Listeria monocytogenes NRRL B-57618 (1/2a), NRRL B-33053 (4b), and NRRL-33466 (1/2b) were obtained from USDA-ARS culture collection [National Center for Agricultural Utilization Research (NRRL), Peoria, IL, United States] and maintained at -80°C in Trypticase Soy Broth (Becton, Dickinson and Company, Sparks, MD, United States) supplemented with 0.6% Yeast Extract (Fisher Scientific, Fair Lawn, NJ, United States) (TSBYE) and 20% (v/v) glycerol. The frozen culture was first activated in TSBYE at 37°C for 24 h statically, and then 1:500 subcultured in TSBYE for additional 24 h at 37°C before use.

Twice-activated bacteria cultures were centrifuged at 8,000 × g for 5 min at 4°C. The resulting bacteria pellets were washed once and then re-suspended in Phosphate Buffered Saline (PBS, pH7.4). Equal volume of each L. monocytogenes strain was combined to make a three-strain cocktail and, subsequently, diluted to ∼6 and 3 Log₁₀ CFU/ml for apple inoculation. At the high inoculation level, there were 6.29 ± 0.04, 6.42 ± 0.04, and 6.01 ± 0.07 Log₁₀ CFU/apple on organic GSA, conventional GSA, and Fuji apples, respectively, immediately after inoculation. At the low inoculation level, 3.01 ± 0.06, 3.04 ± 0.07, and 3.48 ± 0.03 Log₁₀ CFU/apple on organic GSA, conventional GSA, and Fuji apples, respectively, immediately after inoculation.

Whole fresh unwaxed GSA (organic and conventional) and Fuji apples (conventional) were donated by Allan Brothers Inc. and Stemilt Growers LLC. All fruit was of commercial maturity (the optimum maturity for the fresh market or storage) (Girschik et al., 2017). Apples were delivered from the commercial packing houses 1-week prior to inoculation and stored in a walk-in cooler (4°C) temporarily. Medium size apples (with an average weight ∼220 g) without cuts, bruising, or scars were selected and rinsed with cold tap water and dried on a bench top overnight to balance apple temperature to room temperature (RT). The apples were then inoculated with L. monocytogenes by submerging 15 apples each time in 5 L of three-strain L. monocytogenes cocktail inoculum solution at a selected inoculation level, and gently massaged by turning the apples in the inoculum for 10 min so that microorganisms were evenly distributed in the sample. For each variety at each inoculation level, four inocula were independently prepared with a total of ∼100 apples inoculated per inoculum. Inoculated apples were dried for 24 h at RT (∼22.5°C) before being subjected to storage. Twelve apples were sampled (three apples were randomly sampled from each of four inoculation batches) immediately following inoculation and 24 h post-inoculation for microbial enumeration to confirm inoculum uniformity, inoculation level, and the attachment of L. monocytogenes on fresh apples.

Both uninoculated and 24 h post-inoculation apples were placed in cardboard boxes and stored at 1°C, 4°C, 10°C, and RT in lab-scale refrigerated incubators. Temperatures were monitored continuously by the built-in sensors, and double-checked weekly using two calibrated digital thermometers (Omega HH11, Omega Engineering Inc., Stamford, CT, United States) to ensure precision. Actual storage temperatures were 0.93 ± 0.01, 4.17 ± 0.03, 9.76 ± 0.03, and 22.65 ± 0.03°C. Twelve apples (three apples from each of the four inoculation batches) were sampled for microbial analysis from each temperature of each inoculation level (uninoculated, high and low inoculation levels) at selected sampling points. Uninoculated apples were sampled at 2-, 4-, 8-, 12-week of storage for total plate count (TPC) and yeasts/molds (Y/M) enumeration. Apples inoculated with L. monocytogenes were sampled at 1-, 4-, 7- and 14-day intervals for short-term storage, and at 2-, 4-, 8-, and 12-week for long-term cold storage (1, 4, and 10°C).

Each apple was placed into a sterile stomacher bag with 10 ml sterile PBS and hand-rubbed for 1.5 min. Wash solutions from each bag were 10-fold serially diluted with sterile PBS, and 0.1 or 1 ml from appropriate dilutions was plated on duplicate appropriate plates. For inoculated apples, TSAYE (TSBYE with 1.5% agar) plates were used for apples with a L. monocytogenes level higher than 4.0 Log₁₀ CFU/apple where L. monocytogenes counts are much higher than the background flora (<3.0 Log₁₀ CFU/apple). TSAYE overlaid with Modified Oxford agar (MOX, Becton, Dickinson and Company, Sparks, MD, United States) plates were used for the enumeration of L. monocytogenes when its counts are lower than 4.0 Log₁₀ CFU/apple to discern from resident background flora of apples. The detection limit of L. monocytogenes is 10 CFU/apple. For uninoculated apples, rub solutions were plated onto TSAYE for TPC and Potato Dextrose Agar (PDA, Becton, Dickinson and Company, Sparks, MD, United States) plates for Y/M count. TSAYE plates were incubated at 35 ± 1°C for 48 h. PDA plates were incubated at RT for 5 days.

Data was analyzed by GLM from Statistical Analysis Systems (SAS, Cary, NC, United States). Mean values were compared by least significant difference (LSD) multiple-comparison test. P-values less than 0.05 were considered significant. Four batches of inocula were independently used to inoculate apples at each inoculation level for each variety. Each individual apple was an experimental unit. At each sampling point, three apples were randomly sampled from each batch (N = 12). Results were reported as mean ± SEM (standard error of mean).

Immediately after inoculation, organic GSA exhibited attachment of 6.29 ± 0.04 and 3.01 ± 0.06 Log₁₀ CFU/apple of L. monocytogenes, respectively (Figures 1A,B). After 24 h attachment at RT, bacterial counts were 6.31 ± 0.06 and 3.51 ± 0.08 for their respective inoculation levels (Figures 1A,B). There was a significant increase of 0.5 Log₁₀ CFU/apple for the low inoculation level (Figure 1B, P < 0.05). During 2 weeks of cold storage at 1, 4, and 10°C, bacterial populations did not significantly change (Figures 1C,D). On apples stored at 22°C, however, bacterial counts were reduced by 1.17 and 0.66 Log₁₀ CFU/apple for the high and low inoculation levels, respectively (Figures 1C,D). L. monocytogenes established on fresh apples at either high or low level remained stable during cold storage. At both inoculation levels, only a 0.5–1.5 Log₁₀ CFU/apple reduction on organic GSA during the 3-month cold storage was achieved (Figures 1E,F).

Apples from different agricultural practice or different varieties may behave differently in terms of bacterial adhesion and inactivation of the attached microorganisms to their surface. Therefore, we evaluated the fate of L. monocytogenes established on conventional GSA during storage. At the high inoculation level, L. monocytogenes maintained the same population size 24 h post-inoculation (Figure 2A). At the low inoculation level, the bacterial count was increased by ∼0.7 Log₁₀ CFU/apple 24 h post-inoculation (Figure 2B, P < 0.05). After 2 weeks of cold storage, L. monocytogenes counts were slightly decreased for the high inoculation level, while no reduction was observed for the low inoculation level (Figures 2C,D). L. monocytogenes counts on apples stored at RT were reduced by 1.1 and 0.6 Log₁₀ CFU/apple for high and low inoculation level, respectively (Figures 2C,D). After 3-month cold storage, a 0.9–2.0 Log₁₀ CFU/apple reduction was achieved for both inoculation levels with 1°C showing the least reduction and 10°C the most (Figures 2E,F). Given the similar behavior of L. monocytogenes on organic and conventional GSA, the fate of L. monocytogenes was only accessed on conventional Fuji apples. Similar results were observed on inoculated conventional Fuji apples (Figure 3). After 2 weeks of cold storage, L. monocytogenes population on fresh Fuji apples was slightly reduced. There was about ∼1.7 and 0.7 Log₁₀ CFU/apple reduction of L. monocytogenes on Fuji apples with high and low inoculation level, respectively, after 2-week of RT storage (Figures 3C,D), a slightly higher reduction than that on GSA. After 3-months, L. monocytogenes populations on Fuji apples were decreased by 0.8, 0.9, and 1.4 Log₁₀ CFU/apple at 1, 4, and 10°C, respectively, for the high inoculation level (Figure 3E). For the low inoculation level, L. monocytogenes counts on Fuji apples declined by 1.4, 1.5, and 1.8 Log₁₀ CFU/apple at 1, 4, and 10°C, respectively (Figure 3F).

Bacteria, mold, and yeast can cause postharvest decay of apples (Janisiewicz and Korsten, 2002), reducing apple quality and causing a potential chemical (mycotoxin) hazard. Storage temperature has been reported to play a role in fruit decay (Mir et al., 2001). Therefore, TPC, in addition to Y/M count was performed on uninoculated apples of the selected varieties stored under different temperatures. The initial TPC was 3.57 ± 0.06, 3.16 ± 0.07, and 3.55 ± 0.06 Log₁₀ CFU/apple for organic GSA, conventional GSA, and conventional Fuji apples, respectively (Figures 4A–C). After 3-month storage, TPC was significantly reduced by 0.2–0.4 Log₁₀ CFU/apple for organic GSA, and 0.5–0.6 Log₁₀ CFU/apple for conventional GSA and Fuji apples (Figures 4A–C). There was no difference in TPC on apples stored under different temperatures.

The initial Y/M count of organic GSA, conventional GSA, and conventional Fuji apples were 4.74 ± 0.05, 3.67 ± 0.10, and 4.73 ± 0.07 Log₁₀ CFU/apple, respectively (Figures 4D–F). For both GSA (organic and conventional) and Fuji apples, Y/M count stayed the same throughout the cold storage with only a slight decline on apples stored at 22°C (Figures 4D–F).

At the inoculation level of ∼6 Log₁₀ CFU/apple, L. monocytogenes population on fresh apples did not change during the 24 h establishment on the apple surface at RT, regardless of apple varieties or agricultural practice. Our results were corroborated with other previously published studies where the population of L. monocytogenes inoculated on whole strawberries at ∼7.5 Log₁₀ CFU/berry was not significantly changed 24 h post-inoculation at 21°C (Flessa et al., 2005). Interestingly, at the low inoculation level, the population size of L. monocytogenes on apples slightly increased. Similarly, L. monocytogenes inoculated at ∼3 Log₁₀ CFU/g on whole tomatoes were increased by ∼1.5 Log₁₀ CFU/g after 2-day storage at 21°C (Beuchat and Brackett, 1991). While the mechanism for the observed difference between high and low inoculation levels is unknown, it might be due to the competition for the limited nutrients on apple surface at the high inoculation level.

A consumer survey study indicated that 42% of consumers have been reported to store apples at RT (Li-Cohen and Bruhn, 2002). Therefore, we evaluated behavior of L. monocytogenes on fresh apples during 2-week storage at RT. L. monocytogenes on apples of different cultivation practices (organic and conventional) or different varieties (GSA and Fuji apples) behaved similarly with 0.5–1.7 Log₁₀ CFU/apple reduction regardless of the inoculation level after 2-week storage under RT. Consistent with our findings, L. monocytogenes inoculated onto a GSA surface at ∼5 Log₁₀ CFU/apple was reduced by ∼1.5 Log₁₀ CFU/apple after 15-day storage at 25°C (Salazar et al., 2016). The population of L. monocytogenes on whole strawberries at 7.1 Log₁₀ CFU/berry was declined by 1.0 Log₁₀ CFU/berry after 2-day storage under 24°C (Flessa et al., 2005). There was a 1.5 Log₁₀ CFU/cm² reduction of L. monocytogenes inoculated on whole cantaloupe surfaces at ∼3.5 Log₁₀ CFU/cm² during a 15-day storage at 20°C (Ukuku and Fett, 2002).

Fresh apples are commercially stored at ∼1°C for extended periods of time (Mattheis et al., 1991). Apples are also held at 4°C in the packing house and consumer’s refrigerators at home (Duvenage and Korsten, 2016). During the transportation, apples may be exposed to temperature abuse of 10°C (Huang et al., 2015). Therefore, all three temperatures were tested in this study for extended storage of 3 months. Our results showed that L. monocytogenes on apples was stable during the first 2 weeks at all three temperatures regardless of inoculation level, apple variety, or growing practice. A similar phenomenon was observed when L. monocytogenes (5 Log₁₀ CFU/g) was inoculated onto whole Golden Delicious apples; there was no L. monocytogenes reduction during a 9-day storage at 4°C (Rodgers et al., 2004). Similarly, the population size of L. monocytogenes inoculated on whole tomatoes at ∼3.5 Log₁₀ CFU/g remained at the same level throughout the 21-day storage at 10°C (Beuchat and Brackett, 1991).

Survival of L. monocytogenes was similar among 1, 4, and 10°C, which was higher than that of RT storage. It might because apples stored at RT produced more volatile compounds including those with antimicrobial effects such as alcohols, aldehydes, ketones, and esters compared with apples at refrigerated storage (HamiltonKemp et al., 1996; Matich et al., 1996; Dixon and Hewett, 2000), which facilitated L. monocytogenes death under RT storage. On molecular level, cold storage might increase survival through altering fatty acid composition of the bacterial membrane (Mastronicolis et al., 2005), which protects L. monocytogenes against other environmental stresses during storage (Lou and Yousef, 1997). Cold temperatures may have also induced the expression of general stress sigma factor σ^B, facilitating L. monocytogenes survival (Hecker and Volker, 1998; Becker et al., 2000). However, L. monocytogenes inoculated on whole strawberries was reduced by ∼3 Log₁₀ CFU/berry after 7-day storage at 4°C (Flessa et al., 2005). These data indicate that a different food matrix and/or surface properties impacts L. monocytogenes survival at refrigerated temperature. The reduction of L. monocytogenes in strawberries might also be associated with the high moisture and low pH value of the fruit surface.

During 3 months of extended cold storage, reduction of L. monocytogenes on apples was temperature-dependent with relatively more reduction at 10°C. In support of our data, less L. monocytogenes were recovered from stainless steel surfaces stored at 10°C than those at 4°C (Redfern and Verran, 2017). This might due to the induction of a more fierce stress response and accumulation of more low molecular weight solutes at lower temperatures (NicAogain and O’Byrne, 2016). The phenomenon could also resulted from temperature-dependent alteration in apple fruit quality during long-term storage (Mir et al., 2001) with more antimicrobial volatile compounds production at a higher cold storage temperature (Dixon and Hewett, 2000). Inoculation level, apple variety, or agricultural practice had minor influences on L. monocytogenes survival on apple surfaces during cold storage.

Resident bacteria, yeasts, and molds on apples contribute to fruit decay (Janisiewicz and Korsten, 2002). Thus, their fate was further studied over storage. TPC and Y/M counts stayed at the similar level throughout the 3-month storage under different temperatures for apples of the selected varieties and practices. In support of our finding, mesophilic aerobic microorganism, and Y/M counts were not changed on raw carrots during 18-day storage at 5°C (Beuchat and Brackett, 1990). However, inconsistent with our results, TPC and Y/M counts of raw carrots were increased by ∼0.5 and 1.5 Log₁₀ CFU/g, respectively, after the 7-day storage at 15°C (Beuchat and Brackett, 1990). In another study, TPC as well as Y/M counts on whole tomatoes increased during the 20-day storage at 10°C and the 8-day storage at 21°C (Beuchat and Brackett, 1991). The difference in background flora behavior might due to different crop surfaces. The low-moisture condition on apple surface in this study might prevent the growth of resident bacteria, yeasts, and molds.