Effectiveness of Calcium Hypochlorite on Viral and Bacterial Contamination of Alfalfa Seeds

Abstract

Alfalfa sprouts have been involved in numerous foodborne outbreaks, which has increased the awareness for seed and sprout safety. This study compared the effectiveness of calcium hypochlorite (Ca(OCl)₂) on the inactivation of bacteria and viruses on alfalfa seeds and in the presence of a simulated organic load. Alfalfa seeds were inoculated with human norovirus (huNoV) genogroup II (GII), murine norovirus (MNV), Tulane virus (TV), Escherichia coli O104:H4, and Salmonella enterica serovar Agona. Seeds were treated with Ca(OCl)₂ (2000 ppm or 20,000 ppm with the average of free chlorine 1388±117 mg/L and 11,472±1500 mg/L, respectively, pH adjusted to 7.00). The reduction of huNoV genomic copies indicated that huNoV was relatively resistant to Ca(OCl)₂ regardless of concentrations. Significant reductions were observed in the order of TV<Salmonella Agona<MNV<E. coli O104:H4 at 20,000 ppm Ca(OCl)₂. A similar trend was found at 2000 ppm Ca(OCl)₂ in the order of TV, Salmonella Agona, MNV<E. coli O104:H4. Ca(OCl)₂ at 20,000 ppm was more effective than 2000 ppm for all the organisms tested. This trend was also observed in samples containing an artificial organic material load. Ca(OCl)₂ activity on virus inactivation decreased as the organic load increased. Reduction was greater in fetal bovine serum–containing samples compared to alfalfa seeds, indicating a close relationship between the organisms and alfalfa seeds. Ca(OCl)₂ could not completely inactivate bacteria or viruses inoculated on seeds, and high levels of E. coli O104:H4 and Salmonella Agona were present on sprouts from sanitized seed samples following a 7-day germination period.

Introduction

Sprouted seeds have been involved in numerous foodborne outbreaks across the world, and since 1990 more than 30 reported outbreaks were linked to the consumption of raw or lightly cooked alfalfa sprouts in the United States (IFSN, 2009; Bari et al., 2011; Erdozain et al., 2011; CDC, 2012). The number of foodborne illnesses associated with these outbreaks ranged from as few as a single number to as large as thousands including dozens of deaths, as observed with the large outbreak in Germany in 2011 (IFSN, 2009; Buchholz et al., 2011; Erdozain et al., 2011). Many sources are identified as routes for sprout contamination, including pathogen-tainted seeds, contact with soil, fertilizer, irrigation water, harvesting, storing, processing, distribution, and contamination during food preparation (NACMCF, 1999; Erdozain et al., 2013; Yang et al., 2013). Importantly, contaminated seeds were identified as the major source in most sprout-associated outbreaks, and germination is a key step for sprout safety and challenges the sprout industry (NACMCF, 1999). During sprouting, seeds are first soaked in water and then placed in a warm and humid environment, which is ideal for bacterial growth (NACMCF, 1999). If bacteria, such as pathogenic Escherichia and Salmonella spp., both identified as major etiologies in outbreaks associated with sprouted seeds (IFSN, 2009; Erdozain et al., 2011), are present on seeds before germination, they can easily proliferate. Seed disinfection is a preventive approach to reduce the risk associated with contaminated seeds. Soaking seeds in 20,000 ppm calcium hypochlorite (Ca(OCl)₂) solution before sprouting is considered as an appropriate treatment (NACMCF, 1999), while the Canadian Food Inspection Agency (CFIA) also describes a treatment with 2000 ppm of Ca(OCl)₂ or sodium hypochlorite for 15–20 min for use as an antimicrobial treatment for seeds (CFIA, 2008). It is necessary to evaluate seed disinfection treatments, and many studies have aimed to investigate the efficacy of Ca(OCl)₂ on foodborne bacteria on seeds (Lang et al., 2000; Brooks et al., 2001; Holliday et al., 2001; Suslow et al., 2002; Gandhi and Matthews, 2003; Kim et al., 2003; Liao, 2009; Buchholz and Matthews, 2010; Zhao et al., 2010); however, no substantial research has been conducted assessing viruses. Within this study, the effectiveness of treatment on virus is put in perspective by comparative evaluation of bacteria.

The U.S. Centers for Disease Control and Prevention estimate that human norovirus (huNoV) causes the greatest number of illnesses associated with a known pathogen each year in the United States (5.5 million), accounting for up to 58% of foodborne illnesses (Scallan et al., 2011). In addition, recent studies revealed that the total number of illnesses caused by norovirus in United States each year was 19–21 million, and an average of 5 episodes of norovirus gastroenteritis would be experienced by each individual in a lifetime (Hall et al., 2013). Noroviruses are classified into at least five genogroups I–V (GI–GV), and a novel genogroup VI containing canine norovirus was recently proposed (Mesquita et al., 2010). The strains relevant to human disease belong to genetic clusters within GI, GII, and GIV (Zheng et al., 2006). GII is the most common genogroup, causing 73% of all reported norovirus outbreaks from 1997 to 2000 in the United States (Fankhauser et al., 2002). The fact that norovirus has a low infectious dose of 10–100 particles with a median of 18 particles reinforces how foodborne illnesses can occur easily (Teunis et al., 2008; Patel et al., 2009). Due to the lack of cell culture or animal models for routine study of huNoV, surrogates are relied on for the study of huNoV. Two surrogates were selected for use in this study, including murine norovirus (MNV) and Tulane virus (TV) (Cannon et al., 2006; Hirneisen and Kniel, 2013a). Previous studies showed that viruses, especially MNV and TV, persist and survive for up to 50 days in alfalfa seeds (Wang et al., 2013).

During seed disinfection, the presence of organic material may alter the effectiveness of Ca(OCl)₂ on the seeds. Previous studies showed that relatively low levels of organics, such as 0.5% bovine serum albumin (BSA), did not substantially interfere with the antimicrobial activity of sodium hypochlorite (NaOCl) (Sassone et al., 2003); however, it was demonstrated that high concentrations of BSA significantly reduced the antimicrobial activity of NaOCl, calcium hydroxide, and iodine potassium iodide in bacterial inactivation (Portenier et al., 2001; Pappen et al., 2010). It is hypothesized that the high organic loads could substantially decrease the effectiveness of Ca(OCl)₂.

In this study, alfalfa seeds were selected as a model to better understand the efficacy of seed treatment with Ca(OCl)₂. HuNoV GII and its surrogates MNV and TV, and two bacterial sprout isolates Escherichia coli O104:H4 and Salmonella Agona, were used to assess microbial inactivation on alfalfa seeds by Ca(OCl)₂, and the effect of organic loads was also investigated. Comparative disinfection parameters were observed for the microorganisms tested. Additionally, postdisinfection, bacterial growth was assessed following a 7-day germination period.

Materials and Methods

Virus cultivation and infectivity

Murine norovirus (MNV-1) (a gift from Dr. Herbert Virgin, Washington University School of Medicine, St. Louis, MO) was cultured in RAW 264.7 cells (ATCC# TIB-71) in Dulbecco Modified Eagle Medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Mediatech, Manassas, VA), 100 U/mL penicillin G–streptomycin–0.25 μg/mL Amphotericin B (Hyclone, Logan, UT), 2 mM l-alanine-l-glutamine (Gibco, Carlsbad, CA), and 1 mM sodium bicarbonate (Cellgro, Manassas, VA). TV (a gift from Dr. Xi Jiang, University of Cincinnati College of Medicine, Cincinnati, OH) was propagated in LLC-MK2 cells (ATCC# CCL-7) in medium 199 (Hyclone) supplemented with 10% FBS, and 100 U/mL penicillin G–streptomycin–0.25 μg/mL Amphotericin B. After typically 48 h infection of 80%–90% confluent monolayers for both MNV and TV, complete cytopathic effect (CPE) was observed. Viruses were obtained following three cycles of freeze-thawing infected cells, and centrifugation at 2000×g for 15 min. The supernatant was filtered through a 0.2-μm membrane filter (Thermo, Rochester, NY) before storing viruses at −80°C until use.

MNV and TV plaque assays were performed similarly to previous studies, with slight modifications (Wobus et al., 2004; Farkas et al., 2008). In brief, RAW 264.7 and LLC-MK2 cells were grown to 80%–90% confluency in 6-well plates (Castar, Corning, NY), and 100 μL of 10-fold serial dilutions of each virus sample prepared in Hank's balanced salt solution (HBSS; Cellgro, Manassas, VA) was dispensed over monolayers in duplicate. The plates were incubated at 37°C with 5% CO₂ for 1–3 h with gentle agitation every 15 min followed by the addition of 2-mL overlays. MNV-1 overlays consisted of 0.5% agarose (SeaKem LE, Lonza Inc., Rockland, ME) with complete Eagle's medium (Hyclone) supplemented with 2% FBS, 100 U/mL penicillin G–streptomycin–0.25 μg/mL Amphotericin B, 2 mM l-alanine–l-glutamine, and 1 mM sodium pyruvate. TV overlays consisted of 0.5% agarose with complete medium 199 supplemented with 2% FBS, and 100 U/mL penicillin G–streptomycin–0.25 μg/mL Amphotericin B. After the incubation period (typically 48 h for MNV), 1 mL of 0.2 g/L neutral red (Fisher Scientific, Fair Lawn, NJ) was added into each well followed by a 2–5-h incubation. After typically 48–72 h of the incubation period for TV, 2 mL of 3.7% formaldehyde (Fisher Scientific, Fair Lawn, NJ) in phosphate-buffered saline (PBS; pH 7.2) was added into each well, followed by at least 2-h incubation, stained with 0.05% (wt/vol in 10% ethanol) crystal violet (Fisher Scientific, Kalamazoo, MI). Titers of virus were determined and expressed by plaque-forming units (PFU).

huNoV preparation

Human norovirus genogroup II (huNoV GII) was supplied by Megan Davis, South Carolina Department of Health and Environmental Control. HuNoV purification from stool samples was performed similarly to the protocols in previous studies with slight modifications (Lewis and Metcalf, 1988; Hirneisen and Kniel, 2013b). Stool samples were added into 0.01 M PBS to make 10% (vol/vol) slurry. After being vortexed vigorously, the suspension was centrifuged at 2000×g for 20 min to remove the solids. The supernatant was retained and polyethylene glycol (PEG) (Fisher Scientific, Waltham, MA) was then added to make the final concentration of 8% (wt/vol). The suspension was stirred for 4 h at 4°C and then centrifuged at 10,000×g for 30 min. PEG supernatant was discarded and the pellet was suspended in 0.15 M Na₂HPO₄ (pH 9.0) and shaken for 20 min at 250 rpm. After another centrifugation at 10,000×g for 30 min, the supernatant was processed through a 0.2-μm membrane filter (Thermo, Rochester, NY) to remove bacteria and debris. The filtrate was diluted in PBS and stored in aliquots before freezing at −80°C.

HuNoV genome quantification by real-time reverse transcription polymerase chain reaction (RT-PCR)

The level of huNoV was quantified by real-time RT-PCR. To generate a standard curve for each virus type, 1 mL of virus stock with known genomic copies (10⁷ genomic copies/mL for huNoV) was 10-fold serially diluted with HBSS. RNA of virus samples was extracted and reverse transcribed into cDNA by using QIAamp Viral RNA Mini kit (Qiagen, Valencia, CA) and Omniscript RT kit (Qiagen) as reference protocols, respectively. The primers used for huNoV GII were shown as follows: forward primer VP1-FP3 (5′-TGGGTGCTCCCAAGTTATTC-3′) and reverse primer VP1-RP3 (5′-CTGGAGCTGCCTCTTGGTAG-3′) (Hirneisen and Kniel, 2013b). Real-time PCR was performed in a total reaction volume of 20 μL containing 10 μL SYBR-Green PCR Master Mix (Qiagen), 2 μL cDNA, and same set of primers with the protocol from QuantiTect SYBR Green PCR Kit (Qiagen) on 384-well plates. Reactions were run on the Applied Biosystems 7900 HT Sequence Detection system (Applied Biosystems, Foster City, CA) with the following thermal conditions: 95°C for 10 min followed by 40 cycles of 94°C for 15 s, annealing step at 56°C for 30 s, and extension step at 72°C for 30 s, followed by a dissociation step at 60°C for 15 s and 90°C for 15 s. SYBR green signals were read in every cycle, and the logarithm of the increment in fluorescence was plotted versus the cycle number with fixed threshold level for all runs. Virus quantity was then determined by comparison to a standard curve and expressed as genomic copies. The standard curve was generated in duplicates in each run of quantitative PCR. The detection limits for all virus types were determined to be ∼100 genomic copies/mL. HuNoV in HBSS served as positive controls, and negative controls consisted of the seed and HBSS sample without virus as well as PCR blanks.

Bacterial growth and quantification

E. coli O104:H4 (ATCC# BAA-2326) and Salmonella enterica serovar Agona (ATCC# 51957), which were both previously implicated in foodborne outbreaks associated with sprouts, were used in this study. They were cultured at 37°C in 10 mL of LB broth (Fisher Scientific, Fair Lawn, NJ) overnight. Cells of each strain were collected by centrifuge at 2000×g for 15 min at room temperature (22±1°C). Supernatant was then discarded, and cells were resuspended in equal volume of HBSS to remove the entire organic load in growth medium. Bacteria were prepared fresh before use. After treatments, samples including controls were 10-fold diluted in HBSS, and plated 100 μL in duplicate on XLT-4 Agar (BD, Sparks, MD) and Sorbitol MacConkey agar (BD) for Salmonella Agona and E. coli O104:H4, respectively. Plates were incubated at 37°C for 24 h. Colonies were enumerated and determined by colony-forming units (CFU).

Ca(OCl)₂ preparation

Ca(OCl)₂ (Fisher Scientific, Fair Lawn, NJ) was prepared fresh by dissolving 0.400 g or 4.00 g in 200 mL deionized water to make the final concentrations of 2000 ppm and 20,000 ppm, respectively. pH was adjusted to 7.00 by adding 0.1 M HCl. The free chlorine concentrations were measured using the High Range Chlorine Test Kit (HACH, Loveland, CO) with the average of 1388±117 mg/L and 11,472±1500 mg/L, respectively. The Ca(OCl)₂ solutions were used immediately after preparation.

Alfalfa seed preparation

Alfalfa seeds (Medicago sativa) (Johnny's, Winslow, ME) were sterilized by submerging seeds in 70% ethanol for 5 min followed by soaking in a 10% bleach solution for 20 min. Seeds were rinsed with deionized water and then dried under the laminar flow hood at room temperature overnight before dividing into 1-g samples in 15-mL centrifuge tubes prior to inoculation. After treatment, little effect was observed visually on sprouting percentage compared with untreated sprout seeds. Every 1-g seed sample was individually inoculated with 500 μL of huNoV GII, TV, MNV, E. coli O104:H4, and Salmonella Agona, respectively. In order to determine the effectiveness of Ca(OCl)₂, the initial titers of pathogens and surrogates inoculated on alfalfa seeds were 7.70±0.01 log genomic copies of huNoV GII, 7.04±0.18 log PFU of MNV, 6.16±0.23 log PFU of TV, 9.72±0.12 log CFU of E. coli O104:H4, and 9.19±0.65 log CFU of Salmonella Agona, respectively. Inoculum was then allowed to dry visually for 1 h at 20°C. In order to measure the survival rates of the microorganisms inoculated on seeds after drying, seeds were vortexed for 1 min to eluted virus/bacterium with 5 mL HBSS after 1 h drying. For each type of virus/bacterium and concentration level of Ca(OCl)₂, three treatment seed samples and one recovery control were prepared for each of three trials of experiment (a total of nine treatment samples and three recovery controls). One seed sample was also included in each trial by adding 500 μL HBSS without virus inoculation, and it served as controls for Ca(OCl)₂ neutralization and cytotoxicity testing.

Effects of Ca(OCl)₂ on bacteria or viruses inoculated on alfalfa seeds and on bacteria or viruses in the presence of organic materials

After the virus inoculum or HBSS was visibly dry on 1-g alfalfa seeds, 1 mL Ca(OCl)₂ was added into inoculated seed samples and uninoculated controls. All the samples were carefully placed on the shaking platform at 150 rpm to mix thoroughly for 20 min. After 20-min seed treatments, the free chlorine was measured again with the values of ∼250 mg/L and ∼5000 mg/L, respectively. To neutralize any available disinfectant, 4 mL FBS was added immediately to each treated sample after 20 min. To study the effects of Ca(OCl)₂ in the presence of an organic load, a similar experiment was conducted. Viruses were prepared in HBSS containing FBS to make a final concentration of 10%, 30%, and 50%, respectively. Five hundred microliters of Ca(OCl)₂ was added to 500 μL FBS-containing virus sample for 20 min, and all the samples were shaken at 150 rpm. HBSS containing 10%, 30%, and 50% FBS without viruses was included in each trial, which served as controls for neutralization and cytotoxicity testing. To neutralize any available disinfectant, FBS was added immediately after 20 min to 100 μL of each 2000 ppm and 20,000 ppm Ca(OCl)₂ treated sample (90%–99% final concentration). Neutralization buffers for treated seeds without viruses were tested for cell cytotoxicity. Viruses or bacteria mixed with the series 10-fold dilutions of neutralization buffers served as neutralization controls. Samples were then tested for quantification. The log reduction was obtained by subtracting the amount of bacteria/viruses recovered after Ca(OCl)₂ treatments from the amount of bacteria/viruses recovered from samples that did not undergo treatments. If the sample volume was large due to neutralization, Amicon ultra centrifuge filters of 100 kDa (Millipore, Billerica, MA) were used to concentrate samples in a small volume followed the protocol provided. Recovery control was included, and no significant difference was observed between virus samples with and without concentration step (p>0.05).

Germination of bacteria-inoculated seeds after Ca(OCl)₂

Another set of bacteria-inoculated seeds with or without Ca(OCl)₂ treatments was allowed to germinate in the sprout growth chambers (Victorio, Orem, UT) by watering daily. The growth chamber had three trays: the top tray was empty and was used for watering; the middle tray had rings to distribute seeds evenly and was used for germination; and the bottom tray was a container to collect spent irrigation water. During the 7-day germination period, 500 mL municipal tap water (free chlorine was under the detection limit <10 mg/L) was added daily on the top tray, and siphoned over seeds/sprouts, and finally drained and collected in the bottom tray. The humidity and temperature inside of the growth chambers containing uninoculated seeds/sprouts were measured and recorded daily using a Traceable Therm./Clock/Humidity Monitor (Fisher, Pittsburgh, PA). The humidity in the growth chamber averaged >70%, with a range from 36% to >90%, and the temperature was 20.0±1.27°C. After a 7-day germination period, sprouts (approximately 12 g) germinated from 1-g seeds sample were collected in a 50-mL centrifuge tube containing 10 mL HBSS. Samples were vortexed for 1 min to elute bacteria from the sprouts to investigate bacterial growth during germination.

Statistical analysis

All experiments were conducted three times, and the reported results are means and standard deviations. Data were analyzed by analysis of variance on JMP software (Version 10.0, SAS Institute Inc., Cary, NC). Significant differences in least-squares means were indicated if p<0.05.

Results

Virus and bacterial recovery from alfalfa seeds after inoculation

In this study, the survival rates of microorganisms (both viruses and bacteria) on alfalfa seeds were determined after drying seeds for 1 h at room temperature. The log reductions of all the pathogens and surrogates tested are listed in Table 1. After 1 h drying, the levels of huNoV GII, TV, and Salmonella Agona recovered from alfalfa seeds decreased significantly regardless of differences in initial titers (p<0.05). The reductions ranged from approximately 1 to 2 logs as shown in Table 1. Little difference was observed in titers of MNV and E. coli O104:H4 before and after drying in alfalfa seeds (p>0.05).

Table 1.

Log Reduction of huNoV GII, MNV, TV, Escherichia coli O104:H4, and Salmonella Agona Postdrying (1 h) at Room Temperature (22±1°C)

Viruses and bacteria	Initial titer	Log reduction due to drying ^a
huNoV GII (log genomic copies/g seeds)^*	7.70±0.01	1.43±0.21
MNV (log PFU/g seeds)	7.04±0.18	0.27±0.20
TV (log PFU/g seeds)^*	6.16±0.23	0.92±0.07
E. coli (log CFU/g seeds)	9.72±0.12	0.16±0.06
Salmonella Agona (log CFU/g seeds)^*	9.19±0.65	1.09±0.52

Values are means±standard deviation of three replicates; virus or bacterium noted with an asterisk indicates significant difference (p<0.05) when comparing the levels of viruses or bacteria before and after drying.

huNoV GII, human norovirus genogroup II; MNV, murine norovirus; PFU, plaque-forming units; TV, Tulane virus; CFU, colony-forming units.

Inactivation of viruses and bacteria from contaminated alfalfa seeds by Ca(OCl)₂ treatments

Significant reductions were observed for viruses and bacteria after either 2000 ppm or 20,000 ppm Ca(OCl)₂ treatments (Table 2). Following treatment with 2000 ppm Ca(OCl)₂, huNoV GII had ∼1 log reduction in genomic copies; whereas the infectivity of its surrogates TV and MNV had significantly higher reductions with ∼1.7 log PFU/g seeds (p<0.05). Similar trends were observed at 20,000 ppm Ca(OCl)₂ treatment; however, the reduction of MNV was significantly greater than that of TV by approximately 1.5 log PFU/g seeds (p<0.05). E. coli O104:H4 achieved significantly greater reductions than Salmonella Agona at both concentration levels of Ca(OCl)₂ (p<0.05, Table 2). When comparing the inactivation of both viruses and bacteria treated by either 2000 ppm or 20,000 ppm Ca(OCl)₂, E. coli O104:H4 had significantly greater reductions than any other pathogens (p<0.05); however, the genomic copies of huNoV were most stable (p<0.05). To be more exact, significant log reductions were observed in the order of TV<Salmonella Agona<MNV<E. coli O104:H4 at 20,000 ppm Ca(OCl)₂, and the order was TV, Salmonella Agona, MNV<E. coli O104:H4 at 2000 ppm Ca(OCl)_2. In addition, Ca(OCl)₂ at 20,000 ppm was more effective than 2000 ppm for all viruses and bacteria, but complete inactivation was not obtained for the pathogens and surrogates studied, which may be in part due to the high titers inoculated in alfalfa seeds in this study to best assess inactivation.

Table 2.

Reduction of huNoV GII (log Genomic Copies/g Seeds), MNV, and TV (log PFU/g Seeds), and Escherichia coli O104:H4 and Salmonella Agona (log CFU/g Seeds) on Inoculated Alfalfa Seeds After Ca(OCl)₂ Treatments

		Ca(OCl)₂ treatments (log genomic copies/g seeds, log PFU/g seeds, or log CFU/g seeds) ^a
Viruses/bacteria	Initial titer	2000 ppm (free chlorine 1388±117 mg/L)	20,000 ppm (free chlorine 11,472±1500 mg/L)
huNoV GII	6.27±0.23	1.08±0.59^A	1.65±0.40^B
MNV	6.77±0.32	a 1.74±0.35^A	c 3.75±0.42^B
TV	5.24±0.26	a 1.78±0.32^A	a 2.29±0.16^B
E. coli O104:H4	9.63±0.02	b 3.85±0.25^A	d 5.97±0.17^B
Salmonella Agona	8.11±0.01	a 1.84±0.23^A	b 3.10±0.36^B

Values are means±standard deviation of three replicates; values in columns with the same preceding letter indicate no significant difference (p>0.05) when comparing murine norovirus (MNV), Tulane virus (TV), E. coli O104:H4, and Salmonella Agona inactivation after treatments; values in rows with the same following letter indicate no significant difference (p>0.05) when comparing between treatments for each virus/bacterium.

huNoV GII, human norovirus genogroup II; PFU, plaque-forming units; CFU, colony-forming units.

Effects of organic load in Ca(OCl)₂ inactivation of viruses and bacteria

The activity of Ca(OCl)₂ in the presence of artificial organic loads was investigated (Table 3). Bacteria and viruses inoculated in FBS-containing HBSS were slightly diluted, with initial titers of 7.10±0.01 log genomic copies of huNoV GII, 5.01±0. 30 log PFU of TV, 6.46±0.26 log PFU of MNV, 8.42±0.07 log CFU of E. coli O104:H4, and 8.06±0.01 log CFU of Salmonella Agona, respectively. The reductions of pathogens and surrogates in the presence of FBS were >2 log greater than that inoculated in seeds. Microbial inactivation as a result of Ca(OCl)₂ treatment substantially decreased as the concentration of FBS increased, especially in the presence of FBS at >30%.

Table 3.

Reduction of huNoV GII (log Genomic Copies/mL), MNV and TV (log PFU/mL), and Escherichia coli O104:H4 and Salmonella Agona (log CFU/mL) by Ca(OCl)₂ Treatments in the Presence of an Artificial Organic Load

		2,000 mg/L Ca(OCl)₂ (free chlorine 1,388±117 mg/L)		20,000 ppm Ca(OCl)₂ (free chlorine 11,472±1500 mg/L)
Viruses/bacteria	Organic load	Reduction (log genomic copies/mL, log PFU/mL, or log CFU/mL) ^a	Ratio ^b	Reduction (log genomic copies/mL, log PFU/mL, or log CFU/mL) ^a	Ratio ^b
HuNoV GII	10% FBS	a 4.51±0.23^A		a 4.51±0.05^A
	30% FBS	b 3.12±0.14^A		a 4.36±0.28^B
	50% FBS	b 2.80±0.06^A		a 4.02±0.03^B
MNV	10% FBS	a 5.71±1.10^A		a 6.27±0.22^A
	30% FBS	a 5.35±0.99^A		a 6.18±0.46^B
	50% FBS	a 5.17±1.21^A		a 5.96±0.85^A
TV	10% FBS	a 3.45±0.69^A		a 4.99±0.62^B
	30% FBS	ab 3.09±0.52^A		ab 4.45±0.46^B
	50% FBS	b 2.79±0.52^A		b 4.16±0.68^B
E. coli O104:H4	10% FBS	a 8.07±1.20^A		a 8.62±0.54^A
	30% FBS	ab 7.87±1.44^A		a 8.44±0.92^A
	50% FBS	b 6.91±1.72^A		a 7.79±1.43^A
Salmonella Agona	10% FBS	a 8.26±0.50^A		a 8.27±0.50^A
	30% FBS	a 8.09±0.81^A		a 8.27±0.50^A
	50% FBS	a 8.06±1.15^A		a 8.27±0.50^A

Values are means±standard deviation of three replicates; values in columns with the same preceding letter indicate no significant difference (p>0.05) when comparing the effect of organic loads for each virus/bacterium after treatments; values in rows with the same following letter indicate no significant difference (p>0.05) when comparing between treatments for each virus/bacterium.

Ratio is the number of positive samples/total number of samples tested.

huNoV GII, human norovirus genogroup II; MNV, murine norovirus; PFU, plaque-forming units; TV, Tulane virus; CFU, colony-forming units; FBS, fetal bovine serum.

All viruses had great reduction in genomic copies or infectivity regardless of the concentration of FBS present, but complete inactivation was not obtained at 2000 or 20,000 ppm Ca(OCl)₂. Viral genetic material from huNoV GII was detected in all samples treated with both 2000 ppm and 20,000 ppm Ca(OCl)₂. MNV compared with TV was inactivated more readily, as shown by the number of positive samples/total number of samples tested (Table 3). Greater reductions were obtained for MNV, with more than 40% of samples under the detection limit at 2000 ppm Ca(OCl)₂, and with limited increase in inactivation at 20,000 ppm. Viruses were much more resistant compared to bacteria.

Overall, the majority of bacterial samples were decreased to below the detection limit, as shown by the number of positive samples/total number of samples tested (Table 3). The addition of FBS and enhanced organic loads had no effect on bacterial reduction, as the Ca(OCl)₂ was very effective in inactivating bacteria inoculated in HBSS with different levels of FBS; also, the ratio of the number of positive samples/total number of samples tested indicated that most samples were under the detection limit.

Bacterial growth on alfalfa seeds/sprout during germination after Ca(OCl)₂ treatments

Following Ca(OCl)₂ treatments, alfalfa seeds were germinated in the growth chamber over 7 days with daily watering. At the conclusion of the 7-day period, bacterial levels present on sprouts were determined, and compared with those from germinated untreated sprouts (Table 4). Bacteria were not completely eliminated by disinfection treatments in this study. After the treatments, titers of E. coli O104:H4 and S. Agona were found to be 5.78±0.24 and 6.27±0.22 CFU/g seeds following treatment at 2000 ppm Ca(OCl)₂ and 3.67±0.16, and 5.01±0.36 CFU/g seeds from seeds treated with 20,000 ppm. No significant differences in levels of E. coli O104:H4 were found between untreated and 2000 ppm treated samples, nor between 2000 ppm and 20,000 ppm treated samples (p>0.05). However, the level of E. coli O104:H4 was significantly greater in sprouts germinated from untreated seeds than that from seeds treated with 20,000 ppm treated ones (p<0.05). For Salmonella Agona, significant differences were observed between untreated, 2000 ppm, and 20,000 ppm Ca(OCl)₂ treatments. The alfalfa sprouts germinated from 20,000 ppm Ca(OCl)₂ treated seeds had a significantly lower level of Salmonella Agona than that from 2000 ppm Ca(OCl)₂-treated seeds (p<0.05); the difference (∼0.6 log CFU/g seeds) was slight though. Similar differences observed with Salmonella Agona also existed between sprouts germinated from untreated seeds and that from 2000 ppm Ca(OCl)₂ treated seeds.

Table 4.

Escherichia coli O104:H4 and Salmonella Agona Growth on Sprouts from 1-g Seed Samples at 7-Day Postinoculation After Ca(OCl)₂ Treatments

	Treatments
Bacteria	Untreated	Ca(OCl)₂ 2000 ppm (free chlorine 1388±117 mg/L) ^a	Ca(OCl)₂ 20,000 ppm (free chlorine 11,472±1500 mg/L) ^a
E. coli O104:H4	9.33±0.04^A	9.00±0.29^A,B	8.68±0.40^B
Salmonella Agona	9.50±0.26^A	8.87±0.17^B	8.45±0.10^C

Values are means±standard deviation of three replicates; values in rows with the same following letter indicate no significant difference (p>0.05) when comparing between treatments for each bacterium.

Discussion

As the National Advisory Committee on the Microbiological Criteria for Foods mentioned in its guidelines, soaking seeds in 20,000 ppm Ca(OCl)₂ before sprouting is an appropriate procedure used in seed decontamination (NACMCF, 1999). Recently, the Food Safety Modernization Act also proposed rules regarding enhanced safety for sprout production requiring treatment of seeds immediately before sprouting to reduce pathogenic microorganisms. However, these guidelines do not contain details on pH, temperature, time of treatment, testing for free chlorine levels, concentration of Ca(OCl)₂, nor any recommendation for physical force required. With these gaps in knowledge, the inactivation rates of bacterial pathogens have been investigated (Lang et al., 2000; Brooks et al., 2001; Holliday et al., 2001; Suslow et al., 2002; Gandhi and Matthews, 2003; Kim et al., 2003; Buchholz and Matthews, 2010; Liao, 2009; Zhao et al., 2010), but little-to-no knowledge exists about the effectiveness of Ca(OCl)₂ decontamination on viruses.

In this study, we showed Ca(OCl)₂ could significantly reduce the levels of huNoV GII, MNV, TV, Salmonella Agona, and E. coli O104:H4 inoculated on alfalfa seeds, but complete inactivation was not achieved. Incomplete reduction indicated limited efficacy of Ca(OCl)₂, in part likely due to high levels of inoculums. Alfalfa seeds treated with higher concentrations of Ca(OCl)₂ resulted in greater reductions in both viruses and bacteria. Bacteria that survived the treatments grew to >8 log CFU in alfalfa sprouts after a 7-day germination period, and the levels were close to those without treatment.

Recovery of microorganisms from alfalfa seeds after drying varies, depending on bacteria or virus type. Significant reduction of huNoV genomic copies and TV infectivity was observed, but nearly full infectivity remained in the MNV recovered from alfalfa seeds. Similar results in previous studies showed that TV recovery from alfalfa seeds after inoculation significantly decreased, whereas little reduction of MNV was observed (Wang et al., 2013). For bacteria, little reduction was found in E. coli O104:H4 after recovery from alfalfa seeds, but ∼1 log reduction of Salmonella Agona was observed. Similarly, higher reductions of Salmonella on inoculated alfalfa seeds compared to E. coli were previously observed (Zhao et al., 2010). The variations in recovery rates could be explained by loss of genomic copies or inactivation of infectivity during the drying step, and the surface properties of each type of pathogens and surrogates attached to alfalfa seeds. The factors that affect attachment between microorganisms and alfalfa seeds include electrostatic and hydrophobic forces, as well as environmental conditions (Vega et al., 2008; Wang et al., 2011). After Ca(OCl)₂ inactivation and neutralization, the log titers recovered from alfalfa seeds were determined and subtracted from recovery values to calculate log reductions for each microorganism.

Ca(OCl)₂ has previously resulted in an average reduction of 2.5 log CFU/g seeds at 2000 mg/L (2000 ppm), and 3.0∼3.5 log CFU/g seeds at 20,000 mg/L (20,000 ppm), in enteric bacterial pathogens at room temperature (Montville and Schaffner, 2004; Ding et al., 2013). Here, we showed similar reduction levels and obtained similar conclusions that antimicrobial activity of hypochlorite increased with increasing concentrations (Erkmen, 2003, 2010). We found the reductions of Salmonella Agona were 1.84 and 3.10 log CFU/g seeds at different levels of Ca(OCl)_2, respectively. Inactivation rates were similar to results obtained by Nei et al., where ∼3 log CFU/g of Salmonella in alfalfa seeds were inactivated by 20,000 ppm Ca(OCl)₂ after 20-min treatment (Nei et al., 2011). Other studies indicated a range of reductions; Zhao et al. found >6.0 log CFU/g reduction of Salmonella Typhimurium on alfalfa seeds after 20-min treatment of 20,000 ppm Ca(OCl)₂ at 21°C (Zhao et al., 2010), whereas Buchholz et al. showed only ∼1.5 log CFU/g reduction of Salmonella Stanley in alfalfa seeds at 20,000 ppm for 15 min with rotary shaking (100 rpm) at a temperature range from 21°C to 23°C (Buchholz and Matthews, 2010). The variation in Salmonella inactivation could be attributed to the experiment protocols, such as use of specific strains, inoculation level and procedure, source of alfalfa seeds, treatment time, temperature, and physical force.

Interestingly, E. coli O104:H4 showed greater inactivation compared to Salmonella with 3.10 and 5.97 log CFU/g seeds at 2000 and 20,000 ppm treatment, respectively. Previous studies focused on the inactivation of E. coli O157:H7 by Ca(OCl)₂, while little is known about E. coli O104:H4. Taormina et al. found that 2000 ppm Ca(OCl)₂ resulted in only ∼2.0 log CFU/g reduction of E. coli O157:H7 (Taormina and Beuchat, 1999), and Beuchat et al. and Holliday et al. both showed the ∼2.0 log CFU/g reduction of E. coli O157:H7 at 20,000 ppm Ca(OCl)₂ in alfalfa seeds (Beuchat et al., 2001; Holliday et al., 2001). The reduction levels of E. coli O104:H4 by Ca(OCl)₂ shown here were generally higher than those of E. coli O157:H7 in previous studies. These discrepancies may be explained by differences in experiment design and by the characteristics of E. coli strain types. As an enteroaggregative E. coli with Shiga toxin and some enterohemorrhagic E. coli characteristics (Mellmann et al., 2011; Pierard et al., 2012), the E. coli O104:H4 strain is perhaps different in its response to Ca(OCl)₂.

Viruses were generally more resistant to Ca(OCl)₂ treatments than bacteria. The oval shape with uniform surfaces composed of hills and narrow valleys (Fransisca and Feng, 2012) were not likely to protect bacteria from disinfectants, but could provide numerous hiding sites for viruses. The accessibility of chlorine to react with pathogens hidden in crevices or between cotyledon and seed coat of alfalfa seeds could be the major reason (Ding et al., 2013; Yang et al., 2013). The number of genomic copies is determined; however, as huNoV cannot be cultivated in cell culture, infectivity may not be inferred from these data. The numbers of genomic copies of huNoV GII were relatively stable and more resistant to the treatments, with limited reductions observed (<2 log genomic copies/g seeds). Shin et al. assessed the norovirus resistance to chlorine and found only a 2 log reduction in water containing a 1 mg/L (1 ppm) dose of free chlorine (Shin and Sobsey, 2008). Another study showed that a 3.75 mL/L (3.75 ppm) dose of chlorine was not effective to inactivate norovirus, which remained infectious to volunteers (Keswick et al., 1985). It is worth mentioning that the presence of genomic copies certainly does not equal infectivity, nor does it represent infectivity. Ca(OCl)₂ is a strong oxidizer and likely causes damage to the viral capsid (Nuanualsuwan and Cliver, 2003); however, the integrity of the norovirus capsid is unknown, which might be measured by RNase treatment or cell binding assay in the future (Topping et al., 2009; Li et al., 2012), and the genomic copies detected in this study were small segments, which are not representative of the whole genome. Approximate reductions of 1.7 log PFU/g of both MNV and TV in alfalfa seeds were observed following 2000 ppm Ca(OCl)₂, but a significantly higher reduction of MNV (∼4 log PFU/g) was observed following treatment with 20,000 ppm. Previous studies obtained a much higher reduction by hypochlorite; it was found that both TV and MNV had >5 log PFU reductions after treatment of 2000 ppm Ca(OCl)₂ in water (Hirneisen and Kniel, 2013a). Belliot et al. showed that 36.4 mM NaOCl resulted in at least a 4 log drop in MNV infectivity after only 0.5 min of exposure time (Belliot et al., 2008). The greater inactivation could be explained by the different matrix and levels of organic materials as well as the crevices on seed coats, which provide sites protecting viruses from disinfectants. It is likely that both MNV and TV behave similarly at lower level of hypochlorite; however, MNV is more sensitive to chlorine than TV at relatively high levels of Ca(OCl)₂. Thus, MNV may not be the worst-case model for estimating huNoV inactivation. TV was found to be more robust than MNV to disinfectant inactivation, indicating it could be another possible surrogate for huNoV.

The effect of organic materials on antimicrobial activity of Ca(OCl)₂ was also evaluated. The inactivation of each microorganism inoculated on alfalfa seeds was significantly lower than that in FBS-containing HBSS, indicating that alfalfa seeds contain more organic materials and more protection reducing direct interaction with Ca(OCl)₂ and that it is not the organic load alone. Generally, the antimicrobial activity of Ca(OCl)₂ in FBS-containing HBSS decreased as the organic load increased when the FBS was >30% for viruses. A similar conclusion was obtained showing that organic materials could inhibit the chlorine inactivation of bacteria including Listeria monocytogenes, Salmonella, Staphylococcus, and E. coli (Valderrama et al., 2009; Pappen et al., 2010; Buncic and Sofos, 2012). In another study, the reaction between hypochlorite and egg albumin resulted in degradation of protein and reduction of hypochlorite with two to nine molecules of hypochlorite interacting with each amino acid residue attached (Baker, 1947). Given this information, bacteria were inactivated beyond the detection limit in this study regardless of FBS concentration. Bacterial inhibition is affected by both chlorine and organic concentrations.

Ca(OCl)₂ treatments (2000 ppm and 20,000 ppm) were not able to completely inactivate either Salmonella Agona or E. coli O104:H4 inoculated on alfalfa seeds. The bacteria survived after the treatments could grew to >8 log CFU in sprouts after a 7-day germination period, close to those without treatments. Fransisca et al. found that E. coli that survived the 20,000 ppm chlorine treatment in radish seeds with 3.21 log grew to as high as 6.2 log within 3 days of sprouting (Fransisca et al., 2011). Gandhi et al. reported that Salmonella Stanley reached >7.0 log on sprouts grown from Salmonella Stanley–inoculated alfalfa seeds that were treated with 20,000 ppm Ca(OCl)₂ (Gandhi and Matthews, 2003). The results revealed that a high level of chlorine treatment might kill most of the natural microflora on seeds, which allows both Salmonella Agona and E. coli O104:H4 to replicate quickly without competition. Following treatment, while it seemed that large amounts of bacteria were inactivated, upon germination these treated bacteria recovered and increased numbers were observed on sprouts. It has been shown previously that Salmonella enterica and E. coli could grow to higher levels without competition from other bacteria (Cooley et al., 2003; Liao, 2008).

These results suggest that more effective strategies are urgently needed to control sprout safety. Alternative treatments and multihurdle approaches should be considered to decontaminate seeds prior to germination as well as throughout the process of sprouting, such as inclusion of organic acids (Lang et al., 2000; Zhao et al., 2010), H₂O₂ (Holliday et al., 2001), combinations of heat and chemicals (Bari et al., 2009; Bang et al., 2011), electrolyzed water (Bari et al., 2003; Jadeja et al., 2013), ozone or ozonated water (Sharma et al., 2002, 2003), irradiation (Bari et al., 2003; Waje and Kwon, 2007), high pressure (Neetoo et al., 2009; Neetoo and Chen, 2010), or competitive inhibition (Cooley et al., 2003; Liao, 2008). Processing methods, such as dipping, soaking, spraying, or fumigation, may also be considered to help target the disinfectant at the microorganisms tightly bound to the seeds.

Footnotes

Acknowledgment

This work was funded in part by the USDA-NIFA NoroCORE grant 2011-68003-30395.

Disclosure Statement

No competing financial interests exist.

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Effectiveness of Calcium Hypochlorite on Viral and Bacterial Contamination of Alfalfa Seeds

Abstract

Introduction

Materials and Methods

Virus cultivation and infectivity

huNoV preparation

HuNoV genome quantification by real-time reverse transcription polymerase chain reaction (RT-PCR)

Bacterial growth and quantification

Ca(OCl)2 preparation

Alfalfa seed preparation

Effects of Ca(OCl)2 on bacteria or viruses inoculated on alfalfa seeds and on bacteria or viruses in the presence of organic materials

Germination of bacteria-inoculated seeds after Ca(OCl)2

Statistical analysis

Results

Virus and bacterial recovery from alfalfa seeds after inoculation

Inactivation of viruses and bacteria from contaminated alfalfa seeds by Ca(OCl)2 treatments

Effects of organic load in Ca(OCl)2 inactivation of viruses and bacteria

Bacterial growth on alfalfa seeds/sprout during germination after Ca(OCl)2 treatments

Discussion

Footnotes

Acknowledgment

Disclosure Statement

References

Ca(OCl)₂ preparation

Effects of Ca(OCl)₂ on bacteria or viruses inoculated on alfalfa seeds and on bacteria or viruses in the presence of organic materials

Germination of bacteria-inoculated seeds after Ca(OCl)₂

Inactivation of viruses and bacteria from contaminated alfalfa seeds by Ca(OCl)₂ treatments

Effects of organic load in Ca(OCl)₂ inactivation of viruses and bacteria

Bacterial growth on alfalfa seeds/sprout during germination after Ca(OCl)₂ treatments