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Biologia

通过受激拉曼散射成像对氘掺入单个细菌进行快速抗菌药敏试验

Published: February 14, 2022
doi:
10.3791/62398
, ,
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

Summary

该协议通过D 2 O代谢的单细胞刺激拉曼散射成像在2.5小时内提供快速抗菌药敏试验（AST）测定。该方法适用于尿液或全血环境中的细菌，对于临床上的快速单细胞表型AST具有变革性。

Abstract

为了减缓和防止抗微生物药物耐药性感染的传播，迫切需要快速抗菌药敏试验（AST）来定量确定对病原体的抗菌作用。通过基于长期培养的常规方法完成 AST 通常需要数天时间，并且它们不直接适用于临床样本。在这里，我们报告了一种通过氧化氘（D2O）代谢掺入的受激拉曼散射（SRS）成像实现的快速AST方法。通过SRS成像监测D2O在生物质中的代谢掺入和暴露于单个细菌水平的抗生素时的代谢活性抑制。暴露于抗生素时细菌的单细胞代谢失活浓度（SC-MIC）可以在总共2.5小时的样品制备和检测后获得。此外，这种快速AST方法直接适用于复杂生物环境中的细菌样品，例如尿液或全血。氘掺入的SRS代谢成像对于临床中的快速单细胞表型AST具有变革性意义。

Introduction

抗微生物药物耐药性（AMR）是有效治疗传染病的一个日益严重的全球威胁1。据预测，如果不采取行动对抗抗生素耐药细菌，到2050年，抗微生物药物耐药性每年将造成1000万人死亡，全球GDP损失100万亿美元1，2。这强调了对感染性细菌抗生素敏感性检测（AST）的快速和创新诊断方法的迫切需要，以减缓抗生素耐药细菌的出现并降低相关死亡率3。为了确保最佳的临床结果，在24小时内引入有效的治疗至关重要。然而，目前的金标准方法，如盘式扩散法或肉汤稀释法，通常需要至少24小时用于临床样品的预孵育程序，另外需要16-24小时才能获得最小抑制浓度（MIC）结果。总体而言，这些方法过于耗时，无法指导临床上传染病治疗的立即决策，从而导致抗菌素耐药性的出现和传播4。

基因型AST方法，例如基于聚合酶链反应（PCR）的技术5，已被开发用于快速检测。这些技术测量特定的抗性基因序列，以提供快速的AST结果。它们不依赖于耗时的细胞培养;然而，只测试具有耐药性的特定已知基因序列。因此，其应用仅限于各种细菌种类或不同的抗性机制。此外，他们无法为治疗决策提供MIC结果6，7。此外，正在开发用于快速AST的新型表型方法以克服这些限制8，包括微流控器件9，10，11，12，13，光学器件14，15，16，量化核酸拷贝数的表型AST17，18和拉曼光谱方法19，20，21，22，23，24.这些方法减少了指导AST结果的时间，然而，它们中的大多数仅适用于细菌分离株，不直接适用于临床标本，并且仍然需要长时间的预孵育。

在这项工作中，我们提出了一种通过SRS成像监测细胞代谢活性来快速确定尿液和全血中细菌敏感性的方法。水（H2O）参与了活细胞中绝大多数基本的生物分子合成过程。作为水的同位素，通过NADPH中的氧化还原活性氢原子和D2O中的D原子之间的酶催化H / D交换反应，氘可以掺入电池内的生物质中25，26。氘代脂肪酸合成反应由氘标记的NADPH介导。D2O掺入氨基酸（AA）的反应中导致氘代蛋白产生26（图1）。通过这种方式，在单个微生物细胞中新合成的含C-D键的生物分子可以作为一般的代谢活性标志物进行检测。为了读出从头合成的C-D键，拉曼光谱是一种多功能分析工具，可提供生物分子的特异性和定量化学信息，广泛用于确定抗菌药敏性，并将测试时间显着缩短至几个小时27，28，29，30.然而，由于拉曼散射过程固有的低效率，自发拉曼光谱的检测灵敏度较低。因此，使用自发拉曼光谱获得实时图像结果具有挑战性。相干拉曼散射（CRS），包括相干反斯托克斯拉曼散射（CARS）和受激拉曼散射（SRS），由于相干光场产生的光场比自发拉曼光谱大几个数量级，因此达到了很高的检测灵敏度，从而在单细胞水平上呈现高速、特异性和定量的化学成像31，32，33，34，35，36，37，38，39.

在这里，基于我们最近的工作40，我们提出了一种协议，用于通过飞秒SRS C-D成像快速确定代谢活性和抗菌敏感性，在单细胞水平上正常培养基，尿液和全血环境中的细菌掺入D2O。飞秒SRS成像有助于在2.5小时内监测单细胞代谢失活浓度（SC-MIC）在单个细菌水平上对抗抗生素。SC-MIC结果通过肉汤微量稀释的标准MIC测试进行验证。我们的方法适用于确定细菌尿路感染（UTI）和血流感染（BSI）病原体的抗菌敏感性，与传统方法相比，测定时间大大缩短，这为临床上单细胞水平的快速表型AST提供了机会。

Protocol

人类血液标本的使用符合波士顿大学IRB和美国国立卫生研究院（NIH）的指导方针。具体来说，标本来自银行，并且完全去识别化。波士顿大学机构审查委员会（IRB）办公室不认为这些标本是人类受试者。 1.细菌和抗生素储备溶液的制备 制备浓度为 1 mg/mL 的抗生素（硫酸庆大霉素或阿莫西林）储备溶液，溶解在 1.5 mL 微管中的无菌 1x 磷酸盐缓冲盐水 （PBS） 或二甲基亚?…

Representative Results

孵育时间对氘掺入的影响是通过C-D（2070至2250 cm-1）和C-H（2，800至3，100 cm-1）区域的自发拉曼显微光谱法测量的（图4a）。在含有70%D 2 O的培养基中培养的铜绿假单胞菌的延时单细胞拉曼光谱显示CD/CH强度在孵育时间从0分钟增加到180分钟（图4b）单个微生物细胞中C-D丰度的增加表明D2O被掺入细胞内的氘代生物分子中…

Discussion

快速AST可以通过在样品到SC-MIC结果的2.5小时内使用单细胞SRS代谢成像评估细菌代谢活动对抗生素治疗的反应来获得。通过使用C-D键的SRS成像监测用于生物分子合成的D2O的代谢掺入，可以检测细菌代谢活性和抗菌敏感性的反应。由于水在活细胞中无处不在，SRS代谢成像为快速AST提供了一种通用方法。快速AST方法适用于检测复杂生物环境中的细菌，例如尿液或全血在单个细菌水平上。SC-MIC可?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项工作得到了NIH R01AI141439至J.-X.C和M.S.以及R35GM136223至J.-X.C的支持。

Materials

Acousto-optic modulation Gooch&Housego R15180-1.06-LTD Modulating stokes laser beam
Amoxicillin Sigma Aldrich A8523-5G
Bandpass filter Chroma HQ825/150m Block the stokes laser beam before the photodiode
Calcium chloride Sigma Aldrich C1016-100G Cation adjustment
Cation-adjusted Mueller-Hinton Broth Fisher Scientific B12322 Antimicrobial susceptibility testing of microorganisms by broth dilution methods
Centrifuge Thermo Scientific 75002542
Cover Glasses VWR 16004-318
Culture tube with snap cap Fisher brand 149569B
Daptomycin Acros A0386346
Deuterium oxide 151882 Organic solvent to dissolve antibiotics
Deuterium oxide-d6 Sigma Aldrich 156914 Organic solvent as a standard to calibrate SRS imaging system
Escherichia coli BW 25113 The Coli Genetic Stock Center 7636
Eppendorf polypropylene microcentrifuge tubes 1.5 mL Fisher brand 05-408-129
Gentamicin sulfate Sigma Aldrich G4918
Hydrophilic Polyvinylidene Fluoride filters Millipore-Sigma SLSV025NB pore size 5 µm
ImageJ software NIH Version: 2.0.0-rc-69/1.52t Image processing and analysis
Incubating orbital shaker set at 37 °C VWR 97009-890
Inoculation loop Sigma BR452201-1000EA
InSight DeepSee femtosecond pulsed laser Spectra-Physics Model: insight X3 Tunable laser source and fixed laser source at 1045 nm for SRS imaging
Lock-in amplifier Zurich Instrument HF2LI Demodulate the SRS signals
Oil condenser Olympus U-AAC NA 1.4
Pseudomonas aeruginosa ATCC 47085 (PAO1) American Type Culture Collection ATCC 47085
Photodiode Hamamatsu S3994-01 Detector
Polypropylene conical tube 15 mL Falcon 14-959-53A
Polypropylene filters Thermo Scientific 726-2520 pore size 0.2 µm
Sterile petri dishes Corning 07-202-031
Syringe 10 mL Fisher brand 14955459
UV/Vis Spectrophotometer Beckman Coulter Model: DU 530 Measuring optical density at wavelength of 600 nm
Vortex mixer VWR 97043-562
Water objective Olympus UPLANAPO/IR 60×, NA 1.2
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Riferimenti

  1. O’Neill, J. Tackling drug-resistant infections globally: final report and recommendations. The review on Antimicrobial Resistance. , (2016).
  2. Sugden, R., Kelly, R., Davies, S. Combatting antimicrobial resistance globally. Nature Microbiology. 1 (10), 16187 (2016).
  3. Kumar, A., et al. Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Critical Care Medicine. 34 (6), 1589-1596 (2006).
  4. Reller, L. B., Weinstein, M., Jorgensen, J. H., Ferraro, M. J. Antimicrobial susceptibility testing: a review of general principles and contemporary practices. Clinical Infectious Diseases. 49 (11), 1749-1755 (2009).
  5. Frickmann, H., Masanta, W. O., Zautner, A. E. Emerging rapid resistance testing methods for clinical microbiology laboratories and their potential impact on patient management. BioMed Research International. 2014, 375681 (2014).
  6. Avesar, J., et al. Rapid phenotypic antimicrobial susceptibility testing using nanoliter arrays. Proceedings of the National Academy of Sciences. 114 (29), 5787-5795 (2017).
  7. Schoepp, N. G., et al. Digital quantification of DNA replication and chromosome segregation enables determination of antimicrobial susceptibility after only 15 minutes of antibiotic exposure. Angewandte Chemie International Edition. 55 (33), 9557-9561 (2016).
  8. van Belkum, A., et al. Innovative and rapid antimicrobial susceptibility testing systems. Nature Reviews Microbiology. 18 (5), 299-311 (2020).
  9. Hou, Z., An, Y., Hjort, K., Sandegren, L., Wu, Z. Time lapse investigation of antibiotic susceptibility using a microfluidic linear gradient 3D culture device. Lab on a Chip. 14 (17), 3409-3418 (2014).
  10. Choi, J., et al. Rapid antibiotic susceptibility testing by tracking single cell growth in a microfluidic agarose channel system. Lab on a Chip. 13 (2), 280-287 (2013).
  11. Lu, Y., et al. Single cell antimicrobial susceptibility testing by confined microchannels and electrokinetic loading. Analytical Chemistry. 85 (8), 3971-3976 (2013).
  12. Kim, S. C., Cestellosblanco, S., Inoue, K., Zare, R. N. Miniaturized antimicrobial susceptibility test by combining concentration gradient generation and rapid cell culturing. Antibiotics. 4 (4), 455-466 (2015).
  13. Choi, J., et al. A rapid antimicrobial susceptibility test based on single-cell morphological analysis. Science Translational Medicine. 6 (267), (2014).
  14. Baltekin, &. #. 2. 1. 4. ;., Boucharin, A., Tano, E., Andersson, D. I., Elf, J. Antibiotic susceptibility testing in less than 30 min using direct single-cell imaging. Proceedings of the National Academy of Sciences. 114 (34), 9170-9175 (2017).
  15. Fredborg, M., et al. Real-time optical antimicrobial susceptibility testing. Journal of Clinical Microbiology. 51 (7), 2047-2053 (2013).
  16. Choi, J., et al. A rapid antimicrobial susceptibility test based on single-cell morphological analysis. Science Translational Medicine. 6 (267), (2014).
  17. Barczak, A. K., Hung, D. T. RNA signatures allow rapid identification of pathogens and antibiotic susceptibilities. Proceedings of the National Academy of Sciences. 109 (16), 6217-6222 (2012).
  18. Schoepp, N. G., et al. Rapid pathogen-specific phenotypic antibiotic susceptibility testing using digital LAMP quantification in clinical samples. Science Translational Medicine. 9 (410), (2017).
  19. Novelli-Rousseau, A., et al. Culture-free antibiotic-susceptibility determination from single-bacterium Raman spectra. Scientific Reports. 8 (1), 1-12 (2018).
  20. Schröder, U. -. C., et al. Detection of vancomycin resistances in enterococci within 3 1/2 hours. Scientific Reports. 5, 8217 (2015).
  21. Liu, C. -. Y., et al. Rapid bacterial antibiotic susceptibility test based on simple surface-enhanced Raman spectroscopic biomarkers. Scientific Reports. 6 (1), 1-15 (2016).
  22. Chang, K. -. W., et al. Antibiotic susceptibility test with surface-enhanced raman scattering in a microfluidic system. Analytical Chemistry. 91 (17), 10988-10995 (2019).
  23. Galvan, D. D., Yu, Q. surface-enhanced raman scattering for rapid detection and characterization of antibiotic-resistant bacteria. Advanced Healthcare Materials. 7 (13), 1701335 (2018).
  24. Kirchhoff, J., et al. Simple ciprofloxacin resistance test and determination of minimal inhibitory concentration within 2 h using raman spectroscopy. Analytical Chemistry. 90 (3), 1811-1818 (2018).
  25. Zhang, Z., Chen, L., Liu, L., Su, X., Rabinowitz, J. D. Chemical basis for deuterium labeling of fat and NADPH. Journal of the American Chemical Society. 139 (41), 14368-14371 (2017).
  26. Shi, L., et al. Optical imaging of metabolic dynamics in animals. Nature Communications. 9 (1), 2995 (2018).
  27. Berry, D., et al. Tracking heavy water (D2O) incorporation for identifying and sorting active microbial cells. Proceedings of the National Academy of Sciences. 112 (2), 194-203 (2015).
  28. Tao, Y., et al. Metabolic-activity-based assessment of antimicrobial effects by D2O-labeled single-cell raman microspectroscopy. Analytical Chemistry. 89 (7), 4108-4115 (2017).
  29. Yang, K., et al. Rapid antibiotic susceptibility testing of pathogenic bacteria using heavy water-labeled single-cell raman spectroscopy in clinical samples. Analytical Chemistry. 91 (9), 6296-6303 (2019).
  30. Song, Y., et al. Raman-Deuterium Isotope Probing for in-situ identification of antimicrobial resistant bacteria in Thames River. Scientific reports. 7 (1), 16648 (2017).
  31. Freudiger, C. W., et al. Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy. Science. 322 (5909), 1857-1861 (2008).
  32. Cheng, J. -. X., Xie, X. S. Vibrational spectroscopic imaging of living systems: An emerging platform for biology and medicine. Science. 350 (6264), (2015).
  33. Zhang, C., Zhang, D., Cheng, J. -. X. Coherent Raman scattering microscopy in biology and medicine. Annual Review of Biomedical Engineering. 17, 415-445 (2015).
  34. Yue, S., Cheng, J. -. X. Deciphering single cell metabolism by coherent Raman scattering microscopy. Current Opinion in Chemical Biology. 33, 46-57 (2016).
  35. Hu, F., Shi, L., Min, W. Biological imaging of chemical bonds by stimulated Raman scattering microscopy. Nature Methods. 16 (9), 830-842 (2019).
  36. Ji, M., et al. Rapid, Label-free detection of brain tumors with stimulated Raman scattering microscopy. Science Translational Medicine. 5 (201), (2013).
  37. He, R., Liu, Z., Xu, Y., Huang, W., Ma, H., Ji, M. Stimulated Raman scattering microscopy and spectroscopy with a rapid scanning optical delay line. Optics Letters. 42 (4), 659-662 (2017).
  38. Suzuki, Y., et al. Label-free chemical imaging flow cytometry by high-speed multicolor stimulated Raman scattering. Proceedings of the National Academy of Sciences. 116 (32), 15842-15848 (2019).
  39. Camp, C. H., et al. High-Speed Coherent Raman Fingerprint Imaging of Biological Tissues. Nature Photonics. 8, 627-634 (2014).
  40. Zhang, M., et al. Rapid determination of antimicrobial susceptibility by stimulated raman scattering imaging of D2O metabolic incorporation in a single bacterium. Advanced Science. 7 (19), 2001452 (2020).
  41. Michael, I., et al. A fidget spinner for the point-of-care diagnosis of urinary tract infection. Nature Biomedical Engineering. 4 (6), 591-600 (2020).
  42. Bhattacharyya, R. P., et al. Simultaneous detection of genotype and phenotype enables rapid and accurate antibiotic susceptibility determination. Nature Medicine. 25 (12), 1858-1864 (2019).
  43. Stupar, P., et al. Nanomechanical sensor applied to blood culture pellets: a fast approach to determine the antibiotic susceptibility against agents of bloodstream infections. Clinical Microbiology and Infection. 23 (6), 400-405 (2017).
  44. Barber, A. E., Norton, J. P., Spivak, A. M., Mulvey, M. A. Urinary Tract Infections: Current and Emerging Management Strategies. Clinical Infectious Diseases. 57 (5), 719-724 (2013).
  45. Cohen, J., et al. Sepsis: a roadmap for future research. The Lancet Infectious Diseases. 15 (5), 581-614 (2015).
  46. Choi, J., et al. rapid antimicrobial susceptibility test from positive blood cultures based on microscopic imaging analysis. Scientific Reports. 7 (1), 1148 (2017).
  47. Gherardi, G., et al. Comparative evaluation of the Vitek-2 Compact and Phoenix systems for rapid identification and antibiotic susceptibility testing directly from blood cultures of Gram-negative and Gram-positive isolates. Diagnostic Microbiology and Infectious Disease. 72 (1), 20-31 (2012).
  48. Machen, A., Drake, T., Wang, Y. F. Same day identification and full panel antimicrobial susceptibility testing of bacteria from positive blood culture bottles made possible by a combined lysis-filtration method with MALDI-TOF VITEK mass spectrometry and the VITEK2 system. Plos One. 9, 87870 (2014).
  49. Simon, L., et al. Direct identification of 80 percent of bacteria from blood culture bottles by matrix-assisted laser desorption ionization-time of flight mass spectrometry using a 10-minute extraction protocol. Journal of Clinical Microbiology. 57 (2), 01278 (2019).
  50. Leekha, S., Terrell, C. L., Edson, R. S. General principles of antimicrobial therapy. Mayo Clinic Proceedings. 86 (2), 156-167 (2011).
  51. Johnson, L., et al. Emergence of fluoroquinolone resistance in outpatient urinary Escherichia coli isolates. The American Journal of Medicine. 121 (10), 876-884 (2008).
  52. Van Belkum, A., et al. Developmental roadmap for antimicrobial susceptibility testing systems. Nature Reviews Microbiology. 17 (1), 51-62 (2019).
  53. Dubourg, G., Lamy, B., Ruimy, R. Rapid phenotypic methods to improve the diagnosis of bacterial bloodstream infections: meeting the challenge to reduce the time to result. Clinical Microbiology and Infection. 24 (9), 935-943 (2018).

Tags

Rapid Antimicrobial Susceptibility TestingStimulated Raman ScatteringDeuterium IncorporationSingle BacteriumBacterial ConcentrationOptical DensityColony Forming UnitsAntibioticMHB MediumSerial DilutionPositive ControlNegative ControlIncubationHomogenization

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Zhang, M., Seleem, M. N., Cheng, J. Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium. J. Vis. Exp. (180), e62398, doi:10.3791/62398 (2022).
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