biostudies-arrayexpressAmit VikramPseudomonas fluorescenshttps://www.ebi.ac.uk/biostudies/studies/E-GEOD-64448The objective of the current study was to understand the glutaraldehyde resistance mechanisms in P. fluorescens and P. aeruginosa biofilms. Glutaraldehyde is a common biocide used in various industries to control the microbial growth. Recent reports of emergence of glutaraldehyde resistance in several bacterial species motivated this study to understand the genetic factors responsible got glutaraldehyde resistance. Using a combination of phenotypic assays, chemical genetic assays and RNA-seq, we demonstrate that novel efflux pump, polyamine biosynthesis, lipid biosynthesis and phosphonate degradation play significant role in glutaraldehyde resistance and post-glutaraldehyde recovery of Psudomonad biofilms. Examination of P. fluorescens 72 h biofilm transcriptome was elucidated upon exposure to glutaraldehyde. The results were confirmed using qRT--PCR and chemical genetic appraoches in P. fluorescens and P. aeruginosa.biostudies-arrayexpressSample Treatment - P. fluorescens 72 h old biofilms were exposed to 62.5 ppm or 0 ppm glutaraldehyde for 10 min at room temperature.Growth Protocol - The P. fluorescens was maintained on cetrimide agar plates. Overnight broth cultures of P. fluorescens were diluted 100 fold in fresh LB broth and palced in 6 well plates. Biofilms were grown for 72 h.Library Construction - RNA was extracted using TRIZOL according to manufacturer's protocol and processed using ScriptSeq Complete kit for Bacteria. RNA-sequencing was performed on MiSeq. RNA libraries were prepared using ScriptSeq Complete Kit for Bacteria (Epicenter, Madison, WI).OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesData Transformation - Illumina Casava1.7 software used for basecalling. The sequenced were adapter trimmed. The resulting files were then trimmed for low quality scores (q30) and length (<70 nt discarded) on CLC Genomics Workbench 7.5.1 RNAseq analysis in CLC Genomics Workbench v6.5.1 was conducted by merging both R1 (Forward) and R2 (Reverse) reads for each sample and obtaining single output files for 62.5 ppm and 0 ppm glutaraldehdye exposure. The reads mapping to rRNA were manually removed and RPKM was recalculated. The data was further normalized using scaling based normalization method in CLC Genomics Workbench. Differentially expressed genes were identified by comparing normalized gene reads between PBS and glutaraldehyde treated biofilm samples using Baggerley’s test on proportions for replicated experiment. FDR was calculated and genes with FDR corrected p ≤ 0.001 were considered significant. Genome_build: NC_012660UnknownTranscriptomicsGenomicsProteomicsA major challenge in microbial biofilm control is biocide resistance. Phenotypic adaptations and physical protective effects have been historically thought to be the primary mechanisms for glutaraldehyde resistance in bacterial biofilms. Recent studies indicate the presence of genetic mechanisms for glutaraldehyde resistance, but very little is known about the contributory genetic factors. Here, we demonstrate that efflux pumps contribute to glutaraldehyde resistance in Pseudomonas fluorescens and Pseudomonas aeruginosa biofilms. The RNA-seq data show that efflux pumps and phosphonate degradation, lipid biosynthesis, and polyamine biosynthesis metabolic pathways were induced upon glutaraldehyde exposure. Furthermore, chemical inhibition of efflux pumps potentiates glutaraldehyde activity, suggesting that efflux activity contributes to glutaraldehyde resistance. Additionally, induction of known modulators of biofilm formation, including phosphonate degradation, lipid biosynthesis, and polyamine biosynthesis, may contribute to biofilm resistance and resilience. Fundamental understanding of the genetic mechanism of biocide resistance is critical for the optimization of biocide use and development of novel disinfection strategies. Our results reveal genetic components involved in glutaraldehyde resistance and a potential strategy for improved control of biofilms.RNA-seq of coding RNAPseudomonas fluorescensEfflux as a Glutaraldehyde resistance mechanism in Pseudomonas fluorescens and Pseudomonas aeruginosa biofilms.Jennifer BombergerVikram A, Bomberger J, Bibby KJKyle BibbyAmit VikramfalseGlutaraldehyde resistance mechanisms in Pseudomonas fluorescens and Pseudomonas aeruginosa biofilmsThe objective of the current study was to understand the glutaraldehyde resistance mechanisms in P. fluorescens and P. aeruginosa biofilms. Glutaraldehyde is a common biocide used in various industries to control the microbial growth. Recent reports of emergence of glutaraldehyde resistance in several bacterial species motivated this study to understand the genetic factors responsible got glutaraldehyde resistance. Using a combination of phenotypic assays, chemical genetic assays and RNA-seq, we demonstrate that novel efflux pump, polyamine biosynthesis, lipid biosynthesis and phosphonate degradation play significant role in glutaraldehyde resistance and post-glutaraldehyde recovery of Psudomonad biofilms. Examination of P. fluorescens 72 h biofilm transcriptome was elucidated upon exposure to glutaraldehyde. The results were confirmed using qRT--PCR and chemical genetic appraoches in P. fluorescens and P. aeruginosa.2015-04-06T00:00:00Z2023-09-11T18:15:53.658Z2022-01-31T17:22:16.606ZE-GEOD-6444825824217SRP051482EFO_000373810.1128/AAC.05152-14