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

Patient complexity in quality comparisons for glycemic control: An observational study

<p>Abstract</p> <p>Background</p> <p>Patient complexity is not incorporated into quality of care comparisons for glycemic control. We developed a method to adjust hemoglobin A1c levels for patient characteristics that reflect complexity, and examined the effect of using adjusted A1c values on quality comparisons.</p> <p>Methods</p> <p>This cross-sectional observational study used 1999 national VA (US Department of Veterans Affairs) pharmacy, inpatient and outpatient utilization, and laboratory data on diabetic veterans. We adjusted individual A1c levels for available domains of complexity: age, social support (marital status), comorbid illnesses, and severity of disease (insulin use). We used adjusted A1c values to generate VA medical center level performance measures, and compared medical center ranks using adjusted versus unadjusted A1c levels across several thresholds of A1c (8.0%, 8.5%, 9.0%, and 9.5%).</p> <p>Results</p> <p>The adjustment model had R²= 8.3% with stable parameter estimates on thirty random 50% resamples. Adjustment for patient complexity resulted in the greatest rank differences in the best and worst performing deciles, with similar patterns across all tested thresholds.</p> <p>Conclusion</p> <p>Adjustment for complexity resulted in large differences in identified best and worst performers at all tested thresholds. Current performance measures of glycemic control may not be reliably identifying quality problems, and tying reimbursements to such measures may compromise the care of complex patients.</p

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

Strains used in this study.

<p>^a Minimum inhibitory concentration of tobramycin for <i>P</i>. <i>aeruginosa</i> strains as measured by Biomerieux E-test strips according to manufacture’s instructions.</p><p>Strains used in this study.</p

Environmentally Endemic Pseudomonas aeruginosa Strains with Mutations in lasR Are Associated with Increased Disease Severity in Corneal Ulcers.

The Steroids for Corneal Ulcers Trial (SCUT) was a multicenter, international study of bacterial keratitis in which 101 Pseudomonas aeruginosa infections were treated. Twenty-two of 101 P.&nbsp;aeruginosa isolates collected had a colony morphology characteristic of a loss-of-function mutation in lasR, the gene encoding a quorum-sensing master regulator. Ulcers caused by these 22 strains were associated with larger areas of corneal opacification, worse vision, and a lower rate of vision recovery in response to treatment than ulcers caused by the other isolates. The lasR sequences from these isolates each contained one of three nonsynonymous substitutions, and these strains were deficient in production of LasR-regulated protease and rhamnolipids. Replacement of lasR with either of the two most common lasR alleles from the SCUT isolates was sufficient to decrease protease and rhamnolipid production in PA14. Loss of LasR function is associated with increased production of CupA fimbriae, and the LasR-defective isolates exhibited higher production of CupA fimbriae than LasR-intact isolates. Strains with the same lasR mutation were of the same multilocus sequence type, suggesting that LasR-deficient, environmental P.&nbsp;aeruginosa strains were endemic to the area, and infections caused by these strains were associated with worse patient outcomes in the SCUT study. (This study has been registered at ClinicalTrials.gov under registration no. NCT00324168.) IMPORTANCE The LasR transcription factor is an important regulator of quorum sensing in P.&nbsp;aeruginosa and positively controls multiple virulence-associated pathways. The emergence of strains with lasR loss-of-function alleles in chronic disease is well described and is thought to represent a specific adaptation to the host environment. However, the prevalence and virulence of these strains in acute infections remain unclear. This report describes observations revealing that lasR mutants were common among isolates from a large, multicenter clinical study of keratitis and were associated with worse clinical outcomes than LasR-intact strains despite reduced production of LasR-regulated factors. Additionally, these lasR mutants were closely related strains or clones, as determined by molecular analysis. Because bacterial keratitis is community acquired, these data indicate infection by endemic, LasR-deficient strains in the environment. These results suggest that the conventional paradigm regarding the role for LasR-mediated regulation of virulence is more complex than previously appreciated

Environmentally Endemic Pseudomonas aeruginosa Strains with Mutations in lasR Are Associated with Increased Disease Severity in Corneal Ulcers

The Steroids for Corneal Ulcers Trial (SCUT) was a multicenter, international study of bacterial keratitis in which 101 Pseudomonas aeruginosa infections were treated. Twenty-two of 101 P. aeruginosa isolates collected had a colony morphology characteristic of a loss-of-function mutation in lasR, the gene encoding a quorum-sensing master regulator. Ulcers caused by these 22 strains were associated with larger areas of corneal opacification, worse vision, and a lower rate of vision recovery in response to treatment than ulcers caused by the other isolates. The lasR sequences from these isolates each contained one of three nonsynonymous substitutions, and these strains were deficient in production of LasR-regulated protease and rhamnolipids. Replacement of lasR with either of the two most common lasR alleles from the SCUT isolates was sufficient to decrease protease and rhamnolipid production in PA14. Loss of LasR function is associated with increased production of CupA fimbriae, and the LasR-defective isolates exhibited higher production of CupA fimbriae than LasR-intact isolates. Strains with the same lasR mutation were of the same multilocus sequence type, suggesting that LasR-deficient, environmental P. aeruginosa strains were endemic to the area, and infections caused by these strains were associated with worse patient outcomes in the SCUT study. (This study has been registered at ClinicalTrials.gov under registration no. NCT00324168.) IMPORTANCE The LasR transcription factor is an important regulator of quorum sensing in P. aeruginosa and positively controls multiple virulence-associated pathways. The emergence of strains with lasR loss-of-function alleles in chronic disease is well described and is thought to represent a specific adaptation to the host environment. However, the prevalence and virulence of these strains in acute infections remain unclear. This report describes observations revealing that lasR mutants were common among isolates from a large, multicenter clinical study of keratitis and were associated with worse clinical outcomes than LasR-intact strains despite reduced production of LasR-regulated factors. Additionally, these lasR mutants were closely related strains or clones, as determined by molecular analysis. Because bacterial keratitis is community acquired, these data indicate infection by endemic, LasR-deficient strains in the environment. These results suggest that the conventional paradigm regarding the role for LasRmediated regulation of virulence is more complex than previously appreciated