scholarly journals ISOLATION AND IDENTIFICATION OF BACTERIOCIN PRODUCING MICROBES USING BIOCHEMICAL AND MOLECULAR TOOLS AND ANALYSIS OF ITS BIOPRESERVATION POTENTIAL

2016 ◽  
Vol 9 (9) ◽  
pp. 278
Author(s):  
Parmar Keshri Nandan ◽  
Anshita Nagar
Keyword(s):  
16S Rrna ◽  
Molecular Characterization ◽  
Developed Countries ◽  
Diffusion Method ◽  
Dilution Method ◽  
Rrna Gene ◽  
Accession Number ◽  
Isolation And Identification ◽  
16S Rrna Analysis ◽  
Urease Test

ABSTRACTObjective: Food safety is a matter of utmost importance in developing countries as well as in developed countries, so keeping this in mind this researchwork deals with the identification and characterization of bacteriocin producing microbes by using biochemical and molecular characterization. This study has also covered the biopreservation potential of bacteriocin produced by these microbes against sapodilla, tomato and banana as well.Methods: For the purpose of sample collection and isolation, samples of milk, curd and gangajal water were taken and bacteriocin producing microbes were isolated by using serial dilution method. Screening of bacteriocin producing microbe was done by antibacterial sensitivity test using agar well diffusion method against Bacillus amyloliquefaciens, Escherichia Coli, Staphylococcus aureus and Pseudomonas aeruginosa by determining their zone of inhibition. Biochemical characterization was done by using different tests, such as, catalase test, mannitol test, citrate test, gelatin test, maltose test, indole test, urease test, lactose test etc. Molecular characterization was done by using 16S rRNA gene sequencing. Preservative action of bacteriocinwas observed on fruits that comprise sapodilla, tomato and banana by spraying bacteriocin on them and analyzing their activities shows for at least10 days.Results: Microbes were found to be Enterococcus faecalis (Accession number KX011874) and Bacillus cereus (Accession number KX011875). Periodicobservatory studies reflect that using bacteriocin, banana can be preserved for nearly 6-7 days while sapodilla for 8-9 days and tomato for 9-10 days.Conclusion: From present study we would like to conclude that bacteriocins produced by microbes which is found in milk or curd can also be used asbiopreservatives for these defined fruits that is sapodilla, tomato and banana.Keywords: Bacteriocin, Biopreservation, 16S rRNA analysis.

scholarly journals Molecular Characterization and Detection of Antibiotic Resistance Genes in Pseudomonas Species Isolated from Tympanotonus fuscatus

2020 ◽  
pp. 37-45
Author(s):  
T. Sampson ◽  
N. P. Akani ◽  
I. O. Hakam
Keyword(s):  
16S Rrna ◽  
Molecular Characterization ◽  
Plasmid Dna ◽  
Antibiotic Resistance Genes ◽  
West African ◽  
Diffusion Method ◽  
Rrna Gene ◽  
State University ◽  
Pseudomonas Species ◽  
Rivers State

Aim: This was carried out to characterize Pseudomonas species isolated from the West African Mud Creeper (Tympanotonus fuscatus) molecularly and as well detect the possible presence of inducible AmpC gene that mediates resistance to cephalosporins and most penicillins. Sample: Tympanotonus fuscatus (West African Mud Creeper), a gastropod mollusc found in brackish waters of West Africa was used for the study. Place and Duration of Study: This study was carried out between February and August 2019 at the Department of Microbiology, Rivers State University, Port Harcourt, Nigeria. Methodology: Thirty two (32) Pseudomonas species were isolated and identified culturally from T. fuscatus. Pseudomonas species isolates were subjected to a group of ten (10) antibiotics using the Kirby-Bauer disc diffusion method and resistant isolates were screened molecularly for the presence of resistance gene (AmpC). AmpC screening was carried out in a step wise process of DNA extraction, quantification, amplification of ampC gene using appropriate primer and Agarose gel electrophoresis to reveal which DNA extracts had ampC genes amplified. The two most resistant isolates had their 16S rRNA sequenced, identified and were also profiled for plasmids by extracting plasmid DNA. Results: Results revealed that 96.67% of isolates had MAR index greater than 0.2 indicating high a risk source of contamination where antibiotics are often used. Results also showed the presence of ampC gene in seven (7) out of the twelve (12) isolates screened for ampC gene. Molecular characterization via sequencing of the 16S rRNA gene of the two (2) most resistant isolates confirmed that both isolates were strains of Pseudomonas aeruginosa. Profiling of plasmids also revealed the presence of plasmid DNA of about 10 kilo base pairs in both isolates profiled. Conclusion: This study has revealed the resistance ability of Pseudomonas and some reasons behind this resistance. Appropriate investigation into antimicrobial resistance is recommended for the administration of drugs for the treatment of food-mediated Pseudomonas infections.

Screening and Molecular Characterization of Cellulase Producing Actinobacteria from Litchi Orchard

2019 ◽  
Vol 13 (1) ◽  
pp. 90-101
Author(s):  
Sanju Kumari ◽  
Utkarshini Sharma ◽  
Rohit Krishna ◽  
Kanak Sinha ◽  
Santosh Kumar
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Molecular Characterization ◽  
Biochemical Characterization ◽  
Evolutionary Relationship ◽  
Gene Sequencing ◽  
Cellulase Activity ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Rrna Gene Sequencing

Background: Cellulolysis is of considerable economic importance in laundry detergents, textile and pulp and paper industries and in fermentation of biomass into biofuels. Objective: The aim was to screen cellulase producing actinobacteria from the fruit orchard because of its requirement in several chemical reactions. Methods: Strains of actinobacteria were isolated on Sabouraud’s agar medium. Similarities in cultural and biochemical characterization by growing the strains on ISP medium and dissimilarities among them perpetuated to recognise nine groups of actinobacteria. Cellulase activity was measured by the diameter of clear zone around colonies on CMC agar and the amount of reducing sugar liberated from carboxymethyl cellulose in the supernatant of the CMC broth. Further, 16S rRNA gene sequencing and molecular characterization were placed before NCBI for obtaining recognition with accession numbers. Results: Prominent clear zones on spraying Congo Red were found around the cultures of strains of three groups SK703, SK706, SK708 on CMC agar plates. The enzyme assay for carboxymethylcellulase displayed extra cellulase activity in broth: 0.14, 0.82 and 0.66 µmol mL-1 min-1, respectively at optimum conditions of 35°C, pH 7.3 and 96 h of incubation. However, the specific cellulase activities per 1 mg of protein did not differ that way. It was 1.55, 1.71 and 1.83 μmol mL-1 min-1. The growing mycelia possessed short compact chains of 10-20 conidia on aerial branches. These morphological and biochemical characteristics, followed by their verification by Bergey’s Manual, categorically allowed the strains to be placed under actinobacteria. Further, 16S rRNA gene sequencing, molecular characterization and their evolutionary relationship through phylogenetics also confirmed the putative cellulase producing isolates of SK706 and SK708 subgroups to be the strains of Streptomyces. These strains on getting NCBI recognition were christened as Streptomyces glaucescens strain SK91L (KF527284) and Streptomyces rochei strain SK78L (KF515951), respectively. Conclusion: Conclusive evidence on the basis of different parameters established the presence of cellulase producing actinobacteria in the litchi orchard which can convert cellulose into fermentable sugar.

Molecular characterization of the bacterial communities present in sheep's milk and cheese produced in South Brazilian Region via 16S rRNA gene metabarcoding sequencing

LWT ◽  
2021 ◽  
Vol 147 ◽  
pp. 111579
Author(s):  
Creciana M. Endres ◽  
Ícaro Maia S. Castro ◽  
Laura D. Trevisol ◽  
Juliana M. Severo ◽  
Michele B. Mann ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Molecular Characterization ◽  
Bacterial Communities ◽  
Rrna Gene

scholarly journals Evaluation of Commercial Disinfectants against Staphylococcus lentus and Micrococcus spp. of Poultry Origin

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Otun Saha ◽  
Nadira Naznin Rakhi ◽  
Arif Istiaq ◽  
Israt Islam ◽  
Munawar Sultana ◽  
...  
Keyword(s):  
Micrococcus Luteus ◽  
Bacterial Species ◽  
16S Rrna Gene Sequencing ◽  
Average Diameter ◽  
Diffusion Method ◽  
Dilution Method ◽  
Rrna Gene ◽  
Poultry Farms ◽  
Rrna Gene Sequencing ◽  
Selection Of

Introduction. Effective sanitation strategies for poultry farms require an appropriate selection of the disinfectant based on the contaminants present and their sensitivity to the disinfectants. Aim. The current study investigated the prevalence of streptococci/micrococci in poultry farms of Bangladesh and the efficacy of commercial disinfectants (Savlon, Lysol, Quatovet, Virkon S, and Virocid) along with alcohol against these pathogens to adopt appropriate strategies. Materials and Methods. Conventional approaches and the 16S rRNA gene sequencing were performed to confirm the isolates at the species level along with microtiter biofilm assay to determine their biofilm-forming ability. Efficacy of the disinfectants was tested against those isolates using agar well diffusion and minimum inhibitory concentration (MIC) test by broth dilution method using different dilutions of the disinfectants. Results. Staphylococcus lentus (n = 32), Micrococcus luteus (n = 7), and Micrococcus aloeverae (n = 4) were confirmed among 102 presumptively screened streptococci/micrococci isolates from 43 samples. No single disinfectant showed equally high efficacy against all three bacterial species in agar well diffusion test, although Virocid showed the lowest MIC against all three of them. Lysol was least effective among the commercial disinfectants by both MIC and diffusion method, although each commercial disinfectant was more effective than alcohol. Considering both the average diameter of the inhibition zones and the MIC values, efficacy can be interpreted as Virocid > Quatovet > Savlon > Virkon S > Lysol. Although the efficacy decreased with decreasing concentration, the disinfectants retained a satisfactory level of efficacy at 50% concentration. Among test pathogens, M. aloeverae was the most sensitive to the disinfectants and the weakest biofilm producers, whereas 4/14 S. lentus and 1/5 M. luteus were strong biofilm producers, which may cause more reduction in the efficacy in environmental conditions. Conclusion. As no ideal disinfectant was found in the study, the efficacy of the disinfectants should be routinely evaluated and validated to ensure the sanitation standards in the poultry sector.

scholarly journals Isolation and Identification of Resistant Bacteria from Petroleum Producing Vicinity by Gene Amplification and Sequencing

2021 ◽  
pp. 1-8
Author(s):  
O. Aleruchi ◽  
O. Obire
Keyword(s):  
16S Rrna ◽  
Phylogenetic Study ◽  
Resistant Bacteria ◽  
Rrna Gene ◽  
Bacterial Isolates ◽  
Antibiotic Resistant Bacteria ◽  
Isolation And Identification ◽  
Antibiotic Resistant ◽  
Disinfection Process ◽  
Adaptive Mechanisms

This investigation focuses on molecular identification of antibiotic resistant bacteria isolated from petroleum producing vicinity using 16S rRNA sequencing based technique. The bacterial 16s rRNA gene sequences were amplified using polymerase chain reaction, sequenced,  characterized and compared by using primers which has been compared to national center for biotechnology information (NCBI) sequence database. The presence of the plasmid mediated antibiotic resistance determinants CTX-M and QNRB genes in the bacterial isolates were analyzed. A total of four bacterial isolates that were resistant to all the antibiotic agents used were identified molecularly. The BLAST results showed 100 % similarity and phylogenetic study indicated that the genes were evolutionarily related to Morganella morganii, Pseudomonas xiamenensis, Chryseobacterium cucumeris and Staphylococcus sp., respectively. The genes obtained were submitted to the NCBI gene bank and were assigned accession number; MN094330, MN094331, MN094332 and MN094333, respectively. CTX-M and QNRB genes were however absent in the bacterial isolates. The result identified some peculiar abilities of the bacterial isolates to be resistant to antibiotics and suggests a correlation with resistance and hydrocarbon utilizing bacteria. The level of resistance could be as a result of the disinfection process during wastewater treatment procedure or the same adaptive mechanisms possessed by the isolates to control the hydrocarbon concentration in their cell. The study also clearly indicates that these wastewaters, when discharged into the environment directly may pose a risk for the spread of antibiotic resistant bacteria.

scholarly journals First Detection of Pathogenic Escherichia coli Isolates Associated With Donkey Foals’ Diarrhea in Northern China

2020 ◽  
Author(s):  
Liu Wen-qiang ◽  
Xia Nan ◽  
Zhang Jing-wen ◽  
Wang Ren-hu ◽  
Jiang Gui-miao
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Virulence Factors ◽  
Influence Factor ◽  
Genetic Relationships ◽  
Northern China ◽  
Multi Drug Resistance ◽  
Rrna Gene ◽  
Isolation And Identification ◽  
Antibiotics Sensitivity

ABSTRACTObjectiveThe aim of this study was to identify the biological features, influence factor and Genome-wide properties of pathogenic donkey Escherichia coli (DEC) isolates associated with severe diarrhea in Northern China.MethodsThe isolation and identification of DEC isolates were carried out by the conventional isolation、automatic biochemical analysis system、serotype identification、16S rRNA test、animal challenge and antibiotics sensitivity examination. The main virulence factors were identified by PCR. The complete genomic re-sequence and frame-sequence were analyzed.Results216 strains of DEC were isolated from diarrhea samples, conforming to the bacterial morphology and biochemical characteristics of E.coli. The average size of the pure culture was 329.4 nm×223.5 nm. Agglutination test showed that O78 (117/179, 65.4%) was the dominant serotype and ETEC(130/216, 60.1%) was the dominant pathogenic type. Noticeable pathogenic were observed in 9 of 10 (90%) randomly selected DEC isolates caused the death of test mice (100%, 5/5) within 6h∼48h, 1 of 10 (10%) isolates caused the death of test mice (40%, 2/5) within 72h. Our data confirmed that DEC plays an etiology role in dirarrea/death case of donkey foal. Antibiotics sensitivity test showed significant susceptibility to DEC isolates were concentrated in Nor、EFT、ENR、CIP and AMK,while the isolates with severe antibiotic resistance was AM、TE、APR、FFC、RL and CN. Multi-drug resistance was also observed. A total of 15 virulence gene fragments were determined from DEC(n=30) including OMPA (73%), safD (77%), traTa (73%), STa(67%), EAST1 (67%), astA (63%), kspII (60%), irp2 (73%), iucD (57%), eaeA (57%), VAT (47%), iss (33%), cva (27%), ETT2 (73%) and K88 (60%) respectively. More than 10 virulence genes from 9 of 30(30%) DEC strains were detected, while 6 of 30(20%) DEC strains detected 6 virulence factors. phylogenetic evolutionary tree of 16S rRNA gene from different isolates shows some variability. The original data volume obtained from the genome re-sequencing of DEC La18 was 2.55G and Genome framework sequencing was carried out to demonstrate the predicted functions and evolutionary direction and genetic relationships with other animal E.coli.ConclusionsThese findings provide firstly fundamental data that might be useful in further study of the role of DEC and provide a new understanding of the hazards of traditional colibacillosis due to the appear of new production models.

scholarly journals Nocardia jejuensis sp. nov., a novel actinomycete isolated from a natural cave on Jeju Island, Republic of Korea

2006 ◽  
Vol 56 (3) ◽  
pp. 559-562 ◽  
Author(s):  
Soon Dong Lee
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
Jeju Island ◽  
Republic Of Korea ◽  
16S Rrna Gene Sequence ◽  
Phylogenetic Position ◽  
Dilution Method ◽  
Rrna Gene ◽  
Rrna Gene Sequence

A novel actinomycete, strain N3-2T, was isolated from a natural cave on Jeju Island, Republic of Korea, using a dilution method and was subjected to polyphasic taxonomy. The almost complete 16S rRNA gene sequence was determined by direct sequencing of the purified PCR product and was compared with those of representatives of the genus Nocardia. It was revealed from the phylogenetic analysis that the organism forms a distinct clade between the Nocardia salmonicida cluster and the Nocardia alba branch within the evolutionary radius occupied by the genus Nocardia of the family Nocardiaceae. The organism showed 16S rRNA gene sequence similarity of 97·4 % with its nearest phylogenetic neighbours, namely N. salmonicida and N. alba. The chemotaxonomic properties, such as the principal amino acid of peptidoglycan, predominant menaquinone and polar lipids, supported the classification in the genus Nocardia. The organism was readily differentiated from Nocardia species with validly published names by a broad set of phenotypic properties and its unique phylogenetic position; the name Nocardia jejuensis sp. nov. is proposed, with N3-2T (=JCM 13281T=NRRL B-24430T) as the type strain.

scholarly journals Molecular Characterization of anEndozoicomonas-Like Organism Causing Infection in the King Scallop (Pecten maximusL.)

2017 ◽  
Vol 84 (3) ◽  
Author(s):  
Irene Cano ◽  
Ronny van Aerle ◽  
Stuart Ross ◽  
David W. Verner-Jeffreys ◽  
Richard K. Paley ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Molecular Characterization ◽  
Mass Mortality ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Content Type ◽  
The 16S Rrna Gene

ABSTRACTOne of the fastest growing fisheries in the UK is the king scallop (Pecten maximusL.), also currently rated as the second most valuable fishery. Mass mortality events in scallops have been reported worldwide, often with the causative agent(s) remaining uncharacterized. In May 2013 and 2014, two mass mortality events affecting king scallops were recorded in the Lyme Bay marine protected area (MPA) in Southwest England. Histopathological examination showed gill epithelial tissues infected with intracellular microcolonies (IMCs) of bacteria resemblingRickettsia-like organisms (RLOs), often with bacteria released in vascular spaces. Large colonies were associated with cellular and tissue disruption of the gills. Ultrastructural examination confirmed the intracellular location of these organisms in affected epithelial cells. The 16S rRNA gene sequences of the putative IMCs obtained from infected king scallop gill samples, collected from both mortality events, were identical and had a 99.4% identity to 16S rRNA gene sequences obtained from “CandidatusEndonucleobacter bathymodioli” and 95% withEndozoicomonasspecies.In situhybridization assays using 16S rRNA gene probes confirmed the presence of the sequenced IMC gene in the gill tissues. Additional DNA sequences of the bacterium were obtained using high-throughput (Illumina) sequencing, and bioinformatic analysis identified over 1,000 genes with high similarity to protein sequences fromEndozoicomonasspp. (ranging from 77 to 87% identity). Specific PCR assays were developed and applied to screen for the presence of IMC 16S rRNA gene sequences in king scallop gill tissues collected at the Lyme Bay MPA during 2015 and 2016. There was 100% prevalence of the IMCs in these gill tissues, and the 16S rRNA gene sequences identified were identical to the sequence found during the previous mortality event.IMPORTANCEMolluscan mass mortalities associated with IMCs have been reported worldwide for many years; however, apart from histological and ultrastructural characterization, characterization of the etiological agents is limited. In the present work, we provide detailed molecular characterization of anEndozoicomonas-like organism (ELO) associated with an important commercial scallop species.

scholarly journals Studies on Antibacterial Activity and Diversity of Cultivable Actinobacteria Isolated from Mangrove Soil in Futian and Maoweihai of China

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Feina Li ◽  
Shaowei Liu ◽  
Qinpei Lu ◽  
Hongyun Zheng ◽  
Ilya A. Osterman ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Fluorescent Protein ◽  
Protein Biosynthesis ◽  
Antibacterial Activities ◽  
Antagonistic Activity ◽  
Diffusion Method ◽  
Rrna Gene ◽  
Reporter System ◽  
Mangrove Soil

Mangrove is a rich and underexploited ecosystem with great microbial diversity for discovery of novel and chemically diverse antimicrobial compounds. The goal of the study was to explore the pharmaceutical actinobacterial resources from mangrove soil and gain insight into the diversity and novelty of cultivable actinobacteria. Consequently, 10 mangrove soil samples were collected from Futian and Maoweihai of China, and the culture-dependent method was employed to obtain actinobacteria. A total of 539 cultivable actinobacteria were isolated and distributed in 39 genera affiliated to 18 families of 8 orders by comparison analysis of partial 16S rRNA gene sequences. The dominant genus was Streptomyces (16.0 %), followed by Microbacterium (14.5 %), Agromyces (14.3 %), and Rhodococcus (11.9 %). Other 35 rare actinobacterial genera accounted for minor proportions. Notably, 11 strains showed relatively low 16S rRNA gene sequence similarities (< 98.65 %) with validly described species. Based on genotypic analyses and phenotypic characteristics, 115 out of the 539 actinobacterial strains were chosen as representative strains to test their antibacterial activities against “ESKAPE” bacteria by agar well diffusion method and antibacterial mechanism by the double fluorescent protein reporter system. Fifty-four strains in 23 genera, including 2 potential new species, displayed antagonistic activity in antibacterial assay. Meanwhile, 5 strains in 3 genera exhibited inhibitory activity on protein biosynthesis due to ribosome stalling. These results demonstrate that cultivable actinobacteria from mangrove soil are potentially rich sources for discovery of new antibacterial metabolites and new actinobacterial taxa.

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