Project Narrative

(ii) Executive Summary and table of Content

1. Application of predatory bacteria Bdellovibrios bacteriovorus for enhancing the microbial safety of fresh organic products

2. Integrated Project Proposals: (c) Targeted Proposal

3. Legislatively defined goals being addressed: This study specifically addresses the sixth goal of the Farm Bill “Conducting advanced on-farm research and development that emphasizes observation of, experimentation with, and innovation for working organic farms, including research relating to production, marketing, food safety, socioeconomic conditions, and farm business management.” All research effort/funds (100 %) will be dedicated towards this goal.

4. Distribution of percentage of effort between research, education and extension

5. Program Staff and their role:

Prashant Singh (PD)

Assistant Professor, Nutrition Food and Exercise Science, Florida State University, 120 Convocation Way, Tallahassee, FL, E-mail: psingh2@fsu.edu

Francisco Diez-Gonzalez (Co-PD)

Professor and Director, Center for Food Safety, University of Georgia, 1109 Experiment St., Griffin, GA 30223, E-mail: fdiez@uga.edu

Michelle D. Danyluk (Co-PD)

Associate Professor, IFAS Citrus Research and Education Center, University of Florida, 700 Experiment Station, Road Lake Alfred, FL, E-mail: mddanyluk@ufl.edu

Daniel E Kadouri (Co-PD)

Associate Professor, Rutgers School of Dental Medicine, 110 Bergen Street, Newark, NJ, E-mail: kadourde@sdm.rutgers.edu

6. A brief summary of the critical stakeholder needs addressed by the project and the project’s long-term goals (provide cross-references to full descriptions in the narrative).

7. A brief summary (2-3 sentences) of the outreach plan proposed by the project (provide a cross-reference to the full description in the narrative).

8. A brief summary (2-3 sentences) describing potential economic, social, and other benefits

(Who benefits and how will it be measured?).

9. A brief summary (2-3 sentences) describing stakeholder engagement throughout the project (provide a cross-reference to the full description in the narrative).

(iii) Outcome from previous awards:

PI has not previously received any funding from OREI or ORG. PI is a new faculty at Florida State University.

(iv) Introduction

The long-term goal of this study is to assess the efficacy of Bdellovibrios bacteriovorus strains against Gram-negative foodborne pathogens; validate the effectiveness of selected strains as a post-harvest intervention treatment for enhancing the safety of organically grown fresh produce and organic food processing facilitesy.

The specific objectives of this proposal are to:

1) Determine the antimicrobial efficacy of predatory bacteria Bdellovibrios bacteriovorus strains against Gram-negative foodborne pathogens and spoilage bacteria.

2) Assess the antimicrobial efficacy of predatory bacteria for mitigating Gram-negative foodborne pathogens attached to the surface of fresh produce.

3) Verify the antimicrobial efficacy of predatory bacteria for enhancing the safety of sprouts.

4) Evaluateion of predatory bacteria for mitigating biofilm formed by foodborne pathogens on food processing equipment.

5) Assess the compatibility of Bdellovibrios bacteriovorus strains for organic production system by conducting in field farms and processing facilities.

1. Literature Review

The United States ranks first in the world for consumption of organic foods (Ref). The sales of organic food in the United States hasve increased from $24.8 billion in 2012 to $47 billion in 2016. Organically grown fruits and vegetables are collectivelyis the largest component and accounts for almost 40% ($15.6 billion) of all organic food sales. Overall, organic fruits and vegetables accounts for almost 15% of fresh produce that Americans consume (OTA, 2016).

1.

2.

2.1. Microbial safety of fresh produce:

Fresh produce isare an important source of vitamins, minerals and dietary fibers. As a result, the consumption of fresh produce has been increasing in the United States. This increase in fresh produce consumption has been paralleled with an increasing number of fresh produce- associated foodborne infections (Cui, Liu, & Chen, 2018). In between 1973 and 2012, fresh leafy greens were associated with a total of 606 outbreaks, resulting in 20,000 illnesses, 1,030 hospitalizations and 19 deaths (Herman, Hall, & Gould, 2015),; making fresh produce as the most common vehicle for transmitting foodborne illness in the United States (Fischer, 2015). Each year, the number of produce-associated outbreaks ranges from 23 to 60 (Callejón et al., 2015). Therefore, leafy vegetables are considered theas most common cause of foodborne illness in the United States (Sivapalasingam, Friedman, Cohen, & Tauxe, 2004) (Crowe et al., 2015).

Virulent strains of Salmonella, Shiga-toxin Escherichia coli, Campylobacter, Clostridium and Yersinia are the bacterial pathogens associated with fresh produce outbreaks (Ref). Salmonella is the most common bacterial pathogen associated with fresh produce and accounts for nearly 53% of produce related outbreaks (Sivapalasingam et al., 2004). So far, a total of 26 Salmonella serotypes associated with produce/food have been identified by the CDC (2013) and European Food Safety Authority (EFSA, 2013) database. Out of these 26 Salmonella serotypes, Typhimurium, Enteritidis and Newport are most frequently reported (Callejón et al., 2015; Herman et al., 2015) and have been associated with salad, leafy vegetables, tomato, and melon outbreaks in the United States (Callejón et al., 2015). Produce-associated outbreaks caused by pathogenic strains of E. coli (12.2%) are also prevalent, and virulent strains of E. coli O157 are most commonly reported (Sivapalasingam et al., 2004). Recently, other pathogenic strains of E. coli (O145 and O121) have been also implicated (Ref). Moreover, produce outbreaks caused by Campylobacter strains are less frequent (2.4%) (Ref).

Bacterial contamination (Salmonella, E. coli) of fresh fruit and vegetable can occur at any stage of production, harvesting and processing (Murray, Wu, Shi, Jun Xue, & Warriner, 2017). Fresh produce can get contaminated on a farm by use of unprocessed animal manure, contaminated irrigation water or foraging feral animals. Contamination can also occur at the processing stage, e.g., washing, cutting, storage (FDA, 2013). Bacterial pathogens can get internalized into the plant tissue, thus evadinge sanitizer treatments (USDA, 2013). Bacterial cells can also firmly adhere to cut surfaces of fresh produce and multiply. Previously, post-harvest washing was considered as an effective means of decontamination, but step is now viewed as a high-risk step. Post-harvest washing of fresh produce under in a commercial setting has very limited decontamination efficacy due to varying organic loads of wash water, which in turn affects the sanitizer efficacy, and can potentially lead to cross-contamination of produce (Barrera, Blenkinsop, & Warriner, 2012; Gombas et al., 2017). As evident from previous three large produce-associated outbreaks, Oon-farm as well asand processing cross-contamination, as evident from three previous large, produce-associated outbreaks, can during processing both can potentially lead to a large, multi-state outbreak (Wheeler et al., 2005) (CDC, 2006; CDC, 1999). Therefore, a more effective post-harvest decontamination treatment that can supplement current practices is required (Meireles, Giaouris, & Simões, 2016). This highlights the continued need for enhancing the safety of fresh produce and warrants research on novel approaches for reducing the burden of produce related foodborne outbreaks.

2.1.1. Food safety risk associated with organic produce

As organically- and conventionally- grown produce are cultivated in open environment, chances of bacterial contamination in both agriculturale systems are very similar. Therefore, both production systems have very similar food safety challenges. Smith-Spangler et al., (2012) analyzed data from five studies and compared the frequency of bacterial contamination in organic withand conventionally- grown produce and observed that bacterial contamination for organic produce was marginally higher, but the results were not significantly different. Multiple sanitizers can be used for postharvest intervention treatment for conventionally- grown produce, but only a few NOP- approved sanitizers are available for organically- grown produce. This lack of sanitizer options may deter organic growers from using any sanitizer in postharvest rinse water, especially when using portable water. A recent study conducted on an organic farm in Maryland, indicated that the produce wash step was a potential source of contamination due to lack of sanitizer in the wash tank water (Xu, Pahl, Buchanan, & Micallef, 2015). The study also reported data from one organic farm where application of peracetic acid in wash tank water showed no significant reduction in the indicator organism counts. Therefore, organic produce can represent either equal or higher risk due to the lack of a wide range of postharvest intervention options (Oliveira et al., 2010).

2.1.2. Post-harvest intervention and challenges

The post-harvest intervention treatment of fresh -produce serves two main functions. First, it removes the soil and other debris associated with produce after harvest, and second, it is also supposed to remove field associated contaminants (Barrera et al., 2012).

The eEfficacy of post-harvest intervention treatments are initially validated in aunder laboratory setting. Validation studies are conducted by inoculating fresh produce with a high concentration (7-9log CFU) of target pathogen and produce sample after attachment period are washed with a sanitizer. In these controlled laboratory conditions, a high sanitizer efficacy is achieved, which can vary from 2-6 log CFU based on produce texture. A higher reduction (4-6 log CFU) is achieved for produce samples with smoother texture (e.g. tomato), whereas a lower sanitizer efficacy (2-3 log CFU) is observed for produce with rough texture (e.g., leafy green, cantaloupes) (Singh, Hung, & Qi, 2018). However, under commercial processing conditions actual reduction is limited to 1-2 log CFU, regardless of sanitizer and wash time applied (Barrera et al., 2012). This reflects one of the limitations of the currently used sanitizer treatments. The strength of bacterial attachment to the produce surface is one of the factors limiting efficacy of sanitizer treatment beyond a certain point. Other factors, such as biofilm formation and internalization of pathogen into the inner vascular tissue of plant, can lower sanitizer efficacy. Hydrocooling, a process, which uses chilled water to cool down harvested produce, has been reported to assist internalization of bacteria cells into plant tissue (Li, Tajkarimi, & Osburn, 2008). The organic load of the wash tank is another critical component affecting sanitizer efficacy. The organic material in the wash tank provides physical protection to the bacterial cells and neutralizes the antimicrobial components (e.g. free-chlorine) of the sanitizers, enabling pathogen survival in wash water (Shen, Luo, Nou, Wang, & Millner, 2013). This depletion of antimicrobial components (e.g. free-chlorine), caused by high organic load of wash tanks, has been also associated with cross-contamination of different batches of produce where the sanitizer solution was reused (Montibus et al., 2016). Therefore, in the recent past, there has been a transition in food-safety philosophy, from attempting to decontaminate fresh produce to preventing cross-contamination in wash tanks (Gombas et al., 2017). Comment by Monica Key: Unclear

2.1.3. Approved sanitizers for organic food processing

Chlorine based sanitizers (e.g. sodium hypochlorite) are widely used by conventional growers for postharvest intervention treatments, as they are easy to use. Currently, only a few non-rinse food contact sanitizers (hydrogen peroxide, ozone and peracetic acid) are listed for organic processing. Under Section 205.605 of the National List of Allowed and Prohibited Substances, use of chlorine is also allowed for disinfection and sanitizing food contact surfaces in organic processing. The residual chlorine level in wash water for organic processing is limited to 4 mg/L (EPA, Safe Drinking Water Act), which is not effective to sanitize food contact surfaces. Wash water with higher chlorine concentrations can be used for produce with higher organic load (e.g., apple, salad greens), but these produce must receive a final rinse with water containing no more than 4 mg/L chlorine (Guide for organic producer www.ams.usda.gov/nop). Ozone is very a very powerful oxidant and an attractive alternative for organic operations. As ozone has a very low stability (20 min), and it must be generated onsite. Thus, it involves a high initial setup and operating cost, making it less attractive for small farmers. Food-grade hydrogen peroxide (0.5 to 1%) and peracetic/peroxyacetic acid are other approved sanitizers. Peracetic acid has been recently approved with great expectations, as it reportedly has good antimicrobial efficacy in water dump tanks and flume water. However, the fact that its formulation typically contains hydroxyethylidene-1,1-diphosphonic acid (HEDP) or other chemical stabilizers not listed in the NOL, may limit its widespread application. The NOL also allows the use of citric and lactic acid for processing, but compared to typical oxidant sanitizers their effectiveness is very low.

Because of the limited choice of approved sanitizers for organic food processing, most certification agencies have allowed the use of unlisted synthetic sanitizers as long as they are rinsed with water. The rationale for allowing the use of unapproved sanitizers is based on an interpretation of the National Organic Program regulations, sections 7CFR205.270 and 205.272 (27). The practice of rinsing sanitizers, can not only can pose serious safety risks as also leadsleading to product contamination, but it seriously compromises the principles of organic production systems. It is therefore critical to identify and develop strategies that would not only be allowable by organic regulatory agencies, but that could have the potential to be produced according to organic regulations.

1.1.

1.2. Sprouts

Sprouts have a high nutritive value and consumption of raw sprouts is a popular choice among health- consciouscise people. This increased consumption of various types of sprouts (e.g., alfalfa, fenugreek, mung bean, radish) and their mass production at a commercial scale has resulted in a concurrent increase in outbreaks associated with sprouted seeds (Dechet et al., 2014; Taormina, Beuchat, & Slutsker, 1999). A total of 11 sprout related outbreaks, resulting in 687 illnesses were reported in the United States, between 2006 and 2017 (CDC, 2017). The largest sprout related outbreak was reported from Japan, where radish sprouts contaminated with Escherichia coli O157:H7 sickened 9,451 people, resulting in 12 deaths (Michino et al., 1999). More recently in Germany, fenugreek sprouts contaminated with highly virulent E. coli O104:H4 wereas responsible for 3,842 illnesses and claimed 53 lives (Muniesa, Hammerl, Hertwig, Appel, & Brüssow, 2012). Sprout safety is a persistent public health safety challenge and further research needs to be carried out to enhance the safety of sprouts.

Salmonella, E. coli O157, non-O157 shiga-toxin producing E. coli (STEC) and Listeria have been frequently associated with sprout-related outbreaks (Ref). There are multiple routes by which sprouts get contaminated by foodborne pathogens, e.g., contaminated seeds, contamination during growing process, use of contaminated water, dirty equipment (Bang et al., 2016). Contaminated seeds are considered as the most likely source of foodborne pathogens (National Advisory Committee on Microbiological Criteria for Foods, 1999; U.S. Food and Drug Administration, 2014). In the past, extensive research has been performed to develop chemical and physical intervention methods for decontaminating seeds before the sprouting process. The National Advisory Committee on Microbiological Criteria for Foods recommends the use of calcium hypochlorite at 20,000 ppm for decontaminating seeds (National Advisory Committee on Microbiological Criteria for Foods, 1999). Results of this treatment are variable and generate on an average 2.5 log CFU/g reduction on seeds (Montville & Schaffner, 2004). Due to the presence of very conducive bacterial growth condition during the sprouting process, even a small number of surviving pathogenic bacteria cells (e.g. 1 log CFU/g) can recover and quickly multiply in large numbers (Choi, Beuchat, Kim, & Ryu, 2016). As current seed sanitation treatments are not always effective in eliminating bacterial contamination of seeds, there is a need for developing a new intervention treatment for enhancing the safety of organic sprouts. In this proposed study we will evaluate the efficacy of Bdellovibrios (possesses ability to prey on broad-range of Gram-negative bacteria) for enhancing safety of sprouts.

1.3. Biofilm formation on food-contact surfaces

Poor sanitation of food contact surfaces, equipment and food-processing environment promotes soil and moisture buildup, resulting in the development of biofilm. Pathogenic microorganisms in biofilm formed on food-contact surfaces can cause cross-contamination of food (Chmielewski & Frank, 2003). As the biofilm matures, their biotransfer potential also increases and they can resist stronger sanitizer treatments (Garrett, Bhakoo, & Zhang, 2008). Conventional methods for controlling biofilm are: (і) oxidizing agents such as chlorine, PAA and ozonated water (іі) quaternary ammonium compounds (ііі) iodophores. Chlorine is readily inactivated in the presence of organic material, whereas quaternary ammonium compounds leave residues which are not acceptable for organic operations (ref). As the demand for fresh, organic, and RTE food increases, more environmentally- friendly strategies are needed for mitigating biofilm formation on food-contact surfaces in the organic production system.Comment by Monica Key: Should there be another word after this?

1.4. Bdellovibrios

Bdellovibrios belongs to a group of predatory bacteria which attack Gram-negative bacteria. Once they recognize a Gram-negative bacterium they penetrating bacterial cell, multiply inside by forming bdelloplast, finally resulting in lyses of prey organism and release of new Bdellovibrios cells which continue their predatory life-cycle (Sockett, 2009). The attachment and penetration of Bdellovibrios to Gram-negative bacteria cells is non-specific, which confers them the ability to attack a broad range of Gram-negative bacteria (e.g., Alcaligenes, Campylobacter, Erwinia, Escherichia, Helicobacter, Pseudomonas, Salmonella, Legionella and Shigella) (El-Shanshoury, Abo-Amer, & Alzahrani, 2016; Negus et al., 2017; Sockett, 2009). Additionally, they have been reported to prey on antibiotic- resistantce strains of Gram-negative bacteria (Dharani, Kim, Shanks, Doi, & Kadouri, 2018). Comment by Monica Key: Unclear

Bdellovibrios are widely distributed in various natural marine and soil habitats e.g., fresh water, brackish water, seawater, water pipes, water reservoirs, soil, rhizospere of plants roots, crab’s gills and shells, oyster shells, zooplankton and submerged plants (El-Shanshoury et al., 2016; Medina & Kadouri, 2009). Furthermore, Bdellovibrios haves also been isolated from the intestinal content of live animals and humans (Schoeffield, Williams, Turng, & Falkler, 1996). Seafood growing in marine environments and fresh produce which growings in direct contact with soil are commonly associated with strains of Bdellovibrios, making them a bacteria genus directly associated with our food.

The sSafety of Bdellovibrios has been extensively evaluated on human cell lines (Gupta, Tang, Tran, & Kadouri, 2016), human epithelial cells (Monnappa, Bari, Choi, & Mitchell, 2016), immunological responses (Schwudke et al., 2003) and also at the gut microbiome level (Iebba et al., 2013; Shatzkes et al., 2017). They are also being used to for treating various bacterial infections e.g. ocular infections and lungs infections in animal models (Dashiff & Kadouri, 2011; Romanowski et al., 2016; Shanks et al., 2013; Shatzkes et al., 2016).

To date, limited studies have been conducted involving Bdellovibrios in food safety research. Strains of Bdellovibrios have been shown to reduce the number of foodborne pathogens (E. coli, Salmonella, Shigella, Yersinia) in pure culture (Fratamico & Whiting, 1995) and also in biofilm (Kadouri & O’Toole, 2005). Foodborne pathogens can evade sanitizer treatment by forming biofilm;, Bdellovibrios strains have been shown to attack and kill Gram-negative bacteria entrapped in biofilm (Kadouri & O’Toole, 2005). Further, Bdellovibrios strains has been also used to: (1) fight Aeromonas infection in fish (Chu & Zhu, 2010), (2) mitigate Salmonella ion tilapia fish fillets (Lu & Cai, 2010), (3)Vibrio contamination in oyster (Richards et al., 2012), (4) prevent Salmonella infection in young chicken (Atterbury et al., 2011). Comment by Monica Key: Cause? Prevent?

Previously, extensive research has been performed using predatory bacterial strains to treat bacterial infections in animal models, but their application in the area of produce and sprouts safety is completely unexplored. Therefore, we hypothesize that strains of Bdellovibrios can be used as a biocontrol agent against Gram-negative foodborne pathogens and can be used to improve the shelf-life of sprouts and produce.

2. Rationale and Significance

Postharvest sanitation of fresh produce and food processing equipment is of a critical step in the processing of wholesome and safe food, but organic production systems currently lack organically approved sanitizers. Because ofDue to this major limitation, organic food processors have had to resort to the use of other synthetic sanitizers to kill bacteria contaminants on the surfaces of fresh produce and equipment. Additionally, the efficacy of commonly used sanitizer varies a lot greatly depending on the level and texture of texture, level of organic matter (Parish et al., 2003).

Since the National Organic Standards Board allows only hydrogen peroxide, peracetic acid and ozonated water as non-rinse food contact sanitizers at effective antimicrobial concentrations, producers that use other prohibited sanitizers are required to give a final rinse with water. This practice markedly diminishes the effectiveness of the sanitizing treatment and increases the risk of further contamination or cross-contamination during the final rinsing process. In addition, the use of ozone and peracetic acid in food processing facilities entails worker safety risks to operators. Ozonated water must be generated on site, and is costly and energy intensive (29). Bacteriophages have been also used for mitigating foodborne pathogens, but they have a very narrow host range and can target only a specific bacterial species or serotype (Jurkevitch, 2005). Those critical limitations of organic food processing are putting the organic industry at a serious disadvantage compared to conventional counterparts and have the potential to cause a serious foodborne outbreak that could gravely damage the reputation of the whole entire organic food segment.

Considering the limitations of currently approved sanitizer treatments, there is a need for novel, and environmentally friendly alternatives that could complement with current practices and enhance the safety of organically grown produce.

Pure culture

Organic

Produce

Organic

Sprouts

Assess efficacy of predatory bacteria against foodborne pathogens

Evaluate efficacy of BALO for improving safety of organic fruits and vegetables

Assess efficacy of BALO for mitigating foodborne pathogens in sprouts

Efficacy of BALO for mitigating biofilm on food contact surfaces

Objective 1

Objective 2

Objective 3

Objective 4

This proposal presents a unique biocontrol approach using strains of Bdellovibrios for to controlling foodborne pathogens on fresh produce and biofilm formation caused by Gram-negative bacteria on food contact surfaces.

This proposed study is significant to the USDA-OREI 2018 priority area “For both plant and animal–based organic products: evaluate, develop, and improve allowable post-harvest handling, processing, and food safety practices to reduce toxins and microbial contamination, while increasing shelf-life, quality, and other economically important characteristics”. Current data show the predatory potential of Bdellovibrios against Gram-negative foodborne pathogens. Further research in this area will enhance the ability of organic food producers and processors to continue to grow and produce organic products with fewerless synthetic inputs.

Bdellovibrios strains are commonly associated with fresh water, brackish water, water reservoirs, soil and the rhizosphere of plants roots. Food growing in aquatic environments and produce in direct contact withof soil can be associated with Bdellovibrios strains. Thus, use of these natural predatory bacteria has a high probability offor enhancing the safety of organically grown produce.

This integrated project presents a balanced combination of research activities with extension and outreach efforts. Since the ECA water technology based on sodium chloride has been extensively tested experimentally, outreach trials will be conducted from the very start of the project and will continue throughout its duration.

Approach

 

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Antibiotic Resistance

The presence of antibiotic resistant strains of foodborne pathogens has been extensively studied in food of animal origin. However, the widespread application of antibiotics in the agricultural sector has become an important emerging problem for the produce sector, especially fresh fruits, vegetables and herbs that are consumed raw. Since ready-to-eat (RTE) fruits, vegetables and herbs do not undergo a heat treatment before consumption, they can potentially carry a high microbial burden. Only a limited number of studies have been performed to evaluate the burden of ARBs on fruits, vegetables and herbs. Recent studies from the U.S., Portugal, tThe Netherlands and Switzerland indicate that raw vegetables and salads can harbor ARB (Bae et al., 2015;Campos et al., 2013; Nüesch-Inderbinen et al. 2015; Raphael et al., 2011; Reuland et al., 2014; Zurfluh et al., 2015). According to a recent survey conducted by Liu and Kilonzo-Nthenge (2017), ABR strains of Salmonella, Shigella, E. coli, Enterobacter, Klebsiella, and Erwinia were also isolated from fresh produce collected in Tennessee, USA. The study also reported produce as a reservoir of antibiotic resistant bacteria. Bae et al. (2015) reported the isolation of 110 non-typhoidalSalmonella (NTS) strains from 3,840 imported food samples. These NTS strain were mostly isolated from seafood (44.5%), vegetables (28.2%), and freshwater fishes (17.3%), which were imported from India, Taiwan, Vietnam, and Indonesia. Therefore, in efforts to protect public health, research to evaluate the safety of imported food with regards to ARBs is critically warranted and necessary at this time.

References

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Liu, S., &Kilonzo-Nthenge, A. (2017). Prevalence of Multidrug-Resistant Bacteria from US-Grown and Imported Fresh Produce Retailed in Chain Supermarkets and Ethnic Stores of Davidson County, Tennessee. Journal of food protection, 80(3), 506-514.

Bae, D., Cheng, C. M., & Khan, A. A. (2015). Characterization of extended-spectrum β-lactamase (ESBL) producing non-typhoidalSalmonella (NTS) from imported food products. International journal of food microbiology, 214, 12-17.

Zurfluh, K., Nüesch-Inderbinen, M., Morach, M., Berner, A. Z., Hächler, H., & Stephan, R. (2015a). Extended-spectrum-β-lactamase-producing Enterobacteriaceae isolated from vegetables imported from the Dominican Republic, India, Thailand, and Vietnam. Applied and environmental microbiology, 81(9), 3115-3120.

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