#109_CANSIAN_bozza ! Ital. J. Food Sci., vol 28, 2016 - 733 PAPER LISTERIA MONOCYTOGENES ADHESION TO FOOD PROCESSING SURFACES (BONING KNIVES) AND THE REMOVAL EFFICACY OF DIFFERENT SANITIZERS J. BARBOSA1, V. GRZYBOWSKI1, M. CUPPINI1, J. FLACH1, C. STEFFENS*1, G. TONIAZZO1 and R. L. CANSIAN1 1 Department of Food Engineering, URI Erechim, Av. 7 de Setembro, 1621, CEP 99700-000, Erechim RS, Brazil *Corresponding author. Tel.: +55 5435209000; fax: +55 5435209090 E-mail address: claristeffens@yahoo.com.br ABSTRACT The adhesion and biofilm formation of Listeria monocytogenes on new and used boning knives (handle and blade) were observed. The number of pathogens on both surfaces increased with the contact time, forming a biofilm after 2 h. Peracetic acid or hot water effectively removed adhered L. monocytogenes at each of the contact times and concentrations evaluated. Biguanide showed lower removal efficacy on used surfaces, but had increased effectiveness at concentrations of 2.0%. The new knife blades had a lower roughness and flattened morphology in comparison with used surface Keywords: bacterial adhesion, sanitizer, Listeria monocytogenes, nactivation, boning knives ! Ital. J. Food Sci., vol 28, 2016 - 734 1. INTRODUCTION Food contact surfaces constantly must pass a microbiological evaluation for efficiency control and sanitizing procedures, in order to avoid contamination of the food produced (PINHEIRO et al., 2010; ALMEIDA et al., 2013). The exposure of surfaces to pathogens may take place either by direct contact with contaminated objects or indirectly through airborne particles. According to KUSUMANINGRUM et al. (2003), many bacteria, including L. monocytogenes may survive on utensils for hours or days after the initial contamination and can persist on industrial equipment and installations with high potential for adhesion and biofilm formation at low temperatures (GIAOURIS et al., 2014; CARPENTIER and CERF, 2011). Cell surface hydrophobicity and production of extracellular polymeric substances influence the rate and extent of attachment of microbial cells. Another factor that can also influence bacterial adhesion is the surface roughness (RODRIGUEZ et al., 2008; SKOVAGER et al., 2013). Bacterial adhesion to stainless steel, glass, rubber and polypropylene surfaces is a potential source of contamination that may lead to disease transmission (CHAVANT et al., 2007). The adhered cells are highly resistant to acids present in sanitizers, to desiccation and to heat. Tolerance to increased sub-lethal concentrations of disinfectants or resistance to lethal concentrations are documented, because sessile cells produce exopolysaccharides that protect against chemical agents (JOSEPH et al., 2001; CARPENTIER and CERF 2011; SILVA et al., 2010). Several sanitizers are available with different uses, but they do not always eliminate bacteria to the expected level (BELTRAME et al., 2012). In this way, it is very important to know the factors involved in the adherence of biofilms to surfaces, which could be useful to improve methods for disinfection of food processing surfaces (boning knives). Therefore, the purpose of this study was to investigate adhesion and biofilm formation of L. monocytogenes on the handle and blade of new and used boning knives. This study also evaluated the removal of bacteria on the surface of the knife using different contact times and concentrations of chemical sanitizers (peracetic acid and biguanide) or hot water. 2. MATERIALS AND METHODS In this study, bacterial adhesion and inactivation were evaluated on new and used boning knives using Gram-positive L. monocytogenes (ATCC 7644). The bacterial culture was purchased from the Instituto Oswaldo Cruz, mantained at -80°C and revitalized in Luria Bertani broth (LB Merck, Darmstadt, Germany) at 30°C, 24 hours before the experiments. 2.1. Bacterial adhesion and quantification of adhered cells In order to assess bacterial adhesion, the polypropylene (PP) handle and stainless steel (SS) blade of new boning knives (Professional line Mundial® model 5315-6) and the same model of used knives (use in a cattle slaughterhouse for 45 to 60 days) were studied. The entire knife surface (handle and blade) was prepared according to the follows steps: cleaning by manual rubbing with water and neutral liquid detergent, rinsing with water and then by sterile distilled water and air drying. For sanitization, the surfaces were exposed to ultraviolet light (254 nm) for one hour. After cleaning, the entire knife surface was swabbed in order to confirm the absence of initial contamination (negative control), before carrying out the experiments. ! Ital. J. Food Sci., vol 28, 2016 - 735 The bacteria were incubated in LB broth for 24 h at 35-37°C. Next, 10 mL of inoculum (3 log CFU/mL) was inoculated into 1.5 L LB broth that had been previously poured into a polyvinylchloride (PVC) tube with a 10 cm diameter to accommodate the entire knife, and the samples were incubated for 24 h at 35-37°C. Afterward, the knives (new or used) were removed from the PVC tube with sterile forceps and rinsed with water to remove the planktonic cells. The entire surface of the knife handle and knife blade were swabbed separately to assess contamination. Dilutions were performed in peptone water, plated onto LB agar, and incubated at 35-37°C for 24 h. The number of adherent cells was assessed in intervals of 0.1, 0.5, 1, 2, 6, 24 and 48 h at 35°C. These times were selected in order to simulate the factory production time, which is the period that knives remain in contact with products without sanitization. 2.2. Characterization of the boning knife surface by contact angle The hydrophobicity and hydrophilicity of the new and used blades with and without L. monocytogenes adhesion for 6 h, were determined by contact angle with a drop of water using a contact angle metre (KSV Instruments, Helsinki, Finland). The measurements were performed at 25°C and 45% humidity, by depositing 4.0 !L of water with a Hamilton syringe. The handles were not evaluated in this analysis because the texture of the handle produces differences in surface that did not permit the stable formation of a drop on the surface. 2.3. Characterization of the boning knife surface by Atomic Force Microscopy (AFM) The morphology and average roughness (Ra) of the new and used blades with and without L. monocytogenes adhesion for 6 h were analyzed with a Dimension V (Veeco Instruments Inc.) AFM, using a silicon nitride tip, with a spring constant of 42 N/m and resonance frequency of 285 kHz. All images were obtained in tappingTM mode at a scan rate of 1 Hz. The images were processed with the aid of Gwydion© 2.1 data analysis software. The handles were not evaluated due to the characteristics of the non-slip coating that had very large differences in height, which did not permit a surface scan. The Ra value (arithmetic mean deviation of the profile) is the most common measure used to define the surface roughness (VERRAN et al., 1991). AFM scans were performed in 500 x 500 nm2 areas on each surface. The roughness was calculated from three scans in different areas. 2.4. L. monocytogenes inactivation In this study, peracetic acid (Pluron 461 AP®) and biguanide (Pluron 463 AP®) sanitizers were studied; they were prepared in sterilized water immediately prior to testing, according to the supplier’s instructions. In inactivation experiments, the entire knife was analyzed at intervals of 1, 2 and 6 h, simulating industrial conditions. After rinsing with deionized water, the knives were immersed in beakers containing 500 mL of the respective sanitizer solution at concentrations of 0, 0.2, 0.5, 1.0 or 2.0% (v/v) for 10 min at 25°C, to evaluate their efficacy against cell attachment. The hot water treatment was performed by immersing the knife surface in a hot water bath (82.2°C) for 15 s, according to 175/2005/MAPA method 2 (BRASIL 2005). Bacterial presence was quantified by the enumeration method as previously described (KIM et al., 2008) using swabs of the knives. ! Ital. J. Food Sci., vol 28, 2016 - 736 2.5. Statistical analysis The results of L. monocytogenes counts were converted to decimal logarithmic values (log CFU/cm2) and subjected to Tukey's test at a 5% significance level using Statistica 8.0 software (StatSoftInc®, USA). All experiments were run in triplicate and repeated with three separate Knives. 3. RESULTS AND DISCUSSIONS 3.1. Listeria monocytogenes adhesion The adhesion of L. monocytogenes on new and used boning knives (handles and blades) is shown in Fig. 1. A rapid bacterial growth was observed for the first 6 h, with a tendency to stabilize at 48 h. Adherence occurred on handles and blades and the adhesion velocities were similar for used and new materials. However, statistical analysis showed a significant difference in adherence to new and used polyethylene handles between 6 and 48 h (Fig. 1a). The knives a non-slip coating on the surface of the handle, which conveys firmness and grip during handling, however, this feature promoted easy adhesion of bacteria on used surfaces. Figure 1: Adhesion kinetics of L. monocytogeneson(A) new polyethylene handles (NPP) and usedpolyethylene handles (UPP) and (B) newstainless steel blades (NSS) and usedstainless steel blades(USS). Biofilm formation with 3.0 Log CFU/cm2. Means (± standard deviations), for each time tested, who having the * symbol are significantly different (p<0.05). ! Ital. J. Food Sci., vol 28, 2016 - 737 L. monocytogenes showed a greater capacity for adhesion onto SS than to PP (Fig. 1) from 6 h in new, and 24 h in used knives. These results confirm those by Teixeira et al. (2008), who observed greater adhesion on SS as compared with the extent of adhesion on PP (cutting board). RONNER and WONG (1993) defined biofilm formation as a recovery of greater than 3.0 log CFU/cm2 adhered cells. Thus, according to this criterion, L. monocytogenes biofilm growth occurred after 1 h of contact on the handles (new and used), and from 30 min (new) to 1 h (used) on the SS surface. 3.2. Characterization of the surface of the boning knife by contact angle Water contact angle is a quantitative measurement of surface wettability, and also can be used to evaluate the cleanliness of the material surface. Throughout this study, a contact angle of 70±1.0 and 82±1.0 degrees was obtained for new stainless steel (NSS) and used stainless steel (USS) surfaces without microbial adhesion, respectively. These results agree with those found by BERNARDES et al. (2010), who found a value of 70±7.9 on SS surfaces. After 6 h of bacteria exposure, contact angles of 19±1.4 and 30±2.1 were obtained on NSS and USS respectively, showing that the surfaces became more wettable after microbial adhesion. These results corroborate with those of CHAVANT et al. (2007), who observed better adhesion and biofilm formation of L. monocytogenes on hydrophilic (SS) rather than on hydrophobic (PP) surfaces. These decreases in the value of contact angle from 70 down to 19 (NSS) and 80 down to 30 (USS) may be attributed to the L. monocytogenes surface composition (molecules such as proteins, teichoic and lipoteichoic acids) (Bereksi et al., 2002). 3.3. Morphological and roughness characterization The morphology of new and used blades was analyzed through use of AFM before and after L. monocytogenes adhesion (Fig. 2). Roughness values (Ra) of 5.1±2.3, 14.3±1.7, 17.4±2.4 and 28.8±2.1 nm were obtained from new and used blades without and with L. monocytogenes adhesion, respectively. New blades had a low roughness and flattening morphology compared with used surfaces, which is very important to note because studies have shown that an increase in Ra value will cause a corresponding increase in microbial adhesion on surfaces (Whitehead et al., 2004). This increase may be due to protective cells present in microscopic niches. Roughness values of 800 nm or less are generally used to describe a hygienic surface (FLINT et al., 1997). These values were found for all surfaces evaluated in this work (Fig. 2). Teixeira et al. (2008), observed that SS is a material with high surface roughness, similar to what was measured in this study for the surface of the knife blade (Mundial®). TAYLOR et al. (1998) observed a significant increase in the attachment of Pseudomonas aeruginosa and Staphylococcus epidermidis on polymethyl methacrylate when the surfaces had a small increase in roughness (40-1240 nm). Bacterial adhesion has been found to increase numerically with surface roughness, by comparing bacterial adhesion on used versus abraded SS and for Ra value increases from 602 to 706 nm or from 484 to 698 nm (Holah and Thorpe, 1990). Thus, it was found that the roughness data obtained after the adherence of L. monocytogenes are in agreement with those from the literature, but no correlation was demonstrated between Ra and the number of adhering cells. From this evaluation of the surface characteristics of boning knives, it can be observed that with an increase of the surface roughness, there was a decrease in the contact angle. ! Ital. J. Food Sci., vol 28, 2016 - 738 Figure 2: Roughness values on NSS and USS with and withoutL. monocytogenes adhesion. Mean values and standard deviations (error bars) are indicated. Observations correspond to on 500 x 500 nm scan area. 3.4. Efficacy of different sanitizers on food processing surfaces To evaluate their ability to sanitize new and used knives, biguanide and peracetic acid sanitizers were used at different concentrations (0.2, 0.5, 1.0 and 2.0% v/v) for 10 min, or hot water (82.2°C) was used for 15 s. The boning knives remained in contact with L. monocytogenes for different contact times (1, 2 and 6 h) to simulate industrial conditions for adhesion. L. monocytogenes was resistant to biguanide sanitizer under the different contact times studied (Table 1). Similar efficacy levels have been obtained by other researchers. MARTÍN-ESPADA et al. (2014) observed that 1.61% peracetic acid was effective against P. aeruginosa biofilms formed on polystyrene surfaces, inhibiting almost 100% of the microbial population. Similarly, BELTRAME et al. (2015) evaluated the efficacy of 0.5% peracetic acid at inactivating L. monocytogenes cells adhered to cutting boards and observed that it was able to reduce the amount of adhered cells by 100%, after 3 h of contact time with bacteria. Cabeça et al. (2012) observed adhesion of L. monocytogenes on SS surfaces and studied the inactivation after treatment with 0.5% biguanide and 0.5% peracetic acid. The authors verified an initial count of 6.2 log CFU/cm2 with reduction to 2.9 log CFU/cm2 and 1.1 log CFU/cm2 using biguanide and peracetic acid, respectively, showing that peracetic acid was more effective against L. monocytogenes cells. For the sanitizer evaluated, the suppliers recommend a maximum disinfection concentration of 0.5% for both biguanide and peracetic acid. Thus, there was a variation in the effectiveness of the procedures, based on decimal reductions, in microbial effect conveyed by the different sanitization process investigated, as demonstrated in Table 2. In the maximum contact time (6h) between the knife and the microorganism, the low concentration of peracetic acid (0.2%), lower than that recommended by the supplier, showed reliable results from the point of microbiology safety. Similar results were observed for hot water, where the presence of surviving L. monocytogenes was not observed. On the other hand, the maximum concentration of biguanide recommended by ! Ital. J. Food Sci., vol 28, 2016 - 739 the supplier was not able to remove adherent cells, with maximum efficacy of only 50% on NPP (Table 2). Table 1: Reduction of the L. monocytogenes count (Log CFU/cm2) as a function of contact time (1, 2 and 6 h) and the Biguanide sanitizer concentration (0.2, 0.5, 1.0 and 2.0%).Results in Log CFU/cm2. Biguanide Concentration (%) Contact time Boning knives Initial count R* 0.2% % R* 0.5% % R* 1.0% % R* 2.0% % 1h NPP 3.5 2.0±0.2c 57,1 2.7±0.1b 77.1 3.3±0.1a 94,3 3.5±0.3a 100 UPP 3.7 0.5±0.1c 14.3 1.7±0.1b 45,9 1.8±0.2b 48.6 3.0±0.1a 81.1 NSS 3.4 0.6±0.1c 17.6 1.3±0.1b 38.2 3.2±0.1a 94.1 3.4±0.1a 100 USS 4.1 0.2±0.1d 4.9 1.7±0.1c 41.5 2.8±0.1b 68.3 4.0±0.1a 97.6 2h NPP 3.7 1.7±0.1b 4.6 2.4±0.2ab 64.9 2.8±0.2a 75.7 2.7±0.1a 73.0 UPP 3.8 0.8±0.1c 21.0 1.8±0.1b 47.4 1.8±0.1b 47.4 2.7±0.1a 71.0 NSS 3.5 0.2±0.1d 5.7 0.9±0.1c 25.7 1.9±0.1b 54.3 3.3±0.1a 94.3 USS 4.1 0.6±0.1d 14.6 1.6±0.1c 39.0 2.1±0.1b 51.2 3.3±0.1a 80.5 6h NPP 4.2 1.1±0.1c 26.2 2.1±0.1b 50.0 2.5±0.2a 59.5 2.8±0.1a 66.7 UPP 4.8 0.1±0.1c 2.1 2.3±0.2b 47.9 2.7±0.2ab 56.2 3.1±0.2a 64.6 NSS 4.7 1.0±0.1d 21.3 2.0±0.1c 42.6 2.7±0.1b 57.4 3.9±0.1a 83.0 USS 4.8 0.7±0.1c 14.6 0.5±0.1c 10.4 2.4±0.1b 50.0 3.7±0.1a 77.1 NPP: new handles; UPP: used handles, NSS: new blades; USS: used blades. *R Decimal reduction (Log CFU initial population-Log CFU end population submitted to the sanitizers application). Values followed for the same letters in the lines do not differ statistically according to theTukey Test, with 95% confidence range. Table 2: Reduction of the Listeria monocytogenes count (Log CFU/cm2) in boning knives after hot water and sanitizer treatments (6 h of contact time, sanitizer concentrations - 0.5% biguanide and 0.2% peracetic acid). Boning Knives Initial Count Biguanide R* % Peracetic acid R* % Hot water R* % NPP 4.2 2.1±0.1 50.0 4.2±0 100 4.1±0.1 100 UPP 4.8 2.3±0.2 47.9 4.8±0 100 4.8±0 100 NSS 4.7 2.0±0.1 42.6 4.7±0 100 4.7±0 100 USS 4.8 0.5±0.1 10.4 4.8±0 100 4.8±0 100 NPP: new handles; UPP: used handles; NSS: new blades; USS: used blades. * Decimal reduction (initial population Log CFU - end population Log CFU). Thus, this work confirms that the most effective treatments were the hot water and peracetic acid sanitizer, whereas biguanide showed lower performance. Additional studies should be performed to improve the action of biguanide on removal of L. monocytogenes from food preparation surfaces. The treatments with hot water and peracetic acid were shown to be effective for the inactivation of bacteria that had initially adhered. This result can be associated to the disinfectant activity of peracetic acid based on the release of active oxygen, which may ! Ital. J. Food Sci., vol 28, 2016 - 740 disrupt the chemiosmotic function of the lipoprotein cytoplasmic membrane and transport through the dislocation or rupture of cell walls. This can also be effective against outer membrane lipoproteins, facilitating its action against Gram-negative cells. The intracellular peracetic acid may also oxidize essential enzymes, which may damage the bases of DNA molecules (KITIS, 2004). Moreover this sanitizing agent has low environmental hazards and does not produce toxic compounds upon reaction with organic materials. Likewise, the application of hot water was also an effective means of bacterial inactivation. The mechanism of action of hot water treatment is multifactorial. The exposure to this high temperature is likely to affect most components of the bacterial cell including the cell wall, cell membrane, enzymes and proteins, DNA and RNA (PHUA et al., 2014). Differences between the bactericidal effect of biguanide solutions and peracetic acid were observed, corroborating data obtained by SILVA et al. (2010), who showed that peracetic acid was a more effective bactericidal agent than the quaternary ammonium compound (whose mechanism of action is similar to biguanide). (A) (B) Figure 3: AFM characterization of the knives surface in 3D (A) new without bacterial adhesion, (B) used without bacterial adhesion, (C) new with bacterial adhesion and (D) used with bacterial adhesion. ! Ital. J. Food Sci., vol 28, 2016 - 741 Sodium dichloroisocyanurate, hydrogen peroxide and peracetic acid have been evaluated for their abilities to inactivate the biofilm formed by S. aureus on SS and glass surfaces; peracetic acid showed a significant difference (p < 0.05) compared with the three disinfectants used (MARQUES et al., 2007). The higher efficacy of peracetic acid was explained by its high capacity to oxidize cellular molecules. The survival of bacteria after cleaning and sanitizing is a potential danger to the food industry and the consumer (RODGERS et al., 2001). This study demonstrates the need for specific tests in order to select products to be used to clean surfaces that come into contact with food. Furthermore, bacteria can obtain high resistance through adaptation, genetic elements, stress response and biofilm formation (BRIDIER et al., 2011). It should be noted that an appropriate and effective cleaning process is extremely important, since the American Public Health Association (APHA) recommends a maximum tolerated limit of 2 CFU/cm2 in order to consider a food contact surface appropriate (VANDERZANT and SPLITTSSTOESSER, 1992), whereas the World Health Organization (WHO) suggests limits of 30 CFU/cm2. Based on the obtained results, this study confirms that hot water and peracetic acid was fully effective in removing L. monocytogenes cells, which had adhered onto new and used boning knives, whereas biguanide was not efficient in removing the bacterial cells. 4. CONCLUSIONS Adherence of L. monocytogenes occurred on handles and blades, and the adhesion velocities were similar between the used and new materials. Regarding the morphology and contact angle of the surfaces, an increase of the wettability and roughness on the used stainless steel surface in relation to the new stainless steelsurface was observed. Disinfection with peracetic acid was effective at all contact times and concentrations evaluated; no surviving bacteria was found after sanitizer application in all conditions investigated. The hot water treatment (82.2ºC for 15 s) also was effective in reducing L. monocytogenes adhesion on the surfaces tested. Biguanide showed lower efficacy on new and used handles and blades, but had increased effectiveness at concentrations of 2.0% with 1 h of contact time. ACKNOWLEDGEMENTS The authors thank CNPq, CAPES, FAPERGS, Science and Technology Secretary RS and URI-Erechim for the financial support for this research. REFERENCES Almeida G. et al. 2013. Foci of contamination of Listeria monocytogenes in different cheese processing plants. Int. J. Food Microbiol. 167:303-309 doi:http://dx.doi.org/10.1016/j.ijfoodmicro.2013.09.006 Beltrame C.A. et al. 2012. 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Biodeterioration and Biodegradation 54:143-151 doi:http://dx.doi.org/10.1016/j.ibiod.2004.03.010 Paper Received March 21, 2015 Accepted June 12, 2015