Micro-organisms that attach to a surface and form a biofilm are usually found to be highly protected from killing by antimicrobial agents (Stewart et al. 2000a). Even when challenged with a seemingly brute force agent such as free chlorine, micro-organisms in biofilms survive treatments that rapidly eradicate freely suspended cells (Ronner and Wong 1993; Oie et al. 1996; Ntsama-Essomba et al. 1997; Stewart et al. 1998). Chlorine is a benchmark biocide that is used to control biofilm contamination and fouling in diverse application areas, including drinking water, food processing, hospital sanitization, industrial water systems and household cleaning. The basis for biofilm resistance to chlorine remains incompletely understood, but evidence that chlorine penetration into biofilms can be profoundly retarded has been presented recently (de Beer et al. 1994; Chen and Stewart 1996; Xu et al. 1996).
The poor penetration of hypochlorite/hypochlorous acid into biofilm is due to reactive neutralization of the active chlorine in the outermost regions of the biofilm matrix (Chen and Stewart 1996; Xu et al. 1996). In other words, the chlorine is consumed by reaction with organic matter in the surface layers of the biofilm faster than it can diffuse into the biofilm interior. The reaction–diffusion theory that explains this phenomenon predicts that a less reactive biocide should penetrate biofilm more effectively (Stewart and Raquepas 1995; Stewart 1997). This raises the possibility that stabilized forms of hypochlorite (unipositive chlorine), which may react at a slower rate with biofilm organic constituents than the highly reactive hypochlorite species, might penetrate more readily into a biofilm. Examples of halogen-stabilizing agents include ammonia, amines and hydantoins. A weakly reactive disinfectant that penetrates a biofilm may outperform, at least in terms of microbial killing, a stronger disinfectant that fails to penetrate fully. Exactly this scenario seems to be responsible for reports of superior biofilm antimicrobial efficacy by monochloramine compared with equivalent doses of free chlorine (LeChevallier et al. 1990; Neden et al. 1992; Griebe et al. 1993; Samrakandi et al. 1997; Stewart et al. 2000a). Monochloramine is known to be a weaker disinfectant than free chlorine when tested against suspended bacteria.
We have been interested in the potential utility of chlorosulfamates as alternative biofilm control agents. Mono- and dichlorosulfamate (ClNHSO3– and Cl2NSO3–, respectively) form rapidly upon reaction of hypochlorite or hypochlorous acid with sulfamate over a wide range of pH (about pH 1–10). Although the chlorosulfamates are known to be weaker disinfectants than hypochlorous acid (Delaney and Morris 1972), they have several compensatory advantages: chlorosulfamates can be formulated at mild pH (typically pH 5–6) whereas hypochlorite is only stable for prolonged periods at highly alkaline pH (> 10·5); these chlorosulfamate formulations are quite stable and they produce no objectionable fumes and, finally, lower reactivity of chlorosulfamates with soil and biofilm is anticipated compared with free chlorine. Lower reactivity with organics is predicted to facilitate penetration into a biofilm. These features of chlorosulfamates make them particularly attractive as potential biocidal systems in consumer products.
There were two main objectives to our work. The first was to compare biofilm penetration and disinfection efficacy of chlorosulfamates with alkaline hypochlorite. The second was to determine how much of the biofilm resistance observed with either disinfectant could be attributed to poor penetration.