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Very rapid flow cytometric assessment of antimicrobial susceptibility during the apparent lag phase of microbial (re)growth

Srijan Jindal, Harish Thampy, Philip J. Day, Douglas B. Kell
doi: https://doi.org/10.1101/480392
Srijan Jindal
1School of Chemistry, The University of Manchester, 131 Princess St, Manchester M1 7DN, UK
2Manchester Institute of Biotechnology, The University of Manchester, 131 Princess St, Manchester M1 7DN, UK
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Harish Thampy
3Firsway Health Centre, 121 Firs Way, Sale, Manchester M33 4BR, UK
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Philip J. Day
2Manchester Institute of Biotechnology, The University of Manchester, 131 Princess St, Manchester M1 7DN, UK
4Faculty of Faculty of Biology, Medicine and Health, The University of Manchester, Manchester M13 9PT, UK
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Douglas B. Kell
1School of Chemistry, The University of Manchester, 131 Princess St, Manchester M1 7DN, UK
2Manchester Institute of Biotechnology, The University of Manchester, 131 Princess St, Manchester M1 7DN, UK
3Firsway Health Centre, 121 Firs Way, Sale, Manchester M33 4BR, UK
5Present address: Dept of Biochemistry, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Crown St, Liverpool L69 7ZB, UK
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  • For correspondence: dbk@manchester.ac.uk
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Abstract

Cells of E. coli were grown in LB medium, taken from a stationary phase of 2-4h, and reinoculated into fresh media at a concentration (105.mL-1 or lower) characteristic of bacteriuria. Flow cytometry was used to assess how quickly we could detect changes in cell size, number, membrane energisation (using a carbocyanine dye) and DNA distribution. It turned out that while the lag phase observable macroscopically via bulk OD measurements could be as long as 4h, the true lag phase could be less than 15-20 min, and was accompanied by many observable biochemical changes. Antibiotics to which the cells were sensitive affected these changes within 20 min of reinoculation, providing the possibility of a very rapid antibiotic susceptibility test, on a timescale compatible with a visit to a GP clinic. The strategy was applied successfully to genuine potential Urinary Tract Infection (UTI) samples taken from a doctor’s surgery. The methods developed could prove of considerable value in ensuring the correct prescription and thereby lowering the spread of antimicrobial resistance.

Abbreviations
DiBAC4(3)
bis-(1,3-dibutyl-barbituric acid) trimethine oxonol
di-S-C3(5)
3,3′-dipropylthiadicarbocyanine iodide
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Posted November 29, 2018.
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Very rapid flow cytometric assessment of antimicrobial susceptibility during the apparent lag phase of microbial (re)growth
Srijan Jindal, Harish Thampy, Philip J. Day, Douglas B. Kell
bioRxiv 480392; doi: https://doi.org/10.1101/480392
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Very rapid flow cytometric assessment of antimicrobial susceptibility during the apparent lag phase of microbial (re)growth
Srijan Jindal, Harish Thampy, Philip J. Day, Douglas B. Kell
bioRxiv 480392; doi: https://doi.org/10.1101/480392

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