Fig. 1: Proposed workflow for using ribosome phenotypes in a rapid antibiotic susceptibility test.

a Workflow: First the clinical isolate is treated with antibiotics at a standard concentration for 30 min. Then, a standard FISH protocol is used to label the ribosomes with ssDNA fluorescent probes; in this case, EUB338-Cy3 binds a conserved region in the 30S subunit. The samples are imaged on a fluorescence microscope before neural networks use the ribosome signal to segment and then classify the cells as susceptible or resistant to the prescribed antibiotic treatment. b Ribosome Phenotypes: Representative fluorescence images are shown of E. coli MG1655 with and without antibiotic treatment (magenta, DNA stained with DAPI; green, ribosomes labelled with EUB338-Cy3 probes; combined DNA and ribosome signal). The scale bar is 2 µm. The ribosome density can be seen to anti-correlate with the DNA-dense regions. The untreated panel shows fixed cells with no antibiotic treatment. The chloramphenicol panel shows cells treated with 8 mg/L chloramphenicol (1 × EUCAST breakpoint) for 30 min before fixation. The ciprofloxacin panel shows the same, treated with 0.5 mg/L ciprofloxacin (1 × EUCAST breakpoint). The gentamicin panel shows the same, treated with 40 mg/L gentamicin (20 × EUCAST breakpoint). The carbenicillin panel shows the same, treated with 24 mg/L carbenicillin (3 × EUCAST breakpoint).