Fig. 1

A persistent isolate of NTHI exhibits increased biofilm height and transformation efficiency. a Schematic representation of environmental heme–iron restriction. 86-028NP was cultured in defined iron source (DIS) medium in the presence (+) or absence (−) of 2 µg/mL heme–iron for 24 h to generate “replete” or “restricted” populations, respectively, adapted from Hardison et al.³³ These two populations were subcultured into DIS medium containing 2 µg/mL heme–iron. The “continuously exposed” or “transiently restricted” cultures were continuously incubated under static microaeration with no fresh medium added. Viability was determined by plating for CFU/mL every 24 h, each bar indicates the CFU on that day for a representative experiment. The persistent isolate, RM33, was isolated from the transiently restricted culture on day 33 (arrow). b Representative image of a biofilm of 86-028NP (pGM1.1) grown for 48 h with orthogonal views of the three-dimensional rendering of a 20.4 µm high biofilm. c Representative image of a biofilm of RM33 (pKM1.1) grown for 48 h with orthogonal views of the three-dimensional rendering of a 38.4 µm high biofilm. d Representative image of a 13.8 µm biofilm with a 1:1 mixture of 86-028 NP (pGM1.1) and RM33 (pKM1.1) grown for 48 h with orthogonal views of the three-dimensional rendering. e Quantitative assessment of the height of biofilms formed by either the parental strain [86-028NP (pGM1.1), circle] and [RM33 (pKM1.1), square]. The height of the biofilm was measured at ten random locations in technical duplicate and performed on six independent occasions (n = 120 per strain). Each point represents an individual measurement. Statistical significance was determined by a two-tailed unpaired Student’s t test. f Quantitative assessment of the height of each strain within a mixed biofilm was measured as described in e. g The phosphodiesterase activity of whole-cell lysates prepared from 48 h biofilms was determined as described in “Methods” section. The mean and standard error of the mean (s.e.m.) are indicated for the results of five independent assays. Statistical significance was determined using a two-tailed unpaired Student’s t test. h Exogenous DNA was added to a 48 h biofilm of either 86-028NP or RM33. Biofilms were physically disrupted and the efficiency of DNA uptake is reported as the number of antibiotic-resistant colonies within the total population. The mean and standard error of the mean (s.e.m.) are indicated for the results of four independent assays. Statistical significance was determined using a two-tailed unpaired Student’s t test. Scale bar indicates 25 μm