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Strains were obtained from the culture collection of the Bradford Infection Group. Bacteria were harvested from overnight plates and suspended in sterile saline to McFarland turbidity standard 0.5. Suspensions were used to make confluent lawns of each strain on Iso-Sensitest agar (Oxoid Ltd). Sterile filter paper discs (0.6 cm diameter) were impregnated with 5 μg of ciprofloxacin stock or conjugate. Commercially available 5 μg ciprofloxacin discs (Oxoid) were also used to determine if strains were ciprofloxacin resistant, as defined by CLSI criteria. Discs were placed on the agar surface, and plates incubated for 18-20 h at 37 °C, and the diameter of zones of inhibition were then recorded.
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Cultures of BW25113 and its isogenic ompF mutant grown aerobically in M9 minimal medium with either 1 mM or 1 μM ferric citrate were subcultured into 20 ml M9 medium with either 1 mM or 1 μM ferric nitrate and grown to mid-log phase, then harvested by centrifugation, and washed three times in M9. 120 μL of each culture was mixed with 7 mL bacteriological agar which was poured as an overlay on a bottom layer of M9 agar. 0.1 μg of ciprofloxacin or 0.15 μg of 1 were spotted on the surface of the plates and then the plates were incubated at 37 °C overnight before measuring the diameter of the zone of inhibition.
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