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Quantitative Determination of Beta-Lactamase mRNA in the RNA Transcripts of Antibiotic-Resistant Bacteria Using Colorimetric Biochips

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Abstract

A method for the quantitative determination of mRNA of TEM-type serine beta-lactamases in bacteria resistant to beta-lactam antibiotics has been developed. The method includes several stages: the isolation of a total RNA fraction from a bacterial culture and the production of the beta-lactamase target DNA in sequential reverse transcription and polymerase chain reaction with the incorporation of a biotin label, and the hybridization of the target DNA on colorimetric biochips. Sample preparation conditions were optimized to increase the yield of the analyzed target DNA. A calibration curve for determining the amount of mRNA was constructed using a standard sample of beta-lactamase TEM-1 mRNA obtained by transcription in vitro. The advantage of using a standard sample corresponding to the full-length blaTEM-1 gene is that it passes through all the stages of analysis in parallel with the test samples with the same efficiency. The detection limit for TEM-1 beta-lactamase mRNA was 0.40 ± 0.05 amol/mL; the range of determined concentrations was from 1.0 amol/mL to 2000 fmol/mL, and the relative standard deviation did not exceed 12%. The duration of analysis after the production of a bacterial culture was about 7 h. The developed method can be used to study the conditions for the expression of beta-lactamase genes in bacteria resistant to antimicrobial drugs.

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REFERENCES

  1. Eichenberger, E.M. and Thaden, J.T., Antibiotics, 2019, vol. 8, no. 2, p. 97.

    Article  Google Scholar 

  2. World Health Organization. Antibiotic resistance. http://www.who.int/news-room/fact-sheets/detail/ antibiotic-resistance. Accessed July 1, 2021.

  3. Mulani, M.S., Kamble, E.E., Kumkar, S.N., Tawre, M.S., and Pardesi, K.R., Front. Microbiol., 2019, vol. 10, p. 539.

    Article  Google Scholar 

  4. Wright, G.D., Adv. Drug Delivery Rev., 2005, vol. 57, no. 10, p. 1451.

    Article  CAS  Google Scholar 

  5. Egorov, A.M., Ulyashova, M.M., and Rubtsova, M.Y., Acta Nat., 2018, vol. 10, no. 4, p. 33.

    Article  CAS  Google Scholar 

  6. Klein, E.Y., Van Boeckel, T.P., Martinez, E.M., Pant, S., Gandra, S., Levin, S.A., Goossens, H., and Laxminarayan, R., Proc. Natl. Acad. Sci. U. S. A., 2018, vol. 115, no. 15, p. 3463.

    Google Scholar 

  7. King, D.T., Sobhanifar, S., and Strynadka, N.C.J., in Handbook of Antimicrobial Resistance, New York: Springer, 2017, p. 177.

    Google Scholar 

  8. Bush, K., Antimicrob. Agents Chemother., 2018, vol. 62, no. 10, e01076.

    Article  CAS  Google Scholar 

  9. Edwards, T., Williams, C., Teethaisong, Y., Sealey, J., Sasaki, S., Hobbs, G., Cuevas, L.E., Evans, K., and Adams, E.R., Diagn. Microbiol. Infect. Dis., 2020, vol. 97, no. 4. 115076.

    Article  CAS  Google Scholar 

  10. Dallenne, C., Da, CostaA., Decre, D., Favier, C., and Arlet, G., J. Antimicrob. Chemother., 2010, vol. 65, no. 3, p. 490.

    Article  CAS  Google Scholar 

  11. Band, V.I. and Weiss, D.S., PLoS Pathog., 2019, vol. 15, no. 6, e1007726.

    Article  CAS  Google Scholar 

  12. Andersson, D.I., Nicoloff, H., and Hjort, K., Nat. Rev. Microbiol., 2019, vol. 17, no. 8, p. 479.

    Article  CAS  Google Scholar 

  13. Kjeldsen, T.S.B., Overgaard, M., Nielsen, S.S., Bortolaia, V., Jelsbak, L., Sommer, M., Guardabassi, L., and Olsen, J.E., J. Antimicrob. Chemother., 2015, vol. 70, no. 1, p. 62.

    Article  CAS  Google Scholar 

  14. Maurya, A.P., Chanda, D.D., Bora, D., Talukdar, A.D., Chakravarty, A., and Bhattacharjee, A., Microb. Drug Resist., 2017, vol. 23, no. 2, p. 133.

    Article  CAS  Google Scholar 

  15. Balabanian, G., Rose, M., Manning, N., Landman, D., and Quale, J., Microb. Drug Resist., 2018, vol. 24, no. 7, p. 877.

    Article  CAS  Google Scholar 

  16. Lietard, J., Ameur, D., Damha, M.J., and Somoza, M.M., Angew Chem., Int. Ed. Engl., 2018, vol. 57, no. 46, p. 15257.

    Article  CAS  Google Scholar 

  17. Wang, Y., Quc, J., Baa, Q., Donga, J., Zhang, L., Zhanga, H., Wue, A., Wangf, D., Xia, Z., Peng, D., Shuf, Y., Caoc, B., and Jiang, T., J. Virol. Methods, 2016, vol. 234, p. 178.

    Article  CAS  Google Scholar 

  18. Bustin, S. and Nolan, T., Eur. J. Clin. Invest., 2017, vol. 47, no. 10, p. 756.

    Article  Google Scholar 

  19. Hughes, T.R., Marton, M.J., Jones, A.R., Roberts, C.J., Stoughton, R., Armour, C.D., and Friend, S.H., Cell, 2000, vol. 102, no. 1, p. 109.

    Article  CAS  Google Scholar 

  20. Feng, J., Billal, D.S., Lupien, A., Racine, G., Winstall, E., Legare, D., and Ouellette, M., J. Proteome Res., 2011, vol. 10, no. 10, p. 4439.

    Article  CAS  Google Scholar 

  21. Al-Rubaye, D.S., Henihan, G., Al-Abasly, A.K.A., Seagar, A.L., Al-Attraqchi, A.A.F., Schulze, H., Hashim, D.S., Kamil, J.K., Laurenson, I.F., and Bachmann, T.T., J. Med. Microbiol., 2016, vol. 65, no. 2, p. 114.

    Article  CAS  Google Scholar 

  22. Moure, R., Tudó, G., Medina, R., Vicente, E., Caldito, J.M., Codina, M.G., Coll, P., Español, M., Gonzalez-Martin, J., Rey-Jurado, E., Salvadó, M., Tórtola, M.T., and Alcaide, F., Tuberculosis, 2013, vol. 93, no. 5, p. 508.

    Article  CAS  Google Scholar 

  23. Naas, T., Cuzon, G., Truong, H., Bernabeu, S., and Nordmann, P., Antimicrob. Agents Chemother., 2010, vol. 54, no. 8, p. 3086.

    Article  CAS  Google Scholar 

  24. Rubtsova, M.Yu., Ulyashova, M.M., Edelstein, M.V., and Egorov, A.M., Biosens. Bioelectron., 2010, vol. 26, no. 4, p. 1252.

    Article  CAS  Google Scholar 

  25. Rubtsova, M.Yu., Ulyashova, M.M., Pobolelova, Yu.I., Presnova, G.V., and Egorov, A.M., Appl. Biochem. Microbiol., 2020, vol. 56, no. 2, p. 130.

    Article  CAS  Google Scholar 

  26. Díaz-González, M., Pablo Salvador, J., Bonilla, D., Pilar Marco, M., Fernández-Sánchez, C., and Baldi, A., Biosens. Bioelectron., 2015, vol. 74, p. 698.

    Article  Google Scholar 

  27. Palzkill, T., Front. Mol. Biosci., 2018, vol. 5, p. 1.

    Article  Google Scholar 

  28. Pimenta, A.C., Fernandes, R., and Moreira, I.S., Mini Rev. Med. Chem., 2014, vol. 14, no. 2, p. 111.

    Article  CAS  Google Scholar 

  29. Singh, N.S., Singhal, N., Kumar, M., and Virdi, J.S., Infect. Genet. Evol., 2021, vol. 90.

  30. Hubbard, A.T.M., Mason, J., Roberts, P., Parry, C.M., Corless, C., van Aartsen, J., Howard, A., Bulgasim, I., Fraser, A.J., Adams, E.R., Roberts, A.P., and Edwards, T., Nat. Commun., 2020, vol. 11, no. 1, p. 4915.

    Article  CAS  Google Scholar 

  31. Grigorenko, V.G., Andreeva, I.P., Rubtsova, M.Yu., Deygen, I.M., Antipin, R.L., Majouga, A.G., Egorov, A.M., Beshnova, D.A., Kallio, J., Hackenberg, C., and Lamzin, V.S., Biochimie, 2017, vol. 132, p. 45.

    Article  CAS  Google Scholar 

  32. http://www.eucast.org/fileadmin/src/media/PDFs/ EUCAST_files/Breakpoint_tables/v_10.0_Breakpoint_Tables.pdf. Accessed July 1, 2021.

  33. Birnboim, H.C. and Doly, J., Nucleic Acid Res., 1979, vol. 7, p. 1513.

    Article  CAS  Google Scholar 

  34. Greisen, K., Loeffelholz, M., Purohit, A., and Leong, D., J. Clin. Microbiol., 1994, vol. 32, no. 2, p. 335.

    Article  CAS  Google Scholar 

  35. Nossal, N.G. and Heppel, L.A., J. Biol. Chem., 1966, vol. 241, no. 13, p. 3055.

    Article  CAS  Google Scholar 

  36. Schenborn, E.T. and Mierendorf, R.C., Nucleic Acid Res., 1985, vol. 13, no. 17, p. 6223.

    Article  CAS  Google Scholar 

  37. Verma, I.M., in The Enzymes, Boyer, P.D.N.Y, Ed.,

  38. Pobolelova, Yu.I., Ulyashova, M.M., Rubtsova, M.Yu., and Egorov, A.M., Biochemistry (Moscow), 2014, vol. 79, no. 6, p. 566.

    Article  CAS  Google Scholar 

  39. Livak, K.J. and Schmittgen, T.D., Methods, 2001, vol. 25, no. 4, p. 402.

    Article  CAS  Google Scholar 

  40. Bustin, S.A., Biomol. Detect. Quantif., 2014, vol. 2, p. 35.

    Article  Google Scholar 

  41. Bustin, S.A., Benes, V., Garson, J., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G., et al., Nat. Methods, 2013, vol. 10, p. 1063.

    Article  CAS  Google Scholar 

  42. Kurbatov, L.K. and Zgoda, V.G., Biomed. Khim., 2016, vol. 62, no. 6, p. 715.

    Article  CAS  Google Scholar 

  43. Bustin, S.A., J. Mol. Endocrinol., 2002, vol. 29, p. 23.

    Article  CAS  Google Scholar 

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Funding

This work was supported by the Moscow State University (state contract no. АААА-А21-121011290089-4) and the Russian Foundation for Basic Research (grant no. 19-34-50071). 

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Authors and Affiliations

  1. Chemistry Department, Moscow State University, 119991, Moscow, Russia

    M. Yu. Rubtsova, A. A. Filippova, V. G. Grigorenko, G. V. Presnova, M. M. Ulyashova & A. M. Egorov

  2. State Research Center for Applied Microbiology and Biotechnology, 142279, Serpukhov, Moscow oblast, Russia

    N. K. Fursova

Authors
  1. M. Yu. Rubtsova
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  2. A. A. Filippova
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  3. N. K. Fursova
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  4. V. G. Grigorenko
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  5. G. V. Presnova
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  6. M. M. Ulyashova
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  7. A. M. Egorov
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Correspondence to M. Yu. Rubtsova.

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Translated by V. Makhlyarchuk

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Rubtsova, M.Y., Filippova, A.A., Fursova, N.K. et al. Quantitative Determination of Beta-Lactamase mRNA in the RNA Transcripts of Antibiotic-Resistant Bacteria Using Colorimetric Biochips. J Anal Chem 77, 519–530 (2022). https://doi.org/10.1134/S1061934822050124

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  • DOI: https://doi.org/10.1134/S1061934822050124

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