Fig 1.
Optical density measurements of K. pneumonia biofilm growth show inhibition by macrolides.
The inhibition of K. pneumonia ATCC 10031 biofilm growth by eight macrolides was investigated by biofilm assays with optical density (OD570) measurements after 24 h incubation. Mean relative OD570 ± SE (n = 3) is shown, with the no-macrolide (control) treatment indicated by dark circles and 1–9 mg/L macrolide treatments by light circles (trends are suggested by dashed curves). Means not connected by the same letters within panels are significantly different (LSMeans Differences Tukey HSD, alpha = 0.05 for individual macrolide mixed-effects models; note no significant differences for ROX). Triangles at the bottom of panels indicate biofilm minimum inhibitory concentrations (MIC) where relative OD570 was first significantly reduced by macrolide treatment. Macrolides not connected by the same letters indicated in parentheses in the top-right of each panel are significantly different (LSMeans Differences Tukey HSD, alpha = 0.05 for combined macrolide mixed-effects model of relative OD570).
Fig 2.
MTT assays of K. pneumonia biofilm growth show inhibition by macrolides.
The inhibition of K. pneumonia ATCC 10031 biofilm growth by eight macrolides was investigated by biofilm assays using the metabolic indicator dye MTT with absorbance (A570) measurements after 24 h incubation. Mean relative A570 ± SE (n = 3) is shown, with the no-macrolide (control) treatment indicated by dark circles and 1–9 mg/L macrolide treatments by light circles (trends are suggested by dashed curves). Means not connected by the same letters within panels are significantly different (LSMeans Differences Tukey HSD, alpha = 0.05 for individual macrolide mixed-effects models). Triangles at the bottom of panels indicate biofilm minimum inhibitory concentrations (MIC) where relative A570 was first significantly reduced by macrolide treatment. Macrolides not connected by the same letters indicated in parentheses in the top-right of each panel are significantly different (LSMeans Differences Tukey HSD, alpha = 0.05 for combined macrolide mixed-effects model of relative OD570; note no significant differences for ROX and TYL).
Fig 3.
AZM and CMS synergistically inhibit K. pneumonia planktonic and biofilm growth.
The effect of azithromycin (AZM) and colistin methanesulfonate (CMS) on K. pneumonia ATCC 10031 (A) planktonic and (B) biofilm growth was investigated by optical density (OD570) measurements after 16 h and 24 h incubation, respectively. Data are shown as mean relative OD570 ± SE (n = 3), with no-AZM (control) treatments indicated by dark circles, 3 mg/L AZM by grey circles and 9 mg/L AZM by white circles (trends are suggested by dashed curves). Means not connected by the same letters are significantly different (LSMeans Differences Tukey HSD, alpha = 0.05 for planktonic and biofilm growth mixed-effects models of relative OD570). Small triangles indicate planktonic and biofilm minimum inhibitory concentrations (MIC) where relative OD570 was first significantly reduced by CMS for each AZM treatment.
Fig 4.
MTT assays of K. pneumonia biofilm growth confirms synergistic action of AZM and CMS.
The effect of azithromycin (AZM) and colistin methanesulfonate (CMS) on K. pneumonia ATCC 10031 biofilm growth was investigated using the metabolic indicator dye MTT with absorbance (A570) measurements after 24 h incubation. Mean relative A570 ± SE (n = 3) is shown, with no-AZM (control) treatments indicated by dark circles, 3 mg/L AZM by grey circles and 9 mg/L AZM by white circles (trends are suggested by dashed curves). Means not connected by the same letters are significantly different (LSMeans Differences Tukey HSD, alpha = 0.05 for mixed-effects model of relative A570). Small triangles indicate planktonic and biofilm minimum inhibitory concentrations (MIC) where relative A570 was first significantly reduced by CMS for each AZM treatment.
Fig 5.
AZM and CMS synergistically inhibit biofilm growth of K. pneumonia hospital strains.
The effect of azithromycin (AZM) and colistin methanesulfonate (CMS) on biofilm growth of multiple-drug resistant (MDR) and non-MDR K. pneumonia Ukrainian Hospital Isolate (UHI) strains was investigated using biofilm assays and with optical density (OD570) measurements after 24 h incubation. Mean relative OD570 ± SE (n = 3) is shown, with no-AZM (control) treatments indicated by dark circles and 9 mg/L AZM treatments by light circles, (trends are suggested by dashed curves). Means not connected by the same letters are significantly different (LSMeans Differences Tukey HSD, alpha = 0.05 for individual UHI strain mixed-effects models of relative OD570). Strains not connected by the same letters indicated in parentheses in the top-right of each panel are significantly different (LSMeans Differences Tukey HSD, alpha = 0.05 for combined UHI strain mixed-effects model of relative OD570). *, UHI 1090 and UHI 1633 were found to be resistant to COL in the broth dilution assay.
Fig 6.
Synergistic inhibition of colony growth of K. pneumonia hospital strains by AZM and polymyxin B.
The inhibition of colony growth of multiple-drug resistant (MDR) and non-MDR K. pneumonia Ukrainian Hospital Isolate (UHI) strains was investigated using polymyxin B disks on azithromycin (AZM) plates with colony inhibition zone measurements after 48 h incubation. Mean relative inhibition (mm) ± SE (n = 3) is shown, with no-AZM (control) treatments indicated by dark circles and 3–9 mg/L AZM treatments by light circles (trends are suggested by dashed curves). Means not connected by the same letters within panels are significantly different (LSMeans Differences Tukey HSD, alpha = 0.05 for individual mixed-effects models of relative inhibition for UHI 329, 509, 520, 1090 & 1667; individual general linear models for UHI 486, 489, 509, 519 & 1633; asterisk indicates significant difference between no AZM and 9 mg/L AZM treatments; Wilcoxon, UHI 117, ChiSquare, DF1 = 4.1, p = 0.04; UHI 1609, ChiSquare DF1 = 4.5, p = 0.03). Spearman’s correlation (Rho) between relative inhibition and AZM concentration is provided for each strain. Strains not connected by the same letters indicated in parentheses in the top-right of each panel are significantly different (LSMeans Differences Tukey HSD, alpha = 0.05 for combined UHI strain mixed-effects model of relative inhibition; UHI 1633 was omitted from this analysis as there was no variation in relative inhibition across AZM concentrations and was resistant to polymyxin B).