Neutralization of residual antimicrobial processing chemicals in broiler carcass rinse for improved detection of Campylobacter1

Neutralization of residual antimicrobial processing chemicals in broiler carcass rinse for... SUMMARY The USDA-Food Safety and Inspection Service (FSIS) has established pathogen reduction performance standards for Campylobacter on broiler carcasses. Processors may apply antimicrobial processing aids as a spray or immersion to lower contamination on carcasses. In the United States, broiler carcasses are generally sampled by whole carcass rinse and the potential exists for residual levels of antimicrobial processing aid to be carried over into the rinsate. It has been shown that, if un-mitigated, such carryover can interfere with the detection of Salmonella. In the current study, we demonstrate that unmitigated carryover of antimicrobial treatment also can interfere with the detection and recovery of Campylobacter in broiler carcass rinse samples. We tested traditional buffered peptone water and found that it did not offer enough neutralizing capability to counteract residual antimicrobial activity of some post-chill processing aids (peroxyacetic acid, cetylpyridinium chloride, acidified sodium chloride, or a blend of acids) to allow full recovery of Campylobacter. A recently reported formulation for a neutralizing buffered peptone water (currently being used by FSIS) outperformed the traditional carcass rinse medium and allowed significantly improved recovery of Campylobacter even in the presence of 3 of the 4 tested antimicrobial processing aids. Performance of the new carcass rinse medium with the fourth antimicrobial processing aid (acidified sodium chloride) was not different from the traditional formulation. Neutralizing buffered peptone water represents a significant improvement in the broiler carcass rinse method for detection of Campylobacter. DESCRIPTION OF PROBLEM Campylobacter is a human pathogen and a leading cause of bacterial foodborne illness in the United States [1]. Campylobacter can be readily found in association with live broilers and broiler meat products [2, 3]. The ability of commercial broiler processors to meet the established Campylobacter performance standard is assessed by the USDA-Food Safety and Inspection Service (FSIS). A broiler processor that is found to have a positive rate exceeding the performance standard for this organism on fully processed carcasses or parts may face regulatory action [4]. A variety of chemicals are approved for use as antimicrobial broiler processing aids to control human pathogens such as Campylobacter and Salmonella [5]. Processing aid chemicals are generally diluted with water and may be applied by immersion or spray before regulatory verification samples are collected. In earlier work, the potential for water applied to a chilled broiler carcass to remain with the carcass after treatment was examined [6]. Bourassa et al. [6] reported that carryover of antimicrobial treatment fluid was possible, and the volume can vary depending on application method, drip time, and carcass orientation. Subsequently, research was conducted to examine the potential for carryover of bactericidal effect during transport of broiler carcass rinse samples prior to commencement of culture activities. Operating in a close to worst-case scenario of allowable concentration and carryover volume, Gamble et al. [7] found that several processing aids—peroxyacetic acid (PAA), cetylpyridinium chloride (CPC), acidified sodium chloride (ASC), and a blend of acids (BOA)—interfered with detection of Salmonella in broiler carcass rinsate. These findings raise the concern of false negative results. Gamble et al. [8] developed a formula for a broiler carcass rinse medium that effectively neutralizes carryover anti-Salmonella properties of PAA, CPC, ASC, and BOA. This formula is based on buffered peptone water (BPW) with the addition of soy lecithin, sodium thiosulfate, and sodium bicarbonate and shows marked improvement for recovery of Salmonella in the presence of residual antimicrobial chemicals [8]. The new carcass rinse medium has been adopted by FSIS for regulatory broiler carcass sampling and is called neutralizing-BPW (n-BPW) [9]. It is unknown, however, how n-BPW may affect detection and recovery of Campylobacter from broiler carcass rinses with residual antimicrobial processing aids. The objective of the current study was to compare recovery of Campylobacter from broiler carcass rinsate made with BPW to rinsate made with n-BPW in the presence of residual activity from PAA, CPC, ASC, and BOA. MATERIALS AND METHODS Carcass Rinse with Residual Antimicrobial Chemicals On each of 3 replicate sample d, 18 eviscerated broiler carcasses were collected from the line in a commercial broiler slaughter plant. Carcasses were removed from the shackle line immediately prior to application of the inside/outside washing system. Carcasses were transported warm to the laboratory and within 30 min were subjected to a mechanized whole carcass rinse procedure. Nine carcasses were rinsed each in 400 mL of BPW, and 9 were rinsed in a n-BPW as described by Gamble et al. [8]. All rinsates were collected from each group of 9 carcasses, and pooled into separate vessels for BPW and n-BPW. Rinsate was held on ice until antimicrobial chemicals were introduced to simulate residual levels on post-chill broiler carcasses. A prepared stock solution of each processing aid (PAA, CPC, ASC, and BOA) had been prepared at maximum allowable concentration as previously described [8] in order to simulate carryover potential from a post-chill application of antimicrobial chemicals. Briefly, PAA stock solution was prepared by diluting 5.13 mL 39% PAA in acetic acid [10] to 1 L using deionized water (dH20) for a final concentration of 2,000 ppm at pH 3.00; CPC stock solution was prepared by dissolving 8.0 g CPC [10] and 12.0 g of propylene glycol [10] in 1 L of dH2O with a final concentration of 8,000 ppm CPC at pH 6.55; ASC stock solution was prepared by dissolving 1.2 g of sodium chlorite [11] in 250 mL dH2O and adding 750 mL of a 0.053 M citric acid solution for a final concentration of 1,200 ppm sodium chlorite at a pH of 2.3; BOA stock solution was prepared by diluting 5.0 mL of 10 M hydrochloric acid to 450 mL with dH2O followed by addition of 15 g citric acid and further dilution using dH2O to a final pH of 1.0. As previously reported [8], the concentration of antimicrobial chemicals in each stock solution was verified with chemical analysis [12]. In previous work, we determined that carcasses treated by immersion dip in a liquid could result in carryover of 60 mL of the immersion liquid to a whole carcass rinse bag, which would normally hold 400 mL of rinse medium [6, 7]. For the purpose of the current study, we diluted 30 mL of each stock solution to 200 mL using carcass rinse with both BPW and n-BPW, thereby simulating a worst-case scenario of 60 mL in a full 400 mL carcass rinse. Untreated control samples (CON) were prepared by diluting 30 mL of dH2O to 200 mL with rinse. Triplicate samples were prepared in each rinse medium (BPW and n-BPW) for each treatment (PAA, CPC, ASC, BOA, and CON). Prepared rinse samples with residual levels of antimicrobial chemicals were held on ice until inoculated within 30 minutes. The pH of each rinsate was measured [13] and recorded upon initial mixing and again after 24 h of cold storage. Campylobacter Inoculation and Recovery A gentamicin resistant strain of C. coli (Ccgr) originally isolated from processed poultry [14] was used as a marker strain for inoculation to prevent interference by naturally occurring Campylobacter. Inocula were prepared for each replication. Tubes containing 10 mL Campylobacter enrichment broth (CEB), Bolton formulation [15], were inoculated from a 24 h culture of Ccgr. Tubes were incubated 24 h at 42°C in a re-sealable bag flushed with microaerobic gas (5% O2, 10% CO2, and 85% N2). Each mL of 24 h growth in CEB had approximately 108Campylobacter CFU [16]; incubated CEB was serially diluted in PBS and used to inoculate prepared rinsate with addition of residual antimicrobial chemicals to a final cell concentration of approximately 106 CFU per mL of rinse sample. Inoculum was verified by plate count on Campy-Cefex agar (CCAg) [17] with the addition of 200 ppm gentamicin [10]; plates were incubated at 42°C for 48 h in re-sealable bag flushed with microaerobic gas. Characteristic Campylobacter colonies were counted, and the CFU Campylobacter/mL of inoculum was calculated. Following inoculation, all rinse samples were stored at 4°C for 24 h to simulate cold shipping from a processing plant sample site to a remote laboratory. Rinses were examined after 24 h cold storage to compare Campylobacter numbers per mL of control (no residual antimicrobial) to that detected per mL of treated (antimicrobial residual) samples. Serial dilutions of all samples were plated onto the surface of CCAg plates, which were incubated 48 h at 42°C in re-sealable bags flushed with microaerobic gas. Characteristic colonies were counted and confirmed as Campylobacter by observation of cellular morphology and motility under phase contrast microscopy. Statistical Analysis Three replications of the experiment were conducted, each with triplicate samples for each residual intervention treatment (n = 9). All colony counts were log10 transformed, and geometric means were subjected to a general linear model (GLM). Means were further separated by Tukey's Honest Significant Difference (HSD) test. Significance was assigned at P < 0.05. The pH data were collected for every rinsate immediately after mixing and after 24 h of cold storage. The pH data were subjected to GLM and mean separation by Tukey's HSD; significance was assigned at P < 0.05. All statistical analyses were conducted with Statistica 12 [18]. RESULTS AND DISCUSSION The pH values of prepared BPW and n-BPW rinsate with and without residual antimicrobial chemicals are presented in Table 1. The pH of BPW without antimicrobial residue was near neutral and ideal for recovery of bacteria. However, the addition of potential carryover volumes of acidic antimicrobial processing aids resulted in lower pH values. The most extreme drop in pH was noted for the BOA treatment. This pH (below 4) was maintained during 24 h cold storage and would likely prove problematic for recovery of Campylobacter [19]. The pH of n-BPW rinsates was significantly (P < 0.05) higher than BPW in all cases, even when no residual antimicrobial chemical was present. Generally, the pH of n-BPW rinsates were within about 1 pH unit of neutral; in the case of BOA, the pH of n-BPW rinsates was far more hospitable for Campylobacter than was the pH in rinsates prepared with BPW. In earlier work, the pH of n-BPW with the same residual antimicrobials allowed successful recovery of Salmonella [8]. Overall, n-BPW outperforms traditional BPW for controlling pH of broiler carcass rinsate when residual acidic antimicrobial processing aids are present. Table 1. pH ± 95% confidence interval of broiler carcass rinsate with addition of simulated residual antimicrobial at initial mixing and after 24 h 4°C storage (n = 9).1 pH initial pH 24 h Residual2 BPW3 n-BPW4 BPW n-BPW None 7.09 ± 0.06F 7.96 ± 0.06B 7.14 ± 0.10F 8.13 ± 0.12A,B ASC 6.26 ± 0.08J 7.36 ± 0.06E 6.28 ± 0.11I,J 7.62 ± 0.17C,D CPC 7.11 ± 0.06F 8.00 ± 0.06A,B 7.12 ± 0.10F 8.18 ± 0.12A PAA 6.49 ± 0.07 G,H,I 7.45 ± 0.05D,E 6.50 ± 0.10 G,H 7.70 ± 0.11C BOA 3.54 ± 0.09K 6.36 ± 0.09H,I,J 3.55 ± 0.10K 6.66 ± 0.13G pH initial pH 24 h Residual2 BPW3 n-BPW4 BPW n-BPW None 7.09 ± 0.06F 7.96 ± 0.06B 7.14 ± 0.10F 8.13 ± 0.12A,B ASC 6.26 ± 0.08J 7.36 ± 0.06E 6.28 ± 0.11I,J 7.62 ± 0.17C,D CPC 7.11 ± 0.06F 8.00 ± 0.06A,B 7.12 ± 0.10F 8.18 ± 0.12A PAA 6.49 ± 0.07 G,H,I 7.45 ± 0.05D,E 6.50 ± 0.10 G,H 7.70 ± 0.11C BOA 3.54 ± 0.09K 6.36 ± 0.09H,I,J 3.55 ± 0.10K 6.66 ± 0.13G 1Triplicate samples in each of 3 replicate trials. 2Residual intervention: ASC: acidified sodium chloride; CPC: cetylpyridinium chloride; PAA peroxyacetic acid, BOA: blend of acid. 3Buffered peptone water carcass rinse. 4Neutralizing buffered peptone water rinse. A–KValues with no like superscripts are significantly different by Tukey's Honest Significant Difference test (P < 0.05). View Large Table 1. pH ± 95% confidence interval of broiler carcass rinsate with addition of simulated residual antimicrobial at initial mixing and after 24 h 4°C storage (n = 9).1 pH initial pH 24 h Residual2 BPW3 n-BPW4 BPW n-BPW None 7.09 ± 0.06F 7.96 ± 0.06B 7.14 ± 0.10F 8.13 ± 0.12A,B ASC 6.26 ± 0.08J 7.36 ± 0.06E 6.28 ± 0.11I,J 7.62 ± 0.17C,D CPC 7.11 ± 0.06F 8.00 ± 0.06A,B 7.12 ± 0.10F 8.18 ± 0.12A PAA 6.49 ± 0.07 G,H,I 7.45 ± 0.05D,E 6.50 ± 0.10 G,H 7.70 ± 0.11C BOA 3.54 ± 0.09K 6.36 ± 0.09H,I,J 3.55 ± 0.10K 6.66 ± 0.13G pH initial pH 24 h Residual2 BPW3 n-BPW4 BPW n-BPW None 7.09 ± 0.06F 7.96 ± 0.06B 7.14 ± 0.10F 8.13 ± 0.12A,B ASC 6.26 ± 0.08J 7.36 ± 0.06E 6.28 ± 0.11I,J 7.62 ± 0.17C,D CPC 7.11 ± 0.06F 8.00 ± 0.06A,B 7.12 ± 0.10F 8.18 ± 0.12A PAA 6.49 ± 0.07 G,H,I 7.45 ± 0.05D,E 6.50 ± 0.10 G,H 7.70 ± 0.11C BOA 3.54 ± 0.09K 6.36 ± 0.09H,I,J 3.55 ± 0.10K 6.66 ± 0.13G 1Triplicate samples in each of 3 replicate trials. 2Residual intervention: ASC: acidified sodium chloride; CPC: cetylpyridinium chloride; PAA peroxyacetic acid, BOA: blend of acid. 3Buffered peptone water carcass rinse. 4Neutralizing buffered peptone water rinse. A–KValues with no like superscripts are significantly different by Tukey's Honest Significant Difference test (P < 0.05). View Large Recovery of inoculated Campylobacter from BPW and n-BPW carcass rinsates with and without addition of residual post-chill antimicrobial processing aids is presented in Table 2. All rinsate samples were inoculated with approximately 6.0 log CFU/mL, which was verified by plate count. After 24 h cold storage at 4°C, the control samples, without antimicrobial residue, had 5.3 and 5.0 log CFU/mL in BPW and n-BPW, respectively. This finding indicates a loss of about 1 log CFU/mL Campylobacter due just to 24 h cold storage. No Campylobacter was recovered from BPW rinsate samples with residual levels of PAA and BOA. In earlier work, we found that Salmonella also was inhibited by PAA in a BPW broiler carcass rinse [7]; however, in that previous study, residual BOA did not inhibit Salmonella to the same extent as it did Campylobacter in the current study. The low pH of carcass rinse with BOA seems to be more damaging to Campylobacter than Salmonella. When those same antimicrobial residues were present in n-BPW, Campylobacter recovery was not impeded. The same number of Campylobacter was found in n-BPW samples with PAA and BOA as in n-BPW samples with no antimicrobial residue. This suggests that the increased buffering capacity of n-BPW controlled the pH of carcass rinse with BOA, such that Campylobacter could survive cold storage. Residual PAA in n-BPW carcass rinsate was neutralized, allowing Campylobacter to survive. These findings are similar to what was reported for Salmonella, which also survived well in similar n-BPW carcass rinse [8]. Table 2. Mean log CFU Campylobacter per mL rinsate ± 95% confidence interval after 24 h 4°C storage with simulated residual antimicrobial chemical (n = 9).1 Log CFU Campylobacter per mL Residual2 BPW3 n-BPW4 None 5.3 ± 0.2A 5.0 ± 0.2A,B ASC 1.2 ± 0.7C 1.1 ± 0.3C CPC 0.1 ± 0.1D 4.4 ± 0.7B PAA 0.0 ± 0.0D 5.1 ± 0.2A BOA 0.0 ± 0.0D 5.2 ± 0.3A Log CFU Campylobacter per mL Residual2 BPW3 n-BPW4 None 5.3 ± 0.2A 5.0 ± 0.2A,B ASC 1.2 ± 0.7C 1.1 ± 0.3C CPC 0.1 ± 0.1D 4.4 ± 0.7B PAA 0.0 ± 0.0D 5.1 ± 0.2A BOA 0.0 ± 0.0D 5.2 ± 0.3A 1Triplicate samples in each of 3 replicate trials. 2Residual intervention: ASC: acidified sodium chloride; CPC: cetylpyridinium chloride; PAA peroxyacetic acid, BOA: blend of acid. 3Buffered peptone water carcass rinse. 4Neutralizing buffered peptone water rinse. A–DValues with different superscripts are significantly different by Tukey's Honest Significant Difference test ( P< 0.05). View Large Table 2. Mean log CFU Campylobacter per mL rinsate ± 95% confidence interval after 24 h 4°C storage with simulated residual antimicrobial chemical (n = 9).1 Log CFU Campylobacter per mL Residual2 BPW3 n-BPW4 None 5.3 ± 0.2A 5.0 ± 0.2A,B ASC 1.2 ± 0.7C 1.1 ± 0.3C CPC 0.1 ± 0.1D 4.4 ± 0.7B PAA 0.0 ± 0.0D 5.1 ± 0.2A BOA 0.0 ± 0.0D 5.2 ± 0.3A Log CFU Campylobacter per mL Residual2 BPW3 n-BPW4 None 5.3 ± 0.2A 5.0 ± 0.2A,B ASC 1.2 ± 0.7C 1.1 ± 0.3C CPC 0.1 ± 0.1D 4.4 ± 0.7B PAA 0.0 ± 0.0D 5.1 ± 0.2A BOA 0.0 ± 0.0D 5.2 ± 0.3A 1Triplicate samples in each of 3 replicate trials. 2Residual intervention: ASC: acidified sodium chloride; CPC: cetylpyridinium chloride; PAA peroxyacetic acid, BOA: blend of acid. 3Buffered peptone water carcass rinse. 4Neutralizing buffered peptone water rinse. A–DValues with different superscripts are significantly different by Tukey's Honest Significant Difference test ( P< 0.05). View Large Residual CPC in BPW carcass rinsate severely impacted recovery of Campylobacter. However, residual CPC in n-BPW carcass rinses was far less disruptive, allowing close to the same recovery of Campylobacter as noted with the antimicrobial free control. These findings are similar to what was noted for Salmonella with residual CPC in earlier work with BPW [7] and n-BPW [8]. When residual ASC was present in BPW carcass rinse, Campylobacter was detected but at significantly lower numbers than found in the antimicrobial free control rinsate. When residual ASC was present in n-BPW carcass rinse, Campylobacter was still negatively affected; no difference in recovery was noted compared to traditional BPW. This specific finding is different from what we had previously noted with Salmonella. Salmonella was readily recovered in n-BPW carcass rinse, even in the presence of ASC [8]. There may be unknown factors other than the neutralization of pH that are important for Campylobacter recovery. More research is planned to identify such factors and further improve n-BPW for optimal recovery of Campylobacter in the presence of residual ASC. CONCLUSIONS AND APPLICATIONS Traditional broiler carcass rinse with BPW is not completely adequate to recover Campylobacter in the presence of simulated worst-case concentration of residual post-chill antimicrobial processing aid. A new formulation of rinse medium (n-BPW) is significantly more effective than traditional BPW for recovery of Campylobacter from broiler carcass rinses with residual CPC, PAA, and BOA. Four to five log CFU/mL improvement was noted, representing more than a 99.99% improvement. No difference in Campylobacter recovery was noted in n-BPW broiler carcass rinse with residual ASC compared to traditional BPW carcass rinse. Footnotes 1 Mention of trade names or commercial products in the publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. Primary Audience: Poultry Processors, Regulatory Officials, Poultry Microbiology Researchers REFERENCES AND NOTES 1. https://www.cdc.gov/media/releases/2017/p0420-campylobacter-salmonella.html . 2. Berrang M. E. , Meinersmann R. J. , Ladely S. R. , Cox N. A. . 2016 . Campylobacter detection in broiler ceca at processing – A three year 211 flock survey . J. Appl. Poult. Res. 26 : 154 – 158 . 3. Berrang M. E. , Oakley B. B. , Meinersmann R. J. . 2016 . Detection of Campylobacter on the outer surface of retail broiler meat packages and from product within . Food Prot. Trends . 36 : 176 – 182 . 4. https://www.gpo.gov/fdsys/pkg/FR-2016-02-11/pdf/2016-02586.pdf . 5. https://www.fsis.usda.gov/wps/wcm/connect/bab10e09-aefa-483b-8be8-809a1f051d4c/7120.1.pdf?MOD=AJPERES . 6. Bourassa D. V. , Wilson K. M. , Bartenfeld L. N. , Harris C. E. , Howard A. K. , Ingram K. D. , Hinton A. Jr. , Adams E. S. , Berrang M. E. , Feldner P. W. , Gamble G. R. , Frye J. G. , Johnston J. J. , Buhr R. J. . 2016 . Carcass orientation and drip time affect potential surface water carryover for broiler carcasses subjected to a post-chill water dip or spray . Poult. Sci. 96 : 241 – 245 . Google Scholar CrossRef Search ADS PubMed 7. Gamble G. R. , Berrang M. E. , Buhr R. J. , Hinton A. Jr. , Bourassa D. V. , Johnston J. J. , Ingram K. D. , Adams E. S. , Feldner P. W. . 2016 . Effect of simulated sanitizer carry-over on recovery of Salmonella from broiler carcass rinsates . J. Food Prot. 79 : 710 – 714 . Google Scholar CrossRef Search ADS PubMed 8. Gamble G. R. , Berrang M. E. , Buhr R. J. , Hinton A. Jr. , Bourassa D. V. , Ingram K. D. , Adams E. S. , Feldner P. W. , Johnston J.J. . 2017 . Neutralization of unintended antimicrobial carry-over for optimal recovery of Salmonella from broiler carcass rinse samples . J. Food Prot. 80 : 685 – 691 . Google Scholar CrossRef Search ADS PubMed 9. https://www.fsis.usda.gov/wps/wcm/connect/2cb982e0-625c-483f-9f50-6f24bc660f33/41-16.pdf?MOD=AJPERES . 10. Sigma Aldrich, St. Louis, MO . 11. Pfaltz and Bauer, Waterbury, CT . 12. Chemical analyses for verification of antimicrobial concentration. CPC: verified by titration based on polyvinylsulfate with toluidine blue indicator (Chemetrics, Midland VA). PAA: verified by iodine-diethylphenylenediamine coloimetric measurement (Chemetrics). ASC: verified by anion chromatography using an ICS-3000 (Dionex, Sunnyvale, CA) . 13. Thermo Scientific, Beverly, MA . 14. Cox N. A. , Richardson L. J. , Berrang M. E. , Fedorka-Cray P. J. , Buhr R. J. . 2009 . Campylobacter coli naturally resistant to elevated levels of gentamicin as a marker strain in poultry research . J. Food Prot. 72 : 1288 – 1292 . Google Scholar CrossRef Search ADS PubMed 15. Neogen Corp., Lansing, MI . 16. Dacosta K. J. , Berrang M. E. , Meinersmann R. J. , Cox N. A. , Knapp S. W. . 2017 . Cell concentration of Campylobacter coli/jejuni in 24 hour broth culture . Proceedings of the Annual Meeting of Southern Poultry Science Society. Atlanta GA Jan . 30 – 31 . 17. Stern N. J. , Wojton B. , Kwiatek K. . 1992 . A differential selective medium and dry ice generated atmosphere for recovery of Campylobacter jejuni . J. Food Prot. 55 : 514 – 517 . Google Scholar CrossRef Search ADS 18. StatSoft, Tulsa, OK . 19. Shaheen B. W. , Miller M. E. , Oyarzabal O. A. . 2007 . In vitro survival at low pH and acid adaptation response of Campylobacter jejuni and Campylobacter coli . J. Food Safety 27 : 326 – 343 . Google Scholar CrossRef Search ADS Acknowledgments The authors wish to acknowledge expert technical assistance by Eric S. Adams and Peggy W. Feldner. Published by Oxford University Press on behalf of Poultry Science Association 2018. This work is written by (a) US Government employee(s) and is in the public domain in the US. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Applied Poultry Research Oxford University Press

Neutralization of residual antimicrobial processing chemicals in broiler carcass rinse for improved detection of Campylobacter1

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Abstract

SUMMARY The USDA-Food Safety and Inspection Service (FSIS) has established pathogen reduction performance standards for Campylobacter on broiler carcasses. Processors may apply antimicrobial processing aids as a spray or immersion to lower contamination on carcasses. In the United States, broiler carcasses are generally sampled by whole carcass rinse and the potential exists for residual levels of antimicrobial processing aid to be carried over into the rinsate. It has been shown that, if un-mitigated, such carryover can interfere with the detection of Salmonella. In the current study, we demonstrate that unmitigated carryover of antimicrobial treatment also can interfere with the detection and recovery of Campylobacter in broiler carcass rinse samples. We tested traditional buffered peptone water and found that it did not offer enough neutralizing capability to counteract residual antimicrobial activity of some post-chill processing aids (peroxyacetic acid, cetylpyridinium chloride, acidified sodium chloride, or a blend of acids) to allow full recovery of Campylobacter. A recently reported formulation for a neutralizing buffered peptone water (currently being used by FSIS) outperformed the traditional carcass rinse medium and allowed significantly improved recovery of Campylobacter even in the presence of 3 of the 4 tested antimicrobial processing aids. Performance of the new carcass rinse medium with the fourth antimicrobial processing aid (acidified sodium chloride) was not different from the traditional formulation. Neutralizing buffered peptone water represents a significant improvement in the broiler carcass rinse method for detection of Campylobacter. DESCRIPTION OF PROBLEM Campylobacter is a human pathogen and a leading cause of bacterial foodborne illness in the United States [1]. Campylobacter can be readily found in association with live broilers and broiler meat products [2, 3]. The ability of commercial broiler processors to meet the established Campylobacter performance standard is assessed by the USDA-Food Safety and Inspection Service (FSIS). A broiler processor that is found to have a positive rate exceeding the performance standard for this organism on fully processed carcasses or parts may face regulatory action [4]. A variety of chemicals are approved for use as antimicrobial broiler processing aids to control human pathogens such as Campylobacter and Salmonella [5]. Processing aid chemicals are generally diluted with water and may be applied by immersion or spray before regulatory verification samples are collected. In earlier work, the potential for water applied to a chilled broiler carcass to remain with the carcass after treatment was examined [6]. Bourassa et al. [6] reported that carryover of antimicrobial treatment fluid was possible, and the volume can vary depending on application method, drip time, and carcass orientation. Subsequently, research was conducted to examine the potential for carryover of bactericidal effect during transport of broiler carcass rinse samples prior to commencement of culture activities. Operating in a close to worst-case scenario of allowable concentration and carryover volume, Gamble et al. [7] found that several processing aids—peroxyacetic acid (PAA), cetylpyridinium chloride (CPC), acidified sodium chloride (ASC), and a blend of acids (BOA)—interfered with detection of Salmonella in broiler carcass rinsate. These findings raise the concern of false negative results. Gamble et al. [8] developed a formula for a broiler carcass rinse medium that effectively neutralizes carryover anti-Salmonella properties of PAA, CPC, ASC, and BOA. This formula is based on buffered peptone water (BPW) with the addition of soy lecithin, sodium thiosulfate, and sodium bicarbonate and shows marked improvement for recovery of Salmonella in the presence of residual antimicrobial chemicals [8]. The new carcass rinse medium has been adopted by FSIS for regulatory broiler carcass sampling and is called neutralizing-BPW (n-BPW) [9]. It is unknown, however, how n-BPW may affect detection and recovery of Campylobacter from broiler carcass rinses with residual antimicrobial processing aids. The objective of the current study was to compare recovery of Campylobacter from broiler carcass rinsate made with BPW to rinsate made with n-BPW in the presence of residual activity from PAA, CPC, ASC, and BOA. MATERIALS AND METHODS Carcass Rinse with Residual Antimicrobial Chemicals On each of 3 replicate sample d, 18 eviscerated broiler carcasses were collected from the line in a commercial broiler slaughter plant. Carcasses were removed from the shackle line immediately prior to application of the inside/outside washing system. Carcasses were transported warm to the laboratory and within 30 min were subjected to a mechanized whole carcass rinse procedure. Nine carcasses were rinsed each in 400 mL of BPW, and 9 were rinsed in a n-BPW as described by Gamble et al. [8]. All rinsates were collected from each group of 9 carcasses, and pooled into separate vessels for BPW and n-BPW. Rinsate was held on ice until antimicrobial chemicals were introduced to simulate residual levels on post-chill broiler carcasses. A prepared stock solution of each processing aid (PAA, CPC, ASC, and BOA) had been prepared at maximum allowable concentration as previously described [8] in order to simulate carryover potential from a post-chill application of antimicrobial chemicals. Briefly, PAA stock solution was prepared by diluting 5.13 mL 39% PAA in acetic acid [10] to 1 L using deionized water (dH20) for a final concentration of 2,000 ppm at pH 3.00; CPC stock solution was prepared by dissolving 8.0 g CPC [10] and 12.0 g of propylene glycol [10] in 1 L of dH2O with a final concentration of 8,000 ppm CPC at pH 6.55; ASC stock solution was prepared by dissolving 1.2 g of sodium chlorite [11] in 250 mL dH2O and adding 750 mL of a 0.053 M citric acid solution for a final concentration of 1,200 ppm sodium chlorite at a pH of 2.3; BOA stock solution was prepared by diluting 5.0 mL of 10 M hydrochloric acid to 450 mL with dH2O followed by addition of 15 g citric acid and further dilution using dH2O to a final pH of 1.0. As previously reported [8], the concentration of antimicrobial chemicals in each stock solution was verified with chemical analysis [12]. In previous work, we determined that carcasses treated by immersion dip in a liquid could result in carryover of 60 mL of the immersion liquid to a whole carcass rinse bag, which would normally hold 400 mL of rinse medium [6, 7]. For the purpose of the current study, we diluted 30 mL of each stock solution to 200 mL using carcass rinse with both BPW and n-BPW, thereby simulating a worst-case scenario of 60 mL in a full 400 mL carcass rinse. Untreated control samples (CON) were prepared by diluting 30 mL of dH2O to 200 mL with rinse. Triplicate samples were prepared in each rinse medium (BPW and n-BPW) for each treatment (PAA, CPC, ASC, BOA, and CON). Prepared rinse samples with residual levels of antimicrobial chemicals were held on ice until inoculated within 30 minutes. The pH of each rinsate was measured [13] and recorded upon initial mixing and again after 24 h of cold storage. Campylobacter Inoculation and Recovery A gentamicin resistant strain of C. coli (Ccgr) originally isolated from processed poultry [14] was used as a marker strain for inoculation to prevent interference by naturally occurring Campylobacter. Inocula were prepared for each replication. Tubes containing 10 mL Campylobacter enrichment broth (CEB), Bolton formulation [15], were inoculated from a 24 h culture of Ccgr. Tubes were incubated 24 h at 42°C in a re-sealable bag flushed with microaerobic gas (5% O2, 10% CO2, and 85% N2). Each mL of 24 h growth in CEB had approximately 108Campylobacter CFU [16]; incubated CEB was serially diluted in PBS and used to inoculate prepared rinsate with addition of residual antimicrobial chemicals to a final cell concentration of approximately 106 CFU per mL of rinse sample. Inoculum was verified by plate count on Campy-Cefex agar (CCAg) [17] with the addition of 200 ppm gentamicin [10]; plates were incubated at 42°C for 48 h in re-sealable bag flushed with microaerobic gas. Characteristic Campylobacter colonies were counted, and the CFU Campylobacter/mL of inoculum was calculated. Following inoculation, all rinse samples were stored at 4°C for 24 h to simulate cold shipping from a processing plant sample site to a remote laboratory. Rinses were examined after 24 h cold storage to compare Campylobacter numbers per mL of control (no residual antimicrobial) to that detected per mL of treated (antimicrobial residual) samples. Serial dilutions of all samples were plated onto the surface of CCAg plates, which were incubated 48 h at 42°C in re-sealable bags flushed with microaerobic gas. Characteristic colonies were counted and confirmed as Campylobacter by observation of cellular morphology and motility under phase contrast microscopy. Statistical Analysis Three replications of the experiment were conducted, each with triplicate samples for each residual intervention treatment (n = 9). All colony counts were log10 transformed, and geometric means were subjected to a general linear model (GLM). Means were further separated by Tukey's Honest Significant Difference (HSD) test. Significance was assigned at P < 0.05. The pH data were collected for every rinsate immediately after mixing and after 24 h of cold storage. The pH data were subjected to GLM and mean separation by Tukey's HSD; significance was assigned at P < 0.05. All statistical analyses were conducted with Statistica 12 [18]. RESULTS AND DISCUSSION The pH values of prepared BPW and n-BPW rinsate with and without residual antimicrobial chemicals are presented in Table 1. The pH of BPW without antimicrobial residue was near neutral and ideal for recovery of bacteria. However, the addition of potential carryover volumes of acidic antimicrobial processing aids resulted in lower pH values. The most extreme drop in pH was noted for the BOA treatment. This pH (below 4) was maintained during 24 h cold storage and would likely prove problematic for recovery of Campylobacter [19]. The pH of n-BPW rinsates was significantly (P < 0.05) higher than BPW in all cases, even when no residual antimicrobial chemical was present. Generally, the pH of n-BPW rinsates were within about 1 pH unit of neutral; in the case of BOA, the pH of n-BPW rinsates was far more hospitable for Campylobacter than was the pH in rinsates prepared with BPW. In earlier work, the pH of n-BPW with the same residual antimicrobials allowed successful recovery of Salmonella [8]. Overall, n-BPW outperforms traditional BPW for controlling pH of broiler carcass rinsate when residual acidic antimicrobial processing aids are present. Table 1. pH ± 95% confidence interval of broiler carcass rinsate with addition of simulated residual antimicrobial at initial mixing and after 24 h 4°C storage (n = 9).1 pH initial pH 24 h Residual2 BPW3 n-BPW4 BPW n-BPW None 7.09 ± 0.06F 7.96 ± 0.06B 7.14 ± 0.10F 8.13 ± 0.12A,B ASC 6.26 ± 0.08J 7.36 ± 0.06E 6.28 ± 0.11I,J 7.62 ± 0.17C,D CPC 7.11 ± 0.06F 8.00 ± 0.06A,B 7.12 ± 0.10F 8.18 ± 0.12A PAA 6.49 ± 0.07 G,H,I 7.45 ± 0.05D,E 6.50 ± 0.10 G,H 7.70 ± 0.11C BOA 3.54 ± 0.09K 6.36 ± 0.09H,I,J 3.55 ± 0.10K 6.66 ± 0.13G pH initial pH 24 h Residual2 BPW3 n-BPW4 BPW n-BPW None 7.09 ± 0.06F 7.96 ± 0.06B 7.14 ± 0.10F 8.13 ± 0.12A,B ASC 6.26 ± 0.08J 7.36 ± 0.06E 6.28 ± 0.11I,J 7.62 ± 0.17C,D CPC 7.11 ± 0.06F 8.00 ± 0.06A,B 7.12 ± 0.10F 8.18 ± 0.12A PAA 6.49 ± 0.07 G,H,I 7.45 ± 0.05D,E 6.50 ± 0.10 G,H 7.70 ± 0.11C BOA 3.54 ± 0.09K 6.36 ± 0.09H,I,J 3.55 ± 0.10K 6.66 ± 0.13G 1Triplicate samples in each of 3 replicate trials. 2Residual intervention: ASC: acidified sodium chloride; CPC: cetylpyridinium chloride; PAA peroxyacetic acid, BOA: blend of acid. 3Buffered peptone water carcass rinse. 4Neutralizing buffered peptone water rinse. A–KValues with no like superscripts are significantly different by Tukey's Honest Significant Difference test (P < 0.05). View Large Table 1. pH ± 95% confidence interval of broiler carcass rinsate with addition of simulated residual antimicrobial at initial mixing and after 24 h 4°C storage (n = 9).1 pH initial pH 24 h Residual2 BPW3 n-BPW4 BPW n-BPW None 7.09 ± 0.06F 7.96 ± 0.06B 7.14 ± 0.10F 8.13 ± 0.12A,B ASC 6.26 ± 0.08J 7.36 ± 0.06E 6.28 ± 0.11I,J 7.62 ± 0.17C,D CPC 7.11 ± 0.06F 8.00 ± 0.06A,B 7.12 ± 0.10F 8.18 ± 0.12A PAA 6.49 ± 0.07 G,H,I 7.45 ± 0.05D,E 6.50 ± 0.10 G,H 7.70 ± 0.11C BOA 3.54 ± 0.09K 6.36 ± 0.09H,I,J 3.55 ± 0.10K 6.66 ± 0.13G pH initial pH 24 h Residual2 BPW3 n-BPW4 BPW n-BPW None 7.09 ± 0.06F 7.96 ± 0.06B 7.14 ± 0.10F 8.13 ± 0.12A,B ASC 6.26 ± 0.08J 7.36 ± 0.06E 6.28 ± 0.11I,J 7.62 ± 0.17C,D CPC 7.11 ± 0.06F 8.00 ± 0.06A,B 7.12 ± 0.10F 8.18 ± 0.12A PAA 6.49 ± 0.07 G,H,I 7.45 ± 0.05D,E 6.50 ± 0.10 G,H 7.70 ± 0.11C BOA 3.54 ± 0.09K 6.36 ± 0.09H,I,J 3.55 ± 0.10K 6.66 ± 0.13G 1Triplicate samples in each of 3 replicate trials. 2Residual intervention: ASC: acidified sodium chloride; CPC: cetylpyridinium chloride; PAA peroxyacetic acid, BOA: blend of acid. 3Buffered peptone water carcass rinse. 4Neutralizing buffered peptone water rinse. A–KValues with no like superscripts are significantly different by Tukey's Honest Significant Difference test (P < 0.05). View Large Recovery of inoculated Campylobacter from BPW and n-BPW carcass rinsates with and without addition of residual post-chill antimicrobial processing aids is presented in Table 2. All rinsate samples were inoculated with approximately 6.0 log CFU/mL, which was verified by plate count. After 24 h cold storage at 4°C, the control samples, without antimicrobial residue, had 5.3 and 5.0 log CFU/mL in BPW and n-BPW, respectively. This finding indicates a loss of about 1 log CFU/mL Campylobacter due just to 24 h cold storage. No Campylobacter was recovered from BPW rinsate samples with residual levels of PAA and BOA. In earlier work, we found that Salmonella also was inhibited by PAA in a BPW broiler carcass rinse [7]; however, in that previous study, residual BOA did not inhibit Salmonella to the same extent as it did Campylobacter in the current study. The low pH of carcass rinse with BOA seems to be more damaging to Campylobacter than Salmonella. When those same antimicrobial residues were present in n-BPW, Campylobacter recovery was not impeded. The same number of Campylobacter was found in n-BPW samples with PAA and BOA as in n-BPW samples with no antimicrobial residue. This suggests that the increased buffering capacity of n-BPW controlled the pH of carcass rinse with BOA, such that Campylobacter could survive cold storage. Residual PAA in n-BPW carcass rinsate was neutralized, allowing Campylobacter to survive. These findings are similar to what was reported for Salmonella, which also survived well in similar n-BPW carcass rinse [8]. Table 2. Mean log CFU Campylobacter per mL rinsate ± 95% confidence interval after 24 h 4°C storage with simulated residual antimicrobial chemical (n = 9).1 Log CFU Campylobacter per mL Residual2 BPW3 n-BPW4 None 5.3 ± 0.2A 5.0 ± 0.2A,B ASC 1.2 ± 0.7C 1.1 ± 0.3C CPC 0.1 ± 0.1D 4.4 ± 0.7B PAA 0.0 ± 0.0D 5.1 ± 0.2A BOA 0.0 ± 0.0D 5.2 ± 0.3A Log CFU Campylobacter per mL Residual2 BPW3 n-BPW4 None 5.3 ± 0.2A 5.0 ± 0.2A,B ASC 1.2 ± 0.7C 1.1 ± 0.3C CPC 0.1 ± 0.1D 4.4 ± 0.7B PAA 0.0 ± 0.0D 5.1 ± 0.2A BOA 0.0 ± 0.0D 5.2 ± 0.3A 1Triplicate samples in each of 3 replicate trials. 2Residual intervention: ASC: acidified sodium chloride; CPC: cetylpyridinium chloride; PAA peroxyacetic acid, BOA: blend of acid. 3Buffered peptone water carcass rinse. 4Neutralizing buffered peptone water rinse. A–DValues with different superscripts are significantly different by Tukey's Honest Significant Difference test ( P< 0.05). View Large Table 2. Mean log CFU Campylobacter per mL rinsate ± 95% confidence interval after 24 h 4°C storage with simulated residual antimicrobial chemical (n = 9).1 Log CFU Campylobacter per mL Residual2 BPW3 n-BPW4 None 5.3 ± 0.2A 5.0 ± 0.2A,B ASC 1.2 ± 0.7C 1.1 ± 0.3C CPC 0.1 ± 0.1D 4.4 ± 0.7B PAA 0.0 ± 0.0D 5.1 ± 0.2A BOA 0.0 ± 0.0D 5.2 ± 0.3A Log CFU Campylobacter per mL Residual2 BPW3 n-BPW4 None 5.3 ± 0.2A 5.0 ± 0.2A,B ASC 1.2 ± 0.7C 1.1 ± 0.3C CPC 0.1 ± 0.1D 4.4 ± 0.7B PAA 0.0 ± 0.0D 5.1 ± 0.2A BOA 0.0 ± 0.0D 5.2 ± 0.3A 1Triplicate samples in each of 3 replicate trials. 2Residual intervention: ASC: acidified sodium chloride; CPC: cetylpyridinium chloride; PAA peroxyacetic acid, BOA: blend of acid. 3Buffered peptone water carcass rinse. 4Neutralizing buffered peptone water rinse. A–DValues with different superscripts are significantly different by Tukey's Honest Significant Difference test ( P< 0.05). View Large Residual CPC in BPW carcass rinsate severely impacted recovery of Campylobacter. However, residual CPC in n-BPW carcass rinses was far less disruptive, allowing close to the same recovery of Campylobacter as noted with the antimicrobial free control. These findings are similar to what was noted for Salmonella with residual CPC in earlier work with BPW [7] and n-BPW [8]. When residual ASC was present in BPW carcass rinse, Campylobacter was detected but at significantly lower numbers than found in the antimicrobial free control rinsate. When residual ASC was present in n-BPW carcass rinse, Campylobacter was still negatively affected; no difference in recovery was noted compared to traditional BPW. This specific finding is different from what we had previously noted with Salmonella. Salmonella was readily recovered in n-BPW carcass rinse, even in the presence of ASC [8]. There may be unknown factors other than the neutralization of pH that are important for Campylobacter recovery. More research is planned to identify such factors and further improve n-BPW for optimal recovery of Campylobacter in the presence of residual ASC. CONCLUSIONS AND APPLICATIONS Traditional broiler carcass rinse with BPW is not completely adequate to recover Campylobacter in the presence of simulated worst-case concentration of residual post-chill antimicrobial processing aid. A new formulation of rinse medium (n-BPW) is significantly more effective than traditional BPW for recovery of Campylobacter from broiler carcass rinses with residual CPC, PAA, and BOA. Four to five log CFU/mL improvement was noted, representing more than a 99.99% improvement. No difference in Campylobacter recovery was noted in n-BPW broiler carcass rinse with residual ASC compared to traditional BPW carcass rinse. Footnotes 1 Mention of trade names or commercial products in the publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. Primary Audience: Poultry Processors, Regulatory Officials, Poultry Microbiology Researchers REFERENCES AND NOTES 1. https://www.cdc.gov/media/releases/2017/p0420-campylobacter-salmonella.html . 2. Berrang M. E. , Meinersmann R. J. , Ladely S. R. , Cox N. A. . 2016 . Campylobacter detection in broiler ceca at processing – A three year 211 flock survey . J. Appl. Poult. Res. 26 : 154 – 158 . 3. Berrang M. E. , Oakley B. B. , Meinersmann R. J. . 2016 . Detection of Campylobacter on the outer surface of retail broiler meat packages and from product within . Food Prot. Trends . 36 : 176 – 182 . 4. https://www.gpo.gov/fdsys/pkg/FR-2016-02-11/pdf/2016-02586.pdf . 5. https://www.fsis.usda.gov/wps/wcm/connect/bab10e09-aefa-483b-8be8-809a1f051d4c/7120.1.pdf?MOD=AJPERES . 6. Bourassa D. V. , Wilson K. M. , Bartenfeld L. N. , Harris C. E. , Howard A. K. , Ingram K. D. , Hinton A. Jr. , Adams E. S. , Berrang M. E. , Feldner P. W. , Gamble G. R. , Frye J. G. , Johnston J. J. , Buhr R. J. . 2016 . Carcass orientation and drip time affect potential surface water carryover for broiler carcasses subjected to a post-chill water dip or spray . Poult. Sci. 96 : 241 – 245 . Google Scholar CrossRef Search ADS PubMed 7. Gamble G. R. , Berrang M. E. , Buhr R. J. , Hinton A. Jr. , Bourassa D. V. , Johnston J. J. , Ingram K. D. , Adams E. S. , Feldner P. W. . 2016 . Effect of simulated sanitizer carry-over on recovery of Salmonella from broiler carcass rinsates . J. Food Prot. 79 : 710 – 714 . Google Scholar CrossRef Search ADS PubMed 8. Gamble G. R. , Berrang M. E. , Buhr R. J. , Hinton A. Jr. , Bourassa D. V. , Ingram K. D. , Adams E. S. , Feldner P. W. , Johnston J.J. . 2017 . Neutralization of unintended antimicrobial carry-over for optimal recovery of Salmonella from broiler carcass rinse samples . J. Food Prot. 80 : 685 – 691 . Google Scholar CrossRef Search ADS PubMed 9. https://www.fsis.usda.gov/wps/wcm/connect/2cb982e0-625c-483f-9f50-6f24bc660f33/41-16.pdf?MOD=AJPERES . 10. Sigma Aldrich, St. Louis, MO . 11. Pfaltz and Bauer, Waterbury, CT . 12. Chemical analyses for verification of antimicrobial concentration. CPC: verified by titration based on polyvinylsulfate with toluidine blue indicator (Chemetrics, Midland VA). PAA: verified by iodine-diethylphenylenediamine coloimetric measurement (Chemetrics). ASC: verified by anion chromatography using an ICS-3000 (Dionex, Sunnyvale, CA) . 13. Thermo Scientific, Beverly, MA . 14. Cox N. A. , Richardson L. J. , Berrang M. E. , Fedorka-Cray P. J. , Buhr R. J. . 2009 . Campylobacter coli naturally resistant to elevated levels of gentamicin as a marker strain in poultry research . J. Food Prot. 72 : 1288 – 1292 . Google Scholar CrossRef Search ADS PubMed 15. Neogen Corp., Lansing, MI . 16. Dacosta K. J. , Berrang M. E. , Meinersmann R. J. , Cox N. A. , Knapp S. W. . 2017 . Cell concentration of Campylobacter coli/jejuni in 24 hour broth culture . Proceedings of the Annual Meeting of Southern Poultry Science Society. Atlanta GA Jan . 30 – 31 . 17. Stern N. J. , Wojton B. , Kwiatek K. . 1992 . A differential selective medium and dry ice generated atmosphere for recovery of Campylobacter jejuni . J. Food Prot. 55 : 514 – 517 . Google Scholar CrossRef Search ADS 18. StatSoft, Tulsa, OK . 19. Shaheen B. W. , Miller M. E. , Oyarzabal O. A. . 2007 . In vitro survival at low pH and acid adaptation response of Campylobacter jejuni and Campylobacter coli . J. Food Safety 27 : 326 – 343 . Google Scholar CrossRef Search ADS Acknowledgments The authors wish to acknowledge expert technical assistance by Eric S. Adams and Peggy W. Feldner. Published by Oxford University Press on behalf of Poultry Science Association 2018. This work is written by (a) US Government employee(s) and is in the public domain in the US.

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Journal of Applied Poultry ResearchOxford University Press

Published: Sep 1, 2018

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