Long-term antibacterial efficacy of disinfectants based on benzalkonium chloride and sodium hypochlorite tested on surfaces against resistant gram-positive bacteria
Introduction
Foodborne diseases are a widespread public health problem that affect millions of people worldwide (Buzby & Roberts, 2009; EFSA, 2017; WHO, 2015, pp. 1–268). There are several factors that contribute to this kind of diseases, but one of the major factors is bacterial cross-contamination from surfaces to food (Kusumaningrum, Riboldi, Hazeleger, & Beumer, 2003). The food industry is greatly concerned with providing hygienic conditions to ensure the provision of safe, quality food with an acceptable shelf-life. Disinfection is the final step in hygienic procedures to prevent the growth of microorganisms on surfaces. However, if disinfection is insufficient, environmental free-living (planktonic) bacterial cells are able to adhere to surfaces and can form biofilms, which protect microorganisms from adverse conditions and result in the transmission of foodborne disease (DeVere & Purchase, 2007; Reij, Den Aantrekker, & ILSI Europe Risk Analysis in Microbiology Task Force, 2004; Satpathy, Sen, Pattanaik, & Raut, 2016).
Gram-positive bacteria belonging to the genera Staphylococci and Enterococci have a great ability to survive long periods in adverse conditions and have been involved in an increase in the presence of bacterial resistance to different antimicrobial products (Chaibenjawong & Foster, 2011; Kim, Ryu, Park, & Ryu, 2017; Neely & Maley, 2000; Rice, 2006; Ríos-Castillo, González-Rivas, & Rodríguez-Jerez, 2017). The lack of rigor in the rotation of disinfectants during their use and their wrong concentration in the formulations has been noted among the causes of increasing bacterial resistance (Langsrud, Sidhu, Heir, & Holck, 2003a; Meyer, 2006).
To demonstrate the effectiveness of commercial disinfectants under practical conditions, disinfectant products are tested according to standard methods using indicator organisms of food contamination. The term ‘indicator organisms’ has been used for nearly a century to assess the microbiological status of food production and food control systems. It includes evaluating the quality or safety of raw or processed food products and validating the effectiveness of microbial control measures (Halkman & Halkman, 2014; Tortorello, 2003).
Staphylococcus aureus is a common indicator of food contamination because this bacterium has been isolated in a variety of environments as an important source of cross-contamination (Le Loir, Baron, & Gautier, 2003; Rode, Langsrud, Holck, & Møretrø, 2007). This bacterium has been implicated in numerous outbreaks and cases of food poisoning worldwide (Grace & Fetsch, 2018). Likewise, it has a high ability to survive under adverse conditions for long periods, at least for 96 h on dry surfaces (Kusumaningrum et al., 2003), for more than 90 days in polyethylene (Neely & Maley, 2000), and 318 days in screw-top bottles (Wagenvoort, Sluijsmans, & Penders, 2000). Enterococcus spp can persist in food-processing facilities for a long time, so this genus could be monitored mainly as an indicator of process hygiene or food quality. Enterococcus counts are more reliable than coliform counts as an index of sanitary quality because the former is more adapt at surviving in adverse conditions (Byappanahalli, Nevers, Korajkic, Staley, & Harwood, 2012; Devriese, Pot, Van Damme, Kersters, & Haesebrouck, 1995; Halkman & Halkman, 2014; Jay, Loessner, & Golden, 2005). The characteristic to survive in adverse conditions, the potent and unique ability to exchange genetic material confer to Enterococcus an intrinsic and acquired resistance to practically all the antibiotics currently in use (Giraffa, 2014; Hollenbeck & Rice, 2012). Enterococcus is capable of colonizing both raw and heat-treated food. Therefore, their presence in food and other environmental sources may play a significant role in the dissemination of antimicrobial resistance and other virulence traits (Giraffa, 2014).
Products based on benzalkonium chloride and sodium hypochlorite are disinfectants commonly used in the food industry (Fazlara & Ekhtelat, 2012; Fukuzaki, 2006; Ríos-Castillo et al., 2017). Benzalkonium chloride is a quaternary ammonium compound (QACs) widely used in the food processing industry because it is more stable in the presence of organic matter and temperature fluctuations than other disinfectant products (Bessems, 1998; Gilbert & Moore, 2005; McDonnell & Russell, 1999; Taylor, Rogers, & Holah, 1999). Likewise, benzalkonium chloride is effective in preventing the bacterial adhesion and the process of biofilm formation at conventional in-use concentrations (Houari & Di Martino, 2007; Oosterhof, Buijssen, Busscher, van der Laan, & van der Mei, 2006). Sodium hypochlorite (NaClO) is the most widely used disinfectant in the food industry and has a broad spectrum against Gram-positive and Gram-negative bacteria, viruses and spores (Fukuzaki, 2006). Besides, this product has the property to remove adhered organisms from surfaces (McDonnell & Russell, 1999).
The European standard EN 13697:2015 is a quantitative test of contamination on non-porous surfaces for assessing the microbiological efficacy of chemical disinfectants using indicator organisms in the food industry. According to this standard, a reduction of surface attached bacteria of ≥4 log10 is required to rate a disinfectant as effective (Anonymous, 2015; Vestby et al., 2010). However, despite tests to rate the effectiveness of disinfectants in reducing microbial contamination, their lasting effects are relatively brief. Between 20 min and 3 h after disinfection, surfaces were shown to revert to a state of contamination in places with conditions favorable for the growth of microorganisms (Scott, Bloomfield, & Barlow, 1984).
Controlling microbial colonization on surfaces represents one of the challenges in food environments where microbial communities are problematic. Adapting different strategies, such as (1) surface impregnation with antimicrobial agents (Myszka & Czaczyk, 2011) and (2) developing disinfectants with long-term properties are measures to prevent bacterial adhesion and growth on surfaces. Today, many disinfectant products are available that assure effective protection against a posterior possible bacterial adhesion on surfaces.
Nevertheless, there is no method to evaluate the long-term antibacterial efficacy of disinfectant products to demonstrate whether they retain their effectiveness over time. The objective of this study was to adapt the European standard EN 13697:2015 to evaluate the long-term antibacterial efficacy of disinfectant products (benzalkonium chloride and sodium hypochlorite), comparing the results of their initial activity against Staphylococcus aureus and Enterococcus hirae obtained using the regular standard method on surfaces.
Section snippets
Test material
Stainless steel (as non-porous surfaces) type 1.4301 (also known as AISI 304) 2.0 cm in diameter and 1.2 mm thick were used. Prior to performing the study, the surfaces were cleaned by immersion in an aqueous detergent (Dipol® D.I.S. DINO S.L., Madrid, Spain) for 1 h, rinsed, and introduced into a solution of 70% isopropyl alcohol (2-propanol) (Panreac Química S.L.U., Castellar del Vallès, Spain) for 15 min. Then, the surfaces were sterilized using an autoclave at 121 °C for 15 min (Anonymous,
Benzalkonium chloride based products
The products based on benzalkonium chloride in the different dilutions of the study showed an initial activity at 0 h (≥4 log10 reduction) and long-term antibacterial efficacy at 24 h (≥3 log10 reduction) for both bacteria tested, S. aureus and E. hirae (Table 2). The maximum initial activities at 0 h on S. aureus were observed with products B3 (6.93 log10 and 6.96 log10 at 1:1 or 1:2 dilutions, respectively) and B2 (6.81 log10 at 1:4 dilution). For E. hirae, the maximum initial activity at 1:1
Conclusions
The rotation of disinfectants and the use of products with long-term activity should be considered in practical conditions to prevent bacterial adherence on surfaces. Benzalkonium chloride in combination with sodium hydroxide or amine oxide in appropriate concentrations demonstrated long-term antibacterial efficacy against S. aureus and E. hirae. Sodium hypochlorite as a sole compound did not demonstrate long-term activity, but this activity was achieved when it was formulated with other
Acknowledgments
The authors thank Dolors Busquets Soler for her technical assistance and to Mrs. Sarah Davis for English grammar revision.
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