Mannitol Does Not Enhance Tobramycin Killing of Pseudomonas aeruginosa in a Cystic Fibrosis Model System of Biofilm Formation

Cystic Fibrosis (CF) is a human genetic disease that results in the accumulation of thick, sticky mucus in the airways, which results in chronic, life-long bacterial biofilm infections that are difficult to clear with antibiotics. Pseudomonas aeruginosa lung infection is correlated with worsening lung disease and P. aeruginosa transitions to an antibiotic tolerant state during chronic infections. Tobramycin is an aminoglycoside currently used to combat lung infections in individuals with CF. While tobramycin is effective at eradicating P. aeruginosa in the airways of young patients, it is unable to completely clear the chronic P. aeruginosa infections in older patients. A recent report showed that co-addition of tobramycin and mannitol enhanced killing of P. aeruginosa grown in vitro as a biofilm on an abiotic surface. Here we employed a model system of bacterial biofilms formed on the surface of CF-derived airway cells to determine if mannitol would enhance the antibacterial activity of tobramycin against P. aeruginosa grown on a more clinically relevant surface. Using this model system, which allows the growth of robust biofilms with high-level antibiotic tolerance analogous to in vivo biofilms, we were unable to find evidence for enhanced antibacterial activity of tobramycin with the addition of mannitol, supporting the observation that this type of co-treatment failed to reduce the P. aeruginosa bacterial load in a clinical setting

Identification of Streptococcus bovis Biotype I Strains among S. bovis Clinical Isolates by PCR

Streptococcus bovis causes 24% of all streptococcal infective endocarditis cases. There are many reports linking both S. bovis bacteremia and endocarditis with various forms of gastrointestinal disease (primarily colonic cancers). S. bovis is divided into two biotypes: I and II. The biotype I strain is much more frequently isolated from patients with endocarditis, gastrointestinal disease, or both. We describe here the isolation of biotype I-specific DNA sequences and the development of a PCR test which can identify S. bovis biotype I strains among S. bovis clinical isolates

Mannitol does not sensitize non-mucoid, laboratory strain <i>P</i>. <i>aeruginosa</i> PA14 to tobramycin.

<p>A. Mannitol is minimally cytotoxic to CFBE cells. Normalized cytotoxicity as measured by fraction of LDH release. Cytotoxicity was measured after 24 hours of treatment with 0, 40 or 60 mM mannitol as indicated. Cells lysed with Triton X-100 served as a control to determine total lysis. Columns indicate mean of at least three biological replicates, error bars indicate standard deviation (S.D.). **, P<0.01, comparison of indicated sample to total lysis control by ordinary one-way ANOVA with Tukey’s post test for multiple comparisons. B. Viability of <i>P</i>. <i>aeruginosa</i> PA14 grown as a biofilm on CFBE cells after treatment with 0 μg/mL tobramycin (open bars), 8 μg/mL tobramycin (hatched bars), 0 mM mannitol (white bars), 60 mM mannitol (gray bars) or co-treatment with 8 μg/mL tobramycin and 60 mM mannitol, as indicated. Columns indicate mean of at least three biological replicates, error bars indicate S.D. ***, P<0.001 by ordinary one-way ANOVA with Tukey’s post test for multiple comparisons. There is no significant difference between <i>P</i>. <i>aeruginosa</i> PA14 treated with tobramycin +/- mannitol.</p

Mannitol does not sensitize <i>P</i>. <i>aeruginosa</i> clinical isolates grown as biofilms on CF airway cells to tobramycin.

<p>A. Viability of <i>P</i>. <i>aeruginosa</i> clinical isolates grown as biofilms on CFBE cells and treated with 0 μg/mL tobramycin (open bars), 8 μg/mL tobramycin (hatched bars), 0 mM mannitol (white bars), 60 mM mannitol (gray bars) or co-treatment with 8 μg/mL tobramycin and 60 mM mannitol, as indicated. Columns indicate mean of at least three biological replicates, error bars indicate S.D. **, P<0.01 by ordinary one-way ANOVA with Tukey’s post test for multiple comparisons. B. The viability of strains <i>P</i>. <i>aeruginosa</i> PAO1 (left) and FRD1 (right) as biofilms on CFBE cells and treated with 0 μg/mL tobramycin (open bars), 8 μg/mL tobramycin (hatched bars), 0 mM mannitol (white bars), 60 mM mannitol (gray bars) or co-treatment with 8 μg/mL tobramycin and 60 mM mannitol, as indicated. **, P<0.01 or ***, P<0.001 by ordinary one-way ANOVA with Tukey’s post test for multiple comparisons. ns, not significant compared to tobramycin treatment in the absence of mannitol. C. The viability of strain <i>P</i>. <i>aeruginosa</i> PAO1 as a biofilm on plastic and treated with 0 μg/mL tobramycin (open bars), 80 μg/mL tobramycin (hatched bars), 0 mM mannitol (white bars), 60 mM mannitol (gray bars) or co-treatment with 80 μg/mL tobramycin and 60 mM mannitol, as indicated. *, P<0.05 compared to treatment with 80 μg/mL tobramycin with no mannitol. **, P<0.01 or ***, P<0.001 by ordinary one-way ANOVA with Tukey’s post test for multiple comparisons.</p