scholarly journals Investigation of Mutations in the Rifampin-Resistance-Determining Region of the rpoB Gene of Brucella melitensis by Gene Analysis

2021 ◽  
Vol 14 (2) ◽  
Author(s):  
Maryam Dadar ◽  
Nasim Bazrgari ◽  
Ghasem Ali Garosi ◽  
Sameer Hassan
Keyword(s):  
Genetic Diversity ◽  
Synovial Fluid ◽  
Binding Sites ◽  
Brucella Melitensis ◽  
Gene Sequencing ◽  
Rpob Gene ◽  
Gene Analysis ◽  
Disk Diffusion ◽  
Rifampin Resistance ◽  
Susceptibility Tests

Background: RNA polymerase beta subunit (rpoB) gene analysis in bacterial communities is known as a method for determining rifampin sensitivity and genetic diversity among Brucella spp. Detection of antibiotic resistance among Brucella isolates can be a critical approach to control brucellosis. However, rpoB gene analysis of Brucella melitensis for assessing rifampicin resistance has not yet been performed in Iran, which is considered an endemic area for brucellosis. Objectives: The aim of this study was to analyze the whole sequence of rpoB genes of different B. melitensis isolates from humans to identify the single-nucleotide polymorphisms (SNPs) and mutations related to rifampin resistance and to analyze the genetic diversity of these bacteria in Iran. Methods: Between 2017 and 2019, a total of 156 blood samples along with 12 synovial fluid specimens were collected from brucellosis patients in different Iranian provinces and subjected to bacterial culture in Brucella selective media. Brucella identification was carried out using classical biotyping and molecular examinations. Polymerase chain reaction (PCR)-based amplification of the rpoB gene was performed by specific rpoB primers for whole gene sequencing. The antimicrobial susceptibility of Brucella isolates was assessed using disk diffusion susceptibility tests and minimal inhibitory concentration (MIC) methods. The presence of rifampin-binding sites and SNPs were investigated through rpoB whole gene sequencing. Results: Clinical B. melitensis isolates were obtained from blood (13) and synovial fluid (1) samples of patients from different regions of Iran. The results of MIC and disk diffusion susceptibility tests showed that all the isolates were sensitive to rifampin except for one isolate showing intermediate rifampin resistance based on the standards defined for slow-growing bacteria by the Clinical and Laboratory Standards Institute (CLSI). Gene analysis for identifying the mutations related to rifampin resistance and investigating genetic diversity showed that none of the B. melitensis isolates had missense mutations, confirming the susceptibility of all the studied isolates to rifampin. Conclusions: The present study revealed that rpoB gene analysis could be used for the efficient and precise identifying of the mutations related to rifampin resistance, investigating rifampin binding sites, and genotyping Brucella species. Furthermore, the identification of B. melitensis isolates with intermediate resistance to rifampicin highlighted the importance of periodically carrying out antibiotic susceptibility testing. The molecular detection of rpoB mutations in different Brucella isolates may help to prevent the spread of rifampin-resistant Brucella spp. among humans and livestock.

scholarly journals Amplification Refractory Mutation System – Polymerase Chain Reaction for Rapid Detection of rpoB Gene Mutations in Mycobacterium tuberculosis

2017 ◽  
Vol 5 (1) ◽  
pp. 81-85 ◽  
Author(s):  
Hemanta Kumari Chaudhary ◽  
Mitesh Shrestha ◽  
Prakash Chaudhary ◽  
Bal Hari Poudel
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Rpob Gene ◽  
Amplification Refractory Mutation System ◽  
Chain Reaction ◽  
Arms Pcr ◽  
Mutation System ◽  
Mdr Tb ◽  
Total Dna ◽  
Polymerase Chain ◽  
Rifampin Resistance

Multidrug-resistant tuberculosis (MDR-TB) has become a serious worldwide threat including in Nepal. MDR-TB refers to the pathological condition whereby Mycobacterium tuberculosis becomes resistant to the first line of drug treatment i.e. rifampin and isoniazid. Resistance to rifampin (RIF) is mainly caused by the mutations in the rpoB gene which codes for the β-subunit of RNA polymerase. In this study, Amplification Refractory Mutation System – Polymerase Chain Reaction (ARMS – PCR) technique has been used to detect mutations in the rpoB gene of Mycobacterium tuberculosis. Total DNA samples of 34 phenotypic MDR-TB were subjected to ARMS – PCR using three different codon specific primers (516, 526 and 531). These three codons occupy large portion of total mutation responsible for rifampin resistance. Out of the total DNA samples, all were bearing mutation in at least one of the three codons mentioned. Of those bearing mutation, the highest number had mutation in codon 531 (97.05 %) followed by codon 516 (17.64 %) and finally in codon 526 (11.76%) respectively. Hence, ARMS – PCR may be used as an alternative diagnostic technique for detection of rifampin resistance in Mycobacterium tuberculosis strains, especially for a developing country like Nepal.Int. J. Appl. Sci. Biotechnol. Vol 5(1): 81-85

scholarly journals Molecular Analysis of Codon 548 in therpoBGene Involved in Mycobacterium tuberculosis Resistance to Rifampin

2014 ◽  
Vol 59 (3) ◽  
pp. 1542-1548 ◽  
Author(s):  
Yu-Tze Horng ◽  
Wen-Yih Jeng ◽  
Yih-Yuan Chen ◽  
Che-Hung Liu ◽  
Horng-Yunn Dou ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Bacterial Resistance ◽  
Drug Susceptibility ◽  
Content Type ◽  
Stable Hydrogen Bond ◽  
Resistance Patterns ◽  
Resistant Strains ◽  
Rifampin Resistance ◽  
Susceptibility Tests ◽  
Dna Complex

ABSTRACTMostMycobacterium tuberculosisrifampin-resistant strains have been associated with mutations in an 81-bp rifampin resistance-determining region (RRDR) in the generpoB. However, if this region alone were targeted, rifampin-resistant strains with mutations outside the RRDR would not be detected. In this study, among 51 rifampin-resistant clinical isolates analyzed by sequencing 1,681-bp-long DNA fragments containing the RRDR, 47 isolates contained mutations within the RRDR, three isolates contained mutations both within and outside the RRDR, and only one isolate had a single missense mutation (Arg548His) located outside the RRDR. A drug susceptibility test of recombinantMycobacterium smegmatisandM. tuberculosisisolates carrying mutatedrpoB(Arg548His) showed an increased MIC for rifampin compared to that of the control strains. Modeling of the Arg548His mutant RpoB-DNA complex revealed that the His548 side chain formed a more stable hydrogen bond structure than did Arg548, reducing the flexibility of the rifampin-resistant cluster II region of RpoB, suggesting that the RpoB Arg548His mutant does not effectively interact with rifampin and results in bacterial resistance to the drug. This is the first report on the relationship between the mutation in codon 548 of RpoB and rifampin resistance in tuberculosis. The novel mutational profile of therpoBgene described here will contribute to the comprehensive understanding of rifampin resistance patterns and to the development of a useful tool for simple and rapid drug susceptibility tests.

Rifampin resistance mutations in the rpoB gene of Enterococcus faecalis impact host macrophage cytokine production

Cytokine ◽  
2022 ◽  
Vol 151 ◽  
pp. 155788
Author(s):  
Darya V. Urusova ◽  
Joseph A. Merriman ◽  
Ananya Gupta ◽  
Liang Chen ◽  
Barun Mathema ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Cytokine Production ◽  
Rpob Gene ◽  
Resistance Mutations ◽  
Host Macrophage ◽  
Rifampin Resistance ◽  
Macrophage Cytokine

scholarly journals New Genus Fibralongavirus in Siphoviridae Phages of Staphylococcus pseudintermedius

Viruses ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1143
Author(s):  
Michal Zeman ◽  
Pavol Bárdy ◽  
Veronika Vrbovská ◽  
Pavel Roudnický ◽  
Zbyněk Zdráhal ◽  
...  
Keyword(s):  
New Genus ◽  
Bacterial Species ◽  
Gene Sequencing ◽  
Rpob Gene ◽  
Global Alignment ◽  
Significant Similarity ◽  
Staphylococcus Pseudintermedius ◽  
The Family ◽  
Genomic Analyses ◽  
Taxonomical Classification

Bacteriophages of the significant veterinary pathogen Staphylococcus pseudintermedius are rarely described morphologically and genomically in detail, and mostly include phages of the Siphoviridae family. There is currently no taxonomical classification for phages of this bacterial species. Here we describe a new phage designated vB_SpsS_QT1, which is related to phage 2638A originally described as a Staphylococcus aureus phage. Propagating strain S. aureus 2854 of the latter was reclassified by rpoB gene sequencing as S. pseudintermedius 2854 in this work. Both phages have a narrow but different host range determined on 54 strains. Morphologically, both of them belong to the family Siphoviridae, share the B1 morphotype, and differ from other staphylococcal phage genera by a single long fibre at the terminus of the tail. The complete genome of phage vB_SpsS_QT1 was sequenced with the IonTorrent platform and expertly annotated. Its linear genome with cohesive ends is 43,029 bp long and encodes 60 predicted genes with the typical modular structure of staphylococcal siphophages. A global alignment found the genomes of vB_SpsS_QT1 and 2638A to share 84% nucleotide identity, but they have no significant similarity of nucleotide sequences with other phage genomes available in public databases. Based on the morphological, phylogenetic, and genomic analyses, a novel genus Fibralongavirus in the family Siphoviridae is described with phage species vB_SpsS_QT1 and 2638A.

scholarly journals Clonal diversity of Haemophilus influenzae carriage isolated from under the age of 6 years children

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Fahimeh Shooraj ◽  
Bahman Mirzaei ◽  
Seyed Fazlollah Mousavi ◽  
Farzaneh Hosseini
Keyword(s):  
Genetic Diversity ◽  
Gel Electrophoresis ◽  
Genomic Analysis ◽  
Clonal Diversity ◽  
Invasive Disease ◽  
Pulsed Field Gel Electrophoresis ◽  
Genomic Diversity ◽  
Biochemical Tests ◽  
Genetic Origin ◽  
Susceptibility Tests

Abstract Objectives Pharyngeal carriers such as H. influenzae seem to constitute the only reservoir and probably the only transmission vehicle of the invasive disease. The aims of this study were to estimate the prevalence of H. influenzae carriage, to characterize antibiotic susceptibility, and to explore genetic diversity of H. influenzae isolates. Sampling was carried out as nasopharynx swabs among children less than 6 years old volunteers. After traditional biochemical tests, isolates were confirmed by targeting omp6 sequence. Following the susceptibility tests, genomic diversity of strains was analyzed by Pulsed-Field Gel Electrophoresis procedure. Results Out of 328 nasopharynx swabs, 73 strains were identified as H. influenzae. Among H. influenzae isolates, resistance to chloramphenicol (42%) and ampicillin (43%) was observed. Levofloxacin is the most effective antibiotic and the least effect belonged to tetracycline. By genomic analysis of selected H. influenza, 28 PFGE patterns were achieved among which 11 patterns included at least 2 strains. All strains clustered into 25 different clones. The dendrogram analysis of the isolated H. influenzae strains showed that some of these strains had a clonal relationship and common genetic origin. According to our results, antibiotic resistance didn’t show any significant correlation with the clonality of strains.

scholarly journals Genome-Scale Phylogeny and Evolutionary Analysis of Ross River Virus Reveals Periodic Sweeps of Lineage Dominance in Western Australia, 1977–2014

2019 ◽  
Vol 94 (2) ◽  
Author(s):  
Alice Michie ◽  
Vijaykrishna Dhanasekaran ◽  
Michael D. A. Lindsay ◽  
Peter J. Neville ◽  
Jay Nicholson ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Western Australia ◽  
Evolutionary History ◽  
Gene Analysis ◽  
Ross River Virus ◽  
Whole Genome ◽  
Evolutionary Analysis ◽  
Genotype 4 ◽  
E2 Gene ◽  
Genome Scale

ABSTRACT Ross River virus (RRV), an alphavirus of the Togaviridae family, is the most medically significant mosquito-borne virus of Australia. Past RRV phylogenetic and evolutionary analyses have been based on partial genome analyses only. Three geographically distinct RRV lineages, the Eastern, the Western, and the supposedly extinct North-Eastern lineage, were classified previously. We sought to expand on past phylogenies through robust genome-scale phylogeny to better understand RRV genetic diversity and evolutionary dynamics. We analyzed 106 RRV complete coding sequences, which included 13 genomes available on NCBI and 94 novel sequences derived for this study, sampled throughout Western Australia (1977–2014) and during the substantial Pacific Islands RRV epidemic (1979–1980). Our final data set comprised isolates sampled over 59 years (1959–2018) from a range of locations. Four distinct genotypes were defined, with the newly described genotype 4 (G4) found to be the contemporary lineage circulating in Western Australia. The prior geographical classification of RRV lineages was not supported by our findings, with evidence of geographical and temporal cocirculation of distinct genetic groups. Bayesian Markov chain Monte Carlo (MCMC) analysis revealed that RRV lineages diverged from a common ancestor approximately 94 years ago, with distinct lineages emerging roughly every 10 years over the past 50 years in periodic bursts of genetic diversity. Our study has enabled a more robust analysis of RRV evolutionary history and resolved greater genetic diversity that had been previously defined by partial E2 gene analysis. IMPORTANCE Ross River virus (RRV) causes the most common mosquito-borne infection in Australia and causes a significant burden of suffering to infected individuals as well as being a large burden to the Australian economy. The genetic diversity of RRV and its evolutionary history have so far only been studied using partial E2 gene analysis with a limited number of isolates. Robust whole-genome analysis has not yet been conducted. This study generated 94 novel near-whole-genome sequences to investigate the evolutionary history of RRV to better understand its genetic diversity through comprehensive whole-genome phylogeny. A better understanding of RRV genetic diversity will enable better diagnostics, surveillance, and potential future vaccine design.

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