Nanodiagnostic Method for Colorimetric Detection of Mycobacterium tuberculosis 16S rRNA

Numerous assays which use conserved DNA or rRNA sequences as targets for amplification have been described for the diagnosis of tuberculosis. However, these techniques have not been applied successfully to the monitoring of therapeutic efficacy owing to the persistence of amplifiable nucleic acid beyond the point at which smears and cultures become negative. Semiquantitative analysis of rRNA has been used to reduce the time required for antimicrobial susceptibility testing of Mycobacterium tuberculosis, although growth for up to 5 days in the presence of some drugs is still required to discriminate resistant strains. The purpose of the present study was to determine whether quantitative analysis of M. tuberculosis mRNA could be used to assess bacterial viability and to illustrate the application of this technique to rapid determination of drug susceptibility. Levels of mRNA encoding the 85B protein (α-antigen), IS6110 DNA, and 16S rRNA were compared in parallel cultures of M. tuberculosis that were treated with either no drug, 0.2 μg of isoniazid per ml, or 1 μg of rifampin per ml. Exposure of sensitive strains to isoniazid or rifampin for 24 h reduced the levels of 85B mRNA to <4 and <0.01%, respectively, of those present in control cultures without drug. In contrast, the levels of IS6110 DNA and 16S rRNA did not diminish over the same period. Strains which were resistant to either isoniazid or rifampin demonstrated no reduction in 85B mRNA in the presence of the drug to which they were nonresponsive. Quantitative analysis of 85B mRNA offers a potentially useful tool for the rapid determination of M. tuberculosis drug susceptibility and for the monitoring of therapeutic efficacy.

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Novel mutation in 16S rRNA associated with streptomycin dependence in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.39.3.769 ◽

1995 ◽

Vol 39 (3) ◽

pp. 769-770 ◽

Cited By ~ 49

Author(s):

N. Honore ◽

G. Marchal ◽

S. T. Cole

Keyword(s):

Mycobacterium Tuberculosis ◽

16S Rrna ◽

Novel Mutation

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A Mutation in the 16S rRNA Decoding Region Attenuates the Virulence of Mycobacterium tuberculosis

Infection and Immunity ◽

10.1128/iai.00417-16 ◽

2016 ◽

Vol 84 (8) ◽

pp. 2264-2273 ◽

Cited By ~ 5

Author(s):

Shinya Watanabe ◽

Kazunori Matsumura ◽

Hiroki Iwai ◽

Keiji Funatogawa ◽

Yuji Haishima ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

16S Rrna ◽

Binding Sites ◽

Mycolic Acid ◽

Acid Biosynthesis ◽

Mutation Site ◽

Content Type ◽

Antigen 85 ◽

Decoding Region

Mycobacterium tuberculosiscontains a single rRNA operon that encodes targets for antituberculosis agents, including kanamycin. To date, only four mutations in the kanamycin binding sites of 16S rRNA have been reported in kanamycin-resistant clinical isolates. We hypothesized that another mutation(s) in the region may dramatically decreaseM. tuberculosisviability and virulence. Here, we describe an rRNA mutation, U1406A, which was generatedin vitroand confers resistance to kanamycin while highly attenuatingM. tuberculosisvirulence. The mutant showed decreased expression of 20% (n= 361) of mycobacterial proteins, including central metabolic enzymes, mycolic acid biosynthesis enzymes, and virulence factors such as antigen 85 complexes and ESAT-6. The mutation also induced three proteins, including KsgA (Rv1010; 16S rRNA adenine dimethyltransferase), which closely bind to the U1406A mutation site on the ribosome; these proteins were associated with ribosome maturation and translation initiation processes. The mutant showed an increase in 17S rRNA (precursor 16S rRNA) and a decrease in the ratio of 30S subunits to the 70S ribosomes, suggesting that the U1406A mutation in 16S rRNA attenuatedM. tuberculosisvirulence by affecting these processes.

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Functional Role of Methylation of G518 of the 16S rRNA 530 Loop by GidB in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00905-13 ◽

2013 ◽

Vol 57 (12) ◽

pp. 6311-6318 ◽

Cited By ~ 16

Author(s):

Sharon Y. Wong ◽

Babak Javid ◽

Balasubrahmanyam Addepalli ◽

Grzegorz Piszczek ◽

Michael Brad Strader ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

16S Rrna ◽

Functional Role ◽

High Performance ◽

Protein Translation ◽

Titration Calorimetry ◽

Wild Type ◽

Content Type ◽

Wobble Position

ABSTRACTPosttranscriptional modifications of bacterial rRNA serve a variety of purposes, from stabilizing ribosome structure to preserving its functional integrity. Here, we investigated the functional role of one rRNA modification in particular—the methylation of guanosine at position 518 (G518) of the 16S rRNA inMycobacterium tuberculosis. Based on previously reported evidence that G518 is located 5 Å; from proline 44 of ribosomal protein S12, which interacts directly with the mRNA wobble position of the codon:anticodon helix at the A site during translation, we speculated that methylation of G518 affects protein translation. We transformed reporter constructs designed to probe the effect of functional lesions at one of the three codon positions on translational fidelity into the wild-type strain, H37Rv, and into a ΔgidBmutant, which lacks the methyltransferase (GidB) that methylates G518. We show that mistranslation occurs less in the ΔgidBmutant only in the construct bearing a lesion in the wobble position compared to H37Rv. Thus, the methylation of G518 allows mistranslation to occur at some level in order for translation to proceed smoothly and efficiently. We also explored the role of methylation at G518 in altering the susceptibility ofM. tuberculosisto streptomycin (SM). Using high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS), we confirmed that G518 is not methylated in the ΔgidBmutant. Furthermore, isothermal titration calorimetry experiments performed on 70S ribosomes purified from wild-type and ΔgidBmutant strains showed that methylation significantly enhances SM binding. These results provide a mechanistic explanation for the low-level, SM-resistant phenotype observed inM. tuberculosisstrains that contain agidBmutation.

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Detection and identification of Mycobacterium species

10.51429/ejmm30110 ◽

2021 ◽

Vol 30 (1) ◽

pp. 79-86

Author(s):

Asaad Gaber ◽

Hazem Hamed

Keyword(s):

Mycobacterium Tuberculosis ◽

16S Rrna ◽

16S Rrna Gene ◽

Rrna Gene ◽

Mycobacterium Species ◽

Mycobacterial Infections ◽

Rapid Pcr ◽

Detection And Identification ◽

Pcr Method ◽

Dna Fragment

Background: Successful diagnosis and effective treatment for mycobacterial infections are mainly depending on a rapid and sensitive identification method. Objective: To detect and identify the Mycobacterium species. Methodology: PCR and LCD-microarry techniques were compared with the classical methods of Ziehl-Neelsen staining (ZN) and culturing. Two primers based on two conservative regions within the mycobacterium 16S rRNA gene were designed and amplified a DNA fragment of about 1350 bp for both complex of Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM). Results: Regarding to the standard method of culture, 57 positive individuals were identified out of 100 urine samples. The PCR showed 96.30 % sensitivity and 96.70% specificity, while ZN gave Se = 67.50 % and Sp = 100 %. The LCD-microarray analysis exhibited 100 % sensitivity and specificity. One species of MTB was determined as M. tuberculosis and positively represented by 12.3% (n=7). Five species of NTM were determined and represented as M. kansasii 36.8 % (n=21), M. celatum 21 % (n=12), M. gordonae 12.2% (n=7), M. chelonae 10.5 % (n=6), and M. phlei 7% (n=4). Conclusion: The results recommended utilizing the simple and rapid PCR method for early mycobacteria detection. Also, the fast LCD-microarray protocol is very beneficial for identification and differentiation between MTB and of NTM species.

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