Presence of sub-inhibitory concentrations of quaternary ammonium compounds in water: effects on bacteria and their inactivation by ozone and monochloramine

Disinfection agents have been present in our daily lives for several decades in detergents or in personal care products, where their role is to prevent the spread of pathogenic microor-ganisms. One of the largest class of disinfection agents is quaternary ammonium compounds (QACs). Within this class, cetyltrimethylammonium chloride (CTMA) and benzalkonium chlo-ride (BAC) are widely used. QACs have a broad-spectrum effect on bacteria, where they pri-marily target the membrane. After use, QACs are partially discharged to municipal wastewaters, where they are diluted to concentrations in the sub-inhibitory range. These low concentrations of QACs can trigger resistance mechanisms in bacteria, including membrane modifications and the expression of efflux pumps.The aim of this thesis was to investigate the consequences and the impact of the presence of low concentrations of QACs in wastewater and surface water on the survival of bacteria. It could be demonstrated that strains of P. aeruginosa long-term exposed to constant sub-inhibitory concentrations of either BAC or CTMA showed decreased susceptibility to both QACs, regardless of which QAC the bacteria had been exposed to. However, no resistance to selected antibiotics was found after this exposure. Investigations of the membrane using atomic force microscopy showed an increase in surface roughness following exposure to both BAC and CTMA, demonstrating that QACs affect bacterial membranes.QACs are poorly biodegradable and their presence, even at constant sub-inhibitory concen-trations, affect bacteria. QACs are poorly reactive with chemical oxidants commonly used for water disinfection, which aims to inactivate microorganisms. Therefore, QACs remain present during water and wastewater treatment. Several methods of disinfection exist, where ozona-tion and monochloramination are widely applied worldwide. The effect of a pre-exposure of E. coli to CTMA and of the presence of CTMA during disinfection with ozone (O3) and mono-chloramine (NH2Cl) were investigated. The pre-exposure to CTMA led to contrasting results: inactivation of E. coli was enhanced when using ozone but decreased when using monochlo-ramine. This result demonstrates the different modes of inactivation of bacteria for ozone and monochloramine. Moreover, the role of efflux pumps during inactivation was tested with two strains of E. coli, expressing different efflux pump systems. These two strains showed contrasting behaviors when treated with either ozone or monochloramine. Nevertheless, we were unable to resolve the role of efflux pumps in this system. The presence of CTMA en-hanced the inactivation when concentrations were higher than 2 mgL-1 for ozone and 2.25 mgL-1 for monochloramine.Standard methods to determine the minimum inhibitory concentration use ideal conditions for bacterial growth, which do not occur in natural water. Therefore, the susceptibility of E. coli to CTMA in PBS, lake water (Lake Geneva, Switzerland) and a wastewater effluent was investigated and compared to nutrient broth. It could be shown that E. coli was significantly more susceptible to CTMA in dilute aqueous solutions compared to broth. These results are relevant to assess the effect of CTMA and potentially other biocides on bacteria under realis-tic conditions, as it is probably overestimated using the current standard methods for MICs determination.