Inactivation of Pseudomonas aeruginosa MDC by isothiazolones and biocide stabilizing agents

Highlights

• Isothiazolones (IT) stabilizing agents: CuSO₄ stronger biocide than IT, Mg(NO₃)₂ or MgCl₂ no effect on cells.

• Cell inactivation contact time (CICT) was dose dependent, 10 ppm active ingredients no effect.

• CICT 100 ppm: 3,5h (CuSO₄), 24h (IT); CICT 1000 ppm: 2h (CuSO₄), >6h (IT).

• IT drastically reduced respiration and INT reduction but did not lyse cells.

• Isothiazolones treated cells were injured and they recovered in rich media, copper killed the cells.

Abstract

5-chloro-2-methyl-4-isothiazolin-3-one (CMIT)/2-methyl-4-methyl-isothiazolin-3-one (MIT) biocides or preservation agents are chemically stabilized with MgCl₂, Mg(NO₃)₂ or CuSO₄. The role of these stabilization agents in cell inactivation was investigated with Pseudomonas aeruginosa MDC isolated from a water-based paint tank biofilm. 10 ppm/active ingredient (ai) of CMIT/MIT commercial formulation or CuSO₄ alone did not lead to elimination of viable cell counts. Inactivation times of 3.5h (CuSO₄) or 24h (CMIT/MIT) with 100 ppm/ai biocides were further reduced to 2h (CuSO₄) and 6h (CMIT/MIT), respectively, when the dose was increased to 1000 ppm/ai, with no synergism observed between these two biocides. Copper from cells exposed to high doses was remobilized back into the solution in quantities sufficient to become biocidal to fresh cells whilst EDTA added prior to copper prevented cell inactivation. Mg(NO₃)₂ was not biocidal. Cell death defined as the inability to recover biocide-treated cells after 120h incubation in the medium used for growth required minimum contact times of 2.5h (1000 ppm/ai) or 3h (100 ppm/ai) with copper sulfate and 24h with 100 ppm/ai or 1000 ppm/ai CMIT/MIT. Pseudomonas aeruginosa MDC were not lysed after exposure to either copper or CMIT/MIT but only copper made the membrane permeable to propidium iodide. Both copper and CMIT/MIT inhibited the activity of the electron transport chain and reduced INT activity in Pseudomonas aeruginosa MDC to residual levels. The latter effect was also observed with Mg(NO₃)₂. Preventing microbial growth in products protected by CMIT/MIT requires maintenance of effective biocide doses over the entire shelf life.

Introduction

Isothiazolinones are a class of biocides and preservatives with a major share of the market for industrial water treatment, particularly in water-based paints and varnishes, metalworking fluids, cosmetics and household cleaning products (Williams, 2007). These molecules are unstable at pH ≥ 8 and at temperatures >55 °C (Paulus, 2005). They react rapidly with primary and secondary amines, with compounds containing reduced sulfur species (sulfites and bissulfites) and with proteins such as keratin or collagen, which are frequently added to cosmetic formulations (Collier et al., 1990a). The most commonly used product is a 3:1 ratio of 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT) and 2-methyl-4-isothiazolin-3-one (MIT), which has broad spectrum efficacy against bacteria, algae and fungi (Williams, 2007). CMIT/MIT formulations for extended use in humid or wet environments need to be stabilized with magnesium or copper salts to prevent the chemical modification of the active biocide molecules (Barman and Preston, 1992; Miller and Weiler, 1975). Other isothiazolones such as 1–2, Benzisothiazol-3-one do not require stabilization.

Metal salts used for isothiazolone stabilization are usually classified as inert ingredients in the product literature, but several of these components may also directly or indirectly affect bacterial viability. Copper and nitrates used in the formulations, for example, are potentially antimicrobial. Copper inactivates microorganisms by the permeabilization of cell envelopes, the denaturation of nucleic acids, the inactivation of proteins by either direct reaction or displacement of essential metal cations and by membrane lipid peroxidation (Borkow, 2012). In addition, copper is one of the metals capable of undergoing fast redox cycling inside microbial cells. Reduction of Cu²⁺ catalyzed by the superoxide anion radical or by biological reducing agents such as glutathione or ascorbic acid generates Cu¹⁺, which in turn may produce hydroxyl radicals in a Fenton reaction with hydrogen peroxide (Jomova and Valko, 2011). Hydroxyl radicals are potent generic oxidation agents, which react with practically all types of organic molecules present in microbial cells. The association of copper with isothiazolones was reported to increase the antimicrobial efficacy of the latter (Riha et al., 1990; Law and Lashen, 1991). Nitrate alone has no significant biocidal activity. This compound is widely used as a curing agent in the meat industry, but the microbial control associated with its use is due almost entirely to its microbial conversion to nitrite, the actual biocidal agent in the product (Major et al., 2010).

Biocide dosage in a specific application is determined by means of challenge tests such as the minimal inhibitory concentration determination procedure, where the amount of biocide is progressively increased in growth media until growth of the test organism ceases (Russell, 2003). In order to ensure adequate microbiological control of the final product over the desired shelf life and due to the impossibility of conducting challenge tests with the entire diversity of microbes present in environmental samples, the choice of organism for use in such tests becomes critical. Product regulatory guidelines often prescribe the microbial species to be used in the challenge tests. Pseudomonas aeruginosa, for example, is mandated in tests of biocide efficiency in bottled drinking water, cosmetics, paints, etc. (ASTM D 2574-06, 2012 ASTM D 2574 : 2016 : R2020 : EDT 1, 2020); European Standard EN 13697, 2015, United States Pharmacopeia (2003). Pseudomonas aeruginosa, is capable of growth in most natural and man-made environments (Khan et al., 2007; Moradali et al., 2017). It is resistant against most antibiotics (El Zowalaty et al., 2015) and biocides (Meyer and Cookson, 2010; Grobe et al., 2002; McDonnel and Russell, 1999; Bridier et al., 2011) and it is a common opportunistic pathogen (Kerr and Snelling, 2009). Biocide resistance in this organism is further increased when it grows as a biofilm (Grobe et al., 2002; Bridier et al., 2011).

One important disadvantage of culture-dependent biocide tests is their inability to define whether cell inactivation in the presence of the biocide was due to killing of the cell, or to one of the potentially reversible mechanisms of cell injury or transition into the viable but non culturable (VBNC) state (Trevors, 2012). Cell injury is a reversible type of cell damage where the initial loss of cell viability is overcome over time when the stressors (biocide molecules) are removed or when their amounts drop below a threshold concentration, allowing the cells to grow again (Wesche et al., 2009). The VBNC state is a condition where a microbial cell is still alive, but it can only be resuscitated under specific circumstances different from those used for cultivating the cell (Oliver, 2016). Understanding the mode of cell inactivation by a biocide is important because product protection often needs to be warranted for prolonged periods. If cells are killed by the biocide, then the focus of prolonged protective action shifts to avoiding contamination of the product by undesired organisms. If cells are only injured, additional care must be taken to ensure that biocidal doses persist during the entire time required for microbial control. This is challenging because biocidal molecules are often reactive and may be consumed by non-target side-reactions in the product.

This work aimed at investigating the role of CuSO₄ and Mg(NO₃)₂ stabilizing agents either alone or in combination with CMIT/MIT in the inactivation and/or injury of planktonic cells of Pseudomonas aeruginosa MDC isolated from the surfaces of a water-based paint storage tank. The mechanism of inactivation by the different isothiazolone stabilizing agent combinations or by the stabilizing agents alone was assessed by cell integrity analysis and physiological viability assays. Cell recovery after biocide treatment was also investigated.