Control of Salmonella in food related environments by chemical disinfection

Food Research International 45 (2012) 532–544

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Food Research International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s

Control of Salmonella in food related environments by chemical disinfection Trond Møretrø a,⁎, Even Heir a, Live L. Nesse b, Lene K. Vestby b, Solveig Langsrud a a b

Nofima Mat AS, Osloveien 1, N-1430 Aas, Norway National Veterinary Institute, Pb750 Sentrum, N-0106 Oslo, Norway

a r t i c l e

i n f o

Article history: Received 30 November 2010 Accepted 8 February 2011 Keywords: Salmonella Disinfection Sanitation Resistance Cross-contamination Biofilm

a b s t r a c t Salmonella may be transferred to food through cross-contamination during processing and preparation. To minimise the risk of cross-contamination, proper cleaning and disinfection is essential for the food industry. Recently, disinfection of areas for preparation and storage of food has also gained increased popularity in households. There is a range of disinfectants available with different properties and usage areas, and care must be taken to choose the proper disinfectant for the specific application. There are many methods for testing the antimicrobial effect of disinfectants. To evaluate whether a disinfectant will be effective in practical settings, the test method should model real-life situations. Most disinfectants are effective against Salmonella at recommended user concentration in suspension tests. However, a number of factors may reduce the biocidal effect of disinfectants under practical conditions. This include properties of the surface to be disinfected, presence of soiling on the surface, the physiological state of the bacteria exposed to disinfection, including bacteria embedded in biofilms, and the effects of other stresses (e.g. desiccation, starvation and temperature). Here we review the effects of disinfectants used in food related areas in industries and in households against Salmonella. A general overview is given for disinfectants in use and methods used to evaluate effects. Effects of disinfectants against Salmonella in suspension and on surfaces, including biofilms, are presented and compared. Novel control strategies such as use of electrolysed water, antimicrobial surfaces, and anti-biofilm compounds are also covered. Finally, we review the ability of Salmonella to gain reduced susceptibility to disinfectants through adaptation and other physiological responses like biofilm formation. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction In the food industry, hygienic measures normally keep the Salmonella prevalence at a low level, but breach in routines may lead to Salmonella contamination on equipment or other surfaces (Evans, Tromans, Dexter, Ribeiro, & Gardner, 1996; Hennessy et al., 1996). Cross-contamination during food processing is an important source of microbes in food (Reij & Aantrekker, 2004; Todd, Greig, Bartleson, & Michaels, 2009). Cross-contamination has been found to be the cause of a number of foodborne outbreaks with Salmonella. For several of the outbreaks the cause is believed to be lack of or insufficient cleaning and disinfection of equipment and surfaces (Ellis et al., 1998; Evans et al., 1996; Giraudon et al., 2009; Hennessy et al., 1996; Llewellyn, Evans, & Palmer, 1998; Podolak, Enache, Stone, Black, & Elliott, 2010; Reij & Aantrekker, 2004; Rowe et al., 1987). An example was an outbreak of salmonellosis in USA associated with the consumption of ice cream, where an ice cream premix was transported in insufficiently cleaned tanker trailers that had previ⁎ Corresponding author. Tel.: +47 64970100; fax: +47 64970333. E-mail addresses: trond.moretro@nofima.no (T. Møretrø), even.heir@nofima.no (E. Heir), [email protected] (L.L. Nesse), [email protected] (L.K. Vestby), solveig.langsrud@nofima.no (S. Langsrud). 0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2011.02.002

ously hauled nonpasteurised liquid eggs containing S. Enteritidis (Hennessy et al., 1996). In an outbreak of S. Ealing associated with infant dry milk, the source was traced to the factory spray-drier, which had a hole in its inner lining, allowing escape of powder and its return from contaminated insulation (Rowe et al., 1987). Food contact surfaces in households may also be sites of crosscontamination with Salmonella. In kitchens where chicken had been prepared, the prevalence of Salmonella was 60% for cutting boards and 10% for door handles, cupboards, ovens, sink-rims and refrigerators (Cogan, Bloomfield, & Humphrey, 1999). In a survey of homes with Salmonella illness, 12% of dishcloths contained the same serotype as found in faeces of ill persons (Scott, Gaber, & Cusack, 2001). In another study of homes with Salmonella illness, genetically similar strains of S. Typhimurium and S. Arizonae were found on kitchen surfaces and in patients (Schutze, Sikes, Stefanova, & Cave, 1999). It has been shown that although cleaning may reduce the number of Salmonella (0–5 log10), the surface may still retain a small number of Salmonella, indicating that disinfection may be necessary (Barker, Naeeni, & Bloomfield, 2003; Cogan, Slader, Bloomfield, & Humphrey, 2002; Kusumaningrum et al., 2003). It has been estimated that 80 000 cases of foodborne illness could be prevented in USA by targeted disinfection in kitchens (Duff et al., 2003), and use of biocides in risk areas in the domestic environment has been recommended (Bloomfield et al.,

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2010). However, the use of disinfectants in the domestic sector is debated, especially due to fear of resistance development and environmental consequences (Gilbert & McBain, 2001, 2003; Levy, 2001). To obtain sufficient bacterial reduction through disinfection, knowledge and caution are necessary. In general, surfaces should be cleaned properly before disinfection, and the combinatory choice of cleaning and disinfectant agents may be important for the sanitary effect. Numerous types of disinfectants exist, e.g. oxidative disinfectants, tenside based disinfectants and alcohols, with different properties and areas of use. The effect of disinfectants may be influenced by environmental conditions like temperature, soiling and type of surface or medium to be disinfected. In addition, disinfectant effects may vary between target microorganisms. Finally, there are various disinfection strategies used in practise such as surface application, fogging, cleaning-in-place (CIP) or use of antibacterial wipes. Thus the disinfectant and the disinfection method should be carefully chosen based on the specific application to obtain the required antimicrobial effect. Disinfection is important for breaking the infection chain of Salmonella by reducing the risk of cross-contamination. In this review we will focus on chemical disinfection of Salmonella on surfaces in the food industry and in kitchens.

2. Disinfectants in general A disinfectant can be defined as a product that reduces the number of viable microorganisms (but not spores) on a surface, to a level specified as appropriate for further use (adopted from McDonnell (2007)). An ideal surface disinfectant for food preparation or production environments should be safe to use (toxicity, allergenic and inflammability), have no negative impact on surface materials (corrosiveness, staining and reactivity), be stable during storage and over a wide range of pH and temperatures, be robust to environmental factors (soil, hard water and dilution) and have a broad spectrum of activity. Also, the disinfectant should be environmentally friendly and cost efficient. There is no single disinfectant able to fulfil all these criteria and the factors considered most important will also be dependent on the type of application. For example, for some applications wettability is considered positive but this property may be contradictive to rinsability: In equipment/tanks used in fermentation processes, surface active compounds, which are relatively stable and have good wettability, are avoided as residual disinfectant can inhibit the starter culture. Different disinfectants are often used in open and closed processes. In closed CIP operations, it is possible to use disinfectants that are more aggressive or are at higher temperatures, as the probability of direct contact with personnel is less likely. Also, foaming is often not wanted in CIP operations in contrary to open disinfection. Stability is important to be able to store the disinfectants for a long time and to ensure sufficient exposure time. On the other hand, the components should be easily degradable after use. Ozone is regarded as an environmentally friendly disinfectant as it rapidly disintegrates into water and oxygen. On the other hand, it

has to be made in place because of its instability and may react and disintegrate before reaching the target organism. A range of different disinfectants are used and the main groups and some of their properties are given in Table 1. Chlorine-releasing compounds such as hypochlorite are widely used in the food industry and in the domestic environment. Chlorine has a broad spectrum of activity, acts fast and is usually cheap. It is in general more efficient at lower pH-values, but because of the formation of toxic gaseous chlorine and increased corrosiveness at lower pH it is often used at alkaline pH. Disinfectants containing hydrogen peroxide or peracetic acid are regarded as environmentally friendly because they decompose into oxygen and water (or acetic acid). They have a broad spectrum of activity and act fast. Peracetic acid is relatively stable in presence of organic compounds compared to other disinfectant types. Some main limitations of hydrogen peroxide are its ability to decompose at high temperatures and the adverse effect of organic material on activity. Tenside-based disinfectants such as quaternary ammonium compounds (QACs) are widely used in the food industry. They are active against a range of vegetative bacteria and can be used over a wide temperature range. They are usually not used in CIP because of foaming and their activity is reduced in the presence of hard water. Also, the degradability in the environment is slow and residues may contribute to resistance development in bacteria. Disinfectants based on alcohols are effective against a range of microorganisms and relatively robust in the presence of organic material. However, their use is limited due to safety reasons (health and flammability) and relatively high price. Alcohols are therefore mainly used for hand disinfection and on equipment that do not stand water. For further reading about disinfectants the reader is referred to Block (2001a), Grinstead (2009), Lelieveld, Mostert, and Holah (2005), Marriott (1995) and McDonnell (2007). 3. Methods to study effects of disinfectants A number of methods for measuring the antibacterial effect of disinfectants are used, both for research and regulatory purposes. The wide range of areas of applications makes it impossible to establish one standard test that can cover all aspects of disinfectant effects or measure antibacterial effect in practical use. Therefore, the methodology used ranges from basic simple laboratory tests to tests simulating in-use conditions. Important test parameters of disinfectant efficacy tests are concentration and volume of disinfectant, temperature, exposure time, organic load, pH, water hardness, neutralisation of disinfectant after exposure and bacterial concentration, phenotype, mode of bacterial growth (planktonic, attached to a surface and enclosed in biofilm), recovery and detection (Maillard, 2005). Because of the huge variability in test conditions it is often difficult to compare results from different studies. Currently, there are no international recognised standard protocols for testing disinfectants, and some countries have established their own national standards. In Europe, the European Committee for Standardisation (CEN) has developed several standard test methods for documentation of biocidal effect of disinfectants sold in the European Community

Table 1 Properties of active compounds in disinfectants used in food industry and domestic environments. Biocide class

Active agent

Oxidisers

Chlorine-releasing compounds − (hypochlorous acid, chlorine dioxide) Hydrogen peroxide, peracetic acid, ozone −

Surfactants Quaternary ammonium compounds, amphoteric tensides, acid anionic Alcohols Ethanol

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Foam Corrosive Activity reduced by hard water

Activity reduced Mechanism of action by soil

++

−

++

++

+

++

+

−

++

+

Oxidation of thiolgroups in enzymes and proteins, inhibition of DNA synthesis Formation of free radicals reacting with thiolgroups of enzymes and proteins, DNA strand breakage Membrane damage, leaking of cellular constituents

−

−

−

+

Membrane damage, denaturation of proteins

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member countries. The protocols follow two phases: phase one is a screening for antibacterial activity, phase two is an advanced suspension test including more realistic conditions (soil, temperatures, etc.) and a bactericidal surface test. In this review, methodology will be discussed shortly. For further reading about test methods the reader is referred to Cremieux, Freney, and Davin-Regli (2001), Maillard (2005) and McDonnell (2007). 3.1. Suspension tests In suspension tests the bacteria are suspended in the disinfectant for a given exposure time, the disinfectant is neutralised or removed and the number of survivors is measured, usually by plating on nutrient agar. Bactericidal suspension tests are for example used to verify bactericidal effect in development, verification and registration of new biocidal agents/formulations, testing the effect of environmental factors on biocidal activity or comparing susceptibility of different bacterial strains. The bacterial suspension is usually made in a nutrient broth or nutrient agar over-night and diluted to an appropriate concentration. The disinfectant solution is diluted in water, hard water, water added soil (proteins, fat, dependent on intended application) or nutrient broth. The disinfectant is neutralised or removed by filtration after a fixed exposure time (or several exposure times for time-series). The number of survivors is usually determined by plate counts, but methods based on conductance (Dhaliwal, Cordier, & Cox, 1992; Johnston & Jones, 1995), microscopy (Yu, Pyle, & McFeters, 1993), flow cytometry (Langsrud & Sundheim, 1996) and most-probable number (Oblinger & Koburger, 1975) have also been used. The bactericidal effect is calculated by the difference in viable bacteria before and after treatment, or the difference between cells treated the same way without exposure to disinfectant. Examples of standardised protocols for suspension test are European norm (EN) 1040 (CEN, 1997a), EN1276 (CEN, 1997b) and Association of Official Analytical Chemists (AOAC) Official Method 955.11 (AOAC, 1995a) and American Society for Testing and Materials (ASTM) E1891-97 (ASTM, 2002a). Despite that disinfection has been defined as antimicrobial reduction of the number of viable bacteria (McDonnell, 2007), the effect of disinfectants is often tested by measuring the minimum inhibitory concentration (MIC) for growth. In MIC-methods Salmonella is exposed to the disinfectant in nutrient suspension or nutrient agar, and growth is determined after incubation for a specific time (Andrews, 2001). The methods are mostly used in research, for example for screening of strain collections for resistance to biocides. The main advantages of these methods are that they are easy to perform and many strains or disinfectants can be tested in the same experiment. There are however several limitations of these methods. The test conditions are usually far from conditions in practical use, as they are restricted to conditions supporting growth of Salmonella. Thus, the disinfectants are tested in a bacterial growth medium (agar or broth), at a temperature and pH allowing growth and using an exposure time long enough to enable detection (usually measured turbidimetrically or visually). Another limitation is that disinfectants may be neutralised or react with components of the growth medium and that the concentration of active component during exposure is unknown. Since the conditions are restricted to conditions where Salmonella can grow, MIC-tests are not suitable for determination of the influence of environmental conditions on disinfection effect. 3.2. Surface tests In surface tests the bacteria on a surface are exposed to the disinfectant either by immersing the material in disinfectant solution or applying disinfectant on the surface. The bacteria are exposed to the disinfectant for a given time, the disinfectant is neutralised and/or

removed and the number of survivors is measured. Bactericidal surface tests are used to verify effect of surface disinfectants in conditions closer to practical use than suspension tests, testing hygienic quality of different surface materials, determination of tolerance of bacteria in biofilm to disinfectants, etc. The bacteria to be tested are either grown in a nutrient broth or agar followed by drying suspended bacteria on a surface or cultivating the bacteria on the surface as a biofilm. Salmonella is relatively resistant to desiccation (Kramer, Schwebke, & Kampf, 2006), but a limitation of the methods based on drying is that a fraction of the Salmonella may die during the drying step. There are no internationally recognised standard for testing the effect of disinfectants on bacteria in biofilm. A comparison between different methods for producing biofilms for disinfectant efficacy tests is given by Buckingham-Meyer, Goeres, and Hamilton (2007). It should however be noted that Salmonella does not produce biofilm at all conditions, e.g. little if any biofilm will be produced on surfaces submerged in liquid, and the method should be optimised to produce a biofilm with enough cells to be able to detect several log reduction (Møretrø et al., 2009). The disinfectant can be applied in fixed volumes on the surface or the surface (for example a coupon with biofilm) can be immersed in disinfectant solution (static) or exposed to the disinfectant in a fluid flow system (dynamic). At the end of the exposure time, bacterial survival is assessed as in the suspension test after suspending attached bacteria by e.g. swabbing (Joseph, Otta, Karunasagar, & Karunasagar, 2001), sonication (Møretrø et al., 2009) or whirl-mixing with glass beads (Giaouris & Nychas, 2006). Alternatively, the number of survivors can be determined by contact plates or microscopy (Bore & Langsrud, 2005; Wood et al., 1998). A limitation of the latter methods is that it is difficult to detect more than 1–2 log10 reduction. Examples of standard surface tests are EN 13687 (CEN, 2002), ASTM 2111 (ASTM, 2002b) and AOAC Official method 991.47 (AOAC, 1995b). 4. Factors influencing the antibacterial effects of disinfectants in practical use In practical use, the antibacterial efficacy of chemical disinfectants is affected by a number of factors related to environmental conditions and by the sensitivity of the target organisms. Lack of the expected efficacy of a disinfection process can often be explained by not using the disinfectants as intended. Four main factors influencing the effect of disinfectants are user-concentration, temperature, exposure time and the presence of organic material. Manufacturers of disinfectants usually provide in-use recommendations with regard to how these factors affect the efficacy of the specific disinfectant. In general, the efficacy of disinfectants increases with temperature (Weavers & Wickramanayake, 2001b). When disinfecting cold rooms using higher concentrations or longer contact time may compensate for lower temperature. The effect of disinfectants is concentration dependent. If the user-concentration given by the manufacturer is based on simple laboratory tests measuring efficacy in suspension and without additives e.g. soil, this may not be enough to kill Salmonella attached to a surface (Møretrø et al., 2009). Also, in a practical setting, the disinfectant may be diluted during use due to residual water after the cleaning process. To avoid dilution effects, it is important to have hygienic design on equipment and facilities allowing water to run off surfaces instead of accumulating. Furthermore, surfaces should be allowed to dry reasonable up before disinfection. Failure in cleaning prior to disinfection will have a high impact on the disinfection process as the effect of most disinfectants is reduced in presence of organic material. This may be a particular challenge in production of dry foods or feed, where there often is limited cleaning before disinfection, due to the fear of microbial growth if humidity is introduced to the production environment. Organic material may react with oxidative disinfectants and may also neutralise tensidebased disinfectants. The reduction of S. Enteritidis by two QACs was

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2–3 log10 lower in presence of 5% faeces compared to water (Berchieri & Barrow, 1996). S. Typhimurium had 2–3 log10 lower reduction after exposure to peracetic acid (7 mg/l) for 7 min in sewage effluent water compared to distilled water (Jolivet-Gougeon, Sauvager, ArturoSchaan, Bonnaure-Mallet, & Cormier, 2003). Also, some disinfectants, such as QACs may be neutralised by anionic surfactants in cleaning compounds. This demonstrates the importance of proper cleaning and rinsing before disinfection. The antibacterial effect of disinfectants may be dependent on the type, number and physiological state of the target microorganisms. In most studies the number of Salmonella strains tested is too limited to be able to compare the tolerance between strains. However, Salmonella isolates from feed industry of the serovars Agona, Kentucky, Montevideo and Senftenberg has been shown to have higher tolerance to disinfectants than the reference collection strain S. Typhimurium ATCC 13311 (Møretrø, Midtgaard, Nesse, & Langsrud, 2003), which is recommended as a test strain in the European suspension test (CEN, 1997b). In the study the majority of the feed isolates showed a 3–5 log10 reduction for five disinfectants tested (hypochlorite, tensides and formaldehyde/organic acids), while for S. Typhimurium N5 log10 reduction was obtained for all disinfectants (Møretrø et al., 2003)(Møretrø et al., unpublished). The total number of target cells may influence the disinfection results. A high initial number of bacteria may result in surviving cells. In cases where the concentration of the antimicrobial compound of the disinfectant is limited, the bactericidal effect of the disinfectant may be reduced in the presence of high numbers of bacteria. In general, disinfectants are less effective against bacteria attached to surfaces, especially biofilm-associated bacteria, compared to freeliving bacteria (Section 5.2, this review). Also the growth phase of bacteria will influence the susceptibility of bacteria towards disinfectants. It is known that bacteria in exponential phase of growth are more sensitive to disinfectants than stationary phase bacteria (Gilbert, Brown, & Costerton, 1987). 5. Laboratory studies of disinfection effect 5.1. Effect of disinfectants on Salmonella in suspension Salmonella as a Gram-negative bacterium has in general higher tolerance to disinfectants compared to Gram-positive bacteria (Merianos, 2001; Russell, 2001). This can be shown as higher tolerance in MIC tests, and when disinfectants are tested at low concentrations in bactericidal suspension tests. However, when disinfectants are tested at their recommended user concentration in suspension tests, the differences in effect against bacteria are often not apparent, as the majority of disinfectants have a high bactericidal effect under these conditions. Despite the general opinion that Gramnegatives, including Salmonella, have higher intrinsic resistance than Gram-positives, there are some contradictory reports. Salmonella has been shown to be less tolerant to hypochlorite (Kusumaningrum et al., 2003) and vinegar (low pH) (Rutala, Barbee, Aguiar, Sobsey, & Weber, 2000) than Staphylococcus aureus, and less tolerant to hydrogen peroxide than Listeria monocytogenes (Yang, Kendall, Medeiros, & Sofos, 2009). 5.1.1. Oxidative disinfectants Reduction of Salmonella to below detection levels (N5 log10 reduction) in suspension tests at user-concentrations has been shown for Virkon S (10 g/l, 5 min), and other oxidative disinfectants (Møretrø et al., 2009). At low concentrations, Dhir and Dodd (1995) found log10 reductions of about 0.5, 1 and 1.8 after 15 min exposure to 0.1, 0.2 of 0.3 g/l Virkon S, respectively. Exposure to 100 g/l hydrogen peroxide or 0.3 g/l peracetic acid, resulted in N6.4 log10 reduction of S. Typhimurium (Block, 2001b). For S. Typhimurium in sewage water effluent it was shown that treatment

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with 0.015 g/l peracetic acid for 1 h resulted in no colony forming units. However, viable but not culturable cells with retained virulence ability were present (Jolivet-Gougeon et al., 2003). Yang et al. (2009) reported complete reduction (N5 log10) of S. Typhimurium after 1 min exposure to 15 g/l hydrogen peroxide. Møretrø et al. (2009) found N5 log10 reduction of four Salmonella strains after exposure to 0.57 g/l sodium hypochlorite for 5 min. For S. Enteritidis, exposure for 10 min resulted in about 2.5, 3 and 4 log10 reduction, at 0.2, 0.3, 0.4 g/l sodium hypochlorite, respectively (Kusumaningrum et al., 2003). Wong, Townsend, Fenwick, Trengove, and O'Handley (2010) reported survival of S. Typhimurium after 1 min exposure to 1.3 g/l sodium hypochlorite, but no surviving bacteria were observed after 5 min exposure. Yang et al. (2009) reported no surviving bacteria of S. Typhimurium after 1 min exposure to 0.3 g/l sodium hypochlorite. In conclusion, a sodium hypochlorite concentration above 0.5 g/l appears to be sufficient to obtain 5 log10 kill of Salmonella in suspension after 5 min exposure in suspension. Chlorine dioxide (ClO2) of concentrations 5, 10 and 20 mg/l, against a mixture of S. Enteritidis, S. Newport and S. Typhimurium in water, resulted in complete reduction (N5 log10) in 6, 4 and 2 s, respectively (Pao, Kelsey, Khalid, & Ettinger, 2007). Exposure of S. Typhimurium to 0.23–0.26 mg/l ozone at 24 °C, resulted in 99.995% reduction after 1.67 min (Weavers & Wickramanayake, 2001a). In another study N5 log10 reduction of S. Typhimurium was observed after 1 min exposure to 0.19 mg/l ozone at room temperature (Restaino, Frampton, Hemphill, & Palnikar, 1995). In conclusion, the oxidative disinfectants that have been tested are effective at user-concentrations against Salmonella in suspension tests. 5.1.2. Surfactants Møretrø et al. (2009) reported N5 log10 reduction of four Salmonella strains after exposure to user-concentrations of two cationic tenside based disinfectants after 5 min in presence of 3 g/l BSA. In another study, where 22 strains of Salmonella were tested for 5 min in presence of 3 g/l BSA, 19 and 16 of the strains were reduced by 3–5 log10 by two disinfectants with cationic and anionic tensides, respectively, tested at 80% of their recommended user concentration (Møretrø et al., 2003). Rutala et al. (2000) reported complete reduction (N5.7 log10) of S. choleraesuis after 0.5 min to a household disinfectant containing ionic and non-ionic surfactants. When tested at low concentrations, differences in effect between different QACs have been observed. In suspension tests with S. Typhi (10 min, 20 °C), QACs with carbon length in the range 12–18 showed best antibacterial effect, with C14 as the most efficient (Merianos, 2001). Many different types of disinfectants based on QACs exist, and most results in complete reduction (N5 log10) of Salmonella after 0.5–1 min exposure in suspension tests (Berchieri & Barrow, 1996; Rutala et al., 2000). Møretrø et al. (2003) tested three commercial disinfectants with QACs against 22 strains of Salmonella, mostly from the feed industry. When tested at 80% of recommended user concentration for 5 min in presence of 3 g/l BSA, one of the QAC resulted in complete reduction (N5 log10) of all strains, while for two of the QACs some strains had 3–5 log10 or lower reduction (Møretrø et al., 2003). In conclusion, the surfactants reported are effective against Salmonella in suspension tests at their recommended user-concentrations, however some disinfectants may be less robust to further dilution. 5.1.3. Alcohol based disinfectants Ethanol in high concentrations is effective against vegetative bacteria. 70% (700 g/l) ethanol results in complete reduction (N5 log10) of Salmonella in suspension tests (Møretrø et al., 2009; Rutala et al., 2000; Wong, Townsend, Fenwick, Trengove, & O'Handley, 2010). It has also been shown that at acidic conditions, ethanol can be

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bactericidal at concentrations as low as 10–15%. A combination of 150 g/l (15%) ethanol, 1.5 g/l malic and 6 g/l tartaric acid at pH 3, resulted in N6log10 reduction of S. Typhimurium in 3 min (Møretrø & Daeschel, 2004). Similar reductions were observed for wine (Møretrø & Daeschel, 2004), and use of wine as a disinfectant has been suggested (Just & Daeschel, 2003). In conclusion ethanol is effective against Salmonella at concentrations normally used for disinfection (70%), and may under certain conditions also be effective at lower concentrations. 5.2. Effect of disinfectants against sessile Salmonella cells Salmonella may be present on food contact surfaces in the food industry and in households such as on cutting boards, equipment, conveyor belts, drains, floors and walls (Schutze et al., 1999; Scott et al., 2001). On open surfaces that dry up easily like e.g. counter tops and cutting boards, Salmonella will be present in a dry state on the surface. At humid conditions and in presence of nutrients, e.g. in drains, Salmonella may grow and form a biofilm. In general, bacteria attached to surfaces are less sensitive to disinfection than their planktonic counterparts, especially when associated with a biofilm (Costerton, Lewandowski, Caldwell, Korber, & Lappin-Scott, 1995). 5.2.1. Disinfection of attached Salmonella cells For Salmonella dried on or attached to a surface, tolerance to disinfectants is higher than Salmonella in suspension. Møretrø et al. (2009) found that only one (containing 70% ethanol) out of nine disinfectants eliminated (N4 log10 reduction) Salmonella dried on stainless steel, while all nine disinfectants resulted in elimination of Salmonella (N5 log10 reduction) in suspension tests. In the surface test, a disinfectant containing 700 g/l (70%) ethanol was most effective with N4 log10 reduction of viable bacteria. Tensides, oxidative disinfectants and chlorine had limited effect on Salmonella (Møretrø et al., 2009). Ramesh, Joseph, Carr, Douglass, and Wheaton (2002) tested different disinfectants containing QACs, sodium chlorite and hypochlorite against a mixture of S. Typhimurium, S. Thompson, S. Berta, S. Hadar, and S. Johannesburg strains dried on surfaces with a mixture of organic material, and found that it was a tendency towards sodium chlorite containing disinfectants being more effective (4–5 log10 reduction) than the QACs (0.6–2.4 log10 reduction) and hypochlorite (1.1 log10 reduction). A disinfectant containing H2O2 and low levels of surfactants, completely reduced (N5 log10) S. chloleraesuis dried on glass in 30 s (Omidbakhsh & Sattar, 2006). Cleaning cloths are often used to wipe surfaces in the food industry and in kitchens. Antimicrobial cloths containing ethanol or other disinfectants are present on the market. Kusumaningrum et al. (2003) performed experiments where a cloth containing anionic surfactants and control cloths without antimicrobial were used for wiping surfaces artificially contaminated with S. Enteritidis. Directly after wiping with the antimicrobial cloth, the count on contact agar plates from the sampled surfaces was about 0.5 log10 lower than when using control cloths. After usage, the control cloths contained 4 log10–5 log10 cfu/100 cm2, while the counts of the cloth with anionic surfactants were below the detection limit (b2 log10 cfu/100 cm2). A cleaning cloth may also act as a surface where bacteria may attach and accumulate, making the cloth a potential vehicle of cross contamination (Scott et al., 2001). For disinfection of cloths, higher concentrations of hypochlorite were needed for decontamination of S. Enteritidis in cloths compared to in suspension tests (Kusumaningrum et al., 2003). Exposure of cloths to 0.5 g/l and 0.8 g/l sodium hypochlorite for 60 min, did reduce the number of Salmonella by 0.5 and 3 log10, respectively. It was found that repeated exposure to hypochlorite on following days increased the susceptibility of Salmonella to hypochlorite (Kusumaningrum et al., 2003). Boiling

may be an alternative to chemical disinfection of cloths. The use of disposable cloths or paper cloths is often recommended to avoid cross-contamination. The results from testing commercial disinfectants against sessile bacteria indicate that the in-use concentrations of disinfectants are often based on 5 log reduction in suspension tests and not efficacy in surface tests. The low number of studies and diverging results, partly due to differences in in-use-concentrations makes it difficult to give recommendations for disinfection of surfaces. Disinfectants based on 70% ethanol seem to be effective, but use of ethanol in large-scale open disinfection in the food industry may not always be possible due to health, safety and economic reasons. 5.2.2. Disinfection of Salmonella cells in biofilm Bacteria that are attached to a surface may develop biofilm at humid conditions if a minimum of nutrients are available. Biofilms are microbial sessile communities that are attached to a substance, to an interface and/or to each other. In the biofilm, the cells are embedded in a self produced matrix which may act as chemical and mechanical protection against environmental stressors including chemical disinfectants (Costerton et al., 1995; LeChevallier, Cawton, & Lee, 1988). In general, disinfectants are less effective against bacteria in biofilm (Costerton, 1999), and this has also been observed with Salmonella (Joseph et al., 2001; Scher, Römling, & Yaron, 2005). Salmonella is capable of forming biofilm at room temperature on different contact surfaces used in food production like glass, polymers and stainless steel (Hood & Zottola, 1997; Joseph et al., 2001; Møretrø et al., 2009; Solano et al., 1998; Vestby, Møretrø, Ballance, Langsrud, & Nesse, 2009; Vestby, Møretrø, Langsrud, Heir, & Nesse, 2009; Woodward, Sojka, Sprigings, & Humphrey, 2000) and can also form biofilm (pellicle) at liquid-air interfaces (Römling & Rohde, 1999; Scher et al., 2005; Solano et al., 2002; Vestby, Møretrø, Langsrud, et al., 2009; White & Surette, 2006). Bacteria can survive within the biofilm for several months in a dry and nutrient depleted environment (Vestby, Møretrø, Ballance, et al., 2009; White, Gibson, Kim, Kay, & Surette, 2006). Vestby, Møretrø, Langsrud, et al. (2009) reported a statistically significant correlation between the ability of Salmonella clones to form biofilm in laboratory tests at room temperature and long time persistence in feed factories, indicating that biofilm forming abilities also contribute to persistence of Salmonella in natural environments. A relatively small number of publications deal with the effects of disinfectants on Salmonella in biofilm. Møretrø et al. (2009) compared the effect of nine commercial disinfectants at recommended userconcentration against S. Agona and S. Senftenberg wildtype strains isolated from feed factories and grown as a biofilm for two days on stainless steel. The biofilms were exposed to disinfectants at recommended user-concentrations added 3 g/l BSA for 5 min before neutralisation. No surviving bacteria (N4 log10 reduction) was observed after exposure for the disinfectant with 70% ethanol, two peroxygen based agents, as well as Virkon S. Chlorine and a disinfectant containing both glutaraldehyde and ethanol gave a low reduction (0.5–1 log10) of Salmonella, while tenside based agents showed intermediate effects (1.5–4 log10) (Møretrø et al., 2009). In another study, 13 commercial disinfectants were evaluated for their capacities to eliminate Salmonella in biofilm on galvanised steel (Ramesh et al., 2002). Two of the disinfectants, one containing sodium hypochlorite (0.5 g/l) and the other a sodium chlorite and an alkaline peroxide compound, eliminated Salmonella serovars Typhimurium, Thompson, Berta, Hadar, and Johannesburg on the biofilm-covered surfaces. These compounds provided N7 log10 reductions within 2 min. The disinfectants containing QACs were less effective (1–3 log10 reduction) (Ramesh et al., 2002). Wong, Townsend, Fenwick, Trengove, and O'Handley (2010) tested six different compounds (sodium hypochlorite, citric acid, BC, a QAC based disinfectant, chlorhexidine gluconate and ethanol)

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against a S. Typhimurium strain in three day old biofilms on plastic pegs. At 1 min exposure, only sodium hypochlorite reduced the bacteria to undetectable levels (log10 reduction N7) at the recommended user concentration (1.31 g/l). Interestingly, higher doses of sodium hypochlorite (26.3 and 56.5 g/l) were not as effective. At 5 min exposure, citric acid (32 g/l) and sodium hypochlorite were effective at recommended user concentrations, and BC and the QAC based disinfectant were effective at higher concentrations (7.5 g/l and 23.5 g/l, respectively). Chlorhexidine gluconate (1–50 mg/l) and ethanol (70%) failed to eliminate the bacteria. Joseph et al. (2001) studied the sensitivity of wild type S. Weltevreden ten day old biofilm cells grown on plastic, cement and stainless steel to different levels of hypochlorite for varying exposure times. To obtain a complete reduction using hypochlorite, 0.1 g/l Cl2 had to be used for 20 min on plastic (log10 reduction N7) and cement (log10 reduction N6), or for 15 min on steel (log10 reduction N5). Vestby, Møretrø, Ballance, et al. (2009) studied the effect of 0.5 g/l sodium hypochlorite and 0.2 g/l BC on two day old biofilms of six wild type S. Agona isolates on glass. After 5 min exposure, mean log10 reductions were 3.2 for hypochlorite and 1.3 for BC. When studying S. Typhimurium biofilms on glass and rubber, Vieira et al. (2005) found that the bacteria in the biofilm survived for as long as 45 min submerged in 10 g/l sodium hypochlorite, whereas 700 g/l (70%) ethanol needed only 10 min to reduce the number of bacteria below detectable levels. However, the initial numbers of bacteria in the biofilms were not reported. Consequently, the magnitude of the log10 reductions in this experiment is unknown. Exposure of S. Typhimurium on agar plates to aerosolised peracetic acid and hydrogen peroxide for 1 h, resulted in 8 log10 reduction of the viable count compared to untreated control (Oh, Gray, Dougherty, & Kang, 2005). There is a wide variation between different studies in the conditions used for testing the effect of disinfectants on bacteria in biofilm. Consequently, a number of factors which may affect the results differ between the reported experiments. There has been a general consensus that bacteria in biofilm show increased survival after exposure to antimicrobials with increasing age of the biofilm. Most Salmonella studies use biofilms within an age range of two to seven days. Wong et al. (2010) concluded that the age of biofilm did not contribute to resistance towards disinfectants after comparing effects on 3-, 5- and 7-day-old biofilms of S. Typhimurium in a microtiter plate based assay. On the other hand, Ramesh et al. (2002) found that both a quaternary ammonium compound and an enzymatic compound were less effective against five different Salmonella serovars in four day old biofilms as compared to three day old biofilms on galvanised steel. This may have been due to an increase in both biofilm thickness and the number of cfu/cm2 in the biofilm which was observed over time. Korber, Choi, Wolfaardt, Ingham, and Caldwell (1997) observed similar responses when testing trisodium phosphate against 48- and 72-hour old biofilms of S. Enteritidis on glass. We conclude that it may be important to have the age of the biofilms in mind when evaluating the effect of disinfectants. Another important factor is the surface material the biofilm is attached to. In a study where two day old biofilms of S. Typhimurium were exposed to a disinfectant containing chlorine and an anionic acid-based disinfectant, there was considerably less reduction of biofilm on buna-n-rubber (1.5–2 log10) compared to on stainless steel (4–5 log10). The reason behind this was not clear, but the authors speculate that the porous nature of rubber may reduce the efficacy, or that the bacteriostatic properties of the rubber may have altered the physiological state of Salmonella, making them more tolerant to disinfectants (Ronner & Wong, 1993). Additional factors that vary between experiments and influence the results are the test strains/serovars, the number of bacteria in the biofilm, the concentration and volume of the disinfectant and the

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exposure time. Addition of organic material like BSA and neutralisation of disinfectant residues before enumeration of viable bacteria are often included in standardised protocols to avoid overestimation of efficacy. However, this is rarely reported used in biofilm disinfection experiments. All these variations make it difficult to compare the results from different experiments, and are probably also the reason for the seemingly contradicting results regarding the effects of some compounds on Salmonella in biofilm, e.g. hypochlorite, ethanol and BC. Consequently, standard protocols for testing the efficacy of disinfectants on bacteria in biofilm are clearly needed, but making standard tests for biofilms may be more challenging than suspension tests as different bacterial species and strains do not produce similar biofilms at the same conditions. Based on reported results, it is difficult to draw conclusions regarding the efficacy of the different compounds and provide recommendations as to which disinfectants to use. However, the results do show that effective disinfection of Salmonella in biofilm poses a challenge. Therefore, it is important to prevent biofilm build up on food related surfaces, both by regular cleaning and by using equipment without cracks and crevices and parts that cannot be kept properly clean. In addition, cleaning prior to disinfection is important to reduce any amounts of biofilm material as much as possible. 6. In-situ disinfection studies In the food processing industry, hygienic practises, including cleaning and disinfection should keep the prevalence of pathogenic bacteria at the lowest possible level. To our knowledge there are no published studies on the effect of disinfection on Salmonella in the food processing environments. The absence of such studies is probably due to generally low prevalence of Salmonella in most food processing environments. The specific effects of disinfection will therefore be challenging to measure requiring large sampling numbers and strict control of other factors affecting Salmonella prevalence. However, such studies exist in primary production and in slaughterhouses where the prevalence of Salmonella in many cases are as high as 10–30%, thus the effect of disinfection may be easier studied. In animal houses, other disinfectants are commonly used than in the food industry. In one study, commercial disinfectants containing formaldehyde was more effective that disinfectants containing a mixture of glutaraldehyde and quaternary ammonium compounds, which again was more effective than disinfectants containing hydrogen peroxide and/or peracetic acid (Mueller-Doblies, Carrique-Mas, Sayers, & Davies, 2010). Also in other studies formalin/ formaldehyde was the most effective disinfectant (Carrique-Mas, Marin, Breslin, McLaren, & Davies, 2009; Davies & Wray, 1995). However, formaldehyde is not used in the food industry due to its carcinogenic properties. Disinfection in slaughter houses reduces the risk of crosscontamination of Salmonella (Delhalle et al., 2008). Salmonella has been shown to persists in lairage pens for months, resulting in a risk of cross-contamination (Schmidt, O'Connor, McKean, & Hurd, 2004; Small et al., 2006). Cleaning with alkaline chloride followed by disinfection with a combination of hydrogen peroxide and peracetic acid reduced the prevalence of Salmonella in swabs from these environments, however the prevalence of Salmonella in pigs in the same environments were not significantly reduced (Schmidt et al., 2004). Barker et al. (2003) tested the effect of cleaning and chlorine disinfection of Salmonella in a study reflecting food contact surfaces in a kitchen environment. Although detergent-based cleaning had some effect, Salmonella remained on surfaces in low numbers. Cleaning followed by disinfection with 0.5 g/l sodium hypochlorite for 1 and 5 min, reduced the percentage of contaminated surfaces from 100% to 77 and 60%, respectively. Disinfection with 5 g/l sodium hypochlorite for 1 min resulted in 7% contaminated surfaces.

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7. Novel disinfection methods There is an ongoing effort among researchers and manufacturers of disinfectants in improving existing and developing new disinfectants. Factors driving this process are often customers need for products that for examples have effect at dirty conditions, work in low volumes, have low costs and reduce corrosion. Also the food industry, consumers and governmental agencies are increasingly asking for agents less toxic to the environment and which do not lead to the development of microbial resistance. “Natural” products as grape fruit seed extract has been introduced into the market, but in general, their effects are limited compared to conventional disinfectants (Møretrø et al., 2003; Parnes, 1997; Sundheim & Langsrud, 1995) or the documentation of bactericidal effects is scarce. In the EU, the Biocides directive (98/8/EC)(Anonymous, 1998) requires substantial documentation for approval of new disinfectants, and this will lead to high costs for companies launching new disinfectants to the market. There is a trend internationally that more documentation is needed for approval of novel products, and this will likely reduce the number of new disinfectants released to the market. 7.1. Electrolysed water Considerably research has been done during the last decade regarding possible applications of electrolysed water in the food area (Hricova, Stephan, & Zweifel, 2008; Huang, Hung, Hsu, Huang, & Hwang, 2008). By electrolysis, a dilute sodium chloride solution dissociates into acidic electrolysed water (AEW; pH between 2 and 3; oxidation-reduction potential of N1100 mV, and an active chlorine content of 10 to 90 mg/l), and basic electrolysed water (BEW; pH between 10 and 13, and oxidation-reduction potential of −800 to −900 mV) (Hricova et al., 2008). AEW is more antimicrobial than BEW, and a combination of BEW and AEW is more efficient than AEW alone. Neutral electrolysed water (NEW; pH 7–8) is produced by adding hydroxyl ions to AEW or by using a single chamber (Hricova et al., 2008). In general electrolysed water is considered environmental friendly as it is generated from water and dilute salt solution, and reverts to water after use. As electrolysed water in generally has short lifetime, usually a generator onsite is necessary. Electrolysed water is in general effective against vegetative bacteria in suspension. Results from suspension tests show that S. Enteritidis was eliminated (N6 log10 reduction) after 5 min exposure to AEW (Venkitanarayanan, Ezeike, Hung, & Doyle, 1999a) or NEW (Deza, Araujo, & Garrido, 2003). Exposure of S. Typhimurium to NEW for 10 min lead to a reduction of N6 log10 (Guentzel, Lam, Callan, Emmons, & Dunham, 2008). In the food sector electrolysed water has been tested for decontamination of food and food processing surfaces. As for chlorine, the antibacterial effect of AEW is reduced in presence of organic residues (Park, Alexander, Taylor, Costa, & Kang, 2009). To our knowledge, there are no reports on the effect of electrolysed water against Salmonella at abiotic surfaces, however several studies of the taxonomic related Escherichia coli exist. For Escherichia coli on plastic, exposure to AEW for 20 min at 35 °C resulted in a reduction of 8 log10/100 cm2 (Venkitanarayanan, Ezeike, Hung, & Doyle, 1999b). For Escherichia coli on glass and stainless steel, the reduction was N6 log10/50 cm2 after 1 min exposure to AEW at 23 °C (Lee et al., 2004). 7.2. Antimicrobial materials During the last decade a range of antimicrobial materials intended for the food area has been developed, and released to the market. This is materials where an antimicrobial (disinfectant) is incorporated or coated as a film on the material. Such materials are in general marketed as an additional hygienic barrier, not a substitute for good

hygienic practises or regular sanitation. Examples of products are cutting boards, boxes for transport of food, knife handles, flooring materials, conveyor belts and refrigerators (Cutter, 1999; Junker & Hay, 2004; Kampmann et al., 2008; Møretrø, Sonerud, Mangelrød, & Langsrud, 2006). The most common antimicrobials in use are triclosan and silver. Triclosan is incorporated in plastic polymers under the trade name Microban® (www.microban.com). There has been some concern over the increased use of triclosan in domestic sector, due to fear of development of resistant bacteria (Levy, 2001; Webber, Randall, Cooles, Woodward, & Piddock, 2008) (see Section 8, this review). Silver has a long term use as an antimicrobial but its use declined after the discovery of antibiotics. However, it is regarded as safe to use, and are increasingly added to materials as silver or nanosilver. AgION® is a silver zeolite matrix coated on stainless steel marketed for use in food industry. The zeolite releases silver in exchange for cations from the environment (Cowan, Abshire, Houk, & Evans, 2003). In general there is limited scientific literature assessing the effect of such materials. We are only aware of three studies where the effect of materials containing antimicrobials has been tested against Salmonella. Plastic containing 1.5 g/kg triclosan showed inhibition of growth of S. Typhimurium in an agar plate assay. However, when beef was vacuum packed in the plastic containing triclosan no effect on S. Typhimurium on meat compared to control was observed for incubation at 4 °C and 12 °C, up to 14 days (Cutter, 1999). De Muynck, De Belie, and Verstraete (2010) investigated the antimicrobial effect against Salmonella of concrete mortars containing triclosan fibres or zeolites containing 35 g/kg silver and 65 g/kg copper. No antimicrobial effect against Salmonella was observed for mortars containing triclosan after 24 h incubation at 4 or 20 °C. Mortars containing 30 g/kg silver copper zeolite showed a 2 and 4 log10 reduction in cell count compared to control after incubation for 24 h at 4 and 20 °C, respectively. Recently we tested the antibacterial effect of commercially available cutting boards containing triclosan or silver (concentrations not disclosed by the manufacturers) on Salmonella after incubation for 24 and 72 h at 25 °C under dry and humid conditions. While the three boards containing silver had no effect on Salmonella compared to control boards without antimicrobials an antibacterial effect of 1–4 log reduction was observed for the triclosan-containing cutting board dependent on experimental conditions (Møretrø, Høiby-Pettersen, Habimana, Heir, & Langsrud, 2011). In general, the antimicrobial effect varies between different products. The type of material and the choice and concentration of antimicrobial have impact on the antimicrobial efficiency. Especially for silver containing products the antimicrobial effect seem to be neutralised at soiled conditions (Chaitiemwong, Hazeleger, & Beumer, 2010; Fernandez et al., 2009). Also, release of the antimicrobial from the product, may limit the lifetime of the antimicrobial activity (Vasilev, Cook, & Griesser, 2009). In conclusion, materials with added antimicrobials may have some antimicrobial effect on Salmonella under certain conditions, but their effects are usually much lower than traditional cleaning and disinfection. 7.3. Anti-biofilm specific compounds A rapid increase in resistance to antibiotics among pathogenic bacteria has been observed and this has resulted in attempts to find alternative agents often referred to as anti-pathogenic drugs. Antipathogenic drugs selectively block virulence, quorum sensing and/or biofilm formation at concentrations not affecting planktonic growth of the bacteria (Jagusztyn-Krynicka & Wyszynska, 2008; Rasmussen & Givskov, 2006; Steenackers et al., 2010). Furthermore, it has been suggested that since the target for anti-pathogenic drugs is thought to be inhibition of quorum sensing, biofilm formation or virulence rather than exerting a selection pressure on the organism by inhibiting

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growth or killing the organism, these anti-pathogenic drugs are less likely to promote antimicrobial resistance (Costerton, Montanaro, & Arciola, 2007; Lönn-Stensrud, Petersen, Benneche, & Scheie, 2007). For Salmonella, little research is available, but one such class of antipathogenic drug has been shown to be a promising candidate for this purpose; halogenated furanones (Janssens et al., 2008; Steenackers et al., 2010; Vestby et al., 2010). Halogenated furanones were originally extracted from the macro alga Delisea pulchra (de Nys, Givskov, Kumar, Kjelleberg, & Steinberg, 2006; Kjelleberg & Steinberg, 2001), but several different synthetic furanones and analogues have been produced and show similar effect as the natural furanones (de Nys et al., 2006). For Salmonella, it has been found that synthetic halogenated furanones have an inhibitory effect on biofilm formation at a concentration that did not statistically significantly affect planktonic growth (approximately 50 μM in both studies)(Janssens et al., 2008; Vestby et al., 2010). Another favourable effect of the furanones is that they have been reported to potentiate the effect of disinfectants and antibiotics (Janssens et al., 2008; Vestby et al., 2010). Vestby et al. (2010) found that a furanone potentiated the effect of 0.5 g/l sodium hypochlorite and 0.2 g/l BC. For two days old biofilm of S. Agona it was found that pre-exposure to 48 μM furanone resulted in about 2 log10 additional reduction by hypochlorite treatment compared to hypochlorite treatment alone. A less evident effect, but still statistically significant, was seen when pre-exposing biofilms with furanone before treatment with BC. No statistically significant effect of the furanone on viable bacterial counts in the biofilm was observed (Vestby et al., 2010). For other bacteria the antimicrobial effect of furanones has been shown to be regulated through interference with microbial communication systems (quorum sensing) (Dworjanyn, De Nys, & Steinberg, 1999; Kjelleberg & Steinberg, 2001), however there are to date no evidence that synthetic bromated furanones interfere with any known quorum sensing system in Salmonella (Janssens et al., 2008; Vestby et al., 2010). Synthetic halogenated furanones may be promising new candidates in the control of unwanted pathogens such as Salmonella, but there are still several questions regarding e.g. mechanisms and toxicity that need to be answered to fully understand the potential and limitations with the use of these compounds. The potentiated effect these compounds seem to have on traditional disinfectants and antibiotics may prove to be valuable, but it is of no use if the compounds are toxic and render useless for human or environmental use. 8. Resistance of Salmonella to chemical disinfectants 8.1. Disinfectant resistance — definitions and terms The term disinfectant resistance is used to describe bacteria that are not killed by recommended user concentration of disinfectant, but may also be applied to bacteria being able to grow and survive in higher concentrations of disinfectants than other bacteria within a species (Russell, 1999). MICs are determined to detect and describe the latter state. The purpose of disinfection is to kill bacteria and as there is no universal relation between MIC and the minimum bactericidal concentration, determinations of MICs have often limited relevance as measures of effects of disinfectants in food processing environments. In such environments, bacteria with reduced susceptibility to recommended user concentrations of disinfectants are of special attention as this may have a direct effect on the hygienic and microbial safety of environments and products. The increasing use of disinfectants in both food processing industry and in homes with possible frequent exposure of bacteria to sub-lethal concentrations of disinfectants have gained concerns regarding adaptation, selection and resistance to antimicrobial

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compounds (McDonnell & Russell, 1999; Sheldon, 2005; Yazdankhah et al., 2006). Here, an overview of current knowledge on Salmonella disinfectant resistance, effects of sub-lethal exposure and disinfectant resistance mechanisms in Salmonella associated with the food chain are presented. 8.2. Resistance of Salmonella to commonly used disinfectants In general, little resistance to disinfectants has been detected in Salmonella field isolates. The resistance level of wild type strains reported is in most cases based on elevated MIC which is well below recommended user concentrations of the disinfectants. Gradel, Randall, Sayers, and Davies (2005) showed no associations between disinfectant tolerance (MICs) in 286 Salmonella isolates, mainly from broiler houses, and the use of five specified disinfectants, indicating that disinfection did not result in selection of tolerant or resistant clones or adaptation. Similarly, Møretrø et al. (2003) reported no difference in survival after disinfection of feed industry Salmonella isolates compared to isolates of other origin when a total of nine commercial disinfectants based on QAC, hypochlorite, cationic and anionic tensides, grape fruit seed extracts and formaldehyde were tested (Møretrø et al., 2003). A 4% prevalence of Salmonella with decreased susceptibility to triclosan was reported among 428 isolates of human and animal origin (Copitch, Whitehead, & Webber, 2010). The MIC levels of tolerant strains were low (MICs 0.25–4 mg/l) and similar to findings by Randall, Cooles, Sayers, and Woodward (2001). No significant difference in the susceptibility to disinfectants (MICs, QAC included) of Salmonella isolates from poultry and swine in Thailand was reported (Chuanchuen, Pathanasophon, Khemtong, Wannaprasat, & Padungtod, 2008). The distribution of MICs of disinfectants was close to previous data (Aarestrup & Hasman, 2004). The reports concluded that the Salmonella isolates studied had no or only a limited degree of resistance to disinfectants. A number of Salmonella isolated from a poultry abattoir were reported resistant to hypochlorous acid (HOCl; Mokgatla, Brözel, & Gouws, 1998; Mokgatla, Gouws, & Brozel, 2002). Resistant isolates were able to grow in broth containing 28 mg/l free available chlorine while 50–200 mg/l chlorine solutions are commonly used in food industry disinfection (Beuchat, 1998). Thus the reported resistance was based on increased MIC levels and differences in bactericidal concentrations of HOCl among resistant and sensitive isolates were not reported. A connection between disinfection resistance and Salmonella persistence in food or feed processing facilities has been hypothesised, but were not verified through several studies (Davison et al., 2003; Gradel et al., 2005; Møretrø et al., 2003). Neither could increased MICs of disinfectants for certain isolates be linked to the frequent use of different types of disinfectants (Gradel et al., 2005). Biofilms are also potential sources of persistent Salmonella. Surface attached and biofilm-embedded Salmonella are more tolerant to antimicrobials than planktonic cells (Joseph et al., 2001; Wong, Townsend, Fenwick, Trengove, & O'Handley, 2010). The protection of bacteria in biofilms against disinfectants may be due to chemical and mechanical protection by the matrix and/or the altered physiological properties displayed by the bacteria in biofilm (Donlan & Costerton, 2002; Kumar & Anand, 1998; Scher et al., 2005). The majority of wild type Salmonella strains display one of two different matrix phenotypes in laboratory tests, appearing only to differ by the presence or absence of cellulose (Vestby, Møretrø, Ballance, et al., 2009). It has been suggested that cellulose protects the bacterial cells against the bactericidal effects of disinfectants (Solano et al., 2002; White et al., 2006). However, no significant differences in the susceptibility to hypochlorite or BC were observed when comparing wild type S. Agona strains in biofilms with and without cellulose (Vestby, Møretrø, Ballance, et al., 2009). Similar results were also reported in a study using an alcohol based disinfectant and hypochlorite (Møretrø et al., 2009).

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Laboratory studies have shown that exposure of Salmonella to sublethal concentrations of disinfectants can provide increased tolerance to disinfectants, but most studies have been performed using methods based on MIC (Braoudaki & Hilton, 2004; Karatzas et al., 2007). Increased D-values using the European suspension test have been demonstrated after single time exposure of Salmonella to three types of disinfectants (based on an oxidising compound, aldehydes and QAC) (Randall et al., 2007). Exposure of Salmonella to disinfectants may also lead to cross-tolerance to other antimicrobials. Braoudaki and Hilton (2004) reported the adaptive capacity to BC (a QAC) to be strain dependent, but increased MIC to both BC and antibiotics were reported (Braoudaki & Hilton, 2004). Exposure of Salmonella to subinhibitory concentrations of triclosan provided a 2000-fold increase in MIC to triclosan but no changes in MIC to certain other commercial disinfectants (Karatzas et al., 2007). Randall, Cooles, Piddock, and Woodward (2004) reported a 10–100 fold increase in the frequency of mutation to antibiotics or cyclohexane resistance (marker for the multiple antibiotic resistance (MAR) phenotype) for Salmonella after growth at sub-MIC levels of a phenolic farm disinfectant or triclosan (Randall et al., 2004). The obtained mutants showed low-level resistance to antibiotics (4–8 fold increase in MIC). Their tolerance (MIC) to disinfectants after exposure increased by up to 32-fold for triclosan and was just below the lowest in use concentration for the phenolic disinfectant. Mangalappalli-Illathu, Vidovic, and Korber (2008) showed that S. Enteritidis biofilms adapted to BC showed higher biofilm biomass compared to biofilms not pre-exposed to BC during regrowth without BC-stress. The report also indicated that S. Enteritidis biofilms continuously exposed to sublethal concentrations of BC provided improved adaptive capacities compared to intermittently treated biofilms (Mangalappalli-Illathu et al., 2008). The use of triclosan in a large array of domestic products including toothpastes, soaps, cosmetics, kitchen utensils and cutting boards along with being used in industrial settings such as floors in food processing plants (see Section 7.2, this review) has increased the environmental exposure to triclosan. Triclosan has multiple cellular targets and mechanisms of action. However, reports on specific resistance mechanisms and cross-resistance to antibiotics have raised concerns on the widespread and increasing use of triclosan (Aiello & Larson, 2003; Birosova & Mikulasova, 2009; Levy, 2001; McMurry, Oethinger, & Levy, 1998b). Triclosan resistant Salmonella mutants are readily obtained in laboratory conditions although large differences in triclosan resistance (MICs) have been reported (Birosova & Mikulasova, 2009; Braoudaki & Hilton, 2004; Karatzas et al., 2007; Webber, Coldham, Woodward, & Piddock, 2008). The above reports show that Salmonella in general are readily adapted to grow in higher concentrations of disinfectants under appropriate in vitro conditions. Salmonella adaptive responses require exposure well below the recommended user concentration of disinfectants. Different disinfectants also differ in their potential to select for Salmonella mutants with increased tolerance to disinfectants and antibiotics. Gradel et al. (2005) reported no increase in resistance (MICs) after adaptation to five disinfectants commonly used in the poultry premises. This also reflects the complexity of factors affecting the results obtained and the difficulties in comparison of data obtained in different studies. In practical use, dilution effects including presence of substances known to reduce the efficacy of disinfectants may provide environments where such mutants are able to survive brief exposure to the disinfectants. A key consideration is therefore to assess the risks of adapted and mutated strains to survive throughout the food production chain. Relevant here is the finding that disinfectant tolerant mutant strains were not compromised in their ability to survive and persist in chicks (Randall et al., 2007). Also relevant are the situations in real life where Salmonella and other bacteria are unlikely to be exposed to antibacterials in pure cultures but are often present in multibacterial consortia and biofilms. This

may affect both their tolerance to disinfectants and the selection of insusceptible populations (Braoudaki & Hilton, 2004; Gilbert & McBain, 2003; McDonnell & Russell, 1999; Russell, 2002). Various stresses (e.g. chemical stress, desiccation and starvation) experienced by surface-associated bacteria may further provide enhanced resistance to disinfection by Salmonella (Kieboom et al., 2006; Leyer & Johnson, 1997; Mangalappalli-Illathu et al., 2008).

8.3. Mechanisms of disinfectant tolerance in Salmonella Variations in disinfectant susceptibility among bacteria have typically been associated with intrinsic bacterial membrane properties affecting uptake of antibacterial compounds (Russell & Chopra, 1996; Sheldon, 2005). In Gram-negative bacteria, the outer membrane with its LPS-layer is a main barrier against outer detrimental conditions such as chemical disinfectants (Nikaido, 2003). For Salmonella important reported antimicrobial mechanisms are efflux, enzymatic degradation and mutations in specific antimicrobial targets (e.g. mutations in fabI providing triclosan resistance, see details below) or over-expression of target proteins. Efflux pumps are membrane integrated proteins that utilise cellular energy to extrude antimicrobials actively out of the cell (Nishino, Nikaido, & Yamaguchi, 2009). In Salmonella, at least nine drug efflux gene systems exist of which three (AcrAB, AcrEF and MdsABC) are known to transport BC out of the cell. Studies and knowledge on the role of efflux in tolerance of Salmonella to disinfectants is limited compared to their known role in multiple antibiotic resistance (MAR). Types of disinfectants that could be substrates for Salmonella efflux pumps are therefore largely unknown. Randall et al. (2007) showed that efflux was required for resistance of S. Typhimurium to three disinfectants based on a QAC, a tar oil phenol and an aldehyde, respectively. Exposure to an oxidative disinfectant did not activate efflux systems. Although not specifically studied, the data also indicated activation of other efflux systems. Thorrold et al. reported antibiotic resistant Salmonella to be less susceptible to three household disinfectants compared to antibiotic sensitive strains and that efflux systems were responsible for increased tolerance to disinfectants below user concentrations (Thorrold, Letsoalo, Duse, & Marais, 2007). Increased expression of the AcrAB-TolC efflux system has been described as a main mechanism in reduced susceptibility to antimicrobials including disinfectants. Various proteins are involved in the regulation of the efflux systems. Mutations in the regulatory genes may lead to over-expression of efflux pumps and increased tolerance to multiple drugs, including development of the MAR phenotype. Efflux systems are often induced through various stressors, such as low pH, osmotic changes, disinfectants and/or compounds produced by the host, but only a few signals (e.g. bile and indole) that induce multidrug efflux pumps in Salmonella have been identified (Nikaido, Yamaguchi, & Nishino, 2008; Nishino et al., 2009). Of 20 Salmonella isolated from a poultry abattoir, 50% were termed resistant to hypochlorous acid (HOCl) showing growth in 72 mg/l HOCl compared to the sensitive isolates where no growth was obtained (Mokgatla et al., 1998; Mokgatla et al., 2002). A study of one isolate able to grow in 28 mg/l HOCl indicated that resistance was achieved by decreasing the levels of reactive moieties that could react with HOCl and generate toxic, reactive oxygen radicals. This was obtained by increase in catalase activity while the level of membrane bound dehydrogenases decreased. There are major differences in the lifestyle of planktonically grown Salmonella compared to Salmonella grown in biofilms. These differences also affect their tolerance to disinfectants. In a recent study, upregulations of proteins and shift in fatty-acid composition conferred enhanced survival of BC-adapted S. Enteritidis in biofilms relative to BC-adapted planktonic cells (Mangalappalli-Illathu et al., 2008).

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Triclosan is a substrate for multidrug efflux pumps (McMurry, Oethinger, & Levy, 1998a; Webber, Randall, et al., 2008). A marked association between decreased triclosan susceptibility and multidrug resistance obtained through increased efflux activity and acrB expression has been reported (Copitch et al., 2010). This suggests that low levels of triclosan promotes selection of multidrug efflux which is of clinical relevance and therefore represent a major concern (Birosova & Mikulasova, 2009; Copitch et al., 2010). Mutations in a specific target of triclosan, known as enoyl–acyl carrier protein (ACP) reductase (FabI enzyme in Salmonella) involved in fatty acid biosynthesis are also involved in resistance to triclosan in Salmonella and other bacteria (McMurry et al., 1998b; Webber, Randall, et al., 2008). To obtain high-level resistance to triclosan (MIC ≥ 32 mg/l) in Salmonella, a number of resistance mechanisms act in synergy (Tabak et al., 2007; Webber, Coldham, et al., 2008; Webber, Randall, et al., 2008). Mechanisms involved include efflux, mutations in fabI, overexpression of wild type fabI, increased fatty acid synthesis and down regulations of outer membrane porins (Webber, Coldham, et al., 2008; Webber, Randall, et al., 2008) as well as low diffusion of triclosan in biofilms (Tabak, Scher, Chikindas, & Yaron, 2009). Triclosan resistant S. Typhimurium mutants were able to colonise and persist in similar manners as their parent strains. This indicates the good ability of such mutants to survive. The use of triclosan in areas where the benefits of use are absent or questionable should thus be avoided including use in domestic products and in the food and feed processing industry. As disinfectants have multiple modes of action, selection of Salmonella mutants resistant to user concentrations of disinfectants will probably require multiple disinfection exposures and selection events. Based on current knowledge this may be unlikely. However, reported links between disinfectant exposure and induction of the MAR phenotype indicate that certain disinfectants may have a role in increased selective pressure towards antibiotic resistant microorganisms. Bacterial stress responses caused by disinfection exposure may also render Salmonella susceptible to certain types of stresses (Kieboom et al., 2006; Leyer & Johnson, 1997; Wang et al., 2010). This knowledge may promote the development of more effective interventions to reduce the risk of Salmonella adaptation and contamination in the food supply. Studies have indicated a good ability of disinfectant resistant Salmonella mutants to survive and be able to persist in the food chain (Randall et al., 2007; Webber, Randall, et al., 2008). For more complete assessments of the risks of adaptive responses of Salmonella to disinfectants, further research with focus on real life practical applications are needed. The role of e.g. efflux mechanisms for providing Salmonella with increased ability to survive user-concentrations of disinfectants is not clear. A more complete understanding of other physiological functions of key antimicrobial resistance mechanisms including efflux is also needed. These include recent findings that some drug efflux pumps have physiological substrates that could be linked to virulence properties of the strains. Bacteria with active efflux systems may promote invasion and virulence. How efflux pumps and their substrates modulate Salmonella virulence properties will provide new and important information (for a review see Nishino et al. (2009)). Overall, with our knowledge so far, adhering to the manufacturer's instruction on use of the disinfectants should reduce the risks of obtaining strains with reduced susceptibility to disinfectants and antibiotics.

9. Conclusions Proper disinfection will reduce the risk of cross-contamination of food with Salmonella. The data published on effects of disinfection on Salmonella generally support what is known for other bacteria and which is the basis for common guidelines and recommendations for disinfection.

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There exist many methods for testing the effect of disinfectants. To evaluate whether a disinfectant will be effective in practical situations, the test method should model such situations. Most disinfectants are effective against Salmonella at recommended user concentration in suspension tests. However, there are a number of factors that may reduce the effect of disinfection on Salmonella, e.g. presence of organic compounds and high number of cells. Several disinfectants appear to have reduced effect at their recommended user-concentrations against Salmonella on surfaces including Salmonella in biofilms. Salmonella is not particularly tolerant to disinfectants compared to closely related bacteria, but have as a Gram-negative bacterium higher tolerance to some disinfectants, e.g. quaternary ammonium compounds, compared to Gram-positives. Salmonella may also gain reduced susceptibility to certain disinfectants through adaptation and other physiological responses like biofilm formation. The knowledge on the relevance of increased disinfectant tolerance in practical situations is far from complete. There is also a growing concern that multidrug efflux systems induced by certain disinfectants can act as a stepping stone to mutants with antibiotic resistance. Novel disinfectants and disinfection measures such as electrolysed water, use of antimicrobial surfaces, and anti-biofilm compounds have been introduced. Although they may have some applications, the control of Salmonella still depends on the proper use of conventional chemical disinfectants.

Acknowledgements This work was funded by The Foundation for Research Levy on Agricultural Products and the National Veterinary Institute of Norway.

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