Diversity of Bacillus Species Isolated from Okpehe, a Traditional Fermented Soup Condiment from Nigeria

2010 ◽  
Vol 73 (5) ◽  
pp. 870-878 ◽  
Author(s):  
FOLARIN A. OGUNTOYINBO ◽  
MELANIE HUCH ◽  
GYU-SUNG CHO ◽  
ULRICH SCHILLINGER ◽  
WILHELM H. HOLZAPFEL ◽  
...  
Keyword(s):  
Restriction Analysis ◽  
Restriction Enzymes ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Bacillus Species ◽  
Fermented Foods ◽  
Potential Health ◽  
Enterotoxin Genes ◽  
Bacillus Spp ◽  
Dna Restriction

The diversity of Bacillus species isolated from the fermented soup condiment okpehe in Nigeria was studied using a combination of phenotypic and genotypic methods. Fifty strains presumptively characterized as Bacillus spp. using the API 50 CHB test were further identified by PCR of randomly amplified polymorphic DNA (RAPD) and by amplified ribosomal DNA restriction analysis (ARDRA) genotyping methods. ARDRA fingerprinting with HhaI, HinfI, and Sau3AI restriction enzymes did not allow successful differentiation between the Bacillus species, except for distinguishing B. cereus from other Bacillus species. This problem was overcome with the combination of RAPD PCR and ARDRA genotypic fingerprinting techniques. Sequencing of 16S rRNA genes of selected strains representative of the major clusters revealed that the Bacillus strains associated with this fermentation were B. subtilis, B. amyloliquefaciens, B. cereus, and B. licheniformis (in decreasing order of incidence). The presence of enterotoxin genes in all B. cereus strains was demonstrated by multiplex PCR. The high incidence of detection (20%) of possibly pathogenic B. cereus strains that contained enterotoxin genes indicated that these fermented foods may constitute a potential health risk.

scholarly journals Extracellular hydrolytic enzyme production by proteolytic bacteria from the Antarctic

2013 ◽  
Vol 34 (3) ◽  
pp. 253-267 ◽  
Author(s):  
Mauro Tropeano ◽  
Susana Vázquez ◽  
Silvia Coria ◽  
Adrián Turjanski ◽  
Daniel Cicero ◽  
...  
Keyword(s):  
Extracellular Enzymes ◽  
Restriction Analysis ◽  
Hydrolytic Enzyme ◽  
Hydrolytic Enzymes ◽  
Extracellular Enzyme ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Potter Cove ◽  
Starting Point ◽  
Dna Restriction

AbstractCold−adapted marine bacteria producing extracellular hydrolytic enzymes are important for their industrial application and play a key role in degradation of particulate organic matter in their natural environment. In this work, members of a previously−obtained protease−producing bacterial collection isolated from different marine sources from Potter Cove (King George Island, South Shetlands) were taxonomically identified and screened for their ability to produce other economically relevant enzymes. Eighty−eight proteolytic bacterial isolates were grouped into 25 phylotypes based on their Amplified Ribosomal DNA Restriction Analysis profiles. The sequencing of the 16S rRNA genes from representative isolates of the phylotypes showed that the predominant culturable protease−producing bacteria belonged to the class Gammaproteobacteria and were affiliated to the genera Pseudomonas, Shewanella, Colwellia, and Pseudoalteromonas, the latter being the predominant group (64% of isolates). In addition, members of the classes Actinobacteria, Bacilli and Flavobacteria were found. Among the 88 isolates screened we detected producers of amylases (21), pectinases (67), cellulases (53), CM−cellulases (68), xylanases (55) and agarases (57). More than 85% of the isolates showed at least one of the extracellular enzymatic activities tested, with some of them producing up to six extracellular enzymes. Our results confirmed that using selective conditions to isolate producers of one extracellular enzyme activity increases the probability of recovering bacteria that will also produce additional extracellular enzymes. This finding establishes a starting point for future programs oriented to the prospecting for biomolecules in Antarctica.

scholarly journals APLIKASI METODE ARDRA DALAM IDENTIFIKASI ISOLAT Bacillus thuringiensis ENDOGENIK SEBAGAI PENGENDALI HAMA KUBIS (Crocidolomia binotalis)

EUGENIA ◽  
2011 ◽  
Vol 17 (2) ◽  
Author(s):  
Christina L. Salaki ◽  
Langkah Sembiring
Keyword(s):  
Profile Analysis ◽  
Restriction Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Universal Primers ◽  
Chromosomal Dna ◽  
Pcr Products ◽  
Agarose Electrophoresis ◽  
Electrophoresis Method ◽  
Dna Restriction

ABSTRACT Indonesian indigenous bacterial isolates of B. thuringiensis pathogenic to cabbage pest (C. binotalis) were molecularly characterized and identified using DNA fingerprinting method of ARDRA (Amplified Ribosomal DNA Restriction Analysis). Chromosomal DNA of 10 selected isolates (SLK2.3, SRNG4.2, TKO1, TK9, YPPA1, UG1A, BLPPN8.2, YWKA1, BAU3.2, LPST1) and 2 reference strains (B. thuringiensis serovar kurstaki HD1 & B. thuringiensis serovar israelensis H14) were isolated and purified by standard method. 16S rRNA genes were amplified by PCR method using universal primers of 27f and 1529r. PCR products were digested by 4 restriction endonucleases (EcoR1, HindIII, Pst1 dan HaeIII), and separated by agarose electrophoresis method to generate ARDRA profiles. Results of study showed that only ARDRA profiles generated by Hae III digestion were found to be meaningful and therefore used to identify the isolates. The ARDRA profile analysis indicated that the reference strain of B. thuringiensis serovar kurstaki HD1 could be clearly separated with B. thuringiensis serovar israelensis H14. In fact, those two strains have been widely recognized to be different in terms of their pathogenic specifity against insects. B. thuringiensis serovar kurstaki HD1 has been known to be specifically pathogenic to Lepidopteran whereas B. thuringiensis serovar israelensis H14 has been known to be specifically pathogenic to Dipteran. Key words : application, ARDRA, indigenous, B. thuringiensis, C. binotalis  

scholarly journals High Levels of Endemicity of 3-Chlorobenzoate-Degrading Soil Bacteria

1998 ◽  
Vol 64 (5) ◽  
pp. 1620-1627 ◽  
Author(s):  
R. R. Fulthorpe ◽  
A. N. Rhodes ◽  
J. M. Tiedje
Keyword(s):  
South African ◽  
Boreal Forests ◽  
Soil Bacteria ◽  
Restriction Analysis ◽  
Mediterranean Ecosystems ◽  
Soil Samples ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Dna Restriction ◽  
Repetitive Extragenic Palindromic Pcr

ABSTRACT Soils samples were obtained from pristine ecosystems in six regions on five continents. Two of the regions were boreal forests, and the other four were Mediterranean ecosystems. Twenty-four soil samples from each of four or five sites in each of the regions were enriched by using 3-chlorobenzoate (3CBA), and 3CBA mineralizers were isolated from most samples. These isolates were analyzed for the ability to mineralize 3CBA, and genotypes were determined with repetitive extragenic palindromic PCR genomic fingerprints and restriction digests of the 16S rRNA genes (amplified ribosomal DNA restriction analysis [ARDRA]). We found that our collection of 150 stable 3CBA-mineralizing isolates included 48 genotypes and 44 ARDRA types, which formed seven distinct clusters. The majority (91%) of the genotypes were unique to the sites from which they were isolated, and each genotype was found only in the region from which it was isolated. A total of 43 of the 44 ARDRA types were found in only one region. A few genotypes were repeatedly found in one region but not in any other continental region, suggesting that they are regionally endemic. A correlation between bacterial genotype and vegetative community was found for the South African samples. These results suggest that the ability to mineralize 3CBA is distributed among very diverse genotypes and that the genotypes are not globally dispersed.

scholarly journals Genetic and Biochemical Diversity among Isolates ofPaenibacillus alvei Cultured from Australian Honeybee (Apis mellifera) Colonies

2000 ◽  
Vol 66 (3) ◽  
pp. 1098-1106 ◽  
Author(s):  
Steven P. Djordjevic ◽  
Wendy A. Forbes ◽  
Lisa A. Smith ◽  
Michael A. Hornitzky
Keyword(s):  
Apis Mellifera ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Restriction Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Whole Cell ◽  
Restriction Profiles ◽  
Dna Profiles

ABSTRACT Twenty-five unique CfoI-generated whole-cell DNA profiles were identified in a study of 30 Paenibacillus alvei isolates cultured from honey and diseased larvae collected from honeybee (Apis mellifera) colonies in geographically diverse areas in Australia. The fingerprint patterns were highly variable and readily discernible from one another, which highlighted the potential of this method for tracing the movement of isolates in epidemiological studies. 16S rRNA gene fragments (length, 1,416 bp) for all 30 isolates were enzymatically amplified by PCR and subjected to restriction analysis with DraI, HinfI,CfoI, AluI, FokI, andRsaI. With each enzyme the restriction profiles of the 16S rRNA genes from all 30 isolates were identical (one restriction fragment length polymorphism [RFLP] was observed in theHinfI profile of the 16S rRNA gene from isolate 17), which confirmed that the isolates belonged to the same species. The restriction profiles generated by using DraI,FokI, and HinfI differentiated P. alvei from the phylogenetically closely related speciesPaenibacillus macerans and Paenibacillus macquariensis. Alveolysin gene fragments (length, 1,555 bp) were enzymatically amplified from some of the P. alvei isolates (19 of 30 isolates), and RFLP were detected by using the enzymesCfoI, Sau3AI, and RsaI. Extrachromosomal DNA ranging in size from 1 to 10 kb was detected in 17 of 30 (57%) P. alvei whole-cell DNA profiles. Extensive biochemical heterogeneity was observed among the 28 P. alvei isolates examined with the API 50CHB system. All of these isolates were catalase, oxidase, and Voges-Proskauer positive and nitrate negative, and all produced acid when glycerol, esculin, and maltose were added. The isolates produced variable results for 16 of the 49 biochemical tests; negative reactions were recorded in the remaining 30 assays. The genetic and biochemical heterogeneity inP. alvei isolates may be a reflection of adaptation to the special habitats in which they originated.

scholarly journals Integrated Meta-omics Approaches To Understand the Microbiome of Spontaneous Fermentation of Traditional Chinese Pu-erh Tea

mSystems ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Ming Zhao ◽  
Xiao Q. Su ◽  
Bo Nian ◽  
Li J. Chen ◽  
Dong L. Zhang ◽  
...  
Keyword(s):  
Phenolic Compounds ◽  
Gallic Acid ◽  
Glycoside Hydrolases ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Fermented Foods ◽  
Catechol Dioxygenase ◽  
Spontaneous Fermentation ◽  
Food Fermentation

ABSTRACT The microbiome in fermentation has direct impacts on the quality of fermented foods and is of great scientific and commercial interest. Despite considerable effort to explain the microbial metabolism associated with food fermentation, the role of the microbiome in pu-erh tea fermentation remains unknown. Here, we applied integrated meta-omics approaches to characterize the microbiome in two repeated fermentations of pu-erh tea. Metabarcoding analysis of bacterial 16S rRNA genes showed a decrease in the proportion of Proteobacteria and an increase in the abundance of Firmicutes during fermentation. Metabarcoding analysis of fungal internal transcribed spacer (ITS) sequence demonstrated that Rasamsonia, Thermomyces, and Aspergillus were dominant at the intermediate stage, whereas Aspergillus was dominant at other stages in fermentation. Metaproteomics analysis assigned primary microbial metabolic activity to metabolism and identified microbial carbohydrate-active enzymes involved in the degradation of polysaccharides including cellulose, xylan, xyloglucan, pectin, starch, lignin, galactomannan, and chitin. Metabolomics and high-performance liquid chromatography analysis revealed that levels of phenolic compounds, including gallates, decreased whereas contents of gallic acid and ellagic acid significantly increased after fermentation (P < 0.05). The changes in levels of gallates and gallic acid were associated with the hydrolysis of tannase. Glycoside hydrolases, phenol 2-monooxygenase, salicylaldehyde dehydrogenase, salicylate 1-monooxygenase, catechol O-methyltransferase, catechol dioxygenase, and quercetin 2,3-dioxygenases were hypothesized to be related to oxidation, conversion, or degradation of phenolic compounds. We demonstrated microbiota in fermentation and their function in the production of enzymes related to the degradation of polysaccharides, and metabolism of phenolic compounds, resulting in changes in metabolite contents and the quality of pu-erh tea. IMPORTANCE Fermented foods play important roles in diets worldwide and account for approximately one-third of all foods and beverages consumed. To date, traditional fermentation has used spontaneous fermentation. The microbiome in fermentation has direct impacts on the quality and safety of fermented foods and contributes to the preservation of traditional methods. Here, we used an integrated meta-omics approach to study the microbiome in the fermentation of pu-erh tea, which is a well-known Chinese fermented food with a special flavor and healthful benefits. This study advanced the knowledge of microbiota, metabolites, and enzymes in the fermentation of pu-erh tea. These novel insights shed light onto the complex microbiome in pu-erh fermentation and highlight the power of integrated meta-omics approaches in understanding the microbiome in food fermentation ecosystems.

scholarly journals Formation of Pseudo-Terminal Restriction Fragments, a PCR-Related Bias Affecting Terminal Restriction Fragment Length Polymorphism Analysis of Microbial Community Structure

2003 ◽  
Vol 69 (5) ◽  
pp. 2555-2562 ◽  
Author(s):  
Markus Egert ◽  
Michael W. Friedrich
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
16S Rrna ◽  
Length Polymorphism ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Restriction Fragments ◽  
Restriction Fragment Length

ABSTRACT Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using MspI and AluI as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called “pseudo-T-RFs” since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs.

The difference of the gut microbiota of gastric cancer in relation to Helicobacter pylori negativity and positivity.

2019 ◽  
Vol 37 (4_suppl) ◽  
pp. 10-10
Author(s):  
Mijin Seol ◽  
Yu Ra Lee ◽  
Kyung Mi Kim ◽  
Cheol Min Shin ◽  
Hyuk Yoon ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Helicobacter Pylori ◽  
Relative Abundance ◽  
Fecal Sample ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Bacillus Species ◽  
Sequencing Analysis ◽  
Metagenomic Sequencing ◽  
Microbial Composition

10 Background: Helicobacter pylori (HP) is a major risk factor for gastric cancer, however, only 1-2% of HP(+) people develop adenocarcinoma. In this study, we have compared the intestinal microbiota composition related to HP status among gastric cancer patient using 16SrRNA gene-based metagenomic sequencing analysis and culture-based method. Methods: Stool samples were collected from 18 gastric cancer patients. 16S rRNA genes were sequenced on the Illumina Miseq platform and further analyzed to evaluate the gut bacterial community. The bacteria strains of fecal sample were isolated in aerobic and anaerobic condition. Results: Metagenomics analysis of fecal sample showed four major phyla; Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria were dominant. Firmicutes were the most dominant phylum. Within this phylum, the relative abundance of Clostridiales including Ruminococcus was higher in the HP(-) group, whereas Lactobacillales including streptococcus was higher in HP(+) group. In addition the relative abundance of Bacteroidetes in HP(-) group and Actinobacteria (especially, genus Bifidobacterium) in HP(+) group was observed highly. In the bacterial culture-based approach, bacteria strains belonged to Clostridiales such as Clostridium perfringens, Ruminococcus feacis, Blautia sp., Coprococcus comes were isolated in HP(-) sample. In HP(+) sample, Klebsiella, Bacteroides, Bifidobacterium were isolated. Bacillus species, Escherichia/Shigella was enriched regardless of HP exist. Streptococcus was not cultivated in HP(+) group, but isolated in HP(-) group in contrast with metagenome data. Conclusions: We found the intestinal bacterial diversity in the HP(+) group was lower than those in the HP(-) and the microbial composition was different between HP(+) and HP(-). Metagenome analysis showed the order Clostridiales of the phylum Firmicutes were enriched in the HP(-) group while the order Lactobacillales (specially, Streptococcus) were enriched in the HP(+) group. Compared to isolates between two groups, bacteria species belonged to the order Clostridiales such as Clostridium, Ruminococcus , Blautia , Coprococcus were cultivated particularly in HP(-) sample.

Detection of Enterotoxin Genes in Bacillus species Isolated from Cassava Processing Environment in Nigeria.

2022 ◽  
Vol 02 ◽  
Author(s):  
Nkem Torimiro ◽  
Oluwafemi B. Daramola ◽  
Richard K. Omole ◽  
Ifeyimika Z. Adesina
Keyword(s):  
Disease Outbreaks ◽  
Soil Samples ◽  
Bacillus Species ◽  
Frequency Of Occurrence ◽  
Foodborne Disease ◽  
Staple Food ◽  
Lethal Effects ◽  
Enterotoxin Genes ◽  
Bacillus Spp ◽  
Hemolysin Bl

Introduction: The health challenges associated with cassava products as a common staple food for approximately 70% of Africans and part of Asia pose a looming danger due to Bacillus enterotoxins’ presence in the processing environment. Objective: This study investigated the presence of enterotoxigenic genes namely, Bacillus cereus enterotoxin T (bceT), hemolysin bl (hblC, hblD) and non-haemolytic enterotoxin (nheA, nheB and nheC) from Bacillus species isolated from soil of cassava processing environment. Methods: Soil samples from 20 cassava processing sites in Ile-Ife and Modakeke, Nigeria were collected and cultured on Nutrient agar at 37 ºC for 24 hours. Colonies phenotypically identified as Bacillus were identified using Bacillus-specific 16S rRNA-targeted PCR technique. Screened Bacillus spp were assessed for the presence of enterotoxigenic genes using PCR with previously reported primers. Results: A total of 100 Bacillus isolates were selected from this study with Bacillus macerans (33 %) showing the highest frequency of occurrence among the identified species, however, 74 isolates were molecularly confirmed as Bacillus. Amongst the 74 molecularly confirmed Bacillus isolates, 28 (37.84%), 35 (47.30 %) and 37 (50 %) has nhe, hbl and bceT genes respectively. Investigation showed that 42 (56.76 %) of the Bacillus species encoded at least one of the screened enterotoxin genes. Conclusion: The presence of these 3 sets of enterotoxin genes in Bacillus isolated from cassava processing sites calls for immediate attention as they could be pivotal in the release of toxins in cassava products, cause lethal effects via consumption. This study demonstrates the possibility of foodborne disease outbreaks in Bacillus toxin-laden cassava products processed under unhygienic conditions.

scholarly journals Diversity of Lactic Acid Bacteria Associated with Fish and the Fish Farm Environment, Established by Amplified rRNA Gene Restriction Analysis

2007 ◽  
Vol 73 (9) ◽  
pp. 2947-2955 ◽  
Author(s):  
Christian Michel ◽  
Claire Pelletier ◽  
Mekki Boussaha ◽  
Diane-Gaëlle Douet ◽  
Armand Lautraite ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Opportunistic Infections ◽  
Restriction Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Fish Food ◽  
Group A ◽  
Gene Restriction

ABSTRACT Lactic acid bacteria have become a major source of concern for aquaculture in recent decades. In addition to true pathogenic species of worldwide significance, such as Streptococcus iniae and Lactococcus garvieae, several species have been reported to produce occasional fish mortalities in limited geographic areas, and many unidentifiable or ill-defined isolates are regularly isolated from fish or fish products. To clarify the nature and prevalence of different fish-associated bacteria belonging to the lactic acid bacterium group, a collection of 57 isolates of different origins was studied and compared with a set of 22 type strains, using amplified rRNA gene restriction analysis (ARDRA). Twelve distinct clusters were delineated on the basis of ARDRA profiles and were confirmed by sequencing of sodA and 16S rRNA genes. These clusters included the following: Lactococcus raffinolactis, L. garvieae, Lactococcus l., S. iniae, S. dysgalactiae, S. parauberis, S. agalactiae, Carnobacterium spp., the Enterococcus “faecium” group, a heterogeneous Enterococcus-like cluster comprising indiscernible representatives of Vagococcus fluvialis or the recently recognized V. carniphilus, V. salmoninarum, and Aerococcus spp. Interestingly, the L. lactis and L. raffinolactis clusters appeared to include many commensals of fish, so opportunistic infections caused by these species cannot be disregarded. The significance for fish populations and fish food processing of three or four genetic clusters of uncertain or complex definition, namely, Aerococcus and Enterococcus clusters, should be established more accurately.

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