Effects of Polyphosphate Additives on Campylobacter Survival in Processed Chicken Exudates

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2010, p Vol. 76, No /10/$12.00 doi: /aem Copyright 2010, American Society for Microbiology. All Rights Reserved. Effects of Polyphosphate Additives on Campylobacter Survival in Processed Chicken Exudates Nereus W. Gunther IV* U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Microbial Food Safety Research Unit, Wyndmoor, Pennsylvania Received 27 November 2009/Accepted 10 February 2010 Campylobacter spp. are responsible for a large number of the bacterial food poisoning cases worldwide. Despite being sensitive to oxygen and nutritionally fastidious, Campylobacter spp. are able to survive in food processing environments and reach consumers in sufficient numbers to cause disease. To investigate Campylobacter persistence on processed chicken, exudates from chickens produced for consumer sale were collected and sterilized. Two types of exudates from chicken products were collected: enhanced, where a marinade was added to the chickens during processing, and nonenhanced, where no additives were added during processing. Exudates from enhanced chicken products examined in this study contained a mixture of polyphosphates. Exudate samples were inoculated with Campylobacter jejuni or Campylobacter coli strains and incubated under a range of environmental conditions, and viable bacteria present in the resultant cultures were enumerated. When incubated at 42 C in a microaerobic environment, exudates from enhanced chicken products resulted in increased survival of C. jejuni and C. coli compared with that in nonenhanced exudates in the range of <1 to >4 log CFU/ml. Under more relevant food storage conditions (4 C and normal atmosphere), the exudates from enhanced chicken products also demonstrated improved Campylobacter survival compared with that in nonenhanced exudates. Polyphosphates present in the enhanced exudates were determined to be largely responsible for the improved survival observed when the two types of exudates were compared. Therefore, polyphosphates used to enhance chicken quality aid in sustaining the numbers of Campylobacter bacteria, increasing the opportunity for disease via cross-contamination or improperly cooked poultry. Campylobacter species are the major causative agent of foodborne gastrointestinal bacterial infections in the developed world (6, 11, 21). Poultry products are a major source for the introduction of Campylobacter into the food supply (15, 16). Improperly cooked poultry and cross-contamination of other foods by raw poultry are common methods for transmission of Campylobacter to humans (5). However, Campylobacter spp. are nutritionally fastidious organisms that are sensitive to the oxygen levels present in a normal environment (O %) (21). Therefore, Campylobacter appears an unlikely candidate to persist within poultry processing and storage environments at levels sufficient to cause human disease. This conundrum directly leads to a question: what then are the elements that contribute to the ability of Campylobacter to survive through poultry processing and cold storage? To investigate this question, a food-relevant environment consisting of chicken weepage or exudate can be used to perform survival experiments on Campylobacter species. Strains of Campylobacter jejuni and Campylobacter coli were used for the survival studies since these two species are responsible for the vast majority of human cases of campylobacteriosis (20, 28). Chicken exudate is the fluid that seeps out from processed poultry carcasses and is often found to be contaminated with considerable numbers of Campylobacter bacteria. It is comprised of water, blood, fats, and other materials added to the * Mailing address: Eastern Regional Research Center, Microbial Food Safety Research Unit, 600 E. Mermaid Lane, Wyndmoor, PA Phone: (215) Fax: (215) jack.gunther@ars.usda.gov. Published ahead of print on 19 February poultry during processing. Sterilized poultry exudates make for a convenient experimental material that is also relevant to the conditions which Campylobacter will experience as a contaminant of processed poultry (2, 3). Two different types of chicken exudates were collected from commercial producers, one from chickens processed without additives (nonenhanced) and the other from chickens that were treated with a commercial marinade to increase the quality and appeal of the meat at market (enhanced). The commercial poultry marinades contain a significant amount of polyphosphate additives. Polyphosphates comprise a group of food additives that are utilized within poultry processing to enhance the moisture absorbance, color, and flavor and to reduce product shrinkage of poultry (24, 29 32). Polyphosphates have also been shown to have an antimicrobial effect on several different bacterial species (8, 10, 12). The goal of the research was to determine if polyphosphates contribute to the ability of Campylobacter to survive and persist through the supply chain, thus directly increasing the opportunity for Campylobacter-mediated food poisoning of consumers. MATERIALS AND METHODS Bacterial strains. C. coli RM1403, RM4764, and RM1529 and C. jejuni RM1221, RM5032, RM1188, RM1285, RM1449, RM1464, RM3194, and RM1246 strains were kindly provided by Robert Mandrell (USDA, Agricultural Research Service, Western Regional Research Center, Albany, CA). C. jejuni strains and 9368 were kindly provided by Norman J. Stern (USDA, Agricultural Research Service, Richard B. Russell Agricultural Research Center, Athens, GA), and C. jejuni strain was supplied by Patricia Guerry (Naval Medical Research Center, Silver Spring, MD). Strains were cultured from frozen stocks ( 80 C) directly onto brucella (Becton Dickinson, Sparks, MD) agar plates (1.5% agarose). Overnight cultures (O/N) were grown in brucella (Becton 2419

2 2420 GUNTHER APPL. ENVIRON. MICROBIOL. FIG. 1. Three C. coli and 11 C. jejuni strains were incubated for 24 h at 42 C microaerobically. The CFU present in the samples incubated in chicken exudate (light gray bars) or enhanced chicken exudates (dark gray bars) were determined, and the mean value for multiple samples was plotted as a graph along with standard deviations. Sample pairs labeled with different letters were determined to differ with statistical significance (P 0.05) in regards to the number of culturable Campylobacter cells present in each. Dickinson) broth and incubated in a microaerobic chamber (MACS-VA; Don Whitley, United Kingdom) (5% O 2, 10% CO 2, 85% N 2 ) at 42 C. Chicken exudate collection and preparation. The exudates from both enhanced and nonenhanced chicken products were collected at the processing plants of three separate commercial poultry producers prior to packaging. Both types of exudates were initially collected into plastic containers directly off the processing line and from multiple chicken carcasses. The nonenhanced chicken products were chicken carcasses that had been prepared for consumer sale and cleaned and chilled with water only. Enhanced chicken products were chicken carcasses prepared for consumer sale that were cleaned and chilled with water and then treated with a marinade consisting of, in the case of producer 1, a mixture of polyphosphates (1:1 [wt/wt] pentasodium tripolyphosphate and sodium hexametaphosphate; final concentration, 0.5%) and dehydrated chicken broth powder (final concentration, 0.06%) dissolved in a water base and applied within a vacuum tumbler apparatus. Both forms of exudate were initially filtered through cheesecloth to remove large pieces of chicken meat and fat. The exudates were then aliquoted into conveniently sized containers and frozen at 80 C. The frozen aliquots were then irradiated to sterility using a self-contained 137 Cs gamma irradiator (Lockheed Georgia Company, Marietta, GA) at a dose rate of kgy/min (27). The temperature during irradiation was maintained at 0 C through the introduction of gas-phase liquid nitrogen directly into the top of the sample chamber. Conditions for survival experiments. For all survival experiments, with the exception of cold storage experiments (see Fig. 4A and B), 5 l of overnight brucella broth cultures (at concentrations of CFU/ml for C. jejuni strains and CFU/ml for C. coli strains) was used to inoculate 5 ml of the appropriate chicken exudate. For the cold storage experiments (see Fig. 4A and B), the initial inoculants into the exudates from the overnight cultures were 50 l in volume to accommodate the increased experimental periods. The polyphosphate mixture and the dehydrated chicken broth powder were supplied by poultry producer 1. The polyphosphate mixture and dehydrated chicken broth powder were reported by the producer to be used at final concentrations of 0.5% and 0.06%, respectively, and these concentrations were used as a starting point for the experiments presented in this paper. The chicken exudate cultures were incubated under a variety of conditions and for a variety of time periods. Cultures were incubated at 42 C in a microaerobic chamber (described above) whereas incubation at 4 C in a normal atmosphere was accomplished in a refrigerated incubator (Innova 4230; New Brunswick Scientific, Edison, NJ). Regardless of condition or medium, all Campylobacter cultures were incubated statically to replicate commercial conditions as closely as possible. Upon completion of the incubation period, the viable Campylobacter bacteria present in each culture were enumerated using the 6 6 drop plate method with 6 replicates per dilution (7). Statistical analyses. An analysis of variance was performed using the Mixed procedure of the SAS software system to determine the effects and interactions of species and conditions (23). Mean separations were performed using the Bonferroni least significant difference (LSD) technique (19). Analyses were performed separately for grouped C. jejuni strains and grouped C. coli strains. All statistical tests of significance were performed at the P 0.05 level. RESULTS Differences in Campylobacter survival when incubated in chicken exudates versus enhanced chicken exudates. Analyses of several strains of C. coli and C. jejuni incubated in chicken exudates and enhanced chicken exudates showed statistically significant (P 0.05) differences with regard to Campylobacter survival in the two different exudates (Fig. 1). All three of the C. coli strains investigated demonstrated a significantly (P 0.05) greater number of culturable cells present in the chicken exudates from the enhanced chicken products than in the nonenhanced chicken exudates after 24 h in a microaerobic environment at 42 C. Each of the three C. coli strains averaged an 3- to 4-log increase in the number of culturable cells present in enhanced chicken exudates compared with that in nonenhanced chicken exudates. The C. jejuni strains demonstrated a greater range of effects than did the C. coli strains when cultured in the enhanced and nonenhanced chicken exudates. Ten of the 11 C. jejuni strains investigated produced significantly (P 0.05) greater numbers of culturable cells in the chicken exudates from the enhanced chicken products than in the nonenhanced chicken exudates after 24 h in a microaerobic environment at 42 C. Six of the 11 C. jejuni strains exhibited a 2-log increase in culturable cells present in the enhanced chicken exudates over those in the nonenhanced chicken exudates. Of the remaining five strains, two strains demonstrated 1-log increases, two strains showed 1-log increases in culturable cells present in enhanced exudates over those in nonenhanced exudates, and the one remaining strain provided no

3 VOL. 76, 2010 EFFECT OF POLYPHOSPHATES ON CAMPYLOBACTER SURVIVAL 2421 FIG. 2. The resulting mean CFU and standard deviations for C. jejuni strain RM1221 (A) and C. coli strain RM1403 (B) incubated for 24 h at 42 C microaerobically in chicken exudates with varied concentrations of added polyphosphate mixture (1:1 [wt/wt] pentasodium tripolyphosphate and sodium hexametaphosphate). Samples labeled with different letters were determined to differ with statistical significance (P 0.05) from one another in regards to the number of culturable Campylobacter cells present in each. statistically significant survival difference between the two conditions. Effect of polyphosphates on Campylobacter survival. To determine if the effect on Campylobacter survival was due to the polyphosphates (pentasodium tripolyphosphate and sodium hexametaphosphate) or to the dried chicken broth powder, the two materials which compose the enhanced chicken product marinade, they were added individually to exudates derived from nonenhanced chicken products. A representative strain of C. jejuni, RM1221, and one of C. coli, RM1403, were inoculated into the two different exudates to determine the material(s) responsible for the effects observed in Fig. 1. C. jejuni strain RM1221 (Fig. 2A) and C. coli strain RM1403 (Fig. 2B) were observed to be positively affected by the polyphosphate mixture, resulting in significantly (P 0.05) more culturable Campylobacter cells present in the exudates with polyphosphate (0.5% concentration) added than in exudates without polyphosphate after a microaerobic incubation of 24 h at 42 C. The effect of the polyphosphates was also observed to be concentration dependent. FIG. 3. The resulting mean CFU and standard deviations for C. jejuni strain RM1221 incubated for 24 h at 42 C microaerobically in chicken exudates with varied concentrations of added dried chicken broth powder. Samples labeled with different letters were determined to differ with statistical significance (P 0.05) from one another in regards to the number of culturable C. jejuni cells present in each. Effect of dried chicken broth powder on Campylobacter survival. Chicken exudates containing only dried chicken broth powder as an additive demonstrated effects on the C. jejuni strain RM12221 similar to those observed for the chicken exudates containing only polyphosphates. The addition of dried chicken broth powder, 0.06% concentration, resulted in significantly (P 0.05) more culturable C. jejuni cells present in the exudates than in the exudates without dried chicken broth powder when cultures were incubated for 24 h, microaerobically at 42 C (Fig. 3). The observed effects of the dried chicken broth powder were also concentration dependent. However, the dried chicken broth powder demonstrated no positive effects on the culturable cell numbers of C. coli strain RM1403. This lack of positive effect of dried chicken broth powder on C. coli culturable cell numbers was further confirmed in several other C. coli strains (data not shown). Effects of polyphosphates on Campylobacter survival under cold storage conditions. The effects of polyphosphates were also investigated under more realistic food storage conditions: a series of experiments were performed at commercial refrigeration temperature (4 C) and within a normal atmosphere (O 2 content of 21%). For C. jejuni strain RM1221, the enhanced chicken exudates were observed by 168 h postinoculation to have a significantly (P 0.05) larger number of culturable C. jejuni cells than did the nonenhanced chicken exudates (Fig. 4A). Similar results were observed for C. coli strain RM1403 when incubated under the same conditions as those for the C. jejuni strain. Significantly (P 0.05) larger numbers of culturable C. coli cells were present in the enhanced chicken exudates than in the nonenhanced exudates by 168 h postinoculation (Fig. 4B). When the data for the two Campylobacter species were compared over the entire 3-week period, the experiments showed a statistically significant (P 0.05) survival advantage for C. jejuni and C. coli when incubated in enhanced chicken exudate compared with nonenhanced chicken exudate under conditions in which consumers would store chicken intended for consumption.

4 2422 GUNTHER APPL. ENVIRON. MICROBIOL. FIG. 4. C. jejuni strain RM1221 (A) and C. coli strain RM1403 (B) incubated in a normal atmosphere at 4 C for 504 h in chicken exudates derived from chicken products with and without added marinade. Samples were collected at 24, 168, 336, and 504 h after inoculation and used to determine the mean CFU and standard deviations for both Campylobacter species incubated either in chicken exudate (black squares) or in chicken exudate plus marinade (gray triangles) at each time point. Time point sample pairs labeled with an asterisk were determined to differ with statistical significance (P 0.05) in regards to the number of culturable Campylobacter cells present under each condition. Effects of polyphosphates on Campylobacter incubated in laboratory media. It should be noted that both enhanced and nonenhanced chicken exudates do not appear to be good growth media for Campylobacter strains, resulting in moderate growth in two groups of experiments (Fig. 1 and 3) and no growth in three other groups of experiments (Fig. 2, 4, and 5). To observe the effects of polyphosphates on growing Campylobacter strains, a C. jejuni strain and a C. coli strain were incubated with and without 0.5% polyphosphates under conditions that are known to allow for good Campylobacter growth, brucella broth at 42 C in a microaerobic environment. After 24 h under these conditions, the Campylobacter strains were no longer culturable from brucella broth containing the polyphosphates but were readily culturable from the brucella broth alone (data not shown). However, when this experiment was repeated under conditions that would not allow Campylobacter replication (24 h at 4 C, normal atmosphere), there FIG. 5. C. jejuni strain RM1221 and C. coli strain RM1403 incubated for 24 h at 4 C under a normal atmosphere in chicken exudates from chicken products with and without added marinades from poultry producers 2 and 3. The CFU present in the samples incubated in chicken exudates (light gray bars) or chicken exudates enhanced through marinade addition (dark gray bars) to poultry were determined, and the mean value for multiple samples was plotted as a graph along with standard deviations. Sample pairs labeled with different letters were determined to differ with statistical significance (P 0.05) in regards to the number of culturable Campylobacter cells present in each. were statistically equal numbers of culturable Campylobacter cells in cultures of brucella broth plus polyphosphates and in the brucella broth alone (data not shown). Effects on Campylobacter survival of enhanced chicken exudates from additional poultry processors. Each supplier uses a proprietary blend of additives and processing procedures. In an effort to address the variation in processing, we examined nonenhanced and enhanced chicken exudates from two additional poultry processors (processors 2 and 3). C. jejuni strain RM1221 and C. coli strain RM1403 were incubated for 24 h in both types of exudates under food storage conditions (4 C, normal atmosphere). Both Campylobacter strains incubated in the nonenhanced chicken exudates collected from producer 2 yielded a slight statistically significant (P 0.05) increase in the numbers of culturable cells over those in the enhanced exudates from the same producer (Fig. 5). This is directly contrary to the results observed with exudates from producer 1. However, experiments using the enhanced and nonenhanced chicken exudates from processor 3 produced results similar to those seen with producer 1. Both C. jejuni RM1221 and C. coli RM1403 demonstrated significantly (P 0.05) greater numbers of culturable cells present in the enhanced chicken exudates than in the nonenhanced chicken exudates (Fig. 5). It was also observed that the ph differences between the nonenhanced chicken exudates and the enhanced chicken exudates from producers 1 and 3 were more similar, with increases in the ph of the enhanced over the unenhanced exudates of 0.66 and 1.40, respectively (Table 1). Conversely, for the exudates from producer 2, there was a 0.10 decrease in ph of the enhanced exudate compared to that of the unenhanced exudate.

5 VOL. 76, 2010 EFFECT OF POLYPHOSPHATES ON CAMPYLOBACTER SURVIVAL 2423 Poultry producer TABLE 1. phs of chicken exudates by producer Chicken exudate DISCUSSION ph Chicken exudate plus marinade Campylobacter bacteria are responsible for the greatest percentage of cases of food-borne illness caused by bacteria in the developed world (6, 11, 21). This study examines if polyphosphates added to poultry exudates during processing are a factor in enhancing Campylobacter persistence in food in sufficient numbers to cause disease. In this study, evidence that the processing additives known as polyphosphates, and, possibly to a lesser extent, dehydrated chicken broth powder, are factors that contribute to the ability of Campylobacter to persist in hostile environments is presented. While some research on food pathogens often uses artificial media and less-than-relevant incubation conditions; this paper presents effects that were observed for Campylobacter incubated in chicken exudates, the organism s common environment, under normal food storage conditions, thus making the results more applicable to food safety concerns (2, 3, 26). However, these results are surprising given previous research that had shown that polyphosphates exhibited antimicrobial action against many food pathogens (1, 8, 10, 12). Previous research also suggested that a possible mode of action for polyphosphate-mediated bacterial inhibition was the ability of polyphosphates to chelate divalent cations needed for bacterial cell wall formation (17). The nature of the proposed antimicrobial effect of polyphosphates would make them decidedly less effective against slow-growing or nondividing cells (18). This conclusion is supported by the observation that polyphosphates appear lethal only against rapidly growing Campylobacter cells. However, this research further demonstrates a new observation that slow-growing or nondividing Campylobacter cells in chicken exudates are not simply resistant to the antimicrobial effects of polyphosphates; rather, polyphosphates serve to enhance the survival of these cells by some unknown mode of action. Since polyphosphates can act as chelating agents, it is conceivable that the polyphosphates could be chelating an agent normally found in chicken exudate that is potentially harmful to Campylobacter. Alternatively, it could be the subtle shifts in the exudates ph that were caused by the addition of the phosphate-containing marinades during processing which are responsible for the enhanced survival. It was observed that the addition of marinades shifted the ph of the exudates from producer 1 from 5.76 to 6.42 and the ph of the exudates from producer 3 from 6.00 to 7.40 (Table 1). These changes in ph may be slight, but both changes move the exudate toward the optimal ph range for C. jejuni growth of 6.5 to 7.5 (9, 25). Conversely, the addition of marinades by producer 2 resulted in a decrease of exudates ph from 6.80 to 6.70, most likely resulting from the preexisting elevated ph levels of the nonenhanced exudates. This suggests a possible reason why the addition of polyphosphate-containing marinade did not increase Campylobacter survival in the case of producer 2. Therefore, the ph increases created by the addition of phosphatecontaining marinades may be responsible for the enhanced survival of Campylobacter in chicken exudates. Additionally, it should be noted that there are several different inorganic polyphosphates considered safe for use in foods, and their effects on solution ph differ significantly. It may therefore be possible that the positive effects on Campylobacter survival are not a universal effect of all polyphosphates. Therefore, the beginning of any search for the mode of action of the enhanced Campylobacter survival may lie in more fully determining if all commonly used polyphosphates affect the ph of chicken exudates and Campylobacter survival to the same degree. In this way, it may be possible to more safely use certain polyphosphates for the benefits that they supply in food processing while avoiding other polyphosphates that have the potential of enhancing the survival of Campylobacter spp. within a food environment. Finally, this paper shows how the addition of polyphosphates to processed chicken products has the potential to increase the survival of C. jejuni and C. coli strains in these products by 2 logs. The infectious dose for C. jejuni has been shown to be as little as 500 organisms; while it has been demonstrated that on average 5 logs of Campylobacter can be easily recovered from washes of whole chicken carcasses (4, 13, 14). Additional research has shown that increases in Campylobacter survival in the range of 2 logs are expected to cause a disproportionally greater health risk when those chicken products reach consumers homes (22). The same study showed that a 2-log decrease in Campylobacter numbers on a chicken carcass is predicted to result in a 30-fold reduction in incidents of campylobacteriosis. Such predictions make understanding the effects that polyphosphates have on bacterial survival during food processing and storage imperative. 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