Stress Tolerance of Multiple Salmonella enterica Strains Associated with Foodborne Outbreaks

Salmonella, stress tolerance, foodborne outbreak 9 10

We thank and acknowledge researchers at the NY State Agency of 11 Agriculture, the USDA-ARS, FDA CFSAN, and the Cornell Food Safety Lab, who provided 12 isolates for this research. We also thank and acknowledge Tom Ford from Ecolab for providing a 13 sample of PAA-based sanitizer to test, and John Johnston of USDA for facilitating transfer of 14 isolates from USDA-ARS-NRRL in Illinois. This work was supported by the USDA National 15 Institute of Food and Agriculture (NIFA) Agriculture and Food Research Initiative Strengthening 16 and New Investigator Food Safety and Defense Program, project award no. 2019-06903. 17 Additional salary support was received from the USDA Hatch funding mechanism via the 18 Vermont Agricultural Experiment Station and gift funds provided to the Department of Animal 19 Sciences at the University of Vermont for C. Patch’s contributions. Any opinions, findings, 20 conclusions, or recommendations expressed in this publication are those of the author(s) and 21 should not be construed to represent any official USDA or U.S. Government determination or 22 policy. 23 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint 2 24

25 Salmonella enterica is a foodborne pathogen commonly found in food processing 26 environments. While control methods such as heat treatment and sanitizers are often used, S. 27 enterica has evolved strategies for survival and persistence to overcome pathogen control. This 28 study assessed 43 outbreak-associated (OA) and non-outbreak associated (NOA) S. enterica 29 isolates from serovars Enteritidis, Heidelberg, Newport, Typhimurium, and monophasic 30 Typhimurium (I 4,[5],12:i:-) for enhanced stress tolerance. Heat shock at 56˚C, minimum 31 inhibitory concentrations (MICs) for sanitizers sodium hypochlorite (NaOCl) and peracetic acid 32 (PAA), and crystal violet microtiter assays were used to evaluate heat tolerance, sanitizer 33 tolerance, and attachment capacity, respectively. Most isolates (n =34/43) carried at least one 34 antimicrobial resistance gene, and nearly half (n =21/43) displayed genotypic and/or phenotypic 35 resistance to ampicillin, ciprofloxacin, or ceftriaxone. Most isolates carried genes conferring 36 resistance to gold (n =43/43) and arsenic (n= 41/43), and tolerance to mercury, copper, and silver 37 was common among monophasic Typhimurium and Heidelberg isolates. Efflux pump 38 qacEdelta1 was detected among eight Heidelberg isolates. We found enhanced stress tolerance 39 (i.e. an unusually high ability to survive and adapt to various environmental stresses) to sanitizers 40 and enhanced attachment capacity, indicating biofilm formation. Isolates evaluated for heat 41 tolerance survived at least 15 min at 56˚C and three survived >60 min. Overall, we found 42 evidence of enhanced tolerance to individual stresses across both OA and NOA S. enterica. 43 There were no strong patterns based upon serovar or OA/NOA status; however, we did find that 44 specific enhanced stress tolerance profiles may have contributed to outbreak characteristics. 45 46 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint 3

47 Non-typhoidal S. enterica (NTS) is a Gram-negative foodborne pathogen commonly 48 found in poultry, eggs, pork, beef, nuts, and produce [1]. NTS is responsible for numerous 49 foodborne outbreaks annually in the United States and is a common contaminant in processing 50 facilities [2]. Salmonellosis also carries a high economic burden, with the loss of over $3.3 51 billion annually in healthcare costs, lost productivity, and mortality in the U.S. [3]. NTS can 52 exhibit high rates of antimicrobial resistance (AMR), which is cited by the Centers for Disease 53 Control and Prevention (CDC) as a serious public health concern [4]. More specifically, 54 fluoroquinolone resistant NTS is considered a “high concern” pathogen by the World Health 55 Organization [5]. 56 To limit bacterial growth and prevent foodborne outbreaks, various pathogen control 57

may be used in the food processing industry, including temperature control (e.g., heat 58 shock) and sanitizers such as sodium hypochlorite (NaOCl) or peracetic acid (PAA) [6-9]. Often, 59 multiple techniques are used in combination to prevent the proliferation of pathogenic 60 microorganisms [10]. Heat treatment is commonly used and highly effective [11], as S. enterica 61 can grow on foods held between 4˚C and 60˚C (40-140˚F; i.e. the “temperature danger zone”) 62 [12]. In poultry processing, scalding is commonly used, either by steam-spraying or immersion 63 scalds, with temperature and duration varying [13]. Current FSIS guidelines recommend 30-75 64 seconds of exposure at 59-64˚C for hard scalds and 90-120 seconds at 51-54˚C for soft scalds for 65 chickens, while turkey is typically scalded for 50-125 seconds at 59-63˚C [14]. Hard scalds are 66 more common in poultry processing than soft scalds [14]. In pork processing, scalding for 8 67 minutes at 60-62˚C, hot water decontamination for 12-15 seconds at 80˚C, and steam 68 pasteurization at 70˚C are commonly used [15]. Temperature controls are also employed in 69 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint 4 peanut and tree nut processing to reduce S. enterica [16-18]. Almonds should be dry roasted at 70 120-148˚C for 9 min to 3 h, oil roasted at 126˚C for 2 minutes or blanched at 80-90˚C [16-18]. 71 Dry roasting peanuts at 154˚C for 15 min and oil roasting at 150˚C for 1.5 min have been 72 sufficient in reducing Salmonella by over 5.4 log CFU/g and 6.0 log CFU/g, respectively [19]. 73 S. enterica has developed various mechanisms for survival and persistence in the food 74 processing industry [11, 20], including sanitizer, heat tolerance, and biofilm formation [21]. 75 Exposure to one stressor can lead to cross-tolerance to other stressors [21], further enhancing S. 76 enterica’s ability to survive and persist. For instance, heat tolerance in S. enterica is influenced 77 by factors such as pre-exposure to stress and starvation, growth phase during heat shock, and 78 expression of heat shock proteins [22-25]. Additionally, thermal resistance mechanisms can play 79 a role in modulating virulence [22]. Repeated exposure to antimicrobials, such as sanitizers, can 80 lead to elevated minimum inhibitory concentration (MIC), or the level of sanitizer required to 81 inhibit the growth of or kill the bacteria; however, development of true resistance is rare [26, 27]. 82 S. enterica may also form biofilms, which are difficult to eliminate and provide protection 83 against sanitizers, desiccation, antibiotics, and some host defenses [28-31]. These biofilms often 84 form on untreated or mechanically sanded steel surfaces, even when dry and lacking a steady 85 nutrient source [32]. Biofilms are an especially critical target, as an estimated 80% of all U.S. 86 bacterial infections are linked to foodborne pathogens residing in biofilms [31]. 87 Previous research by Etter et al., 2019 highlighted that six Salmonella Heidelberg isolates 88 from a 2013-2014 chicken poultry outbreak displayed enhanced heat tolerance and attachment 89 capacity under stressful conditions [23]. This research laid the groundwork for this study, which 90 aims to further investigate enhanced stress tolerances, variation by serovar, and overall 91 mechanisms that may contribute to outbreak characteristics across multiple S. enterica serovars 92 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint 5 and outbreaks. Further, understanding strain variation can improve processing techniques and 93 consumer risk modeling to reduce foodborne illness [11]. The objectives of this research were to 94 characterize (i) AMR and stress associated genes, (ii) sanitizer tolerance, (iii) heat tolerance, and 95 (iv) attachment capacity of S. enterica isolates of various serovars to understand intrinsic 96 characteristics contributing to differences between serovars and isolates associated with 97 historically relevant outbreaks, as well as non-outbreak associated isolates. 98 99

100 Acquisition of Culture Collection 101 48 strains were included in this study (Table 1); 25 were obtained from the USDA-ARS 102 Culture Collection (NRRL), six from the Food and Drug Administration (FDA), three from the 103 New York State Food Laboratory, eight from the Cornell Food Safety Lab, and one strain was 104 ordered from the American Type Culture Collection (ATCC). 105 Upon acquisition, cultures were transferred into sterile trypticase soy broth (TSB; BD, 106 Franklin Lakes, NJ) and incubated (37°C, 200 rotations per minute (rpm), 24 hours), then 107 streaked out for isolation onto trypticase soy agar (TSA; BD, Franklin Lakes, NJ) and incubated 108 (37°C, 24 hours). Isolates were stored in sterile cryovials in 25% glycerol at -80°C as a working 109 stock for future use. 110 111 Genomic Characteristics 112 Antimicrobial resistance genes, stress tolerance genes, and heavy metal resistance genes 113 for each isolate were extracted from the NCBI Isolates Browser platform, which detected these 114 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint 6 genes using AMRFinderPlus (v3.8.4) [33]. A single nucleotide polymorphism (SNP)-based 115 phylogenetic tree was created to assess isolate relationships using CSIphylogeny (v1.4) [34]. 116 117 Sanitizer Tolerance 118 Minimum Inhibitory Concentrations (MICs) for sodium hypochlorite (NaOCl) and 119 peroxyacetic acid (PAA) sanitizer tolerances were determined as follows, using a procedure 120 adapted from methods previously described [23, 35]. 121 Preparation of Cultures: Isolates were recovered from working stock solutions; 122 approximately 15-25 isolated colonies per strain were selected via sterile swab and suspended in 123 3 mL of TSB. Optical density at 600nm (OD600) was read and adjusted to 0.600 – 0.800. 124 Adjusted culture was diluted 1:100 into 1 mL of either 2X TSB or 1/10X TSB to achieve a final 125 concentration of 1/20X (nutrient depletion) or 1X (nutrient abundance) upon sanitizer addition. 126 An NaOCl solution (4.275% NaOCl; Clorox, Oakland, CA) was prepared to achieve a 127 concentration of 400 ppm, then serially diluted into phosphate buffered saline (PBS) to 6.25-200 128 ppm (0.000625-0.02%) upon addition of bacterial culture. A PAA solution (Inspexx™ 250, Saint 129 Paul, MN) was prepared to a concentration of 400 ppm and serially diluted 1:1 into PBS to 25-130 200 ppm (0.0025-0.02%) upon addition of bacterial cultures. 131 Plate Inoculation and Incubation: Polystyrene microtiter plates were prepared at 1/20X 132 and 1X conditions per strain in triplicate. Plates were read immediately following inoculations to 133 determine a baseline OD600 and read again at 24 hours to measure growth at room temperature 134 (22oC), where the MIC was the concentration of sanitizer at which no growth occurred in the 135 wells. 136 137 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint 7 Heat Shock 138 Growth curves and heat shock assays were performed to determine heat tolerance using 139 procedures adapted from methods previously described by Etter et al. [23]. Isolates were 140 recovered from the working stock solutions, inoculated on TSA, and incubated (37˚C, 24 hours). 141 A single colony was inoculated into 10 mL TSB and incubated (37˚C, 200 rpm, 16 hours) before 142 serial dilution in duplicate by a factor of 10-5 and incubation (37˚C, 200 rpm, 8 hours). 143 For growth curves, 1 mL aliquots were collected each hour for eight hours and serially 144 diluted into PBS. Dilutions were spread-plated onto TSA in duplicate and incubated (37˚C, 36 145 hours). For heat shock, 1 mL aliquots were transferred to preheated 56˚C water baths (hard scald 146 temperature [23, 36]) for heat shock, staggering incubation by 3 minutes per strain. At 0, 3, 6, 9, 147 15, 30, 45, and 60 minutes, 1 mL aliquots were removed and serially diluted into PBS. Dilutions 148 were immediately pour-plated with 10 mL liquid TSA in duplicate. At 15, 30, 45, and 60 149 minutes, non-diluted cultures were pour-plated in 100 µL aliquots in duplicate and 250 µL 150 aliquots in quadruplicate to capture low titer cultures, and plates were incubated (37˚C, 36 151 hours). Colonies were counted using a countable range of 30-300 colonies/plate, all counts from 152 250 µL plates were used, and the limit of detection was 1 CFU/mL. 153 154 Biofilm Attachment Assays 155 Attachment to polystyrene plates was evaluated using a crystal violet staining procedure 156 adapted from methods previously described [23, 35]. Polystyrene 96-well plates were prepared 157 under four conditions: 1/20X TSB, 4°C (nutrient depletion, refrigeration); 1/20X TSB, RT 158 (22°C) (nutrient depletion, room temperature); 1X TSB, 4°C (nutrient abundance, refrigeration); 159 and 1X TSB, RT (nutrient abundance, room temperature) in triplicate per strain. Absorbance at 160 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint 8 600nm was recorded to determine crystal violet (CV) retention as an approximation of cell 161 density (biomass) with an Epoch microplate spectrophotometer at 24, 72, and 120 hours (Agilent 162 Technologies, Santa Clara, CA). Growth was not confirmed via enumeration. All CV attachment 163 assays were repeated in their entirety in triplicate. 164 165 Statistical Methods 166 All significant differences for the crystal violet attachment assays, the minimum 167 inhibitory concentration assays, and the heat shock assays were assessed using Analysis of 168 Variance (ANOVA), followed by Tukey’s honest significance test in R v4.2.1 [37]. 169 170

and Discussion 171 Genotypic Profiles 172 A SNP-based phylogenetic tree of all isolates, aligned against reference Salmonella Heidelberg 173 SL476 is available in figure S1. All Salmonella Heidelberg genomes, both outbreak and non-174 outbreak associated isolates, were within 130 SNPs of each other. Isolates from the Kosher 175 Broiled chicken liver [38, 39] outbreak were zero SNPs different from each other, while isolates 176 from the 2013-2014 poultry outbreak, which involved six strains [40], were 1-122 SNPs 177 different. Monophasic Salmonella isolates were 1-102 SNPs different, including the NOA 178 isolate. The two Salmonella Typhimurium isolates from the 2008-2009 Peanut Butter outbreak 179 [41] were 73 SNPs different, despite sharing the same two-enzyme PFGE patterns 180 (JPXX01.0459/JPXA26.0462). The Salmonella Enteritidis almond outbreak isolate [42] was the 181 most different from its paired NOA isolates; while the NOA isolates were 44-45 SNPs different 182 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint 9 from each other, they were 2440-2449 SNPs different from the OA isolate. This may have been 183 due to source; the NOA isolates were from produce and soil. 184 185 Antimicrobial Resistance Genes 186 Antimicrobial resistance is a concern for S. enterica outbreaks as resistance to medically 187 relevant antibiotics (defined as ampicillin, ciprofloxacin, azithromycin, ceftriaxone, and 188 amoxicillin can complicate the treatment of severe clinical cases) and AMR genes often travels 189 on plasmids which can carry virulence, AMR, and sanitizer efflux genes [4, 43-46]. We were 190 interested in whether AMR might be connected to stress tolerance in outbreak-associated S. 191 enterica 192 Most isolates (n = 34/43) carried at least one gene conferring AMR (Figure 1). Nearly 193 half (n =21/43) harbored genes associated with resistance to at least one medically important 194 antibiotic. Three Heidelberg isolates from the 2013-2014 chicken outbreak and all monophasic 195 Typhimurium isolates from the 2015 roast pork outbreak carried blaTEM-1, conferring 196 ampicillin resistance. Additionally, blaTEM-1 was detected in one NOA isolate each from 197 serovars Heidelberg, monophasic Typhimurium, and Enteritidis. Newport isolate FML-M2-0046 198 (NOA, water), contained blaCMY-2, linked to third-generation cephalosporin resistance [47, 48]. 199 NOA monophasic Typhimurium isolate FML-M2-0025 (NOA, pork feet) contained blaSHV-12 200 (linked to ampicillin and cephalosporin resistance) [49] and qnrB19 (intermediate quinolone 201 resistance, defined as 0.12–0.5 mg/L [50]); it was the only isolate to carry either gene. 202 Phenotypic resistance data (Table S1, available for a limited number of isolates) showed that 203 monophasic Typhimurium isolate FML-M2-0025 (NOA, pork feet) also exhibited intermediate 204 ciprofloxacin resistance and Enteritidis isolate FML-M2-0028 (NOA, chicken breast) exhibited 205 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint 10 intermediate resistance to amoxicillin/clavulanic acid. As for genotypic resistance among NOA 206 isolates, half (n =7/14) contained no AMR genes, while AMR genes were particularly prevalent 207 among Heidelberg isolate FML-M2-0026 (NOA, ground turkey), Enteritidis isolate FML-M2-208 0028 (NOA, chicken breast), and Newport isolate FML-M2-0046 (NOA, water). 209 Efflux pump qacEdelta1, encoding a quaternary ammonium compound (QAC) efflux 210 pump, was detected in eight Heidelberg chicken outbreak isolates (FML-M2-0001 through FML-211 M2-0004, FML-M2-0006, FML-M2-0007, FML-M2-0009, and FML-M2-0010). This gene has 212 been linked to tolerance to QACs, and it may be selected for in environments where QAC-based 213 sanitizers are used [51, 52]. The asr gene (anaerobic sulfite reduction), encoding an acid shock 214 protein for survival in acidic conditions (such as acid-based antimicrobials [53]), was present in 215 all but two NOA isolates (Enteritidis FML-M2-0028, NOA, raw chicken breast; Typhimurium 216 var. 5- FML-M2-0029, NOA, chicken breast). 217 Higher resistance levels in some OA strains have been previously reported; Etter et al. 218 found 1-10 AMR genes among seven Heidelberg strains from the 2013-2014 chicken outbreak 219 [23], while phenotypic testing of 2015 roast pork outbreak monophasic Typhimurium strains 220 showed most were multi-drug resistant [54]. Conversely, Procura et al. found few AMR genes in 221 S. enterica from chicken livers, except erythromycin resistance in all isolates and streptomycin 222 resistance in 22% [55]. In a study comparing prevalence of AMR in animal and nut products, the 223 authors found most S. enterica from raw almonds (n =73/83) were resistant to two or fewer of 15 224 antimicrobials, with 52 being pan susceptible [56]. 225 226 Heavy Metal Tolerance Genes 227 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint 11 Heavy metal carriage has been previously linked with AMR gene carriage in livestock 228 and human salmonellosis and may be carried on plasmids or other transferable elements [57-60]. 229 Consequently, we assessed carriage of heavy metal resistance genes in our isolates. Carriage of 230 genes conferring gold and arsenic tolerance was nearly ubiquitous among S. enterica isolates 231 (Figure 1). All contained golST for gold tolerance, and all but two (Enteritidis FML-M2-0028, 232 NOA, chicken breast; Typhimurium var. 5- FML-M2-0029, NOA, chicken breast) carried arsR 233 or arsABCDR for arsenic tolerance. Copper (pcoABCDERS or pcoACDRS) and silver 234 (silABCEFPRS) genes were found in all monophasic Typhimurium isolates (FML-M2-0011 235 through FML-M2-0025) and five 2013-2014 chicken outbreak Heidelberg isolates (FML-M2-236 0001, FML-M2-0003, FML-M2-0006, FML-M2-0009, and FML-M2-0010). Tellurium tolerance 237 (terDWZ) was present in these five Heidelberg isolates and monophasic Typhimurium isolate 238 FML-M2-0025 (NOA, pork feet). Mercury tolerance (merAPR, merABDPRT, or merACDEPRT) 239 was observed in three chicken outbreak Heidelberg isolates (FML-M2-0002, FML-M2-0004, and 240 FML-M2-0007), all roast pork monophasic Typhimurium isolates, and one kosher broiled 241 chicken liver outbreak Heidelberg isolate (FML-M2-0030). Mercury tolerance did not co-occur 242 with tellurium, copper, or silver tolerance among Heidelberg isolates, nor with tellurium 243 tolerance among monophasic Typhimurium isolates. 244 Overall, monophasic Typhimurium isolates carried the greatest number of heavy metal 245 tolerance genes, consistent with previous research [61]. Acquisition of these genes facilitates 246 bacterial survival and has been noted to contribute to outbreak severity [61]. Furthermore, the 247 highest frequency of both heavy metal tolerance and AMR genes was observed in Heidelberg 248 isolates from the chicken outbreak and monophasic Typhimurium isolates from the roast pork 249 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint 12 outbreak, which was previously reported for S. enterica from livestock, animal foods and carcass 250 samples in the EU, where heavy metals are often used as growth promoters [57]. 251 252 Sanitizer Tolerance 253 Sodium hypochlorite (NaOCl) 254 Acquired tolerance to sanitizers used in food processing environments can contribute to 255 survival and persistence of S. enterica [62], increasing the risk of potential outbreaks. Exposure 256 to sublethal doses can lead to bacterial repair mechanisms and allow for subsequent survival in 257 extreme conditions [62]. Sanitizer tolerances are shown in Figure 1 with detailed values in 258 Table S2. NaOCl MIC among isolates FML-M2-0001 through FML-M2-0048 averaged >200 259 ppm in 1X TSB and 113 ppm in 1/20X TSB, indicating that nutrient concentration influences 260 MIC values (p <0.05). MIC varied by serovar (p 200 ppm), followed by Enteritidis (125 ppm), Heidelberg (109 ppm), Newport 262 (111 ppm), and monophasic Typhimurium and Typhimurium var. 5- (100 ppm). Typhimurium 263 var. 5- isolates had a higher MIC than monophasic Typhimurium averaged across both 264 conditions (padj <0.05). Isolates from the roast pork (FML-M2-0011 through FML-M2-0023), 265 chicken (FML-M2-0001 through FML-M2-0010), kosher broiled chicken liver (FML-M2-0030 266 through FML-M2-0032), raw almond (FML-M2-0033), and peanut butter (FML-M2-0047 267 through FML-M2-0048) outbreaks had mean NaOCl MIC values at 1/20X TSB of 92 ppm, 95 268 ppm, 158 ppm, 150 ppm, and >200 ppm, respectively, though differences were not significant 269 (padj >0.05). NOA isolates averaged 122 ppm in 1/20X TSB. 270 Previous studies have also reported high NaOCl tolerance among S. enterica isolates [63-271 66]. Xiao et al. found >256 ppm MIC when using 11.92% NaOCl for most poultry supply chain-272 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint 13 derived isolates (n =161/172) [63], while Obe et al. observed MIC values of 500-1,000 ppm 273 using 12.5% NaOCl [64]. Humayoun et al. reported a mean MIC of 3,152 ppm [65] with a lower 274 active chlorine concentration (5.25-6.15% NaOCl). Sublethal NaOCl concentrations (50 ppm) 275 were marginally more effective against Heidelberg than Typhimurium and Enteritidis isolates 276 [67], consistent with this study. 277 Higher NaOCl tolerance has been linked to AMR and the qacEdelta1 efflux pump [63]; 278 however, Heidelberg isolates in this study carrying both (n =8; Figure 1) had a lower mean MIC 279 than other isolates. The high and moderate MIC values of nut-associated isolates (>200 ppm in 280 1X, Typhimurium FML-M2-0047 and FML-M2-0048, OA, peanut butter; 150 ppm in 1X, 281 Enteritidis FML-M2-0033, OA, almond) may be due to their low-moisture food matrices, which 282 have been associated with cross-tolerance to NaOCl [68]. The concerningly high NaOCl 283 tolerance observed in this study and others may contribute to S. enterica persistence in the food 284 processing environments where this sanitizer is used. 285 286 Peracetic Acid (PAA) 287 PAA MIC values for isolates FML-M2-0001 through FML-M2-0029 averaged 176 ppm 288 in 1X TSB and 26 ppm in 1/20X TSB; isolates FML-M2-0030 through FML-M2-0048 were not 289 tested. In 1/20X TSB, all isolates had MIC values of 25 ppm, except for monophasic 290 Typhimurium FML-M2-0011 (50 ppm; OA, roast pork). Nutrient concentration influenced MIC 291 values (p <0.05), with nearly all isolates exhibiting higher MIC values in 1X TSB than 1/20X 292 (padj <0.05). MICs varied little by serovar or outbreak, remaining between 25-27 ppm in 1/20X 293 and 170-200 ppm in 1X TSB. Monophasic Typhimurium had the lowest MIC in 1X (173 ppm), 294 while Enteritidis and Typhimurium var. 5- had the highest (>200 ppm). However, serovar was 295 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint 14 not a significant determinant of MIC (p >0.05). By outbreak status, the lowest MIC in 1X TSB 296 was among Heidelberg chicken outbreak isolates (170 ppm), while NOA isolates had the highest 297 (183 ppm), though differences were not significant (padj >0.05). Overall, PAA tolerance was 298 lower than NaOCl, though nutrient concentration appeared more influential in PAA tolerance. 299 PAA tolerance findings aligned with previous studies. Etter et al. reported an average 300 MIC of 73.6 ppm among MDR Salmonella Heidelberg isolates from the 2013-2014 chicken 301 outbreak testing concentrations of 25-250 ppm (0.0025-0.025%) [23]. Mourao et al. found an 302 MIC of 60-70 ppm in S. enterica from chicken meat using 5-90 mg/L PAA (0.0005-0.009%) 303 [69]. In contrast, Jolivet-Gougeon et al. reported an MIC of 7 ppm PAA (0.0007%) for 304 Salmonella Typhimurium LT2 [70]. Humayoun et al. found a much higher average MIC of 880 305 ppm in 88 MDR S. enterica isolates using PAA concentrations of 80-15,104 μ g ml-1 (0.01-1.5%) 306 [65], but found no association between MDR and increased PAA tolerance. Micciche et al. 307 reported MICs of 500 ppm for household PAA (0.00625-0.4%), and 1,000 ppm for industrial 308 grade PAA (0.00625-0.4%) in Salmonella Typhimurium derived from animals [71]. 309 Salmonella Typhimurium can withstand potentially lethal acid shock (pH < 4.0) 310 following adaptation to milder acidic conditions [72], which may partially explain the high MIC 311 values among Typhimurium isolates. Acid tolerance can also be partially enhanced by pre-312 exposure to other stressors [73], which may be more prevalent in food processing environments 313 [74]. This study did not evaluate PAA tolerance among non-food processing derived isolates (i.e. 314 soil, water, and farm swabs: FML-M2-0036 through FML-M2-0038 and FML-M2-0043, FML-315 M2-0044, and FML-M2-0047), limiting evaluation of food processing stress effects on PAA 316 tolerance. It is important to note that MIC values observed in all previous studies were below the 317 2,000 ppm PAA limit set by the USDA-FSIS [75], though advisable concentrations may vary by 318 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint 15 product. These findings emphasize the importance of using sanitizers at full working 319 concentrations to avoid tolerance and avoid inducing antimicrobial resistance [76]. 320 321 Heat Shock 322 Development of heat tolerance can allow for bacterial survival of S. enterica through 323 food processing and potentially incomplete cooking, as well as provide cross-protection to other 324 stresses [24]. We had previously identified unusual heat tolerance in an outbreak-associated S. 325 enterica serovar Heidelberg, including a strain associated with illness from ready-to-eat rotisserie 326 chicken [23, 40]. Consequently, we investigated whether this might be a common strategy in our 327 outbreak-associated isolates in this study. We assessed the heat tolerance of 11 OA isolates 328 (FML-M2-0009 through FML-M2 0013, FML-M2-0030 through FML-M2-0033, FML-M2-329 0047 and FML-M2-0048) from four serovars at 56°C (Figure 2) after conducting growth curves 330 (data not shown) to confirm comparability of growth profiles. All isolates survived at least 15 331 minutes of heat shock, 10 survived past 30 minutes, nine past 45 minutes, and three past 60 332 minutes. The most heat tolerant isolates (Heidelberg FML-M2-0010, OA, chicken and 333 monophasic Typhimurium FML-M2-0012, OA, roast pork) had 30.6 CFU/mL and 575.0 334 CFU/mL, respectively, after 60 minutes. Serovar (p <0.05) and time point (p 0.05). Bacterial counts (CFU/mL) did not 336 significantly decrease after 3 minutes scald (padj >0.05) but decreased after 6 minutes (padj <0.05). 337 Enteritidis isolate (FML M2-0033, OA, raw almonds) was more heat tolerant than monophasic 338 Typhimurium (FML-M2-0011 through FML-M2-0013, OA, roast pork; padj <0.05) and 339 Typhimurium isolates (FML M2-0047 and FML M2-0048, OA, peanut butter; padj <0.05). 340 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint 16 Survival of poultry-associated strains at 56°C is concerning, where scalds in poultry 341 processing typically last <2 minutes at 51-54°C for soft scalds and 59-64°C for hard scalds [14]. 342 Three isolates from the 2013-2014 chicken outbreak (FML-M2-0002/R1-002, FML-M2-343 0005/R1-0004, FML-M2-0008/R1-0007) previously displayed enhanced heat tolerance [23], 344 whereas only FML-M2-0010 did in the present study. However, all strains survived beyond 345 typical scald durations. Inadequate cooking, as noted in the kosher broiled liver outbreak (FML-346 M2-0030 through FML-M2-0032 in Figure 2) [39], and possibly the roast pork (FML-M2-0011 347 through FML-M2-0013) and chicken outbreaks (FML-M2-0009 and FML-M2-0010), may have 348 contributed to survival. Dawoud et al. reported that sublethal heat exposure can enhance bacterial 349 thermal resistance, suggesting that prior heat exposure during food processing or cooking may 350 have contributed to the survival observed in these outbreaks [22]. Our in vitro heat shock assay 351 supports that inherent heat tolerance may have played a role. 352 Burns et al. found comparable heat tolerance in monophasic Typhimurium isolates from 353 pig feed production, with one highly heat tolerant strain with the ASSuT AMR profile 354 (resistances to ampicillin (A), streptomycin (S), sulfisoxazole (Su), and tetracycline (T)); isolates 355 with the same AMR profile were implicated in the roast pork outbreak [54, 77]. However, only 356 two of our three heat tolerant isolates were MDR, and prior research found no consistent AMR-357 heat tolerance association among poultry outbreak Heidelberg isolates [23]. 358 While the heat tolerance of peanut butter and raw almond isolates was high, this 359 temperature is substantially lower than that used in nut processing (>95oC) [16-18]. Other studies 360 have demonstrated S. enterica survival in peanut butter at temperatures up to 120°C [78], and 361 thermal resistance in peanut butter is well documented [79]. Ma et al. found that Salmonella 362 Tennessee outbreak strains survived for 50 minutes at 90°C, with a 1-log CFU/g reduction, and 363 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint 17 were only undetectable after 120 minutes [80]. Similarly, Shachar and Yaron observed that 364 peanut butter derived isolates of Salmonella Agona, Enteritidis and Typhimurium only had a 3.2-365 log reduction after 50 minutes at 90°C, with even lower efficacy at 80°C and 70°C [81]. 366 Furthermore, Salmonella in dry products, such as nuts, are more heat resistant [82]. We found 367 contradictory thermal resistance among our isolates from dry products; the raw almond isolate 368 FML-M2-0033 was more tolerant compared to peanut butter isolates FML-M2-0047 and FML-369 M2-0048 (p <0.05). Regulations regarding almond processing changed following the almond 370 outbreaks to require a minimum of 4-log CFU reduction of Salmonella from heat treatments 371 [83], necessitated following large influxes of illness. 372 Previous studies have observed variation in heat tolerance by serovar and strain, similar 373 to our findings. Two studies reported that Salmonella Enteritidis isolates were generally more 374 resistant than Typhimurium [6, 82], while another study did not find differences by serovar 375 substantial [6]. Hosts with higher internal body temperatures (e.g., chicken: 42°C) may activate 376 thermal stress resistance mechanisms [22], which may explain enhanced heat tolerance among 377 poultry-derived Heidelberg. Enhanced survival has also been observed in human illness-378 associated Enteritidis PT4 strains [84]. Lastly, we found potential cross-tolerances between heat 379 stress and other environmental stresses. Induction of heat tolerance is known to provide 380 subsequent cross-resistance to other stresses [24, 72], and we found that heat tolerant Heidelberg 381 isolate FML-M2-0031 (OA, kosher broiled chicken liver) survived past 60 minutes of scald at 382 56°C, attached well in 1/20X TSB at 22°C, and also had an NaOCl MIC ≥ 200ppm. Heidelberg 383 isolate FML-M2-0010 (OA, chicken) survived past 60 minutes of heat shock and attached 384 strongly in 1X TSB at 22°C, but did not display additional phenotypic stress tolerances. 385 Monophasic Typhimurium isolate FML-M2-0012 (OA, roast pork) had the highest CFU/mL 386 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint 18 after 60 minutes of heat shock and displayed enhanced attachment capacity but had relatively 387 low NaOCl tolerance (50 ppm in 1/20X, 200 ppm in 1X). 388 Overall, isolates’ enhanced heat tolerance presents a concern for food processing 389 facilities. According to the USDA-FSIS Salmonella Framework for Raw Poultry Products, a 390 product is considered adulterated if it contains 10 CFU/mL or more of a serovar of public health 391 concern [85]. After 60 minutes of a 56°C scald, significantly longer than used in processing for 392 most products, we found two isolates were still above this level of contamination (Heidelberg 393 FML-M2-0010, OA, chicken and monophasic Typhimurium FML-M2-0012, OA, roast pork), 394 which is extremely concerning. 395 396 Biofilm Attachment Assays 397 As the majority (80%) of bacterial infections in the U.S. are linked to foodborne 398 pathogens residing in biofilms [31] and biofilms have been involved in several foodborne 399 outbreaks [86-89], understanding attachment and biofilm formation capacity is crucial to 400 identifying potential attributes to outbreaks. We evaluated all isolates at 24 h, 72 h, and 120 h for 401 biomass (OD600) as an indication of attachment capacity and biofilm formation under four 402 conditions: 1/20X 4°C (nutrient depletion, refrigeration); 1/20X RT (22°C) (nutrient depletion, 403 room temperature); 1X 4°C (nutrient abundance, refrigeration); and 1X RT (22°C) (nutrient 404 depletion, room temperature) (Figure 3). 405 In 1/20X 4°C, biomass varied by serovar (p <0.05), with Newport, Typhimurium, and 406 Enteritidis having the greatest biomass, and monophasic Typhimurium with the least (padj <0.05). 407 Isolates Heidelberg FML-M2-0031 (OA, kosher broiled chicken liver) and Typhimurium FML-408 M2-0048 (OA, peanut butter) had the highest biomass (padj <0.05). Biomass was unaffected by 409 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint 19 time point (24, 72, or 120 hours) (p <0.05) (Figure 3A). In 1X TSB, 4°C, biomass varied by 410 serovar, time point, isolate, and serovar-time interactions (p <0.05). Biomass peaked at 72 h for 411 all serovars except monophasic Typhimurium, which peaked at 120 h, outperforming all other 412 serovars (padj <0.05). Monophasic Typhimurium isolates had the highest biomass compared to all 413 other serovars over all time points (padj <0.05), and Typhimurium FML-M2-0012 (OA, roast 414 pork) had a particularly high biomass (padj <0.05). NOA isolates had greater biomass at 72 h than 415 OA isolates, but OA isolates had greater biomass at 24 and 120 h, though not significant (Figure 416 3B). 417 At 1/20X RT, biomass varied by serovar, outbreak, time, isolate number, and serovar-418 time interactions (p <0.05). Enteritidis had the highest biomass (padj < 0.05), while monophasic 419 Typhimurium had the lowest (padj <0.05). Biomass was highest at 72 h (padj <0.05). At 72 h, 420 Enteritidis had particularly high biomass (padj <0.05), whereas monophasic Typhimurium had 421 particularly low biomass, but exceeded all other serovars at 120 h. NOA isolates tended to form 422 stronger biofilms compared to OA isolates, though not significant (Figure 3C). In 1X TSB RT, 423 biomass varied by serovar, outbreak, time point, isolate number, and serovar-time interactions (p 424 <0.05). Biomass was highest at 120 h compared to 72 h (padj <0.05). Monophasic Typhimurium 425 had the highest biomass across all serovars (padj <0.05). OA isolates had higher biomass at all 426 time points compared to NOA isolates (p <0.05). Heidelberg isolates had higher biomass than 427 Enteritidis, Newport, and Typhimurium (padj <0.05). The three kosher broiled chicken liver 428 outbreak Heidelberg isolates (FML-M2-0030 through FML-M2-0032) had lower biomass 429 compared to chicken outbreak Heidelberg isolates (FML-M2-0001 through FML-M2-0010). 430 Specifically, FML-M2-0003 outperformed eight other isolates (padj <0.05) (Figure 3D). 431 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint 20 Overall, biomass was highest in 1/20X RT, followed by 1X RT. By serovar, 432 Typhimurium isolates had the highest mean biomass in all conditions, followed by Newport, 433 Enteritidis, Heidelberg, and monophasic Typhimurium. Monophasic Typhimurium isolates had 434 the lowest biomass in 1/20X TSB, but the highest in 1X TSB. Enteritidis isolates had particularly 435 high biomass values in 1X RT. Serovars Newport and Typhimurium also had particularly high 436 biomass values in the high stress conditions of 1/20X 4°C. At room temperature, biomass at 72 437 and 120 h exceeded 24 h, suggesting that initial attachment is less temperature-dependent and 438 maturation of biofilms is enhanced at RT compared to 4°C. OA isolates had greater biomass in 439 1X, RT at all time points (padj 0.05). NOA isolates had higher biomass in 1/20X, 4°C at all time points (padj <0.05), 441 marginally higher biomass in 1/20X RT at all time points, and marginally higher biomass in 1X 442 4°C at 72 h. 443 Consistent with our findings, Stepanović et al. found that Salmonella biofilm formation in 444 1/20X TSB was more effective than 1X TSB across 122 strains [90]. Salmonella’s ability to form 445 biofilms in response to starvation stress [91] is concerning, as 1/20X TSB conditions mimic the 446 food processing industry [90]. Additionally, Stepanović et al. found that isolate source does not 447 impact biofilm forming ability [90], which contradicts our findings where differences in biomass 448 in 1X TSB were influenced by outbreak (padj <0.05), though serovar distribution may be 449 confounding. Agarwal et al. tested 151 strains across 69 serovars and found that over half formed 450 moderate or strong biofilms [92]. Enteritidis outperformed Typhimurium in 1X 4°C, consistent 451 with our results, and the highest biomass was observed at 48 h and 72 h, respectively [92]. 452 Interestingly, Burns et al., noted biofilm formation as a potential future direction of research in 453 their study evaluating heat tolerance among monophasic Typhimurium isolates from pig feed 454 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint 21 (Burns et al., 2016). Our findings suggest that monophasic Typhimurium may take longer to 455 form mature biofilms, which could have contributed to persistence observed in the 2015 roast 456 pork outbreak, as biofilm formation is crucial for environmental survival [93]. Further 457 investigation into swine associated monophasic Typhimurium biofilm capacity is warranted. 458 Lastly, Wang et al. linked sanitizer tolerance to biofilm forming ability [94]. In this study, 459 Typhimurium – the strongest biofilm formers across all conditions— consistently exhibited the 460 highest NaOCl tolerance, while the one Typhimurium isolate tested (FML-M2-0029) had the 461 highest PAA tolerance, along with Enteritidis isolates. 462 463

464 We assessed 43 OA and NOA S. enterica isolates from various serovars for phenotypic 465 stress tolerance to heat and sanitizers, and for attachment capacity under processing-relevant 466 conditions (nutrient abundance vs. depletion, and room temperature vs. refrigeration). Isolates 467 displayed varying patterns of stress tolerance, with some showing potential cross-tolerance to 468 multiple stresses. The three meat and poultry associated outbreaks included at least one isolate 469 with survival following 60 minutes scald, suggesting that heat tolerance may have contributed to 470 these outbreaks, particularly in cases of improper cooking. The short heat treatment used in the 471 meat industry, aimed at preserving quality, emphasizes the concern regarding heat tolerance 472 among meat-associated isolates. Nut-associated isolates also exhibited enhanced stress tolerance: 473 Typhimurium isolates showed high biomass and NaOCl tolerance in 1/20X, and the Enteritidis 474 isolate from almonds had high biomass and moderate NaOCl tolerance in 1/20X, as well as the 475 highest survival across all time points during heat shock. Overall, the characteristics of these 476 isolates likely contributed to the scope and severity of their respective outbreaks, and further 477 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint 22 research regarding the complex and nuanced relationship between specific S. enterica strains, 478 stress tolerance, and their impacts to human health is needed to accurately model risks. 479 480 481 482 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint 1 Table 1: Characteristics of strains used in this study. 483 FML Strain Obtained From† Isolate Identifiers Serovar ‡ Biosample Accession cgMLST Type Outbreak ¶ Specific Source FML-M2-0001 USDA-ARS B-65037, B-59140, FSIS-FY14-1 Heidelberg SAMN02709124 14616 Chicken, 2013-14 Chicken tenderloin FML-M2-0002 USDA-ARS B-65038, B-59141, FSIS-FY14-2 Heidelberg SAMN02709125 14619 Chicken, 2013-14 Chicken thighs FML-M2-0003 USDA-ARS B-65039, B-59142, FSIS-FY14-3 Heidelberg SAMN0270912 14444 Chicken, 2013-14 Rotisserie chicken FML-M2-0004 USDA-ARS B-65040, FSIS-FY14-4 Heidelberg SAMN02709127 14626 Chicken, 2013-14 Chicken breast FML-M2-0005 USDA-ARS B-65042, B-59143, FSIS-FY14-6 Heidelberg SAMN02709129 14621 Chicken, 2013-14 Chicken drumsticks FML-M2-0006 USDA-ARS B-65043, B-59144, FSIS-FY14-7 Heidelberg SAMN02709130 14624 Chicken, 2013-14 Chicken tenderloin FML-M2-0007 USDA-ARS B-65044, FSIS-FY14-8 Heidelberg SAMN02709131 14625 Chicken, 2013-14 Chicken leg quarters FML-M2-0008 USDA-ARS B-65046, B-59146, FSIS-FY14-10 Heidelberg SAMN02709133 14623 Chicken, 2013-14 Raw Intact Chicken FML-M2-0009 USDA-ARS B-65047, FSIS-FY14-11 Heidelberg SAMN02709134 14620 Chicken, 2013-14 Chicken drumsticks FML-M2-0010 USDA-ARS B-65048, FSIS-FY14-12 Heidelberg SAMN02709135 14617 Chicken, 2013-14 Raw Intact Chicken FML-M2-0011 USDA-ARS B-65411, FSIS1503788 m. Typh. SAMN04088947 10418 Roast pork, 2015 Roaster Swine FML-M2-0012 USDA-ARS B-65412, FSIS1503789 m. Typh. SAMN04125694 10418 Roast pork, 2015 Market Swine FML-M2-0013 USDA-ARS B-65413, FSIS1503790 m. Typh. SAMN04125695 8618 Roast pork, 2015 Market Swine FML-M2-0014 USDA-ARS B-65414, FSIS1503791 m. Typh. SAMN04125696 10421 Roast pork, 2015 Roaster Swine FML-M2-0015 USDA-ARS B-65415, FSIS1503792 m. Typh. SAMN04125697 8618 Roast pork, 2015 Market Swine FML-M2-0016 USDA-ARS B-65416, FSIS1503800 m. Typh. SAMN04088949 12051 Roast pork, 2015 Roaster Swine FML-M2-0017 USDA-ARS B-65417, FSIS1503803 m. Typh. SAMN04088950 12050 Roast pork, 2015 Market Swine FML-M2-0018 USDA-ARS B-65418, FSIS1503831 m. Typh. SAMN04088343 8558 Roast pork, 2015 Roaster Swine FML-M2-0019 USDA-ARS B-65419, FSIS1503896 m. Typh. SAMN04054238 8618 Roast pork, 2015 Roaster Swine FML-M2-0020 USDA-ARS B-65420, FSIS1503955 m. Typh. SAMN04160886 8618 Roast pork, 2015 Market Swine FML-M2-0021 USDA-ARS B-65421, FSIS1503956 m. Typh. SAMN04160887 8618 Roast pork, 2015 Market Swine FML-M2-0022 USDA-ARS B-65435, FSIS1606261 m. Typh. SAMN04856237 917 Roast pork, 2015 Pork Sausage FML-M2-0023 USDA-ARS B-65436, FSIS1606262 m. Typh. SAMN04856238 929 Roast pork, 2015 Ground Pork .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint 2 FML-M2-0024 USDA-ARS B-65437, FSIS1606267 m. Typh. SAMN04855045 911 NOA, 2016 Sausage FML-M2-0025 USDA-ARS B-65439, FSIS1606643 m. Typh. SAMN05001758 39962 NOA, 2016 Pork Feet FML-M2-0026 FDA N42462, 12CT11GT04-S Heidelberg SAMN05201972 46955 NOA, 2012 Ground Turkey FML-M2-0027 FDA N15798, 07NM11GT07-S Heidelberg SAMN03842331 11444 NOA, 2007 Ground Turkey FML-M2-0028 FDA N37936, 11NY10CB10-S Enteritidis SAMN05201730 47354 NOA, 2011 Chicken breast FML-M2-0029 FDA N30688, 11NY04CB01-S Typh. v. 5- SAMN05201583 47184 NOA, 2011 Chicken breast FML-M2-0030 NYSAGM 11B09799A-1, CFSAN062518 Heidelberg SAMN06672001 8100 Chicken liver, 2011 Fresh chopped chicken liver FML-M2-0031 NYSAGM 11B09801A-1, CFSAN062519 Heidelberg SAMN06672000 8100 Chicken liver, 2011 Broiled chicken liver FML-M2-0032 NYSAGM 11B09887A-1, CFSAN062520 Heidelberg SAMN06671998 8100 Chicken liver, 2011 Broiled chicken liver FML-M2-0033 ATCC ATCC BAA-1045 Enteritidis SAMN07682529 170461 Almonds, 2003-04 Raw almonds FML-M2-0036 Cornell FSL S10-1621; CFSAN068898 Enteritidis SAMN08048784 35137 NOA, 2011 Produce farm soil FML-M2-0037 Cornell FSL S10-1623 Enteritidis SAMN01902470 35137 NOA, 2011 Produce farm soil FML-M2-0038 Cornell FSL S10-1644 Enteritidis SAMN01902476 35137 NOA, 2011 Produce farm swab FML-M2-0040 Cornell FSL R9-5251; MDD314R Newport SAMN07411290 170519 NOA Tomatoes FML-M2-0041 Cornell FSL R9-5252; MDD314 Newport SAMN07411291 170750 NOA Tomatoes FML-M2-0043 Cornell FSL S10-1020 Newport SAMN01902454 13323 NOA Farm, drag swab FML-M2-0044 Cornell FSL S10-1060 Newport SAMN01902455 35117 NOA Farm, soil FML-M2-0046 Cornell FSL S10-1630 Newport SAMN01902472 35133 NOA Water FML-M2-0047 FDA SAL 9672 Typh. SAMN02678704 17662 Peanut butter, 2008- 09 Peanut paste FML-M2-0048 FDA SAL 10566 Typh. SAMN02846344 4207 Peanut butter, 2008- 09 Peanut butter cheese cracker †Obtained From: USDA-ARS = USDA-ARS Culture Collection, NYSAGM = New York State Department of Agriculture and Marketing, 484 Cornell = Cornell Food Safety Laboratory; ‡Serovar: m. Typh. = monophasic Typhimurium (I 4,5,[12]:i:-), Typh. v. 5- = Typhimurium variant 5-, 485 Typh. = Typhimurium 486 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint 1 487 Figure 1: Heatmap of isolate antimicrobial resistance profiles, relevant genotypic characteristics, 488 and results of environmental stress tolerance experiments. Heatmap values for environmental 489 stress tolerance are based upon MIC for sanitizers, absorption values for biofilm formation 490 (OD600), and the timepoint to which isolates survived 56°C heat shock (30, 45, 60 or >60 491 minutes). 492 493 Figure 2: Heat tolerance of isolates (n=11) in stationary phase undergoing heat shock at 56°C. 494 Time for isolates to reach 56°C from 37°C incubation (i.e. come-up time) was 2 minutes, 21 495 seconds. 496 497 Figure 3: Attachment capacity of isolates, grouped by serovar, on polystyrene plates measured 498 by crystal violet at 24, 72, and 120 hours in (A) 1/20X 4°C; (B) 1X 4°C; (C) 1/20X RT; and (D) 499 1X RT conditions, where a Log10(OD600) value closer to zero (shorter bar) indicates higher 500 biomass and thus better attachment capacity. 501 502 Supplementary Material 503 Figure S1: Phylogenetic tree of isolates included in this study. 504 Table S1: Phenotypic AMR profiles for isolates in this study (from NCBI/isolate metadata). 505 Table S2: Tolerance to sodium hypochlorite (NaOCl) and peracetic acid (PAA) as minimum 506 inhibitory concentrations (MIC; ppm). 507 508 509 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint 2

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Dis., 2017. 753 14(12): p. 687-695. 754 755 .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint BLE FOF QNL Acid Strain Serovar¶ OA/ NOA † aadA1 ant(2")-Ia aac(3)-Via aac(3)-11d aph(3")-Ib aph(3')-Ia aph(3')-IIa aph(6)-Ic aph(6)-Id ble blaCMY-2 blaSHV-12 blaTEM-1 cmlA5 floR fosA7 qnrB19 sul1 sul2 tetA tetB Mercury Tellurium Copper Silver Gold Arsenic asr qacEdelta1 1/20x 1x 1/20x 1x Heat Shock 1/20X, 4C 1/20X, RT 1X, 4C 1X, RT FML-M2-0001 Heidelberg OA a n/a FML-M2-0002 Heidelberg OA a n/a FML-M2-0003 Heidelberg OA a n/a FML-M2-0004 Heidelberg OA a n/a FML-M2-0005 Heidelberg OA a n/a FML-M2-0006 Heidelberg OA a n/a FML-M2-0007 Heidelberg OA a n/a FML-M2-0008 Heidelberg OA a n/a FML-M2-0009 Heidelberg OA a FML-M2-0010 Heidelberg OA a FML-M2-0011 m. Typh. OA b FML-M2-0012 m. Typh. OA b FML-M2-0013 m. Typh. OA b FML-M2-0014 m. Typh. OA b n/a FML-M2-0015 m. Typh. OA b n/a FML-M2-0016 m. Typh. OA b n/a FML-M2-0017 m. Typh. OA b n/a FML-M2-0018 m. Typh. OA b n/a FML-M2-0019 m. Typh. OA b n/a FML-M2-0020 m. Typh. OA b n/a FML-M2-0021 m. Typh. OA b n/a FML-M2-0022 m. Typh. OA b n/a FML-M2-0023 m. Typh. OA b n/a FML-M2-0024 m. Typh. NOA n/a FML-M2-0025 m. Typh. NOA n/a FML-M2-0026 Heidelberg NOA n/a FML-M2-0027 Heidelberg NOA n/a FML-M2-0028 Enteritidis NOA n/a FML-M2-0029 Typh. var 5- NOA n/a FML-M2-0030 Heidelberg OA c n/a n/a FML-M2-0031 Heidelberg OA c n/a n/a FML-M2-0032 Heidelberg OA c n/a n/a FML-M2-0033 Enteritidis OA d n/a n/a FML-M2-0036 Enteritidis NOA n/a n/a n/a FML-M2-0037 Enteritidis NOA n/a n/a n/a FML-M2-0038 Enteritidis NOA n/a n/a n/a FML-M2-0040 Newport NOA n/a n/a n/a FML-M2-0041 Newport NOA n/a n/a n/a FML-M2-0043 Newport NOA n/a n/a n/a FML-M2-0044 Newport NOA n/a n/a n/a FML-M2-0046 Newport NOA n/a n/a n/a FML-M2-0047 Typhimurium OA e n/a n/a FML-M2-0048 Typhimurium OA e n/a n/a †OA/NOA: OA = outbreak -as s oc iated, NOA = non-outbreak -as s oc iated; aChicken outbreak, 2013-14; bRoast pork outbreak, 2015; cKosher broiled chicken liver outbreak, 2011; dRaw almond outbreak, 2003-04; ePeanut butter outbreak, 2008-09; ‡Genotypic AMR (antimicrobial resistance) Profiles: AG = Aminoglycoside, BLE = Bleomycin, β -L = Beta-lactam, CHL = Chloramphenicol, FOF = Fosfomycin, QNL = Quinolones, SN = Sulfonamides, TET = Tetracyclines; ¶ Serovar : Typh. Var. 5- = Typhimurium variant 5-, m. Typh. = monophasic Typhimurium (I 4,5,[12]:i:-) Genotypic AMR Profiles‡ Environmental Stress Tolerance BiofilmNaOCl PAA TET AG β-L CHL SN Heavy Metal Tolerance Stress .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint 0 20 40 60 0 5 10 Minutes Log10(CFU/mL) Isolate 9 Isolate 10 Isolate 30 Isolate 31 Isolate 32 Isolate 11 Isolate 12 Isolate 13 Isolate 33 Isolate 47 Isolate 48 ∇ monophasic Typhimurium lHeidelberg « Enteritidis u Typhimurium .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint Ente ritidi s Heide lbe rg mo nophasicTyphimu rium New port Typ himu rium -4 -3 -2 -1 0 Attachment of S. enterica on polystyrene grown in 1/20X TSB at 4°C Serovar Log10(OD600nm) 24h 72h 120h .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint Ente ritidi s Heide lbe rg mo nophasi cTyp himu rium New port Typ himu rium -4 -3 -2 -1 0 Attachment of S. enterica on polystyrene grown in 1X TSB at 4°C Serovar Log10(OD600nm) 24h 72h 120h .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint Ente ritidi s Heide lbe rg mo nophasi cTyp himu rium New port Typ himu rium -4 -3 -2 -1 0 Attachment of S. enterica on polystyrene grown in 1/20X TSB at 22°C (RT) Serovar Log10(OD600nm) 24h 72h 120h .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint Ente ritidi s Heide lbe rg mo nophasi cTyp himu rium New port Typ himu rium -4 -3 -2 -1 0 Attachment of S. enterica on polystyrene grown in 1X TSB at 22°C (RT) Serovar Log10(OD600nm) 24h 72h 120h .CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (whichthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.23.649775doi: bioRxiv preprint