An investigation into the relative resistances of common bacterial pathogens to quaternary ammonium cation disinfectants

BioscienceHorizons Volume 10 2017 10.1093/biohorizons/hzx008 ................................................................................................................................................................. Research article An investigation into the relative resistances of common bacterial pathogens to quaternary ammonium cation disinfectants Gregory Wickham* Department of Biological Science, University of Chester, Parkgate Road, Chester CH1 4BJ, United Kingdom *Corresponding author: Department of Biological Science, University of Chester, Parkgate Road, Chester CH1 4BJ, United Kingdom. Email: Supervisor: Philip Wood and Chris Davis, Department of Biological Science, University of Chester, Parkgate Road, Chester CH1 4BJ, United Kingdom, (P.W.)/ (C.D.) ................................................................................................................................................................. Benzalkonium chloride is a common quaternary ammonium cation-based disinfectant used as an industrial-grade biocide, but little independent work has been undertaken quantifying the concentrations required for sterilization. This study investigated relative differences in resistance between common Gram-negative and Gram-positive bacterial pathogens and determined the complete sterilization concentrations for each. A membrane filtration methodology was used to quantify an enriched isolate of deionized water, which was subjected to various concentrations of disinfectant incubated on MacConkey agar. The colony forming units at each concentration were compared to an untreated control. Three main trends, defined as ‘phases of inhibition’, were observed across all isolates studied. Phase I occurred from 0 to 1 mL disinfectant/L water and displayed a moderate, consistent rate of inhibition. Phase II occurred from 0.1% to 0.4% biocide in solution and was characterized by a dramatic increase in inhibition and a divergence of inhibition rates for each organism. Phase III occurred from 0.4% biocide in solution onward and was characterized by the gradual decline in rate of inhibition until each organism reached total inhibition. It was found that the Gram-negative group, comprising Escherichia coli and Pseudomonas aeruginosa, was generally more resistant than the Gram-positive group, comprising Enterococcus faecalis and Staphylococcus aureus, p < 0.001, with the individual Gram-negative organisms, having the highest complete sterilization concentrations. It was also observed that a variation in resistance existed between organisms of the same Gram stain group. This resulted in some organisms exhibiting resistances comparable to that of organisms of the opposite group, namely between the E. faecalis and P. aeruginosa, which exhibited no significance difference, p = 0.080. Therefore, a model is proposed in which the Gram stain groups can be generalized as being distinct in terms of intrinsic resistance, but also that the range of resistance exists as a spectrum within each group which can cause a similarity between individual organisms of different groups. Key words: Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, membrane filtration, benzalkonium chloride Submitted on 15 October 2016; editorial decision on 3 July 2017 ................................................................................................................................................................. ................................................................................................................................................................. © The Author 2017. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 1 Bioscience Horizons • Volume 10 2017 Research article ................................................................................................................................................................. Introduction bacterial resistance to QACs and appear to be remaining prevalent in current studies in the field. Quaternary ammonium cations (QACs) are an extremely potent, versatile family of disinfectants commonly used as industrial grade biocides. Biocleanse® is one such established brand of QAC-based disinfectants and utilizes benzalkonium chloride (BAC) as its active antimicrobial agent. BAC possesses strong biocidal activity against most major forms of pathogenic microbes including fungi, encapsulated viruses and bacteria. QACs exhibit antimicrobial properties due to a membrane-active interaction which initiates autolysis and results in the leakage of intracellular constituents (Ioannou, Hanlon, and Denyer, 2007). The first materials to evacuate the cell are usually low molecular weight cytoplasmic bodies and later, degradation of heavier components occurs, such as proteins and nucleic acids. This activates autolytic enzymes that disintegrate the cytoplasmic membrane causing lysis (McDonnell and Russel, 1999). The primary mechanism by which resistance in bacteria develops to QACs is prolonged exposure to sub-inhibitory concentrations due to intensified selective pressure, which is especially significant throughout exposure during the cellular exponential phase (Ibusquiza et al., 2012). This has also been shown to contribute to biofilm formation (Ortiz, López, and Martínez-Suárez, 2014), which is noteworthy as bacteria grown in a biofilm can be up to 1000 times more resistant to biocides than cultured bacteria (Bastian, Alabouvette, and Siaz-Jimenez, 2009). Traditionally, there was a long-held belief that development of bacterial resistance to QACs was extremely unlikely due to its non-specific target of action (Gerba, 2014). The rationale for this being that any attempts by bacteria to evolve a structural or biochemical component to subvert the disinfection mechanism would often be responded to by a different but equally as effective membrane bound reaction on account of the amphiphilic properties of the QAC molecule (Ahlström and Edebo, 1998). Contrary to this, clinical reports of increasingly QAC resistant strains of methicillin-resistant Staphylococcus aureus have been on the rise in recent years, particularly in nosocomial environments (Minbiole et al., 2016). Bacterial resistance to disinfectants involves not just inactivating antimicrobials, or inhibiting their penetration, but also removing non-endogenous molecules from the cell as quickly as possible (Heinzel, 1998). In regards to this, the contribution of plasmid and chromosome encoded efflux pumps has been increasingly stated in recent years (Blair et al., 2015). Complex efflux systems are particularly widespread in Gramnegative bacteria. Most have no known QAC detoxifying prop (...truncated)