1887

Journal of Medical Microbiology logo

Volume 68, Issue 2

A rapid and simple detection method for phenotypic antimicrobial resistance in Escherichia coli by loop-mediated isothermal amplification Free

Abstract

Purpose. In infectious disease therapy, administration of adequate antimicrobial agents is essential for preventing the emergence and spread of resistant bacteria. However, conventional antimicrobial susceptibility testing (AST), based on bacterial growth, is time consuming; therefore, a rapid, simple assay is needed for the timely selection of appropriate antibiotics in clinical laboratories. Here, we established a simple, cost-effective, time-saving and highly sensitive AST assay based on loop-mediated isothermal amplification (LAMP).

Methodology. The targeted bacteria were cultivated for a short period with or without antibiotic before the LAMP reaction. The time to detect a positive reaction with LAMP was used to generate a threshold time (Tt) value, and subtraction of the Tt value for an antibiotic-free sample from the Tt value in an antibiotic-exposed sample generated the ΔTt value, which was used as a marker of antimicrobial susceptibility. The ΔTt value generated using the LAMP-based assay simply and quickly detected antimicrobial resistance in clinical Escherichia coli isolates.

Results. Detection of susceptibility to levofloxacin using the ΔTt value perfectly matched with the results of the conventional assay. In addition, the sensitivity and specificity for the detection of ampicillin, trimethoprim-sulfamethoxazole and fosfomycin resistance were 100 %, 93.8 %, 100 % and 80.0 %, 93.3 %, 97.6 %, respectively.

Conclusion. These results showed that this LAMP-based AST has high sensitivity and specificity for detecting resistant strains and a significant time advantage compared with the conventional method.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): antimicrobial resistance , antimicrobial susceptibility testing , Escherichia coli and loop-mediated isothermal amplification
© 2019 The Authors
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2019-01-09
2024-04-25
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References

  1. O’Neill J. The review on antimicrobial resistance. Tackling drug-resistant infections globally: final report and recommendations. HM Government; 2016 Available at https://amr-review.org/sites/default/files/160518_Final%20paper_with%20cover.pdf
  2. Kerremans JJ, Verboom P, Stijnen T, Hakkaart-van Roijen L, Goessens W et al. Rapid identification and antimicrobial susceptibility testing reduce antibiotic use and accelerate pathogen-directed antibiotic use. J Antimicrob Chemother 2008; 61:428–435 [View Article][PubMed]
     [Google Scholar]
  3. Frickmann H, Masanta WO, Zautner AE. Emerging rapid resistance testing methods for clinical microbiology laboratories and their potential impact on patient management. Biomed Res Int 2014; 2014:1–19 [View Article][PubMed]
     [Google Scholar]
  4. Jorgensen JH, Ferraro MJ. Antimicrobial susceptibility testing: a review of general principles and contemporary practices. Clin Infect Dis 2009; 49:1749–1755 [View Article][PubMed]
     [Google Scholar]
  5. Murakami K, Minamide W, Wada K, Nakamura E, Teraoka H et al. Identification of methicillin-resistant strains of staphylococci by polymerase chain reaction. J Clin Microbiol 1991; 29:2240–2244[PubMed]
     [Google Scholar]
  6. Lemon JK, Khil PP, Frank KM, Dekker JP. Rapid Nanopore sequencing of plasmids and resistance gene detection in clinical isolates. J Clin Microbiol 2017; 55:3530–3543 [View Article][PubMed]
     [Google Scholar]
  7. Louie M, Cockerill FR, Testing S. Phenotypic and genotypic tests for bacteria and mycobacteria. Infect Dis Clin North Am 2001; 15:1205–1226
     [Google Scholar]
  8. Davies J, Davies D. Origins and evolution of antibiotic resistance. Microbiol Mol Biol Rev 2010; 74:417–433 [View Article][PubMed]
     [Google Scholar]
  9. Rolain JM, Mallet MN, Fournier PE, Raoult D. Real-time PCR for universal antibiotic susceptibility testing. J Antimicrob Chemother 2004; 54:538–541 [View Article][PubMed]
     [Google Scholar]
  10. Mezger A, Gullberg E, Göransson J, Zorzet A, Herthnek D et al. A general method for rapid determination of antibiotic susceptibility and species in bacterial infections. J Clin Microbiol 2015; 53:425–432 [View Article][PubMed]
     [Google Scholar]
  11. Schoepp NG, Khorosheva EM, Schlappi TS, Curtis MS, Humphries RM et al. Digital quantification of DNA replication and chromosome segregation enables determination of antimicrobial susceptibility after only 15 minutes of antibiotic exposure. Angew Chem Int Ed Engl 2016; 55:9557–9561 [View Article][PubMed]
     [Google Scholar]
  12. Schoepp NG, Schlappi TS, Curtis MS, Butkovich SS, Miller S et al. Rapid pathogen-specific phenotypic antibiotic susceptibility testing using digital LAMP quantification in clinical samples. Sci Transl Med 2017; 9:eaal3693 [View Article][PubMed]
     [Google Scholar]
  13. Kinnunen P, Mcnaughton BH, Albertson T, Sinn I, Mofakham S et al. Self-assembled magnetic bead biosensor for measuring bacterial growth and antimicrobial susceptibility testing. Small 2012; 8:2477–2482 [View Article][PubMed]
     [Google Scholar]
  14. Liu CY, Han YY, Shih PH, Lian WN, Wang HH et al. Rapid bacterial antibiotic susceptibility test based on simple surface-enhanced Raman spectroscopic biomarkers. Sci Rep 2016; 6:23375 [View Article][PubMed]
     [Google Scholar]
  15. Choi J, Yoo J, Lee M, Kim EG, Lee JS et al. A rapid antimicrobial susceptibility test based on single-cell morphological analysis. Sci Transl Med 2014; 6:267ra174 [View Article][PubMed]
     [Google Scholar]
  16. Baltekin Ö, Boucharin A, Tano E, Andersson DI, Elf J. Antibiotic susceptibility testing in less than 30 min using direct single-cell imaging. Proc Natl Acad Sci USA 2017; 114:9170–9175 [View Article][PubMed]
     [Google Scholar]
  17. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 2000; 28:63e–63 [View Article][PubMed]
     [Google Scholar]
  18. Mori Y, Nagamine K, Tomita N, Notomi T. Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun 2001; 289:150–154 [View Article][PubMed]
     [Google Scholar]
  19. Li Y, Fan P, Zhou S, Zhang L, Amplification L-M. Loop-mediated isothermal amplification (LAMP): a novel rapid detection platform for pathogens. Microb Pathog 2017; 107:54–61
     [Google Scholar]
  20. Neilan BA, Jacobs D, del Dot T, Blackall LL, Hawkins PR et al. rRNA sequences and evolutionary relationships among toxic and nontoxic cyanobacteria of the genus Microcystis . Int J Syst Bacteriol 1997; 47:693–697 [View Article][PubMed]
     [Google Scholar]
  21. Dallenne C, da Costa A, Decré D, Favier C, Arlet G. Development of a set of multiplex PCR assays for the detection of genes encoding important beta-lactamases in Enterobacteriaceae . J Antimicrob Chemother 2010; 65:490–495 [View Article][PubMed]
     [Google Scholar]
  22. Wayne PA. Methods for dilution antimicrobial susceptibility tests for bacteria that grow Aerobically; 9th ed. M07-A9. Clinical and Laboratory Standards Institute. Clinical and Laboratory Standards Institute; 2012
  23. Wayne PA. Performance standards for antimicrobial susceptibility testing; 25th informational supplement. CLSI M100-S25. Clinical and Laboratory Standards Institute. , 7th ed. Wayne, PA: Clinical and Laboratory Standards Institute; 2015
  24. Morrill HJ, Morton JB, Caffrey AR, Jiang L, Dosa D et al. Antimicrobial resistance of Escherichia coli urinary isolates in the Veterans Affairs Health Care System. Antimicrob Agents Chemother 2017; 61:e0223616 [View Article][PubMed]
     [Google Scholar]
  25. Oteo J, Orden B, Bautista V, Cuevas O, Arroyo M et al. CTX-M-15-producing urinary Escherichia coli O25b-ST131-phylogroup B2 has acquired resistance to fosfomycin. J Antimicrob Chemother 2009; 64:712–717 [View Article][PubMed]
     [Google Scholar]
  26. Muratani T, Matsumoto T. Bacterial resistance to antimicrobials in urinary isolates. Int J Antimicrob Agents 2004; 24:28–31 [View Article]
     [Google Scholar]
  27. Kato H, Yokoyama T, Kato H, Arakawa Y. Rapid and simple method for detecting the toxin B gene of Clostridium difficile in stool specimens by loop-mediated isothermal amplification. J Clin Microbiol 2005; 43:6108–6112 [View Article][PubMed]
     [Google Scholar]
  28. Takano C, Seki M, Kim DW, Kilgore PE, Fuwa K et al. Molecular serotype-specific identification of non-type b. Front Microbiol 1877; 2017:8
     [Google Scholar]
  29. Liu W, Zou D, Li Y, Wang X, He X et al. Sensitive and rapid detection of the new Delhi metallo-beta-lactamase gene by loop-mediated isothermal amplification. J Clin Microbiol 2012; 50:1580–1585 [View Article][PubMed]
     [Google Scholar]
  30. Safavieh M, Kanakasabapathy MK, Tarlan F, Ahmed MU, Zourob M et al. Emerging loop-mediated isothermal amplification-based microchip and microdevice technologies for nucleic acid detection. ACS Biomater Sci Eng 2016; 2:278–294 [View Article]
     [Google Scholar]
  31. Kostić T, Ellis M, Williams MR, Stedtfeld TM, Kaneene JB et al. Thirty-minute screening of antibiotic resistance genes in bacterial isolates with minimal sample preparation in static self-dispensing 64 and 384 assay cards. Appl Microbiol Biotechnol 2015; 99:7711–7722 [View Article][PubMed]
     [Google Scholar]
  32. Hayami H, Takahashi S, Ishikawa K, Yasuda M, Yamamoto S et al. Nationwide surveillance of bacterial pathogens from patients with acute uncomplicated cystitis conducted by the Japanese surveillance committee during 2009 and 2010: antimicrobial susceptibility of Escherichia coli and Staphylococcus saprophyticus . J Infect Chemother 2013; 19:393–403 [View Article][PubMed]
     [Google Scholar]
  33. Goss WA, Deitz WH, Cook TM. Mechanism of action of nalidixic acid on Escherichia coli . J Bacteriol 1964; 88:1112–1118[PubMed]
     [Google Scholar]
  34. Wormser GP, Keusch GT, Heel RC. Co-trimoxazole (trimethoprim-sulfamethoxazole): an updated review of its antibacterial activity and clinical efficacy. Drugs 1982; 24:459–518 [View Article][PubMed]
     [Google Scholar]
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A rapid and simple detection method for phenotypic antimicrobial resistance in Escherichia coli by loop-mediated isothermal amplification
J. Med. Microbiol. 68, 169 (2019); https://doi.org/10.1099/jmm.0.000903
/content/journal/jmm/10.1099/jmm.0.000903
/content/journal/jmm/10.1099/jmm.0.000903
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