FIGURE 3.

Acidic pH modulates ESKAPE pathogens biofilm formation and impairs antibiotic biofilm prevention against P. aeruginosa. All P. aeruginosa strains (2 lab strains and 12 clinical strains) were incubated in pH adjusted m63 medium for 18 h. The crystal violet staining method was used to quantify biofilm formation (n = 6). (A) P. aeruginosa biofilm formation without antibiotic treatment. Biofilm formation in pH 7.5 served as a positive control. (B–D) P. aeruginosa biofilm formation was treated by ceftazidime, ciprofloxacin, and tobramycin at the concentrations of 1x MIC of each P. aeruginosa strain (MIC in normal pH 7.5 condition). The percentage of antibiotic-treated biofilm formation was calculated by comparing it to each untreated P. aeruginosa strain in pH 7.5. (E) Biofilm formation of other bacterial species of the ESKAPE bacterial pathogens. Five isolates from each of the other ESKAPE species were grown at pH 6.5 and 7.5 (as a control) in biofilm mode. E.a. and E.c. are abbreviations for Enterobacter aerogenes and Enterobacter cloacae, respectively. The color scale bars represent the percentage of biofilm formation compared to appropriate control groups. Red scale: increased biofilm formation (100%–200%); white: no change (100%); blue scale: decreased biofilm formation (0–100%). Data were collected from three independent experiments. Unpaired t-test was used for statistical analysis between each pH 6.5 and pH 7.5 conditions. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001; otherwise not significant.