Food Control 137 (2022) 108971 Contents lists available at ScienceDirect Food Control journal homepage: www.elsevier.com/locate/foodcont Detection by real-time PCR and conventional culture of Salmonella Typhimurium and Listeria monocytogenes adhered to stainless steel surfaces under dry conditions Abel Guillermo Ríos-Castillo , Carolina Ripolles-Avila , José Juan Rodríguez-Jerez * Human Nutrition and Food Science Area, Departament de Ciència Animal i dels Aliments, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193, Barcelona, Spain A R T I C L E I N F O A B S T R A C T Keywords: This study evaluated the capacity of real-time PCR and conventional culture methods to detect Salmonella S. Typhimurium enterica serovar Typhimurium and Listeria monocytogenes adhered to stainless steel surfaces used as food contact L. monocytogenes surfaces. The adhesion of the microorganisms to the surfaces was performed under dry conditions to represent Real-time PCR the stress to which these pathogens can be subjected in the food processing environment. The samples were Adhesion dry conditions analyzed with various pre-enrichment times: S. Typhymurium (0, 6, and 18 h) and L. monocytogenes (0, 6, and 25 Surfaces h) and with procedures concentrating or not concentrating the samples after the pre-enrichments. The results showed that real-time PCR obtained increased capacity than the conventional method to detect a low number of both pathogens, and real-time PCR even detected samples without pre-enrichment. However, pre-enrichment is recommended to avoid the detection of false positives from dead cells during adhesion and to ensure the absence of false negatives due to low initial concentrations. The concentration of the adhering bacteria increased the frequency in the detection of positive results for S. Typhimurium, but this effect was not observed in the case of L. monocytogenes. 1. Introduction Salmonella spp. and Listeria monocytogenes are two pathogens that can adhere to food contact materials and are involved in outbreaks The capacity of pathogenic bacteria to adhere to surfaces represents associated with the ingestion of contaminated food. Salmonella spp. can a potential risk because it may lead to bacterial transfer to food or survive in dry processing environments (Margas et al., 2014). It has been subsequent adhesion and biofilm formation on surfaces that are more reported that this pathogen can survive on surfaces under dry condi­ resistant to disinfection than free-living bacteria (Bae et al., 2012; tions, and survival can even be prolonged at lower temperatures (Iibuchi González-Rivas et al., 2018). Even when the bacterial adhesion to sur­ et al., 2010). In the case of food production lines, when these objects are faces is followed by adverse conditions for their growth, such as dryness, contaminated with Salmonella spp., this pathogen can remain on they can survive being protected under a biofilm structure, and there­ equipment and facility surfaces (Gounadaki et al., 2008). In the case of fore, be a source of contamination (Fuster-Valls et al., 2008; Lang et al., L. monocytogenes, it is a ubiquitous pathogen in the environment and 2004). Previous works have indicated the relationship between the resistant to diverse environmental conditions, representing a significant survival of pathogens on surfaces and cross-contamination of food in problem in the food industry because it causes numerous outbreaks foodborne outbreaks (Kusumaningrum et al., 2003; Muhterem-Uyar throughout the world (Lepe, 2020; Mazaheri et al., 2021). It has been et al., 2015). These outbreaks are associated with sanitation protocol shown that L. monocytogenes can be transferred from food to surfaces and failure, poor hygiene, and other environmental factors. In the food in­ vice versa, possessing a substantial ability to survive desiccation, adhere dustry, stainless steel is widely used as food contact surfaces for its ease to surfaces such as stainless steel, and form biofilms (Hingston et al., of cleaning. However, these surfaces also tend to become contaminated 2013; Truelstrup Hansen & Vogel, 2011). when hygienic conditions are not adequate, causing bacteria to persist Microbiological detection in food production and preparation envi­ on them (Truelstrup Hansen & Vogel, 2011). ronments guarantee the commercialization of safe food for consumption * Corresponding author. Facultat de Veterinària, Universitat Autònoma de Barcelona, Travessera dels Turons s/n. Bellaterra, 08193, Barcelona, Spain. E-mail address: josejuan.rodriguez@uab.cat (J.J. Rodríguez-Jerez). https://doi.org/10.1016/j.foodcont.2022.108971 Received 22 October 2021; Received in revised form 14 March 2022; Accepted 15 March 2022 Available online 17 March 2022 0956-7135/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). A.G. Ríos-Castillo et al. Food Control 137 (2022) 108971 (Ríos-Castillo et al., 2021). Given that currently in the food industry, cm in diameter, and 1 mm thick. Surfaces were cleaned and sterilized at food production can be on a large scale with short times between pro­ 121 ◦ C for 15 min before their use in the tests. Once the concentration of duction, an effective and rapid detection of bacterial pathogens on food the bacterial suspensions was determined by microscopy, the surfaces contact surfaces and other surfaces involved in the production is were inoculated in quadruple with 10, 100, and 1000 cells in volumes necessary in order to take immediate hygienic and sanitary measures less than 50 μL. Three surfaces of each concentration were used for the (Margas et al., 2014). The presence of Salmonella spp. and study to obtain levels of contamination of 30, 300, and 3000 CFU/10 L. monocytogenes is based on standard microbiological culture methods cm2 and one was analyzed by microscopy, as it is described below in that include pre-enrichment, selective enrichment, isolation, and Section 2.3. An inoculum control for each inoculated concentration was microbiological characterization, all of which take several days. In this carried out ten times in TSA plates. context, fast and accurate methods to identify pathogenic bacteria are of significant value in the food industry to shorten the analysis time. 2.3. Dry conditions Real-time PCR is considered a fast and sensitive molecular method to detect and quantify bacterial pathogens. As in the case of conventional Immediately after the inoculation of surfaces, they were dried in a methods, an enrichment treatment can be combined with PCR to laminar flow cabinet at 37 ◦ C for a time not exceeding 1 h to facilitate enhance assay sensitivity by ensuring the presence of viable pathogens microbial adherence to the test surfaces. After the drying time elapsed, because they often occur in very low numbers (Ferretti et al., 2001; Park one of the four disc surfaces per concentration group was randomly et al., 2014). Although real-time PCR has been widely used for detecting chosen and analyzed by microscopy (Olympus BX51/BX52) using the pathogens in food, its effectiveness in determining their presence on Live/Dead® BacLight™ kit. A pre-enrichment step was applied to the surfaces is not particularly accurate. The objective of this study was to three remaining inoculated disc surfaces. determine the capacity to detect Salmonella enterica serovar Typhimu­ rium and Listeria monocytogenes adhered to stainless steel surfaces under 2.4. Pre-enrichment dry conditions by real-time PCR, comparing it with the conventional methodology recommended. The study also considered various The remaining three disc surfaces of each concentration group were pre-enrichment times and the concentration of the samples obtained placed in parallel in flasks and in 50 mL conical tubes with 10 mL of pre- from the surfaces. enrichment broth: TSBYE-T80 (TSBYE with 1 g/L of Tween 80) for S. Typhimurium and DFraser-T80 (Demi-Fraser Broth with 1 g/L of Tween 2. Materials and methods 80) for L. monocytogenes. The flasks and tubes were allowed to rest for 5 min and then mixed by vortex for 1 min at maximum speed. The flasks 2.1. Bacterial strains and test surfaces and tubes with the discs were then left to rest for different pre- enrichment times: 0 h (immediately after mixing by vortex), 6 h, and Salmonella enterica serovar Typhimurium (S. Typhimurium) (CCUG 18 h for S. Typhimurium (incubated at 37 ◦ C), and 0 h, 6 h, and 25 h for 29478) and Listeria monocytogenes (L. monocytogenes) (CCUG 15526) L. monocytogenes (incubated at 30 ◦ C). Once the pre-enrichment times were the standard bacterial strains used for this study (Culture Collec­ had elapsed, the flasks and tubes were mixed by vortex for 1 min, and tion of the University of Göteborg, Sweden). The strains, conserved on the contents were recovered by either without concentrating or cryoballs at − 20 ◦ C, were resuspended in tryptone soy broth with 6 g/L concentrating the microorganisms. The procedure followed to recover of yeast extract (TSBYE; Becton Dickinson and Co., Le Pont De Claix, the target pathogens without concentrating the microorganisms con­ France) and incubated at 37 ◦ C for 24 h. Afterwards, they were cultured sisted of taking the aliquots directly from the flasks after being mixed. on tryptone soy agar (TSA; Becton Dickinson and Co.) and incubated at The recovery procedure, concentrating the microorganisms, was per­ 37 ◦ C for 24 h. Once the incubation time had elapsed, isolated colonies formed from the tubes removing the discs previously. Then, the tubes of each microorganism were transferred to 10 mL of TSBYE and incu­ were centrifuged at 12,000×g for 5 min. The supernatants were dis­ bated at 37 ◦ C for 18–24 h, obtaining bacterial cultures in the stationary carded, and the pellets were resuspended in 3 mL TSBYE-T80 broth for S. phase. The cultures were centrifuged at 9800×g for 10 min and washed Typhimurium and 3.5 mL DFraser-T80 broth for L. monocytogenes. The twice using saline solution (8.5 g/L NaCl). The resulting pellets were discs from the tubes were recovered to determine the efficiency of resuspended in 8.5 mL of saline-tryptone solution (8.5 g/L NaCl, 1.0 g/L bacterial detachment from the surfaces. For this stage, surfaces were tryptone, pH 7.0 ± 0.2) to obtain bacterial suspensions with an washed with 10 mL of phosphate-buffered saline (PBS; 8.0 g/L sodium approximate concentration of 108 colony-forming units per milliliter chloride, 0.2 g/L sodium potassium, 1.15 g/L bisodium phosphate, 0.2 (CFU/mL). The concentration of the bacterial suspensions was quanti­ g/L monopotassium phosphate; pH 7.3). Once washed, they were fied by inoculating 20 μL in the center of stainless steel disc surfaces that transferred to Petri dishes and cultured with TSA with 6 g/L yeast were subsequently analyzed by direct epifluorescence microscopy extract (TSAYE; Becton Dickinson and Co) at 37 ◦ C for 24–48 h. (DEM, Olympus BX51/BX52; Olympus, Tokyo, Japan). A Live/Dead® BacLight™ Kit (L-13152, Molecular Probes, Inc., Oregon, USA) was used 2.5. Count and detection of microorganisms for microscopic viable cell counting. The kit contains two fluorescent nucleic acid-binding stains: SYTO 9 and propidium iodide. SYTO 9 2.5.1. Plate count penetrates the cells with intact and damaged cell membranes and dyes To determine the total viable cell counts, a 1 mL aliquot of TSBYE- them green. On the other hand, propidium iodide penetrates only cells T80 and DFraser-T80 broths was cultured in duplicate in TSAYE and with damaged cell membranes, reducing the SYTO 9, and stains non- incubated at 37 ◦ C for 24–48 h. viable cells red. The kit was used by adding 20 μL of the stain to each disc surface with the corresponding cell concentrations. The surfaces 2.5.2. Conventional culture were then left in the dark at room temperature for 15 min to allow the The cultures for detecting and enumerating microorganisms were stains to penetrate the bacterial cells. Ten microscopic images were prepared according to ISO 6579-1:2017/AMD 1:2020 (ISO, 2020) for S. acquired to determine the viable cells in green color using the software Typhimurium and ISO 11290-1:2017 (ISO, 2017) for L. monocytogenes. analySIS Auto 3.2 (Soft Imaging System GmbH, Münster, Germany). The procedure for S. Typhimurium involved transferring 0.1 mL aliquot of TSBYE-T80 to 10 mL of Rappaport-Vassiliadis selective enrichment 2.2. Characteristics of the surfaces and inoculation broth (bioMérieux, Marcy-l’Étoile, France) and incubating at 42 ◦ C for 24 h. Subsequently, loops of the enrichment broth were streaked on The test surfaces were stainless steel discs type 304 grade 2B finish, 2 chromID Salmonella agar plates (bioMérieux, France) and incubated at 2 A.G. Ríos-Castillo et al. Food Control 137 (2022) 108971 37 ◦ C for 24–48 h. In the case of L. monocytogenes, 0.1 mL aliquot of microorganism recovery method without a concentration of the samples DFraser-T80 was transferred to 10 mL of Fraser selective enrichment were compared using the Analysis of Variance and the subsequent post broth (bioMérieux, France) and incubated at 37 ◦ C for 48 h. Loops of hoc Student-Newman-Keuls (SNK) test with a significance level of α = DFraser-T80 and Fraser broths were streaked onto PALCAM (bio­ 0.05. Ct values were presented as mean ± standard deviations. The Mérieux, France) and ALOA (AES Laboratoire, Combourg, France) agar relationship between Ct values and the viable cell count obtained by plates and incubated at 37 ◦ C for 24–48 h. TSAYE was compared by regression analysis. Statistical analyses were performed using the SAS statistical program (version 9.1; SAS Institute, 2.5.3. Real-time PCR analysis Inc., USA). Real-time PCR analysis was performed with iQ-Check™ Salmonella and iQ-Check™ L. monocytogenes kits (Bio-Rad Laboratories, Inc; Her­ 3. Results and discussion cules, CA, USA) according to the manufacturer’s instructions. The pro­ cedure consisted of using aliquots of 1.0 mL of TSBYE-T80 for S. 3.1. Detection of surface-adhered S. Typhimurium Typhimurium and 1.5 mL of DFraser-T80 for L. monocytogenes. Aliquots were transferred to Eppendorf tubes and centrifuged at 11,700×g for 5 The results for S. Typhimurium adhered to surfaces in dry conditions min. Subsequently, the supernatants were removed, and the pellets were are shown in Table 1. When the contamination level was 30 CFU/10 mixed with the lysis reagents (supplied with the iQ-Check kits): 200 μL cm2, without pre-enrichment (0 h) and without concentrating or for S. Typhimurium and 250 μL for L. monocytogenes. The obtained concentrating the samples, S. Typhimurium was not detected by the mixtures were vortexed for 30 s at full speed. Then, the tubes were conventional ISO method (0/6), whereas real-time PCR detected 5 of 6 placed in a water bath at 100 ◦ C for 15 min, cooled at room temperature samples (5/6). At this same level of contamination, with a pre- for 5 min, and again vortexed at maximum speed for 15 s. The tubes enrichment time of 6 h, real-time PCR detected 2/6 without concen­ were again centrifuged at 11,700×g for 5 min. Then, the DNA was trating the samples, and the detection was total (6/6) after concen­ amplified by adding 45 μL of PCR mix that contained the amplification trating the samples. With 18 h of pre-enrichment, the detection was 4/6 solutions and fluorescent probes to 5 μL of the supernatants on well by the conventional method and total (6/6) by real-time PCR using both plates. Thereafter, the well plates were sealed with optical caps and recovery procedures. When the level of contamination was increased to introduced into the thermocycler Chromo4™ RT-PCR Detection (Bio- 300 and 3000 CFU/10 cm2, the detection of the conventional method Rad Laboratories, Inc). The thermocycler program steps were: 1st at was only total with the pre-enrichment times of 6 and 18 h using both 50 ◦ C for 2 min, 2nd at 95 ◦ C for 10 min, and 3rd (repeated 50 times) at recovery procedures. In the case of real-time PCR, the detection was 95 ◦ C for 20 s, 55 ◦ C for 20 s, and 72 ◦ C for 30 s. The obtained threshold total with all the pre-enrichment times (0, 6, and 18 h) for both pro­ cycle (Ct) values were analyzed using Opticon Monitor software v 3.1 cedures. According to the results, the detection limit without concen­ (Bio-Rad Laboratories, Inc). trating the samples for the real-time PCR was 300 CFU/10 cm2 without pre-enrichment, and it was reduced to 30 CFU/10 cm2 with 18 h of pre- 2.6. Statistical analysis enrichment. In the case of the procedure concentrating the samples, the real-time PCR detection limit was 300 CFU/10 cm2 without pre- The tests were carried out in quadruple and repeated three times on enrichment and 30 CFU/10 cm2 with 6 h pre-enrichment. Although no different days (n = 12). Each test to compare the frequency of detection differences were observed between the detection limits without pre- by the conventional method and real-time PCR was performed for each enrichment with both recovery procedures, it was possible to reduce microorganism, recovery method, contamination levels, and pre- the time to 6 h for detecting 30 CFU/10 cm2 with the procedure enrichment times. Viable cell counts in TSAYE were expressed in deci­ concentrating the samples. Our results indicate that the detection of low mal logarithmic units (log). The results obtained by TSAYE by the S. Typhimurium contamination adhered to surfaces was more sensitive Table 1 Comparison of conventional culture method (ISO 6579-1) and real-time PCR for detection of S. Typhimurium adhered under dry conditions to stainless steel with different levels of contamination, pre-enrichment times, and recovery methods. Recovery method Contamination level (CFU/10 cm2)a Pre-enrichment time (h) TSAYE (log CFU/mL)b Ct (Threshold cycle)c Detection frequencyd ISO 6579-1 Real-time PCR Without concentration 30 0 0.05c ± 0.12 38.50 ± 1.31 0/6 5/6 6 1.08b ± 1.05 37.68 ± 0.57 3/6 2/6 18 8.85a ± 0.46 15.21 ± 0.73 4/6 6/6 300 0 0.78c ± 0.31 36.34 ± 0.66 2/6 6/6 6 3.68b ± 0.47 30.73 ± 1.94 6/6 6/6 18 8.97a ± 0.24 15.51 ± 0.49 6/6 6/6 3000 0 1.56c ± 0.30 33.30 ± 0.36 4/6 6/6 6 4.37b ± 0.44 28.25 ± 0.83 6/6 6/6 18 9.03a ± 0.20 14.79 ± 0.69 6/6 6/6 Concentration 30 0 36.78 ± 2.29 0/6 5/6 6 36.25 ± 1.56 6/6 6/6 18 13.63 ± 0.45 4/6 6/6 300 0 34.01 ± 2.96 3/6 6/6 6 31.18 ± 1.13 6/6 6/6 18 13.96 ± 0.33 6/6 6/6 3000 0 32.45 ± 0.45 4/6 6/6 6 29.91 ± 1.04 6/6 6/6 18 13.86 ± 0.07 6/6 6/6 a–c Mean values with different lowercase letters are statistically different (p < 0.05). a Total contamination level corresponding to three stainless steel discs. b Mean values of cell count ± standard deviation; n = 6. c Mean values of Ct ± standard deviation; n = 6. d Number of positive samples/number of samples analyzed. 3 A.G. Ríos-Castillo et al. Food Control 137 (2022) 108971 using real-time PCR than the conventional method. In addition, the re­ BPW were 10, 1, and 0.1 CFU/g, respectively. sults of the conventional culture method were inconsistently positive in As in our study, other works carried out on food and environmental samples without pre-enrichment for all levels of contamination, and a samples have shown that although rapid methods are highly efficient in minimum enrichment period of 6 h was necessary for all samples with detecting Salmonella spp., pre-enrichment increases efficacy. This 300 CFU/10 cm2 to give positive results. This observation agrees with improvement particularly occurs when bacteria such as Salmonella spp. other works, which described that PCR has the same or increased grow in a low concentration or in a sub-lethal state (Park et al., 2014; sensitivity compared to the culture method when tests were performed Taskila et al., 2012). Lin et al. (2004) observed that through a preculture from environmental samples and food processing facilities (Hyeon et al., step with selenite cystine broth for 8 h previous to the PCR assay, Sal­ 2019; Nadin-Davis et al., 2019). monella spp. serovars from milk and raw chicken meat could be detected The S. Typhimurium count in TSAYE in the samples without con­ at concentrations as low as 1–9 CFU/g. Ferretti et al. (2001) demon­ centration showed that even though this microorganism was subjected strated that PCR with 6 h non-selective enrichment using BPW detected to stress through desiccation on the surfaces prior to pre-enrichment, it Salmonella spp. in food at the level of 1 CFU/100 mL. In our study, with a recovered when the level of contamination and the times of pre- concentration procedure by centrifugation after sampling, a low number enrichment increased (p < 0.05). Thus, the contamination level of of Salmonella spp. was detected using real-time PCR without 3000 CFU/10 cm2 showed the highest counts in TSAYE: 1.56 log CFU/ pre-enrichment. mL (0 h), 4.37 log CFU/mL (6 h), and 9.03 log CFU/mL (18 h). A high correlation was also observed between the Ct values obtained by real- 3.2. Detection of surface-adhered L. monocytogenes time PCR and the viable cell counts in TSAYE (R2 = 0.9718; p < 0.0001) (Fig. 1). The increase in S. Typhimurium count, while the Ct The results of L. monocytogenes detection adhered to surfaces in dry values were decreasing, indicated that the recovery with the pre- conditions are provided in Table 2. With a contamination level of 30 enrichment times was adequate because the Ct values were inversely CFU/10 cm2, the conventional ISO method did not detect proportional to higher amounts of target DNA in the sample (Pérez et al., L. monocytogenes when the procedure was used without concentration of 2013). samples and with any of the pre-enrichments (0, 6, and 25 h). However, The infective dose of Salmonella spp. to cause illness associated with at 0 and 6 h of pre-enrichment and concentrating the samples, L. mono­ food consumption is low. Salmonellosis has been reported to result from cytogenes was detected for both in 1/6 samples (1/6). When the samples less than 10 cells/100 g in naturally contaminated cheese (Ratnam & were evaluated by real-time PCR at this level of contamination, March 1986). Surfaces are one of the most important sources from which L. monocytogenes was detected at 0 (5/6), 6 (5/6), and 25 h pre- bacterial pathogens contaminate food; hence, surface detection becomes enrichment (4/6) without concentrating the samples; detection using important. The detection limits previously reported for Salmonella spp. real-time PCR was total (6/6) at 0 and 6 h when concentrating the by real-time PCR, although not on surfaces but on different foods and samples. With a contamination level of 300 CFU/10 cm2, the conven­ environmental samples, are comparable to our results. Malorny et al. tional method partially detected L. monocytogenes at 6 (1/6) and 25 h (3/ (2004) studied Salmonella Enteritidis and detected <3 CFU/50 mL 6) without concentrating the samples and 2/6 at 25 h when concen­ carcass rinse from whole chicken or 10 mL whole eggs with real-time trating the samples. In the case of detection by real-time PCR, the PCR, preceded by a pre-enrichment step (18–24 h) in BPW, with the detection was complete with all pre-enrichment times, whether or not sensitivity and specificity of this method being 100% equivalent to the the samples were concentrated. When the level of contamination was traditional culture-based detection method. In addition (Hyeon et al., increased to 3000 CFU/10 cm2, the results by the conventional method 2019), noted that the detection limits of Salmonella spp. carried out by a showed that the detection was not total after 25 h pre-enrichment (5/6) combination of immunomagnetic separation, multiple displacement without concentrating the samples. By concentrating the samples, amplification, and real-time PCR with 0, 4 to 6, and 8 h enrichment with L. monocytogenes was partially detected at 0 (2/6), 6 (1/6), and 25 h pre- Fig. 1. Relationship between counts in TSAYE and Ct values obtained for S. Typhimurium adhered to stainless steel surfaces. 4 A.G. Ríos-Castillo et al. Food Control 137 (2022) 108971 Table 2 Comparison of conventional culture method (ISO 11290-1) and real-time PCR of L. monocytogenes detection adhered under dry conditions to stainless steel surfaces with different levels of contamination, pre-enrichment times, and recovery methods. Recovery method Contamination level (CFU/10 cm2) a Pre-enrichment time (h) TSAYE (log CFU/mL) b Ct (Threshold cycle) c Detection frequency d ISO 11290-1 Real-time PCR Without concentration 30 0 < LD 40.27 ± 1.34 0/6 5/6 6 < LD 39.99 ± 0.61 0/6 5/6 25 < LD 40.06 ± 0.93 0/6 4/6 300 0 0.23b ± 0.36 36.24 ± 1.56 0/6 6/6 6 0.39b ± 0.61 36.96 ± 0.54 1/6 6/6 25 2.48a ± 0.2.8 31.42 ± 6.61 3/6 6/6 3000 0 0.30b ± 0.27 33.52 ± 0.66 0/6 6/6 6 0.23b ± 0.27 33.82 ± 1.07 0/6 6/6 25 3.45a ± 2.03 24.58 ± 1.88 5/6 6/6 Concentration 30 0 37.48 ± 1.91 1/6 6/6 6 39.03 ± 1.47 1/6 6/6 25 38.80 ± 0.78 0/6 5/6 300 0 36.47 ± 0.79 0/6 6/6 6 36.18 ± 0.91 0/6 6/6 25 32.02 ± 6.03 2/6 6/6 3000 0 31.89 ± 1.91 2/6 6/6 6 33.27 ± 0.95 1/6 5/6 25 31.69 ± 6.85 4/6 5/6 < LD: Lower than detection limits. a–b Mean values with different lowercase letters are statistically different (p < 0.05). a Total contamination level corresponding to three stainless steel discs. b Mean values of cell count ± standard deviation; n = 6. c Mean values of Ct ± standard deviation; n = 6. d Number of positive samples/number of samples analyzed. enrichment (4/6). Using real-time PCR, the detection was total with the Gattuso et al. (2014), who observed that real-time PCR showed higher three pre-enrichment times without concentrating the samples, and performance in detecting L. monocytogenes compared to the standard when concentrating the samples, the detection was total (6/6) at 0 h and method using reduced volumes of half-Fraser broth. The inconsistency 5/6 at 6 and 25 h. in the positive results by the conventional method can be explained According to the results, the real-time PCR detection limit for because the pre-enrichment broth (DFraser-T80) could have suppressed L. monocytogenes with the procedure without concentrating the samples the recovery of cells that were sublethally injured during drying. The and without pre-enrichment was 300 CFU/10 cm2. When concentrating finding is also explained because when applying the sample without the samples, the detection limit was not established because inconsistent concentration procedure, the viable cell counts in TSAYE did not show positive results were obtained. Furthermore, the results showed that the differences at 0 and 6 h (p > 0.05) with contamination levels of 300 and detection of L. monocytogenes by real-time PCR was more sensitive than 3000 CFU/10 cm2, and the count only increased with a pre-enrichment by the conventional method (Table 2). These findings agree with time of 25 h. Besides, the real-time PCR results showed a low correlation Fig. 2. Relationship between counts in TSAYE and Ct values obtained for L. monocytogenes adhered to stainless steel surfaces. 5 A.G. Ríos-Castillo et al. Food Control 137 (2022) 108971 between Ct values and viable cell counts in TSAYE (R2 = 0.5755, p < washing with 10 mL of PBS could not recover. Furthermore, in our 0.0001). In this sense, the samples using 0 and 6 h of pre-enrichment study, the time required for adhesion was not exceeding 1 h. In this showed a higher dispersion compared to the results obtained after 25 sense, Lang et al. (2004) observed that Salmonella spp. and h, where the values were observed mainly related to the viable cell L. monocytogenes could adhere to tomato surfaces in 1 h at 20 ◦ C under counts (Fig. 2). This observation demonstrates that the pre-enrichment dry conditions. In another study, carried out on fruit skin, 1 h after medium is essential, significantly influencing the sensitivity of detec­ Salmonella inoculation, extensive fixation structures were formed that tion methods and increasing the probability of recovering injured cells helped to irreversibly anchor the pathogen to the surface (Mathew et al., (Nam et al., 2005). 2018). Although the infective dose of L. monocytogenes is considered high Salmonella spp. and Listeria monocytogenes can survive under various (103 CFU), the initial contamination with low numbers of this pathogen stress conditions, such as desiccation (Lang et al., 2004; Ríos-Castillo can be potentially dangerous due to its ability to grow at low tempera­ et al., 2020). The fact that both studied pathogens could remain viable tures (Bhagwat, 2003; Walker et al., 1990). Hence, it is important that on dry surfaces represents a potential recontamination risk. The capacity the detection limit of rapid methods fulfills this characteristic for prac­ of Salmonella spp. to adhere and form biofilms at room temperature is of tical application purposes in food processing plants. Liming and Bhag­ particular interest to the food industry. This pathogen even has the wat (2004) indicated a detection limit of L. monocytogenes of 4–7 ability to survive on food contact surfaces after long periods of time CFU/25 g of mixed salad, with 25 h enrichment time and using the under dry conditions (Gruzdev et al., 2011; Margas et al., 2014). iQ-Check protocol; however, the inoculated cells were not subjected to Therefore, regular and adequate cleaning of surfaces against Salmonella any stress process, and the samples were processed within 10 min after spp. is necessary (Humphrey, 2004; Ripolles-Avila et al., 2020). In the inoculation. Rodríguez-Lázaro et al. (2005) observed that real-time PCR case of L. monocytogenes, this microorganism can easily enter with a previous procedure that involved filtration and DNA purification food-processing environments, adhere to surfaces, and multiply even at could detect 10 CFU/g L. monocytogenes from smoked salmon, showing low temperatures, leading to an increased risk of cross-contamination the high accuracy of real-time PCR compared to the standard plate count from surfaces to food (Carpentier & Cerf, 2011; Muhterem-Uyar et al., method. In another study carried out in 11 laboratories, 10 CFU/25 g 2015). L. monocytogenes were detected in artificially contaminated cheese Today, standard pathogen detection methods can determine the samples using real-time PCR with enrichment and DNA extraction in 27 presence of a low number of viable cells after pre- and selective h, demonstrating that this procedure was more sensitive than the ISO enrichment steps (Chapela et al., 2015). However, when pathogens are reference method (Gianfranceschi et al., 2014). Oravcová et al. (2007), subjected to stressful conditions, the detection methods’ sensitivities can using a two-step enrichment involving a 24-h incubation in half-Fraser be interfered with and, in turn, be ineffective (Myint et al., 2006). The broth followed by a 6-h subculture in Frasger broth, detected 1 CFU/g ability of real-time PCR to detect DNA from dead bacteria as false pos­ with real-time PCR from 61 food samples contaminated at a level of 100 itives is considered a disadvantage (de Boer et al., 2015; Wolffs et al., CFU per 25 g. Moreover, in the same work, real-time PCR detected 2005). However, false-positive PCR signals can also occur due to the L. monocytogenes in all samples; by contrast, the conventional ISO presence of a high initial number of the pathogen and the time that method only detected 58 positive samples. elapses between bacterial death and analysis (Wolffs et al., 2005; Young et al., 2007). In this sense, if DNA degradation is rapid, it minimizes the 3.3. S. Typhimurium and L. monocytogenes detection under dry risk of a false positive; however, when a partial release of DNA occurs or conditions is protected by dead cells, the risk increases (Wolffs et al., 2005). Therefore, it is essential to ensure optimal enrichment to guarantee For both microorganisms, real-time PCR showed a better detection bacterial recovery and growth during PCR detection analyses (Myint limit of S. Typhimurium and L. monocytogenes under dry conditions et al., 2006). In addition, PCR is more sensitive than conventional compared to the conventional culture method. Similar results were methods because microorganisms can be presented in a viable but observed by Mafu et al. (2009), who indicated that real-time PCR was non-culturable state (VBNC), and the growth of target cells can be more sensitive and faster than the standard microbiological method for inhibited by the presence of other bacteria during enrichment (Patel detecting Salmonella spp., L. monocytogenes, and Escherichia coli O157: et al., 2006). The PCR detection of cells in the VBNC state is an advan­ H7 when at least 103 CFU/mL bacteria were present on surfaces of wood, tage because certain microorganisms have the capacity to repair the polypropylene, and stainless steel using a single enrichment medium. damage caused by environmental factors and remain in a potentially Under natural conditions, bacteria can be found in various states of infectious VBNC state (Fakruddin et al., 2013; Jackson et al., 2009). stress and with sublethal damage, whereas when grown in pure cultures Food processing can lead to bacterial stress and result in damaged or under laboratory conditions, viability may not necessarily be affected dead bacteria; therefore, the detection in these states indicates the and therefore, microbiological results may be overestimated. This fact previous presence or even the permanence of bacteria in a VBNC state, was considered in the present study, applying dry conditions during which is of concern (Ceuppens et al., 2014; de Boer et al., 2015). Ales­ bacterial adhesion to represent real growth conditions. The maximum sandria et al. (2010) observed that real-time PCR detected more positive time required for drying was 1 h at 37 ◦ C. When the Live/Dead® kit was samples of L. monocytogenes than a modified ISO method in samples of applied to verify the vitality of the bacteria after the drying process, it cheese brines and environmental samples from a dairy processing plant, was observed that most of the cells were dead or damaged. When where bacteria have to respond to several stresses, such as disinfectants, comparing the TSAYE counts of the microorganisms inoculated on the starvation, and dried conditions, and therefore, these bacteria could be surfaces, before and after desiccation, a significant reduction (p < 0.05) in a VBNC state. In our study, the detection of pathogens was carried out slightly greater than one log unit was observed at all levels of contam­ on clean surfaces; therefore, we consider it necessary to perform addi­ ination (30, 300, and 3000 cells) for S. Typhimurium and L. mono­ tional studies to evaluate the detection limit of these pathogens on cytogenes (data not shown). In addition, the efficiency of the procedure surfaces with interfering substances that simulate the presence of food to verify the bacterial recovery adhered to the test surfaces when the residues in the food industry. samples were concentrated, performed with washes with PBS and seeding in TSAYE, did not show counts at 0 and 6 h of pre-enrichment for 4. Conclusions either microorganism. However, counts of 3 log CFU/mL at 18 h for S. Typhimurium and 0.47 log CFU/mL at 25 h for L. monocytogenes were Real-time PCR provided a better detection limit than the ISO con­ obtained. These results could be explained due to the increase in the ventional culture method of a low level of contamination of S. Typhy­ number of microorganisms with a longer pre-enrichment time and that murium and L. monocytogenes adhered in dry conditions to stainless steel 6 A.G. Ríos-Castillo et al. 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