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Survival of Escherichia Coli O157:H7 and Campylobacter Jejuni in Bottled Purified Drinking Water Under Different Storage Conditions

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Survival of Escherichia Coli O157:H7 and Campylobacter Jejuni in Bottled Purified Drinking Water Under Different Storage Conditions 0 Journal of Food Protection, Vol. 000, No. 000, 0000, Pages 000–000 doi:10.4315/0362-028X.JFP-10-368 Copyright G, International Association for Food Protection Survival of Escherichia coli O157:H7 and Campylobacter jejuni in Bottled Purified Drinking Water under Different Storage Conditions HAMZAH M. AL-QADIRI,1* XIAONAN LU,2 NIVIN I. AL-ALAMI,3 AND BARBARA A. RASCO2 1Department of Nutrition and Food Technology, Faculty of Agriculture, The University of Jordan, Amman 11942, Jordan; 2School of Food Science, Box 646376, Washington State University, Pullman, Washington 99164-6376, USA; and 3Water and Environment Research and Study Center, The University of Jordan, Amman 11942, Jordan MS 10-368: Received 1 September 2010/Accepted 15 October 2010 ABSTRACT ;< Survival of Escherichia coli O157:H7 and Campylobacter jejuni that were separately inoculated into bottled purified drinking water was investigated during storage at 22, 4, and 218uC for 5, 7, and 2 days, respectively. Two inoculation levels were used, 1 and 10 CFU/ml (102 and 103 CFU/100 ml). In samples inoculated with 102 CFU/100 ml, C. jejuni was not detectable (.2-log reduction) after storage under the conditions specified above. E. coli O157:H7 was detected on nonselective and selective media at log reductions of 1.08 to 1.25 after storage at 22uC, 1.19 to 1.56 after storage at 4uC, and 1.54 to 1.98 after storage at 218uC. When the higher inoculation level of 103 CFU/100 ml was used, C. jejuni was able to survive at 22 and 4uC, with 2.25- and 2.17-log reductions observed on nonselective media, respectively. At these higher inoculation levels, E. coli O157:H7 was detectable at 22, 4, and 218uC, with log reductions of 0.76, 0.97, and 1.21 achieved on nonselective media, respectively. Additionally, E. coli O157:H7 showed significant differences in culturability (P , 0.05) on the nonselective and selective culture media under the different storage conditions, with storage at 218uC for 2 days being the treatment most inhibiting. The percentage of sublethal injury of E. coli O157:H7 ranged from ,33 to 75%, indicating that microbial examination of bottled water must be done carefully, otherwise false-negative results or underestimation of bacterial numbers could pose a health risk when low levels of pathogens are present. The most common and widespread health risk associated with as few as 10 cells able to cause human illness and with drinking water is microbial contamination, resulting in potentially, death (3, 27, 43). Monitoring for the presence of waterborne diseases of high morbidity and mortality rates. At this pathogen in drinking water is essential, since water- least 6 billion cases of gastrointestinal illness are estimated to borne outbreaks due to E. coli O157:H7 still occur (19), and occur annually from contaminated water, especially in several outbreaks due to the presence of this pathogen in children, the elderly, and the immunocompromised (2, 3, drinking water have been recently reported (7, 25, 43). 11, 42). Accordingly, an increase in the demand for treated, Additionally, E. coli O157:H7 can survive in bottled purified bottled drinking water is a direct result of public drinking water stored at room temperature (23, 41), and health concerns about the safety of main supply water, and can readily adapt to, and survive under, different environ- due in part to the recent trend of consuming purified water mental conditions, particularly when exposed to sublethal (20, 23, 31). Many believe that bottled water is safer than stresses related to changes in temperature (i.e., freeze and municipal supplies are, and that bottled water does not cold stresses), availability of nutrients, and exposure to contain microorganisms; however, bottled drinking water is chlorine (43). Accordingly, E. coli O157:H7 might not be rarely absolutely free from microbial contaminants, and it can recovered when exposed to such sublethal injuries (22, 30), support the growth of bacterial pathogens (36). More than and for that reason it is important to determine the presence 50% of Americans drink bottled water, and worldwide sales of sublethally injured E. coli O157:H7 in drinking water, of bottled water exceed $5.7 billion (36). As reported by since injured cells could retain the ability to produce Shiga Rosenberg (36), confirmed outbreaks related to consumption toxin (44) and thus pose a risk for the consumption of of bottled drinking water could be linked to water contaminated bottled water. contamination rather than to source water quality. Campylobacter is the most commonly reported bacterial Enterohemorrhagic Escherichia coli O157:H7 is con- cause of gastrointestinal illness worldwide (7, 16, 28). C. sidered one of the most important foodborne pathogens, jejuni is a food and waterborne pathogen responsible for millions of campylobacteriosis cases (enteritis and diarrhea, * Author for correspondence. Tel: z962-6-5355000, Ext. 22422; Fax: z962- followed by rapid recovery) and causes hundreds of fatalities 6-5300806; E-mail: [email protected]; [email protected]. annually. An estimated 250,000 cases of campylobacteriosis Journal of Food Protection food-74-02-22.3d 16/11/10 15:20:17 1 Cust # JFP-10-368R 0 AL-QADIRI ET AL. J. Food Prot., Vol. 000, No. 000 infection occur annually in the United States (37); 183,961 campylobacter enrichment broth (10, 33) consisting of campylo- laboratory–confirmed campylobacteriosis cases recorded in bacter nutrient broth no. 2 (CM0067, Oxoid, Ltd., Basingstoke, the European Union in 2004 (39); however, the etiology of C. UK) and supplemented with campylobacter growth supplement jejuni remains only partly understood (16). Although (SR0232E, Oxoid, Ltd.). C. jejuni broth was then incubated in campylobacteriosis is generally classified as a self-limiting anaerobic jar at 42uC for 24 h under microaerophilic conditions (10% CO ,5% O , and 85% N ) by using an MGC-Pack- disease (14), it can result in life-threatening sequelae, among 2 2 2 MicroAero culture box (Mitsubishi Gas Chemical America, Inc., which is Guillain-Barre´ syndrome (4). New York, NY). One of the main transmission routes for campylobac- After 24 h incubation, 10 ml of broth of each strain was teriosis is water (24); Campylobacter spp. can survive as transferred to a 50-ml sterile centrifuge tube. The tubes were then viable but nonculturable in drinking water. Waterborne centrifuged for 15 min at 5,000 rpm (3,380 | g) (AccuSpin model outbreaks due to C. jejuni have occurred recently in the 400 bench top centrifuge, Fisher Thermo Scientific, Pittsburgh, PA) United States (7) and Wales (35). Bottled water has been to harvest bacterial cells. To eliminate any effect of broth reported as a possible risk factor for C. jejuni infection (15). components and bacterial metabolites, the resultant pellets were Evans et al. (12) found that Campylobacter spp. infection resuspended in 10 ml of sterile 0.85% (wt/vol) saline solution and due to the consumption of bottled drinking water has a risk centrifuged as before. After the second centrifugation, the factor of 12% as compared with eating chicken (31%) and supernatant was decanted, and the pellets were resuspended in 10 ml of the same sterile, bottled water used for the survival study salad vegetables (21%). Accordingly, C. jejuni could and recentrifuged as above. After centrifugation, the resulting accidentally contaminate bottled drinking water, in which washed pellets were then resuspended in sterile 10-ml aliquots of the it could survive and grow during storage, creating a health same bottled water, corresponding to approximately 108 CFU/ml. risk concern. However, most of research performed to date has Inoculation of water samples for survival studies. Using focused on survival of these pathogens in bottled water appropriate serial decimal dilutions, E. coli O157:H7 and C. jejuni during storage at ambient temperature by using high were separately inoculated into the 500-ml water samples to achieve inoculum levels, 103 to 106 CFU/ml (16, 23, 38, 40, 41). two different inoculum concentrations, 1 and 10 CFU/ml, which is 2 3 Therefore, this current research aimed at investigating the equivalent to 10 and 10 CFU/100 ml, respectively. These survival of E. coli O157:H7 and C. jejuni that were inoculum densities were applied because they might best represent separately inoculated into purified bottled drinking water at the realistic contamination levels of bottled water worldwide. Immediately after inoculation, bacterial viable counts were mea- low inoculum levels (1 and 10 CFU/ml) during storage at sured in duplicate by using the membrane filtration technique (5) 22, 4, and 218uC. described previously. Water samples were then stored at 22, 4, and 218uC for 5, 7, and 2 days, respectively. These storage intervals MATERIALS AND METHODS were selected because some consumers keep unsealed bottled water Water samples. Purified drinking water (treated by reverse at room temperature for 2 to 5 days, whereas others prefer to osmosis, microfiltration, and ozone sterilization) bottled in polyeth- refrigerate bottled water and consume it within a week from ylene terephthalate containers was purchased from a local grocery opening. The 218uC treatment was examined to investigate the store. Water was examined within 2 weeks of its production date. As survival of inoculated bacteria in ice. Uninoculated control water labeled, this type of water contained insignificant amounts of sodium samples were also included in this study, stored as above. bicarbonate and calcium chloride (to preserve fresh taste). To examine the microbial quality of purchased bottled water, all water Recovery of bacteria and culture media. To recover samples used for bacterial inoculation were microbially examined in surviving bacteria, water samples were examined in duplicate by duplicate by using a membrane filtration technique (5). Under the membrane filtration technique (5) detailed above. Water aseptic conditions, 100 ml of drinking water sample was filtered samples were gently mixed with a rolling motion to detach through a gridded, sterile, cellulose–nitrate membrane filter (0.45- adhered bacterial cells from bottles surfaces.
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