Quantification of extracellular indole concentrations

Extracellular indole concentration was measured on bacterial cultures grown overnight with and without antibiotics using the Kovacs reagent (Saint-Ruf et al., 2014). First, we established an indole concentration standard curve using 1 ml culture medium (without bacteria) supplemented with indole 0 to 1000 μM (100 μM steps). After adding 500 μl KOVACS reagent, 100 μl of the top layer of the reaction was mixed with 800 μl isoamyl-HCl and OD was read at 570 nm. For indole measurement on bacterial samples, 1 ml of culture was subjected at the same protocol. Measured indole concentration was normalized to the bacterial dry mass based on the assumption that for an OD 600 nm = 1 bacterial dry mass is 0.3 mg/ml (Soini et al., 2008). We detected no indole production in MH medium in any condition. In order to quantify secreted indole in the presence and absence of sub-MIC antibiotics, we thus used the defined rich MOPS transparent medium (Teknova EZ rich defined medium), where MIC TOB is 0.75 μg/ml. All the following experiments (persistence, growth) were conducted in MH, as it allows to study the impact of defined indole concentrations added to the media.

Quantification of fluorescent neomycin uptake was performed as described (S.A.P. et al., unpublished data; Okuda, 2015; Sabeti Azad et al., 2020). Neo-cy5 is an aminoglycoside coupled to the fluorophore Cy5, and has been shown to be active against Gram- bacteria (Okuda, 2015; Sabeti Azad et al., 2020). Briefly, overnight cultures were diluted 100-fold in rich MOPS (Teknova EZ rich defined medium). When the bacterial strains reached an OD 620 nm of ∼0.25, they were incubated with 0.4 μM of Cy5 labeled Neomycin for 15 minutes at 37°C. 10 μl of the incubated culture were then used for flow cytometry, diluting them in 250 μl of PBS before reading fluorescence. WT V. cholerae, was incubated simultaneously without Neo-Cy5 as a negative control. Flow cytometry experiments were performed as described (Baharoglu et al., 2010) and repeated at least 3 times. For each experiment, 100,000 events were counted on the Miltenyi MACSquant device.