OxyR Is Required for Serratia Marcescens Biofilm Formation

Abstract

Purpose:: Serratia marcescens is an opportunistic pathogen capable of causing sight-threatening keratitis. Biofilm formation is a major component in S. marcescens pathogenesis. A genetic approach was adopted to identify factors required for bacterial attachment to abiotic surfaces.

Methods:: An S. marcescens transposon library was generated and screened for biofilm formation on microtiter plates using LB as a growth medium. Relative biofilm formation was determined by staining biofilms with crystal violet (CV) and the absorbance of solubilized CV was read at 590 nm (A590). Mutations were mapped using arbitrary PCR. Sensitivity to oxidative stress agents was determined using disk diffusion assays. Fimbriae function was assessed by yeast agglutination and autoagglutination in peptone glycerol medium.

Results:: The largest class of biofilm deficient mutations mapped to predicted fimbriae structural genes. One transposon mapped to a gene with 90% identity to the oxyR gene from E. coli. OxyR is required for full levels of virulence in several bacterial species and is required for oxidative stress survival. In E. coli, OxyR regulates several surface adhesins and inhibits biofilm formation. In contrast, the S. marcescens oxyR mutant was defective in biofilm formation (biofilm formation (A590): wild type (WT) = 0.5 ± 0.05, oxyR = 0.03 ± 0.03, P < 0.0001)). The oxyR mutant was hypersensitive to H2O2 and cumene hyperoxide (zones of clearing in mm: for H2O2, wt = 16 ± 1.3, oxyR = 21.5 ± 0.9, for cumene hyperoxide, wt = 13 ± 1.3, oxyR = 16.3 ± 1.3, P 60s, P < 0.0001; % agglutination measured spectrophotomectrically, the WT = 89.7 ± 1% agglutination whereas the oxyR mutant = 14 ± 5.7% agglutination, P < 0.001) and autoagglutination (WT = 48.1 ± 5%, oxyR-1 = 6.6 ± 6.0%, P < 0.001). Targeted mutagenesis of oxyR conferred similar defects to the WT strain. The oxyR gene was cloned and was able to complement the defects of oxyR mutants (complemented oxyR mutant biofilm formation A590 = 0.4 ± 0.01, agglutination time = 4.1 ± 0.6s, % agglutination = 89.5 ± 0.5).

Conclusions:: Our data supports a model in which OxyR contributes to S. marcescens biofilm formation as a positive regulator of fimbriae expression or function. Ongoing studies are aimed at determining the role of OxyR in S. marcescens pathogenesis using rabbit keratitis models.

Keywords: microbial pathogenesis: experimental studies • genetics • keratitis