Long-Term Survival Phase Cells of Salmonella Typhimurium ATCC 14028 Have Significantly Greater Resistance to Ultraviolet Radiation in 0.85% Saline and Apple Juice

Abstract

Nonendospore-forming pathogenic bacteria in the long-term survival (LTS) phase can remain viable for months or years and may show reduced susceptibility to various antimicrobial interventions. In the present study, we investigated the response of LTS phase Salmonella enterica serovar Typhimurium (ATCC 14028) to ultraviolet (UV) radiation in 0.85% (w/v) saline and apple juice and the extent of sublethal injury in LTS phase survivors. The LTS-phase Salmonella Typhimurium cells were cultured at 35°C for 14 days in tryptic soy broth with 0.6% (w/v) yeast extract (TSBYE). Exponential- and stationary-phase cells, cultured in TSBYE (35°C) for 2.5 and 18 h, respectively, served as control samples. Cells (10⁷ CFU [colony-forming unit]/mL) from each physiological state were exposed to UV light in saline (80 μW/cm²) and apple juice (1500 μW/cm²). The Salmonella Typhimurium survivors were plated for enumeration on either tryptic soy agar with 0.6% yeast extract or xylose-lysine-tergitol 4 (XLT4) agar and colonies counted after incubation (35°C, 24 h). Of all the growth phases tested, LTS phase cells were consistently impacted the least by UV treatment (p < 0.05). In saline, D-values of exponential, stationary, and LTS Salmonella Typhimurium were 0.35, 0.38, and 0.49 min, respectively. D-values in apple juice at pH 3.63 and pH 5.65 were 2.52, 3.19, and 3.57 min and 3.24, 3.50, and 4.18 min, respectively. UV radiation (80 μW/cm²) of Salmonella Typhimurium in saline for 2.5 min reduced the number of exponential- and stationary-phase cells by ∼7.19 and 6.30 log₁₀ CFU/mL, respectively. In contrast, LTS cells were only reduced by 5.08 log₁₀ CFU/mL. Among the three physiological states, LTS phase cells had the least sublethal injury in the surviving population (p < 0.05). These results indicate that the LTS state cross-protects Salmonella Typhimurium against UV radiation and should be considered in determination of the UV radiation D-value for this pathogen.

Introduction

Among the top five most problematic foodborne pathogens, nontyphoidal Salmonella enterica (NTS) were identified as having the highest estimated annual cost of illness and quality-adjusted life year loss in the United States (Batz et al., 2012). The total estimated annual cost of foodborne illness from Salmonella is greater than $3.6 billion, and (USDA Economic Research Service, 2014) global estimates for gastroenteritis due to NTS include 93.8 million cases and 155,000 deaths (Majowicz et al., 2010). The NTS shed in animal feces can survive for long periods in contaminated environments, and their prevalence in samples from farm environments (soil, bedding, litter, feedstuffs, etc.) ranges from 10% to 26% (Rodriguez et al., 2006). Due to numerous animal reservoirs (Dione et al., 2011) and widespread distribution of NTS in the environment, Salmonella can contaminate a wide variety of foods (Noël et al., 2010; Pui et al., 2011).

Ultraviolet (UV) irradiation is an attractive alternative process for juices, which may undergo undesirable nutritional and sensory changes during heat pasteurization. Compared to thermal treatment, UV radiation had a minimal effect on quality characteristics of juice (Noci et al., 2008). The U.S. Food and Drug Administration (FDA) approved UV irradiation as an alternative to thermal pasteurization of fresh juice (Donahue et al., 2004; Food and Drug Administration, HHS, 2012). During UV irradiation, a food is exposed to germicidal light having a wavelength between 220 and 300 nm. UV radiation dimerizes adjacent pyrimidine nucleotide bases in DNA or RNA, which ultimately causes death when the extent of dimerization overwhelms cellular repair mechanisms (Sizer and Balasubramaniam, 1999). Therefore, the extent of pathogen inactivation is directly dependent on the UV radiation dose.

The effect of growth phase on bacterial resistance to UV radiation and ionizing radiation has been reported. For example, Escherichia coli in the stationary phase exhibited an increased resistance to UV radiation, compared to exponential-phase cells (Abedi-Moghaddam et al., 2004). Stationary phase Listeria monocytogenes were more resistant than exponential-phase cells to electron beam irradiation in 0.85% saline (NaCl) and ground pork (Mendonça et al., 2004). The inherent hardiness of stationary phase bacterial cells has encouraged their routine use as target organisms in challenge studies to validate the antimicrobial efficacy of food processes.

The bacterial life cycle is typically characterized by four phases, namely the lag phase, the exponential phase, the stationary phase, and the death phase (Kolter et al., 1993). However, others have posited a fifth phase, in which bacteria display long-term survival (LTS). That phase occurs after the death phase and was referred to by Steinhaus and Birkeland (1939) as the “senescent phase” and by Finkel (2006) as the “long-term stationary phase.” The LTS phase was also described in L. monocytogenes whereby culturable cells remained at 10⁸ CFU/mL for over 30 d in tryptic soy broth with 0.6% (w/v) yeast extract (TSBYE) at 35°C (Wen et al.,, 2009). The LTS L. monocytogenes were predominantly coccoid and exhibited significantly greater resistance to heat and high pressure compared to both stationary- and exponential-phase cells. Also they germinated rapidly to vegetative (reproducing) cells when placed into fresh TSBYE (Wen et al., 2009).

While there is an emerging body of knowledge on the enhanced resistance of LTS L. monocytogenes to food processes such as thermal processing and high pressure (Wen et al., 2009), there are no published reports describing the LTS phase in Salmonella and its effect on the pathogen's resistance to UV irradiation. Accordingly, the purpose of the present study was to evaluate the resistance of LTS Salmonella Typhimurium to UV radiation in 0.85% saline and in apple juice. An additional objective was to compare the extent of sublethal injury in exponential-, stationary-, and LTS phase survivors following UV radiation.

Materials and Methods

Bacterial strain and culture conditions

Salmonella Typhimurium ATCC 14028 was obtained from the culture collection of the Microbial Food Safety Laboratory of Iowa State University. The culture was maintained as frozen (−80°C) stock in brain-heart infusion broth (BHI; Difco; Becton, Dickinson and Company, Sparks, MD) containing 10% (v/v) glycerol. The frozen stock culture was thawed and resuscitated in TSBYE (35°C). Before each experiment at least two consecutive 24 h transfers of the resuscitated stock culture were performed in TSBYE (35°C) to prepare a working culture.

Preparation of exponential-phase, stationary-phase, and LTS cells

A portion (1.0 mL) of Salmonella Typhimurium working culture was transferred to TSBYE (100 mL) in a screw-capped 250 mL Erlenmeyer flask. The inoculated medium was incubated at 35°C with shaking (150 revolutions per minute [rpm]) in a gyrorotary shaker incubator (New Brunswick Scientific Co., Inc., Edison, NJ) for 2.5 and 18 h to obtain exponential- and stationary-phase cells, respectively. LTS phase cells were harvested after 14 d.

Determination of cell culturability

Serial dilutions (10-fold) of Salmonella Typhimurium cultures from each of the three physiological states were prepared in buffered peptone water (BPW; Difco), and 0.1-mL aliquots of appropriate dilutions were surface plated on both tryptic soy agar (TSA; Difco) supplemented with 0.6% yeast extract (TSAYE) and xylose-lysine-tergitol 4 (XLT4) agar (Difco). All inoculated agar plates were incubated aerobically at 35°C, and bacterial colonies were counted at 24 h.

Preparation and inoculation of saline and apple juice

Pasteurized, clarified apple juice (pH 3.63) was purchased from a local grocery in Ames, Iowa and the pH of a portion of that juice was adjusted to 5.65 with 1 M aqueous NaOH. The juice samples were filter sterilized using a bottle-top vacuum filtration system with 0.22 μm pore size filter (Corning, Amsterdam, Netherlands) and stored at 4°C before inoculation. Exponential, stationary, and LTS cells in TSBYE (35°C) were harvested by centrifugation (10,000 × g, 10 min, 22°C) using a Sorvall Super T21 ultracentrifuge (Sorvall Product, L.P., Newtown, CT). The pelleted cells were washed (by vortexing) in 0.85% (w/v) NaCl (saline), harvested by centrifugation, then suspended in fresh saline or sterilized apple juice (pH 3.63 or 5.65) to obtain ∼10⁷ CFU/mL.

UV irradiation treatment

The two UV radiation intensities (μW cm⁻²) used in this study were achieved by adjusting the distance between the UV lamp and samples and measuring the intensity using an UV meter (Model DM-254XA; Spectronics Corporation, Westbury, NY). The UV lamp was a Spectroline Model XX-15F (Spectronics Corporation) emitting UV light (254 nm) and working at 120 V, 60 Hz, and 0.7 A. Aliquots (5-mL) of Salmonella Typhimurium cell suspensions in saline or apple juice were dispensed into sterile 60 × 15 mm plastic petri dishes (Falcon, Tewksbury, MA). Cell suspensions (each 2.0 mm deep) in petri dishes were positioned on an electrical stirrer (Thermolyne Cimarec 2, Dubuque, IA) and stirred at 5 rpm. Cells in saline were exposed to UV radiation (80 μW/cm²) for 0 (control) to 3 min, whereas cells in apple juice were exposed to 1500 μW/cm² for 0 (control) to 12 min. For stable radiation intensity the lamp was warmed up for 10 min before each experiment.

Microbiological analysis

Serial dilutions (10-fold) of control and UV-treated Salmonella Typhimurium in saline and apple juice were prepared in BPW, and aliquots (1.0 or 0.1 mL) of appropriate dilutions were surface plated, in duplicate, on TSAYE and XLT4 plates. In instances when survivors were less than 10 CFU/mL, 1.0-mL samples of nondiluted saline or juice were plated directly onto agar media. All inoculated agar plates were incubated at 35°C, and colonies were counted at 24 h. Only colony counts on TSAYE were used in developing survivor curves of the pathogen to derive D-values.

Calculation of D-values

Survivor curves were prepared by plotting survivors (log₁₀ CFU/mL) versus time of exposure (minutes) to UV radiation using Microsoft Excel 2010 Software (Microsoft, Inc., Redmond, WA). The line of best fit for the data was obtained by linear regression analysis (Ostle and Mensing, 1975). The D-values (exposure times at a specified UV radiation intensity that produced a 90% reduction in the initial population of culturable cells) were determined by calculating the negative reciprocal of the slopes of the regression curves.

Determination of sublethal injury

Survivor curves based on recovery of bacterial colonies on TSAYE and XLT4 were prepared. Reduction in culturability was expressed as the logarithm of the reduction factor (RF). The RF is the ratio of colony counts (CFU/mL) of control to CFU/mL of the treated sample (Wuytack et al., 2003). The log of RF from CFU on XLT4 was plotted on the y-axis against the log of RF from CFU on TSAYE, and linear regression lines were fitted through the data points. The extent to which each treatment caused sublethal injury was compared using the slopes of the regression lines for each treatment (Wuytack et al., 2003).

Statistical analysis

All experiments were replicated thrice, and results are reported as averages. The D-values and linear regression model were prepared using SAS software (SAS version 8.2; SAS Institute, Cary, NC). Tests were carried out at a 5% significance level.

Results and Discussion

Cell culturability

Figure 1 shows exponential phase (A), stationary phase (B), death phase (C), and LTS phase (D) of Salmonella Typhimurium growth cycle in TSBYE (35°C). The exponential phase lasted for ∼13 h until the population reached ∼9.82 log₁₀ CFU/mL (stationary phase; B). The duration of the stationary phase was about 24 h before the cells entered into death phase (C), and the culturable population decreased to ∼7.90 and 6.50 log₁₀ CFU/mL after 2 and 30 d, respectively. After 30 d, numbers of culturable cells remained stable in TSBYE for more than 36 months. There was no significant difference between cell counts (CFU/mL) of LTS Salmonella Typhimurium obtained at set times from 30 to 1200 d (p > 0.05). Even though a few minor aspects—such as length of the lag and exponential phases and highest cell concentration attained—may vary due to factors such as bacterial species or specific growth conditions, the profile of the five-phase life cycle observed for Salmonella Typhimurium was similar to that described elsewhere (Steinhaus and Birkeland, 1939; Finkel, 2006).

FIG. 1.

Growth of Salmonella Typhimurium (ATCC 14028) in TSBYE at 35°C for different times to yield exponential-phase (A), stationary-phase (B), death-phase (C), or long-term survival phase (D) cells. The data points and error bars represent averages and standard deviations, respectively, based on three replications of the experiment. TSBYE, tryptic soy broth with 0.6% (w/v) yeast extract.

UV resistance in saline and apple juice

Figure 2 shows survival curves of exponential-, stationary-, and LTS phase cells of Salmonella Typhimurium following UV irradiation (80 μW/cm²) in 0.85% (w/v) saline at 23°C ± 1°C. Irrespective of growth phase, cell culturability declined with increasing exposure to UV radiation. A significantly (p < 0.05) higher concentration of LTS cells remained culturable after 2.5 and 3.0 min. After 2.5 min, no exponential cells could be detected (detection limit 1 CFU/mL), whereas numbers of stationary phase and LTS survivors were 1.20 and 2.60 log₁₀ CFU/mL, respectively. After 3.0 min neither exponential- nor stationary-phase cells were detected, representing a 7.0 log₁₀ CFU reduction in initial numbers of the pathogen. In contrast about 2.04 log₁₀ CFU of LTS survivors remained, reflecting a 4.96 log₁₀ CFU reduction. Radiation (UV) D-values for survivors from the three growth phases indicate that the D-value for LTS cells in 0.85% saline was significantly (p < 0.05) higher (Table 1).

FIG. 2.

Culturability of exponential-, stationary-, and long-term survival phase cells of Salmonella Typhimurium ATCC 14028 following UV irradiation in 0.85% (w/v) saline at 23°C ± 1°C. The data points and error bars represent averages and standard deviations, respectively, derived from three replications of the experiment. UV, ultraviolet.

Table 1.

Radiation (Ultraviolet) D-Values for Exponential, Stationary, and Long-Term Survival Cells of Salmonella Typhimurium in 0.85% Saline and Apple Juice at pH 3.63 and 5.65 (pH-Adjusted)

Life cycle phase	0.85% Saline 80 μW/cm²	Apple juice (pH 3.63) 1500 μW/cm²	Apple juice (pH 5.65) 1500 μW/cm²
Exponential	0.35 ± 0.00Ax	2.52 ± 0.12Ay	3.24 ± 0.06Az
Stationary	0.38 ± 0.00Bx	3.19 ± 0.05By	3.50 ± 0.07Bz
LTS	0.49 ± 0.01Cx	3.57 ± 0.06Cy	4.18 ± 0.06Cz

Survivors were recovered on tryptic soy agar supplemented with 0.6% yeast extract. Values are averages ± standard deviations from three replications of the experiment. Averages with different letters (A, B, C) within each column or within each row (x, y, z) are significantly different (p < 0.05).

LTS, long-term survival.

This high resistance of LTS cells to UV radiation (1500 μW/cm²) was also observed in apple juice irrespective of juice pH (Table 1). For both 0.85% saline and apple juice the order of UV radiation resistance of Salmonella Typhimurium was: exponential < stationary < LTS (p < 0.05). After 4 through 12 min of UV irradiation in apple juice, irrespective of juice pH, log₁₀ CFU reductions were significantly lower (p < 0.05) for LTS cells (Fig. 3A, B). These results indicate that LTS Salmonella was impacted by UV radiation to a much lesser extent than were exponential- or stationary-phase cells.

FIG. 3.

Reduction in populations of culturable exponential, stationary, and long-term survival cells of Salmonella Typhimurium following UV irradiation in apple juice (pH 3.63) (A) and pH-adjusted apple juice (pH 5.65) (B). Reductions in populations are average values from three replications of the experiment. Error bars represent standard deviations. For each exposure time point, bars that do not share a common letter are significantly different (p < 0.05). UV, ultraviolet.

Our results are consistent with those of Wen et al., 2009 who demonstrated enhanced resistance of LTS L. monocytogenes to high pressure and thermal treatment compared to the pathogen in other phases of the life cycle (p < 0.05). In addition, bacteria that cease growing and remain viable without nutrients for extended times demonstrate enhanced resistance to antimicrobial interventions. For example, E. coli O157:H7 and L. monocytogenes Scott A that survived for 10 d in 0.85% saline exhibited enhanced resistance to electron beam radiation (p < 0.05) compared to exponential- and stationary-phase cells (Mendonça et al., 2004; Hong et al., 2014).

In the natural environment, conditions that allow constant growth of bacteria as observed in nutrient-rich laboratory broth are rarely found. In fact, due to limited nutrients and harsh conditions in natural environments, bacteria may enter a prolonged stationary phase and exhibit low metabolic rate or dormancy (Kolter et al., 1993). For example, cell dormancy induced by nutrient limitation has been reported in marine bacteria (Novitsky and Morita, 1978). While some species of Gram-positive bacteria produce dormant spores, many Gram-negative bacteria produce seemingly dormant vegetative cells under such conditions. Therefore, it is likely that LTS cells enter a dormant state that reduces their sensitivity to antimicrobial interventions. This explanation seems plausible because “persister” cells of clinically important bacteria are described as dormant nondividing cells with reduced metabolism, and those cells, for reasons unclear, have a very high tolerance for antibiotics (Shah et al., 2006; Lewis, 2010). Further research is needed to determine if LTS Salmonella Typhimurium are dormant and, if so, to explore the connections between dormancy and their physiological resiliency. This research could have significant ramifications for the design and validation of effective antimicrobial interventions in foods where LTS cells may be present.

UV radiation-induced sublethal injury

Table 2 shows, as an index of sublethal injury, the linear regression slopes from plots of reduction in culturability for exponential, stationary, and LTS Salmonella Typhimurium on selective versus nonselective media following UV irradiation (1500 μW/cm²) in apple juice. The logarithm of reduction in pathogen culturability, as calculated for XLT4 selective agar, was plotted against the log of reduction in culturability for TSAYE agar, and linear regression lines were fitted through the data points (Wuytack et al., 2003). The extent to which each treatment caused sublethal injury was compared using the slopes of the lines shown in Table 2. When the slope is equal to 1, there is no sublethal injury, since the same reduction in culturability is observed on both selective and nonselective agars. A slope that is >1 indicates sublethal injury, since a higher reduction in culturability is observed on the selective plates than on the nonselective plates.

Table 2.

Linear Regression Slopes from Plots of Viability Reduction (Reduction Factor) for Exponential, Stationary, and Long-Term Survival Cells of Salmonella Typhimurium on Selective Versus Nonselective Media Following Ultraviolet Irradiation (1500 μW/cm ² ) in Apple Juice

Life cycle phase	Apple juice (pH 3.63)	Apple juice (pH 5.65)
Exponential	1.16 ± 0.11Aa^a	1.04 ± 0.07Cb
Stationary	1.10 ± 0.01Ba	1.00 ± 0.09Db
LTS	1.05 ± 0.02Ca	0.99 ± 0.04Da

Values are averages ± standard deviations from three replications of the experiment. Averages with the same uppercase letter in the same column or with the same lowercase letter in the same row are not significantly different (p > 0.05).

Cells of Salmonella Typhimurium from all three growth phases exhibited less sublethal injury in pH-adjusted apple juice (pH 5.65) compared to apple juice with pH of 3.63; however, this difference was only significant (p < 0.05) for exponential- and stationary-phase cells but not for LTS cells (Table 2). In apple juice (pH 5.65) the difference in sublethal injury in stationary phase cells and LTS cells was not significant (p > 0.05). The highest and lowest levels of sublethal injury were observed in exponential-phase cells and LTS cells, respectively, irrespective of the juice pH (p < 0.05).

Sublethally injured cells formed colonies on nonselective agar (TSAYE) but not on selective agar (XLT4). Such cells most likely endured UV radiation-induced damage which they were unable to repair on selective agar. The primary cellular target of UV radiation is the DNA, resulting in photodimerization of pyrimidine bases, which ultimately interferes with DNA replication and cell division (Lindahl, 1993), although other cellular structures may be affected. Similar to our findings, Abedi-Moghaddam et al. (2004) reported that stationary E. coli cells were more resistant than exponential cells to UV radiation. Those authors suggested an upregulation of DNA repair mechanisms or a lower DNA replication rate in stationary phase, which allowed more time for DNA repair. Possible explanations for our results showing an increased resiliency of LTS cells to UV irradiation may include some means of DNA protection against UV damage by these cells, more efficient repair mechanisms, or a reduction or stoppage in DNA replication, allowing more time for repair of DNA. This latter explanation is reminiscent of theories proposed for the tolerance of persister cells to antibiotics in the clinical realm, raising the possibility that LTS cells in foods are functionally similar (or identical) to persister cells which are problematic in clinical microbiology (Wood et al., 2013).

Conclusions

The LTS state substantially reduces susceptibility of Salmonella Typhimurium to UV radiation and results in less sublethal injury in those cells compared to exponential- or stationary-phase cells. Stationary phase bacterial cells are used in validation of food processes for inactivation of pathogens. However, our findings indicate that stationary phase Salmonella Typhimurium is less resistant to UV radiation than LTS phase cells. Consequently, food processing or preservation methods that are validated using stationary phase cells may overestimate the utility of such interventions and, therefore, may be inadequate to reduce the food safety risk from LTS Salmonella. If salmonellae occur in the LTS state in food processing environments, particularly in hard-to-clean growth niches (Tompkin, 2002) or in biofilms (Rodrigues et al., 2011), they could represent a constant source of contamination to food products. Our results suggest that LTS-induced protection of pathogens against food UV irradiation should be considered when determining D-values to ensure the microbial safety of foods treated with UV radiation.

Footnotes

Acknowledgments

The authors thank Dr. Stephen Knabel for critical review of the article and Yutong Zhang for technical assistance. This work is supported by AFRI Food Safety Challenge Area grant No. 2013-67005-21271/project accession No. 1001300 from the USDA National Institute of Food and Agriculture and by Iowa Agriculture and Home Economics Experiment Station Project No. IOW03902, sponsored by Hatch Act and State of Iowa funds.

Disclosure Statement

No competing financial interests exist.

References

Abedi-Moghaddam

, Bulic

, Herderson

, Lam

. Survival of Escherichia coli to UV irradiation during exponential and stationary phases of growth. J Exp Microbiol Immunol, 2004; 5:44–49.

Batz

, Hoffmann

, Morris

Jr . Ranking the disease burden of 14 pathogens in food sources in the United States using attribution data from outbreak investigations and expert elicitation. J Food Prot, 2012; 75:1278–1291.

Dione

, Ikumapayi

, Saha

, Mohammed

, Geerts

, Ieven

, Adegbola

, Antonio

. Clonal differences between non-typhoidal Salmonella (NTS) recovered from children and animals living in close contact in the Gambia. PLoS Negl Trop Dis, 2011; 5:e1148.

Donahue

, Canitez

, Bushway

. UV inactivation of E. coli O157: H7 in apple cider: Quality, sensory and shelf-life analysis. J Food Process Preserv, 2004; 28:368–387.

Finkel

. Long-term survival during stationary phase: Evolution and the GASP phenotype. Nat Rev Microbiol, 2006; 4:113–120.

Food and Drug Administration HHS. Irradiation in the production, processing and handling of food. Final rule. Fed Regist, 2012; 77:71312–71320.

Hong

, Mendonça

, Daraba

, Shaw

. Radiation resistance and injury in starved Escherichia coli O157: H7 treated with electron-beam irradiation in 0.85% saline and in apple juice. Foodborne Pathog Dis, 2014; 11:900–906.

Kolter

, Siegele

, Tormo

. The stationary phase of the bacterial life cycle. Annu Rev Microbiol, 1993; 47:855–874.

Lewis

. Persister cells. Annu Rev Microbiol, 2010; 64:357–372.

10.

Lindahl

. Instability and decay of the primary structure of DNA. Nature, 1993; 362:709–715.

11.

Majowicz

, Musto

, Scallan

, Angulo

, Kirk

, O'Brien

, Jones

, Fazil

, Hoekstra

, International Collaboration on Enteric Disease “Burden of Illness” Studies. The global burden of nontyphoidal Salmonella gastroenteritis. Clin Infect Dis, 2010; 50:882–889.

12.

Mendonça

, Romero

, Lihono

, Nannapaneni

, Johnson

. Radiation resistance and virulence of Listeria monocytogenes Scott A following starvation in physiological saline. J Food Prot, 2004; 67:470–474.

13.

Noci

, Riener

, Walkling-Ribeiro

, Cronin

, Morgan

, Lyng

. Ultraviolet irradiation and pulsed electric fields (PEF) in a hurdle strategy for the preservation of fresh apple juice. J Food Eng, 2008; 85:141–146.

14.

Noël

, Hofhuis

, De Jonge

, Heuvelink

, De Jong

, Heck

, De Jager

, van Pelt

. Consumption of fresh fruit juice: How a healthy food practice caused a national outbreak of Salmonella Panama gastroenteritis. Foodborne Pathog Dis, 2010; 7:375–381.

15.

Novitsky

, Morita

. Possible strategy for survival of marine bacteria under starvation conditions. Mar Biol, 1978; 48:289–295.

16.

Ostle

, Mensing

. Statistics in Research, 3rd edition. Ames, IA: Iowa State University Press, 1975.

17.

Pui

, Wong

, Chai

, Nillian

, Ghazali

, Cheah

, Nakaguchi

, Nishibuchi

, Radu

. Simultaneous detection of Salmonella spp., Salmonella Typhi and Salmonella Typhimurium in sliced fruits using multiplex PCR. Food Control, 2011; 22:337–342.

18.

Rodrigues

, Teixeira

, Oliveira

, Azeredo

. Salmonella enterica Enteritidis biofilm formation and viability on regular and triclosan-impregnated bench cover materials. J Food Prot, 2011; 74:32–37.

19.

Rodriguez

, Pangloli

, Richards

, Mount

, Draughon

. Prevalence of Salmonella in diverse environmental farm samples. J Food Prot, 2006; 69:2576–2580.

20.

Shah

, Zhang

, Khodursky

, Kaldalu

, Kurg

, Lewis

. Persisters: A distinct physiological state of E. coli . BMC Microbiol, 2006; 6:53.

21.

Sizer

, Balasubramaniam

. New intervention processes for minimally processed juices. Food Technol, 1999; 53:64–67.

22.

Steinhaus

, Birkeland

. Studies on the life and death of bacteria: I. The senescent phase in aging cultures and the probable mechanisms involved. J Bacteriol, 1939; 38:249.

23.

Tompkin

. Control of Listeria monocytogenes in the food-processing environment. J Food Prot, 2002; 65:709–725.

24.

USDA Economic Research Service.. Cost Estimates of Foodborne Illness. 2014. Available at www.ers.usda.gov/data-products/cost-estimates-of-foodborne-illnesses, accessed October 1, 2017 .

25.

Wen

, Anantheswaran

, Knabel

. Changes in barotolerance, thermotolerance, and cellular morphology throughout the life cycle of Listeria monocytogenes . Appl Environ Microbiol, 2009; 75:1581–1588.

26.

Wood

, Knabel

, Kwan

. Bacterial persister cell formation and dormancy. Appl Environ Microbiol, 2013; 79:7116–7121.

27.

Wuytack

, Phuong

, Aertsen

, Reyns

, Marquenie

, De Ketelaere

, Masschalck

, Van Opstal

, Diels

, Michiels

. Comparison of sublethal injury induced in Salmonella enterica serovar Typhimurium by heat and by different nonthermal treatments. J Food Prot, 2003; 66:31–37.