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.

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.

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 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.

scholarly journals Effects of Antibiotics on the Bacterial Community, Metabolic Functions and Antibiotic Resistance Genes in Mariculture Sediments during Enrichment Culturing

2020 ◽  
Vol 8 (8) ◽  
pp. 604
Author(s):  
Meng-Qi Ye ◽  
Guan-Jun Chen ◽  
Zong-Jun Du
Keyword(s):  
Antibiotic Resistance ◽  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
Antibiotic Resistance Genes ◽  
Control Group ◽  
Rrna Gene ◽  
Metabolic Functions ◽  
Culture Dependent

The effect of antibiotics on the diversity and functioning of indigenous microorganisms in the environment has attracted much attention. In this study, effects of exposure to six different antibiotics on the bacterial community, metabolic functions and antibiotic resistance genes (ARGs) in marine sediments during enrichment culturing were investigated. Classical culture-dependent method and high-throughput 16S rRNA gene sequencing method were both applied. In the culture-dependent analysis, the obtained 1549 isolates belonged to four phyla (Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria) and 155 genera. Proteobacteria and Firmicutes were the dominant phyla. The diversity and abundance of obtained bacteria after antibiotic processing exhibited different degrees of decrease. Enrichment culturing for different time could also affect the bacterial community composition. Some genera of bacteria were not isolated in the control group, but they could be isolated in the antibiotic-treated groups. In high-throughput 16S rRNA gene amplicon sequencing analyses, all the effective reads were clustered into 2822 OTUs at 97% similarity cutoff; they were annotated to 49 phyla, 103 class, 220 orders, 347 families, 624 genera and 1122 species. An alpha diversity analysis indicated that the community diversity and richness decreased under antibiotic exposure. The changes at the genus level were much more obvious. Only 48 genera of 129 genera were shared by all the samples. A total of 29 genera which were not detected in the initial control sample could be detected in at least one antibiotic-treated group. SIMPER analysis showed that OTU2543 and OTU1450 were the most common taxa to the dissimilarity of bacterial community between antibiotic-treated groups and the control group. OTU2034 and OUT2543 were the most contributive taxa to dissimilarity of groups incubating for different time. Metabolism was the predominant bacterial function. A total of 30 ARGs were detected in the samples. This study mainly focused on the changes of microbiota under the selective pressure of antibiotics for different time and the results demonstrated that the antibiotic could affect the bacterial diversity and richness in the marine ecosystem.

scholarly journals Antibiotic susceptibility and phylogenetic analyses for the origins and serotypes of Listeriamonocytogenes strains isolated from ice cream and cream cakes

2020 ◽  
Vol 44 (5) ◽  
pp. 1100-1109
Author(s):  
Orkun BABACAN
Keyword(s):  
16S Rrna ◽  
Antibiotic Susceptibility ◽  
Phylogenetic Analyses ◽  
Ice Cream ◽  
Penicillin G ◽  
Diffusion Method ◽  
Rrna Gene ◽  
Gene Sequences ◽  
Serotype 1 ◽  
Hlya Gene

Listeria monocytogenes is a zoonotic bacterium which also infects humans. The aim of this study was to isolate this organism from cream cakes and ice cream and obtain 16S rRNA and hlyA gene sequences from isolates in order to perform phylogenetic analyses. Serotypes and antibiotic susceptibility were also determined. The cream cake and ice cream samples were examined for L. monocytogenes according to ISO 11290-1 and using the mini Vidas LMO 2 kit procedure. Antibiotic susceptibilities were investigated using the disc diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI) standards. The Sanger DNA sequencing method for phylogenetic analysis was used for L. monocytogenes isolates. A total of 16 (n =128, 12.50%) L. monocytogenes strains, 9 (12.16%) from cream cake samples and 7 (12.96%) from ice cream samples, were isolated. This was the first investigation involving sequencing of L. monocytogenes isolated from cream cakes and ice creams in Turkey. All isolates were susceptible to sulfamethoxazole/trimethoprim, tetracycline, streptomycin, gentamicin, meropenem, and erythromycin. The numbers of isolates resistant to sulbactam/ampicillin, penicillin G, chloramphenicol, and ampicillin were 16, 2, 1, and 1, respectively. Moreover, 3 isolates showed intermediate resistance to amikacin and 2 to ciprofloxacin. The hlyA gene sequences of 11 of the L. monocytogenesisolates isolated from milk were closely related to the hlyA gene sequences of the GenBank reference strains. The comparison of the 16s rRNA gene sequences of the L. monocytogenes strains with the GenBank reference serotypes identified 1 isolate as serotype 1/2c, 1 as serotype 1/2a, and 1 as serotype 4. Nucleic acid sequencing is useful for identification of L. monocytogenes. The 16S rRNAand hlyA sequences can be used to determine the origin and relationship between L. monocytogenesisolates, as well as the serotype.

scholarly journals MOLECULAR CHARACTERIZATION OF GENE 16S rRNA MICRO SYMBIONTS IN SPONGE AT MELAWAI BEACH, EAST KALIMANTAN

2018 ◽  
Author(s):  
Ismail Marzuki ◽  
Alfian Noor ◽  
Nursiah La Nafie
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Molecular Characterization ◽  
Morphological Identification ◽  
Rrna Gene ◽  
Isolation And Purification ◽  
East Kalimantan ◽  
Degree Of Similarity ◽  
The 16S Rrna Gene

Molecular characterization studies have been conducted 16S rRNA gene micro symbiont of sponge origin Melawai Beach, Balikpapan in East Kalimantan. Objective analysis of histo- morphological research, isolation-purification, molecular characterization of micro-symbiont genes in order to search symbiont bacteria that can live in extreme environments contaminated hydrocarbon waste. The research method that morphological identification, isolation-purification and molecular characterization of the 16S rRNA gene with Chain Reaction Polymerization method. The results of histo-morphological analysis concluded sponge samples with species of Callyspongia sp. Isolation and purification mikro symbionts of sponge obtained 2 (two) isolates. Characteristics of Isolates 1; spherical shape, colonize and creamy, while isolates 2; jagged shape, oval and white colonies. Molecular characterization of the 16S rRNA gene by PCR, Bacillus subtilis strain BAB-684 identification for isolates one is the number of nucleotide pairs reached 899 bp and the degree of similarity in GenBank reached 89% homologous, while the second is a Bacillus flexus strain PHCDB20 isolates the number reached 950 bp nucleotide pairs with the degree of similarity in GenBank reached 99% homologous

Bakteri Simbion Karang Porites dari Perairan Gunungkidul, Yogyakarta dan Aktivitas Antibakteri terhadap Bakteri Patogen Staphylococcus aureus dan Escherichia coli

2018 ◽  
Vol 7 (1) ◽  
pp. 43
Author(s):  
Rivan Novianto Madilana ◽  
Diah Permata Wijayanti ◽  
Agus Sabdono
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Antibacterial Activity ◽  
16S Rrna ◽  
Bacillus Pumilus ◽  
Coral Tissue ◽  
Diffusion Method ◽  
Rrna Gene ◽  
E Coli ◽  
Porites Coral

Porites merupakan genus karang yang memiliki persebaran luas di Indonesia, termasuk perairan Gunungkidul, Yogyakarta. Penelitian menunjukkan bahwa bakteri simbion karang Porites memiliki potensi antibakteri dalam menanggulangi bakteri patogen. Penelitian ini betujuan untuk mengetahui jenis bakteri simbion karang Porites dari Perairan Gunungkidul Yogyakarta yang memiliki aktivitas antibakteri patogen S. aureus dan E. coli. Bakteri simbion diisolasi dari fragmen jaringan karang dengan pengenceran bertingkat, kemudian uji aktivitas antibakteri dilakukan dengan menggunakan metode overlay dan difusi paperdisk. Delapan dari 64 isolat aktif menghambat kedua bakteri patogen Staphylococcus aureus dan Escherichia coli. Dua diantaranya merupakan isolat unggul yang menunjukkan aktivitas antibakteri paling baik. Kedua isolat selanjutnya diidentifikasi karakteristik molekular DNA dengan sekuen gen 16S rRNA. Hasil identifikasi 16S rRNA menunjukkan isolat GKP1.4.3 memiliki kesamaan 99% dengan Bacillus pumilus strain NBRC 12092, dan isolat GKP3.2.2 memiliki kesamaan 99% dengan Vibrio natriegens strain NBRC 15636.Porites is a coral which has distributed widely in Indonesia, including Gunungkidul Waters, Yogyakarta. Research has shown that Porites coral symbiont bacteria have antibacterial potency against pathogenic bacteria.This study aims to determine the type of Porites coral symbiont bacteria collected from the waters of Gunungkidul Yogyakarta which has antibacterial activity of S. aureus and E. coli. Bacteria symbionts were isolated from coral tissue fragments by serial dillution method, while antibacterial activity was performed by using overlay and paperdisk diffusion method. Eight of the 64 active isolates inhibited both pathogenic bacteria S. aureus and E. coli. Two of 8 isolates showed stronger antibacterial activity. The two isolates subsequently identified the molecular characteristics of DNA with the 16S rRNA gene sequence. The identification of 16S rRNA showed that GKP1.4.3 isolate had 99% similarity with Bacillus pumilus strain NBRC 12092, and GKP3.2.2 isolate had 99% similarity with Vibrio natriegens strain NBRC 15636.

Isolation and Molecular Characterization of Riemerella anatipestifer from Domesticated Ducks of Assam, India

2021 ◽  
Author(s):  
M.K. Doley ◽  
S. Das ◽  
R.K. Sharma ◽  
P. Borah ◽  
D.K. Sarma ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Molecular Characterization ◽  
Molecular Identification ◽  
Rrna Gene ◽  
Eastern Region ◽  
Riemerella Anatipestifer ◽  
Field Samples ◽  
Apparently Healthy

Background: Riemerella anatipestifer (R. anatipestifer) is a gram negative, microaerophilic, non-motile, bipolar bacteria. High genetic diversity and molecular differentiation were reported among field isolates. Although the bacterium causes one of the most economically important duck diseases in the north-eastern region of India, little work has been done on isolation, identification and molecular characterization of the bacteria. Hence, the present investigation was undertaken with a view to characterize the R. anatipestifer isolates from ducks of Assam.Methods: Phenotypic and molecular identification of R. anatipestifer isolates from domesticated ducks of Assam, India were carried out during the period from February, 2019 to January 2020. A total of 624 samples (Ocular swab, throat swab, liver, spleen, kidney, brain, heart, lung) from ducks comprising of apparently healthy, ailing and dead ducks were collected from five districts of Assam, India were processed to isolate and identify the bacteria. The tentative identification of the bacteria was done based on phenotypic characteristics viz., colony morphology, growth characteristics and biochemical reactions. All the phenotypically positive isolates were further subjected to molecular identification based on PCR assay targeting 16S rRNA gene and ERIC sequence.Result: The bacteria could be isolated from different field samples. The highest percentage of the samples that yielded the bacteria are from brain (76%) followed by spleen (74%) of dead ducks and less number of ocular swab (33%) from apparently healthy ducks were found positive. Sequencing of the amplified product of the selected R. anatipestifer isolates targeting 16S rRNA gene revealed homology percentage of 96.5-100%. Further, sequences representing five geographical locations were submitted to NCBI gene bank. Phylogenetic studies of the isolates indicated that there is prevalence of at least two genetically different strains of R. anatipestifer in the study area. The study suggested that the R. anatipestifer infection is endemic in Assam causing varying rate of morbidity (39%) and mortality (53%) and molecular based confirmation is necessary besides phenotypic identification.

Sign in / Sign up

Export Citation Format

Share Document