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Biology
単一細菌への重水素取り込みの誘導ラマン散乱イメージングによる迅速抗菌薬感受性試験
単一細菌への重水素取り込みの誘導ラマン散乱イメージングによる迅速抗菌薬感受性試験
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Biology
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JoVE Journal Biology
Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium

単一細菌への重水素取り込みの誘導ラマン散乱イメージングによる迅速抗菌薬感受性試験

Full Text
3,378 Views
12:08 min
February 14, 2022

DOI: 10.3791/62398-v

Meng Zhang1,2, Mohamed N. Seleem3, Ji-Xin Cheng1,2,4,5

1Department of Electrical and Computer Engineering,Boston University, 2Boston University Photonics Center,Boston University, 3Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine,Virginia Polytechnic Institute and State University, 4Department of Biomedical Engineering,Boston University, 5Department of Chemistry,Boston University

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Overview

This study describes a rapid antimicrobial susceptibility testing (AST) assay that can be completed in 2.5 hours using single-cell-stimulated Raman scattering imaging of D2O metabolism. This protocol is significant as it allows for testing of bacterial samples from urine and whole blood, providing a transformative approach to rapid single-cell phenotypic AST in clinical settings.

Key Study Components

Research Area

  • Antimicrobial susceptibility testing
  • Clinical microbiology
  • Rapid diagnostic methods

Background

  • Conventional AST methods require longer processing times.
  • Rapid methods could lead to timely and targeted treatments for patients.
  • Single-cell analysis provides detailed insights into bacterial behavior.

Methods Used

  • Single-cell-stimulated Raman scattering (SRS) imaging
  • Bacteria from urine and whole blood
  • Sequential dilution and antibiotic treatment of bacterial samples

Main Results

  • Successful implementation of rapid AST within 2.5 hours.
  • Effectiveness demonstrated in monitoring bacterial metabolic activity.
  • Conclusions aligned with the potential for clinical application.

Conclusions

  • This study demonstrates a rapid and effective method for AST using SRS imaging.
  • The approach has the potential to significantly improve the speed and accuracy of bacterial diagnostics in clinical microbiology.

Frequently Asked Questions

What is the advantage of this protocol over traditional methods?
This protocol significantly reduces the time required for antimicrobial susceptibility testing from several hours to just 2.5 hours.
Can this method be applied to different bacterial species?
Yes, this method is adaptable to various bacterial species present in urine and blood.
What technology is primarily used in this study?
The primary technology used is single-cell-stimulated Raman scattering imaging.
What type of samples were tested in this study?
The study involved bacterial samples spiked in urine and whole blood.
How does this method measure bacterial metabolism?
It measures metabolic activity through deuterium incorporation and Raman scattering imaging.
What are the implications of this technology in a clinical setting?
The rapid results can lead to more timely and effective treatments for infections.
Is there a specific antibiotic concentration used in the testing?
Yes, the assay uses a serial dilution method to test various concentrations of antibiotics.

このプロトコルは、D2O代謝の単一細胞刺激ラマン散乱イメージングによる2.5時間以内の迅速な抗菌剤感受性試験(AST)アッセイを提示します。この方法は、尿または全血環境の細菌に適用され、診療所での迅速な単一細胞表現型ASTに変化します。

尿や血液中で2.5時間以内に迅速な抗菌薬感受性試験が可能となり、従来のブロス微量希釈法と比較して分析時間が大幅に短縮できると考えられます。全血などの複雑な環境で細菌の代謝活動を監視できます。まず、波長600ナノメートルの光度計で光学密度を測定して、サンプルから細菌濃度を確認します。

ミリリットル当たり10〜第5コロニー形成単位(CFU)の8倍の最終細胞濃度に達するには、重水素を含まない通常のMHB培地を用いて細菌溶液を希釈する。細菌細胞をボルテックスで混合した後、7本の1.5ミリリットルマイクロチューブに300マイクロリットルの細菌溶液のアリコートを、1本の1.5ミリリットルマイクロチューブに600マイクロリットルの細菌溶液のアリコートを除去します。次に、4.8マイクロリットルの抗生物質ストック溶液を600マイクロリットルの細菌溶液を含むマイクロチューブに追加して、ミリリットルあたり8マイクログラムの最終抗生物質濃度を達成します。

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