Comparative analysis of Salmonella susceptibility and tolerance to the biocide chlorhexidine identifies a complex cellular defense network.

Orla Condell,Jenny Renaut,Sarah Finn,Kristian Hã¤Ndler,Aine Sheridan,Jay C D Hinton,Catherine M Burgess,Sã©Amus Fanning,Karen A Power,Jarlath E Nally,Kjell Sergeant

doi:10.3389/fmicb.2014.00373

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 (ST24CHX) 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.

Highlights

Chlorhexidine (1,6 bis(4 -chlorophenylbiguanide)hexane) is a cationic biocide of the biguanide class
Based on our current understanding of the modes of action related to various biocides, tolerance to these compounds, including chlorhexidine, typically does not develop following mutation to a particular target gene but rather involves broader cellular changes, such as up-regulated efflux pump activity or alterations in cell wall permeability (Poole, 2002)
The tolerant phenotype that develops may correlate with a reduced susceptibility to other antimicrobial agents, for example, chlorhexidine exposure has been associated with a reduced susceptibility to clinically important antimicrobial agents (Braoudaki and Hilton, 2004; Condell et al, 2012a)

Summary

Introduction

Chlorhexidine (1,6 bis(4 -chlorophenylbiguanide)hexane) is a cationic biocide of the biguanide class It has a broad spectrum of action and is effective against Gram-negative and Gram-positive bacteria as well as yeasts, protozoa and some lipophilic viruses (Jones, 2000). This biocide is used as a disinfectant, an antiseptic and a preservative. Chlorhexidine is composed of a six carbon chain, a structure that is smaller compared to other cationic compounds. In addition it contains an inflexible hydrophobic region. Its size and molecular inflexibility are thought to limit its ability to fold sufficiently to allow its insertion into the membrane www.frontiersin.org

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