Surface-Enhanced Raman Scattering to Improve the Sensitivity of the MTT Test

Cover Page

Cite item

Full Text

Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription Access

Abstract

Currently, empirical therapy regimens are often used in the treatment of infectious diseases that are not based on data on pathogen resistance. One of the main reasons for the unjustified prescription of antibacterial drugs is the lack of rapid and at the same time universal methods of determining antibiotic resistance of the pathogen. The most widely used culture techniques, such as the microdilution method, require a long time to generate the necessary number of bacterial cells. Less time-consuming methods of resistance assessment (genomic or proteomic) are based on the determination of specific markers (resistance genes, overexpression of certain proteins, etc.); in this case, the specific protocol is most often applicable to a narrow number of both microorganism strains and antibiotics. Previously, we have demonstrated the possibility of using Raman spectroscopy (RS) technology for quantitative determination of the product of bacterial cell activity in the MTT аssay, formazan, directly in the cell suspension. The absence of the formazan isolation step simplifies the assay and increases its accuracy. The analysis time did not exceed 2 h while maintaining the versatility of the MTT аssay itself. Limitations of the developed protocol for RS detection of MTT аssay results include a high sensitivity threshold of 107 CFU/mL for bacterial cell concentration, so a preliminary stage of cultivation is necessary for samples with low bacterial content. Here, we propose a method to increase the sensitivity of formazan determination by utilizing the effect of surface-enhanced Raman scattering (SERS) on gold nanoparticles. As a result of the study, the optimal conditions for SERS analysis of formazan in both solution and suspension of Escherichia coli cells are selected. Formazan signal amplification due to the use of SERS on gold nanoparticles instead of RS allowed us to reduce the sensitivity threshold for the number of bacterial cells in the sample at least 30 times, up to 3 × 105 CFU/mL. This sensitivity is not the limit of the SERS technology capabilities because of the introduction of other types of nanoparticles (more optimal in shape, size, concentration, etc.) into the experiment will allow to achieve even higher signal amplification.

Full Text

Restricted Access

About the authors

В. A. Mushenkov

Lomonosov Moscow State University

Author for correspondence.
Email: vladimir.mushenkov@mail.ru
Russian Federation, Moscow, 119991

D. A. Lukyanov

Lomonosov Moscow State University; Skolkovo Institute of Science and Technology

Email: vladimir.mushenkov@mail.ru
Russian Federation, Moscow, 119991; Moscow, 121205

N. F. Meshcheryakova

Lomonosov Moscow State University

Email: vladimir.mushenkov@mail.ru
Russian Federation, Moscow, 119991

V. I. Kukushkin

Osipyan Institute of Solid State Physics of the Russian Academy of Sciences

Email: vladimir.mushenkov@mail.ru
Russian Federation, Chernogolovka, 142432

Е. G. Zavyalova

Lomonosov Moscow State University

Email: vladimir.mushenkov@mail.ru
Russian Federation, Moscow, 119991

References

  1. Walsh T.R., Gales A.C., Laxminarayan R., Dodd P.C. (2023) Antimicrobial resistance: addressing a global threat to humanity. PLoS Med. 20(7), e1004264.
  2. Ranjbar R., Alam M. (2024) Antimicrobial Resistance Collaborators (2022). (2023) Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Evid. Based Nurs. 2023, ebnurs-2022–103540. https://doi.org/ 10.1136/ebnurs-2022-103540
  3. O’Neill J. (2016) Tackling Drug-Resistant Infections Globally: Final Report and Recommendations. https://apo.org.au/node/63983
  4. Klein E.Y., Van Boeckel T.P., Martinez E.M., Pant S., Gandra S., Levin S.A., Goossens H., Laxminarayan R. (2018) Global increase and geographic convergence in antibiotic consumption between 2000 and 2015. Proc. Natl. Acad. Sci. USA. 115(15), E3463–E3470.
  5. Durand G.A., Raoult D., Dubourg G. (2019) Antibiotic discovery: history, methods and perspectives. Int. J. Antimicrob. Agents. 53(4), 371–382.
  6. de Kraker M.E.A., Lipsitch M. (2021) Burden of antimicrobial resistance: compared to what? Epidemiol. Rev. 43(1), 53–64.
  7. Hanberger H., Walther S., Leone M., Barie P.S., Rello J., Lipman J., Marshall J.C., Anzueto A., Sakr Y., Pickkers P. (2011) Increased mortality associated with meticillin-resistant Staphylococcus aureus (MRSA) infection in the Intensive Care Unit: results from the EPIC II study. Int. J. Antimicrob. Agents. 38(4), 331–335.
  8. Yang C.C., Sy C.L., Huang Y.C., Shie S.S., Shu J.C., Hsieh P.H., Hsiao C.H., Chen C.J. (2018) Risk factors of treatment failure and 30-day mortality in patients with bacteremia due to MRSA with reduced vancomycin susceptibility. Sci. Rep. 8(1), 7868.
  9. Dellinger R.P., Levy M.M., Carlet J.M., Bion J., Parker M.M., Jaeschke R., Reinhart K., Angus D.C., Brun-Buisson C., Beale R. (2008) Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008. Crit. Care Med. 36(1), 296–327.
  10. Chen H.C., Lin W.L., Lin C.C., Hsieh W.H., Hsieh C.H., Wu M.H., Wu J.Y., Lee C.C. (2013) Outcome of inadequate empirical antibiotic therapy in emergency department patients with community-onset bloodstream infections J. Antimicrob. Chemother. 68(4), 947–953.
  11. Dickinson J.D., Kollef M.H. (2011) Early and adequate antibiotic therapy in the treatment of severe sepsis and septic shock. Curr. Infect. Dis. Rep. 13, 399–405.
  12. Goneau L.W., Delport J., Langlois L., Poutanen S.M., Razvi H., Reid G., Burton J.P. (2020) Issues beyond resistance: inadequate antibiotic therapy and bacterial hypervirulence. FEMS Microbes. 1(1), xtaa004.
  13. Reller L.B., Weinstein M., Jorgensen J.H., Ferraro M.J. (2009) Antimicrobial susceptibility testing: a review of general principles and contemporary practices. Clin. Infect. Dis. 49(11), 1749–1755.
  14. Khan Z.A., Siddiqui M.F., Park S. (2019) Current and emerging methods of antibiotic susceptibility testing. Diagnostics (Basel). 9(2), 49.
  15. Steingart K.R., Schiller I., Horne D.J., Pai M., Boehme C.C., Dendukuri N. (2014) Xpert® MTB/RIF assay for pulmonary tuberculosis and rifampicin resistance in adults. Cochrane Database Syst. Rev. 2014(1), CD009593.
  16. Kumar P., Nagarajan A., Uchil P.D. (2018) Analysis of cell viability by the MTT assay. Cold Spring Harb. Protoc. 2018(6), pdb-prot095505.
  17. Van Meerloo J., Kaspers G.J.L., Cloos J. (2011) Cell sensitivity assays: the MTT assay. Methods Mol. Biol. 2011, 237–245.
  18. Bahuguna A., Khan I., Bajpai V.K., Kang S.C. (2017) MTT assay to evaluate the cytotoxic potential of a drug. Bangl. J. Pharm. 12(2), 115–118.
  19. Tolosa L., Donato M. T., Gómez-Lechón M. J. (2015) General cytotoxicity assessment by means of the MTT assay. Methods Mol. Biol. 2015, 333–348.
  20. Weichert H., Blechschmidt I., Schröder S., Ambrosius H. (1991) The MTT-assay as a rapid test for cell proliferation and cell killing: application to human peripheral blood lymphocytes (PBL). Allerg. Immunol. (Leipz). 37(3–4), 139–144.
  21. Molaae N., Mosayebi G., Pishdadian A., Ejtehadifar M., Ganji A. (2017) Evaluating the proliferation of human peripheral blood mononuclear cells using MTT assay. Int. J. Basic Sci. Med. 2(1), 25–28.
  22. Cole S.P.C. (1986) Rapid chemosensitivity testing of human lung tumor cells using the MTT assay. Cancer Chemother. Pharmacol. 17(3), 259–263.
  23. Campling B.G., Pym J., Baker H.M., Cole S.P.C., Lam Y.M. (1991) Chemosensitivity testing of small cell lung cancer using the MTT assay. Br. J. Cancer. 63(1), 75–83.
  24. Grela E., Kozłowska J., Grabowiecka A. (2018) Current methodology of MTT assay in bacteria — a review. Acta Histochem. 120(4), 303–311.
  25. Montoro E., Lemus D., Echemendia M., Martin A., Portaels F., Palomino J.C. (2005) Comparative evaluation of the nitrate reduction assay, the MTT test, and the resazurin microtitre assay for drug susceptibility testing of clinical isolates of Mycobacterium tuberculosis. J. Antimicrob. Chemother. 55(4), 500–505.
  26. Mshana R.N., Tadesse G., Abate G., Miörner H. (1998) Use of 3–(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide for rapid detection of rifampin-resistant Mycobacterium tuberculosis. J. Clin. Microbiol. 36(5), 1214–1219.
  27. Moodley S., Koorbanally N.A., Moodley T., Ramjugernath D., Pillay M. (2014) The 3-(4, 5-dimethylthiazol-2-yl)-2, -5-diphenyl tetrazolium bromide (MTT) assay is a rapid, cheap, screening test for the in vitro anti-tuberculous activity of chalcones. J. Microbiol. Methods. 104, 72–78.
  28. Shi L., Ge H.M., Tan S.H., Li H.Q., Song Y.C., Zhu H.L., Tan R.X. (2007) Synthesis and antimicrobial activities of Schiff bases derived from 5-chloro-salicylaldehyde. Eur. J. Med. Chem. 42(4), 558–564.
  29. Brambilla E., Ionescu A., Cazzaniga G., Edefonti V., Gagliani M. (2014) The influence of antibacterial toothpastes on in vitro Streptococcus mutans biofilm formation: a continuous culture study. Am. J. Dent. 27(3), 160–166.
  30. Stevens M.G., Olsen S.C. (1993) Comparative analysis of using MTT and XTT in colorimetric assays for quantitating bovine neutrophil bactericidal activity. J. Immunol. Methods. 157(1–2), 225–231.
  31. Stevens M.G., Kehrli Jr M.E., Canning P.C. (1991) A colorimetric assay for quantitating bovine neutrophil bactericidal activity. Vet. Immunol. Immunopathol. 28(1), 45–56.
  32. Kudelski A. (2008) Analytical applications of Raman spectroscopy. Talanta. 76(1), 1–8.
  33. Kuhar N., Sil S., Umapathy S. (2021) Potential of Raman spectroscopic techniques to study proteins. Spectrochim. Acta A Mol. Biomol. Spectrosc. 258, 119712.
  34. Martinez M.G., Bullock A.J., MacNeil S., Rehman I.U. (2019) Characterisation of structural changes in collagen with Raman spectroscopy. Appl. Spectrosc. Rev. 54(6), 509–542.
  35. Beljebbar A., Bouché O., Diébold M.D., Guillou P.J., Palot J.P., Eudes D., Manfait M. (2009) Identification of Raman spectroscopic markers for the characterization of normal and adenocarcinomatous colonic tissues. Crit. Rev. Oncol. Hematol. 72(3), 255–264.
  36. Depciuch J., Kaznowska E., Zawlik I., Wojnarowska R., Cholewa M., Heraud P., Cebulski J. (2016) Application of Raman spectroscopy and infrared spectroscopy in the identification of breast cancer. Appl. Spectrosc. 70(2), 251–263.
  37. Chan J.W., Taylor D.S., Lane S.M., Zwerdling T., Tuscano J., Huser T. (2008) Nondestructive identification of individual leukemia cells by laser trapping Raman spectroscopy. Anal. Chem. 80(6), 2180–2187.
  38. Pahlow S., Meisel S., Cialla-May D., Weber K., Rösch P., Popp J. (2015) Isolation and identification of bacteria by means of Raman spectroscopy. Adv. Drug. Deliv. Rev. 89, 105–120.
  39. Stöckel S., Kirchhoff J., Neugebauer U., Rösch P., Popp J. (2016) The application of Raman spectroscopy for the detection and identification of microorganisms. J. Raman. Spectrosc. 47(1), 89–109.
  40. Riss T. (2017) Is your MTT assay really the best choice? Promega Corporation website http://www.promega.in/resources/pubhub/is-your-mtt-assay-really-the-best-choice/
  41. Mao Z., Liu Z., Chen L., Yang J., Zhao B., Jung Y.M., Wang X., Zhao C. (2013) Predictive value of the surface-enhanced resonance Raman scattering-based MTT assay: a rapid and ultrasensitive method for cell viability in situ. Anal. Chem. 85(15), 7361–7368.
  42. Mao Z., Liu Z., Yang J., Han X., Zhao B., Zhao C. (2018). In situ semi-quantitative assessment of single-cell viability by resonance Raman spectroscopy. Chem. Commun. (Camb.). 54(52), 7135–7138.
  43. Wilson M.L., Gaido L. (2004) Laboratory diagnosis of urinary tract infections in adult patients. Clin. Infect. Dis. 38(8), 1150–1158.
  44. Lamy B., Dargère S., Arendrup M.C., Parienti J.J., Tattevin P. (2016) How to optimize the use of blood cultures for the diagnosis of bloodstream infections? A state-of-the art. Front. Microbiol. 7, 191111.
  45. Stiles P.L., Dieringer J.A., Shah N.C., Van Duyne R.P. (2008) Surface-enhanced Raman spectroscopy. Annu. Rev. Anal. Chem. 1, 601–626.
  46. Bell S.E.J., Charron G., Cortés E., Kneipp J., de la Chapelle M.L., Langer J., Procházka M., Tran V., Schlücker S. (2020) Towards reliable and quantitative surface‐enhanced Raman scattering (SERS): from key parameters to good analytical practice. Angew. Chem. Int. Ed. Engl. 59(14), 5454–5462.
  47. Lin C., Li Y., Peng Y., Zhao S., Xu M., Zhang L., Huang Z., Shi J., Yang Y. (2023) Recent development of surface-enhanced Raman scattering for biosensing. J. Nanobiotechnology. 21(1), 149.
  48. Eilers P.H.C. (2003) A perfect smoother. Anal. Chem. 75(14), 3631–3636.
  49. Baek S.J., Park A., Ahn Y.J., Choo J. (2015) Baseline correction using asymmetrically reweighted penalized least squares smoothing. Analyst. 140(1), 250–257.
  50. Frens G. (1973) Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nat. Phys. Sci. 241(105), 20–22.
  51. Hong S., Li X. (2013) Optimal size of gold nanoparticles for surface‐enhanced Raman spectroscopy under different conditions. J. Nanomater. 2013(1), 790323.
  52. Gerlier D., Thomasset N. (1986) Use of MTT colorimetric assay to measure cell activation. J. Immunol. Methods. 94(1–2), 57–63.
  53. Baba T., Ara T., Hasegawa M., Takai Y., Okumura Y., Baba M., Datsenko K.A., Tomita M., Wanner B.L., Mori H. (2006) Construction of Escherichia coli K‐12 in‐frame, single‐gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2(1), 2006–2008.
  54. Kumari G., Kandula J., Narayana C. (2015) How far can we probe by SERS? J. Phys. Chem. C. 119(34), 20057–20064.
  55. Xu W., Shi D., Chen K., Palmer J., Popovich D.G. (2023) An improved MTT colorimetric method for rapid viable bacteria counting. J. Microbiol. Methods. 214, 106830.

Supplementary files

Supplementary Files
Action
1. JATS XML
Download
2. Appendix
Download (522KB)
Indexing metadata
3. Fig. 1. MTT reduction reaction.

Download (137KB)
Indexing metadata
4. Fig. 2. Selection of the concentration of gold nanoparticles to enhance the formazan signal in the GCR spectrum.

Download (215KB)
Indexing metadata
5. Fig. 3. Selection of the incubation time of formazane with gold nanoparticles to enhance the signal in the GCR spectrum.

Download (207KB)
Indexing metadata
6. Fig. 4. GCR spectrum of formazane (concentration 1.25 micrograms/ml). The characteristic peaks of formazan are signed; the wavelength of the exciting radiation is 638 nm.

Download (160KB)
Indexing metadata
7. Fig. 5. Comparative analysis of the GCR and CR spectra of formazane.

Download (190KB)
Indexing metadata
8. Fig. 6. Comparison of the sensitivity of GCR and RAMAN spectroscopy to determine the concentration of formazane formed from MTT in viable E. coli cells.

Download (193KB)
Indexing metadata

Copyright (c) 2024 Russian Academy of Sciences