1 Introduction

Listeria monocytogenes is a psychrotropic and environmentally resistant pathogen and, thus, a serious public health threat (EFSA 2021). Through natural occurrence in soil, surface water, wastewater, and plants, Listeria enters agriculture and consequently, the food chain. Their excellent adaptability to environmental conditions such as temperature (-2 to 44 °C), salinity (up to 10%) and pH (4.5 to 9.5) allow them to accumulate in food and enter processing environments for different food types e.g., ready-to-eat (RTE) salads, raw milk products, smoked fish or minced meat (EFSA 2021; Farber and Peterkin 1991). Although 13 serotypes of L. monocytogenes have been described, primarily serotype IIa, IIb and IVb cause the disease listeriosis in humans (Cartwright et al. 2013). This food-associated infection poses a particular risk to young, old, pregnant or immunocompromised people (YOPI). Therefore, L. monocytogenes is legally classified as a food safety criterion in Regulation (EC) No 2073/2005. Acting as biofilm formers, the adherent microbial cells produce an extracellular polymeric matrix which protects them from external environmental influences (Oliveira et al. 2010; Zheng et al. 2021). This protection enables L. monocytogenes to persist permanently in food-producing plants. Predisposed are places which are difficult to access for cleaning and disinfection and permanently moist (e.g., drains, corners, and cracks). Biofilms can also occur in processing, storage or refrigeration rooms (Tompkin 2002). The high attachment ability of L. monocytogenes to food contact surfaces such as stainless steel, polytetrafluoroethylene and high-density polyethylene has already been described (Blackman and Frank 1996; Silva et al. 2008). Cross-contamination of L. monocytogenes between hands, food, and food contact surfaces can also occur (Midelet and Carpentier 2002). Therefore, the most important parts of hygiene monitoring are detection, quantification, and identification of pathogenic microorganisms from surfaces. The earliest possible detection through a suitable sampling plan and sampling procedure is essential. It prevents cross-contamination in food production plants and helps to assess and prevent biological hazards. The selection of the sampling technique depends on material, size, nature and moisture of the sampled surface. General requirements for sampling of surfaces in the food chain environment are regulated in ISO 18593:2018. Studies by Lahou and Uyttendaele (2014) and Faille et al. (2020) confirmed the basic suitability of stick swabs, scratch sponges and contact agar for the detection of L. monocytogenes on various surfaces such as stainless steel, polyvinyl chloride, rubber or wood. However, previous studies were restricted to either qualitative or quantitative comparison of different sampling devices on certain contact surfaces.

Therefore, the aim of this study was to compare the sensitivity of different swab materials (cotton, viscose and nylon-flocked) for qualitative and quantitative detection of L. monocytogenes on typical food contact surfaces (stainless steel, polyvinyl chloride, polytetrafluoroethylene and high-density polyethylene).

2 Materials and methods

2.1 Inoculum preparation

Three L. monocytogenes test strains (serotype IIa, IIb and IVb) were obtained from the national reference laboratory for L. monocytogenes of the Federal Institute for Risk Assessment Berlin and has been originally isolated from industrial surface samples in food processing plants. They were cryopreserved at -80 °C (Cryobank, Mast Group Ltd., Germany). Fresh working cultures were established on trypticase soy yeast agar (TSY; Merck KGaA, Germany) and used for experiments for 4 weeks. A bacterial suspension of all 3 tested strains was prepared for each experiment by adding a single colony of L. monocytogenes from a TSY plate to 4.95 ml tryptone soy broth (TSB; sifin diagnostics GmbH, Germany) using an inoculation loop. Stationary phase and a cell density of 1.0 × 109 CFU/ml were reached after 16 h of incubation at 37 °C. Daily initial concentration of the bacterial suspensions was determined for each individual experiment.

2.2 Surface preparation

Stainless steel (steel; material type V2A; chrome-nickel steel X5CrNi18-10; Hornbach Baumarkt AG, Germany), polyvinyl chloride (PVC, S-Polytec GmbH, Germany), polytetrafluoroethylene (PTFE, Teflon®, food conformity according to Regulation (EU) No 10/2011 and Food and Drug Administration (FDA), S-Polytec GmbH, Germany) and high-density polyethylene (HDPE, food approval according to German Food and Feed Code (LFGB) and Regulation (EC) No 1935/2004, food conformity according to Regulation (EU) No 10/2011 and FDA, S-Polytec GmbH, Germany) were used as food contact surfaces. All surfaces had a length and width of 15 cm and a total area of 225 cm2. Pre-treatment included decontamination for 60 min in 5% Decon 90 solution (Decon™, Fisher Scientific GmbH, Germany), manual cleaning with detergent (MANUDISH, tana-Chemie GmbH, Germany), coarse rinsing with hand-warm tap water (30.0–35.0 °C) and additional rinsing with heated distilled water (85.0–90.0 °C) followed by air drying in a biosafety cabinet. Test surfaces were exposed to UV light for 30 min in a biosafety cabinet before each experiment.

2.3 Surface inoculation

A sampling template (blue square template, Technical Service Consultants Ltd, United Kingdom) marks the sampling area (100 cm2). The initial concentration (1.0 × 109 CFU/ml) of the inoculum was reduced by a decadic dilution series. The applied volume of 400 μl/100 cm2 simulates a contamination of approx. 1.0 × 101-1.0 × 102 CFU/100 cm2 for qualitative detection and approx. 4.0 × 104 CFU/100 cm2 for quantitative detection. Six surfaces of the same material were inoculated simultaneously with 80 drops of 5 μl each using an 8-channel pipette. Inoculated surfaces were incubated for 15 min in a biosafety cabinet.

2.4 Sampling procedure

Sampling was performed by double-swab technique using cotton (C; head diameter 5 mm; length 150 mm; polypropylene stick; Carl Roth GmbH + Co. KG, Germany), viscose (V; head diameter 4.6 mm; length 105 mm; polystyrene stick; Carl Roth GmbH + Co. KG, Germany) and nylon-flocked (N; PurFlock Ultra®; head diameter 7 mm; length 150 mm; polystyrene stick; Check Diagnostics GmbH, Germany) swabs (Fig. 1). The wet swab was moistened with sodium chloride peptone solution (Carl Roth GmbH + Co. KG, Germany) for 5 min before using. The surface sampling was carried out according to ISO 18593:2018. Each surface was swabbed horizontally, vertically and diagonally in a meandering pattern by using the wet swab first followed by the dry swab (Fig. 2). Both swab heads were cut off with sterile scissors and vortexed in a sterile test tube with 2 ml (qualitative detection) or 9 ml (quantitative detection) sodium chloride peptone solution for 30 s at 2500 rpm (IKA®-Werke GmbH & CO. KG, Germany). Swabs were stored for 4 and 24 h at 5 ± 1 °C until further processing.

Fig. 1
figure 1

Scanning electron microscopy images of cotton (a), viscose (b) and nylon-flocked (c) swab. Shown are swab head (left, 96×, bar 1 mm), enlarged view of the sterile (centre, 1.500×, bar 50 μl) and L. monocytogenes contaminated swab fibre (right, 10.000×, bar 10 μm)

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Fig. 2
figure 2

Illustration of sampling procedure. Wet swab was first swept across the surface in meandering pattern horizontally (black arrowheads), vertically (dark grey) and diagonally (light grey), followed by the dry swab in the same way

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2.5 Recovery and enumeration of L. monocytogenes

2.5.1 Qualitative recovery

Recovery was carried out according to ISO 11290-1:2017 as an enrichment method. Swabs were filled with 5 ml of half-Fraser broth (sifin diagnostics GmbH, Germany) and incubated at 30 °C for 24 h as the primary enrichment. Then 100 μl of the primary enrichment was pipetted into 10 ml Fraser broth (sifin diagnostics GmbH, Germany) and incubated at 37 °C for 24 h as the secondary enrichment. After incubation, 100 μl of the secondary enrichment was plated on Listeria agar according to Ottaviani and Agosti (AL; Bio-Rad Laboratories GmbH, Germany) and PALCAM Listeria agar (PAL; sifin diagnostics GmbH, Germany) and incubated for 24 − 48 h at 37 °C. L. monocytogenes typical colony growth (blue-green colonies surrounded by an opaque halo on AL; grey-green colonies with black halo on PAL) on one or both agar plates were regarded as positive detection. Negative results were obtained if both plates showed no or atypical growth.

2.5.2 Quantitative recovery

Stored swabs were vortexed in tubes for 30 s at 2500 rpm. A decadic dilution series in sodium chloride peptone solution was prepared and 100 μl spread on TSY in duplicate. Incubation for 24 h at 37 °C was followed by counting and calculation of the recultivated colonies per 100 cm2 surface.

2.6 Scanning electron microscopy

Images were analysed by scanning electron microscopy (SEM), which was carried out with a digital scanning microscope (EVO LS 15, Carl Zeiss AG, Germany). The preparations were previously coated with a gold/palladium target. Sterile swabs were incubated in 15 ml TSB with a concentration of 1.0 × 109 CFU/ml for 24 h at 25 °C to visualise attached L. monocytogenes on the swab fibres.

2.7 Data analysis

All statistical analyses were performed using Prism9 (GraphPad Software, LL, USA).

2.7.1 Qualitative analysis

A global chi-square test was used to detect significant differences between the swabs used. If the global chi-square test showed statistical significance, all groups were compared pairwise and the p-value was adjusted using Bonferroni correction. The significance level was set at α = 0.05.

2.7.2 Quantitative analysis

Actual bacterial concentrations for inoculation of surfaces were considered, as there were slight differences on different experimental days. All bacterial counts were log10 transformed for statistical analysis. The recovery rate for sampling methods was determined by transforming the average of the inoculum concentration and comparing to the quantitative recovery of the tested surfaces. The mean recovery values were averaged from the tested observations. Data was tested for Gaussian distribution by Shapiro-Wilk test. Normal distribution was confirmed for almost all data sets, so parametric tests were chosen for statistical comparisons. A one-way analysis of variance (ANOVA) was used to determine significant differences between means for each swab material and surface type as well as processing time at a statistical significance of α = 0.05. When the ANOVA indicated a difference between means, Tukey’s multiple range test was used to assess significant differences pairwise between group means.

3 Results

3.1 Qualitative detection

For qualitative detection, surfaces were contaminated with approx. 1.0 × 101-1.0 × 102 CFU/100 cm2 of L. monocytogenes. With the exception of 60% (steel/viscose/4 h) and 70% (steel/nylon-flocked/4 h as well as PVC/viscose/4 h), the detection rates were at least 80%. Particularly high detection rates of 90 and 100% could be achieved on PTFE and HDPE with different swabs and processing times (Fig. 3).

Fig. 3
figure 3

Mean values of qualitative L. monocytogenes detection (n = 10) from steel (a), PVC (b), PTFE (c) and HDPE (d) surfaces by cotton (C, black), viscose (V, dark grey) and nylon-flocked (N, light grey) swabs at 4 and 24 h after sampling

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3.2 Quantitative detection

The initial load of all 9 experiments for quantitative detection of L. monocytogenes was approx. 4.0 × 104 CFU/100 cm2. The results reflect the log-transformed difference of the applied CFU and the recultivated CFU by the swab. This difference is consequently the loss resulting from sampling and recultivation. The smaller the difference, the better the detectability through the swab. For steel, the logarithmic differences were the highest (> 1.50 log10), followed by PVC (1.25–1.50 log10), independent of the swab material and the processing time (psteel=0.239 − 0.469; pPVC=0.164 − 0.463). There was a loss from mean recovery level of inoculum for HDPE also in the range of 1.25 − 1.50 log10 and the only significant difference was observed for cotton/4 h compared to nylonflocked/24 h (pHDPE=0.083–0.179). The smallest loss was observed for PTFE (1.00-1.50 log10; pPTFE=0.103–0.209), for which statistically significant differences were found for all swab time combinations (Table 1) compared to nylon-flocked/24 h (pC4/N24<0.0001; pV4/N24<0.0001; pN4/N24=0.039; pC24/N24=0.021; pV24/N24=0.034).

Table 1 Quantitative detection of L. monocytogenes from stainless steel (steel), polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE) and high-density polyethylene (HDPE) surfaces by cotton (C), viscose (V) and nylon-flocked (N) swabs at 4 and 24 h after sampling. Shown are logarithmic mean values of bacterial loss (± SD) from 9 experiments. Different letters show significant differences within surface groups
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4 Discussion

Persistent L. monocytogenes can be detected by food environment sampling. The prevalence of L. monocytogenes in milking facility environments is up to 19% (Fox et al. 2009) and in the environment of trout processing facilities between 6 and 30% (Autio et al. 1999; Dimitrijevic et al. 2011). Non-food contact surfaces are more often contaminated with L. monocytogenes than food contact surfaces (Dalmasso and Jordan 2013; Kovačević et al. 2012; Tirloni et al. 2020). There also seems to be a correlation between the presence of L. monocytogenes in RTE food and in the environment of these food processing plants (Kovačević et al. 2012). This study detected contamination doses of up to < 3 × 104 CFU/g in RTE salmon products. But most studies report microbial loads in food in a range of < 10 CFU/g or 10–100 CFU/g or detect no positive sample (Andritsos et al. 2013; Angelidis and Koutsoumanis 2006). Therefore, a low contamination dose in the processing environment can be assumed. The swabs were consequently tested for the ability to detect low levels of L. monocytogenes. Additionally, a quantitative detection at a higher concentration was carried out to examine swabs of different materials for differences. Since ISO 11290:1-2017 recommends qualitative detection for L. monocytogenes due to zero tolerance of certain products, swab samples are rarely tested quantitatively for L. monocytogenes. A positive (qualitative) detection is already sufficient to initiate extensive cleaning and disinfection measures in the affected farm.

According to ISO 18593:2018 for the sampling procedure using contact plates or swabs on surfaces in the food industry, the stick swab is the method of choice for sampling hard-to-reach areas. Therefore, the present study investigated the sensitivity of different swab materials for the detection of L. monocytogenes on food contact surfaces. Although the detection rate of L. monocytogenes in swabs was low (Faille et al. 2020; Kang et al. 2007), only the stick swab reached the hard-to-reach areas where L. monocytogenes are mostly detected (Tompkin 2002).

4.1 Influence of swab material

Cotton swabs have been described as having an equal to higher absorbency compared to nylon-flocked swabs. The compact cotton swab, where natural cellulose fibres have high absorbency and are tightly wrapped around the shaft, can absorb more fluid than the loose structure of the nylon-flocked swab (Bruijns et al. 2018; Moore and Griffith 2007). However, the studies of Moore and Griffith (2002) and Brownlow et al. (2012) noted that the absorbed microorganisms were no longer fully released from the cotton swab due to the compact structure, while the open structure of the nylon-flocked swabs was reported to ease the release (Bruijns et al. 2018; Finazzi et al. 2016). Numerous studies hence report an increased recovery rate with nylon-flocked swabs compared to other swab materials (Bruijns et al. 2018; Dalmaso et al. 2008; Dolan et al. 2011; Hedin et al. 2010). The results from PVC, PTFE and HDPE confirm these assumptions. The nylon-flocked swab tended to a higher quantitative detection rate than the cotton or viscose swabs.

4.2 Influence of surface

Regardless of the swab material, fewer cells tended to be detected from stainless steel in the quantitative experiments, compared to the other 3 surface materials. The reasons could be diverse. Silva et al. (2008) already found lower viability of L. monocytogenes on stainless steel compared to polypropylene, while other studies report better attachment ability of L. monocytogenes to stainless steel than to other surfaces (Lahou and Uyttendaele 2014). Preliminary tests also showed that the inoculation volume on stainless steel dries the fastest, regardless of the environmental temperature. The accelerated drying can lead to the loss of viability of individual cells. To circumvent this influence, the incubation time (15 min) in this study was chosen so that a moisture level was still visible on all surfaces, because L. monocytogenes sites are often moist (Tompkin 2002). The sampling of dry surfaces also leads to a significant reduction in the detection rate compared to wet surfaces due to drying loss (Moore and Griffith 2002).

4.3 Influence of processing time

Swabs should be processed within 24 h after sampling according to ISO 18593:2018. Storage at 3 ± 2 °C for up to 48 h is possible in exceptional cases. To simulate different possibilities of official monitoring sample collection and processing, a typical (4 h) and maximum (24 h) processing time was tested. Although few significant differences were detected, the presented results mostly confirm the consistent validity of the swab detection regardless of the processing time, which has already been described by Stewart et al. (2021) for the L. monocytogenes detection on stainless steel.

4.4 Other influences

The pressure exerted by the swab on the surface during sampling is difficult to quantify. In addition to personal differences, the swab design also influences the pressure (Foschino et al. 2003). Therefore, the swabs were selected so that all swab sticks were made of plastic to ensure that all swabs had the same conditions for sampling. More bacteria can be detached from the surface by increasing the mechanical pressure (Moore and Griffith 2002). All the swab samples in the present study were taken by the same person in order to prevent personal differences.

One key moment in swab sample processing is sufficient vortexing (Moore and Griffith 2002). This step is to release the absorbed L. monocytogenes cells from the swab. A longer vortex time of 45 or 60 s does not necessarily release more microorganisms from the swab than 15 or 30 s (Sandle 2017). According to ISO 11290-1:2017, swabs should be vortexed 3 times for 10 s at an appropriate speed. The tested swabs were vortexed after sampling and before further processing at 2500 rpm.

5 Conclusion

Although more effective detection methods than stick swabs have already been described for L. monocytogenes, this work consciously examines swabs for hard-to-reach areas that are inaccessible to scraper sponges, wipes and other sampling devices. This study showed differences in recovery rates between swabs made from the same material. Therefore, it is essential to select the most appropriate sampling device for the intended use before sampling. The insufficient sensitivity of incorrectly selected sampling devices can lead to false negative results and thus misleading conclusions about the cleanliness of the surface and the effectiveness of the cleaning and disinfection measures. Although the observed differences were small and mostly not statistically significant, optimal overall results for detection of L. monocytogenes should be obtained by sampling stainless steel and PVC surfaces with cotton swabs and PTFE and HDPE surfaces with viscose or nylon-flocked swabs.