919. Impact of Hypermutators on Pathoadaptation of Staphylococcus aureus to the Cystic Fibrosis Airway
Session: Poster Abstract Session: Bacterial Pathogenesis
Saturday, October 22, 2011
Room: Poster Hall B1
Background:  Staphylococcus aureus is a leading cause of airway infections in pediatric and adolescent cystic fibrosis (CF) patients.  Prior studies identified S. aureus hypermutators as a possible cause for persistence during chronic infection of the CF airway.  Hypermutators are bacteria with an elevated mutation frequency and for S. aureus these include strains with a mutation frequency > 10-7.  Past efforts to genotypically characterize the S. aureus hypermutator phenotype by mutation to the mismatch repair (MMR) system (mutS and mutL) has failed to define the cause of this phenotype for some strains.  Because the oxidized guanine (GO) system (mutMYT) has been implicated in several other bacterial pathogens as a contributing source to a hypermutable phenotype, we hypothesize that inactivation of the GO system in S. aureus may also facilitate a hypermutator phenotype and contribute, along with impairment of MMR, to pathoadaptation in chronic CF infection by accelerating generation of higher fitness mutants. 

Methods:  GO and MMR mutants were generated in S. aureus NCTC8325 and HG003 by markerless allelic replacement and characterized for:  1) mutation frequency using a phenotypic fluctuation assay (PFA) and 2) biofilm phenotype in a 24-hour static biofilm assay.

Results:  Genes predicted to encode the GO (mutM and mutY) and MMR (mutS and mutL) system in S. aureus NCTC8325 (rsbU-, tcaR-) and HG003 (isogenic to NCTC8325 except for repair in rsbU and tcaR) were inactivated and found in a PFA to increase mutation frequency by 3, 10.5, 18.4, and 24.4 fold, respectively.  Similar results were also obtained for mutants generated in S. aureus HG003.   Surprisingly, repair of rsbU, a positive activator of the stress responsive sigma factor sigB, reduced mutation frequency nearly 4-fold compared to NCTC8325.  Using a static biofilm assay, we demonstrated a 1.5 and 2-fold increase in biofilm biomass for mutM KO and mutY KO, respectively, in comparison to HG003.  Repeat of this assay in the presence of Pseudomonas aeruginosa PA01 demonstrated enhanced survival of mutM (p=0.0168) and mutY KO (p=0.004) as compared to HG003.

Conclusion:  GO system inactivation increases mutation frequency, promotes production of S. aureus biofilm and survival in the presence of P. aeruginosa.


Subject Category: B. Bacterial pathogenesis, studies in animal models, molecular pathogenicity

Gregory Canfield, BS1, Kanitsak Boonanantanasarn, PhD2, Kelly Vore, BS2, Matthew Foley2, Ann Gill, MS2, Mark Sutton, PhD3 and Steven Gill, PhD2, (1)Department of Microbiology and Immunology, University at Buffalo, Buffalo, NY, (2)Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, (3)Department of Biochemistry, University at Buffalo, Buffalo, NY

Disclosures:

G. Canfield, None

K. Boonanantanasarn, None

K. Vore, None

M. Foley, None

A. Gill, None

M. Sutton, None

S. Gill, None

Findings in the abstracts are embargoed until 12:01 a.m. EST Thursday, Oct. 20 with the exception of research findings presented at IDSA press conferences.