Article Figures & Data
Figures
- FIG 1
Cigarette smoke exposure reduces MRSA susceptibility to macrophage killing and lysis while suppressing bacterial growth. (A) Starting with an MOI of 1 × 105, control MRSA was killed by alveolar macrophages over 100 min, while MRSA exposed to 75% CSE resisted killing and overgrew. By 220 min, CFU of control MRSA were 4-fold lower than those of CSE-MRSA. (B) CSE exposure did not change the rate of MRSA-GFP phagocytosis by macrophages. (C) The increased numbers of MRSA during killing assays was not due to increased growth, as CSE decreased the rate of MRSA growth in a dose-dependent manner. (D) CSE exposure tended to increase MRSA resistance to killing by H2O2 (oxygen radicals). (E) CSE induced resistance to MRSA cell lysis in the presence of detergent. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
- FIG 2
Cigarette smoke exposure increases resistance of MRSA to human AMP LL-37. (A) CSE exposure increased the MIC of MRSA to LL-37 from 4 to 8 μM and increased the concentration needed to inhibit metabolic activity from 8 to 16 μM. (B) Growth of control MRSA and MRSA after exposure to 75% CSE was identical during the AMP growth kinetics assays, which were run without CSE present. (C, D, and F) Prior exposure to 75% CSE allowed MRSA to escape killing by LL-37 at 16 μM (C), 8 μM (D), and 2 μM (F). (E and G) These effects were CSE dose dependent, with exposure to 50% through 90% CSE inducing resistance to AMP killing at both 8 μM (E) and 2 μM (G). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
- FIG 3
Cigarette smoke exposure induces MRSA surface changes, including a less negative surface charge and increased hydrophobicity, leading to increased adherence and invasion of epithelial cells. (A) CSE-MRSA binds less of cationic PLL-FITC, consistent with alteration in surface charge (less negative = more positive surface charge) in response to CSE exposure, in a dose-dependent manner. (B) Background fluorescence (no PLL added) was equal among groups. CSE precipitate alone had no impact on surface charge, with equivalent surface binding of 2 μM PLL as control MRSA. However, 24-h-old CSE induced a 2-fold shift in surface charge, but not the 12-fold shift induced by freshly made CSE. (C) Nicotine alone induced shifts in MRSA surface charge leading to less PLL-FITC binding (5-fold by 3 mg and 11-fold by 6 mg), consistent with nicotine contributing to CSE-induced surface charge changes. (D) Surface charge changes induced by CSE exposure persisted after passage into THB two times, for 24 h after exposure, and repetitive daily exposures for 3 and 4 days further enhanced surface charge changes. (E) S. aureus strain SA113 had similar changes in PLL-FITC binding upon exposure to CSE. (F) Exposure to CSE induced increasing hydrophobicity in a dose-dependent manner, as evidenced by fewer bacteria recovered from the aqueous phase as the CSE concentration was increased. (G and H) CSE exposure increased adherence of MRSA to epithelial cells (G) and increased MRSA invasion of and persistence within epithelial cells (H). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
- FIG 4
MRSA exposed to CSE has increased virulence in a mouse model of pneumonia. (A) Lungs harvested at 8 and 20 h postinfection had higher numbers of CSE-MRSA (**, P < 0.01). (B) In a mortality model of pneumonia, 40% of mice infected with CSE-MRSA died, whereas 10% of controls survived (P = 0.021; n = 20 mice per group). (C) Histology of lungs from mice with MRSA pneumonia demonstrated more bacteria and inflammation in CSE-MRSA-infected mice at both the 8- and 20-h time points (n = 5 per group at each time point) (inflammation scores shown in white).
Additional Files
Supplemental material
- Supplemental file 1 -
Fig. S1. MRSA exposed to CSE prior to incubation with human AMP LL-37 had a higher MBC compared to control MRSA (32 μM vs 8 to 16 μM, respectively), as evidenced by live bacteria growing on THA plates after overnight incubation with LL-37. Table S2. Compared to control MRSA, CSE-MRSA were more resistant to LL-37 by MIC, metabolic activity, and MBC assays. Fig. S3. S. aureus strain SA113ΔmprF, in which mprF is knocked out, had no change in PLL-FITC binding after exposure to CSE.
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- Supplemental file 1 -
