Streptococcus pneumoniae in the heart subvert the host response through biofilm-mediated resident macrophage killing
Fig 2
S. pneumoniae within cardiac microlesions exhibit biofilm properties.
(A) In vitro antimicrobial tolerance assays for survival of paired blood-isolated pneumococci (BIP) and heart-isolated pneumococci (HIP) from the same mouse (n = 6). Tolerance to penicillin and erythromycin killing at the designated concentration was tested. Statistical analysis was performed using Mann-Whitney test. (B) Representative high magnification immunofluorescent microscopy images of cardiac microlesions showing presence of biofilm extracellular matrix components: nucleic acids (stained with DAPI [DAPI], blue), capsule (stained with anti-serotype 4 capsule antibody [CPS], red), and mouse extracellular DNA (stained with anti-mH2A histone antibody [mH2A], green). A minimum of 5 stained heart sections were examined. (C) Representative tile-stitched image of whole heart sections from TIGR4 infected mice (n = 5). The cardiac sections (stained with DAPI, blue) were probed for TIGR4 (red), using serotype 4 capsule polysaccharide antisera, and for exposed galactose residues (green), using fluorescein labeled Erythrina crystagalli lectin, within the heart. Inset, Representative tile-stitched image of an uninfected control heart (n = 3). (D) Static biofilm-forming ability of TIGR4 (T4) and T4 ΔspxB was assessed in 48-hour 6-well plate model (n = 3 biological replicates, each with 2 technical replicates). Biofilm biomass was measured using crystal violet staining. Statistical analysis was performed using Student’s t-test. (E) Average number of cardiac microlesions detected per cardiac section in T4 and T4 ΔspxB infected mice post infection. For each time point, cardiac sections from at least 5 mice were examined. Averages were calculated by enumerating the number of microlesions in 3 non-adjacent cardiac sections. Statistical analysis was performed using Mann-Whitney test. P value: * ≤ 0.05, ** ≤ 0.01, **** ≤ 0.0001; data are represented as mean ± SEM.