Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
Enhanced protection of pathogenic Escherichia coli ingested by a soil nematode Caenorhabditis elegans against sanitizer treatments Mi Ri Park, Sangnam Oh, Hyun Sun Yun, Soon Han Kim, Young Ho Ko, JeeHoon Ryu, Min Suk Rhee, Ok Sarah Shin & Younghoon Kim To cite this article: Mi Ri Park, Sangnam Oh, Hyun Sun Yun, Soon Han Kim, Young Ho Ko, Jee-Hoon Ryu, Min Suk Rhee, Ok Sarah Shin & Younghoon Kim (2014) Enhanced protection of pathogenic Escherichia coli ingested by a soil nematode Caenorhabditis elegans against sanitizer treatments, Bioscience, Biotechnology, and Biochemistry, 78:11, 1917-1922, DOI: 10.1080/09168451.2014.940830 To link to this article: http://dx.doi.org/10.1080/09168451.2014.940830
Published online: 23 Jul 2014.
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Date: 05 November 2015, At: 13:57
Bioscience, Biotechnology, and Biochemistry, 2014 Vol. 78, No. 11, 1917–1922
Enhanced protection of pathogenic Escherichia coli ingested by a soil nematode Caenorhabditis elegans against sanitizer treatments Mi Ri Park1,a, Sangnam Oh1,a, Hyun Sun Yun1, Soon Han Kim2, Young Ho Ko3, Jee-Hoon Ryu4, Min Suk Rhee4, Ok Sarah Shin5,6 and Younghoon Kim1,* 1
BK21 Plus Graduate Program, Department of Animal Science and Institute Agricultural Science and Technology, Chonbuk National University, Jeonju, Republic of Korea; 2Food Microbiology Division, National Institute of Food and Drug Safety Evaluation, Cheongju, Republic of Korea; 3Food Safety Risk Assessment Division, National Institute of Food and Drug Safety Evaluation, Cheongju, Republic of Korea; 4Department of Biotechnology, Korea University, Seoul, Republic of Korea; 5Department of Biomedical Sciences, College of Medicine, Korea University, Seoul, Republic of Korea; 6Department of Microbiology, College of Medicine, Korea University, Seoul, Republic of Korea
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Received March 6, 2014; accepted May 18, 2014 http://dx.doi.org/10.1080/09168451.2014.940830
We employed Caenorhabditis elegans as a model to study the effectiveness of sanitizers in killing pathogenic Escherichia coli strains ingested by freeliving nematodes. Adult worms that had fed on six pathogenic E. coli strains (highly persistent in the nematode intestine) were treated with three chemical solutions. In planktonic cells, none of the H2O2 and acetic acid treatments influenced the survival of the pathogenic E. coli strains, whereas sodium hypochlorite critically decreased the viability of the strains. Importantly, the survival of the E. coli strains was dramatically increased by persistence in the C. elegans gut under 0.1% sodium hypochlorite, and several strains could survive at a concentration of 0.5%. In addition, all pathogenic E. coli strains in the C. elegans gut survived on the lettuce for 5 days even though they were washed with 0.1% sodium hypochlorite. Taken together, our results indicate that pathogenic E. coli ingested by C. elegans may be protected against washing treatment with commercial sanitizers on raw food materials. Key words:
Caenorhabditis elegans; pathogenic E. coli; persistence; sanitizer; raw food materials
Safety associated with consuming fresh foods is known to be related, in part, to changes in agronomic, harvesting, processing, and consumption patterns. Fresh products (vegetables and fruits) can be contaminated by foodborne pathogens throughout the food chain system including harvesting, washing, transport, processing, distribution, and marketing, as well as in food service and home settings.1) Specifically, inappropriate washing *Corresponding auhor. Email: [email protected] a There authors contributed equally to this study. © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
of farm products can lead to the growth of foodborne pathogens because the surface of fresh food acts as a physical barrier, preventing or greatly minimizing the penetration of micro-organisms into the interior.2) Typically, washing the surface of raw fruits and vegetables with tap water is used to prevent the contamination of micro-organisms from the soil, but should not be relied upon to disinfect the surface.3) The effectiveness of washing with a sanitizer for the purpose of removing foodborne pathogens is a function of the properties of the sanitizer, treatment conditions, and characteristics of the fruits and vegetables.4) Chlorine is the most widely used chemical sanitizer in the fresh food industry. Water containing 50–200 ppm of free chlorine has been recommended for inactivating enteric pathogens that might be present on fruits and vegetables.5) Besides chlorine alone, the combination with organic acids including acetic, citric, lactic, and malic acids has been shown to reduce microbial populations.6,7) The free-living soil nematode Caenorhabditis elegans has been used extensively in biological studies as an in vivo surrogate host. Recently, it has been reported that free-living nematodes may serve as carriers or vectors of human enteric pathogens from soil resources and these nematodes have been shown to be resistant to free chlorine and to offer protection to ingested pathogens against chemical sanitizers. Indeed, Caldwell et al. reported that Salmonella ingested by nematodes are persistent in the worm’s gut and are protected against inactivation by sanitizers.8) Escherichia coli are the predominant non-pathogenic facultative anaerobic constituent of the human intestinal microbiota. Several E. coli strains, however, have developed the ability to cause diseases of the gastrointestinal, urinary, and central nervous systems in the
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human host. Pathogenic E. coli can be classified into five major categories on the basis of distinct epidemiological and clinical features9): enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC, a cause of dysentery), enterohemorrhagic E. coli (EHEC, a cause of hemorrhagic colitis and hemolytic uremic syndrome), enteropathogenic E. coli (EPEC, an important cause of infant diarrhea), and enterotoxigenic E. coli (ETEC, a major cause of travelers’ diarrhea and infant diarrhea in less developed countries). Among them, EHEC O157: H7 contaminations have been frequently implicated in outbreaks associated with consumption of fruit and vegetables, including tomatoes, lettuce, seed sprouts, parsley, melon, apple, orange, and celery.10) Here, we determined the persistence of pathogenic E. coli in the gut of nematodes. We also investigated the survival of pathogenic E. coli ingested by C. elegans when exposed to sanitizers. In addition, we tested the lethality of sanitizers to pathogenic E. coli internalized by C. elegans on lettuce leaves.
Materials and methods Pathogenic E. coli. Pathogenic E. coli strains including EPEC #29, ETEC #31, EIEC #32, EAEC #34, EHEC #33, and EHEC O157:H7 EDL933 were obtained from the Korea Centers for Disease Control and Prevention (KCDC; Osong, Korea). All bacterial strains were cultured in Luria-Bertani (LB) broth (BD Biosciences, Sparks, MD) at 37 °C for 18 h with 200–250 rpm. For long-term storage, cultures were maintained at −80 °C containing 15% glycerol in cryoprotectant. All strains were sub-cultured two times prior to experimental analysis. C. elegans nematode. C. elegans CF512 fer-15 (b26)II;fem-1(hc17)IV (fer-15;fem-1 worms) strain11) which is infertile at 25 °C, was used in all experiments. The worms were routinely maintained on nematodegrowth media (NGM) plates seeded with non-pathogenic E. coli OP50 using standard procedures.12) Persistence of pathogenic E. coli inside the C. elegans digestive tract. The numbers of bacteria cells in the worms were determined according to the method of Garsin et al.13), with some modifications. After worms were exposed to each of the prepared E. coil lawns on NGM agar plates containing nystatin suspension (50 unit/mL; Sigma–Aldrich, St. Louis, MO) over the course of 5 days, 10 worms were picked randomly, washed twice in M9 buffer and placed on a Brain Heart Infusion (BHI) agar plate containing 100 g/mL of kanamycin and streptomycin (Sigma–Aldrich). We then washed the worms in 5–μl drops of 100 μg/mL gentamicin (Sigma–Aldrich) on agar plates for 5 min to remove surface bacteria. The washed worms were then lysed in M9 buffer with 1% triton X-100 and mechanically disrupted using a motor pestle. The worm lysates were serially diluted in M9 buffer and incubated overnight at 37 °C on LB agar plates. Colonies were quantified and used to calculate the number of bacteria per nematode.
Sanitizer treatment of pathogenic E. coli. Three chemical solutions were evaluated for their effectiveness in killing E. coli ingested by C. elegans. Hydrogen peroxide (H2O2; Junsei Chemical Co., Tokyo, Japan), sodium hypochlorite (Sigma–Aldrich, St. Louis, MO), and acetic acid (J.T Baker Chemical Co., Phillipsburg, NJ) were tested at concentrations of 0.1, 0.5, and 1.0%. Sterile deionized water was used as a control. All chemical treatment solutions were used within 30 min of preparation. (1) Sanitizer treatment for planktonic cells. Overnight-cultured E. coli strains were centrifuged at 13,000 rpm and the supernatants were removed. The bacterial pellets (ca 1.0 × 107 colony forming unit (CFU)/mL) were washed twice with 0.85% NaCl and the three chemical solutions at concentrations of 0.1, 0.5, and 1.0% were added at 25 °C for 10 min. After washing the bacteria five times with 0.85% NaCl, the residual bacteria were serially diluted in 0.85% NaCl and plated on LB agar at 37 °C for 24 h. (2) Sanitizer treatment ingested cells in C. elegans intestines. Synchronization from gravid adults was performed at 25 °C, hatching the eggs overnight in M9 buffer and isolating L1 stage worms. The worms were grown for two days at 25 °C. After this incubation period, L4/ young adult worms were deposited onto the surface of pathogenic E. coli bacterial lawns on NGM agar and incubated at 25 °C for 24 h. The worms were allowed to ingest E. coli for 24 h at 25 °C before subjecting them to sanitizer treatments. After 24 h of exposure, 10 worms were washed twice in M9 buffer and placed in each chemical treatment solution. After 10 min at 25 °C, the supernatant was removed, and 1 ml of M9 buffer was added five times. The washed worms lysed in M9 buffer with 1% triton X-100 were mechanically disrupted using a motor pestle to rupture the cuticle of C. elegans and release ingested E. coli. The worm lysates were serially diluted in M9 buffer and surface plated on LB agar. Plates were incubated and enumerated at 37 °C for 24 h. Sodium hypochlorite treatment of lettuce inoculated with C. elegans that had ingested pathogenic E. coli strains. The effectiveness of sanitizers in killing E. coli strains ingested by C. elegans and deposited onto the surface of lettuce leaves was investigated. Commercial lettuce (Lactuca sativa L.) was purchased from a local supermarket in Jeonju, Korea. An aluminum foil template (14 × 19 cm) was placed on the surface of an inner leaf. Leaves were sterilized with an ultraviolet lamp on a clean bench for 24 h. Worms fed for 24 h on colonies of E. coli strains grown on NGM were prepared as described above. A 10 μL M9 buffer suspension containing ca. 100 worms was placed on a leaf of lettuce and allowed to dry for 1 and 5 days at 25 °C before treating with test chemicals. Each leaf of lettuce inoculated with C. elegans that had ingested E. coli strains was placed in a sterilized bag filter. Ten milliliters of 0.1% sodium hypochlorite was added and the mixture was agitated for 10 min. The lettuce leaf was transferred to 10 mL of 0.85% NaCl and ground using a stomacher (WES-400, Daihan scientific, Seoul,
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Korea) at level 10 for 2 min. The suspensions diluted in M9 buffer were surface plated on LB agar and plates were incubated for 24 h at 37 °C. Statistical analysis. All data represents the results from three independent replicates. Data are expressed as means ± SEM. All data were analyzed by ANOVA and differences among non-pathogenic E. coli OP50 control and pathogenic E. coli groups were tested for significance (p < 0.05) by Duncan’s multiple range tests with the SAS software package (version 9.1; SAS, Inc., Cary, NC).
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Results and discussion Ingestion of pathogenic E. coli by C. elegans We hypothesized that live pathogenic E. coli cells colonize the C. elegans intestinal tract, which protects the bacteria and acts as a natural barrier against a number of stress conditions. In order to investigate this possibility, we initially evaluated the colonization ability of pathogenic E. coli in the nematode intestine. As described in our previous methods,12) worms were exposed to a lawn of pathogenic E. coli. To remove surface bacteria, washes were performed on BHI plates containing kanamycin and gentamicin as described previously.13) As a control, we used non-pathogenic E. coli OP50.14) Interestingly, all pathogens had a high attachment ability and were remarkably more persistent in the nematode gut (p < 0.05) than E. coli OP50, the control strain, on both day 1 and 3. The colony counting results showed that there is no dramatic difference in the colonization in C. elegans intestine among tested pathogenic E. coli strains. Several strains exhibited outstanding attachment to the C. elegans intestine over 4.8 CFU/mL/worm at day 1 and 6.1 CFU/mL/worm at day 3 (Fig. 1). As expected, the attachment of nonpathogenic E. coli OP50 was significantly lower than that of pathogenic E. coli (3.4 CFU/mL/worm and 3.5 CFU/mL/worm at day 1 and 3, respectively). Our finding is consistent with previous reports showing that a number of foodborne pathogens including Grampositive12) and Gram-negative15) strains can survive in the worm intestine. Taken together, these data indicate that pathogenic E. coli are highly resistant to digestion by C. elegans and they can colonize in the nematode intestine.
Efficacy of sanitizers in killing pathogenic E. coli internalized by C. elegans Next, we explored if internalization of pathogenic E. coli in nematodes can enhance their viability compared with planktonic cells treatment with H2O2, sodium hypochlorite, and acetic acid, which are common sanitizers that can be used directly or indirectly on food and are the most widely used and studied sanitizers in Korea.16) As expected, all six strains of pathogenic E. coli as planktonic cells (109 CFU/ml) were completely inactivated by exposure to sodium hypochlorite at concentrations of 0.1, 0.5, and 1.0% for 10 min. However, they were highly resistant to H2O2
Fig. 1. Persistence of pathogenic E. coli in C. elegans intestine for 1 and 3 days. Notes: Persistent cells in the nematode gut were evaluated using CFU studies. Each experiment was repeated in two independent biological replicates. Error bars represent standard deviations. abcd Mean values with different superscripts are significantly different among non-pathogenic E. coli OP50 control and pathogenic E. coli groups for 1 day (p < 0.05). ABCD Mean values with different superscripts are significantly different among non-pathogenic E. coli OP50 control and pathogenic E. coli groups for 5 days (p < 0.05).
(except 1.0%) and acetic acid under the same planktonic conditions (Fig. 2(A)). In addition, C. elegans that had fed on pathogenic E. coli for 24 h were suspended in sanitizer solutions of sodium hypochlorite, hydrogen peroxide, and acetic acid for 10 min, then analyzed for populations of the pathogen that survived. Interestingly, exposure to 0.1, 0.5, and 1.0% H2O2 and acetic acid for 10 min was insufficient to kill all strains of pathogenic E. coli internalized in C. elegans (Fig. 2(B)). However, treatment with sodium hypochlorite successfully reduced populations of pathogenic E. coli compared with other sanitizers (Fig. 2(B)). Consistent with the results from the planktonic cells, treatment with 1.0% sodium hypochlorite caused extermination of treated worms, thus no pathogenic E. coli was detected. Unexpectedly, there was a significantly higher resistance of EHEC EDL933, EPEC #29, EHEC #33, and EAEC #34 killed by the treatment with 0.5% sodium hypochlorite compared with the other strains that were not detected (p < 0.05). Typically, a sodium hypochlorite concentration of 1.0% (approximately 1,000 ppm) is the upper limit used by the produce industry, but most processors commonly use lower concentrations (approximately 0.005–0.2%) for washing fresh vegetables.4) Importantly, the resistance of all tested pathogenic E. coli strains internalized in the worm gut was increased despite treatment with 0.1% sodium hypochlorite, which is a concentration commonly used for food washing (Fig. 2(B)). EAEC #34 in the C. elegans intestine had the lowest susceptibility among all tested strains, surviving after exposure to 0.1 and 0.5% sodium hypochlorite for 10 min (5.6 CFU/mL/worm and 5.4 CFU/mL/worm at 0.1% and 0.5% concentrations of sodium hypochlorite, respectively). Lupi et al.17) suggested that free-living nematodes may release ingested bacteria in a viable form, either through defecation or disintegration. In
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Fig. 2. The survival of Pathogenic E. coli exposed to hydrogen peroxide (H2O2), sodium hypochlorite, and acetic acid for 10 min in planktonic Cells (A) and Cells Ingested by a Nematode (B). Notes: Cell viability was evaluated using CFU studies. Each experiment was repeated in two independent biological replicates. Error bars represent standard deviations.
agreement with our data, Kenny and colleagues indicated that none of the commercial cleaners and sanitizers evaluated was effective in killing all cells of Salmonella when ingested by C. elegans.14) Our finding indicated that the tolerance levels of tested pathogenic E. coli strains were critically different in planktonic cells and ingested cells in worm intestine when they were exposed to the sanitizers. Furthermore, persistent cells in C. elegans were protected by nematode as a natural barrier against sanitizer conditions. To our knowledge, this is the first report that demonstrates the effectiveness of sanitizers in killing pathogenic E. coli in the gut of C. elegans. Efficacy of sanitizers in killing E. coli internalized by C. elegans and inoculated onto lettuce The potential of C. elegans to serve as a contamination vector for the transport of pathogens including pathogenic E. coli in soil to the lettuce surface has been demonstrated.8) Generally, sodium hypochlorite is widely used for reducing the bacterial contamination of fruits, vegetables, and fresh produce.14) The effectiveness of 0.1% sodium hypochlorite in killing pathogenic E. coli that had been ingested by C. elegans and inoculated onto the surface of lettuce leaves was determined over the course of 5 days. As shown in Fig. 3, populations of pathogenic E. coli were recovered from lettuce treated with 0.1% sodium hypochlorite. On day 1, only
ETEC #29 and EAEC #34 were observed compared with the number of strains recovered from lettuce treated with sterile water (Fig. 3(A); 7.5 CFU/mL and 6.2 CFU/mL on ETEC #29 and EAEC #34, respectively). However, all tested strains were highly detected on the surface of lettuce on day 5. This suggests that the number of pathogenic E. coli cells released from the gut of C. elegans (Fig. 3(B)) increased after initial exposure. It has been established that the number of cells recovered from the nematode depends on the bacterial genus/species.18) Previous research indicated that C. elegans are attracted to high virulent strains of Salmonella enterica serotype Poona and Shigella spp. as well as avirulent strains of Listeria welshimeri and Bacillus spp.19) Recently, it was shown that highly virulent foodborne pathogens are more effective at surviving in the intestinal tract because they possess specific attachment/colonization factors (e.g. Salmonella pathogenicity island (SPI) for Salmonella spp.).20) Pathogenic E. coli also retains critical virulence factors for attaching to the intestine surface, such as colonization factor (CF) in ETEC, bundle-forming pilus in EPEC, intimin/translocated intimin receptor in EHEC, and aggregative adherence factor in EAEC.21) In addition, ingested bacterial cells in the C. elegans intestine were also affected by the type of raw food. Caldwell and challengers reported the protection of ingested S. enterica serotype Poona against sanitizers applied to lettuce, but this was not observed in cantaloupe. Pathogenic
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Funding This research was supported by the Ministry of Food and Drug Safety [grant number 13162MFDS045] in 2013.
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References
Fig. 3. Populations of Pathogenic E. coli recovered from Lettuce inoculated with C. elegans-ingested bacteria and incubated for 1 (A) or 5 days (B) at 25 °C before treatment with 0.1% sodium hypochlorite. Notes: Cell viability was evaluated using CFU studies. Each experiment was repeated in two independent biological replicates. Error bars represent standard deviations. abcd Mean values with different superscripts are significantly different among non-pathogenic E. coli OP50 control and pathogenic E. coli groups for 1 day (p < 0.05). ABCD Mean values with different superscripts are significantly different among non-pathogenic E. coli OP50 control and pathogenic E. coli groups for 5 days (p < 0.05).
E. coli is all-rounder in contamination for various raw food materials including meat, milk, vegetable, and fruit22); hence, the application of sanitizers to kill bacteria ingested by nematodes is an important step in reducing pathogenic E. coli on raw food materials destined for the retail market. Our study provides evidence indicating the general ineffectiveness of chemical sanitizers in reducing populations of pathogenic E. coli on lettuce when they are internalized in the C. elegans intestine and that worms serve as a vector of transmission to raw food materials. Our results also suggest that pathogenic E. coli ingested by C. elegans are protected from sanitizer treatments compared with planktonic conditions. In conclusion, we describe an additional contamination route for foodborne disease by pathogenic E. coli.
[1] Doyle MP, Erickson MC. Opportunities for mitigating pathogen contamination during on-farm food production. Int. J. Food Microbiol. 2012;152:54–74. [2] Erickson MC. Internalization of fresh produce by foodborne pathogens. Annu. Rev. Food Sci. Technol. 2012;3:283–310. [3] Mattick K, Durham K, Domingue G, Jørgensen F, Sen M, Schaffner DW, Humphrey T. The survival of foodborne pathogens during domestic washing-up and subsequent transfer onto washing-up sponges, kitchen surfaces and food. Int. J. Food Microbiol. 2003;85:213–226. [4] Cherry JP. Improving the safety of fresh produce with antimicrobials. Food Technol. 1999;53:54–59. [5] Brown AL, Brooks JC, Karunasena E, Echeverry A, Laury A, Brashears MM. Inhibition of Escherichia coli O157:H7 and Clostridium sporogenes in spinach packaged in modified atmospheres after treatment combined with chlorine and lactic acid bacteria. J. Food Sci. 2011;76:M427–M432. [6] Vadlamudi S, Taylor TM, Blankenburg C, Castillo A. Effect of chemical sanitizers on Salmonella enterica Serovar Poona on the surface of cantaloupe and pathogen contamination of internal tissues as a function of cutting procedure. J. Food Prot. 2012;75: 1766–1773. [7] Rodgers SL, Cash JN, Siddiq M, Ryser ET. A Comparison of different chemical sanitizers for inactivating Escherichia coli O157:H7 and Listeria monocytogenes in solution and on apples, lettuce, strawberries, and cantaloupe. J. Food Prot. 2004;67: 721–731. [8] Caldwell KN, Adler BB, Anderson GL, Williams PL, Beuchat LR. Ingestion of Salmonella enterica Serotype Poona by a freeliving nematode, Caenorhabditis elegans, and protection against inactivation by produce sanitizers. Appl. Environ. Microbiol. 2003;69:4103–4110. [9] Clarke SC. Diarrhoeagenic Escherichia coli – an emerging problem? Diagn. Microbiol. Infect Dis. 2001;41:93–98. [10] Kwak TY, Kim NH, Rhee MS. Response surface methodologybased optimization of decontamination conditions for Escherichia coli O157:H7 and Salmonella typhimurium on fresh-cut celery using thermoultrasound and calcium propionate. Int. J. Food Microbiol. 2011;150:128–135. [11] Murphy CT, McCarroll SA, Bargmann CI, Fraser A, Kamath RS, Ahringer J, Li H, Kenyon C. Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. Nature. 2003;424:277–283. [12] Kim Y, Mylonakis E. Caenorhabditis elegans immune conditioning with the probiotic bacterium Lactobacillus acidophilus strain NCFM enhances Gram-positive immune responses. Infect Immun. 2012;80:2500–2508. [13] Garsin DA, Sifri CD, Mylonakis E, Qin X, Singh KV, Murray BE, Calderwood SB, Ausubel FM. A simple model host for identifying Gram-positive virulence factors. Proc. Natl. Acad. Sci. USA. 2001;98:10892–10897. [14] Kenney SJ, Anderson GL, Williams PL, Millner PD, Beuchat LR. Effectiveness of cleaners and sanitizers in killing Salmonella Newport in the gut of a free-living nematode, Caenorhabditis elegans. J Food Prot. 2004;67:2151–2157. [15] Kenney SJ, Anderson GL, Williams PL, Millner PD, Beuchat LR. Persistence of Escherichia coli O157:H7, Salmonella Newport, and Salmonella Poona in the gut of a free-living nematode, Caenorhabditis elegans, and transmission to progeny and uninfected nematodes. Int. J. Food Microbiol. 2005;101: 227–236.
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[16] Park KM, Baek M, Kim HJ, Kim BS, Koo M. Susceptibility of foodborne pathogens isolated from fresh-cut products and organic vegetable to organic acids and sanitizers. J. Food Hyg. Saf. 2013;28:227–233. [17] Lupi E, Ricci V, Burrini D. Recovery of bacteria in nematodes isolated from a drinking water supply. J. Water Supply Res. Technol. AQUA. 1995;44:212–218. [18] Irazoqui JE, Urbach JM, Ausubel FM. Evolution of host innate defence: insights from Caenorhabditis elegans and primitive invertebrates. Nat. Rev. Immunol. 2010;10:47–58. [19] Anderson GL, Caldwell KN, Beuchat LR, Williams PL. Interaction of a free-living soil nematode, Caenorhabditis elegans, with
surrogates of foodborne pathogenic bacteria. J Food Prot. 2003;66:1543–1549. [20] Marcus SL, Brumell JH, Pfeifer CG, Finlay BB. Salmonella pathogenicity islands: big virulence in small packages. Microbes Infection. 2000;2:145–156. [21] Croxen MA, Law RJ, Scholz R, Keeney KM, Wlodarska M, Finlay BB. Recent advances in understanding enteric pathogenic Escherichia coli. Clin. Microbiol. Rev. 2013;26:822–880. [22] Smith JL, Fratamico PM, Gunther NW. Extraintestinal pathogenic Escherichia coli. Foodborne Pathog. Dis. 2007;4: 134–163.
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
Enhanced protection of pathogenic Escherichia coli ingested by a soil nematode Caenorhabditis elegans against sanitizer treatments Mi Ri Park, Sangnam Oh, Hyun Sun Yun, Soon Han Kim, Young Ho Ko, JeeHoon Ryu, Min Suk Rhee, Ok Sarah Shin & Younghoon Kim To cite this article: Mi Ri Park, Sangnam Oh, Hyun Sun Yun, Soon Han Kim, Young Ho Ko, Jee-Hoon Ryu, Min Suk Rhee, Ok Sarah Shin & Younghoon Kim (2014) Enhanced protection of pathogenic Escherichia coli ingested by a soil nematode Caenorhabditis elegans against sanitizer treatments, Bioscience, Biotechnology, and Biochemistry, 78:11, 1917-1922, DOI: 10.1080/09168451.2014.940830 To link to this article: http://dx.doi.org/10.1080/09168451.2014.940830
Published online: 23 Jul 2014.
Submit your article to this journal
Article views: 122
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tbbb20 Download by: [Universite Laval]
Date: 05 November 2015, At: 13:57
Bioscience, Biotechnology, and Biochemistry, 2014 Vol. 78, No. 11, 1917–1922
Enhanced protection of pathogenic Escherichia coli ingested by a soil nematode Caenorhabditis elegans against sanitizer treatments Mi Ri Park1,a, Sangnam Oh1,a, Hyun Sun Yun1, Soon Han Kim2, Young Ho Ko3, Jee-Hoon Ryu4, Min Suk Rhee4, Ok Sarah Shin5,6 and Younghoon Kim1,* 1
BK21 Plus Graduate Program, Department of Animal Science and Institute Agricultural Science and Technology, Chonbuk National University, Jeonju, Republic of Korea; 2Food Microbiology Division, National Institute of Food and Drug Safety Evaluation, Cheongju, Republic of Korea; 3Food Safety Risk Assessment Division, National Institute of Food and Drug Safety Evaluation, Cheongju, Republic of Korea; 4Department of Biotechnology, Korea University, Seoul, Republic of Korea; 5Department of Biomedical Sciences, College of Medicine, Korea University, Seoul, Republic of Korea; 6Department of Microbiology, College of Medicine, Korea University, Seoul, Republic of Korea
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Received March 6, 2014; accepted May 18, 2014 http://dx.doi.org/10.1080/09168451.2014.940830
We employed Caenorhabditis elegans as a model to study the effectiveness of sanitizers in killing pathogenic Escherichia coli strains ingested by freeliving nematodes. Adult worms that had fed on six pathogenic E. coli strains (highly persistent in the nematode intestine) were treated with three chemical solutions. In planktonic cells, none of the H2O2 and acetic acid treatments influenced the survival of the pathogenic E. coli strains, whereas sodium hypochlorite critically decreased the viability of the strains. Importantly, the survival of the E. coli strains was dramatically increased by persistence in the C. elegans gut under 0.1% sodium hypochlorite, and several strains could survive at a concentration of 0.5%. In addition, all pathogenic E. coli strains in the C. elegans gut survived on the lettuce for 5 days even though they were washed with 0.1% sodium hypochlorite. Taken together, our results indicate that pathogenic E. coli ingested by C. elegans may be protected against washing treatment with commercial sanitizers on raw food materials. Key words:
Caenorhabditis elegans; pathogenic E. coli; persistence; sanitizer; raw food materials
Safety associated with consuming fresh foods is known to be related, in part, to changes in agronomic, harvesting, processing, and consumption patterns. Fresh products (vegetables and fruits) can be contaminated by foodborne pathogens throughout the food chain system including harvesting, washing, transport, processing, distribution, and marketing, as well as in food service and home settings.1) Specifically, inappropriate washing *Corresponding auhor. Email: [email protected] a There authors contributed equally to this study. © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
of farm products can lead to the growth of foodborne pathogens because the surface of fresh food acts as a physical barrier, preventing or greatly minimizing the penetration of micro-organisms into the interior.2) Typically, washing the surface of raw fruits and vegetables with tap water is used to prevent the contamination of micro-organisms from the soil, but should not be relied upon to disinfect the surface.3) The effectiveness of washing with a sanitizer for the purpose of removing foodborne pathogens is a function of the properties of the sanitizer, treatment conditions, and characteristics of the fruits and vegetables.4) Chlorine is the most widely used chemical sanitizer in the fresh food industry. Water containing 50–200 ppm of free chlorine has been recommended for inactivating enteric pathogens that might be present on fruits and vegetables.5) Besides chlorine alone, the combination with organic acids including acetic, citric, lactic, and malic acids has been shown to reduce microbial populations.6,7) The free-living soil nematode Caenorhabditis elegans has been used extensively in biological studies as an in vivo surrogate host. Recently, it has been reported that free-living nematodes may serve as carriers or vectors of human enteric pathogens from soil resources and these nematodes have been shown to be resistant to free chlorine and to offer protection to ingested pathogens against chemical sanitizers. Indeed, Caldwell et al. reported that Salmonella ingested by nematodes are persistent in the worm’s gut and are protected against inactivation by sanitizers.8) Escherichia coli are the predominant non-pathogenic facultative anaerobic constituent of the human intestinal microbiota. Several E. coli strains, however, have developed the ability to cause diseases of the gastrointestinal, urinary, and central nervous systems in the
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human host. Pathogenic E. coli can be classified into five major categories on the basis of distinct epidemiological and clinical features9): enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC, a cause of dysentery), enterohemorrhagic E. coli (EHEC, a cause of hemorrhagic colitis and hemolytic uremic syndrome), enteropathogenic E. coli (EPEC, an important cause of infant diarrhea), and enterotoxigenic E. coli (ETEC, a major cause of travelers’ diarrhea and infant diarrhea in less developed countries). Among them, EHEC O157: H7 contaminations have been frequently implicated in outbreaks associated with consumption of fruit and vegetables, including tomatoes, lettuce, seed sprouts, parsley, melon, apple, orange, and celery.10) Here, we determined the persistence of pathogenic E. coli in the gut of nematodes. We also investigated the survival of pathogenic E. coli ingested by C. elegans when exposed to sanitizers. In addition, we tested the lethality of sanitizers to pathogenic E. coli internalized by C. elegans on lettuce leaves.
Materials and methods Pathogenic E. coli. Pathogenic E. coli strains including EPEC #29, ETEC #31, EIEC #32, EAEC #34, EHEC #33, and EHEC O157:H7 EDL933 were obtained from the Korea Centers for Disease Control and Prevention (KCDC; Osong, Korea). All bacterial strains were cultured in Luria-Bertani (LB) broth (BD Biosciences, Sparks, MD) at 37 °C for 18 h with 200–250 rpm. For long-term storage, cultures were maintained at −80 °C containing 15% glycerol in cryoprotectant. All strains were sub-cultured two times prior to experimental analysis. C. elegans nematode. C. elegans CF512 fer-15 (b26)II;fem-1(hc17)IV (fer-15;fem-1 worms) strain11) which is infertile at 25 °C, was used in all experiments. The worms were routinely maintained on nematodegrowth media (NGM) plates seeded with non-pathogenic E. coli OP50 using standard procedures.12) Persistence of pathogenic E. coli inside the C. elegans digestive tract. The numbers of bacteria cells in the worms were determined according to the method of Garsin et al.13), with some modifications. After worms were exposed to each of the prepared E. coil lawns on NGM agar plates containing nystatin suspension (50 unit/mL; Sigma–Aldrich, St. Louis, MO) over the course of 5 days, 10 worms were picked randomly, washed twice in M9 buffer and placed on a Brain Heart Infusion (BHI) agar plate containing 100 g/mL of kanamycin and streptomycin (Sigma–Aldrich). We then washed the worms in 5–μl drops of 100 μg/mL gentamicin (Sigma–Aldrich) on agar plates for 5 min to remove surface bacteria. The washed worms were then lysed in M9 buffer with 1% triton X-100 and mechanically disrupted using a motor pestle. The worm lysates were serially diluted in M9 buffer and incubated overnight at 37 °C on LB agar plates. Colonies were quantified and used to calculate the number of bacteria per nematode.
Sanitizer treatment of pathogenic E. coli. Three chemical solutions were evaluated for their effectiveness in killing E. coli ingested by C. elegans. Hydrogen peroxide (H2O2; Junsei Chemical Co., Tokyo, Japan), sodium hypochlorite (Sigma–Aldrich, St. Louis, MO), and acetic acid (J.T Baker Chemical Co., Phillipsburg, NJ) were tested at concentrations of 0.1, 0.5, and 1.0%. Sterile deionized water was used as a control. All chemical treatment solutions were used within 30 min of preparation. (1) Sanitizer treatment for planktonic cells. Overnight-cultured E. coli strains were centrifuged at 13,000 rpm and the supernatants were removed. The bacterial pellets (ca 1.0 × 107 colony forming unit (CFU)/mL) were washed twice with 0.85% NaCl and the three chemical solutions at concentrations of 0.1, 0.5, and 1.0% were added at 25 °C for 10 min. After washing the bacteria five times with 0.85% NaCl, the residual bacteria were serially diluted in 0.85% NaCl and plated on LB agar at 37 °C for 24 h. (2) Sanitizer treatment ingested cells in C. elegans intestines. Synchronization from gravid adults was performed at 25 °C, hatching the eggs overnight in M9 buffer and isolating L1 stage worms. The worms were grown for two days at 25 °C. After this incubation period, L4/ young adult worms were deposited onto the surface of pathogenic E. coli bacterial lawns on NGM agar and incubated at 25 °C for 24 h. The worms were allowed to ingest E. coli for 24 h at 25 °C before subjecting them to sanitizer treatments. After 24 h of exposure, 10 worms were washed twice in M9 buffer and placed in each chemical treatment solution. After 10 min at 25 °C, the supernatant was removed, and 1 ml of M9 buffer was added five times. The washed worms lysed in M9 buffer with 1% triton X-100 were mechanically disrupted using a motor pestle to rupture the cuticle of C. elegans and release ingested E. coli. The worm lysates were serially diluted in M9 buffer and surface plated on LB agar. Plates were incubated and enumerated at 37 °C for 24 h. Sodium hypochlorite treatment of lettuce inoculated with C. elegans that had ingested pathogenic E. coli strains. The effectiveness of sanitizers in killing E. coli strains ingested by C. elegans and deposited onto the surface of lettuce leaves was investigated. Commercial lettuce (Lactuca sativa L.) was purchased from a local supermarket in Jeonju, Korea. An aluminum foil template (14 × 19 cm) was placed on the surface of an inner leaf. Leaves were sterilized with an ultraviolet lamp on a clean bench for 24 h. Worms fed for 24 h on colonies of E. coli strains grown on NGM were prepared as described above. A 10 μL M9 buffer suspension containing ca. 100 worms was placed on a leaf of lettuce and allowed to dry for 1 and 5 days at 25 °C before treating with test chemicals. Each leaf of lettuce inoculated with C. elegans that had ingested E. coli strains was placed in a sterilized bag filter. Ten milliliters of 0.1% sodium hypochlorite was added and the mixture was agitated for 10 min. The lettuce leaf was transferred to 10 mL of 0.85% NaCl and ground using a stomacher (WES-400, Daihan scientific, Seoul,
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Korea) at level 10 for 2 min. The suspensions diluted in M9 buffer were surface plated on LB agar and plates were incubated for 24 h at 37 °C. Statistical analysis. All data represents the results from three independent replicates. Data are expressed as means ± SEM. All data were analyzed by ANOVA and differences among non-pathogenic E. coli OP50 control and pathogenic E. coli groups were tested for significance (p < 0.05) by Duncan’s multiple range tests with the SAS software package (version 9.1; SAS, Inc., Cary, NC).
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Results and discussion Ingestion of pathogenic E. coli by C. elegans We hypothesized that live pathogenic E. coli cells colonize the C. elegans intestinal tract, which protects the bacteria and acts as a natural barrier against a number of stress conditions. In order to investigate this possibility, we initially evaluated the colonization ability of pathogenic E. coli in the nematode intestine. As described in our previous methods,12) worms were exposed to a lawn of pathogenic E. coli. To remove surface bacteria, washes were performed on BHI plates containing kanamycin and gentamicin as described previously.13) As a control, we used non-pathogenic E. coli OP50.14) Interestingly, all pathogens had a high attachment ability and were remarkably more persistent in the nematode gut (p < 0.05) than E. coli OP50, the control strain, on both day 1 and 3. The colony counting results showed that there is no dramatic difference in the colonization in C. elegans intestine among tested pathogenic E. coli strains. Several strains exhibited outstanding attachment to the C. elegans intestine over 4.8 CFU/mL/worm at day 1 and 6.1 CFU/mL/worm at day 3 (Fig. 1). As expected, the attachment of nonpathogenic E. coli OP50 was significantly lower than that of pathogenic E. coli (3.4 CFU/mL/worm and 3.5 CFU/mL/worm at day 1 and 3, respectively). Our finding is consistent with previous reports showing that a number of foodborne pathogens including Grampositive12) and Gram-negative15) strains can survive in the worm intestine. Taken together, these data indicate that pathogenic E. coli are highly resistant to digestion by C. elegans and they can colonize in the nematode intestine.
Efficacy of sanitizers in killing pathogenic E. coli internalized by C. elegans Next, we explored if internalization of pathogenic E. coli in nematodes can enhance their viability compared with planktonic cells treatment with H2O2, sodium hypochlorite, and acetic acid, which are common sanitizers that can be used directly or indirectly on food and are the most widely used and studied sanitizers in Korea.16) As expected, all six strains of pathogenic E. coli as planktonic cells (109 CFU/ml) were completely inactivated by exposure to sodium hypochlorite at concentrations of 0.1, 0.5, and 1.0% for 10 min. However, they were highly resistant to H2O2
Fig. 1. Persistence of pathogenic E. coli in C. elegans intestine for 1 and 3 days. Notes: Persistent cells in the nematode gut were evaluated using CFU studies. Each experiment was repeated in two independent biological replicates. Error bars represent standard deviations. abcd Mean values with different superscripts are significantly different among non-pathogenic E. coli OP50 control and pathogenic E. coli groups for 1 day (p < 0.05). ABCD Mean values with different superscripts are significantly different among non-pathogenic E. coli OP50 control and pathogenic E. coli groups for 5 days (p < 0.05).
(except 1.0%) and acetic acid under the same planktonic conditions (Fig. 2(A)). In addition, C. elegans that had fed on pathogenic E. coli for 24 h were suspended in sanitizer solutions of sodium hypochlorite, hydrogen peroxide, and acetic acid for 10 min, then analyzed for populations of the pathogen that survived. Interestingly, exposure to 0.1, 0.5, and 1.0% H2O2 and acetic acid for 10 min was insufficient to kill all strains of pathogenic E. coli internalized in C. elegans (Fig. 2(B)). However, treatment with sodium hypochlorite successfully reduced populations of pathogenic E. coli compared with other sanitizers (Fig. 2(B)). Consistent with the results from the planktonic cells, treatment with 1.0% sodium hypochlorite caused extermination of treated worms, thus no pathogenic E. coli was detected. Unexpectedly, there was a significantly higher resistance of EHEC EDL933, EPEC #29, EHEC #33, and EAEC #34 killed by the treatment with 0.5% sodium hypochlorite compared with the other strains that were not detected (p < 0.05). Typically, a sodium hypochlorite concentration of 1.0% (approximately 1,000 ppm) is the upper limit used by the produce industry, but most processors commonly use lower concentrations (approximately 0.005–0.2%) for washing fresh vegetables.4) Importantly, the resistance of all tested pathogenic E. coli strains internalized in the worm gut was increased despite treatment with 0.1% sodium hypochlorite, which is a concentration commonly used for food washing (Fig. 2(B)). EAEC #34 in the C. elegans intestine had the lowest susceptibility among all tested strains, surviving after exposure to 0.1 and 0.5% sodium hypochlorite for 10 min (5.6 CFU/mL/worm and 5.4 CFU/mL/worm at 0.1% and 0.5% concentrations of sodium hypochlorite, respectively). Lupi et al.17) suggested that free-living nematodes may release ingested bacteria in a viable form, either through defecation or disintegration. In
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Fig. 2. The survival of Pathogenic E. coli exposed to hydrogen peroxide (H2O2), sodium hypochlorite, and acetic acid for 10 min in planktonic Cells (A) and Cells Ingested by a Nematode (B). Notes: Cell viability was evaluated using CFU studies. Each experiment was repeated in two independent biological replicates. Error bars represent standard deviations.
agreement with our data, Kenny and colleagues indicated that none of the commercial cleaners and sanitizers evaluated was effective in killing all cells of Salmonella when ingested by C. elegans.14) Our finding indicated that the tolerance levels of tested pathogenic E. coli strains were critically different in planktonic cells and ingested cells in worm intestine when they were exposed to the sanitizers. Furthermore, persistent cells in C. elegans were protected by nematode as a natural barrier against sanitizer conditions. To our knowledge, this is the first report that demonstrates the effectiveness of sanitizers in killing pathogenic E. coli in the gut of C. elegans. Efficacy of sanitizers in killing E. coli internalized by C. elegans and inoculated onto lettuce The potential of C. elegans to serve as a contamination vector for the transport of pathogens including pathogenic E. coli in soil to the lettuce surface has been demonstrated.8) Generally, sodium hypochlorite is widely used for reducing the bacterial contamination of fruits, vegetables, and fresh produce.14) The effectiveness of 0.1% sodium hypochlorite in killing pathogenic E. coli that had been ingested by C. elegans and inoculated onto the surface of lettuce leaves was determined over the course of 5 days. As shown in Fig. 3, populations of pathogenic E. coli were recovered from lettuce treated with 0.1% sodium hypochlorite. On day 1, only
ETEC #29 and EAEC #34 were observed compared with the number of strains recovered from lettuce treated with sterile water (Fig. 3(A); 7.5 CFU/mL and 6.2 CFU/mL on ETEC #29 and EAEC #34, respectively). However, all tested strains were highly detected on the surface of lettuce on day 5. This suggests that the number of pathogenic E. coli cells released from the gut of C. elegans (Fig. 3(B)) increased after initial exposure. It has been established that the number of cells recovered from the nematode depends on the bacterial genus/species.18) Previous research indicated that C. elegans are attracted to high virulent strains of Salmonella enterica serotype Poona and Shigella spp. as well as avirulent strains of Listeria welshimeri and Bacillus spp.19) Recently, it was shown that highly virulent foodborne pathogens are more effective at surviving in the intestinal tract because they possess specific attachment/colonization factors (e.g. Salmonella pathogenicity island (SPI) for Salmonella spp.).20) Pathogenic E. coli also retains critical virulence factors for attaching to the intestine surface, such as colonization factor (CF) in ETEC, bundle-forming pilus in EPEC, intimin/translocated intimin receptor in EHEC, and aggregative adherence factor in EAEC.21) In addition, ingested bacterial cells in the C. elegans intestine were also affected by the type of raw food. Caldwell and challengers reported the protection of ingested S. enterica serotype Poona against sanitizers applied to lettuce, but this was not observed in cantaloupe. Pathogenic
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Funding This research was supported by the Ministry of Food and Drug Safety [grant number 13162MFDS045] in 2013.
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References
Fig. 3. Populations of Pathogenic E. coli recovered from Lettuce inoculated with C. elegans-ingested bacteria and incubated for 1 (A) or 5 days (B) at 25 °C before treatment with 0.1% sodium hypochlorite. Notes: Cell viability was evaluated using CFU studies. Each experiment was repeated in two independent biological replicates. Error bars represent standard deviations. abcd Mean values with different superscripts are significantly different among non-pathogenic E. coli OP50 control and pathogenic E. coli groups for 1 day (p < 0.05). ABCD Mean values with different superscripts are significantly different among non-pathogenic E. coli OP50 control and pathogenic E. coli groups for 5 days (p < 0.05).
E. coli is all-rounder in contamination for various raw food materials including meat, milk, vegetable, and fruit22); hence, the application of sanitizers to kill bacteria ingested by nematodes is an important step in reducing pathogenic E. coli on raw food materials destined for the retail market. Our study provides evidence indicating the general ineffectiveness of chemical sanitizers in reducing populations of pathogenic E. coli on lettuce when they are internalized in the C. elegans intestine and that worms serve as a vector of transmission to raw food materials. Our results also suggest that pathogenic E. coli ingested by C. elegans are protected from sanitizer treatments compared with planktonic conditions. In conclusion, we describe an additional contamination route for foodborne disease by pathogenic E. coli.
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