Received Date: June 10, 2013; Accepted Date: June 15, 2013; Published Date: June 20, 2013
Citation: Khosravi P, Silva J, Sommers CH, Sheen S (2013) Catfish Special Edition: Thermal Inactivation of Non-O157:H7 Shiga Toxin Producing Escherichia coli (STEC) on Catfish Fillets. J Food Process Technol S11:006. doi: 10.4172/2157-7110.S11-006
Copyright: © 2013 Khosravi P, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Non-O157:H7 shiga toxin-producing Escherichia coli (non-O157 STEC) strains have emerged as food borne pathogens which have been involved in numerous food borne illness outbreaks worldwide. Seafood (fish) consumption has increased in recent years: it could become more common that STEC outbreaks may be associated with the non-O157 serovars. However, there is a lack of data on thermal inactivation of non-O157 STECs in fish muscle (e.g. catfish). Catfish fillets were inoculated with a six-isolate cocktail of non-O157 STEC serovars, i.e. O26:H11, O45:H2, O103:H2, O111:NM, O121H19, and O145:RM (the Big 6), to determine the impact of thermal treatment (heat) on their survival or thermal inactivation kinetics. The inoculated catfish fillet samples (108-9 cfu/g) were subjected to isothermal heating at 55, 60, and 65°C. The D- and z- values were determined by using linear regression of the survival data. The D- values were found to be 712 sec (R2 = 0.88), 38.8 sec (R2 = 0.97) and 3.6 sec (R2 = 0.91) at 55, 60 and 65°C, respectively. The z-value was 4.4°C, consistent with reported values for STECs in other food systems. The results of this study showed that the thermal inactivation effect of non-O157 STECs is not the same as of O157 strains in catfish meat, especially at lower temperature (e.g. 55°C), but becomes similar as temperature increases. The findings will assist risk assessors in providing safer finfish products to consumers.
Food borne illness; Sea food; Shiga toxin producing Escherichia coli (STEC); Catfish fillets
Microbial contamination of food products by pathogens is a growing public concern and food borne illnesses is a serious public health threat in the United States [1,2]. The Centers for Disease Control and Prevention (CDC) estimates that 76 million food borne illnesses, including 325,000 hospitalizations and 5,000 deaths, occur in the United States each year resulting in an economical loss of $10- 83 billion annually . Seafood has received increasing attention as a vehicle for food borne illnesses in the United States. When adjusted for the annual per capita consumption (ca. 17 lb), it is responsible for more food borne illnesses than other meats or produce [4,5].
Shiga toxin producing Escherichia coli (STEC) is one of the major pathogens and continue to be a leading cause of food borne illness in the United States . STECs are some of the most important food borne pathogens to emerge within the past two decades, causing illness with major implications for human morbidity and mortality worldwide . Although O157:H7 STEC is recognized as the most important serotype in relation to human infection, non-O157 STEC strains have emerged as important food borne pathogens and implicated as the cause of food borne illness outbreaks and sporadic cases of human infections and life-threatening hemolytic uremic syndrome in the United States and worldwide [8-12]. This suggests that non-O157 STEC infections are as prevalent or more than O157:H7 infections . Scallan  reported that among the total STEC infection cases, 68% of the E. coli O157:H7 infections and 82% of the non-O157 STEC cases are estimated to be food borne. Approximately three-fourths of the disease-inducing non-O157 STEC isolates reported to the Centers for Disease Control and Prevention belonged to six E. coli O serogroups: O26, O45, O103, O111, O121, and O145 . In 2011, due to their increasing public health impact, these six non-O157 STEC serogroups were declared as adulterants if present in raw beef products by the United States Food Safety and Inspection Service (FSIS) . Thus, it appears that the non-O157 STEC are responsible for a large portion of the total STEC food borne infections in the United States [14,15]. Regardless of increased prevalence of these non-O157 STEC associated outbreaks, inactivation practices for these strains in food system is lacking in the literature.
Currently, aquaculture accounts for about 40% of seafood production worldwide . Food borne illnesses due to the consumption of pathogenic E. coli contaminated seafood have been known and occurred around the world [17-22]. Suhalim et al.  evaluated the survival of Escherichia coli O157:H7 in channel catfish pond and reported that channel catfish may become a potential carrier of the pathogens. Although E.coli is a major contaminant of seafood [24,25], illnesses due to STECs from consumption of farm raised catfish in the U.S. is relatively rare [26,27]. However, there are many imported catfish fillets available in U.S. market. With the globalization trend, establishing the inactivation database to cover the potential non-O157 STEC food borne hazard in catfish fillets is highly desirable.
Heat inactivation is the primary means of reducing and eliminating pathogens in food systems and numerous studies have been conducted to investigate the thermal effect or impact on E. coli O157:H7 survival in beef and chicken [28-31], breaded pork patties and sausage [32,33], fresh produce and juices [34-36] and fish . However, it is not yet known if validated thermal inactivation data for E coli O157:H7 would also be as effective to inactivate the non-O157 STEC in food systems. To-date, almost no information available with regard to thermal inactivation kinetics data of STEC other than E.coli O157:H7 in catfish. Quantitative data on the thermal inactivation of pathogens are required for non-O157: H7 STEC to determine whether it meets the lethality performance standards . Our hypothesis is that heat may effectively inactivate or destroy non-O157 STEC in catfish. To test this hypothesis, the thermal inactivation of non-O157 STEC inoculated in catfish fillet meat was performed to determine the decimal reduction time (D- value) and the changes in temperature required if the D-value changed 10-fold or 1 log (z-value). The objective of this study was to evaluate the thermal inactivation curves of non-O157 Shiga-toxin producing Escherichia coli obtained at three different temperatures in catfish fillet meat. The data will allow processors to accurately monitor thermal processing of finfish to ensure safety and quality for consumers and to comply with federal regulations on food safety.
Six Shiga-toxin producing Escherichia coli isolates representing serotypes O26: H11, O45: H2, O103: H2, O111: H11, O121: H19, and O145: RM were obtained from Dr. Rob Mandrell (USDA, Western Regional Research Center, Albany, CA) via Dr. Pina Fratamico (USDA, Eastern Regional Research Center, Wyndmoor, PA). The STECs were propagated on Brain Heart Infusion Agar (BHA, BBL/Difco Laboratories, Sparks, MD) and stored at 4°C. Those procedures are considered standard and similar to those for Escherichia coli O157:H7 collected and used in the ERRC lab.
Each bacterial strain isolate was cultured individually in 10-mL of Brain Heart Infusion Broth (BHIB, BBL/Difco Laboratories, Sparks, MD) in a sterile 50-mL polypropylene tube at 37°C for 18 to 24 h. The cocktail was made by combining 5 ml of each strain (incubated overnight) and centrifuged at 3600 Xg for 10 min (Sorvall Legend™ RT centrifuge, Kendro Laboratory Products, Newtown, CT). The pellet was re-suspended in Butterfield´s phosphate buffer (BPB-6.8 pH; Hardy Diagnostics, Santa Maria, CA) to the original cocktail volume. The cell density of the cocktail was determined by serial dilution in 0.1% peptone water (PW-BBL/Difco Laboratories, Sparks, MD) and plating in duplicate on Aerobic Plate Count (APC). The STEC cocktail at a cell density of 108-9 CFU/mL was used to inoculate the catfish.
Catfish sample preparation
Refrigerated catfish fillets (30 lbs of 3 oz fillets, no additives) were obtained in bulk from a Mississippi-based catfish processor and shipped overnight to the Eastern Regional Research Center. The fillets were then subdivided and frozen at -20°C in sealed polynylon bags (Uline, Inc., Philadelphia, PA). The catfish was allowed to thaw overnight in a refrigerator at 4°C prior to use in thermal inactivation experiments. Then, the catfish fillets were cut into smaller pieces before being homogenized in a sterile laboratory blender (Model 38BL54, Waring, Torrington, CT). Ten grams (10g) of the homogenized catfish were weighed into a re–closable bag, frozen and irradiated frozen (–20°C) with 10 kGy dose sufficient to inactivate background microorganisms . The irradiated samples were kept frozen (–20°C) until used.
Catfish samples (10 g each) were thawed to room temperature and thoroughly mixed with 0.1 ml of STEC cocktail inoculum at 1:100 dilution of the initial inoculum for a final cell concentration of ca.107-8 CFU/g. One gram sample was weighed into stomacher bags (200 ml, Whirl-Pak filter bags, Nasco, Fort Atkinson, WI), then, the fish sample was distributed in the bag to form a thin layer (1-2 mm in thickness) before vacuum sealed (Model C 200, MULTIVAC, Sepp Haggenmüller GmbH & Co. KG, Wolfertschwenden, Germany) to allow the removal of the internal air and to ensure fast and uniform heat transfer across the fish meat layer during the isothermal heating treatment in a water bath. The sample bags were placed in a rack constructed to provide adequate clearance and proper contact with the hot water. The thermal inactivation set-up and procedures originally developed by Huang  and slightly modified to meet our equipment needs was used. All the samples were placed in a re-circulating hot water bath (Model NESLAB RTE17, Thermo Electron Co., Newington, NH) which was equipped with a temperature control unit (Digital One, Thermo Electron Co.). Samples were fully submerged under hot water circulation for thermal inactivation. The water bath temperature was monitored by its built-in device (± 0.5°C) and constantly re-checked by inserting a thermometer probe at the set temperature of 55, 60 or 65°C. The sampling (time) intervals were 0, 120, 360, 600, 720, 960, and 1200 s at 55°C; 0, 10, 20, 40, 60, 80, and 100 s at 60°C; and, 0, 2, 4, 6, 8, 10, 12 s at 65°C. The come-up time (time required for the sample temperature to reach water bath temperature) was about 4-5 s , which was consistent with previous studies. At the determined time intervals, catfish samples were removed from the hot water bath and immediately immersed in iced water (0-1.0°C) to terminate the heating process. The fish samples remained chilled and recovery of the survivors was performed immediately. The thermal inactivation study on the catfish at each temperature was repeated two times and three plate counts (replicates) were attained for each experiment.
Non-O157 STEC enumeration
The bag containing the pre-weighed 1.0g catfish samples was aseptically cleaned, opened and 9 ml of buffered peptone water (Becton, Dickinson and Co.) added to obtain a 1:10 dilution. The samples were stomached for 2 min and serial diluted with peptone water before being surface plated on APC agar to determine survivor counts. The plates were incubated for 24-48 h at 37°C before being enumerated.
Data analysis and determination of thermal D- and z-values
Data entered into a Microsoft Excel spreadsheet (Microsoft, Redmond, WA.) were converted to log10 cfu/g. The survival counts of non-O157 STEC in catfish after each heat treatment were plotted against the heating time and analyzed for D- and z-values by using the DMFit program  or the MS Excel Spreadsheet build-in linear curve fitting analysis option. The thermal D-value which is the time required to reduce the microbial population by 90% was calculated for each heating temperature. The D-value is the negative inverse slope of the plot (i.e. D-value = -1/slope) .
The z-values of the bacterial cocktail were calculated by determining the linear regression of the log10 D-values and temperatures (T). The z-values of the bacteria were calculated from the inverse negative slopes from the graph of the logarithm (base 10) of D- value and the heating temperature or by using the formula : z = (T2 - T1)/(log D1 - log D2), where T1 and T2 are two different temperatures and D1 and D2 are the corresponding D-values, respectively.
The D- and z-values were analyzed by ANOVA to determine the effects of treatments.
The heat inactivation or survival curves of non- O157 STECs in catfish at 55, 60 and 65°C are shown in Figures 1-3. In general, the bacterial survival counts in catfish decreased with heating time and temperature. At lower temperature (55°C), the log counts of non-O157 STEC in catfish reduced by about 0.27 log CFU/g (from 8.31 log10 CFU/g to 8.05 log10 CFU/g) within the first 2 min and by 1.77 log CFU/g at 20 min (Figure 1). In comparison, heating inoculated catfish fillets at 60 and 65°C for 1.7 min (100 seconds) and 0.2 min (12 seconds) resulted in 2.60 and 3.69 log CFU/g reduction from an initial inoculums of 8.31 and 8.61 log10 CFU/g, respectively (Figures 2 and 3). The calculated log reductions per unit of time (min) for non-O157 STEC in catfish was 0.09, 1.5 and 18.4 log CFU/g/min at 55, 60 and 65°C, respectively, showing that inactivation rate increased as temperature rose, which is similar to E. coli O157:H7 strains.
Heat inactivation effect on microorganisms has been described, typically, in the log-linear cell counts vs. process time pattern at isothermal conditions. However, researchers have consistently reported that there may be significant deviations from first-order kinetics of thermal death . Hansen and Riemann  suggested that the deviations from linear survival curves results may be due to the possible heterogeneity of cell population in heat resistance. In this study, the survival curve observed at 60°C (Figure 2) had apparently the most linear trend showing a strong coefficient of correlation (R2=0.97) between the non-O157 STEC survival and time as compared to those observed at 55°C ( R2=0.88). This indicates that the higher linearity may be used to better describe the inactivation kinetics at higher temperatures. At 55°C, the reduction of bacteria did not seem to change significantly at the early stage of the heating process (Figure 1). When the heating time exceeded a certain threshold, which varied with temperature, the rate of bacterial inactivation began to accelerate and became more significant. The typical “shoulder effect”, where the log counts of bacteria did not decrease immediately after the bacteria was exposed to heat, was not observed in this study.
Using DMFit program, non-O157 STEC serotypes showed linear inactivation kinetics at each thermal inactivation temperature and no lag phase was observed in any of the STECs regression curves at the selected temperature range. The D values obtained at 55, 60 and 65°C, and the regression statistics are shown in Table 1. The non-O157 STECs inoculated on catfish had significantly higher (P <0.05) D- value at 55°C which was not observed at the higher temperatures. The D-values were 712.4, 38.8 and 3.6 seconds at 55, 60 and 65°C, respectively, indicating the time required to reduce the non-O157 STEC population on catfish was much lower at the higher temperature. The coefficient of determination (R2) for the linear regression curves were 0.88, 0.97 and 0.91 at 55, 60 and 65°C, respectively. The high R2 value indicates that thermal inactivation of the non-O157 STECs is strongly temperature/ time dependent. Our results are in general agreement with the work by Rajkowski  who used the similar methodologies for thermal inactivation of O157: H7 isolates to determine their thermal resistance in catfish. The magnitude of D- values were 422, 55.5 and 4.2 seconds at 55, 60 and 65°C, respectively.
|Temp (°C)||Non–O157: H7 STECs||O157: H7 STEC#|
|y–axis intercept*||Slope||R2||D–value (second)||D–value (second)||R2|
*: The y–axis is log cfu/g and x–axis is time (second) in the x–y plot
Table 1: The D–values calculation for non–O157: H7 Shiga–toxin producing Escherichia coli and comparison with D–values reported by Rajkowski for O157: H7 strains under similar thermal treatment in catfish.
The similar trends between the results in this study and the work reported by Rajkowski  indicating thermal inactivation for E coli O157: H7 effective in reducing the population of non-O157 STEC in catfish. In both studies, the level of thermal inactivation for E coli O157: H7 and non-O157 STEC increased significantly (P<0.05) with increasing heating temperature from 55°C to 65°C. However, the D-value for non-O157 STEC at 55°C in our study was 41% greater than that of O157: H7 STEC at the same temperature (712 vs. 422 seconds). Approximately, twice as long in time required to inactive non-O157 STEC compared to E.coli O157:H7 in catfish fillet meat. This indicates that E. coli O157:H7 is more heat sensitive than non-O157 STEC at the lower temperature. However, the D-value at 55°C for O26, a non-O157 STEC, evaluated on minced beef was reported at 11.7 min  which was similar to D-value (11.8 min) at 55°C for non-O157 STEC on catfish in our study.
Understanding the heat resistance variations of similar STEC serovars in different food systems is very important in designing or selecting adequate thermal parameter(s) to eliminate STECs in processed foods. There was no significant difference in D-values observed between E. coli O157: H7 reported by Rajkowski  (4.2 seconds) and non-O157 STEC in our study (3.6 seconds) at the higher temperature of 65°C (Table 1). Therefore, it is reasonable to assume that non-O157 STEC strains behave similarly to O157:H7 when exposed to heat at higher temperature stress (e.g. >60°C) in fish meat. The findings may play an important role in if a continuous thermal process deviates from the original temperature/time settings then, a compensation process time will be required to warrant the food safety (e.g. to achieve a 5-log reduction in a thermal process).
Another important parameter for total lethality calculation is the z-value which refers change in temperature (°C) required for the thermal destruction of one log cycle time change in D-value. The z-value was estimated by computing the linear regression of mean log10 D-values against their corresponding heating temperatures . In this study, the z-value for non-O157 STEC in catfish fillets was 4.4°C, which is consistent with the finding by Rajkowski , where a z-value of 4.3°C of E. coli O157:H7 in both catfish and tilapia was reported.
The thermal D-values for non-O157 STEC on catfish at lower temperature (e.g. 55°C) was found much greater than those obtained at higher temperatures (e.g. > 60°C) compared to the O157 serovars. However, the z-values were approximately the same as reported in other studies for fish meat. The results indicated that food borne STEC strains may exhibit different survival behavior in various food systems and temperature ranges. It is of great interest that microbial food safety related data may need to be established to warrant food safety in selecting the proper thermal processing parameters. The results obtained in this study will assist seafood industry in designing acceptance limits on critical control points that ensure safety in eliminating the possible presence of any food borne STEC pathogens.