Quaternary Ammonium Biocides as Antimicrobial Agents Protecting Historical Wood and Brick*

Quaternary ammonium compounds (QACs) are widely used in disinfection of water, surfaces and instruments as well as in textile, leather and food industries because of their relatively low toxicity, broad antimicro-bial spectrum, non-volatility and chemical stability. Due to these advantages, QACs are also used in restoration and can be applied on historical material. The aim of this study was to determine the usefulness of biocides based on quaternary ammonium salts and containing various excipients in the protection of historical materials against microbial growth. The study determined the antimicrobial activity of three biocides against bacteria: Acremonium strictum moulds showed high sensitivity to quaternary ammonium biocides. Historical wood can be effectively disinfected by three applications of biocide A (30% v/v) containing dodecyl dimethyl ammonium chloride (DDAC), citric acid, propiconazole and propanol. Dis-infection of historical brick can be carried out by three applications of 6% v/v solutions of biocide B (based on DDAC and ethylenediaminetetraacetic acid — EDTA) or biocide C (containing a non-ionic surfactant, DDAC and EDTA). Effective protection of historical building materials against microbial growth for a period of seven days can be achieved by the application of biocide A (30% v/v) on the wood surface and biocide B (6% v/v) on the brick surface.


INTRODUCTION
Quaternary ammonium compounds (QACs) are surfactants, and in 2004 the annual consumption of QACs in the world was estimated at 500 000 tons (Tezel & Pavlostathis, 2011).The structure of these compounds is formed by a positively charged nitrogen (N) atom and four alkyl groups R 1 -R 4 (saturated, unsaturated, branched or straight, cyclic or acyclic), one of which is a long alkyl chain having different numbers of carbon atoms and a chloride or bromide anion (X -), according to the formula [R 1 R 2 R 3 R 4 N] + X -.
The antimicrobial effectiveness of QACs was described in the thirties of the twentieth century, indicating their high activity against bacteria, moulds and lipophilic viruses, at very low concentrations (Tischer et al., 2012).The sensitivity of microorganisms to their activity varies depending mainly on concentration and contact time, but also on individual sensitivity of a given strain.It has been found that QACs are more efficient against Grampositive than Gram-negative bacteria.When QACs are used as disinfectants, the concentration applied is almost always below 1000 ppm (Tezel & Pavlostathis, 2011;Lipińska-Ojrzanowska & Walasiuk-Skorupa, 2014).They are used in medicine and many industries (food, cosmetics, paper) as biocides and even drugs (Raghavendra, 2002;Xiao et al., 2008).They are readily biodegradable, not toxic to humans and animals, dissolve well in water and are active in a wide pH range (4-10).In addition, they are used as agents to combat biofilms formed by microorganisms in cooling systems (Rucka et al., 1983).Quaternary ammonium salts are also used in the restoration of historical buildings (Karbowska-Berent et al., 2011;Sterflinger & Piñar, 2013;Stupar et al., 2014), as ingredients of pesticides and wood preservatives (Dubois & Ruddick, 1998;Tezel & Pavlostathis, 2011), where they primarily act as antifungal compounds.They are also used as chemical corrosion inhibitors, plasticizers in the production of textiles and paper, or antistatic agents for materials such as cotton or cellulose fibres (Obłąk & Gamian, 2010;Lipińska-Ojrzanowska & Walasiuk-Skorupa, 2014).
As quaternary ammonium salts show high antimicrobial activity, have no destructive impact on historical material (Sterflinger & Piñar 2013), and are often used as active substances in biocidal products, biocides containing these particular compounds have been selected for the study on the protection of historical materials against microbial growth.The tested biocides contain dodecyl dimethyl ammonium chloride and various excipients that can potentially modulate the lethal activity of the formulations.Environmental strains of microorganisms isolated from historical materials were used in this study.While the activity of biocides against collection microorganisms has been commonly tested in vitro, there are still few studies on their efficacy against specific strains of bacteria and moulds that have strongly adapted to historical nutrient-poor materials.The aim of this study was to select quaternary ammonium biocides for effective eradication of microorganisms inhabiting historical wood and brick in situ.

MATERIALS AND METHODS
Microorganisms.The effectiveness of biocides was tested for six selected strains of bacteria and six strains of moulds isolated from the surfaces of historical wooden and brick barracks at Historical materials.Historical materials in the form of wood and brick fragments measuring 50 × 20 × 10 mm, collected from the Auschwitz II -Birkenau State Museum in Oświęcim, were used in the study (Fig. 1).Samples of the materials were sterilized twice at 121°C for fifteen minutes and then stabilized in a constant climate chamber (Binder) at 28°C and relative humidity (RH) of 80% for seven days.
Biocides.Three commercial biocides designated as A, B, and C were used.The tested formulations contained dodecyl dimethyl ammonium chloride as the active ingredient and various excipients (Table 1).According to their purpose, biocides A and B were applied to the surface of the wood, and biocides B and C on the surface of the brick.Each biocide was tested at three concentrations, according to the manufacturers' recommendations.
Evaluation of antimicrobial activity of biocides in situ on the samples of wood and brick.One millilitre of monocultures of bacteria (10 8 CFU/ml) or moulds (10 6 conidia/ml) in the medium ((NH 4 ) 2 SO 4 0.075%, K 2 HPO 4 0.025%, MgSO 4 × 7 H 2 O 0.125%, yeast extract 0.125% , glucose 0.5%, agar 0.1%, pH 6.0) was applied on the surface of each sample tested.The samples were incubated in a constant climate chamber with a relative humidity of 80% and a temperature of 28°C for 7 days (bacteria) or 21 days (moulds).After incubation, biocides were applied on the surfaces of the wood and brick samples one, two, and three times at an interval of 24 hours, using the spraying method (professional sprayer Mercury Super Pro 360, Quasar).The number of microorganisms after successive applications of each biocide was determined by the contact plate method in TSA medium (Tryptic Soy Agar, Merck) -bacteria, and MEA (Malt Extract Agar, Merck) -moulds.Cultures were incubated at 30°C for 48 hours (bacteria) and at 28°C for 5 days (moulds).
Evaluation of the effectiveness of protection of the historical materials against microbial growth.On the surface of each sterile, conditioned sample of historical wood and brick, the biocides were applied three times at an interval of 30 minutes, followed by an inoculum of bacteria (10 8 CFU/ml) or moulds (10 6 conidia/ml).The samples were incubated in a constant climate chamber (28°C, 80% RH).The effectiveness of the protection of the material against microbial growth was determined by the contact plate method in TSA medium (bacteria) and MEA (moulds) after 7 and 28 days of the treatment, similarly as in the tests of the antimicrobial activity.
The evaluation scale for the antimicrobial activity of the biocides on the surfaces of historical materials.In order to determine the activity of the biocides against bacteria and moulds, a calibration scale was used according to Table 2.

RESULTS AND DISCUSSION
The antimicrobial activity of biocides was determined using microorganisms isolated from historical wood and brick surfaces with visible signs of deterioration (Koziróg et al., 2014;Rajkowska et al., 2014;Piotrowska et al., 2014).Moulds and bacteria colonizing historical materials may cause serious aesthetical destruction, inhabit and penetrate into the materials, resulting in material loss, due to acid corrosion, enzymatic degradation and mechanical attack (Sterflinger & Piñar, 2013).Effective protection of historical material against microorganisms provides a properly conducted disinfection and protection against re-colonization by bacteria and moulds  (Koziróg & Żakowska, 2014).The study was carried out on historical wood and brick samples using three biocides based on quaternary ammonium salts but containing various excipients, i.e. biocide A -DDAC, citric acid, propiconazole and propanol, biocide B -DDAC and EDTA, biocide C -non-ionic surfactant, DDAC and EDTA.
The tested mould and bacteria strains showed varying susceptibility to the biocides.In general, Chaetomium globosum, Penicillium citreonigrum, Aspergillus fumigatus and Cladosporium cladosporioides (II) moulds demonstrated lower sensitivity (Tables 3, 4 and 7).P. citreonigrum and Ch.globosum on wood samples were insensitive to biocide B at all tested concentrations.Ch. globosum was sensitive to 20% and 30% v/v biocide A, but only after its third application.While P. citreonigrum showed sensitivity to preparation A at the lowest concentration of 10% v/v, it also required its three applications (Table 3).On brick samples, A. fumigatus and C. cladosporioides (II) exhibited insensitivity to biocides B and C, both at concentration of 1.5% v/v.C. cladosporioides (II) was highly sensitive to preparations B and C at concentration of 3% v/v after three applications, or 6% v/v after two applications (Table 4).At the same time, for eradication of A. fumigatus from the brick surface, three applications either of biocide B (3% or 6% v/v) or biocide C (3% or 6% v/v) were necessary.
In comparison to the other bacteria tested, less sensitive were two bacterial strains of Pseudomonas fluorescens to biocide B and Bacillus muralis to biocides B and C (Tables 3, 4 and 7).P. fluorescens on wood samples was highly sensitive to 10% v/v biocide A, but only after its third application, or to 20% v/v biocide A already after the first use (Table 3).Still, the complete inhibition of B. muralis growth on brick surface required three applications of 6% v/v biocide C (Table 4).
Quaternary ammonium compounds (QACs) are considered to be membrane active agents (McDonnell & Russell, 1999).In microbial cells exposed to QACs, there are sequential processes of adsorption and penetration of the agent into the cell wall, reaction with the cytoplasmic membrane followed by membrane disorganization, leakage of intracellular low-molecular-weight material, degradation of proteins and nucleic acids and wall lysis caused by the autolytic enzymes (Tischer et al., 2012).
Regardless of the mechanism of action, different types of microorganisms vary in their response to disinfectants.Generally, Gram-negative and spore-forming bacteria show lower sensitivity to biocides (Maillard, 2002).This classification is consistent with the results obtained in this study, indicating that Bacillus muralis and Pseudomonas fluorescens bacteria, but also moulds, are less sensitive to QACs.Lower sensitivity may be attributed, depending on the cell structure, to the outer membrane in Gram- negative bacteria, which acts as a permeability barrier and restricts entry of many types of chemicals, or to the presence of a resistant wall in bacterial endospores and fungal conidia (White & McDermott, 2001).It should be emphasized, however, that microbial susceptibility to antiseptics and disinfectants is a strain-dependent feature.
As for the strains with lower sensitivity to the tested quaternary ammonium biocides, a high or good antimicrobial activity was achieved on wood samples after three applications of 30% v/v biocide A, containing DDAC, citric acid, propiconazole and propanol (Tables 3 and 4).On the other hand, brick samples can be disinfected by three applications of 6% v/v solutions of biocide B (based on DDAC and EDTA) or biocide C (containing a non-ionic surfactant, DDAC and EDTA).Biocide B applied on the wood surface was ineffective in inhibiting the growth of P. fluorescens, Ch. globosum and P. citreonigrum even at the highest tested concentration of 6% v/v (Table 3).
Earlier studies conducted in the museum's environment showed that the total number of microorganisms on the surfaces of the tested historical materials did not exceed 10 5 CFU/100 cm 2 (Koziróg et al., 2014;Rajkowska et al., 2014).Taking into account the number of microorganisms, it can be assumed that these materials can be effectively disinfected using biocides at concentrations corresponding to their good activity.Then, to inhibit the growth of the tested organisms on the wood surface, it should be applied three times for biocide A at a concentration of 20% v/v, and on the brick sample biocide C at a concentration of 3% v/v (Tables 3 and 4).
The effectiveness of the wood and brick surface protection before recolonization of microorganisms was determined after 7 and 28 days.The effectiveness of the protection was dependent on the biocide and the strain.Similarly to the disinfection process, the protection was less effective against P. fluorescens and B. muralis bacteria and Ch.globosum, P. citreonigrum, A. fumigatus moulds (Tables 5 and 6).In the case of wood samples, good or high protection was found after using 30% v/v biocide A, but within a period of 7 days.After 28 days of incubation, this biocide did not inhibit the growth of Ch. globosum (Table 5).Biocide B, used at a concentration of 6% v/v, limited microbial growth on the brick surface for 7 days.In the long term, however, it did not protect the historical brick surface against the growth of B. muralis (Table 6) and the wood surface against the growth of P. fluorescens, Ch. globosum and P. citreonigrum (Table 5).
Inhibition of the growth of five out of six tested microorganisms on the brick samples was observed after use of biocide C (6% v/v).This biocide, however, did not protect the surface against the growth of A. fumigatus, neither within 7 nor 28 days (Table 6).
The efficacy of biocides depends on many factors, including their concentration, the period of contact with microorganisms, the presence of interfering materials (e.g.organic or inorganic soils), temperature as well as the nature and type of target microorganisms (Russell & McDonnell, 2000).In addition, the efficacy of antimicrobial agents depends on and varies significantly with formulation effects, which can often enhance biocidal activity.In addition to dodecyl dimethyl ammonium chloride, the active ingredients of biocide A include a propanol derivative, showing a broad spectrum of antifungal and antibacterial activities, and fungistatic propiconazole (Tang et al., 2013;Picheansathian, 2004).Alcohols cause membrane damage and rapid denaturation of proteins, with subsequent interference in metabolism and cell ly-sis (McDonell & Russell, 1999).They are also known to inhibit sporulation and spore germination, but this effect is reversible (Trujillo & Laible, 1970).Lower concentrations of alcohols are used as preservatives and to potentiate the activity of other biocides (Maillard, 2002).Propiconazole's mode of action is demethylation of C-14 during ergosterol biosynthesis, leading to accumu-  7 Antimicrobial activity of quaternary ammonium salts lation of C-14 methyl sterols and abnormalities in the formation of the cell wall of fungi (Kalawate & Pandey, 2012).In contrast, biocides B and C additionally contain EDTA, a chelating agent that disorganizes phospholipid composition of microorganism membranes, thereby altering the permeability for certain substances and permitting the entry into the cell of chemically unrelated molecules (Al-Bakri et al., 2009).
According to the Regulation of the European Parliament and of the European Council (EU) No 528/2012, concerning the making available on the market and use of biocidal products, labels of biocidal products must contain specified information on every active substance and its concentration.This obligation does not apply to excipients which are present in the formula of biocides and can modulate their antimicrobial activity.Differences in the antimicrobial activity of the tested biocides may thus result from a different content of QACs, the presence of other active ingredients, as well as the presence of additional excipients in the formulations.
Choosing the right concentration of biocide is crucial to ensure proper protection of historical materials against microbial growth, because random multiple detrimental effects have been shown with highly reactive biocides used at high concentrations.When lower concentrations of biocides are used (i.e.inhibitory or sublethal concentrations), the emergence of resistant microorganisms has been reported (Maillard, 2002).
In summary, effective disinfection of historical wood samples against the tested strains of microorganisms can be achieved by three applications of biocide A at a concentration of 30% v/v.On the other hand, historical brick samples can be disinfected by three applications of biocide B or C in a concentration of 6% v/v.Furthermore, the use of biocide A (30% v/v) on the wood surface, and biocide B (6% v/v) on the brick surface, effectively protects these materials against the development of the tested microorganisms for a period of seven days.

Table 7 .
Antimicrobial and protective activity of the investigated biocides based on the example of biocide C used on the historical brick samples against Bacillus muralis and Cladosporium cladosporioides II

Table 3 . Antimicrobial activity of biocides on wood samples
Symbols with respect to biocidal activity: +++ high, ++ good, + low, +/-very low, -no activity Antimicrobial activity of quaternary ammonium salts

Table 6 . The efficacy of brick protection against microbial growth
Symbols with respect to biocidal activity: +++ high, ++ good, + low, +/-very low, -no activity