Attachment of Salmonella Typhimurium and survival on post-harvest produce and seed

Abstract

Salmonella contamination of post-harvest produce has been a persistent problem worldwide. We determined the effects of storage temperature (5 and 25 °C), lauric acid amide pyrrolidine (LAPY), a bacterial competitor (Bacillus subtilis) and produce rinse on attachment and survival of Salmonella on produce. Produce types but not storage temperature influenced the strength of Salmonella attachment (SR-values). SR-values were significantly (p < 0.05) greater on pistachio and soybean seed (0.53–0.76) than on alfalfa and carrot (0.10–0.13). LAPY resulted in significantly (p < 0.05) greater Salmonella reduction on alfalfa, pistachio and soybean (2.10–3.00 logs), but not on carrot (< 1.50 log). Produce rinse with peptone water significantly (p < 0.05) reduced Salmonella attachment and bacteria populations. Mitigation of Salmonella attachment by selective post-harvest decontamination measures may limit Salmonella survival and enhance produce safety.

Introduction

Foodborne pathogens have often contributed to health risks of consumers in United States (Scallan et al., 2011). The predominant pathogens often linked to contamination of processed or post-harvest fruits, vegetable and deli-based foods are Salmonella enterica, Listeria monocytogenes, and Shiga-toxin producing Escherichia coli (Carstens et al., 2019). In the case of United States and other countries, contamination of foods by pathogenic bacteria have resulted in illnesses or health issues and mortality with overall costs in excess over 20 billion dollars (Scharff, 2015). In the United States, specific costs of each major category of food-borne pathogens have been reported as $3.60 billion for Salmonella enterica and $271 million for Escherichia coli O157:H7 (Batz et al., 2004).

To limit contamination, many pathogen control measures and interventions may be utilized. Some control measures that could minimize Salmonella contamination on produce and reduce outbreaks may include limiting bacterial attachment. This is because bacterial contamination of produce surfaces at pre-harvest and post-harvest have resulted from produce contacts with pathogen-contaminated farm soil, irrigation waters, insects, domestic and wild animals, or human handling before harvest, at harvest, and processing activities. Contact equipment is also a major source of contamination during and after harvest (Heaton and Jones, 2008).

Storage temperatures, environmental conditions, the presence of chemical biosurfactant agents such as fatty acid amides and others may influence survival of foodborne bacteria on produce surfaces (Olanya et al., 2014), but effects on bacterial attachment are not precisely known. Post-harvest commodity surfaces and configurations and the physiological characteristics of bacteria may impact bacterial attachment on produce surfaces and simultaneously impede bacterial inactivation (Dickson and Koohmaraie, 1989). Similarly, biological factors which may include inoculum load and bacterial competitors may either mitigate or enhance attachment and survival of pathogens on produce (Ukuku et al. 2016). However, the role of chemical/antimicrobial agents such as fatty acid amides, biosurfactants and others on attachment and survival of Salmonella are not adequately known.

Similarly, although non-thermal interventions such as use of biocontrol predators of foodborne bacteria have been shown to impact microbial growth and survival on produce following applications; their direct effects on bacterial attachment have not been demonstrated (Olanya et al., 2020). Due to the increase in consumer demand for produce with limited processing, nutritional values and ease of preparation, food safety measures and biorational approaches are desperately needed.

Bacterial attachment of post-harvest commodities is a prelude to produce contamination. Therefore, impediment to Salmonella attachment on post-harvest produce would limit contamination and subsequent pathogen outbreaks. Data on Salmonella attachment and its survival on diverse produce such as alfalfa sprouts (Medicago sativa), baby carrot (Daucus carrota), pistachio seed (Pistacia vera), and soybean seed (Glycine max) are needed to design effective controls for Salmonella contamination and possible outbreaks. The extent to which aerobic microbes could impact bacterial attachment on produce is often not well documented. Therefore, the objectives of this research were to: 1) quantify the occurrence of aerobic microbes on produce surfaces types and assess recovery of Salmonella Typhimurium from produce surfaces and 2) determine storage temperature effects on strength of bacterial attachment on produce; and 3) determine the effects of a lauric acid derivative (biorational compound), bacterial competitors, and produce rinse on Salmonella attachment and survival on post-harvest produce.

Materials and Methods

Produce types  Bagged and processed raw baby carrots (Daucus carota), alfalfa sprouts (Medicago sativa), soybean seed (Glycine max), raw, unshelled pistachio (Pistacio vera) were purchased from a grocery store and refrigerated at 5 °C. Produce types were chosen for their diversity in surface architecture, different attachment sites, and based on their historic involvement in Salmonella outbreaks. Salmonella outbreaks have been implicated in contaminated sprouts (Charkowski et al., 2002) and soybean seed was implicated in reports of Salmonella contamination (Hald et al., 2006). It has been used as an important food ingredient in soybean-based diets for animal feed (Wierup and Kristoffersen, 2014). Survival of Salmonella on almond and pistachios have also been reported (Kimber et al., 2012) and Salmonella contamination of carrots has been documented (Babic et al., 1994). Therefore, alfalfa, baby carrots, as well as soybean and pistachio seed were meant to address knowledge gaps on Salmonella attachment and survival. Produce (10 g) was weighed for appropriate treatments. Produce types (10 g each) in stomacher bags were inoculated with 6–7 log CFU of Salmonella Typhimurium by dipping in a 20 mL bacteria suspension in a biosafety cabinet (LABGARD ES Class II) for 5 min and airdried in a biosafety cabinet for 30 min (Olanya et al., 2018).

Salmonella inoculum  The Salmonella enterica Typhimurium strain H3380, obtained from USDA-ARS, Wyndmoor, PA. This strain is a human isolate, has antibiotic resistance profile and was implicated in produce outbreaks (Briggs and Fratamico, 1999). There is limited knowledge on the attachment of this strain on produce. The Salmonella Typhimurium stock cultures, maintenance and transfer in TSB were as previously described (Olanya et al., 2019; Berrios-Rodriguez et al., 2020). The bacterial cells were centrifuged (10 000 ×g, 5 min, 5 °C) and pellets were washed twice with 0.1 % BPW (BBL, Sparks, MD, USA). Pellets were re-suspended in 10 mL of phosphate buffer saline to concentrations of 6–7 log CFU/mL (Berrios-Rodriguez et al., 2022). The Bacillus subtilis (Natto) strain ATCC 25175, used as bacterial competitor was purchased from ATCC and stored as glycerol stocks at −80 °C until use. Bacillus Natto was chosen because its previous competitive ability, biocontrol potential and production of various bioactive antimicrobial compounds (Miyazawa et al., 2022). For its growth, B. subtilis was inoculated in nutrient broth medium (BBL, Sparks, MD, USA) and incubated on a rotary shaker (172 rpm, at 30 °C) for 24 h. Bacterial inoculum were collected as described above.

Storage temperature effects on strength of attachment (S_R-values) of Salmonella Typhimurium (H3380) cells on post-harvest produce  To determine strength of bacterial attachment on post-harvest produce, cells were recovered and quantified on produce following Salmonella inoculations (6 logs inoculum) on produce in sterilized stomacher bags. The experiment had 2 storage temperatures (5 °C and 25 °C) × 4 produce types, and 3 replicates resulting in 24 treatment combinations. Two storage temperatures were used to simulate refrigerated produce storage (5 °C) and produce stored at room temperature (grocery stores, 25 °C) and to quantify Salmonella attachment. Produce inoculated in stomacher bags as described above were stored for 2 h (5 °C and 25 °C).

Bacterial cells with loose attachment were rinsed with phosphate buffer (20 mL) from each inoculated produce type after sample agitation and contents were pipetted into conical flask. The rinse was diluted, plated on XLT4 medium and Salmonella populations quantified. The populations were designated as loosely attached bacteria. Similarly, strongly attached Salmonella populations (strong adherence to produce), were quantified when 20 mL of peptone water was added to inoculated produce types (10 g) in stomacher bags and stomached for 2 min at 230 rpm. Aliquots were serially diluted, plated on XLT4 (37 °C, 2 days), and CFU/g were quantified and recorded. The SR values (attachment strength) were computed as: Strongly attached/[strongly attached + loosely attached], as described (Ukuku et al., 2022).

Post-harvest produce treatment with lauric acid amide pyrrolidine (LAPY) on survival and reduction of Salmonella   Typhimurium The experiment had 2 treatments (LAPY treated & non-treated) × 4 produce types × 1 Salmonella Typhimurium strain in a completely randomized design with 3 replicates. Salmonella Typhimurium inoculum and produce type inoculation were done as described above. Following produce inoculation with Salmonella, 50 μL of LAPY was applied to each produce type (10 g). Samples were agitated and stored for 2 h (25 °C) prior to Salmonella recovery and quantification on XLT4 medium. A control treatment of Salmonella inoculated produce types with no LAPY (chemical biosurfactant) application was also processed as above.

Produce inoculation with Salmonella after treatment with Bacillus subtilis (bacterial competitor) on survival of Salmonella Typhimurium  The effect of B. subtilis on Salmonella attachment and survival was investigated (25 °C) on 2 treatments (bacterial competitor and control) × four produce types (completely randomized design, 3 replicates). B. subtilis was applied on produce prior to inoculation with Salmonella Typhimurium (~6–7 log CFU). Survival and reduction of Salmonella populations were computed (Berrios-Rodriguez et al., 2022).

Produce rinse effects on attachment of Salmonella cells and scanning electron microscopy (SEM) imaging of bacterial cells on produce  Four post-harvest produce types (as described above) were sanitized (10 g each produce) with 200 ppm of free chlorine wash and rinsed with sterile de-ionized water two times. The produce types were inoculated with Salmonella cells (~6 logs) by pipetting 2 mL of inoculum suspension onto produce in stomacher bags and storing for 2 h at 25 °C (room temperature). The produce tissues were either rinsed with peptone water or not rinsed and subjected to SEM analysis (Olanya et al., 2020) to assess Salmonella cell density and attachment on produce tissues. Aliquots of rinses of Salmonella cells loosely and strongly attached were plated on XLT-4 agar for bacterial counts.

Data analyses  Data were analyzed separately for each individual experiment. The means and standard errors for microbiota on produce types were analyzed by Proc Means of the Statistical Analysis System (SAS Institute, Cary, NC). Data on the Salmonella Typhimurium populations recovered from the produce, storage temperature effects on Salmonella attachment (SR-values) were computed by Proc Means. Similarly, effects of LAPY and bacterial competitor (B. subtilis) on Salmonella survival on produce were analyzed by Proc Means and ANOVA to assess significance of treatment effects (SAS, Cary, NC). The effects of produce rinse on Salmonella attachment and density were compared by lsd statistics and SEM imaging.

Results and Discussion

Salmonella  growth on alfalfa sprouts and Swiss chard (Beta vulgaris L. var cicla) microgreens was documented (Reed et al., 2018), noting that Salmonella growth and survival was affected by inoculum level and time. The similarity was explained in low inoculum level (< 3 log CFU), which is considerably less than in this study. In a study on Salmonella inactivation during drying and storage of carrot slices, 7.00 log CFU/g was recovered (Dipersio et al., 2005). In contrast, Salmonella was recovered on other produce such as shelled and in-shell pistachios (< 1 log CFU/g) as described by Hasani et al., 2020, which is consistent with this study.

Storage temperature effects on strength of Salmonella attachment (SR-values) on produce  Storage temperature effects on the strength (SR-values) of Salmonella attachment were not significant (p > 0.05); however, differences were significant (p < 0.05) among produce types within a storage temperature (Table 1). On alfalfa sprout and baby carrot, the attachment SR-values were low (0.10–0.13 at 5 °C and 0.10–0.11 at 25 °C). On pistachio and soybean seed, SR-values were significantly greater (p < 0.05) than on alfalfa sprouts and baby carrots. The SR-values were almost identical and ranged from 0.53 to 0.76 at 5 and 25 °C on pistachio and soybean seed (Table 1). The greater SR-values recorded on pistachio and soybean seed as opposed to alfalfa sprout and baby carrot may be attributed to the background microbiota on alfalfa sprout and baby carrot. Our analysis of background microbiota data and strength of attachment on the two produce types showed inverse relationship, implying that the greater the microbiota on alfalfa sprout and baby carrot surfaces, the lower the Salmonella attachment. In contrast, on pistachio and soybean seed, lower microbiota populations were observed with greater Salmonella attachment.

Table 1. Storage temperature effects on strength of attachment (S_R–Values) of Salmonella Typhimurium (H3380) cells following inoculations on post-harvest produce.

Produce types	Refrigerated Temp (5 °C)X	Room Temp (25 °C)Y
Produce	SR - Values	SR - Values
Alfalfa sprout	0.13 ± 0.04a	0.10 ± 0.00a
Baby carrot	0.10 ± 0.00a	0.11 ± 0.04a
Pistachio	0.56 ± 0.05b	0.53 ± 0.09b
Soybean seed	0.73 ± 0.08b	0.76 ± 0.07b

^x  Strength of Salmonella attachment (SR-values) was quantified on produce contaminated with Salmonella (~ 7 logs) and then stored for 2 h (5°C, refrigerated temperature) and plated on XLT-4 selective medium (37 °C). Data are means and associated standard errors and the same letters within columns are not significantly different (p > 0.05).

^y  Strength of Salmonella attachment (SR-values) on produce stored at 25 °C (room temperature).

Means followed by the same letters within columns are not significantly different (p > 0.05).

Treatment of post-harvest produce with lauric acid amide pyrrolidine (LAPY) on Salmonella attachment and its survival  The mean Salmonella population on alfalfa sprout (4.78 log CFU/g) was significantly (p < 0.05) lower than the counts on carrot, pistachio and soybean seed, which ranged from 5.94–6.95 log CFU/g (Table 2). In previous research, the authors observed that bacterial attachment commenced at 30 min with stability of attachment recorded at 360 mins of storage (Ukuku et al., 2022). In this research, differences in attachment strength may be attributed to variation in produce topography, moisture content, as well as microbiota presence. Variation in the strength of Salmonella attachment such as on cabbage (0.12), iceberg lettuce (0.23) and on Romaine lettuce (0.34) on intact and cut surfaces was recorded. (Patel and Sharma, 2010). However, when LAPY was applied following produce inoculation, Salmonella populations on alfalfa sprout, pistachio and soybean seed ranged from 2.69 to 3.38 log CFU/g and bacterial counts were not significantly (p > 0.05) different among the three produce types, implying that surfactant properties did not differ on the produce types (Table 2). LAPY application on baby carrot led to lower reduction of Salmonella recovered on baby carrot. The presence of high populations of aerobic bacteria on baby carrots could have mitigated Salmonella attachment, resulting in lower bacteria counts.

Table 2. Effects of treatment of post-harvest produce with lauric acid amide pyrollidine (LAPY) on survival and reduction of Salmonella Typhimurium (H3380) populations at 25 °C.

Produce typesW	Salmonella TyphimuriumX	LAPY + Salmonella TyphimuriumY	Salmonella reduction
Produce	Log CFU/g	Log CFU/g	Log CFU/g
Alfalfa sprout	4.78 ± 1.89b	2.69 ± 1.70c	2.09c
Baby carrot	6.95 ± 0.91a	5.60 ± 0.28a	1.35d
Pistachio	6.35 ± 1.16a	3.38 ± 0.04c	2.97c
Soybean seed	5.94 ± 1.61a	3.11 ± 0.45c	2.83c

^W  Produce was purchased from a local grocery store and used immediately for the experiments.

^X  Produce (10 g) was inoculated with Salmonella Typhimurium (6–7 log CFU/g) and stored at 5 °C (refrigerated conditions) for 2 h, prior to recovery on XLT4 selective medium. Data are means and standard errors and same letter means are not significantly different (p<0.05).

^Y  Produce (10 g) was inoculated with Salmonella Typhimurium followed by application of 50μL of fatty acid amide of lauric acid (LAPY) and stored at 25 °C (room temp) for 2 h. Salmonella populations were recovered on XLT4 selective medium. Data refer to means and standard errors, and same letter means within columns are not significantly different (p > 0.05).

In this study, it was hypothesized that LAPY application on produce surfaces would disrupt interfacial force as well as hydrophobicity between Salmonella and produce surfaces, thereby reducing its attachment. But, LAPY treatment exhibited some biosurfactant properties and moderate effect on Salmonella attachment. (Table 2, Fig. 1). In published research on the effects of natural and synthetic surfactants on the inhibition of Staphylococcus aureus biofilms on intact surfaces, it was reported that mano-rhamnolipid (a natural surfactant) and Tween 80 (a synthetic surfactant) resulted in the inhibition of bacterial adhesion by 97 % and inhibition of biofilm formation of S. aureus by 85 % (Allegrone et al., 2021). Similarly, it was reported that a pulsed light and antimicrobial wash combination was effective in inactivating Salmonella on tomato stem scars (Juncai et al., 2020). Therefore, the effects of LAPY in limiting the attachment of Salmonella and its reductions on produce surfaces were moderate, consistent with the above findings. Similarly, in another study on the effects of surfactants on bacterial adhesion, it was noted that in the presence of Tween 20 (a non-ionic surfactant) and lipopeptide biosurfactant (an anionic surfactant), bacterial adhesion to coated sand decreased slightly, compared to the effects of deionized water (Choi et al., 2011).

Fig. 1

Effects of spot application of lauric acid pyrrolidine (LAPY) on attachment of Salmonella Typhimurium (H3380) cells on alfalfa sprouts, baby carrot, pistachio and soybean seed at 2 h storage following pathogen inoculation (25 °C). The images: A – Alfalfa sprout + Salmonella, B – LAPY + Salmonella on Alfalfa sprout, C- baby carrot + Salmonella, D – LAPY + Salmonella on baby carrot; E- Pistachio + Salmonella, F – LAPY + Salmonella on pistachio; G- Soybean + Salmonella, H – LAPY + Salmonella on Soybean.

Treatment of Salmonella inoculated produce with Bacillus subtilis (bacterial competitor) on pathogen attachment and its survival  Populations of Salmonella Typhimurium recovered from post-harvest produce ranged from 5.52–7.48 log CFU/g (Table 3). Salmonella populations recorded were lowest on alfalfa sprouts followed by baby carrots. Salmonella counts on pistachios and soybean seed following its inoculation, were not significantly different (p > 0.05). The presence of the bacterial competitor

Table 3. Application of Bacillus subtilis as a bacterial competitor on attachment and survival of Salmonella Typhimurium (H3380) on post-harvest produce.

Post-harvest produce	Salmonella Typhimurium (25 °C)w	B. subtilis + Salmonella (25 °C)x	Salmonella reduction
	Log CFU/g	Log CFU/g	Log CFU/g
Alfalfa sprouts	5.52 ± 0.28c	<1.00 ± 0.00e (nd)	> 4.00d
Baby carrots	6.47 ± 0.00b	6.12 ± 0.43b	0.35e
Pistachios	7.26 ± 0.36a	6.86 ± 0.19ab	0.40e
Soybean seed	7.48 ± 0.67a	7.32 ± 0.45a	0.16e

^w  Produce was inoculated with Salmonella Typhimurium and quantified on XLT4 selective medium. Data represents means ± standard errors.

^x  Produce was inoculated with bacterial competitor (B. subtilis) at 5 logs prior to application of Salmonella Typhimurium cells and produce storage for 2 h. Salmonella populations were quantified on XLT4 medium. Data are means ± standard errors.

Bacillus subtilis,  in combination with Salmonella Typhimurium, did not result in any significant reductions in bacterial attachment, except on alfalfa sprout (Table 3).

The mean Salmonella populations on alfalfa sprouts treated with B. subtilis, were undetectable (< 1.00 log CFU/g), while on other produce types, Salmonella counts were 6.12–7.32 log CFU/g (Table 3). We hypothesized that addition of bacterial competitor (B. Subtilis) prior to or after produce inoculation with Salmonella would reduce attachment and its subsequent recovery from treated produce, relative to the control (produce inoculated with Salmonella only). However, we recorded no significant inhibition of Salmonella on produce, except on alfalfa sprouts. The limited effect may result from the duration (time) that may be needed for bacterial competitor establishment on produce to mitigate pathogen attachment.

In this research, B. subtilis was applied on Salmonella-inoculated produce with a 2-hr lag time prior to bacterial recovery. This storage time may have been insufficient for colonization and establishment of B. subtilis as a bacterial competitor, to retard Salmonella attachment. Aerobic mesophilic bacteria on alfalfa sprouts indicated high populations (data not shown). It is possible that high populations of mesophilic bacteria on alfalfa sprouts may have adversely impacted Salmonella attachment on alfalfa sprouts, due to competition for nutrients and space. In previous research, Lactobacillus rhamnosus was shown to inhibit Salmonella growth as well as attachment of Salmonella on vegetable seed (Lou et al., 2021).

Produce rinse effects on Salmonella attachment and scanning electron microscopy (SEM) imaging of bacterial cells  Post-harvest rinse following Salmonella inoculations demonstrated that loosely attached bacteria could be considerably reduced (Fig. 2). It appears that produce rinse could easily detach cells that were not capable of developing a strong attachment (or no attachment at all). The rinse process physically removes bacterial cells from produce surfaces and is not totally unexpected. Rinsing is often used in the produce industry as a wash step prior to produce tumbling (Gil et al., 2009), or pre-packaging and has been shown to be cost-effective measure for pathogen removal. Our findings are consistent with previous research in which Salmonella reductions on tomatoes from 2.20 to < 1.00 log CFU/g of produce were recorded (Ukuku et al., 2022). Similarly, on apple surfaces, decreases in Salmonella populations by produce washing from 2.50 logs to < 1.00 log CFU/g of produce were also recorded as evidence of reduced attachment (Ukuku et al., 2022).

Fig. 2

Scanning Electron Micrographs of Salmonella Typhimurium cells attached to alfalfa sprouts, baby carrot, pistachio and soybean seed following inoculations and subsequent storage of produce for 2 hrs (25C) and rinsed with peptone water or not rinsed. The images: A – Alfalfa sprouts (not rinsed), B – Alfalfa sprout – rinsed; C- baby carrot (not rinsed), D – baby carrot – rinsed; E- pistachio (not rinsed), F – pistachio – rinsed; G- soybean (not rinsed), H – soybean (rinsed).

Conclusions

Mitigation of Salmonella attachment is important for reducing pathogen contamination. In this study, produce types, but not storage temperature influenced the strength of Salmonella attachment (SR-values) as significant (p < 0.05) SR-values were recorded on pistachio and soybean more than on alfalfa and carrots. LAPY applied on Salmonella-inoculated produce had similar effects on produce types but, differed significantly (p < 0.05) on baby carrot when compared to the control. B. subtilis application resulted in similar Salmonella occurrence and attachment on produce types except alfalfa sprouts where limited attachment and significant reduction were recorded. Rinsing of Salmonella-inoculated produce with peptone water significantly (p < 0.05) affected bacterial attachment and Salmonella counts on produce. These results suggest that decontamination measures may limit Salmonella attachment and its survival depending on produce types.

Authorship contribution statement  O.M. Olanya: Conceptualization, Investigation, Formal analysis, Writing - original draft; S. Mukhopadhyay: Investigation, Writing - review & editing; D.O. Ukuku: Investigation, Methodology, writing; B.A. Niemira: Investigation, Methodology and editing; J. Uknalis: SEM Methodology and editing.

Acknowledgements  We thank Edlyn Jusino for technical assistance. This research was supported from CRIS Project No. 8072-41420-026-00D, Development of Alternative Intervention Technologies for Minimally Processed Foods and No. 8072-41420-022-00D, Mitigation of foodborne pathogens in water and fresh produce via application of biochar.

Data Availability Statement  Data will be available upon request.

Disclaimer  Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. “USDA is an equal opportunity provider and employer.”

Conflict of interest  There are no conflicts of interest to declare.

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