Contamination by and persistence of pathogenic bacteria in ready-to-eat produce have emerged as significant food safety and public health concerns. Viable produceborne pathogens cope with several stresses (e.g., temperature fluctuations and low-temperature storage) during production and storage of the commodities. In this study, we investigated the impact of transient cold shock on Escherichia coli O157:H7 (EcO157) cells in a produce matrix (romaine lettuce leaf lysate). EcO157 cells were exposed to 25°C for 1 h, 4°C for 1 h, and 4°C for 10 min in lettuce lysate. The expression of selected genes coding for virulence, stress response, and heat and cold shock proteins was quantified by real-time quantitative reverse transcription PCR assay. Treated EcO157 cells adhered to MAC-T mammalian cells were enumerated by in vitro bioassay. Expression of the Shiga toxin 1 gene (stx1a) was upregulated significantly (P < 0.05) upon cold shock treatments, but virulence genes related to EcO157 attachment (eaeA, lpfA, and hcpA) were down-regulated. Two key members of the cold shock regulon, cold shock protein (cspA) and gyrA, were significantly induced (P < 0.05) at the refrigeration temperature (4°C). Significant upregulation of an SOS response gene, recA, was also observed. E. coli heat shock regulon member grpE was induced, but a universal stress protein (uspA) was down-regulated at the refrigeration temperatures in lettuce lysate. The adhesion assay revealed a temperature-dependent reduction in the attachment of cold-shocked EcO157 cells. The results of the current study indicate a reduction in the attachment of cold-shocked EcO157 to epithelial cells and higher levels of Shiga toxin gene expression at the molecular level.

Leafy vegetables are frequently linked to foodborne disease outbreaks. According to epidemiological reports, food commodities of plant origin (including fresh produce) have been associated with the highest number of domestically acquired foodborne illnesses in the United States in recent years (31, 37). Fresh produce may harbor human pathogens as part of their microflora due to contamination from a wide variety of sources spread over the entire processing chain from fields to the consumer's plate (5, 11). The persistence and successful adaptation of these pathogens in the plant environment is possible due to altered gene expressions that trigger a cascade of intracellular stress response mechanisms (7, 28). For example, in produce microecosystems (lettuce leaf lysate), induction of several key genes in Escherichia coli O157:H7 (EcO157) was reported (26), which included stress-response genes such as members of the OxyR regulon associated with oxidative stress (e.g., ahpF, ahpC, grxA, trxC, and katG) and osmotic stress (e.g., betA, betB, caiA, caiB, and fix), genes associated with the locus of enterocyte effacement pathogenicity island (e.g., escR, escC, and sepL), and genes responsible for antimicrobial resistance (e.g., marA). Viable produceborne pathogens cope with additional sublethal stressors (such as temperature fluctuations and low-temperature storage) during the production and storage of various commodities. Transient arrest of cellular growth (3 to 6 h) and differential regulation of genes in bacteria exposed to a down-shift in temperature (refrigeration) has been reported (6, 35). For EcO157, cold exposure impacted genes with diverse functionalities, e.g., acid response, global regulons, osmolarity, and oxidative stress (1, 9, 43). The expression of cold shock proteins (Csp) belonging to the CspA family (CspA, CspB, CspG, and CspI) in E. coli are cold inducible, with a 2- to 10-fold increase in production during down-shifts in temperature from physiological temperatures (18, 22, 35). Several other genes belonging to well-characterized regulons with diverse functions are cold inducible (6): deaD (RNA helicase), grpE (hyperosmotic and heat shock response), gyrA (DNA gyrase), recA (DNA repair and SOS response), rpoS (general stress regulator encoding alternative sigma factor S), and uspA (universal stress protein A) (15, 17, 29, 47, 51). Virulence genes, including stx1 of EcO157, can be upregulated by cold shock (1). Therefore, cold shock and cold adaptation may impact virulence determinants of foodborne pathogens in produce. To address this issue, we used real-time PCR to evaluate the differential expression of selected virulence and stress response genes of EcO157 during refrigerated storage. The genes selected in this study are known to evoke differential responses to temperature changes (such as cold shock). Along with the virulence and stress response gene expression, we also evaluated the host-pathogen interaction by using an in vitro adhesion bioassay with cold-shocked EcO157 cells exposed to romaine lettuce lysate and minimal bacteriological medium.

Bacterial strain and growth condition.

A rifampin-resistant mutant of pathogenic EcO157 strain EDL933 (ATCC 43895) was used in all experiments. For inoculation into lettuce leaf lysate and for cold shock experiments, EcO157 EDL933 cells were grown overnight at 37°C in M9 minimal medium (BD Diagnostic Systems, Sparks, MD) supplemented with 0.2% glucose and sterile rifampin (MP Biomedicals, Santa Ana, CA) at a concentration of 100 μg/ml (M9-G). All experiments were conducted using cells from mid-log phase (determined by cell density at 600 nm) in M9-G medium unless otherwise stated. The M9-G was used to run controls for this study because the chemical complexity of M9-G was more similar to that of lettuce lysate as previously described (26).

Preparation of lettuce leaf lysate.

The romaine lettuce lysate mimicking a model of physicochemical conditions in injured lettuce tissues was prepared by using an electric juicer (Tristar Products, Fairfield, NJ) followed by filtration (8-μm-pore-size grade 2 filter; Whatman, GE Healthcare, Pittsburgh, PA), centrifugation (5,000 × g for 10 min), and filter sterilization (0.22-μm-pore-size filter; EMD Millipore, Billerica, MA) as described previously (26). Sterile rifampin at 100 μg/ml was added to each lettuce leaf lysate before experimentation.

Cold shock treatments.

To study the effect of a down-shift in temperature on EcO157 gene expression and virulence traits, cold shock treatments were performed at 25 and 4°C in lettuce lysate and M9-G. EcO157 cells were washed with phosphate-buffered saline (PBS) at 25 or 4°C and then exposed to lettuce lysate or M9-G at 25°C for 1 h, 4°C for 1 h, and 4°C for 10 min. The short incubation periods (10 or 60 min) in lettuce lysate were used to understand the early response (gene expression) of EcO157 to fluid (lysate) oozing out of the injured plant tissues under refrigerated conditions, as described previously (26). For a control, RNA was isolated from EcO157 in M9-G broth under corresponding storage (time-temperature) conditions.

RNA isolation, quality control, and QRT-PCR.

Bacterial RNA was stabilized using RNAprotect Bacteria Reagent (Qiagen, Valencia, CA) before isolation. Total RNA was extracted with the RNeasy kit (Qiagen) according to the manufacturer's instructions. The RNA samples were treated with RNase-free DNase I (FEREN0521, Thermo Scientific, Waltham, MA) before performing the real-time quantitative reverse transcription PCR (QRT-PCR) assay. The resulting RNA samples were tested for the presence of DNA by running the QRT-PCR assay targeting gapA with RNA as a template. The samples were then analyzed for integrity using a 2100 Bioanalyzer (Agilent, Santa Clara, CA). The quality of the total RNA was evaluated based on the RNA integrity numbers (RIN; Agilent) of the samples. Samples with an RIN of 9.3 or above (mean = 9.67) and the 23S/16S ratio of 1.8 or above (mean =1.96) were selected for QRT-PCR experiments. Stress and virulence gene expression in EcO157 in response to various romaine lettuce storage conditions was evaluated using a comparative QRT-PCR with Brilliant II SYBR Green QPCR Master Mix (Agilent) and a CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA) as described previously (9). The primer sequences for all gene targets and the PCR conditions have been previously reported (see Table 1). Relative mRNA levels were determined according to a separate tube comparative critical threshold (2−ΔΔCT) real-time PCR as described previously (9, 27, 32). Three independent biological replicates in triplicate (technical replicates) for each gene were performed. The housekeeping genes utilized for calculating the relative gene expression were gapA (encoding glyceraldehyde-3-phosphate dehydrogenase), mdh (encoding malate dehydrogenase), and rrsE (encoding 16S ribosomal RNA). These housekeeping genes were selected based on a previously described model (19, 32).

TABLE 1.

Relative gene expression profiles obtained with a QRT-PCR assay of E. coli O157:H7 strain EDL933 subjected to cold shock in lettuce lysatea

Relative gene expression profiles obtained with a QRT-PCR assay of E. coli O157:H7 strain EDL933 subjected to cold shock in lettuce lysatea
Relative gene expression profiles obtained with a QRT-PCR assay of E. coli O157:H7 strain EDL933 subjected to cold shock in lettuce lysatea

Mammalian cell culture and adhesion assay.

Cells from a bovine mammary epithelial cell line (MAC-T; Dr. Mark D. Hanigan, Virginia Tech, Blacksburg) were routinely cultured in Dulbecco's modified Eagle's medium with phenol red (HyClone, Thermo Scientific) supplemented with 10% fetal bovine serum (HyClone, Thermo Scientific) at 37°C in 7% CO2. Adhesion of bacteria to mammalian cells was quantified using a plating method described previously (10) with some modification. Confluent monolayers of MAC-T (5 × 105 CFU per well) grown in 24-well plates were infected with a multiplicity of infection of 10 and 100 (bacteria to host cells). After 1 h of incubation, cells were washed three times with PBS and lysed with 0.5 ml of trypsin in PBS for 10 min. Cell lysate was serially diluted and plated on Luria-Bertani agar, and colonies were enumerated after overnight incubation to compute percent adhesion. To validate the adhesion assay plating data, a Giemsa staining experiment was performed as described previously (4).

Statistical analysis.

The effect of the treatment on virulence and stress gene expression in the bacterial cells was investigated by three independent experimental trials in triplicate. Results are presented as means ± standard error of the mean using Microsoft Excel (version 2010, Microsoft Corp., Redmond, WA). An analysis of variance (ANOVA) was used to determine any significant differences (P < 0.05) among the treatment groups (temperature and exposure time), and all variables were treated as fixed variables.

To understand the impact of short-term cold shock exposure on selected EcO157 gene expression in lettuce leaf lysate under various storage conditions (time and temperature), we used QRT-PCR. The expression of different target genes was normalized based on three housekeeping genes, and the values for gene expression (fold-change) were averaged (Table 1). The housekeeping genes or normalization genes used in this study (gapA, mdh, and rrsE) were selected based on a model described previously (19). These genes have only limited variation (in individual expression) across the investigated treatment conditions. Even after stringent selection of the housekeeping genes, we observed some variations in relative expression of the target genes depending on which gene was used for normalization (Table 1). However, previous reports indicate that this limitation is not uncommon (2, 38, 42, 45), and many previous studies have underscored the importance of using multiple calibrator or housekeeping genes in gene expression studies with RT-PCR experiments (2, 19, 42). In lettuce lysate, we observed temperature-dependent differential expression of several gene targets tested in this study. Among them, stx1a was highly upregulated under each treatment. Levels of expression of the stx1a were significantly higher (P < 0.05) in the 4°C treatments in lettuce lysate, with average fold-changes of 82.6 and 88.1 (4°C for 10 and 60 min, respectively). Genes encoding other subunits of Stx were upregulated under the test conditions. Stx is sensitive to environmental stresses. A well-established link between the SOS response and prophage induction for stx1 and stx2 expression under stressed conditions has been reported (14, 30). In a previous study, Allen et al. (1) reported upregulation of EcO157 stx1 as a result of cold shock (7.5°C).

The expression of major EcO157 adhesion-related genes that are critical to host colonization and virulence was also impacted by the temperature down-shift in lettuce leaf lysate. Among the five adhesion-related genes tested (Table 1), flagellin (fliC) was slightly upregulated, but the other four adhesion-related genes, eaeA (intimin), lpfA1 and lpfA2 (long polar fimbriae [LPF]), and hcpA (hemorrhagic coli pilus [HCP]), were down-regulated under refrigeration conditions. Both up- and down-regulation of E. coli eaeA have been reported under cold conditions (1, 9, 20). The cold-induced down-regulation of eaeA in lettuce lysate observed in our study is in agreement with a previous report by Huang et al. (20) of eaeA down-regulation (by QRT-PCR) and reduced expression of adherence-related proteins (by two-dimensional electrophoresis). Down-regulation of cold-induced eaeA expression was also reported by Elhanafi et al. (12) to be influenced by growth phase and pH. In contrast, Carey et al. (9) reported slight upregulation of eaeA in cells on whole lettuce leaves stored at 4°C for a longer period (up to 9 days). Allen et al. (1) also reported that eaeA was upregulated as a result of cold shock (at 7.5°C in tryptic soy broth). The slight down-regulation of eaeA found in our study can be attributed to the composition of the lettuce lysate, which is significantly different from the surface of whole lettuce leaves and rich bacteriological medium (26). The treatment conditions (4°C in lettuce lysate) impacted the expression of LPF genes; both lpfA1 and lpfA2 were down-regulated. LPF genes are highly prevalent among E. coli strains that are positive for the locus of enterocyte effacement and associated with severe epidemic diseases (39). In previous studies, expression of the EcO157 lpf operon 1 (lpf1) in E. coli K-12 increased adherence of K-12 cells to tissue culture cells with the appearance of long fimbriae (40, 41). The gene hcpA, which is critical to EcO157 adherence to several types of epithelial cells, was also down-regulated at 4°C in lettuce lysate (Table 1). Xicohtencatl-Cortes et al. (48) found that hcpA-mediated attachment of EcO157 was a type III secretion system–associated function and that these functions and HCP act synergistically to mediate EcO157 attachment and colonization. HCP-mediated attachment is independent of Stx-induced attachment mechanisms (36), and the status of stx induction does not impact the hcp-mediated adherence of E. coli cells to host epithelial cells (48). Deletion of hcpA reduced EcO157 adhesion to epithelial cells of various origins (48, 49). The presence of the lpfA, eaeA, and hcpA genes is associated with the colonization efficiency of E. coli strains the cause severe disease outcomes; therefore, down-regulation of these genes may eventually impact pathogen colonization of the host.

Enhancement of low-temperature survival of EcO157 probably occurs through the induction of the heat shock regulon (1, 15, 51). One of the two heat shock regulon members tested, grpE, was induced at all three test temperatures in lettuce lysate with a significant upregulation at 4°C after 60 min. The other heat shock regulon member encoding for a universal stress protein (uspA) was down-regulated at refrigeration temperatures in lettuce lysate (Table 1). In previous studies, differential regulation (induction or repression) of heat shock proteins was also found in cold-exposed EcO157 cells (1, 21). Two key members of the cold shock regulon, cspA and gyrA, were significantly induced (P < 0.05) (as expected) at the refrigeration temperature (4°C) compared with the control treatments (at 37°C) in lettuce lysate. CspA is a key E. coli cold shock protein whose proposed function is as an RNA chaperone responsible for RNA stability upon cold shock, thereby facilitating efficient initiation of translation in prokaryotes (16, 33, 50). Based on deletion analysis, the cspA 5′ untranslated region is presumed to be responsible for the extreme instability of cspA mRNA at 37°C and has a positive affect on mRNA stabilization at low temperature (33, 50). CspA mRNA is dramatically but transiently stabilized (half-life of more than 20 min at 15°C but only 12 s at 37°C) immediately following cold shock. The stability of E. coli CspA mRNA increases greatly after a drop in temperature. In previous studies, gyrA was found to function as a key member of the E. coli cold shock regulon by facilitating the binding of cold shock protein CS7.4 to its promoter, which results in a positive transcriptional response to cold shock (23). The gyrA gene is regulated by CspA at the level of transcription upon cold shock, which is thought to help or stabilize the open complex formation for transcription (50).

Both of the global stress regulators tested in this study, rpoS and phoB, were upregulated in the cold-shocked cells in lettuce leaf lysate. Even though the transcription factor RpoS and the pho operon regulator (phoB) are not typically cold inducible, these regulators have been associated with the expression of genes in response to many stresses, including the shift to low temperature (1, 46). We observed a significant upregulation of recA (Table 1), a critical gene for SOS induction (14), at 4°C in lettuce lysate. RecA-dependent Stx phage induction occurs via degradation of two key repressor proteins, LexA and CI, subsequently leading to the induction of previously silent phage-encoded genes (stx in this case) (24). Our results reveal significant differential expression of EcO157 genes at transcription levels in lettuce lysate at cold temperatures.

To understand whether the differential expression of the major adherence-related genes of EcO157 tested in refrigerated lettuce lysate affects EcO157 adhesion to mammalian cells, we conducted an in vitro bioassay to determine whether there is any change in the ability of cold-shocked EcO157 cells from lettuce leaf lysate to attach to the mammalian epithelial cells. We also wanted to corroborate the results of the transcription analyses, which revealed decreased expressions of eaeA, lpfA, and hcpA, the principal adherence-related virulence factors of EcO157, after cold shock in lettuce lysate. The results of the adhesion assay of EcO157 cells (treated for various times and temperatures in M9-G or lettuce lysate) on MAC-T epithelial cells at a multiplicity of infection of 1:100 and 1:10 did not indicate any significant difference due to growth medium (M9-G or lettuce lysate) for corresponding time-temperature treatments (Fig. 1). This lack of difference might be due to the nutritional similarity between the two media, as reported by Kyle et al. (26). EcO157 multiplies faster on damaged lettuce leaves than on intact leaves, and after a period of acclimation, the pathogen show can respond to the environment by using the substrates available to multiply in injured lettuce tissue (8, 26). Therefore, cells grown in lettuce lysate probably retain key physiological functions similar to those of cells grown in a minimal bacteriological medium such as M9-G. We found a significant difference in the adhesion of EcO157 to MAC-T cells among the different cold shock treatments. EcO157 cells exposed to refrigeration temperature (4°C) were significantly less able (P < 0.05) to attach to MAC-T cells compared with cells at the physiological temperature of 37°C or at room temperature (25°C) (Fig. 1). This cold-induced reduction in attachment of EcO157 to mammalian cells may be attributed to decreased expression of adherence factors coded by adhesion-related genes such as lpfA, eaeA, and hcpA. In our transcription analyses, we observed down-regulation of these genes at 4°C treatments. Therefore, reduced attachment in epithelial cells may indicate a nontransient suppression of these genes in EcO157 exposed to refrigerated lettuce lysate.

FIGURE 1.

Adhesion analysis of E. coli O157:H7 to MAC-T epithelial (mammary) cells in M9-G medium and romaine lettuce lysate (LL) under various time-temperature conditions. E. coli O157:H7 cells were inoculated at a multiplicity of infection of 100 bacterial cells to 1 epithelial cell (1:100) and 10 bacterial cells to 1 epithelial cell (1:10). The adhesion profile is expressed as the mean (+SEM) number of bacterial cells attached per epithelial cell for triplicate experiments. Different uppercase letters above bars indicate significant differences (P < 0.05) between the treatments (37°C for 60 min, 25°C for 60 min, 4°C for 10 min, and 4°C for 60 min) within each growth medium (M9-G or LL). Different lowercase letters above bars indicate significant differences (P < 0.05) between the four treatment conditions across the two media.

FIGURE 1.

Adhesion analysis of E. coli O157:H7 to MAC-T epithelial (mammary) cells in M9-G medium and romaine lettuce lysate (LL) under various time-temperature conditions. E. coli O157:H7 cells were inoculated at a multiplicity of infection of 100 bacterial cells to 1 epithelial cell (1:100) and 10 bacterial cells to 1 epithelial cell (1:10). The adhesion profile is expressed as the mean (+SEM) number of bacterial cells attached per epithelial cell for triplicate experiments. Different uppercase letters above bars indicate significant differences (P < 0.05) between the treatments (37°C for 60 min, 25°C for 60 min, 4°C for 10 min, and 4°C for 60 min) within each growth medium (M9-G or LL). Different lowercase letters above bars indicate significant differences (P < 0.05) between the four treatment conditions across the two media.

Close modal

These data indicate that cold shock resulted in reduced attachment to epithelial cells of EcO157 cells exposed to lettuce leaf lysate and promoted higher levels of stx gene expression at the molecular level. The present study reveals the role of cold shock (refrigeration) in the potential severity of infections caused by sublethally injured EcO157 associated with ready-to-eat produce. However, we evaluated gene expression of only mid-log-phase EcO157 at specific times after cold shock in a food matrix. This pathogen may encounter several other stressors (e.g., low pH in the stomach, bile, and depletion of iron and other micronutrients) in a physiological situation inside a host or in simulated gastric juices. Therefore, appropriate in vivo follow-up studies are needed to understand the overall pathogenicity of EcO157 cells that have encountered cold shock in a produce environment. The stability of many microbial toxins (or toxic by-products) varies widely depending on the immediate surroundings. For example, Shiga toxin molecules (Stx1 and Stx2) become unstable in acidic pH; therefore, Stx that has formed in foods may not reach the site of action in the large intestine before it is denatured (25). Nonetheless, the results of the present study are valuable for understanding EcO157 adaptation to the temperature outside of its host and in lettuce processed and stored at refrigeration temperatures. Our goals for future work include conducting the transcription analyses at various time intervals after cold shock and at environmentally relevant temperatures (e.g., 18 and 20°C) using EcO157 cells at different growth phases. The present study also revealed that a subset of virulence determinants may be induced during cold shock while some other virulence-related genes may be suppressed. The gene transcription status may not reflect events subsequent to transcription (such as posttranscription processing of genes) or exposure to additional stress factors inside the host (e.g., bile and low pH in the stomach). Therefore, when conducting an assessment of the virulence of an enteropathogen such as EcO157 exposed to a stress environment (such as in cold storage), the effects of several virulence factors must be accounted for and the actual biological and physiological implications should be deduced by conducting bioassays such as cytotoxicity assays, metabolic profiling, or mouse virulence tests. This approach will lead to better risk assessment of a pathogen in food matrices under various conditions and will aid in devising appropriate interventions to decrease the burden of foodborne illness.

This research was partly supported by grants from the U.S. Department of Agriculture, National Institute of Food and Agriculture (ALAX-012-0210 and 2010-38821-21448) and the U.S. Food and Drug Administration (1U54FD004330-01) to P. Banerjee and by start-up funds from the University of Memphis. The authors thank Dr. Ramesh Kantety (deceased) for help in sample analyses.

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