scholarly journals Differentiation between Mycobacterium tuberculosis and Mycobacterium avium by Amplification of the 16S-23S Ribosomal DNA Spacer

1998 ◽  
Vol 36 (9) ◽  
pp. 2399-2403 ◽  
Author(s):  
Arunnee Sansila ◽  
Poonpilas Hongmanee ◽  
Charoen Chuchottaworn ◽  
Somsak Rienthong ◽  
Dhanida Rienthong ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterial Infection ◽  
Clinical Isolates ◽  
23S Rrna ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Rrna Gene ◽  
Biochemical Tests ◽  
Specific Primers ◽  
23S Rrna Gene

Differentiation between Mycobacterium tuberculosis andM. avium is helpful for the treatment of disseminated mycobacterial infection in AIDS patients. This can traditionally be done by time-consuming biochemical tests or with Accuprobe. Previously, PCR restriction enzyme analysis (PCR-REA) of the 16S-23S rRNA gene spacer was shown to be able to identify a limited number of strains ofMycobacterium. In this study the method was improved by using more specific primers and was tested with 50 clinical isolates ofM. tuberculosis and 65 clinical isolates of M. avium complex. Probes specific to the spacers ofM. tuberculosis and M. avium were also tested. Both M. tuberculosis and M. avium could be reliably identified either by PCR-REA or by PCR-hybridization, with the results completely agreeing with those obtained by biochemical tests and with the Accuprobe, respectively. The method may therefore be useful as an alternative in-house method for identification of the bacteria.

Antimicrobial susceptibility and mechanisms of high-level macrolide resistance in clinical isolates of Moraxella nonliquefaciens

2014 ◽  
Vol 63 (2) ◽  
pp. 242-247 ◽  
Author(s):  
Shotaro Nonaka ◽  
Kosuke Matsuzaki ◽  
Tomoya Kazama ◽  
Hiroyuki Nishiyama ◽  
Yoko Ida ◽  
...  
Keyword(s):  
Antimicrobial Susceptibility ◽  
Ribosomal Proteins ◽  
Macrolide Resistance ◽  
Clinical Isolates ◽  
23S Rrna ◽  
Rrna Gene ◽  
Efflux System ◽  
Strong Activity ◽  
23S Rrna Gene ◽  
High Level

We investigated antimicrobial susceptibility and the molecular mechanism involved in conferring high-level macrolide resistance in 47 clinical isolates of Moraxella nonliquefaciens from Japan. Antimicrobial susceptibility was determined using Etest and agar dilution methods. Thirty-two erythromycin-non-susceptible strains were evaluated for the possibility of clonal spreading, using PFGE. To analyse the mechanism related to macrolide resistance, mutations in the 23S rRNA gene and the ribosomal proteins, and the presence of methylase genes were investigated by PCR and sequencing. The efflux system was examined using appropriate inhibitors. Penicillin, ampicillin, amoxicillin, cefixime, levofloxacin and antimicrobials containing β-lactamase inhibitors showed strong activity against 47 M. nonliquefaciens isolates. Thirty-two (68.1 %) of the 47 isolates showed high-level MICs to macrolides (MIC ≥128 mg l−1) and shared the A2058T mutation in the 23S rRNA gene. The geometric mean MIC to macrolides of A2058T-mutated strains was significantly higher than that of WT strains (P<0.0001). Thirty-two isolates with high-level macrolide MICs clustered into 30 patterns on the basis of the PFGE dendrogram, indicating that the macrolide-resistant strains were not clonal. In contrast, no common mutations of the ribosomal proteins or methylase genes, or overproduction of the efflux system were observed in A2058T-mutated strains. Moreover, of the 47 M. nonliquefaciens strains, 43 (91.5 %) were bro-1 and 4 (8.5 %) were bro-2 positive. Our results suggest that most M. nonliquefaciens clinical isolates show high-level macrolide resistance conferred by the A2058T mutation in the 23S rRNA gene. This study represents the first characterization of M. nonliquefaciens.

scholarly journals Identification of the genusGeobacillususing genus-specific primers, based on the 16S–23S rRNA gene internal transcribed spacer

2007 ◽  
Vol 277 (2) ◽  
pp. 165-172 ◽  
Author(s):  
Nomeda Kuisiene ◽  
Juozas Raugalas ◽  
Milda Stuknyte ◽  
Donaldas Chitavichius
Keyword(s):  
Internal Transcribed Spacer ◽  
23S Rrna ◽  
Rrna Gene ◽  
Specific Primers ◽  
23S Rrna Gene

scholarly journals Mutations in a 23S rRNA Gene of Chlamydia trachomatis Associated with Resistance to Macrolides

2004 ◽  
Vol 48 (4) ◽  
pp. 1347-1349 ◽  
Author(s):  
O. Y. Misyurina ◽  
E. V. Chipitsyna ◽  
Y. P. Finashutina ◽  
V. N. Lazarev ◽  
T. A. Akopian ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Chlamydia Trachomatis ◽  
Gene Mutations ◽  
Clinical Isolates ◽  
23S Rrna ◽  
Rrna Gene ◽  
In Vitro Susceptibility ◽  
23S Rrna Gene ◽  
Mixed Populations

ABSTRACT For six clinical isolates of Chlamydia trachomatis, in vitro susceptibility to erythromycin, azithromycin, and josamycin has been determined. Four isolates were resistant to all the antibiotics and had the mutations A2058C and T2611C (Escherichia coli numbering) in the 23S rRNA gene. All the isolates had mixed populations of bacteria that did and did not carry 23S rRNA gene mutations.

scholarly journals Molecular detection and evaluation of resistance of Mycoplasma Pneumoniae to macrolide (Erythromycin) due to mutation in the 23S rRNA gene among Iranian Patients with respiratory infections

2020 ◽  
Author(s):  
Mohammad Niakan ◽  
Susan Rostampur ◽  
Reza Mirnejad ◽  
Mehrdad Halaji ◽  
Iman Pouladi
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Molecular Detection ◽  
Inhibitory Concentration ◽  
Respiratory Infections ◽  
Community Acquired Pneumonia ◽  
23S Rrna ◽  
Rrna Gene ◽  
Specific Primers ◽  
23S Rrna Gene ◽  
Iranian Patients

Abstract Objective: Mycoplasma pneumoniae is a common cause of community-acquired pneumonia. The global increased resistance of M. pneumoniae strains to macrolide (ML) has become a worrisome health problem. The widespread use of these drugs has led to increased rate of reported ML-resistant M. pneumoniae (MRMP) throughout the world. Therefore, this study was aimed to evaluate the resistance of M. pneumoniae against erythromycin due to mutations in the 23S rRNA gene of patients with respiratory infections in Iran.Results: According to the findings of the present study, employing specific primers showed that 17 cases (17%) were positive for mycoplasma genus and 6 cases (6%) positive for M. pneumoniae species. Also, analysis of the sequence of 23S rRNA gene, revealed that one of the samples had mutations at positions A2431G and G2491A. Measuring the minimum inhibitory concentration (MIC), revealed that all samples positive for M. pneumoniae with 23S rRNA gene were sensitive to erythromycin, and no ML resistance was reported.

scholarly journals Genotyping of Mycoplasma pneumoniaeClinical Isolates Reveals Eight P1 Subtypes within Two Genomic Groups

2000 ◽  
Vol 38 (3) ◽  
pp. 965-970 ◽  
Author(s):  
J. Wendelien Dorigo-Zetsma ◽  
Jacob Dankert ◽  
Sebastian A. J. Zaat
Keyword(s):  
Sequence Data ◽  
Rflp Analysis ◽  
Clinical Isolates ◽  
23S Rrna ◽  
Rrna Gene ◽  
Intergenic Spacer Region ◽  
Long Pcr ◽  
23S Rrna Gene

Three methods for genotyping of Mycoplasma pneumoniaeclinical isolates were applied to 2 reference strains and 21 clinical isolates. By a modified restriction fragment length polymorphism (RFLP) analysis of PCR products of the M. pneumoniae cytadhesin P1 gene, 5 subtypes were discriminated among 13 P1 type 1 strains and 3 subtypes were discriminated among 8 P1 type 2 strains. Sequence analysis of the 16S-23S rRNA gene spacer region and part of the 23S rRNA gene revealed one nucleotide difference in the intergenic spacer region in 3 of the 21 isolates. In the 23S rRNA gene sequence of the 8 P1 type 2 strains an extra adenosine was present, but it was absent from the 13 P1 type 1 strains. On the basis of M. pneumoniae genome sequence data, primers were designed to amplify large interrepeat fragments by long PCR, and these fragments were subsequently analyzed by RFLP analysis. Only two types, long PCR types 1 and 2, could be discriminated among the M. pneumoniaeisolates. All P1 type 1 strains were assigned to long PCR type 1, and all P1 type 2 strains were assigned to long PCR type 2. These data obtained by three independent typing methods thus confirm the existence of two distinct M. pneumoniae genomic groups but expand the possibility of strain typing on the basis of variations within their P1 genes.

scholarly journals Trends towards Lower Antimicrobial Susceptibility and Characterization of Acquired Resistance among Clinical Isolates of Brachyspira hyodysenteriae in Spain

2011 ◽  
Vol 55 (7) ◽  
pp. 3330-3337 ◽  
Author(s):  
Álvaro Hidalgo ◽  
Ana Carvajal ◽  
Birte Vester ◽  
Märit Pringle ◽  
Germán Naharro ◽  
...  
Keyword(s):  
Antimicrobial Susceptibility ◽  
Molecular Mechanisms ◽  
Tandem Repeats ◽  
Point Mutations ◽  
Clinical Isolates ◽  
23S Rrna ◽  
Rrna Gene ◽  
Content Type ◽  
Brachyspira Hyodysenteriae ◽  
23S Rrna Gene

ABSTRACTThe antimicrobial susceptibility of clinical isolates ofBrachyspira hyodysenteriaein Spain was monitored, and the underlying molecular mechanisms of resistance were investigated. MICs of tylosin, tiamulin, valnemulin, lincomycin, and tylvalosin were determined for 87B. hyodysenteriaeisolates recovered from 2008 to 2009 by broth dilution. Domain V of the 23S rRNA gene and the ribosomal protein L3 gene were sequenced in 20 isolates for which the tiamulin MIC was ≥4 μg/ml, presenting decreased susceptibility, and in 18 tiamulin-susceptible isolates (MIC ≤ 0.125 μg/ml), and all isolates were typed by multiple-locus variable-number tandem repeats analysis. A comparison with antimicrobial susceptibility data from 2000 to 2007 showed an increase in pleuromutilin resistance over time, doubling the number of isolates with decreased susceptibility to tiamulin. No alteration in susceptibility was detected for lincomycin, and the MIC of tylosin remained high (MIC50> 128 μg/ml). The decreased susceptibility to tylosin and lincomycin can be explained by mutations at position A2058 of the 23S rRNA gene (Escherichia colinumbering). A2058T was the predominant mutation, but A2058G also was found together with a change of the neighboring base pair at positions 2057 to 2611. The role of additional point mutations in the vicinity of the peptidyl transferase center and mutations in the L3 at amino acids 148 and 149 and their possible involvement in antimicrobial susceptibility are considered. An association between G2032A and high levels of tiamulin and lincomycin MICs was found, suggesting an increasing importance of this mutation in antimicrobial resistance of clinical isolates ofB. hyodysenteriae.

scholarly journals Identification of Agrobacterium vitis as a causal agent of grapevine crown gall in Serbia

2012 ◽  
Vol 64 (4) ◽  
pp. 1487-1494 ◽  
Author(s):  
N. Kuzmanovic ◽  
Katarina Gasic ◽  
M. Ivanovic ◽  
Andjelka Prokic ◽  
A. Obradovic
Keyword(s):  
Crown Gall ◽  
23S Rrna ◽  
Vitis Vinifera L ◽  
Rrna Gene ◽  
Agrobacterium Vitis ◽  
Biochemical Tests ◽  
Crown Gall Disease ◽  
Multiplex Pcr Assay ◽  
Pcr Product ◽  
23S Rrna Gene

In 2010, a serious outbreak of crown gall disease was observed on grapevines (Vitis vinifera L. cv. Cabernet Sauvignon) in several commercial vineyards located in the Vojvodina province, Serbia. Bacteria were isolated from the young tumor tissue on nonselective YMA medium and five representative strains were selected for further identification. Tumorigenic (Ti) plasmid was detected in all strains by PCR using primers designed to amplify the virC pathogenicity gene, producing a 414-bp PCR product. The strains were identified as Agrobacterium vitis using differential physiological and biochemical tests, and a multiplex PCR assay targeting 23S rRNA gene sequences. In the pathogenicity assay, all strains induced characteristic symptoms on inoculated tomato and grapevine plants. They were less virulent on tomato plants in comparison to the reference strains of A. tumefaciens and A. vitis.

scholarly journals Acidaminococcus intestini sp. nov., isolated from human clinical samples

2007 ◽  
Vol 57 (10) ◽  
pp. 2314-2319 ◽  
Author(s):  
Estelle Jumas-Bilak ◽  
Jean-Philippe Carlier ◽  
Hélène Jean-Pierre ◽  
Francine Mory ◽  
Corinne Teyssier ◽  
...  
Keyword(s):  
Large Scale ◽  
Enzymic Activity ◽  
Clinical Isolates ◽  
23S Rrna ◽  
Clinical Samples ◽  
Rrna Gene ◽  
The Novel ◽  
23S Rrna Gene ◽  
Propionic Acid Production ◽  
Novel Bacteria

Eleven strains of a hitherto unknown, Gram-negative, anaerobic coccus were recovered from various human clinical samples of patients hospitalized in two geographically distant French hospitals. These strains displayed the morphology and growth characteristics of those related to the genus Acidaminococcus. The clinical isolates shared at least 99.9 and 99.7 % of their nucleotide positions in the 16S and 23S rRNA gene sequences, respectively. They displayed 95.6 and 88.9 % 16S and 23S rRNA gene sequence similarities, respectively, with Acidaminococcus fermentans. The 16S rRNA-based phylogeny revealed that all the clinical isolates grouped in a statistically well supported cluster separate from A. fermentans. Enzymic activity profiles as well as metabolic end product patterns, including propionic acid production, differentiated the novel bacteria from A. fermentans. Finally, phenotypic, genotypic and phylogenetic data, including large-scale chromosome structure and DNA G+C content, supported the proposal of a novel species of the genus Acidaminococcus, for which the name Acidaminococcus intestini sp. nov. is proposed. The type strain is ADV 255.99T (=AIP 283.01T=CIP 108586T=CCUG 50930T).

scholarly journals Site-Specific Mutations in the 23S rRNA Gene ofHelicobacter pylori Confer Two Types of Resistance to Macrolide-Lincosamide-Streptogramin B Antibiotics

1998 ◽  
Vol 42 (8) ◽  
pp. 1952-1958 ◽  
Author(s):  
Ge Wang ◽  
Diane E. Taylor
Keyword(s):  
Point Mutations ◽  
Clinical Isolates ◽  
23S Rrna ◽  
Cross Resistance ◽  
Rrna Gene ◽  
Type I ◽  
Type Ii ◽  
Clarithromycin Resistance ◽  
23S Rrna Gene ◽  
H Pylori

ABSTRACT Clarithromycin resistance in Helicobacter pylori is mainly due to A-to-G mutations within the peptidyltransferase region of the 23S rRNA. In the present study, cross-resistance to macrolide, lincosamide, and streptogramin B (MLS) antibiotics (MLS phenotypes) has been investigated for several clinical isolates of H. pylori. Two major types of MLS resistance were identified and correlated with specific point mutations in the 23S rRNA gene. The A2142G mutation was linked with high-level cross-resistance to all MLS antibiotics (type I), and the A2143G mutation gave rise to an intermediate level of resistance to clarithromycin and clindamycin but no resistance to streptogramin B (type II). In addition, streptogramin A and streptogramin B were demonstrated to have a synergistic effect on both MLS-sensitive and MLS-resistant H. pyloristrains. To further understand the mechanism of MLS resistance inH. pylori, we performed in vitro site-directed mutagenesis (substitution of G, C, or T for A at either position 2142 or 2143 of the 23S rRNA gene). The site-directed point mutations were introduced into a clarithromycin-susceptible strain, H. pylori UA802, by natural transformation followed by characterization of their effects on MLS resistance in an isogenic background. Strains with A-to-G and A-to-C mutations at the same position within the 23S rRNA gene had similar levels of clarithromycin resistance, and this level of resistance was higher than that for strains with the A-to-T mutation. Mutations at position 2142 conferred a higher level of clarithromycin resistance than mutations at position 2143. All mutations at position 2142 conferred cross-resistance to all MLS antibiotics, which corresponds to the type I MLS phenotype, whereas mutations at position 2143 were associated with a type II MLS phenotype with no resistance to streptogramin B. To explain that A-to-G transitions were predominantly observed in clarithromycin-resistant clinical isolates, we propose a possible mechanism by which A-to-G mutations are preferentially produced in H. pylori.

scholarly journals Molecular pathways to high-level azithromycin resistance in Neisseria gonorrhoeae

2021 ◽  
Author(s):  
J G E Laumen ◽  
S S Manoharan-Basil ◽  
E Verhoeven ◽  
S Abdellati ◽  
I De Baetselier ◽  
...  
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Ribosomal Proteins ◽  
Efflux Pump ◽  
23S Rrna ◽  
Rrna Gene ◽  
23S Rrna Gene ◽  
Evolution Of Resistance ◽  
High Level ◽  
Azithromycin Resistance ◽  
Level Resistance

Abstract Background The prevalence of azithromycin resistance in Neisseria gonorrhoeae is increasing in numerous populations worldwide. Objectives To characterize the genetic pathways leading to high-level azithromycin resistance. Methods A customized morbidostat was used to subject two N. gonorrhoeae reference strains (WHO-F and WHO-X) to dynamically sustained azithromycin pressure. We tracked stepwise evolution of resistance by whole genome sequencing. Results Within 26 days, all cultures evolved high-level azithromycin resistance. Typically, the first step towards resistance was found in transitory mutations in genes rplD, rplV and rpmH (encoding the ribosomal proteins L4, L22 and L34 respectively), followed by mutations in the MtrCDE-encoded efflux pump and the 23S rRNA gene. Low- to high-level resistance was associated with mutations in the ribosomal proteins and MtrCDE efflux pump. However, high-level resistance was consistently associated with mutations in the 23S ribosomal RNA, mainly the well-known A2059G and C2611T mutations, but also at position A2058G. Conclusions This study enabled us to track previously reported mutations and identify novel mutations in ribosomal proteins (L4, L22 and L34) that may play a role in the genesis of azithromycin resistance in N. gonorrhoeae.

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