title: Comparative analysis of Salmonella susceptibility and tolerance to the biocide chlorhexidine identifies a complex cellular defense network.
abstract: Chlorhexidine is one of the most widely used biocides in health and agricultural settings as well as in the modern food industry. It is a cationic biocide of the biguanide class. Details of its mechanism of action are largely unknown. The frequent use of chlorhexidine has been questioned recently, amidst concerns that an overuse of this compound may select for bacteria displaying an altered susceptibility to antimicrobials, including clinically important anti-bacterial agents. We generated a Salmonella enterica serovar Typhimurium isolate (ST24(CHX)) that exhibited a high-level tolerant phenotype to chlorhexidine, following several rounds of in vitro selection, using sub-lethal concentrations of the biocide. This mutant showed altered suceptibility to a panel of clinically important antimicrobial compounds. Here we describe a genomic, transcriptomic, proteomic, and phenotypic analysis of the chlorhexidine tolerant S. Typhimurium compared with its isogenic sensitive progenitor. Results from this study describe a chlorhexidine defense network that functions in both the reference chlorhexidine sensitive isolate and the tolerant mutant. The defense network involved multiple cell targets including those associated with the synthesis and modification of the cell wall, the SOS response, virulence, and a shift in cellular metabolism toward anoxic pathways, some of which were regulated by CreB and Fur. In addition, results indicated that chlorhexidine tolerance was associated with more extensive modifications of the same cellular processes involved in this proposed network, as well as a divergent defense response involving the up-regulation of additional targets such as the flagellar apparatus and an altered cellular phosphate metabolism. These data show that sub-lethal concentrations of chlorhexidine induce distinct changes in exposed Salmonella, and our findings provide insights into the mechanisms of action and tolerance to this biocidal agent.
authors.affiliation: UCD Centre for Food Safety, School of Public Health, Physiotherapy and Population Science, University College Dublin Belfield, Dublin, Ireland ; European Program for Public Health Microbiology Training, European Centre for Disease Prevention and Control Stockholm, Sweden.
authors.forename: Orla
authors.initials: O
authors.lastname: Condell
authors.affiliation: UCD Centre for Food Safety, School of Public Health, Physiotherapy and Population Science, University College Dublin Belfield, Dublin, Ireland.
authors.forename: Karen A
authors.initials: KA
authors.lastname: Power
authors.affiliation: Department of Microbiology, School of Genetics and Microbiology, Moyne Institute of Preventive Medicine, Trinity College Dublin Dublin, Ireland.
authors.forename: Kristian
authors.initials: K
authors.lastname: Händler
authors.affiliation: UCD Centre for Food Safety, School of Public Health, Physiotherapy and Population Science, University College Dublin Belfield, Dublin, Ireland.
authors.forename: Sarah
authors.initials: S
authors.lastname: Finn
authors.affiliation: Food Safety Department, Teagasc Food Research Centre Ashtown, Dublin, Ireland.
authors.forename: Aine
authors.initials: A
authors.lastname: Sheridan
authors.affiliation: Department of Environment and Agrobiotechnologies (EVA), Centre de Recherche Public-Gabriel Lippmann Belvaux, Luxembourg.
authors.forename: Kjell
authors.initials: K
authors.lastname: Sergeant
authors.affiliation: Department of Environment and Agrobiotechnologies (EVA), Centre de Recherche Public-Gabriel Lippmann Belvaux, Luxembourg.
authors.forename: Jenny
authors.initials: J
authors.lastname: Renaut
authors.affiliation: Food Safety Department, Teagasc Food Research Centre Ashtown, Dublin, Ireland.
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Burgess
authors.affiliation: Department of Microbiology, School of Genetics and Microbiology, Moyne Institute of Preventive Medicine, Trinity College Dublin Dublin, Ireland ; Institute of Integrative Biology, University of Liverpool Liverpool, UK.
authors.forename: Jay C D
authors.initials: JC
authors.lastname: Hinton
authors.affiliation: School of Veterinary Medicine, University College Dublin Belfield, Dublin, Ireland.
authors.forename: Jarlath E
authors.initials: JE
authors.lastname: Nally
authors.affiliation: UCD Centre for Food Safety, School of Public Health, Physiotherapy and Population Science, University College Dublin Belfield, Dublin, Ireland ; Institute for Global Food Security, Queen's University Belfast Belfast, Northern Ireland.
authors.forename: Séamus
authors.initials: S
authors.lastname: Fanning
keywords: SNP typing; Salmonella; biocide tolerance; chlorhexidine; proteomics; transcriptomics; whole genome sequencing