The evolution and spread of antibiotic-resistant pathogens has become a major threat to public health.
Advanced tools are urgently needed to quickly diagnose antibiotic-resistant infections to initiate appropriate treatment. Here we report the development of a highly sensitive flow cytometric method to
probe minority population of antibiotic-resistant bacteria via single cell detection. Monoclonal antibody
against TEM-1 β-lactamase and Alexa Fluor 488-conjugated secondary antibody were used to selectively
label resistant bacteria green, and nucleic acid dye SYTO 62 was used to stain all the bacteria red. A
laboratory-built high sensitivity flow cytometer (HSFCM) was applied to simultaneously detect the side
scatter and dual-color fluorescence signals of single bacteria. By using E. coli JM109/pUC19 and E. coli
JM109 as the model systems for antibiotic-resistant and antibiotic-susceptible bacteria, respectively, as
low as 0.1% of antibiotic-resistant bacteria were accurately quantified. By monitoring the dynamic population change of a bacterial culture with the administration of antibiotics, we confirmed that under the
antimicrobial pressure, the original low population of antibiotic-resistant bacteria outcompeted susceptible strains and became the dominant population after 5 hours of growth. Detection of antibioticresistant infection in clinical urine samples was achieved without cultivation, and the bacterial load of
susceptible and resistant strains can be faithfully quantified. Overall, the HSFCM-based quantitative
method provides a powerful tool for the fundamental studies of antibiotic resistance and holds the
potential to provide rapid and precise guidance in clinical therapies.
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