Advertisement

Microchimica Acta

, Volume 184, Issue 12, pp 4619–4628 | Cite as

Integrated microfluidic platform for rapid antimicrobial susceptibility testing and bacterial growth analysis using bead-based biosensor via fluorescence imaging

  • Pooja Sabhachandani
  • Saheli Sarkar
  • Paola C. Zucchi
  • Betsy A. Whitfield
  • James E. Kirby
  • Elizabeth B. Hirsch
  • Tania Konry
Original Paper

Abstract

The paper describes a droplet-based microfluidic method for phenotypic-based antimicrobial susceptibility testing (AST). In particular, this micro-droplet-based phenotypic assay evaluates susceptibility of different bacterial strains towards antibiotics by tracking effects on individual bacterial cells, including changes in bacterial cell number and morphology. The platform was validated by applying the method to test the responses of E. coli ATCC 25922 and 6937 (a clinical isolate), in spiked urine samples at a concentration of 5 × 104 cfu mL−1, to the antibiotics ceftazidime and levofloxacin. Both E. coli strains showed dose-dependent inhibition of bacterial replication and morphological alteration. These correlated well with minimal inhibitory concentrations determined by the reference broth microdilution method. Discrete bacterial divisions and morphological changes were observed within 20 min of on-chip incubation, demonstrating performance of rapid AST directly on urine samples. As proof-of-concept, specific bead-based biosensors were tested for capture and detection of E. coli for on-bead proliferation. The method has the attractive feature of allowing the detection of at least one bacterium per bead in less than 30 min. It can potentially be used to isolate a specific bacterial strain directly from patient urine samples for AST monitoring.

Graphical Abstract

(A) Schematic of the droplet microfluidic chip for bacterial detection and Antibiotic Susceptibility Testing (AST); (B) Time lapse proliferation images of green fluorescent protein expressing E. coli in droplets. (C) Bacterial proliferation on the bead-based sensor.

Keywords

Droplet microfluidics Bioassay development Biosensors Urinary tract infections E. coli Antibiotic susceptibility Fluorescence microscopy 

Notes

Acknowledgements

The authors would like to acknowledge Abhinav Gupta, Sneha Varghese and Sai Mynampati at Northeastern University for their assistance in fabrication of microfluidic devices. The authors would like to thank Zhi Shen Lin (Northeastern University) for his assistance in bacterial cell culture. The authors would also like to acknowledge the CIMIT grant awarded to Dr. Konry (CIMIT/POCT/NIH/NIBIB/16111315).

Compliance with ethical standards

The author(s) declare that they have no competing interests.

Supplementary material

604_2017_2492_MOESM1_ESM.docx (586 kb)
ESM 1 (DOCX 586 kb)
604_2017_2492_MOESM2_ESM.avi (1.8 mb)
ESM 2 (AVI 1817 kb)

References

  1. 1.
    Levy SB, Marshall B (2004) Antibacterial resistance worldwide: causes, challenges and responses. Nat Med 10:S122–S129CrossRefGoogle Scholar
  2. 2.
    World Health Organization (WHO). Antimicrobial resistance: global report on surveillance (2014) ISBN: 978 92 4 156474 8Google Scholar
  3. 3.
    Barber AE, Norton JP, Spivak AM, Mulvey MA (2013) Urinary tract infections: current and emerging management strategies. Clin Infect Dis 57(5):719–724CrossRefGoogle Scholar
  4. 4.
    Stamm WE, Norrby SR (2001) Urinary tract infections: disease panorama and challenges. J Infect Dis 183(Supplement 1):S1–S4CrossRefGoogle Scholar
  5. 5.
    Aguiar JP, Da Costa FA, Silva PC (2015) Evaluation of Empirical Antibiotic Therapy for the Treatment of Community-Acquired Urinary Tract Infections (CA-UTI). Int Arch Clin Pharmacol 1:002CrossRefGoogle Scholar
  6. 6.
    Walker E, Lyman A, Gupta K, Mahoney MV, Snyder GM, Hirsch EB (2016) Clinical management of an increasing threat: outpatient urinary tract infections due to multidrug-resistant uropathogens. Clin Infect Dis 63(7):960–965CrossRefGoogle Scholar
  7. 7.
    Brennan-Krohn T, Smith KP, Kirby JE (2017) The poisoned well: enhancing the predictive value of antimicrobial susceptibility testing in the era of multidrug resistance. J Clin Microbiol 55:2304–2308CrossRefGoogle Scholar
  8. 8.
    Wiegand I, Hilpert K, Hancock RE (2008) Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc 3(2):163–175CrossRefGoogle Scholar
  9. 9.
    CLSI (2009) Clinical and Laboratory Standards Institute (CLSI) performance standards for antimicrobial disk diffusion susceptibility tests 19th ed. approved standard. Wayne PA. Clinical and Laboratory Standards Institute document M100. S19–29Google Scholar
  10. 10.
    Schoepp NG, Khorosheva EM, Schlappi TS, Curtis MS, Humphries RM, Hindler JA, Ismagilov RF (2016) Digital Quantification of DNA Replication and Chromosome Segregation Enables Determination of Antimicrobial Susceptibility after only 15 Minutes of Antibiotic Exposure. Angew Chem 128(33):9709–9713CrossRefGoogle Scholar
  11. 11.
    Mezger A, Gullberg E, Göransson J, Zorzet A, Herthnek D, Tano E, Nilsson M, Andersson DI (2015) A general method for rapid determination of antibiotic susceptibility and species in bacterial infections. J Clin Microbiol 53(2):425–432CrossRefGoogle Scholar
  12. 12.
    Saint-Ruf, C., Crussard, S., Franceschi, C., Orenga, S., Ouattara, J., Ramjeet, M., Surre, J. and Matic, I., 2016. Antibiotic susceptibility testing of the Gram-Negative bacteria based on flow cytometry. Front Microbiol, 7Google Scholar
  13. 13.
    Zboromyrska Y, Rubio E, Alejo I, Vergara A, Mons A, Campo I, Bosch J, Marco F, Vila J (2016) Development of a new protocol for rapid bacterial identification and susceptibility testing directly from urine samples. Clin Microbiol Infect 22(6):561–5e1CrossRefGoogle Scholar
  14. 14.
    Hrabák J, Chudáčková E, Walková R (2013) Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry for detection of antibiotic resistance mechanisms: from research to routine diagnosis. Clin Microbio Rev 26(1):103–114CrossRefGoogle Scholar
  15. 15.
    Liu CY, Han YY, Shih PH, Lian WN, Wang HH, Lin CH, Hsueh PR, Wang JK, Wang YL (2016) Rapid bacterial antibiotic susceptibility test based on simple surface-enhanced Raman spectroscopic biomarkers. Sci Rep 6:23375CrossRefGoogle Scholar
  16. 16.
    Tang Y, Zhen L, Liu J, Wu J (2013) Rapid antibiotic susceptibility testing in a microfluidic pH sensor. Anal Chem 85(5):2787–2794CrossRefGoogle Scholar
  17. 17.
    Price CS, Kon SE, Metzger S (2014) Rapid antibiotic susceptibility phenotypic characterization of Staphylococcus aureus using automated microscopy of small numbers of cells. J Microbiol Methods 98:50–58CrossRefGoogle Scholar
  18. 18.
    Choi J, Yoo J, Lee M, Kim EG, Lee JS, Lee S, Joo S, Song SH, Kim EC, Lee JC, Kim HC (2014) A rapid antimicrobial susceptibility test based on single-cell morphological analysis. Sci Transl Med 6(267):267ra174CrossRefGoogle Scholar
  19. 19.
    Churski K, Kaminski TS, Jakiela S, Kamysz W, Baranska-Rybak W, Weibel DB, Garstecki P (2012) Rapid screening of antibiotic toxicity in an automated microdroplet system. Lab Chip 12(9):1629–1637CrossRefGoogle Scholar
  20. 20.
    Javid B, Sorrentino F, Toosky M, Zheng W, Pinkham JT, Jain N, Pan M, Deighan P, Rubin EJ (2014) Mycobacterial mistranslation is necessary and sufficient for rifampicin phenotypic resistance. PNAS 111:1132–1137CrossRefGoogle Scholar
  21. 21.
    Kang DK, Ali MM, Zhang K, Huang SS, Peterson E, Digman MA, Gratton E, Zhao W (2014) Rapid detection of single bacteria in unprocessed blood using Integrated Comprehensive Droplet Digital Detection. Nat Commun 5:5427CrossRefGoogle Scholar
  22. 22.
    Choi K, Ng AH, Fobel R, Wheeler AR (2012) Digital microfluidics. Anal Chem 5:413–440Google Scholar
  23. 23.
    Mohan R, Mukherjee A, Sevgen SE, Sanpitakseree C, Lee J, Schroeder CM, Kenis PJ (2013) A multiplexed microfluidic platform for rapid antibiotic susceptibility testing. Biosens Bioelectron 49:118–125CrossRefGoogle Scholar
  24. 24.
    Pulido MR, García-Quintanilla M, Martín-Peña R, Cisneros JM, McConnell MJ (2013) Progress on the development of rapid methods for antimicrobial susceptibility testing. J Antimicrob Chemother 68(12):2710–2717CrossRefGoogle Scholar
  25. 25.
    Mairhofer J, Roppert K, Ertl P (2009) Microfluidic systems for pathogen sensing: a review. Sensors 9(6):4804–4823CrossRefGoogle Scholar
  26. 26.
    Lui C, Cady NC, Batt CA (2009) Nucleic acid-based detection of bacterial pathogens using integrated microfluidic platform systems. Sensors 9(5):3713–3744CrossRefGoogle Scholar
  27. 27.
    Golberg A, Linshiz G, Kravets I, Stawski N, Hillson NJ, Yarmush ML, Marks RS, Konry T (2014) Cloud-enabled microscopy and droplet microfluidic platform for specific detection of Escherichia coli in water. PLoS One 9(1):e86341CrossRefGoogle Scholar
  28. 28.
    Sarkar S, Cohen N, Sabhachandani P, Konry T (2015) Phenotypic drug profiling in droplet microfluidics for better targeting of drug-resistant tumors. Lab Chip 15(23):4441–4450CrossRefGoogle Scholar
  29. 29.
    Wilson ML, Gaido L (2004) Laboratory diagnosis of urinary tract infections in adult patients. Clin Infect Dis 38(8):1150–1158CrossRefGoogle Scholar
  30. 30.
    CLSI (2016) Clinical and Laboratory Standards Institute: Performance standards for antimicrobial susceptibility testing; twenty-sixth informational supplement. Wayne PA. Clinical and Laboratory Standards Institute document M100, S26Google Scholar
  31. 31.
    Hayes MV, Orr DC (1983) Mode of action of ceftazidime: affinity for the penicillin-binding proteins of Escherichia coli K12, Pseudomonas aeruginosa and Staphylococcus aureus. J Antimicrob Chemother 12(2):119–126CrossRefGoogle Scholar
  32. 32.
    Buijs J, Dofferhoff ASM, Mouton JW, Wagenvoort JHT, Van Der Meer JWM (2008) Concentration dependency of β-lactam induced filament formation in Gram-negative bacteria. Clin Microbiol Infect 14(4):344–349CrossRefGoogle Scholar
  33. 33.
    Davis R, Bryson HM (1994) Levofloxacin. Drugs 47(4):677–700CrossRefGoogle Scholar
  34. 34.
    Justice SS, Hunstad DA, Cegelski L, Hultgren SJ (2008) Morphological plasticity as a bacterial survival strategy. Nat Rev Microbiol 6(2):162–168CrossRefGoogle Scholar
  35. 35.
    Dörr T, Lewis K, Vulić M (2009) SOS response induces persistence to fluoroquinolones in Escherichia coli. PLoS Genet 5(12):e1000760CrossRefGoogle Scholar
  36. 36.
    Xu S (2012) Electromechanical biosensors for pathogen detection. Microchim Acta 178(3–4):245–260CrossRefGoogle Scholar
  37. 37.
    Liebsch C, Rödiger S, Böhm A, Nitschke J, Weinreich J, Fruth A, Roggenbuck D, Lehmann W, Schedler U, Juretzek T, Schierack P (2017) Solid-phase microbead array for multiplex O-serotyping of Escherichia coli. Microchim Acta 184(5):1405–1415CrossRefGoogle Scholar
  38. 38.
    Roda A, Mirasoli M, Roda B, Bonvicini F, Colliva C, Reschiglian P (2012) Recent developments in rapid multiplexed bioanalytical methods for foodborne pathogenic bacteria detection. Microchim Acta 178(1–2):7–28CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria 2017

Authors and Affiliations

  1. 1.Department of Pharmaceutical Sciences, School of Pharmacy, Bouve College of Health SciencesNortheastern UniversityBostonUSA
  2. 2.Department of Pharmacy and Health Systems Sciences, School of Pharmacy, Bouve College of Health SciencesNortheastern UniversityBostonUSA
  3. 3.Department of PathologyBeth Israel Deaconess Medical CenterBostonUSA
  4. 4.Department of Experimental and Clinical PharmacologyUniversity of Minnesota College of PharmacyMinneapolisUSA

Personalised recommendations