Sub-Inhibitory Clindamycin and Azithromycin reduce S. aureus Exoprotein Induced Toxicity, Inflammation, Barrier Disruption and Invasion

Abstract

Background: Chronic rhinosinusitis (CRS) is defined as a chronic inflammation of the nose and paranasal sinus mucosa associated with relapsing infections-particularly with S. aureus. Long-term treatments with protein synthesis inhibitor antibiotics have been proposed to reduce inflammation in the context chronic severe inflammatory airway pathologies, including CRS. This study assessed the effect of subinhibitory clindamycin and azithromycin on S. aureus exoprotein induced inflammation, toxicity and invasiveness.

Methods: S. aureus ATCC51650 and two clinical isolates grown in planktonic and biofilm form were treated with subinhibitory clindamycin and azithromycin. Exoproteins were collected and applied to primary human nasal epithelial cells (HNECs) in monolayers and at air-liquid interface. This was followed by lactate dehydrogenase (LDH), enzyme-linked immunosorbent assay (ELISA), Transepithelial Electrical Resistance (TEER) and paracellular permeability assays to assess the effect on cell toxicity, inflammatory cytokine production and mucosal barrier structure and function, respectively. The effect of these treatments was tested as well on the S. aureus invasiveness of HNECs.

Results: Subinhibitory clindamycin reduced S. aureus exoprotein production in planktonic and biofilm form, thereby blocking exoprotein-induced toxicity, reversing its detrimental effects on mucosal barrier structure and function and modulating its inflammatory properties. Sub-inhibitory azithromycin had similar effects-albeit to a lesser extent. Furthermore, clindamycin-but not azithromycin-treated S. aureus lost its invasive capacity of HNECs.

Conclusion: Subinhibitory clindamycin and azithromycin reduce S. aureus exoprotein production, thereby modulating the inflammatory cascade by reducing exoprotein-induced toxicity, inflammation, mucosal barrier disruption and invasiveness.

Keywords: S. aureus; azithromycin; chronic rhinosinusitis; clindamycin; exoprotein; mucosal barrier; sub-inhibitory.

Figure 1

Sub-inhibitory concentrations of clindamycin and azithromycin do not significantly affect bacterial growth following 24 h incubation. The OD600 absorbance values of S. aureus after 24 h culture. ATCC51650, CI1, CI2 were grown without (no antibiotic, grey) or with ½, ¼, ⅛ MIC clindamycin (blue) or azithromycin (green) (p > 0.05). n = 3, bars represent standard error of means.

Figure 2

Protein content of S. aureus exoproteins after sub-inhibitory clindamycin and azithromycin treatment. (a) Protein content measured by NanoOrange (μg/mL) or (b) gel electrophoresis of ATCC 51650, CI1 and CI2 24-h planktonic cultures in the absence (no antibiotic, grey bar) or presence of ½, ¼ or ⅛ MIC clindamycin (blue bars) or azithromycin (green bars). n = 3. * p < 0.05, ** p < 0.01, one-way ANOVA followed LSD post hoc comparison.

Figure 2

Protein content of S. aureus exoproteins after sub-inhibitory clindamycin and azithromycin treatment. (a) Protein content measured by NanoOrange (μg/mL) or (b) gel electrophoresis of ATCC 51650, CI1 and CI2 24-h planktonic cultures in the absence (no antibiotic, grey bar) or presence of ½, ¼ or ⅛ MIC clindamycin (blue bars) or azithromycin (green bars). n = 3. * p < 0.05, ** p < 0.01, one-way ANOVA followed LSD post hoc comparison.

Figure 3

Biofilm protein content after ½ MIC and 1 MIC clindamycin and azithromycin treatment. (a) Protein content measured by NanoOrange (μg/mL) or (b) gel electrophoresis of 48-h biofilms of ATCC 51650, CI1 or CI2 in the absence (no antibiotic, grey bar) or presence of ½ MIC and 1 MIC clindamycin (blue bars) or azithromycin (green bars). (* p < 0.05, ** p < 0.01, one-way ANOVA followed LSD post hoc comparison, n = 3).

Figure 4

Reduction of S. aureus exoprotein-induced cytotoxicity of HNECs by treatment with sub-inhibitory clindamycin or azithromycin. Cell viability relative to negative control (5% tryptic soy broth in cell culture medium) of HNECs treated for 24 h with untreated S. aureus (ATCC 51650, CI1 and CI2) exoproteins (no antibiotic, grey bars) or ½, ¼ or ⅛ MIC clindamycin (blue bars) or azithromycin (green bars) treated exoproteins. Positive control = 10% Triton X-100; Antibiotic control = ½ MIC clindamycin (dark blue) or azithromycin (dark green) in medium. n = 3; * p < 0.05, ** p < 0.01, one-way ANOVA, followed by LSD post hoc comparison).

Figure 5

Sub-inhibitory clindamycin and azithromycin can modulate the inflammatory response of HNECs exposed to S. aureus exoproteins. IL6 (a) and IL8 (b) protein concentration in HNEC culture medium after a 24-h challenge with 5% S. aureus exoproteins untreated (no antibiotic, grey bars) or treated with ½, ¼ or ⅛ MIC clindamycin (blue bars) or azithromycin (green bars) for 24 h. Positive control = 10 μg/mL Poly (I:C) LMW; negative control = 5% tryptic soy broth in cell culture medium; Antibiotic control = ½ MIC clindamycin (dark blue) or azithromycin (dark green) in medium. n = 3. * p < 0.05 one-way ANOVA followed LSD post hoc comparison.

Figure 6

½ MIC clindamycin treatment of S. aureus reduced intracellular infection of HNECs. a: Giemsa staining of HNECs with different antibiotic treated S. aureus ATCC51650 and CI1 showing the presence of intracellular cocci (arrow). b: Colony Forming Units (log CFU/mL) of intracellular S. aureus ATCC 51560 and CI1 in HNECs. ** p < 0.01, one-way ANOVA followed LSD post hoc comparison.

Figure 7

Transepithelial electrical resistance, relative permeability and immunofluorescence staining of S. aureus exoproteins treated HNEC-ALI cultures. (a,b) ½ MIC clindamycin significantly reversed the TEER decrease (a) and the increased HNEC-ALI permeability of FITC-dextrans induced by S. aureus ATCC 51650, CI1 and CI2 (b). (c) Immunofluorescence staining of ZO-1 (green) in HNEC-ALI cultures treated with medium (control) or exoproteins from ATCC 51650, CI1 and CI2 left untreated or treated with ½ MIC clindamycin or ½ MIC azithromyxin. DAPI stains nuclei blue. * p < 0.05, ** p < 0.01. n = 3.

Figure 7

Transepithelial electrical resistance, relative permeability and immunofluorescence staining of S. aureus exoproteins treated HNEC-ALI cultures. (a,b) ½ MIC clindamycin significantly reversed the TEER decrease (a) and the increased HNEC-ALI permeability of FITC-dextrans induced by S. aureus ATCC 51650, CI1 and CI2 (b). (c) Immunofluorescence staining of ZO-1 (green) in HNEC-ALI cultures treated with medium (control) or exoproteins from ATCC 51650, CI1 and CI2 left untreated or treated with ½ MIC clindamycin or ½ MIC azithromyxin. DAPI stains nuclei blue. * p < 0.05, ** p < 0.01. n = 3.