Bacillus subtilis vegetative isolate surviving chlorine dioxide exposure: an elusive mechanism of resistance.

D.J.H Martin,S.P Denyer,G Mcdonnell,R.L Wesgate,J.-Y Maillard

doi:10.1111/jam.12963

Abstract

Oxidizing agents such as chlorine dioxide are widely used microbicides, including for disinfection of medical equipment. We isolated a Bacillus subtilis isolate from a washer-disinfector whose vegetative form demonstrated unique resistance to chlorine dioxide (0·03%) and hydrogen peroxide (7·5%). The aim of this study was to understand the mechanisms of resistance expressed by this isolate. A range of resistance mechanisms were investigated in the B.subtilis isolate and a reference B.subtilis strain (ATCC 6051) to include bacterial cell aggregation, the presence of profuse exopolysaccharide (EPS), and the expression of detoxification enzymes. The basis of resistance of the isolate to high concentrations of oxidizing agents was not linked to the presence of endospores. Although, the presence of EPS, aggregation and expression of detoxification enzymes may play a role in bacterial survival to low concentrations of chlorine dioxide, it is unlikely that the mechanisms helped tested to survive the bactericidal effect of higher oxidizer concentrations. Overall, the mechanisms conferring resistance to chlorine dioxide and hydrogen peroxide remains elusive. Based on recent advances in the mode of action of oxidizing agents and notably hydrogen peroxide, we postulate that additional efficient intracellular mechanisms may be involved to explain significant resistance to in-use concentrations of commonly used high-level disinfectants. The isolation of a highly resistant vegetative Gram-positive bacterium to a highly reactive oxidizing agent is worrying. Understanding the mechanisms conferring such resistance is essential to effectively control such bacterial isolates. Here, we postulate that there are still mechanisms of bacterial resistance that have not been fully characterized.

Highlights

Bacterial resistance to biocides is a subject that has been highlighted by the European Commission (SCENIHR, 2009, 2010) and has culminated with a requirement of biocidal product manufacturers to provide information on the risks of resistance development with their products (Biocidal Product Regulation, 2013)
In 2008, we reported the isolation of a number of Gram-positive vegetative bacteria from an endoscope washer-disinfector that used chlorine dioxide for highlevel disinfection (Martin et al 2008)
This study reported that both strains were heavily embedded in a high concentration of exopolysaccharides (EPS)

Summary

Introduction

Bacterial resistance to biocides is a subject that has been highlighted by the European Commission (SCENIHR, 2009, 2010) and has culminated with a requirement of biocidal product manufacturers to provide information on the risks of resistance development with their products (Biocidal Product Regulation, 2013). The United States Food and Drug Administration (2013) recently required manufacturers of antimicrobial hand washes to provide evidence that their products have no effect on emerging bacterial resistance to antimicrobials. Reports of bacterial resistance (or tolerance) to biocides have not been limited to low level disinfection or preservatives (Scientific Committee on Consumer Safety 2012; Maillard et al 2013), and to highly reactive chemistries (including alkylating and oxidizing agents) used for highlevel disinfection. Similar glutaraldehyde-resistant isolates were later observed to be cross-resistant to other highly reactive biocides such as hypochlorite and oxidizers (Griffiths et al 1997) but not to ortho-phthalaldehyde (Walsh et al 1999; Fraud et al 2001). A report has described the development of mycobacterial (and other bacterial) resistance to glutaraldehyde and/or ortho-phthalaldehyde (Fisher et al 2012)

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Results

Conclusion