PaPer Ital. J. Food Sci., vol. 28 - 2016 57 - Keywords: E. coli O157:H7, environmental stress, leafy green vegetables, rocket leaves, inoculation method - RecoveRy and BehaviouR of StReSSed EschErichia coli o157:h7 cellS on Rocket leaf SuRfaceS inoculated By diffeRent MethodS anaS a. al-naBulSi1*, aMin n. olaiMat2, taReq M. oSaili1, heBa M. oBaidat1, Ziad W. JaRadat3, Reyad R. ShakeR4 and RichaRd a. holley5 1Department of Nutrition and Food Technology, Faculty of Agriculture, Jordan University of Science and Technology, Irbid, Jordan 2Department of Clinical Nutrition and Dietetics, Faculty of Allied Health Sciences, Hashemite University, Zarqa, Jordan 3Department of Biological and Genetic Engineering, Jordan University of Science and Technology, Irbid, Jordan 4Department of Clinical Nutrition and Dietetics, University of Sharjah, Sharjah, United Arab Emirates 5Department of Food Science, Faculty of Agriculture and Food Science, University of Manitoba, Winnipeg, Canada *Corresponding author: Tel. +962 02 7201000, Fax +962 02 7201078, email: anas_nabulsi@just.edu.jo AbstrAct E. coli O157:H7 is an emerging public health concern worldwide because of its low infectious dose and ability to survive under adverse conditions. tests were conducted to determine the abil- ity of unstressed E. coli O157:H7 cells or those stressed by acid, cold, salt exposure or starvation to survive on the surfaces of rocket leaves after contamination by three methods (dip, spray or spot inoculation) and following storage at 10 or 25ºc. E. coli O157:H7 numbers recovered from rocket leaves contaminated by the different techniques were in the order of dip > spot > spray in- oculation. Numbers of stressed E. coli O157:H7 recovered after inoculation by all three methods increased significantly over 7d storage at 10 or 25ºc, while unstressed E. coli O157:H7 only grew following dip inoculation. Exposure to adverse environmental conditions may increase the risk of E. coli O157:H7 survival and spread on leafy green vegetables. mailto:anas_nabulsi%40just.edu.jo?subject= 58 Ital. J. Food Sci., vol. 28 - 2016 INtrOductION Escherichia coli O157:H7 is a facultatively an- aerobic, Gram-negative rod-shaped enteric bac- terium which produces shiga toxins 1 and/or 2 as important virulence factors and emerged as a foodborne pathogen in 1982. E. coli O157:H7 in- fection presents with numerous symptoms in- cluding abdominal pain, watery and bloody di- arrhea, vomiting, mild fever, sometimes leading to hemorrhagic colitis and the hemolytic ure- mic syndrome (Hus) with renal tubular damage (WHO, 2011). scALLAN et al. (2011) and tHOMAs et al. (2013) reported that E. coli O157:H7 is as- sociated with 63,153 and 12,827 foodborne ill- nesses every year in the us and canada, respec- tively. E. coli O157:H7 illness outbreaks have been associated with a variety of foods including ground beef, spinach, lettuce, radishes, vegeta- ble sprouts, fermented sausages, unpasteurized fruit juices, apple cider and raw milk (cHAurEt, 2011). Annually, fresh produce is responsible for 45.9% of foodborne illnesses, 38.1% of hospitali- zations and 22.9% of deaths caused by contami- nated food in the us (PAINtEr et al., 2013). Fur- thermore, E. coli O157:H7 has been associated with repeated illness outbreaks resulting from the consumption leafy vegetables including lettuce, spinach, parsley and rocket leaves (OLAIMAt and HOLLEY, 2012; NYGArd et al., 2008). In recent years contamination of raw or mini- mally processed food products such as fresh pro- duce with E. coli O157:H7 has become a concern worldwide because of its low infectious dose and ability to survive for long periods in the environ- ment (cHAurEt, 2011; dELAQuIs et al., 2007). this contamination may occur on the surface of leafy vegetables due to the transfer of the pathogen from soil or water (dELAQuIs et al., 2007). survival of E. coli O157:H7 on these surfaces is affected by several factors including nutrient availability, com- petition with indigenous microflora, uV radiation and relative humidity (brANdL, 2006). Fresh pro- duce can be contaminated with foodborne patho- gens during pre- or post-harvest processes (OLAI- MAt and HOLLEY, 2012). Agricultural production and post-harvest environments may exert a va- riety of stresses on pathogens, which may affect their survival on the final product. In response, strategies have been developed by exposed path- ogens to reduce the impact of this stress, includ- ing formation of aggregates in protective niches, localization in biofilms, and internalization with- in plant tissues. bacteria have also been shown to respond to these stresses through genetic and/or physiological means including stress adaptation, development of cross-protection mechanisms, conversion to a viable but non-culturable (VbNc) state, through heterogeneous phenotypic expres- sion, and by sheer genetic diversity (dINu et al., 2009). studies have shown that ability of E. coli O157:H7 to exhibit responses to sub-lethal envi- ronmental stresses, which may enable its survival under more severe conditions, enhance its resist- ance to subsequent processing conditions, and/ or enhance its virulence. therefore, understand- ing the effects of environmental stress caused by acid, cold, starvation and abnormal osmotic con- ditions on the survival of E. coli O157:H7 is im- portant in order to assess and minimize the risk of foodborne illnesses caused by this organism (cHuNG et al., 2006). It has been reported that damaged produce supports the growth of foodborne pathogens, however, intact vegetables like lettuce, toma- toes, endive, carrots, cabbage, asparagus, broc- coli and cauliflower may also permit the growth or survival of pathogens (OLAIMAt and HOLLEY, 2012). several studies have investigated growth and survival of E. coli O157:H7 on the surfaces of leafy green vegetables including lettuce (LANG et al., 2004b; MArKLANd et al., 2013; McEVOY et al., 2009; cHANG and FANG, 2007), parsley (IsLAM et al., 2004; LANG et al., 2004b), spinach (MArK- LANd et al., 2013; LuO et al., 2009), basil (MArK- LANd et al., 2013) and thale cress (cOOLEY et al., 2003). However, little information is available on the survival of stressed E. coli O157:H7 cells on the surfaces of leafy greens (McEVOY et al., 2009). since produce contamination may occur dur- ing pre- or post-harvest activities, different tech- niques have been used to reproduce contamina- tion that may occur commercially. spot inocula- tion, where a volume of inoculum containing a known cell density is applied at several locations on produce surfaces, can represent contamina- tion that may occur from contact with soil, work- ers’ hands, or equipment surfaces. dip inocula- tion can represent contamination that may oc- cur from run-off as well as irrigation, flume wa- ter use and water immersion which are common among industry practices. Additionally, spray in- oculation can represent contamination that may result from aerosols (bEucHAt et al., 2003). Given the physical differences among each of the ways produce can become accidentally or deliberately contaminated, it became of interest to determine whether the experimental method used for fresh produce inoculation could influence inoculated pathogen survival or growth. thus, the objective of the current study was to compare the recovery of unstressed or acid-, cold-, starved- and salt- stressed E. coli O157:H7 cells from the surfaces of rocket leaves inoculated by different methods and stored at 10° or 25ºc. MAtErIALs ANd MEtHOds Preparation of bacterial strains and inocula Four clinical isolates of E. coli O157:H7 (00:0304, 02:0627, 02:0628, and 02:3581) now in the department of Nutrition and Food technology, Jordan university of science and technology cul- ture collection were stored individually at -80ºc Ital. J. Food Sci., vol. 28 - 2016 59 in trypticase soy broth (tsb; Oxoid Ltd., basing- stoke, uK) containing 20% (vol/vol) glycerol (sig- ma-Aldrich, st. Louis, MO). Frozen stock cultures were activated by transferring one loopful from each culture to 10 mL of tsb and incubating at 37ºc for 24 h. the strains were streaked on sorb- itol Macconkey Agar (sMAc) plates and stored at 4ºc. One colony was transferred to 10 ml tsb and incubated at 37ºc for 24 h. Equal volumes of each strain were mixed to prepare an E. coli O157:H7 cocktail which was centrifuged at 4,500 rpm for 20 min, the supernatant fluid was removed and the pellet was washed with sterile deionized water and then transferred to 10 mL of sterile deionized water. this suspension was diluted in sterile deionized water to achieve 108 cFu/mL. Preparation of stressed E. coli O157:H7 Acid-stressed cells were prepared by trans- ferring a loopful of each strain to 10 ml of tsb containing 10 g/L glucose and incubated at 37ºc for 18 h where ~ 9 log cFu/mL was reached at a final pH 4.9 ± 0.1 (AL-NAbuLsI et al., 2014; LEENANON and drAKE, 2001). Equal volumes of each strain were mixed in sterile tubes to pre- pare a cocktail mixture containing equal num- bers of each strain. salt-stressed cells were prepared by trans- ferring a loopful from each strain into 10 ml of tsb supplemented with 0.65 M Nacl and incu- bated at 37ºc for 18 h where ~ 9 log cFu/mL was reached. A cocktail was prepared containing equal numbers of each strain as described above and resuspended in 10 ml sterile deionized water (AL-NAbuLsI et al., 2014; HAJMEEr et al., 2006). cold-stressed cells were prepared by inoc- ulating a loopful of each strain into 10 ml of tsb at 37ºc for 18 h where ~ 9 log cFu/mL was reached. A cocktail was prepared contain- ing equal numbers of each strain as described above, was resuspended in 10 ml tsb and in- cubated for 7 d at 5ºc (AL-NAbuLsI et al., 2014; LEENANON and drAKE, 2001). starved cells were prepared by inoculating a loopful of each strain into 10 mL of tsb which was incubated at 37ºc for 18 h. A cocktail was prepared containing equal numbers of each strain as described above in saline solution (0.85% Nacl, pH 6.6) and incubated further for 48 h at 37ºc (AL-NAbuLsI et al., 2014; LEENA- NON and drAKE, 2001). Inoculation of leaf surfaces by spot, spray or dip methods rocket leaves were purchased from a super- market in Irbid, Jordan on the day of each ex- periment. damaged leaves were removed; intact leaves were washed with tap water and dried us- ing a salad spinner. unstressed and stressed E. coli O157:H7 cells were used to inoculate the rocket leaves to obtain an inoculum level of 7.0 log cFu/ leaf. the following procedures were used for inocu- lation of rocket leaves: for spot inoculation, 50 µL cell suspension was added at different places on the surface of each leaf; for dip inoculation leaves were dipped in 100 mL of inocula prepared as de- scribed above for 1 min, and for spray inoculation 50 µL of inocula was sprayed on each leaf using a gas chromatography sample syringe connected to a nitrogen gas supply at 2 psi. Inoculated leaves were placed in a biosafety cabinet for 2 h to dry. After that, the leaves were incubated at 4ºc for 22 h to allow to E. coli O157:H7 cells to attach to the leaf surfaces (LANG et al., 2004), and samples were stored at 10 or 25ºc for 7 d. Microbiological analysis the inoculated leaves were analyzed at 0.5, 1, 3, and 7 d after storage at 10 or 25ºc. the leaves were transferred to a sterile stomacher bag, treated in a stomacher (Easy Mix, AEs Lab- oratoire, France) for 2 min, serially diluted in 0.1 % peptone water and plated on sorbitol Macco- nkey Agar supplemented with 0.05 mg/L cefixi- me and 2.5 mg/L potassium tellurite (ct sMAc). the solidified ct sMAc (20 ml) had been over- laid with 10 ml tsA (thin agar layer format) to facilitate the growth of injured cells. Inoculat- ed plates were incubated at 37ºc for 18-24 h. Statistical analysis data presented are means of three experi- ments with two replicates for each experiment (n=6). Values were analyzed by sPss software, version 19 (IbM Inc., Armonk, NY) using a uni- variate general linear model. differences were considered significant at p ≤ 0.05. rEsuLts Behaviour of unstressed E. coli O157:H7 recovered from leaf surfaces inoculated by spot, dip and spray methods the initial number of E. coli O157:H7 applied to each leaf was ~ 7.0 log cFu/leaf by each of the three methods. However, significantly higher numbers of E. coli O157:H7 cells were recovered from the dip-inoculated rocket leaves (7.10 log cFu/leaf) compared to the spot- or spray-inocu- lated leaves (6.35-6.71 log cFu/leaf). Further, E. coli O157:H7 numbers recovered from the dip-in- oculated leaves significantly increased and by 7 d reached 8.20 or 8.37 log cFu/leaf at 10 or 25°c, respectively. the spray and spot methods did not perform differently from each other, and numbers of the pathogen present on spray- and spot-inoc- ulated leaves also increased during storage; how- ever, changes (0.07-0.4 log cFu/leaf) were signif- icantly smaller than with dip-inoculated samples (1.1-1.3 log cFu/leaf) (table 1). 60 Ital. J. Food Sci., vol. 28 - 2016 Behaviour of acid-stressed E. coli O157:H7 recovered from leaf surfaces inoculated by spot, dip and spray methods the numbers of acid-stressed E. coli O157:H7 recovered from rocket leaves differed with the three methods and were ranked in the order of dip > spray > spot inoculation, although the numbers recovered following all inoculation methods were similar (p > 0.05). during stor- age, E. coli O157:H7 numbers recovered from dip-, spot- or spray-inoculated rocket leaves sig- nificantly increased, and increases by 7 d were 0.9 to 1.3 log cFu/leaf at 10°c and 1.2 to 1.6 log cFu/leaf at 25°c. Meanwhile, the highest E coli O157:H7 numbers present by 7 d storage at both temperatures were on rocket leaves inocu- lated by dipping (8.51-8.78 log cFu /leaf) and by spraying (7.79-7.93 log cFu /leaf) (table 2). Behaviour of cold-stressed E. coli O157:H7 recovered from leaf surfaces inoculated by spot, dip and spray methods E. coli O157:H7 numbers recovered from the dip-inoculated leaves were significantly higher (7.53 log cFu/leaf) than those recovered from either the spot-inoculated (6.65 log cFu/leaf) or spray-inoculated leaves (6.48 log cFu/leaf). dur- ing storage, E. coli O157:H7 numbers recovered from rocket leaves, regardless of the inoculation method used, significantly increased (0.8-1.29 log cFu/leaf) at 10 and 25ºc by 7 d (table 3). Behaviour of starvation-stressed E. coli O157:H7 recovered from leaf surfaces inoculated by spot, dip and spray methods As with cold-stressed cells, numbers of star- vation-stressed E. coli O157:H7 recovered from the dip-inoculated rocket leaves were significantly higher (7.50 log cFu/leaf) than those recovered from the spot- (6.64 log cFu/leaf) or spray-inocu- lated leaves (6.46 log cFu/leaf). during storage at 10 or 25ºc, the numbers of E. coli O157:H7 cells recovered from rocket leaves inoculated using the three methods remained constant for 1 day, but after that there was a significant increase in their numbers (0.7-1.1 log cFu/leaf). spot and spray inoculation methods had the same effect on E. coli O157:H7 numbers during storage; however dip inoculation enabled higher recoveries from the leaves at all storage intervals (table 4). table 1 - Viable count of unstressed E. coli O157:H7 cells on the surface of rocket leaves stored at 10° or 25ºc after inocu- lation by three methods. Inoculation Method 10ºC 25ºC Day Dip Spot Spray Dip Spot Spray 0 7.10±0.75aB 6.71±0.41abAB 6.35±0.51aA 7.10±0.75aB 6.71±0.41abAB 6.35±0.51aA 0.5 7.95±0.08bB 6.74±0.52abA 6.43±0.21aA 7.10±0.44aC 6.71±0.32aB 6.24±0.29aA 1 7.98±0.08bC 7.19±0.58aB 6.43±0.20aA 7.67±0.59aB 7.21±0.62aA 6.35±0.76aA 3 7.92±0.85bB 6.50±0.74bA 6.59±0.83aA 7.72±0.08bB 6.81±0.61aA 6.73±0.46aA 7 8.20±0.44bB 6.78±0.48abA 6.72±0.63aA 8.37±0.11bB 6.89±0.46aA 6.78±0.54aA Values in the same row at each temperature with the same uppercase letter are not significantly different (p ≥ 0.05). Values in the same column with the same lowercase letter are not significantly different (p ≥ 0.05). table 2 - Viable count of acid-stressed E. coli O157:H7 cells on the surface of rocket leaves stored at 10° or 25ºc after inoc- ulation by three methods. Inoculation Method 10ºC 25ºC Day Dip Spot Spray Dip Spot Spray 0 7.23±0.47aA 6.49±0.75aA 6.75±0.84aA 7.23±0.47aA 6.49±0.75aA 6.75±0.84aA 0.5 7.81±0.39bB 6.76±0.26aA 6.89±0.51aA 8.09±0.34aB 7.12±0.24aA 7.16±0.58aA 1 8.07±0.12bC 6.94±0.72abA 7.54±0.51bB 8.27±0.36abB 7.47±0.13abA 7.59±0.16abA 3 8.12±0.08bB 7.42±0.41bA 7.66±0.49bAB 8.50±0.18bcC 7.49±0.24abA 7.82±0.2bB 7 8.51±0.04cC 7.36±0.25bA 7.79±0.11bB 8.87±0.07cC 7.71±0.07bA 7.93±0.04bB Values in the same row at each temperature with the same uppercase letter are not significantly different (p ≥0.05). Values in the same column with the same lowercase letter are not significantly different (p ≥ 0.05). Ital. J. Food Sci., vol. 28 - 2016 61 table 3 - Viable count of cold-stressed E. coli O157:H7 cells on the surface of rocket leaves stored at 10° or 25ºc after inoc- ulation by three methods. Inoculation Method 10ºC 25ºC Day Dip Spot Spray Dip Spot Spray 0 7.53±0.16aB 6.65±0.32aA 6.48±0.25aA 7.53±0.16aB 6.65±0.32aA 6.48±0.25aA 0.5 7.99±0.12bB 7.24±0.13bA 7.01±0.29bcA 7.53±0.51aB 6.90±0.57aA 6.76±0.48aA 1 7.81±0.68abB 7.23±0.15bA 6.69±0.66abA 7.86±0.10bB 7.06±0.57abA 6.97±0.51abA 3 8.15±0.06bcB 7.39±0.04bcA 7.34±0.03cA 8.35±0.04bC 7.52±0.06bcB 7.41±0.09bcA 7 8.43±0.09cB 7.45±0.08cA 7.38±0.09cA 8.39±0.05bC 7.94±0.07cB 7.62±0.07cA Values in the same row at each temperature with the same uppercase letter are not significantly different (p ≥ 0.05). Values in the same column with the same lowercase letter are not significantly different (p ≥ 0.05). table 4 - Viable count of starvation-stressed E. coli O157:H7 cells on the surface of rocket leaves stored at 10° or 25ºc af- ter inoculation by three methods. Inoculation Method 10ºC 25ºC Day Dip Spot Spray Dip Spot Spray 0 7.50±0.19aB 6.64±0.30aA 6.46±0.27aA 7.50±0.19aB 6.64±.30aA 6.46±0.27aA 0.5 7.50±0.47aB 6.74±0.56aA 6.77±0.73abA 7.86±0.52aB 7.01±0.59aA 6.79±0.50aA 1 7.81±0.38aB 6.96±0.53abA 6.79±0.50abA 8.11±0.47abB 7.08±0.39aA 6.93±0.51aA 3 8.14±0.06bC 7.43±0.05cB 7.31±0.07bA 8.32±0.11bcB 7.54±0.07bA 7.44±.0.06bA 7 8.46±0.12bB 7.36±0.06bcA 7.21±0.43bA 8.51±0.06cB 7.54±0.07bA 7.54±0.08bA Values in the same row at each temperature with the same uppercase letter are not significantly different (p ≥ 0.05). Values in the same column with the same lowercase letter are not significantly different (p ≥ 0.05). table 5 - Viable count of salt-stressed E. coli O157:H7 cells on the surface of rocket leaves stored at 10° or 25ºc after inoc- ulation by three methods. Inoculation Method 10ºC 25ºC Day Dip Spot Spray Dip Spot Spray 0 7.10±0.91aA 6.94±0.38aA 6.45±0.26aA 7.10±0.81aA 6.94±0.38aA 6.45±0.26aA 0.5 7.81±0.37bB 6.86±0.49aA 6.82±0.55abA 7.86±0.52aB 6.99±0.60aA 6.81±0.52aA 1 7.86±0.38bcB 6.88±0.53aA 6.88±0.50abA 8.08±0.49aB 7.09±0.57aA 6.97±0.50aA 3 7.89±0.40bcB 7.02±0.50aA 6.94±0.52abA 8.13±0.51aB 7.20±0.53aA 7.30±0.52aA 7 8.44±0.07cB 7.36±0.15aA 7.31±0.17bA 8.32±0.11bB 7.38±0.44aA 7.45±0.06bA Values in the same row at each temperature with the same uppercase letter are not significantly different (p ≥ 0.05). Values in the same column with the same lowercase letter are not significantly different (p ≥ 0.05). Behaviour of salt-stressed E. coli O157:H7 recovered from leaf surfaces inoculated by spot, dip and spray methods Numbers of salt-stressed E. coli O157:H7 were also higher (p < 0.05) on dip-inoculated leaves than those of other treatments. their numbers significantly increased on spray- or dip-inoculated leaves, and by 7 d reached 7.31 and 8.44 log cFu/leaf, respectively, at 10ºc and 7.45 and 8.32 log cFu/leaf, respectively, at 25ºc. However, there was no change in the numbers of E. coli O157:H7 recovered from spot-inoculated rocket leaves (p > 0.05). In- creases during storage were 0.4 log cFu/leaf on samples spot-inoculated, but numbers on leaves from the other treatments increased 1.0 - 1.3 log cFu/leaf at both temperatures (table 5). 62 Ital. J. Food Sci., vol. 28 - 2016 dIscussION different inoculation methods have been used experimentally to contaminate fresh produce in studies of the survival or inactivation of patho- gens (AL-NAbuLsI et al., 2014; LANG et al., 2004 a,b; sINGH et al., 2002). However, it is possible that the method chosen for inoculation may af- fect pathogen behaviours (survival, growth, inju- ry, or death). In the present study three inocula- tion techniques (dip, spot and spray) were used and there was variability in the number of E. coli O157:H7 present on the leaves contaminated. It was found that dipping yielded larger numbers of unstressed or stressed E. coli O157:H7 cells on rocket leaves. this may have been because of the greater exposure of leaf surfaces, including cut surfaces where some cells could have been internalized. these results are similar to those obtained by LANG et al. (2004a) who showed that higher numbers of E. coli O157:H7 and Salmo- nella were recovered from dip-inoculated toma- toes compared to those spot- or spray–inocu- lated. In another study, LANG et al. (2004b) ob- served that applying the cell suspension to the surface of lettuce by dipping enhanced the in- ternalization of bacteria at the cut edge and via stomata which can facilitate bacterial access to internal leaf tissue. the results of the cur- rent study also indicated that bacterial num- bers recovered from spot-inoculated leaves were not significantly different from those recovered from those that were spray-inoculated. similar- ly, LANG et al. (2004b) found that the number of E. coli O157:H7 and Salmonella recovered from lettuce when inoculated by spot or spray meth- ods were similar. However, they recommended using the spot method as the standard for in- oculation in studying the efficacy of sanitizers against pathogenic bacteria. sINGH et al. (2002) found that some sanitizers (thyme oil, aqueous chlorine dioxide, ozonated water) were less ef- fective against E. coli O157:H7 on lettuce inoc- ulated by dipping or sprinkling than by the spot or drop method. It should be noted that even when fresh produce was washed and sanitized using chemical agents such as chlorine, only a 1-2 log microbial reduction was achieved (OLAI- MAt and HOLLEY, 2012). unstressed E. coli O157:H7 cells were able to grow when inoculated by dipping at 10 or 25ºc, but cells inoculated by spraying or spotting sur- vived without significant change in numbers at both temperatures over 7 d storage. these re- sults are similar to those reported by cHANG and FANG (2007) who found that E. coli O157:H7 numbers on lettuce increased by 2.7 log cFu/g at 22ºc, although they decreased by 1.4 log cFu/g at 4ºc. FrANcIs and O’bEIrNE (2001) also reported that E. coli O157:H7 numbers in- creased by up to 2.5 log cFu/g after 12 d on spot-inoculated shredded lettuce, coleslaw and soybean sprouts at 8ºc. LuO et al. (2010) found that storage of spray-inoculated lettuce at 5ºc allowed E. coli O157:H7 to survive, but its growth was limited. At 12ºc there was more than a 2.0 log cFu/g increase in E. coli O157:H7 numbers after 3 d storage. In contrast, MArKLANd et al. (2013) did not detect E. coli O157:H7 cells af- ter 4 d on basil plants that were spray irrigated. the behaviour of microorganisms in food prod- ucts depends on the interaction of intrinsic and extrinsic factors such as temperature, pH, and water activity. bacteria may encounter sub-le- thal stresses in variety of food products, par- ticularly minimally processed food such as fresh produce. responses of bacteria to these stress- es may enhance their survival under more se- vere conditions, enhance their resistance to sub- sequent processing conditions and perhaps en- hance virulence. thus, understanding the ef- fects of environmental stress on the behaviour of pathogens is important in order to assess and minimize the risk of foodborne illness (cHuNG et al., 2006). In E. coli O157:H7 exposure to stress can initiate several mechanisms to min- imize the effects of the challenge. For example, the rpoS gene regulates expression of > 50 pro- teins that are involved in the general stress re- sponse. Also, heat and cold shock genes can play a major role in the level of expression of the re- sponse by stressed E. coli O157:H7. these mech- anisms facilitate adaptation of E. coli O157:H7 to environmental change and increase its surviv- al (cHAurEt 2011). In the current study, num- bers of stressed E. coli O157:H7 recovered by the three different methods increased significantly at 10° and 25ºc. In contrast, McEVOY et al. (2009) found that the behaviour of cold-stressed E. coli O157:H7 was similar to that of unstressed cells on fresh iceberg lettuce where the cold-stressed and unstressed cells grew significantly at 30ºc, but survived without changes in their numbers at the non-permissive growth temperature of 5ºc after 8 d. several factors are likely to affect growth and survival of E. coli O157:H7 on fresh produce including its type (pH, surface smooth- ness, porosity, nutrient availability), physiologi- cal state, moisture, storage temperature > 7ºc, and the identity of the bacterial strain. It was of interest from the present work that stressed cells of a 4 strain E. coli O157:H7 cocktail on rocket leaves were able to increase in numbers during a week of storage at 10º and 25ºc to similar ex- tents, but unstressed cells did not. In conclusion, it appears that the method used for bacterial inoculation of produce leaves can influence the levels of E. coli O157:H7 recov- ered from treated samples. the greatest uptake of cells from the inoculum occurred when leaves were dipped. thus the importance of control- ling the quality of water used in produce plant flumes and for produce immersion becomes ap- parent. spot and spray inoculation yielded low- er but similar levels of contamination. thus pro- duce contact with unclean equipment surfaces, Ital. J. Food Sci., vol. 28 - 2016 63 handling of produce in an unsanitary manner by employees and the occurrence of aerosols dur- ing processing can increase the bacterial bur- den that is likely to occur on the final product. Most importantly, when cells stressed by acid- ic pH, cold, starvation or salt exposure were in- oculated on rocket leaves, cells were able to grow slowly at both 10º and 25ºc, whereas un- stressed cells did not increase in numbers dur- ing 7 d storage. this unanticipated feature of E. coli O157:H7 may enhance its ability to be spread through shipments of produce during distribution, increasing risks associated with this foodborne pathogen. AcKNOWLEdGMENts the authors thank the deanship of scientific research at Jordan university of science and technology (grant 282/2011). rEFErENcEs Al-Nabulsi A.A., Osaili t.M., Obaidat H.M., shaker r.r., Awaisheh s.s. and Holley r.A. 2014. Inactivation of stressed Escherichia coli O157:H7 cells on the surfaces of rocket salad leaves by chlorine and peroxyacetic acid. J. Food Prot. 77: 32-39. beuchat L.r., Farber J.M., Garrett M., Harris L.J., Parsih M.E., suslow t.V. and busta F.F. 2003. standardization of a method to determine the efficacy of sanitizers in in- activating human pathogenic microorganisms on raw fruits and vegetables. compr. rev. Food sci. Food safe- ty 6: 174-178. brandl M.t. 2006. Fitness of human enteric pathogens on plants and implications for food safety. Ann. rev. Phyto- pathol. 44: 367-392. chang J.M. and Fang t.J. 2007. survival of Escherichia coli O157:H7 and Salmonella enterica serovars typhimurium in iceberg lettuce and the antimicrobial effect of rice vine- gar against E. coli O157:H7. Food Microbiol. 24: 745-751. chauret c. 2011. survival and control of Escherichia coli O157:H7 in foods, beverages, soil and water. Virulence 2: 593-601. chung H.J., bang W. and drake M.A. 2006. stress re- sponse of Escherichia coli. compr. rev. Food sci. Food safety 5: 52-64. cooley M.b., Miller W.G. and Mandrell r.E. 2003. coloniza- tion of Arabidopsis thaliana with Salmonella enterica and enterohemorrhagic Escherichia coli O157:H7 and compe- tition by Enterobacter asburiae. Appl. Environ. Microbi- ol. 69: 4915-4926. delaquis P., bach s. and dinu L. 2007. behavior of Escher- ichia coli O157:H7 in leafy vegetables. J. Food Prot. 70: 1966-1974. dinu L.d., delaquis P. and bach s. 2009. Nonculturable response of animal enteropathogens in the agricultur- al environment and implications for food safety. J. Food Prot. 72: 1342-1354. Francis G.A. and O’beirne O. 2001. Effects of vegetable type, package atmosphere and storage temperature on growth and survival of Escherichia coli O157:H7 and Listeria monocytogenes. J. Ind. Microbiol. biotech. 27: 111-116. Hajmeer M., ceylan E., Marsden J.L. and Fung d.Y.c. 2006. Impact of sodium chloride on Escherichia coli O157:H7 and Staphylococcus aureus analysed using transmission electron microscopy. Food Microbiol. 23: 446-452. Islam M., doyle M.P., Phatak s.c., Millner P. and Jiang X.P. 2004. Persistence of enterohemorrhagic Escherichia coli O157:H7 in soil and on leaf lettuce and parsley grown in fields treated with contaminated manure composts or ir- rigation water. J. Food Prot. 67: 1365-1370. Lang M.M., Harris L.J. and beuchat L.r. 2004a. Evalua- tion of inoculation method and inoculum drying time for their effects on survival and efficiency of recovery of Es- cherichia coli O157:H7, Salmonella, and Listeria monocy- togenes inoculated on the surface of tomatoes. J. Food Prot. 67: 732-741. Lang M.M., Harris L.J. and beuchat L.r. 2004b. surviv- al and recovery of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes on lettuce and parsley as af- fected by method of inoculation, time between inocula- tion and analysis, and treatment with chlorinated water. J. Food Prot. 67: 1092-1103. Leenanon b. and drake M.A. 2001. Acid stress, starvation, and cold stress affect poststress behavior of Escherichia coli O157:H7 and nonpathogenic Escherichia coli. J. Food Prot. 64: 970-974. Luo Y., He Q. and McEvoy J.L. 2010. Effect of storage tem- perature and duration on the behavior of Escherichia coli O157:H7 on packaged fresh-cut salad containing ro- maine and iceberg lettuce. J. Food sci. 75: M390-M397. Luo Y., He Q., McEvoy J.L. and conway W.s. 2009. Fate of Escherichia coli O157:H7 in the presence of indigenous microorganisms on commercially packaged baby spinach as impacted by storage temperature and time. J. Food Prot. 72: 2038-45. Markland s.M., shortlidge K.L., Hoover d.G., Yaron s., Pa- tel J., singh A., sharma M. and Kniel K.E. 2013. survival of pathogenic Escherichia coli on basil, lettuce, and spin- ach. Zoonoses Public Health 60: 563-71. McEvoy J.L., Luo Y., Zhou b., Feng H. and conway W.s. 2009. Potential of Escherichia coli O157:H7 to grow on field-cored lettuce as impacted by postharvest storage time and temperature. Int. J. Food Microbiol. 128: 506- 509. Nygard K., Lassen J., Vold L., Andersson Y., Fisher I. and Lofdahl s. 2008. Outbreak of Salmonella thompson in- fections linked to imported rucola lettuce. Foodborne Pat- hog. dis. 5: 165-173. Olaimat A.N. and Holley r.A. 2012. Factors influencing the microbial safety of fresh produce: A review. Food Micro- biol. 32: 1-19. Painter J.A., Hoekstra r.M., Ayers t., tauxe r.V., braden c.r., Angulo F.J. and Griffin P.M. 2013. Attribution of foodborne illnesses, hospitalizations, and deaths to food commodities by using outbreak data, united states, 1998- 2008. Emerg. Infect. dis. 19: 407-415. scallan E., Hoekstra r.M., Angulo F.J., tauxe r.V., Wid- dowson M.A., roy s.L., Jones J.L. and Griffin P.M.. 2011. Foodborne illness acquired in the united states— major pathogens. Emerg. Infect. dis. 17: 7-15. singh N., singh r.K., bhunia A.K. and stroshine r.L. 2002. Efficacy of chlorine dioxide, ozone, and thyme essen- tial oil or a sequential washing in killing Escherichia coli O157:H7 on lettuce and baby carrots. LWt Food sci. techn. 35: 720-729. thomas M.K., Murray r., Flockhart L., Pintar K., Pollari F., Fazil A., Nesbitt A. and Marshall b. 2013. Estimates of the burden of foodborne illness in canada for 30 speci- fied pathogens and unspecified agents, circa 2006. Food- borne Pathog. dis. 10: 639-648. World Health Organization (WHO). 2011. Enterohaemorrhag- ic Escherichia coli (EHEc), Fact sheet N°125. Available at: http://www.who.int/mediacentre/factsheets/fs125/en/ (Accessed on May 30, 2014). Paper Received June 23, 2014 Accepted April 15, 2015 http://www.who.int/mediacentre/factsheets/fs125/en