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 PCR Targeted to the 16S-23S rRNA Gene Intergenic Spacer Region ofClostridium difficile and Construction of a Library Consisting of 116 Different PCR Ribotypes

1999 ◽  
Vol 37 (2) ◽  
pp. 461-463 ◽  
Author(s):  
Simon L. J. Stubbs ◽  
Jon S. Brazier ◽  
Gael L. O’Neill ◽  
Brian I. Duerden
Keyword(s):  
Intergenic Spacer ◽  
Pcr Primers ◽  
23S Rrna ◽  
Rrna Gene ◽  
Ofclostridium Difficile ◽  
Intergenic Spacer Region ◽  
The United Kingdom ◽  
23S Rrna Gene ◽  
Pcr Ribotyping

A reference library of types of Clostridium difficilehas been constructed by PCR ribotyping isolates (n = 2,030) from environmental (n = 89), hospital (n = 1,386), community practitioner (n = 395), veterinary (n = 27), and reference (n = 133) sources. The library consists of 116 distinct types identified on the basis of differences in profiles generated with PCR primers designed to amplify the 16S-23S rRNA gene intergenic spacer region. Isolates from 55% of infections in hospitals in the United Kingdom belonged to one ribotype (type 1), but this type was responsible for only 7.5% of community infections.

scholarly journals Intrinsic Macrolide Resistance in Mycobacterium smegmatis Is Conferred by a Novel erm Gene, erm(38)

2003 ◽  
Vol 47 (10) ◽  
pp. 3053-3060 ◽  
Author(s):  
Kevin A. Nash
Keyword(s):  
Mycobacterium Smegmatis ◽  
Sequence Data ◽  
Macrolide Resistance ◽  
23S Rrna ◽  
Cross Resistance ◽  
Rrna Gene ◽  
Mycobacterium Fortuitum ◽  
Knockout Mutant ◽  
23S Rrna Gene ◽  
High Level

ABSTRACT High-level, acquired macrolide resistance in mycobacteria is conferred by mutation within the 23S rRNA gene. However, several mycobacteria are naturally resistant to macrolides, including the Mycobacterium smegmatis group and Mycobacterium tuberculosis complex. Thus, the aim of this study was to characterize this resistance. Intrinsic macrolide resistance in M. smegmatis was inducible and showed cross-resistance to lincosamides but not to streptogramin B (i.e., ML resistance). A similar phenotype was found with Mycobacterium microti and macrolide-resistant Mycobacterium fortuitum. A search of the DNA sequence data for M. smegmatis strain mc2155 identified a novel erm gene, erm(38), and expression analysis showed that erm(38) RNA levels increased >10-fold after a 2-h incubation with macrolide. Inducible ML resistance was not expressed by an erm(38) knockout mutant, and complementation of this mutant with intact erm(38) in trans resulted in high-level ML resistance (e.g., clarithromycin MIC of >512 μg/ml). Thus, the results indicate that erm(38) confers the intrinsic ML resistance of M. smegmatis. Southern blot analysis with an erm(38)-specific probe indicated that a similar gene may be present in macrolide-resistant M. fortuitum. This finding, with the presence of the erm(37) gene (Rv1988) in the M. tuberculosis complex, suggests that such genes are widespread in mycobacteria with intrinsic macrolide resistance.

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 16S–23S rRNA Gene Intergenic Spacer Region Variability Helps Resolve Closely Related Sphingomonads

2016 ◽  
Vol 7 ◽  
Author(s):  
Sima Tokajian ◽  
Nahla Issa ◽  
Tamara Salloum ◽  
Joe Ibrahim ◽  
Maya Farah
Keyword(s):  
Intergenic Spacer ◽  
23S Rrna ◽  
Rrna Gene ◽  
Intergenic Spacer Region ◽  
23S Rrna Gene

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.

scholarly journals Rapid Detection of the Chlamydiaceae and Other Families in the Order Chlamydiales: Three PCR Tests

1999 ◽  
Vol 37 (3) ◽  
pp. 575-580 ◽  
Author(s):  
Karin D. E. Everett ◽  
Linda J. Hornung ◽  
Arthur A. Andersen
Keyword(s):  
Sequence Data ◽  
Epidemiologic Study ◽  
Intergenic Spacer ◽  
23S Rrna ◽  
Rrna Gene ◽  
Direct Sequence ◽  
Identification Methods ◽  
The Third ◽  
23S Rrna Gene ◽  
Direct Sequence Analysis

Few identification methods will rapidly or specifically detect all bacteria in the order Chlamydiales, familyChlamydiaceae. In this study, three PCR tests based on sequence data from over 48 chlamydial strains were developed for identification of these bacteria. Two tests exclusively recognized theChlamydiaceae: a multiplex test targeting theompA gene and the rRNA intergenic spacer and a TaqMan test targeting the 23S ribosomal DNA. The multiplex test was able to detect as few as 200 inclusion-forming units (IFU), while the TaqMan test could detect 2 IFU. The amplicons produced in these tests ranged from 132 to 320 bp in length. The third test, targeting the 23S rRNA gene, produced a 600-bp amplicon from strains belonging to several families in the order Chlamydiales. Direct sequence analysis of this amplicon has facilitated the identification of new chlamydial strains. These three tests permit ready identification of chlamydiae for diagnostic and epidemiologic study. The specificity of these tests indicates that they might also be used to identify chlamydiae without culture or isolation.

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.

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