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FEMS Microbiology Letters logoLink to FEMS Microbiology Letters
. 2025 May 19;372:fnaf048. doi: 10.1093/femsle/fnaf048

Impact of disinfectant-neutralizing buffers used for sampling methods on the viability of adherent Listeria monocytogenes cells on surfaces

Thomas Brauge 1,2,3,✉, Eglantine Chalivat 4, Guylaine Leleu 5, Anthony Colas 6, Graziella Midelet 7
PMCID: PMC12202139  PMID: 40388299

Abstract

This study assessed the impact of six disinfectant-neutralizing buffers on Listeria monocytogenes adhering to stainless steel surfaces treated with quaternary ammonium or hydrogen peroxide-based disinfectants. The goal was to evaluate potential stressors induced by these buffers during sampling, minimizing false negatives in food industry surface monitoring. Neutralizing buffers are essential in preserving bacterial viability during sample transport by counteracting residual disinfectants. Listeria monocytogenes populations were quantified immediately after sampling and after 24-h incubation at 8°C, simulating transport conditions. While neutralizing buffers had minimal impact on untreated adherent cells, they significantly reduced mortality in disinfectant-treated cells. However, most buffers failed to preserve viable culturable (VC) populations after disinfection, instead promoting viable but non-culturable (VBNC) states. Notably, prolonged incubation in the Sponge neutralizer facilitated VC population recovery, likely through VBNC resuscitation or VC growth. In contrast, other buffers inhibited recovery, suggesting detrimental effects on stressed cells due to their chemical composition. These findings underscore the importance of selecting appropriate neutralizing buffers in L. monocytogenes detection, influencing food safety surveillance and risk assessment protocols.

Keywords: Listeria, disinfectant-neutralizing buffers, disinfectants, adhering cells, sampling, VBNC

This study explores how disinfectant-neutralizing buffers affect Listeria monocytogenes detection, highlighting their potential impact on bacterial recovery in food industry surface monitoring.

Introduction

Listeria monocytogenes is a psychrotrophic pathogen that poses a significant threat to public health. It can grow, accumulate, and transfer within food processing environments for various food types, including refrigerated foods like fruits, vegetables, meat, and cold-stored fish and dairy products (Shamloo et al. 2019, Disson et al. 2021). Consuming food contaminated by L. monocytogenes can lead to listeriosis infection, a rare disease affecting high-risk populations with different symptoms and a high mortality rate. In 2022, 2770 confirmed cases of listeriosis were reported by 30 EU/EEA member states, resulting in a notification rate of 0.52 per 100 000 inhabitants (ECDC 2024). Several listeriosis outbreaks have been associated with fresh products, notably an outbreak linked to the consumption of salmon and trout (Lachmann et al. 2022, Halbedel et al. 2023).

Facing this microbial threat, professionals in the food industry resort to a variety of surface disinfectants, carefully selected to eradicate or inhibit the proliferation of microorganisms, including L. monocytogenes, which can colonize surfaces (Charron et al. 2023). Among the disinfectants commonly used in food industry disinfection protocols are quaternary ammonium compounds, amphoteric compounds, hypochlorites, peroxides (peracetic acid and hydrogen peroxide), aldehydes (such as formaldehyde, glutaraldehyde, and paraformaldehyde), as well as phenolic compounds. Recently, disinfection programs have also integrated alkylamines, chlorine dioxide, and mixtures of quaternary ammonium (Carrascosa et al. 2021). However, the efficacy of these disinfectants may be compromised in the presence of biofilm extracellular polymeric substances that act as a protective barrier, preventing the disinfectant from coming into direct contact with bacteria (Bridier et al. 2011). The study of Norwood and Gilmour (2000) showed that L. monocytogenes biofilms exposed to increasing concentrations of sodium hypochlorite exhibited over 100 times greater tolerance to chlorine than their planktonic counterparts. Furthermore, research carried out by Brauge et al. (2020b) and Overney et al. (2017) demonstrated that quaternary ammonium or peracetic acid-based disinfectants failed to eliminate Listeria cells present in biofilms but instead induced a dormant state known as viable but non-culturable cells (VBNCs). Cells in the VBNC state remain viable but are incapable of dividing, making them undetectable by classical microbiological culture-dependent techniques (Wideman et al. 2021). In addition to the VBNC state, disinfection can cause sublethal injuries, also resulting in the non-detection of L. monocytogenes (Arvaniti and Skandamis 2022). These physiological states potentially lead to false-negative results while retaining a potential risk to food safety.

To detect and enumerate the bacteria in the food processing area, professionals commonly employ surface sampling using various tools such as swabs, sponges, contact plates, or gauze pads (Brauge et al. 2020a). While these sampling tools can be used dry, they are generally associated with a nutrient medium containing disinfectant neutralizing buffers. The addition of neutralizers in the sampling device aims to deactivate any residual disinfectant potentially present on the sampled surface. This precautionary measure aims to prevent the persistence of disinfectant action on sampled bacterial cells during sample storage, ensuring the accuracy of microbial load measurements. Various neutralizing buffers have been identified, including compounds such as lecithin, polysorbate, sodium thiosulfate, sodium bisulfite, and aryl sulfonate complex (EN ISO 18593:2018; Li et al. 2020, Hitchins et al. 2022). Additionally, neutralizing buffers also include agents capable of neutralizing the pH, such as disodium phosphate and monopotassium phosphate. Despite guidelines from authorities like the U.S. Food and Drug Administration and International and European standard EN ISO 18593:2018 providing recommendations on the use of neutralizing buffers in sampling tools, questions remain regarding the safety of these neutralizers concerning L. monocytogenes cells (FDA 2017; EN ISO 18593:2018). International and European standard EN ISO 18593:2018 recommends avoiding the use of neutralizers in samples taken from surfaces not exposed to a disinfectant (EN ISO 18593:2018). On the other hand, L. monocytogenes cells on surfaces can undergo various stresses, such as disinfectant treatment, temperature variations, or nutrient deprivation, factors that can damage cells and affect their viability in the presence of neutralizing buffers (Wiktorczyk-Kapischke et al. 2021).

Previous studies have examined the toxicity of various neutralizing buffers on different bacterial species, but the results remain contradictory, and furthermore, they were conducted on planktonic cultures (Kemp and Schneider 2000, Chen 2011, Zhu et al. 2012, Gilbert et al. 2014, Mohammad et al. 2018, Li et al. 2020, Katerji et al. 2023). Li et al. (2020) investigated the impact of three neutralizers including Dey Engley, Letheen, and HiCap on L. monocytogenes cells adhered to stainless steel surfaces and subjected to desiccation stress and subsequent disinfectant treatment. Although this was not explicitly concluded in their study, their results revealed that even in the absence of disinfectant, the three neutralizing buffers (Dey/Engley, Letheen, and HiCap™) had a mixed impact on the recovery of L. monocytogenes. Specifically, when an initial inoculum of approximately 25 colony-forming unit (CFU)/square was applied without prior exposure to QAC, the number of positive test portions out of 20 varied depending on the neutralizing buffer used. The buffers were stored at 4°C prior to use, and after just 2 h of storage at room temperature, about half of the 20 test portions failed to yield culturable L. monocytogenes. The situation worsened after 72 h of storage at 4°C, with only one-quarter of the samples testing positive. Furthermore, at high disinfectant concentrations, the recovery of culturable L. monocytogenes from sponges pre-moistened with Letheen broth was significantly lower than with Dey/Engley or HiCap™ buffers. However, no study to date has specifically assessed the impact of neutralizing buffers on the viability of adherent L. monocytogenes cells, particularly in relation to the induction of the VBNC state. This information is critical for determining whether disinfectant-neutralizing buffers may themselves contribute to the induction of the VBNC state and thereby lead to an underestimation of culturable L. monocytogenes populations. Therefore, the objective of this study was to assess the impact of six commonly used disinfectant-neutralizing buffers in sampling methods on the recovery and viability of L. monocytogenes cells adhering on stainless steel surfaces for 24 h and treated with disinfectant based on quaternary ammonium or hydrogen peroxide compounds.

Material and methods

Bacterial strain and culture conditions

One bacterial strain was used in the study, L. monocytogene s Lm1 (serogroup 1/2a-3a) (Brauge et al. 2020b, Faille et al. 2020). This strain was isolated from contact surfaces in seafood processing plants. One hundred milliliters of fresh culture in was centrifuged at 5000 g for 10 min at 4°C, and the pellet was washed with 100 ml of a sterile saline solution (8.5 g/ml NaCl). The procedure was repeated twice and the pellet was resuspended in 2.5 ml sterile saline. The inoculum concentration was adjusted with TSBYe (Tryptic soy broth supplemented with yeast extract) to obtain a final concentration of 108 CFU/ml.

Bacterial adherence

Bacteria adhered to stainless steel coupons type 316 L finish 2B (AISI 316 L 2B, Sapim Inox, Loison-sous-Lens, France) measuring 40 mm × 20 mm. These coupons were washed with a 2% (v/v) solution of RBST105 (RBS France, Vallerois-le-Bois, France) and disinfected with a 0.2% (v/v) Oxygal (Ecolab, Arcueil, France, active ingredients: peracetic acid at 2.5% (v/v) and hydrogen peroxide 15% (v/v)) solution for 5 min. The coupons were placed in 60-mm diameter Petri dishes (Nunc, Roskilde, Denmark). Each 60-mm diameter Petri dish was placed in a 140-mm diameter Petri dish (Grosseron, Couëron, France) containing a paper towel soaked with 25 ml sterile distilled water to prevent dehydration during incubation. Ten milliliters of bacterial inoculum at 108 UFC/ml were deposited on each coupon and the coupons were incubated at 20°C for 24 h.

Disinfectant treatment

After incubation, non-adherent bacteria were removed by pouring 25 ml of sterilized saline water over the coupons. The coupons were then treated for 20 min with 12.5 ml of hydrogen peroxide or quaternary ammonium (Active ingredients: quaternary ammonium and glutaraldehyde) solutions at 2% (v/v), as recommended by the supplier (Ecolab, Arcueil, France) or sterilized saline water (control). The disinfectant solutions or water were then removed, the coupons were immersed in 12.5 ml of sterilized saline water. All experiments were carried out at room temperature. Coupons of AISI 316 L 2B stainless steel were then quickly placed in a new 60-mm diameter Petri dishes using sterilized forceps.

Adherent cells sampling and treatment with neutralizing buffers

Six neutralizing buffers were tested: Sponge, Dey-Engley, Universal neutralizer, Tryptic Soy Broth + Egg lecithin (TSB + LT), Tryptic Soy Broth + Egg lecithin + L-Histidine (TSB + LTHTh) and Soy Broth + Egg lecithin + L-Histidine + Sodium thioglycolate (TSB + LTHC) (Table 1). After treatment, adherent cells were removed using a swabbing technique. A swab was plunged into a 5 ml Eppendorf tube containing 4 ml of either one of the six neutralizing buffers, either simple diluent buffered peptone water (BPW) used as a control. The pre-moistened swab was used to recover the adherent cells from the coupon. The swab was then plunged back into the same Eppendorf tube. This procedure was repeated a second time with a new swab plunged into the same Eppendorf tube. Both swabs were broken and the tube was vortexed for 20 s. Once the cells were resuspended, the 4 ml sampling solution was divided into 2 equal parts: 2 ml were analyzed immediately after sampling (post-sampling), and the other 2 ml were incubated for 24 h at 8°C and then analyzed (post-24-h storage at 8°C).

Table 1.

Composition of the neutralizing buffers tested.

Components Sponge neutralizer (pH 7.2 ± 0.2 in 25°C) Dey-Engley (pH 7.6 ± 0.2 in 25°C) Universal neutralizer (pH 8.5 ± 0.2) TSB + LT (pH 7.2–7.5) TSB + LTHC (pH 7.1–7.5) TSB + LTHTh (pH 7.1–7.5)
Tween 20 / / 20 ml 5 ml 30 ml 30 ml
Polysorbate 80 (Tween 80) / 5 g 30 ml / / /
Egg lecithin / 7 g 3 g 3 g 3 g 3 g
Sodium thiosulfate (Na2S2O3) 0.16 g 6 g 7.9 g / / /
L-Histidine / / 1 g/l / 1 g/l 1 g/l
Disodium phosphate (Na2HPO4) / / 75.45 g 2.5 g 2.5 g 2.5 g
Monopotassium phosphate (KH2PO4) 0.0425 g / / / / /
Sodium thioglycolate (C2H3NaO2S) / 1 g / / / 5 g
Sodium bisulfite (NaHSO₃) / 2.5 g / / / /
Sodium / / / 5 g 5 g 5 g
Aryl sulfonate complex 5 g / / / / /
Casein enzyme hydrolysate / 5 g / 17 g 17 g 17 g
Yeast extract / 2.5 g / 2.5 g 2.5 g 2.5 g
Dextrose / 10 g / / / /
Purple bromocresol / 0.02 g / / / /
Soy peptone / / / 3 g 5 g 4 g
Cysteine / / / / 1 g/l /
Distilled water 1000 ml 1000 ml 950 ml 1000 ml 1000 ml 1000 ml
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Quantification of total, viable, and culturable bacteria

Bacterial load in both sampling solutions (post-sampling and post-24-h storage at 8°C) was assessed using quantitative polymerase chain reaction (qPCR), propidium monoazide (PMA)-qPCR, and agar plate enumeration to quantify total, viable, and viable culturable (VC) populations, respectively. The difference between PMA-qPCR and agar plate counts indicated the presence of a VBNC population and/or sublethally injured cells with membrane integrity, while the gap between qPCR and PMA-qPCR quantifications identified dead cells. Enumeration of L. monocytogenes VC populations was conducted on ALOA® agar (BioMérieux, France) and incubated at 37°C for 24 h. Results were normalized by the coupon surface area (8 cm²), with a detection threshold of 0.53 log CFU/cm². Quantification of total and viable DNA by qPCR and PMA-qPCR was performed as described by (Brauge et al. 2020b), with a sensitivity threshold of 3.91 log GE/cm².

Microscopic observation

The viability of the recovered cells, treated either with neutralizing buffers or with BPW (control), was assessed post-sampling and after 24-h storage at 8°C using the LIVE/DEAD® BacLight™ bacterial viability kit (Invitrogen, Carlsbad, CA, USA). In this process, 10 µl of the recovered adherent cells, treated with neutralizing buffers or BPW, was mixed with 10 µl of a working solution containing SYTO™ 9 at a final concentration of 6 µM and propidium iodide at 30 µM. The mixture was placed between a glass slide and a cover slip, followed by a 15-min incubation in the dark at room temperature. Visualization of the stained solution was carried out using an epifluorescence microscope (Imager.Z1, Zeiss, Marly-le-Roi, France) connected to a CCD camera (Axiocam—MRm, Zeiss) at ×40 and ×100 magnifications. A minimum of four random acquisitions were recorded for each condition. Images acquired were merged to create a composing image, allowing the simultaneous visualization of both live and dead or membrane injured cells.

Statistical analysis

Experiments were carried out three times, each having independent triplicates. Differences in cell quantification were evaluated using an analysis of variance (ANOVA) test followed by a Tukey test with Bonferroni correction. Differences were considered statistically significant when P < 0.05 (n = 6). Statistical analysis was performed using RStudio 4.4.1 software (RStudio 2024).

Results

Impact of neutralizing buffers on the viability of adherent L. monocytogenes cells without disinfectant treatment

Firstly, microscopic observations of the contaminated surfaces confirmed that our culture conditions favored the adherence of L. monocytogenes cells to stainless steel coupons (Fig. S1). These adherent cells were mainly viable, stained in green, with a small proportion of dead cells stained in red. Then, we quantified bacterial populations sampled with the six different disinfectant-neutralizing buffers (Fig. 1a). As expected, during post-sampling analyses, the adherent cells sampled in buffer peptone water (BPW, control) consisted exclusively of viable populations (8.12 Log GE/cm2), including 6.36 Log CFU/cm2 of VC population and a proportion of VBNC cells with a difference of 1.76 Log GE/cm2 between viable and VC populations. The six different neutralizing buffers had no impact on the viability of adherent Listeria cells when compared to the control. Incubation for 24 h at 8°C of adherent cells sampled in BPW had no impact on the viability, with populations still exclusively viable, around 8.42 Log GE/cm2, including 6.79 Log CFU/cm2 of VC population, and a proportion of VBNC cells (difference of 1.63 Log GE/cm2 between viable total and VC populations). Similar observations were made for adherent cells sampled with the neutralizers. Similar trends were observed for cells sampled with the neutralizing buffers, regardless of whether the populations were VC, VBNC, or dead. The epifluorescence microscopic observations to assess viability, stained living cells in green and dead or membrane injured cells in red (Fig. 1b). It is important to note that SYTO9 binds non-specifically to nucleic acids and may therefore stain both healthy (live) cells and cells that are not membrane-compromised. In contrast, propidium iodide (PI) only penetrates cells with damaged membranes, allowing differentiation between membrane-intact and membrane-compromised cells. Adherent cells sampled with BPW analyzed post-sampling exhibited mainly living bacterial cells. However, resuspended cells in all neutralizing buffers did not show stained bacteria, while observations in white light confirmed the presence of bacterial cells (data not shown). Interestingly, with Dey-Engley, no bacterial cells were visible, but the solution contained clusters of debris stained in red or green. Adherent cells sampled with BPW and incubated for 24 h at 8°C exhibited living bacterial cells. Consistent with earlier post-sampling observations, adherent cells sampled with all neutralizing buffers and incubated for 24 h at 8°C showed an absence of stained bacteria, except for the Dey-Engley neutralizer, where the solution retained both live and dead stains.

Figure 1.

Figure 1.

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Impact of neutralizing buffers on L. monocytogenes without disinfectant stress. (a) Quantification of VC, viable total (VC and VBNC), and total populations (viable and dead) of 24 h adherent L. monocytogenes cells grown at 20°C and treated with sterilized saline water (control). Subsequently, sampling was performed with swabs pre-moistened either with diluent-neutralizers (six different diluent neutralizers: sponge neutralizer, Dey-Engley, Universal neutralizer, TSB + LT, TSB + LTHTh, and TSB + LTHC) or with a simple diluent (BPW). Analyses of the samples were conducted immediately after sampling (t = 0 h) and after storage for 24 h at 8°C (t = 24 h) to simulate transport timing. (b) Bacterial viability assessment by epifluorescence microscopy of resuspended adherent cells. Adherent cells were stained with the LIVE/DEAD® BacLight™ bacterial viability kit. Live cells are stained by the SYTO 9 dye, while dead or membrane injured cells are stained with the PI stain. The scale bar represents 20 μm.

Impact of neutralizing buffers on the viability of adherent L. monocytogenes cells after treatment with hydrogen peroxide product

Adherent cells treated with hydrogen peroxide were sampled using six different neutralizing buffers (Fig. 2). Post-sampling analyses of cells sampled in (BPW, control) revealed the absence of a VC population (<0.53 Log CFU/cm²), a VBNC population (4.73 Log GE/cm² difference between total viable and VC populations), and a dead population (3.14 Log GE/cm² difference between total and viable populations) (Fig. 2a). No VC populations were quantified when adherent cells were resuspended in neutralizing buffers (<0.53 Log CFU/cm²). Interestingly, all viable populations sampled with neutralizing buffers were significantly higher (P < 0.05) than those sampled without a neutralizing buffer (control), except for the TSB + LT neutralizer. This increase was explained by a higher proportion of VBNC populations in all conditions where adherent cells were resuspended in neutralizing buffers, except for the TSB + LT neutralizer. Additionally, exposure to neutralizing buffers did not affect the total L. monocytogenes population. However, dead populations significantly decreased under all neutralizer conditions, and were absent in samples treated with the Sponge neutralizer. After 24 h of incubation at 8°C, L. monocytogenes adherent cells sampled with BPW exhibited changes in viability, including the appearance of a VC population (0.79 Log CFU/cm²). VC populations were also observed when adherent cells were incubated with the Sponge and Dey-Engley neutralizers (∼1 Log CFU/cm²). Interestingly, incubation of the samples did not alter the total viable populations or overall populations, whether in the control or neutralizer-treated samples. In the control condition, VBNC populations persisted (3.96 Log GE/cm² difference between total viable and VC populations). These VBNC populations remained higher in all conditions where adherent L. monocytogenes cells were incubated with neutralizers, except for the TSB + LT neutralizer. As for the dead populations, they remained stable in the control sample (3.05 Log GE/cm² difference between total and viable populations) as well as in the samples with neutralizers. These dead populations were consistently lower in the neutralizer-treated samples compared to the control. Epifluorescence microscopy post-sampling revealed no visible stained bacteria in any condition (control or samples treated with neutralizing buffers) (Fig. 2b). However, clusters of debris stained red or green were observed again in samples treated with the Dey-Engley neutralizer. After 24 h of incubation at 8°C, adherent cells sampled with BPW displayed green-stained living bacterial cells. In contrast, few or no stained bacteria were observed in adherent cells sampled with all neutralizing buffers, except for the Dey-Engley neutralizer, where colored debris clusters were still present.

Figure 2.

Figure 2.

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Impact of neutralizing buffers on L. monocytogenes with Hydrogen peroxide treatment. (a) Quantification of VC, viable total (VC and VBNC), and total populations (viable and dead) of 24 h adherent L. monocytogenes cells grown at 20°C and treated with a hydrogen peroxide-based disinfectant. Subsequently, sampling was performed with swabs pre-moistened either with diluent-neutralizers (six different diluent neutralizers: sponge neutralizer, Dey-Engley, Universal neutralizer, TSB + LT, TSB + LTHTh, and TSB + LTHC) or with a simple diluent (BPW). Analyses of the samples were conducted immediately after sampling (t = 0 h) and after storage for 24 h at 8°C (t = 24 h) to simulate transport timing. * indicates significant differences between post-sampling and post-24 h storage at 8°C. (b) Bacterial viability assessment by epifluorescence microscopy after Hydrogen peroxide treatment. Adherent cells were stained with the LIVE/DEAD® BacLight™ bacterial viability kit. Live cells are stained by the SYTO 9 dye, while dead or membrane injured cells are stained by the PI stain. The scale bar represents 20 μm.

Impact of neutralizing buffers on the viability of adherent L. monocytogenes cells after treatment with quaternary ammonium product

Adherent cells treated with quaternary ammonium compounds were sampled using six different neutralizing buffers (Fig. 3). Overall, the post-sampling results were similar to those observed with hydrogen peroxide treatments. Adherent cells sampled in buffered peptone water (BPW, control) exhibited no VC population and consisted exclusively of a VBNC population (4.89 Log GE/cm² difference between total viable and VC populations) and a dead population (2.4 Log GE/cm² difference between total and viable populations) (Fig. 3a). Similar trends were observed for adherent cells sampled with neutralizers, showing an absence of VC populations, significantly higher VBNC populations, and lower dead populations (P < 0.05) compared to the control. Notably, the Sponge neutralizer showed no dead population. Incubation for 24 h at 8°C had a measurable impact on the viability of L. monocytogenes populations. In the BPW (control) condition, a VC population emerged (0.95 Log CFU/cm²). Similarly, VC populations appeared when adherent cells were incubated with the Sponge neutralizer (0.54 Log CFU/cm²). However, no VC populations were detected with the other neutralizers (<0.53 Log CFU/cm²). Except for the TSB + LT neutralizer, VBNC populations were significantly higher in all suspensions resuspended with neutralizers, and dead populations were significantly lower compared to the control. The VBNC and dead populations remained unchanged after incubation compared to post-sampling, regardless of the sampling condition (control or neutralizers). Microscopic observations in epifluorescence, both post-sampling and after 24 h of incubation at 8°C, revealed similar outcomes. No stained bacteria were visible under any conditions (control or neutralizing buffers) (Fig. 3b). As previously noted, debris exhibiting both live and dead stains was observed in samples treated with the Dey-Engley neutralizer.

Figure 3.

Figure 3.

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Impact of neutralizing buffers on L. monocytogenes with quaternary ammonium treatment. (a) Quantification of VC, viable total (VC and VBNC), and total populations (viable and dead) of 24 h adherent L. monocytogenes cells grown at 20°C and treated with a quaternary ammonium-based disinfectant. Subsequently, sampling was performed with swabs pre-moistened either with diluent-neutralizers (six different diluent neutralizers: sponge neutralizer, Dey-Engley, Universal neutralizer, TSB + LT, TSB + LTHTh, and TSB + LTHC) or with a simple diluent (BPW). Analyses of the samples were conducted immediately after sampling (t = 0 h) and after storage for 24 h at 8°C (t = 24 h) to simulate transport timing. * Indicates significant differences between post-sampling and post-24 h storage at 8°C. (b) Bacterial viability assessment by epifluorescence microscopy after quaternary ammonium treatment. Adherent cells were stained with the LIVE/DEAD® BacLight™ bacterial viability kit. Live cells are stained green with the SYTO 9 dye, while dead or membrane injured cells are stained by the PI stain. The white scale bar represents 20 μm.

Discussion

This study aimed to assess the impact of six commonly used disinfectant-neutralizing buffers on the recovery and viability of L. monocytogenes adherent cells after 24 h of contact on stainless steel surfaces treated with quaternary ammonium compounds or hydrogen peroxide. The six tested buffers were: Sponge Neutralizer, Dey-Engley, Universal, TSB + LTHC, TSB + LTHTh, and TSB + LT. We first observed the presence of a subpopulation of VBNC cells under control conditions, which was also detected in the conditions involving neutralizing agents. This result could be explained by prolonged incubation in a nutrient-poor environment, leading to starvation stress known to induce the VBNC state in L. monocytogenes (Oliver 2005, Li et al. 2020). Furthermore, Faille et al. (2020) demonstrated that mechanical stress caused by swabbing during sampling may also promote the transition of L. monocytogenes into the VBNC state. However, additional exposure to the neutralizing buffers did not show any further impact on the viability of L. monocytogenes during sampling of untreated adherent cells. These findings are partially consistent with those of Li et al. (2020), who, although they did not explicitly conclude as such, reported that a 2-h exposure at room temperature to Letheen, Dey-Engley, or HiCap buffers led, on average, to the detection of L. monocytogenes by enrichment in only half of the test samples. Our results also align with those of Stewart et al. (2021), who showed that swabs pre-moistened with Butterfield’s phosphate buffer, neutralizing buffer, Letheen broth, or Dey-Engley broth had no negative effect on the detection of L. monocytogenes. We also examined whether the viability of L. monocytogenes was affected by a 24-h incubation at 8°C in the presence of the tested neutralizing buffers, simulating sample storage prior to analysis. This temperature and incubation period were selected because they represent the worst-case scenario recommended by ISO standard 18593:2018, section 8 “Storage and transport”, paragraph 8.2 “Stick swab, sponge/cloth” (EN ISO 18593:2018). We also showed that prolonged exposure to the neutralizing buffers had no significant effect on the viability of L. monocytogenes compared to the control conditions. Although enrichment testing was not conducted in our study, our results contrast with those of Li et al. (2020) and Stewart et al. (2021), who showed that prolonged exposure (72 h at 4°C) to Letheen, Dey-Engley, or HiCap buffers did not consistently allow recovery of culturable L. monocytogenes populations. Microscopic observations of adherent bacterial cells stained with the Live/Dead kit revealed no stained cells in the presence of neutralizing agents, despite confirmed cell presence in fresh conditions (data not shown) and qPCR results. This may be due to chemical incompatibility between neutralizing agents (surfactants, chelating agents, salts) and fluorescent dyes (Fluorescence Excitation and Emission Fundamentals). Additionally, bromocresol purple in Dey-Engley buffer likely interfered with fluorescence detection (Kurzweilová and Sigler 1993).

We resuspended L. monocytogenes adherent cells in six neutralizing buffers after exposure to hydrogen peroxide or quaternary ammonium compounds, two widely used disinfectants in the food industry (Bridier et al. 2021). No VC populations were detected post sampling, either in the BPW control or in the neutralizing buffers, indicating that the cell population, whether in the presence or absence of neutralizers, consisted solely of VBNC cells and/or sublethally injured cells with intact membranes, along with dead cells. These results suggest, first, that prior exposure to disinfectants induced a VBNC state—a phenomenon already documented in several studies on L. monocytogenes biofilms treated with disinfectants of different types (Brauge et al. 2020b, Gu et al. 2020, Arvaniti et al. 2021). Second, they indicate that the neutralizing buffers did not restore cell culturability, which could lead to false-negative results in microbiological analyses. All neutralizers significantly reduced dead cell proportions while increasing VBNC and/or sublethally injured with membrane integrity populations, except for the LT buffer. This suggests two possibilities: neutralizers inactivated disinfectants, preventing further lethality, or they imposed secondary stress, inducing the VBNC state and/or sublethal injuries. Our results are partially consistent with Li et al. (2020), who compared three neutralizing broths (Dey/Engley (D/E), Letheen, and HiCap™) for recovering L. monocytogenes from stainless steel surfaces exposed to high QAC concentrations. At 8000 ppm QAC, Letheen recovered only 1/20 positive samples, while D/E and HiCap™ recovered 10/20 and 13/20, respectively. Although the authors concluded that D/E and HiCap™ were more effective at neutralizing QAC, they did not assess the potential induction of a VBNC state. This remains a possibility, particularly since even without QAC, detection failed in about half of the samples after 2 h at room temperature.

We evaluated the impact of prolonged exposure to neutralizing buffers (24 h at 8°C), simulating transport conditions, on adherent cells previously treated with disinfectants. In the absence of a neutralizer, VC populations reappeared in cells treated with hydrogen peroxide or quaternary ammonium compounds. Similarly, VC populations were observed in cells sampled with the Sponge neutralizer for both treatments and in hydrogen peroxide-treated cells sampled with Dey-Engley. However, other neutralizers failed to preserve VC populations. Notably, Dey-Engley did not restore VC populations in quaternary ammonium-stressed cells, contrasting with Zhu et al. (2012), who reported VC L. monocytogenes populations after 24–72 h in biofilms treated with quaternary ammonium disinfectants. Our results are partially consistent with those of Li et al. (2020), who showed that following a 24-h incubation at 4°C of a sponge sample pre-moistened with Letheen neutralizing broth—used to sample a surface contaminated with 3000 CFU of L. monocytogenes and treated with 8000 ppm of QAC—only 2 out of 20 samples tested positive for the detection of L. monocytogenes. In contrast, at lower surface contamination levels and with milder disinfectant concentrations, detection of L. monocytogenes was nearly systematic. The authors concluded that the Letheen buffer was ineffective at neutralizing high concentrations of QAC. However, another hypothesis could be considered: the Dey-Engley buffer itself may exert stress on L. monocytogenes cells and promote entry into the VBNC state. This hypothesis is supported by additional results from Li et al. (2020) who observed that, even in the absence of QAC treatment, a 2-h incubation at 4°C in the Dey-Engley buffer led to undetectable L. monocytogenes in 11 out of 20 samples. Two hypotheses explain VC resurgence in our study: first, neutralizing media may resuscitate VBNC cells, as previously observed (Oliver 2005, Brauge et al. 2019, Lotoux et al. 2022). Second, initial VC populations below detection thresholds may have grown, though this is unlikely due to L. monocytogenes long generation time at 8°C (∼10 h) (Cordero et al. 2016).

In our study, only the Sponge neutralizer allowed VC L. monocytogenes populations, regardless of whether adherent cells were treated with hydrogen peroxide or quaternary ammonium compounds. This suggests a potential deleterious effect of Universal, TSB + LTHC, TSB + LTHTh, and TSB + LT buffers on already stressed cells, potentially preventing VBNC cells from resuscitating despite the presence of nutrients. One hypothesis is that these buffers contain compounds with toxic effects absent in the Sponge neutralizer. Patel et al. (1995) reported that certain neutralizers, including those with cysteine and sodium thioglycolate, led to increased L. monocytogenes cell damage and lower recovery rates after incubation. Similarly, Kari et al. (1971) found that cysteine inhibited Escherichia coli growth due to cytotoxicity. Nielsen et al. (2016) demonstrated that 0.1% Tween 80 reduced biofilm density in L. monocytogenes, indicating possible detrimental effects on bacterial cells. However, further investigation is needed, as sodium thioglycolate is also present in Dey-Engley, which allowed VC resuscitation after 24 h of incubation at 8°C post-hydrogen peroxide treatment. Some manufacturers state that cysteine inactivates hydrogen peroxide compounds, supporting the hypothesis that certain neutralizers impose additional stress on bacteria. Sutton et al. (2002) reported that most neutralizers tested exhibited toxicity against at least one microorganism, except for Dey-Engley and NIH Thioglycollate Media. Notably, disodium phosphate (a pH buffer) and Tween 20 (a surfactant) are present in all buffers except Sponge and Dey-Engley, which may contribute to differences in L. monocytogenes recovery. Wu et al. (2013) found that Tween 20 inhibited E. coli biofilm formation, whereas Su et al. (2021) showed that low concentrations (2 mg/l) promoted Acidianus manzaensis adhesion and biofilm formation. While most neutralizers except Sponge and Dey-Engley impaired VBNC revival after 24 h at 8°C, we observed a significant increase in VBNC populations after both treatments, except for TSB + LT. This buffer showed no direct impact on inducing VBNC state post-sampling but still prevented VC regrowth after storage, suggesting a negative effect similar to other neutralizers. It was also the least effective at neutralizing disinfectants, showing the highest dead populations post-sampling and after storage. Its composition, lacking histidine, cysteine, and sodium thioglycolate but containing lecithin, Tween 20, and disodium phosphate, may explain these observations.

Our results showed that all tested neutralizers reduced dead L. monocytogenes populations in adherent cell samples, both post-sampling and after 24-h incubation at 8°C. This highlights their efficiency in stopping disinfectant action and limiting cell mortality. While no study has assessed neutralizers’ impact on L. monocytogenes dead populations, our findings align with Sutton et al. (2002), who reported that no universal neutralizer exists due to varying microorganism responses and biocide interactions.

Notably, only the Sponge neutralizer completely eliminated dead cell populations after both treatments and incubation. Its efficiency may be attributed to monopotassium phosphate (buffering capacity) (Garg 2003), aryl sulfonate complex (quaternary ammonium neutralization), and sodium thiosulfate (oxidizing agent neutralization) (Chen 2011). Though no studies confirm these compounds reduce L. monocytogenes mortality, some indicate no toxic effects. Chen (2011) found that sodium thiosulfate and monopotassium phosphate had no impact on L. monocytogenes VC populations after 24 h at 37°C. Similarly, Kemp and Schneider (2000) reported no harmful effects of sodium thiosulfate on E. coli and its effectiveness in neutralizing acidified sodium chlorite. Espigares et al. (2003) found no antibacterial activity in Tween 80, sodium thiosulfate, sodium bisulfate, sodium thioglycolate, lecithin, cysteine, or histidine.

In conclusion, neutralizing buffers had no significant impact on L. monocytogenes viability in untreated cells and reduced mortality in disinfectant-stressed cells. However, most failed to preserve VC populations after quaternary ammonium or hydrogen peroxide treatment. Only the Sponge neutralizer restored VC populations after 24 h, while Dey-Engley was effective only after hydrogen peroxide treatment. Except for TSB + LT, all buffers induced the VBNC state. These findings highlight the critical role of neutralizer selection in L. monocytogenes detection, offering valuable insights into bacterial stress responses in food safety monitoring.

Supplementary Material

fnaf048_Supplemental_Files
fnaf048_supplemental_files.zip (3.2MB, zip)

Contributor Information

Thomas Brauge, Sanitary Safety of Aquatic Origin Food Unit (SANAQUA), Laboratory for Food Safety, French Agency for Food, Environmental and Occupational Health and Safety (ANSES), 62200 Boulogne-sur-Mer, France; Member of the EU Reference Laboratory for  Listeria monocytogenes, Laboratory for Food Safety, French Agency for Food, Environmental and Occupational Health and Safety (ANSES), 94700 Maisons-Alfort, France; Member of the RMT CHLEAN ACTIA Hygienic Design of Lines & Equipment, 75014 Paris, France.

Eglantine Chalivat, Sanitary Safety of Aquatic Origin Food Unit (SANAQUA), Laboratory for Food Safety, French Agency for Food, Environmental and Occupational Health and Safety (ANSES), 62200 Boulogne-sur-Mer, France.

Guylaine Leleu, Sanitary Safety of Aquatic Origin Food Unit (SANAQUA), Laboratory for Food Safety, French Agency for Food, Environmental and Occupational Health and Safety (ANSES), 62200 Boulogne-sur-Mer, France.

Anthony Colas, Sanitary Safety of Aquatic Origin Food Unit (SANAQUA), Laboratory for Food Safety, French Agency for Food, Environmental and Occupational Health and Safety (ANSES), 62200 Boulogne-sur-Mer, France.

Graziella Midelet, Territorial Health and Environment Unit 21 (UTSE 21), Regional Health Agency of Bourgogne-Franche-Comté, 2100 Dijon, France.

Conflict of interest

None declared.

Funding

Funded by the European Union. Views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union or European Health and Digital Executive Agency (HaDEA). Neither the European Union nor HaDEA can be held responsible for them. French National Research Agency (ANR) under the Collaborative research project (PRC) SUBLIM (ANR-22-CE21-0010) also supported this work.

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