Title

Evaluation by Fluctuation Analysis of a Disk Diffusion Method for Identifying Pseudomonas aeruginosa Hypermutators in the CF Lung

Presentation Type

Poster

Abstract

Pseudomonas aeruginosa (PA) hypermutators occur frequently in chronic respiratory infections (CRI) of cystic fibrosis (CF) patients and negatively affect clinical outcome. To attempt to determine whether Staphylococcus aureus SA coinfection affects the prevalence of hypermutable PA in CF-CRI, we sought to validate the disk diffusion method for identifying hypermutators by performing fluctuation analyses on candidate PA.

PA and SA samples were collected during quarterly evaluations between 2008 and 2010 in a 100-patient longitudinal study at Seattle Children’s Hospital. PA were subjected to a preliminary screen for hypermutator status using a disk diffusion assay with five different antibiotics. PA scored as hypermutators and a subset of those scored wild-type were subjected to fluctuation analysis of resistance to rifampicin (Rif) using the Ma-Sandri-Sarkar Maximum Likelihood method. The number of spontaneous mutations to RifR was used to estimate mutation rate, using the number of colonies formed on Mueller-Hinton agar with and without 300 mg/mL Rif. We chose to estimate mutation rate, rather than mutation frequency, because mutation frequency estimates are susceptible to error arising from “jackpot” mutations occurring early in log phase, which become overrepresented in populations assayed at stationary phase. Initial fluctuation analysis results confirm hypermutator status in 24% of strains deemed potential hypermutators by disk diffusion assay. Hypermutators were defined to be strains having mutation rate > 4.00E-8, a value established by converting the mutation frequency cutoff used to validate the disk diffusion method to a mutation rate using previously described formulae.

Our results indicate that the disk diffusion method’s high rate of false positives makes it inadequate for PA hypermutator screening. We aim to improve the predictive value of disk assays so they can be used to enhance patient care and to establish a mutation rate cut-off for the subset of PA hypermutators most closely associated with clinical decline.

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Apr 12th, 11:00 AM Apr 12th, 12:00 PM

Evaluation by Fluctuation Analysis of a Disk Diffusion Method for Identifying Pseudomonas aeruginosa Hypermutators in the CF Lung

UC Ballroom

Pseudomonas aeruginosa (PA) hypermutators occur frequently in chronic respiratory infections (CRI) of cystic fibrosis (CF) patients and negatively affect clinical outcome. To attempt to determine whether Staphylococcus aureus SA coinfection affects the prevalence of hypermutable PA in CF-CRI, we sought to validate the disk diffusion method for identifying hypermutators by performing fluctuation analyses on candidate PA.

PA and SA samples were collected during quarterly evaluations between 2008 and 2010 in a 100-patient longitudinal study at Seattle Children’s Hospital. PA were subjected to a preliminary screen for hypermutator status using a disk diffusion assay with five different antibiotics. PA scored as hypermutators and a subset of those scored wild-type were subjected to fluctuation analysis of resistance to rifampicin (Rif) using the Ma-Sandri-Sarkar Maximum Likelihood method. The number of spontaneous mutations to RifR was used to estimate mutation rate, using the number of colonies formed on Mueller-Hinton agar with and without 300 mg/mL Rif. We chose to estimate mutation rate, rather than mutation frequency, because mutation frequency estimates are susceptible to error arising from “jackpot” mutations occurring early in log phase, which become overrepresented in populations assayed at stationary phase. Initial fluctuation analysis results confirm hypermutator status in 24% of strains deemed potential hypermutators by disk diffusion assay. Hypermutators were defined to be strains having mutation rate > 4.00E-8, a value established by converting the mutation frequency cutoff used to validate the disk diffusion method to a mutation rate using previously described formulae.

Our results indicate that the disk diffusion method’s high rate of false positives makes it inadequate for PA hypermutator screening. We aim to improve the predictive value of disk assays so they can be used to enhance patient care and to establish a mutation rate cut-off for the subset of PA hypermutators most closely associated with clinical decline.