To study listerial biofilms on plant surfaces, we departed from the commonly used models. First, instead of growing L. monocytogenes in rich media that contains peptides, amino acids, lipids, and nucleotides or nucleic acids, we switched to minimal medium containing glucose as carbon source, because such medium better approximates conditions under which listeria interact with plant matter in nature and in fresh produce processing facilities. Second, we incubated autoclaved wood coupons (as substrates mimicking partially degraded wood in the environment) and sterilized pieces of fresh produce in the liquid medium inoculated with L. monocytogenes for extended period, 48 h, to allow biofilm formation.

Following incubation, a significant fraction of biomass of the Pss EPS-synthesizing ΔpdeB/C/D strain was attached to the wood coupons (Figures 1A, B). This contrasted poor colonization of surfaces of the manmade materials, i.e., stainless steel, aluminum or acrylic (Figures 1A, B). This finding is consistent with our earlier observations that listerial EPS-aggregates formed in liquid medium poorly attach to polystyrene and glass surfaces (Chen et al., 2014). To test if surface roughness, as opposed to the nature of the material, played a major role in attachment, we scratched the surfaces of manmade materials thus bringing surfaces roughness closer to the roughness of the wood coupons. This did not improve bacterial attachment (not shown), suggesting that EPS-synthesizing listeria strongly prefer wood over manmade materials.

In contrast to the voluminous biofilms formed on wood coupons by the Pss-synthesizing strain, 48-h old biofilms of the wild-type strain, EGD-e, or the ΔpdeB/C/DΔpssC mutant impaired in EPS synthesis were not visible to the naked eye (Figures 1A, B). To quantify attached cells, the biofilm biomass was scraped off from the surface of wood coupons, vigorously vortexed in liquid medium, and serial dilutions were plated for enumerating colony forming units (CFUs). Figure 1C shows that the Pss EPS strongly, by at least fourfold, promotes colonization of wood coupons. Because prior to biomass scraping, loosely associated aggregates were removed by rinsing in sterile medium, the reported fold-difference is an underestimate.

To assess whether preference for wood is specific to the (unknown) tree species from which coupons were made, we tested coupons made from three known tree types. The EPS-synthesizing strain showed improved attachment to all tested wood coupons, albeit to varying extent (Figure 2). Therefore, listerial Pss increases attachment to various kinds of wood.

Next, we investigated the role of the Pss EPS in listerial colonization of fresh produce using produce types that were associated with the past listeriosis outbreaks. We tested the role of Pss in biofilm formation on cantaloupe rind, the cause of major listeriosis outbreaks in the US and Australia (McCollum et al., 2013; NSW Government, 2018), as well as on cut pieces of fresh produce, which caused the majority of listeriosis outbreaks (Marik et al., 2020; Centers for Disease Control and Prevention, 2022). Sterilized pieces of approximately the same sizes were inoculated with L. monocytogenes in flasks containing minimal medium at 30°C.

After 48-h incubation, the EPS-synthesizing strain formed visible biofilms on cantaloupe rind (Figure 3A). Such biofilms were also visible on the surfaces of celery and lettuce (Figure 3A), most prominently on the cutting edges. No biofilms were visible on the pieces of produce grown in the presence of the wild-type and EPS-impaired strains (Figure 3A). Biofilms of the EPS-synthesizing strain contained ∼2−12-fold higher CFUs, compared to the wild type, and ∼4−22-fold higher, compared to the EPS-impaired strain (Figure 3B), suggesting that EPS strongly promotes colonization of various plant surfaces. The effect of EPS on colonization of cantaloupe rind versus pulp was stronger (12-fold versus 2-fold) (Figure 3B), suggesting that plant surface composition and roughness play important roles in EPS-dependent L. monocytogenes colonization.

In the experiments described above, minimal media contained glucose as a carbon source. To mimic conditions at cantaloupe storage and processing facilities, where water/moisture may be present, but carbon source is likely absent or limiting, we repeated the experiment using minimal medium lacking glucose. Residual bacterial growth under those conditions was possible due to the juice leaking from cantaloupe pieces. The lack of glucose in the medium significantly, by almost 10³-fold, decreased colonization of cantaloupe rind (7.4 × 10⁶ versus 7.0 × 10⁹ CFU, Figures 3B, C). However, the trend in surface colonization was preserved, i.e., the EPS-synthesizing strain colonized cantaloupe rind several-fold more efficiently than the wild-type or the EPS-impaired strains (Figure 3C).

For a closer look at the biofilms on cantaloupe rind, we employed scanning electron microscopy (SEM). All strains formed extracellular appendages on cantaloupe rind, which may represent a combination of eDNA, nanotubes and flagella (Figure 4) and will not be discussed further. The biofilms formed by the EPS-synthesizing strain showed abundant EPS matrix in which listeria were embedded (Figure 4). We were unable to unambiguously determine if the wild type formed any Pss. It showed somewhat higher attachment to plant matter, compared to the EPS-impaired strain, ΔpdeB/C/DΔpssC (Figures 1C, 3B), however, whether this was due to low levels of Pss is unclear.

Earlier we showed that the Pss EPS enhances L. monocytogenes tolerance to desiccation, when cell aggregates grown in liquid medium were compared to planktonically grown cells (Chen et al., 2014). Desiccation is the condition experienced by listeria during storage of fresh produce, such as whole cantaloupes, at the processing facilities and transportation to supermarkets and consumer homes and restaurants (Esbelin et al., 2018). To assess the role of the Pss EPS-biofilms on fresh produce surfaces in desiccation tolerance, we subjected the 48-h old biofilms formed on cantaloupe rind to drying on air at room temperature for up to 3 weeks. For ease of comparisons, we adjusted CFUs of surface-attached bacteria of all strains to approximately the same initial values by mechanically removing ∼90% biofilm of the EPS-synthesizing strain (Figure 3B) prior to the desiccation experiment. We realize that much thinner EPS-biofilms likely resulted in our underestimating the magnitude of desiccation tolerance benefits rendered by the Pss EPS.

After 7 days, CFUs decreased in the wild type from 100 to ∼10%, in the EPS-impaired strain to ∼7.4%, and in the EPS-synthesizing strain to 74%, i.e., by ∼10, ∼13, and 1.35-fold, respectively, (Figure 5). During prolonged desiccation, the EPS-synthesizing strain continued to maintain significant survival advantage over other strains (Figure 5). Desiccation tolerance in this strain was 6−16-fold higher, compared to the wild type, over the whole 3 week time course. This implies that the listerial EPS-biofilms formed on fresh produce could survive storage and transportation of fresh produce much better than biofilms formed by the strains not synthesizing Pss.

Prior to reaching hospitable small intestine, the site of the gastrointestinal tract where L. monocytogenes causes disease, it faces the challenge of stomach (gastric) acid. To mimic the process of consumption of the contaminated produce, we tested acid stress survival of the listerial biofilms formed on the cut fresh produce. For this experiment, we switched from cantaloupe rind as a model to cantaloupe pulp and celery, because rind is not consumed. The pieces of pulp and celery containing 48-h old listerial biofilms were exposed to acid stress, approximating pH of the stomach acid, i.e., pH 1.5−3.5 (Marieb and Hoehn, 2018). One stress regimen involved a 15-min exposure of the produce pieces to pH 2.5. Another regimen involved a 15-min exposure to pH 5.0, followed by 15 min at pH 2.5 (Guerreiro et al., 2022).

As shown in Figures 6A, B, exposure to pH 5 alone resulted in only moderate decreases in bacterial survival in all strains. However, exposure to pH 2.5 uncovered a large difference in survival, i.e., ∼20% for the EPS-synthesizing strain versus ∼1.8% for the wild type and ∼1.4% for the EPS-impaired strain (Figure 6A). Thus gives the EPS-synthesizing strain an ∼11-fold survival advantage over the wild type. Similarly, after a two-step regimen (pH 5.0, then pH 2.5), ∼1% of the EPS-synthesizing strain survived but only ∼0.085% of the wild type and ∼0.081% of the EPS-impaired strain, thus revealing an ∼12-fold advantage of the EPS-synthesizing strain over the wild type. Similar results were obtained when listerial survival in biofilms on the celery pieces was measured (Figure 6B), i.e., ∼25% survival of the EPS-synthesizing strain versus ∼2% for the wild type and ∼1.7% for the EPS-impaired strain following exposure to pH 2.5, and ∼4.7% versus 0.04% and 0.03% survival following a two-step regimen. Thus, the EPS-synthesizing strain has a 12−116-fold survival advantage on celery pieces, compared to the wild-type. These results suggest that from ∼11- to ∼116-fold higher number of L. monocytogenes in the EPS-biofilms on cut fresh produce could survive exposure to stomach acid, compared to the biofilms lacking Pss EPS.

In L. monocytogenes, EPS synthesis is the dominant, albeit not the only phenotype associated with the elevated c-di-GMP levels (Chen et al., 2014; Elbakush et al., 2018). In other bacteria, elevated c-di-GMP levels, in addition to upregulating EPS synthesis, affect such biofilm components as protein adhesins, pili, and fimbria (Römling et al., 2013). To access the impact of c-di-GMP-dependent factors other than Pss EPS that contribute to listerial colonization and survival on plant matter, we constructed the L. monocytogenes c-di-GMP null strain, ΔpdeB/C/DΔdgcA/B/C. In addition to lacking c-di-GMP-specific PDEs, this mutant also lacks all DGCs involved in c-di-GMP synthesis (Chen et al., 2014). We found that the c-di-GMP null mutant behaved similarly to the high c-di-GMP strain impaired in Pss synthesis, ΔpdeB/C/D ΔpssC, in survival assays (Figures 5, 6), thus suggesting that Pss is the key factor affecting stress tolerance. In plant surface colonization assays the c-di-GMP null strain appeared somewhat, ∼2-fold, inferior, compared to the ΔpdeB/C/D ΔpssC strain (Figures 1C, 3B, C). From these experiments, it is evident that the Pss EPS is the dominant c-di-GMP-dependent factor affecting listerial colonization of plant matter and stress tolerance.

Strains and plasmids used in this study are listed in Table 1. The c-di-GMP null strain, ΔpdeB/C/D ΔdgcA/B/C, was constructed from strain ΔpdeB/C/D using sequential deletion of dgcA/B (with the help of pKSV7-ΔdgcAB) and dgcC (with the help of pKSV7-ΔdgcC), as described earlier (Chen et al., 2014). Plasmid pAM-mScarlet expressing the red fluorescent protein mScarlet (Bindels et al., 2017) was constructed to enhance visualization of L. monocytogenes on the surfaces of wood and fresh produce. The mScarlet gene was synthesized with L. monocytogenes-optimized codons (Twist Bioscience) and cloned downstream of the strong Bacillus subtilis Pveg promoter (Guiziou et al., 2016) in the pAM101-derived vector (Fujimoto and Ike, 2001). The listerial strains were grown in the liquid minimum HTM medium (Tsai and Hodgson, 2003) containing 3% glucose and 0.2% yeast extract, HTM/GY, at 30°C under shaking (220 rpm). When strains containing pAM-mScarlet were used, the media was supplemented with 10 μg/mL chloramphenicol (Cm). For enumerating CFUs, cultures were routinely plated onto Brain Heart Infusion (BHI) agar (Millipore Sigma, Burlington, MA, United States) and incubated at 37°C for 36 h.

All coupons (disks for do-it-yourself arts and crafts projects) were purchased on from various vendors, including coupons made from unspecified wood (32 mm diameter × 1.2 mm thickness, Woodpile), birch (50 mm × 8 mm, Woodpeckers), poplar (50 mm × 2.5 mm, Juvale), Schima superba (38 mm × 4 mm, Axe Sickle), stainless steel (38 × 1.55 mm, PH Pandahall, CA, USA), aluminum (38 × 1.55 mm, RPM Stamping Blanks, Rose Metal Products, Springfield, MO, USA), and acrylic (25.4 × 1.8 mm, Tupalizy, PTC-Office). Prior to use, all non-wood coupons were immersed in 70% ethanol for 20 min for disinfection, rinsed twice in sterile deionized water and air dried. The wood coupons were autoclaved (121°C, 30 min). In some experiments, prior to sterilization, surfaces of steel, aluminum and acrylic coupons were roughened up by a scrapper to imitate surface roughness of wood coupons.

The overnight L. monocytogenes cultures were diluted 1:100 into 10 mL HTM/GY medium in 125-mL flasks and grown at 30°C until optical density of ∼ 0.4 at 600 nm. Sterile wood coupons or pieces of fresh produce were added at this point and cultivation was continued for 48 h. The coupons were subsequently withdrawn and rinsed twice in sterile HTM to remove loosely attached biofilms. The biomass attached to the coupons and produce pieces was thoroughly scrapped off into HTM medium using a sterile scalpel, and the suspensions were vortexed. Serial dilutions were plated onto BHI agar plates and grown at 37°C for 36 h followed by CFU enumeration.

Whole cantaloupes, celery, and mixed salads (Iceberg lettuce, red cabbage, carrots) used in this study were purchased from local (Laramie, WY) retail stores. Cantaloupe coupons (20 mm × 4 mm) were obtained by using a cork borer. Some pieces were cut out to contain rind, others were cut out to contain cantaloupe pulp only. Celery was cut in pieces of varying lengths from similar sized stocks. Prior to processing, fresh produce was thoroughly washed. Cutout pieces were sterilized by complete submersion into 8.25% sodium hypochlorite (CloroxPro, Clorox Professional Products Company) in the 50-mL centrifuge tubes placed on a rocker platform for 20 min. Following sterilization, fresh produce pieces were submerged in 50-mL sterile distilled water overnight and subsequently rinsed in fresh sterile distilled water. Sterile pieces were added to listerial cultures, as described above for coupons.

Following 48-h incubation, produce pieces were aseptically removed from the cultures and rinsed by dipping in three sequential beakers with sterile HTM media. To measure listerial colonization, cantaloupe rind was carefully separated from pulp and processed separately, while the remaining pulp was discarded. To measure colonization of cantaloupe pulp, pulp-only pieces lacking rind were used. The produce pieces were mechanically macerated in the Stomacher bags in 5 mL HTM medium and homogenized for 10 min at high speed (Stomacher^® 80 Biomaster, Seward, UK). The serial dilutions of the homogenates were plated onto BHI agar plates for CFU enumeration.

Small (5 mm diameter x 1 mm thickness) pieces of cantaloupe rind containing L. monocytogenes biofilms were used for SEM (Scanning Electron Microscope FEI Quanta 250). Briefly, rind pieces were fixed for 2 h in 2% glutaraldehyde-PBS buffer at room temperature, followed by dehydration steps in a series of ethanol baths (10 min each) containing 25, 50, 75, 95, and 100% (3 times) ethanol, and postfixed by incubation with 1% osmium tetroxide in PBS for 1 h in the dark. The samples were then dried using Balzers Critical Point Dryer (CPD 020) before coating with gold-palladium, as described earlier (Muravnik et al., 2016).

Rind-containing cantaloupe pieces were removed from the liquid L. monocytogenes cultures after 48-h incubation. To adjust biomass levels to approximately the same initial cell numbers, the voluminous EPS-biofilms of the ΔpdeB/C/D strain were gently brushed off cantaloupe rind, resulting in the removal of ∼ 90% biomass. Coupons were then aseptically cut into four equal-sized quarters. One quarter was used to determine initial CFU (day 0) following homogenization in Stomacher. The remaining quarters were placed on sterile blotting paper in open Petri dishes and kept at room temperature inside the biological safety cabinet with the switched off blower. After 7, 14, and 21 days of storage the quarters were placed in Stomacher bags with 5 mL HTM medium, soaked for 1 h, homogenized and serial dilutions were plated for CFU enumeration.

Biofilms formed on the pieces of celery and cantaloupe pulp were used in acid stress experiments. The sizes of celery pieces were adjusted to have similar initial number of listeria in biofilms, as follows: 20 mm (EGD-e), 5 mm (ΔpdeB/C/D), 22 mm (ΔpdeB/C/DΔpssC), and 24 mm (ΔpdeB/C/DΔdgcA/B/C). The 4-mm thick cantaloupe pulp pieces had the following diameters: 18 mm (EGD-e), 15 mm (ΔpdeB/C/D), 19 mm (ΔpdeB/C/DΔpssC), and 20 mm (ΔpdeB/C/DΔdgcA/B/C). Following 48-h incubation in liquid L. monocytogenes cultures, celery or cantaloupe pulp pieces were removed and placed in HTM/GY acidified to pH 5.0 or 2.5 with hydrochloric acid for 15 min at 37°C. A preconditioning protocol involved exposing celery pieces to pH 5.0 for 15 min followed by exposure to pH 2.5 for additional 15 min, as described (Guerreiro et al., 2022). The acid was neutralized to pH 7.0 by addition of the sodium hydroxide solution, celery pieces were homogenized, and serial dilutions were plated for CFU enumeration.