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

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 doctors surgery. The methods developed could prove of considerable value in ensuring the correct prescription and thereby lowering the spread of antimicrobial resistance.