Downloaded from www.microbiologyresearch.org by IP: 52.87.167.114 On: Sun, 13 May 2018 03:30:09 The response of Escherichia coli to exposure to the biocide polyhexamethylene biguanide Michael J. Allen,3 Graham F. White and Andrew P. Morby Correspondence Andrew P. Morby morby@cardiff.ac.uk School of Biosciences, Cardiff University, Museum Avenue, PO Box 911, Cardiff CF10 3US, UK Received 31 October 2005 Revised 4 January 2006 Accepted 10 January 2006 The global response of Escherichia coli to the broad-spectrum biocide polyhexamethylene biguanide (PHMB) was investigated using transcriptional profiling. The transcriptional analyses were validated by direct determination of the PHMB-tolerance phenotypes of derivatives of E. coli MG1655 carrying either insertionally inactivated genes and/or plasmids expressing the cognate open reading frames from a heterologous promoter in the corresponding chromosomally inactivated strains. The results showed that a wide range of genes was altered in transcriptional activity and that all of the corresponding knockout strains subsequently challenged with biocide were altered in tolerance. Of particular interest was the induction of the rhs genes and the implication of enzymes involved in the repair/binding of nucleic acids in the generation of tolerance, suggesting a novel dimension in the mechanism of action of PHMB based on its interaction with nucleic acids. INTRODUCTION Polyhexamethylene biguanide (PHMB) is a broad-spectrum antibacterial agent that has been widely used for many years as an antiseptic in medicine and the food industry, and its current applications also include impregnation of fabrics to inhibit microbial growth (Cazzaniga et al., 2002; Payne & Kudner, 1996); water treatment (Kusnetsov et al., 1997); disinfection of a variety of solid surfaces such as contact lenses (Hiti et al., 2002); as a mouthwash (Rosin et al., 2001, 2002); treatment of hatching eggs to prevent Salmonella infection (Cox et al., 1998, 1999); and as a treatment against fungi (Messick et al., 1999) and Acanthamoeba (Donoso et al., 2002; Gray et al., 1994; Narasimhan et al., 2002) in infective keratitis. Its preparations are mixtures of polymeric biguanides of structure [-(CH 2 ) 6 .NH.C(=NH).NH.C(= NH).NH-] n where n=2–40, with a mean size of n=11, giving a molecular mass range of approximately 400–8000, with various combinations of amino (-NH 2 ), guanide [-NH.C(=NH).NH 2 ] or cyanoguanide [-NH.C(=NH). NH.CN] as end-groups. PHMB is bacteriostatic at low concentrations (typically 1–10 mg l 21 ), but bactericidal at higher concentrations, and inhibition of growth and bactericidal activity both increase with increased polymerization (Broxton et al., 1983; Gilbert et al., 1990a). The lethal action is considered to involve interaction at the cytoplasmic membrane to cause non- specific alterations in membrane integrity. The proposed basis for the polymer-size effect is that PHMB interacts with acidic membrane-lipids to cause phase separation and domain formation; larger PHMB molecules produce larger domains and therefore more disruption (Broxton et al., 1984; Ikeda et al., 1984). This view was refined by Gilbert et al. (1990b) who showed that although the activity increased with increasing length of the polymer, the effect of polymer length was much reduced above n=6. Comparison of whole cells and spheroplasts showed that the cell envelope, while not providing complete protection, provides a significant exclusion barrier. Removal of lipopolysaccharides from the outer envelope markedly increased the activity of high- but not low-molecular-mass fractions. These observations, and the discovery of a strong synergy between low- and high-molecular-mass components in biocidal activity, led Gilbert et al. (1990b) to conclude that the low-activity, low- molecular-mass components enable larger homologues to gain access to sites of action in the cytoplasmic membrane. Acanthamoeba castellanii treated with high concentrations of PHMB contained clusters of densely stained precipitates (Khunkitti et al., 1998). Moreover, PHMB treatment pro- duced increased amounts of phosphorus inside the cells compared with untreated controls, and these accumulations were often confined to cell walls and nuclei (Khunkitti et al., 1999). Reduced membrane permeability causing reten- tion of phosphorus, coagulation of proteins and aggrega- tion of phospholipids have been considered as possible causes of elevated phosphorus but the possibility of asso- ciation between PHMB and nucleic acids has not been 3Present address: Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth PL1 3DH, UK. Original macroarray data have been deposited at the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/projects/geo/index. cgi), accession number GSE2827. Abbreviation: PHMB, polyhexamethylene biguanide. 0002-8643 G 2006 SGM Printed in Great Britain 989 Microbiology (2006), 152, 989–1000 DOI 10.1099/mic.0.28643-0