OTA levels were analyzed using HPLC technique after extraction and filtration using an immunoaffinity column.
Results: Results obtained revealed an overall percentage of fungal contamination between 60-92% in 2009 and 70-90% in 2010. There was no ecological difference in the composition of ochratoxigenic species present in five sites. Coffee beans sampled in 2009 had a colonization incidence of 18-40% A. carbonarius, 12-22% A. niger, 3-15% A. ochraceus while those of 2010 had a colonization incidence of 15-30% A. carbonarius, 35-40% A. niger, and 2-7% A. ochraceus. Fungal diversity was not correlated with the geographical origin, coffee cultivar and processing method. There was no difference between the processes studied in terms of occurrence of ochratoxigenic fungi. OTA levels were mostly below the recommended standards although some isolated cases of extreme contamination were observed in 2009. A higher level of OTA was detected in the presence of A. niger, A. carbonarius and A. ochraceus than when only A. niger was present.
Conclusion: The important fungi with the potential to produce OTA in Cameroonian coffee beans are A. carbonarius and A. niger. These two species were predominant on each type of coffee beans. It was also observed that once a toxigenic strain was isolated from a coffee sample, the sample contained OTA.

Open Access Original Research Article

Antibacterial Activity of Phenolic Compounds Derived from Ginkgo biloba Sarcotestas against Food-Borne Pathogens

Antonio Carraturo, Katia Raieta, Idolo ., Jinwoong Kim, Gian Luigi Russo

Microbiology Research Journal International, Page 18-27
DOI: 10.9734/BMRJ/2014/5623

Aims: We investigated the antibacterial activity of three groups of phenolic compounds obtained from the chloroform (CHCl3) extract of the fleshy seed coat (sarcotestas) of Ginkgo biloba.
Study Design: An experimental study.
Methodology: Inhibition of microbial growth was measured by an agar diffusion method and susceptibility tests were performed by the broth microdilution method. Bactericidal effect of Ginkgo biloba compound 5-7 against Salmonella enterica serovar Typhimurium was assessed by time-kill assay.
Results: Ginkgo biloba compounds 5-7 and 8-10 showed high antimicrobial activity against Gram-positive and Gram-negative bacteria, including several food-borne pathogens. In particular, compounds 5-7 and 8-10, containing phenolic acids and bilobols, respectively, were highly effective against Salmonella enteric serovar Typhimurium, Listeria monocytogenes, Listeria innocua, Streptococcus pyogenes, Escherichia coli, and Shigella dysenteriae. On the opposite, compounds 1-4, containing cardanols, showed little antibacterial activity. Compounds 5-7 exerted a bactericidal and bacteriolytic effect on Salmonella enteric serovar Typhimurium with a Minimal Inhibitory Concentration (MIC) and a Minimal Bactericidal Concentration (MBC) of 8.3 µg ml–1.
Conclusion: The results of this study indicate that phenolic compounds derived from Ginkgo biloba sarcotestas, because of their strong inhibitory characteristics towards food pathogens, can be considered ideal candidates for possible application in food microbiology due to their natural origins.

Open Access Original Research Article

First Isolation and Identification of Listeria monocytogenes Isolated from Frozen and Shock Frozen Dressed Broiler Chicken in Sudan

A. D. I. Alsheikh, G. E. Mohammed, M. A. Abdalla, A. O. Bakhiet

Microbiology Research Journal International, Page 28-38
DOI: 10.9734/BMRJ/2014/2449

Aim: To isolate and identify Listeria monocytogenes from frozen and shock frozen raw dressed broiler chicken in Khartoum State, Sudan.
Place and Duration of Study: Department of Pathology, Microbiology and Parasitology, Sudan University of Science and Technology, Khartoum-North, Sudan, between July 2011 and June 2012.
Methodology: Eight hundred samples were used in this the study. Five hundred frozen (-18º) raw dressed broiler chickens samples were collected from five station chicken abattoirs in Khartoum State. Three hundred samples were collected as fresh, -18ºC and shock frozen (-40ºC) raw dressed broiler chickens. Detection and isolation of L. monocytogenes was carried out using the conventional International Organization for Standardization method.
Results: Out of the 500 samples, 195(39%) were found to be contaminated with Iisteria spp.; L. monocytogenes 64(12.8%), Listeria ivanovii 97(19.4%), Listeria grayi 20(4%), Listeria seeligeri 5(1%), Listeria welshimeri 9(1.8%). Out of the 300 samples, 111(37%) were found to be contaminated with Iisteria spp.; L. monocytogenes 39(13%), Listeria ivanovii 54(18%), Listeria grayi 11(3.6%), Listeria seeligeri 3(1%) and Listeria welshimeri 4(1.3%).
Conclusion: The results presented in this study indicated that L. monocytogenes was found in frozen (-18ºC) raw dressed broiler chicken and shock frozen (-40ºC) raw dressed broiler chickens.

Open Access Original Research Article

Virulence Markers and Antifungal Susceptibility Profile of Candida glabrata: An Emerging Pathogen

Sachin Deorukhkar, Santosh Saini

Microbiology Research Journal International, Page 39-49
DOI: 10.9734/BMRJ/2014/5806

Aims: To study virulence factors and antifungal susceptibility profile of C. glabrata isolated from various clinical specimens.
Study Design: A total of 175 C. glabrata spp. isolated from various clinical specimens were included in the study.
Place and Duration of Study: Department of Microbiology, Rural Medical College, Pravara Institute of Medical Sciences (PIMS), Loni, Maharashtra, India, between March 2008 to March 2013.
Methodology: C. glabrata was identified by sugar assimilation and fermentation tests and colony color on Hichrome Candida agar. HiCandida identification kit supplemented the identification of the isolates. The virulence markers studied were production of extracellular hydrolytic enzymes (phospholipase, proteinase and coagulase), haemolytic activity and biofilm formation. The antifungal susceptibility profile of C. glabrata isolates was determined by Hicomb minimum inhibitory concentration (MIC) test. The antifungal agents used were amphotericin B (range 0.002-32 µg), fluconazole (range 0.016-256 µg), itraconazole (range 0.002-32 µg) and ketoconazole (range 0.002-32 µg).
Results: Maximum number of isolates were obtained from blood culture (36%) followed by urine sample (29.7%). ICU stay followed by HIV infection were the main predisposing factors found to be associated with C. glabrata infection. A total of 53 (30.2%) C. glabrata isolates showed phospholipase activity. Proteinase production was seen in 56 (32%) isolates. 48 (27.4%) isolates were coagulase positive. Haemolytic activity was noted in 43 (24.5%) isolates. Most of C. glabrata produced β- type of haemolysis on sheep blood SDA agar. Biofilm forming ability was noted in 68 (38.8%) isolates. Maximum isolates were resistant to fluconazole (46.8%) and ketoconazole (46.8%) followed by itraconazole (45.7%). Amphotericin B resistance was seen in 58 (33.1%) isolates.
Conclusion: Once considered as a non pathogenic human commensal, C. glabrata has emerged as an important pathogen in various clinical types of candidiasis. C. glabrata is innately resistant to antifungal drugs and various antifungal mechanisms of the body. Present research data available is not satisfactory to understand the pathogenic and other mechanisms involved in the transition of C. glabrata from nonpathogenic commensal to a potential pathogen. Therefore more research studies are needed to explain pathogenesis, host-pathogen interaction and other survival properties of this emerging pathogen.

Open Access Original Research Article

Effects of Gamma Interferon on Intestinal Epithelial Cells Stimulated with Salmonella Enterica serovar Typhimurium and Candida albicans

Florence A. Okafor, Ebony I. Weems, Leonard L. Williams

Microbiology Research Journal International, Page 50-62
DOI: 10.9734/BMRJ/2014/4989

Aims: The objective of this study was to determine the level of secretion of gamma-interferon by interferon-primed and unprimed Caco-2 intestinal epithelial cells and their survival or growth following infection with Salmonella enterica subsp. enterica serovar Typhimurium (ATCC 14028), Salmonella enterica subsp. enterica serovar Typhimurium DT104 (ATCC 700408), and Candida albicans (Robin) Berkhout, anamorph (ATCC 10231) as well as the survival of the test microorganisms following infection.
Study Design: Controlled laboratory experiments were performed using two different species of Salmonella and adenocarcinoma Caco-2 cells. Untreated/ unprimed Caco-2 cells served as control; Caco-2 cells’ growth and interferon production were then determined using, Enzyme Linked Immunosorbent Assay (ELISA) and flow cytometry.
Place and Duration of Study: Department of Biological & Environmental Sciences Alabama A& M University and Center for Excellence in Post-Harvest Technologies, North Carolina A&T University USA April 2008 and February 2010.
Methodology: Cell culture supernatants of Caco-2 intestinal epithelial cells, primed and unprimed with IFN-γ were infected with either wild-type Salmonella Typhimurium 14028, Candida albicans10231 or multi-drug resistant Salmonella Typhimurium DT104 were collected and analyzed. ELISA and flow cytometry were used to determine apoptosis, cell growth and interferon production. The Bioscreen-C Automated Growth Curve Analysis System was used under controlled environment to determine the growth of the microorganisms in the presence of different concentrations of IFN-γ.
Results: Secretion of IFN-γ from Caco-2 cells that were previously treated with 50μg/ml, 20μg/ml, 10μg/ml, 5μg/ml, and 2.5 μg/ml of IFN-γ were not concentration dependent. However, the amount of IFN-γ released from Caco-2 cells was dependent on microbial stimulus type. Cells that were pretreated with 5 μg/ml and 2.5 μg/ml of IFN-γ and then infected with Salmonella Typhimurium DT104 showed an increase in the amount of IFN-γ in the culture medium after 5 minutes. IFN-y induced CaCo-2 cell death was dose-dependent for S.Typhimurium DT104 and Candida albicans. Results are reported as mean ± SEM fortriplicate values from three independent experiments at each time point and IFN-γ dose.
Conclusion: These findings indicate that IFN-γ may serve as alternative antimicrobial compounds to reduce the persistence of multi-drug resistant microorganisms such as S. Typhimurium DT104. Induction of interferon-gamma production may be related to microbial virulence/pathogenicity. The potential of IFN-γ as a natural therapeutic for persistent infections in the immune-compromised populations still needs to be further investigated.

Open Access Original Research Article

Optimization of Culture Conditions for Enhanced Antimicrobial Activity of Rhodococcus erythropolis VLK-12 Isolated from South Coast of Andhra Pradesh, India

Krishna Naragani, Rajesh Kumar Munaganti, Usha Kiranmayi Mangamuri, Vijayalakshmi Muvva

Microbiology Research Journal International, Page 63-79
DOI: 10.9734/BMRJ/2014/5707

Aims: To Isolate and characterize the antimicrobial actinomycetes from the marine habitats of south coast of Andhra Pradesh, India.
Place and Duration of the Study: Marine habitats of south coast of Andhra Pradesh, India, between June 2011 and July 2012.
Methodology: The soil samples were collected, pre-treated and plated on yeast extract- malt extract dextrose agar medium. Identification of the strain was carried out by employing the polyphasic taxonomical studies including the 16S rRNA sequence based analysis. Phylogenetic tree was constructed using the Molecular Evolutionary Genetic Analysis (MEGA) version 5. The influence of culture conditions and the effect of environmental factors on the biomass and antimicrobial activy\ity of the strain was the focus of this study.
Results: A total of 20 actinobacteria were isolated from the marine habitats of south coast of Andhra Pradesh, India, and screened for antimicrobial activity against test bacteria and fungi. The potent bioactive metabolite producing strain was designated as VLK-12. Further polyphasic studies revealed that the Isolate VLK-12 belongs to the genera Rhodococcus. Phylogenetic analysis of 16S rRNA sequencing studies revealed that the strain is closely related to Rhodococcus erythropolis. The crude ethyl acetate extract obtained by culturing the strain on YMD inhibited Gram positive and Gram negative bacteria along with fungi.
Conclusion: Rhodococcus erythropolis isolated from the marine habitats of south coast of Andhra Pradesh exhibited antimicrobial activity against pathogens.

Open Access Original Research Article

Molecular Detection of Giardia lamblia and Cryptosporidium parvum in Different Water Sources of District Bannu, Khyber Pakhtunkhwa, Province of Pakistan

Muhammad Shoaib Alam, Sana Ullah Khan, Sultan Ayaz, Noor ul Akbar, Muhammad Asim Khan, Iftikhar Ahmad, Muhammad Idrees, Muhammad Waqar

Microbiology Research Journal International, Page 80-88
DOI: 10.9734/BMRJ/2014/3947

Background: Cryptosporidium parvum and Giardia lamblia are intestinal parasites that predominantly causes "waterborne" infections that are transmitted through consumption of contaminated water. Both parasites typically cause an acute short-term infections with self-limiting diarrhea as the main symptom in people with intact immune systems. However, in immunocompromised individuals, the symptoms are particularly severe and might be fatal.
Methods: The study was carried out in District Bannu Khyber Pukhtunkhwa, Pakistan for the detection of G. lamblia and C. parvum parasites in drinking water in different villages/localities (Kakki, Jamon Road, Kotka Juma Khan, Sokari, Mandan and Bannu City). Water samples n=75 were collected from different water sources between 1st August 2011 to 30th January 2012. These samples included tap, pond, borewell and hand pump water that were filtrated and residue was subjected to amplify by PCR.
Results: Overall prevalence of parasites was 36% (25/75), containing tap 17.64% (9/51) and pond water 75% (6/8), bore well water 41.66% (5/12) and hand pump water 50% (2/4). Similarly over all prevalence rate of tap water for C. parvum was 7.84% (4/51) while for G. lamblia was 9.80% (5/51) positive. The present study revealed that the people of the area should use the cleaned and filtered water.
Conclusion: Contamination of water with G. lamblia and C. parvum was found in water sources especially the drinking ones, of District Bannu which need proper water treatment to decontaminate and large scale studies are needed.

Open Access Original Research Article

Effect of Solvent Extracts of Some Plants on Ralstonia solanacearum

A. A. Owoseni, T. E. Sangoyomi

Microbiology Research Journal International, Page 89-96
DOI: 10.9734/BMRJ/2014/4179

Aims: To study the efficacy of different solvent extracts (chloroform, ethanol, methanol and hexane) of ten plants on Ralstonia solanacearum the causal organism of bacterial wilt of tomato.
Place and Duration of Study: Departments of Crop Production, Soil and Environmental Management and Biological Sciences, Bowen University, Iwo, Nigeria from August 2011 to April 2012.
Methodology: Ten plants namely Ocimum gratissimum, Vernonia amygdalina, Allium sativum, Zingiber officinale, Cymbopogon citratus, Azadirachta indica, Jatropha curcas, Senna obtusifolia, Senna occidentalis and Senna alata were collected from Iwo, air dried and pulverized. Chloroform, ethanol, methanol and hexane were used to extract active ingredients from the ten plants. The solvent extracts were tested against R. solanacearum the causal organism of bacterial wilt of tomato and other plants using the disc diffusion method on Mueller Hinton agar. The minimum inhibitory concentration (MIC) of the effective extracts was determined.
Results: The plant extracts from chloroform were the most active and this was followed by methanol and ethanol, the lowest activity was recorded from the hexane extracts. The chloroform extracts of J. curcas had the widest zone of inhibition of 15mm followed by O. gratissimum (13mm). All the solvent extracts of A. sativum were active except the hexane extract. The means and standard error of triplicate tests were recorded. The MIC of the active extracts were studied, the MIC of the A. sativum ethanolic extract was 0.25 mg/ml while it was 0.5mg/ml for the V. amygdalina ethanol extract. The MIC of the A. sativum chloroform extract was 0.25mg/ml; J. curcas chloroform extract MIC was 0.125mg/ml, and the MIC for methanolic extract of both extracts were 0.5mg/ml and 0.25mg/ml respectively.
Conclusion: The activities of the different solvent extracts are remarkable when compared with the water extracts. Hence, solvent extracts will enhance the efficacy of these phytochemicals in the management of R. solanacearum infections as opposed to water extracts.

Open Access Original Research Article

Purification, Kinetic Properties and Antitumor Activity of L-Glutaminase from Penicillium brevicompactum NRC 829

Ali Mohamed Elshafei, Mohamed Mohamed Hassan1, Nadia Hussein Ali, Mohamed Abd-Elmontasr Abouzeid, Dalia Ali Mahmoud, Dina Helmy Elghonemy

Microbiology Research Journal International, Page 97-115
DOI: 10.9734/BMRJ/2014/5098

Aim: The aims of the present study were to purify and characterize L-glutaminase from Penicillium brevicompactum NRC 829; and to evaluate the antitumor activity of the purified enzyme against different tumor human cell lines.
Study Design: Testing of antitumor activity of L-glutaminase, purified from a filamentous fungal strain, 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 January 2011 and February 2012.
Methodology: P. brevicompactum NRC 829 was grown and maintained on modified Czapek Dox agar (MCD) medium. Cell-free extract was directly used as the source of crude enzyme. L-glutaminase was purified by heat treatment for 20 min at 50ºC, followed by gel filtration on Sephadex G-100 and G-200 columns.
Results: An intracellular L-glutaminase from Penicillium brevicompactum NRC 829 was purified to homogeneity (162.75 fold) with an apparent molecular mass (Mr) of 71 kDa. The purified enzyme showed its maximal activity against L-glutamine when incubated at pH 8.5 at 50ºC for 30 min. The purified enzyme retained about 92 % of its initial activity after incubation at 70ºC for 30 min indicating the thermo-stability nature of this enzyme. The highest activity was reported towards its natural substrate, L-glutamine, with an apparent Km value of 1.66 mM. The purified enzyme inhibited the growth of human cell line hepatocellular carcinoma (Hep-G2), with IC50 value of 63.3μg/ml.
Conclusion: L-glutaminase purified from Penicillium brevicompactum NRC 829 is a potential candidate in food and pharmaceutical industries.

Open Access Original Research Article

Isolation, Characterization and Protein Profiling of Lead Resistant Bacteria

Sabyasachi Chatterjee, Juthika Das, Saheli Chatterjee, Pratiti Choudhuri, Agniswar Sarkar

Microbiology Research Journal International, Page 116-131
DOI: 10.9734/BMRJ/2014/5862

Aims: To isolate lead resistant bacteria from industrial effluent and characterize them by biochemical tests, effect of physical and chemicals factors on their growth and protein profiling of the isolates on with or without metal stress.
Study Design: Cross-sectional study of related research articles and papers.
Place and Duration of Study: Samples were collected from effluent water of Indian Iron and Steel Company (IISCO), Burnpur, West Bengal, India and studied at The Department of Biotechnology, The University of Burdwan. West Bengal, India, between September 2010 and June 2011.
Methodology: Isolation of the isolates was done by pour plating the diluted effluent water onto the metal (Lead acetate) containing agar plates. The basic characterization of the isolates was done on the basis of morphological, physical, biochemical and antibiogram tests. Quantification of metal absorption was determined by Atomic absorption Spectrophotometric analysis. Protein expressions of the isolates on metal stress were studied by SDS-PAGE.
Results: Two bacterial isolates namely PbrB1 and PbrB2 were isolated; they showed resistance to lead up to 6mM Lead (Lead acetate) and also to different antibiotics. They are able to ferment different sugars even in presence of low concentration of metals. Their protein profiling by SDS-PAGE shows different expressions of proteins upon metal stress.
Conclusion: The isolates can be utilized to bioremediate lead from contaminated environment and further study of molecular mechanisms underlying lead accumulation process can be studied.

Open Access Original Research Article

Molecular Detection of Antimicrobial Resistance Gene Cassettes Associated with Class 2 Integron in Salmonella Serovars Isolated in Iran

Bahareh Rajaei, Nahid Sepehri Rad, Farzad Badmasti, Mohamad Reza Razavi, Mohammad Reza Aghasadeghi, Raheleh Saboohi, Taraneh Rajaei, Arfa Moshiri, Mehdi Nejati, Ahmad Reza Bahremand, Seyed Davar Siadat

Microbiology Research Journal International, Page 132-141
DOI: 10.9734/BMRJ/2014/4639

Aim:Salmonella is an important food-borne pathogen in humans and a broad range of animals. Antimicrobial resistance in Salmonella spp. is a serious health problem in human and veterinary medicine worldwide. The aim of this study was to detect integrons, the natural recombination systems that can be transferred in companion with mobile genetic elements and play a major role in spreading antibiotic resistance genes in clinical isolates.
Place and Duration of Study:Salmonella clinical isolates were provided by a number of institutes and hospitals over the country through the years 2008-2009.
Methodology: Antimicrobial susceptibility testing and serotyping were carried out for eighty four epidemiologically unrelated clinical isolates of Salmonella serovars collected from different provinces of Iran through the years 2008-2009. PCR assays were carried out to detect intI2 gene (integrase I attributed to class 2 integron) and internal variable regions (IVRs) of class 2 integron. These sequences were deposited in EMBL/GenBank database (www.ncbi.nlm.nih.gov).
Results: Eleven isolates (13.1%) which were resistant to at least 4 groups of antimicrobial agents were considered as MDR (multidrug resistant) Salmonella serovars. PCR assays detected intI2 gene (integrase I attributed to class 2 integron) and internal variable regions (IVRs) of class 2 integron in Fourteen (16.7%) and eleven (78.6%) of Salmonella clinical isolates respectively. Analysis of the sequence data revealed 3 gene cassette arrays deposited in Genbank databases including the dhfrA1 (0.75 kb), dfrA14- lsp (1 kb), dhfrA1-sat2-aadA1 (3 kb) with three IVR distribution patterns. An artifact PCR product of 2 kb was reported in this study to be amplified together with IVRs of class 2 integrons which was associated with the fhuE- ptsG genes.
Conclusions: Presence of MDR Salmonella serovars demonstrates that antimicrobial selection pressure is widespread in our clinical settings. Detection of class 2 integron carrying gene cassettes which confer resistance to different classes of antibiotics such as aminoglycosides, and trimethoprim confirms that integron-mediated antimicrobial gene cassettes are prevalent in Salmonella serovars isolated in Iran.