Quantification of Zoonotic Bacterial Pathogens within Commercial Poultry Processing Water Samples Using Droplet Digital PCR

Michael J. Rothrock, Kelli L. Hiett, Brian H. Kiepper, Kim Ingram, Arthur Hinton

.
Poultry Processing and Swine Physiology Research Unit, USDA-Agricultural Research Service, Athens, Georgia.

DOI: 10.4236/aim.2013.35055                

Abstract

Raw poultry and poultry products are a significant source of zoonotic bacterial pathogen transmission; thus the sensitive detection of major zoonotic pathogens (Salmonella spp., Campylobacter jejuni, and Listeria monocytogenes) is a vital food safety issue. Recently, third generation PCR technology, known as droplet digital PCR (ddPCR) has been developed to be more accurate and sensitive to detect genetic targets than current quantification methods, but this technology has not been tested within an industrial setting. There is an on-going study within our laboratory is investigating the effects of sampling times and sampling methods on the cultural and molecular (via qPCR) quantification of dominant zoonotic pathogens within a poultry processing facility. This presents a unique opportunity to compare the quantification resulted from this emerging, third generation technology to traditional quantification methods currently employed by the poultry industry. The results show that ddPCR detected pathogen-specific genes from more pathogen:sampling time combinations than either the qPCR or culturing methods from the final scalder and chiller tanks at three stages of processing (Start, Mid, and End). In fact, both ddPCR and qPCR substantially outperformed culture methods commonly used in poultry processing food safety-related studies, with Salmonella recovered only from the Mid and End sampling times from the scalder tank. While neither C. jejuni nor L. monocytogenes were recovered culturally, ddPCR was able to detect their respective genes commonly throughout the processing day in both the scalder and chiller water samples. Additionally, the use of unfiltered processing water provided significantly greater detection of bacterial and pathogen-specific gene abundances than did an analysis of larger volumes of filtered water. Considering the ddPCR-derived concentrations of the bacterial pathogens were consistent with what was previously found culturally in commercial poultry processing operations, ddPCR represented a significant advancement in poultry processing zoonotic pathogen quantification.

Keywords

ddPCR; Poultry Processing; Zoonotic Pathogens; qPCR

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	S. Buncic and J. Sofos, “Interventions to Control Salmonella Contamination during Poultry, Cattle and Pig Slaughter,” Food Research International, Vol. 45, No. 2, 2012, pp. 641-655. doi:10.1016/j.foodres.2011.10.018
[2]	J. M. Cox and A. Pavic, “Advances in Enteropathogen Control in Poultry Production,” Journal of Applied Microbiology, Vol. 108, No. 3, 2010, pp. 745-755. doi:10.1111/j.1365-2672.2009.04456.x
[3]	S. Finstad, C. A. O’Bryan, J. A. Marcy, P. G. Crandall and S. C. Ricke, “Salmonella and Broiler Processing in the United States: Relationship to Foodborne Salmonellosis,” Food Research International, Vol. 45, No. 2, 2012, pp. 789-794. doi:10.1016/j.foodres.2011.03.057
[4]	C. R. Friedman, R. M. Hoekstra, M. Samuel, R. Marcus, J. Bender, B. Shiferaw, et al., “Risk Factors for Sporadic Campylobacter Infection in the United States: A Case-Control Study in FoodNet Sites,” Clincal Infectious Disease, Vol. 38, Suppl. 3, 2004, pp. S285-S296.
[5]	E. M. Nielsen, V. Fussing, J. Engberg, N. L. Nielsen and J. Neimann, “Most Campylobacter Subtypes from Sporadic Infections Can Be Found in Retail Poultry Products and Food Animals,” Epidemiology & Infection, Vol. 134, No. 4, 2006, pp. 758-767. doi:10.1017/S0950268805005509
[6]	L. M. Lawrence and A. Gilmour, “Incidence of Listeria spp. and Listeria monocytogenes in Poultry Products and the Poultry Processing Environment, and Their Rapid Confirmation by Multiplex PCR,” Applied Environmental Microbiology, Vol. 60, No. 12, 1994, pp. 4600-4604.
[7]	M. G. R. Reiter, C. M. M. Bueno, C. Lopez and R. Jordano, “Occurrence of Campylobacter and Listeriamonocytogenes in a Poultry Processing Plant,” Journal of Food Protection, Vol. 68, No. 9, 2005, pp. 1903-1906.
[8]	M. Baker, “Digital PCR Hits Its Stride,” Nature Methods, Vol. 9, No. 6, 2012, pp. 541-544. doi:10.1038/nmeth.2027
[9]	L. B. Pinheiro, V. A. Coleman, C. M. Hindson, J. Herrmann, B. J. Hindson, S. Bhat, et al., “Evaluation of a Droplet Digital Polymerase Chain Reaction Format for DNA Copy Number Quantification,” Analytical Chemistry, Vol. 84, No. 2, 2012, pp. 1003-1011. doi:10.1021/ac202578x
[10]	B. J. Hindson, K. D. Ness, D. A. Masquelier, P. Belgrader, N. J. Heredia, A. J. Makarewicz, et al., “High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number,” Analytical Chemistry, Vol. 83, No. 22, 2011, pp. 8604-8610. doi:10.1021/ac202028g
[11]	N. J. Stern, B. Wojton and K. Kwiatek, “A Differential-Selective Medium and Dry Ice-Generated Atmosphere for Recovery of Campylobacterjejuni,” Journal of Food Protection, Vol. 55, No. 7, 1992, pp. 514-517.
[12]	APHA, “Standard Methods for the Examination of Water and Wastewater,” 21st Edition, American Public Health Association, Washington DC, 2005,
[13]	D. Hamm, “Characteristics of Effluents in Ten Southeastern Poultry Processing Plants,” Poultry Science, Vol. 51, No. 3, 1972, pp. 825-829. doi:10.3382/ps.0510825
[14]	W. C. Merka, “Processing Water and Wastewater,” In: A. R. Sams, Ed., Poultry Meat Processing, CRC Press LLC, Boca Raton, 2001, pp. 301-310.
[15]	F. L. Bryan and M. P. Doyle, “Health Risks and Consequences of Salmonella and Campylobacter jejuni in Raw Poultry,” Journal of Food Protection, Vol. 58, No. 3, 1995, pp. 326-344.
[16]	Z. Belkot, “Bacterial Contamination of Chicken Carcasses as Influenced by the Time of Slaughter during the Day,” Medycyna Weterynaryjna, Vol. 67, No. 11, 2011, pp. 760-764.
[17]	P. Whyte, K. McGill, C. Monahan and J. D. Collins, “The Effect of Sampling Time on the Levels of Micro-Organisms Recovered from Broiler Carcasses in a Commercial Slaughter Plant,” Food Microbiology, Vol. 21, No. 1, 2004, pp. 59-65. doi:10.1016/S0740-0020(03)00040-6
[18]	M. E. Berrang, J. A. Dickens and M. T. Musgrove, “Effects of Hot Water Application after Defeathering on the Levels of Campylobacter, Coliform Bacteria, and Escherichia coli on Broiler Carcasses,” Poultry Science, Vol. 79, No. 11, 2000, pp. 1689-1693.
[19]	J. A. Cason and A. Hinton Jr., “Coliforms, Escherichiacoli, Campylobacter, and Salmonella in a Counterflow Poultry Scalder with a Dip Tank,” International Journal of Poultry Science, Vol. 5, No. 9, 2006, pp. 846-849. doi:10.3923/ijps.2006.846.849
[20]	M. B. Peyrat, C. Soumet, P. Maris and P. Sanders, “Recovery of Campylobacterjejuni from Surfaces of Poultry Slaughterhouses after Cleaning and Disinfection Procedures: Analysis of a Potential Source of Carcass Contamination,” International Journal of Food Microbiology, Vol. 124, No. 2, 2008, pp. 188-194. doi:10.1016/j.ijfoodmicro.2008.03.030
[21]	G. Figueroa, M. Troncoso, C. Lopez, P. Rivas and M. Toro, “Occurrence and Enumeration of Campylobacter spp. during the Processing of Chilean Broilers,” BMC Microbiology, Vol. 9, 2009, p. 94.
[22]	H. Yang, Y. Li and M. G. Johnson, “Survival and Death of Salmonella typhimurium and Campylobacterjejuni in Processing Water and on Chicken Skin during Poultry Scalding and Chilling,” Journal of Food Protection, Vol. 64, No. 6, 2001, pp. 770-776.
[23]	J. A. Cason, A. Hinton Jr. and K. D. Ingram, “Coliform, Escherichiacoli, and Salmonellae Concentrations in a Multiple-Tank, Counterflow Poultry Scalder,” Journal of Food Protection, Vol. 63, No. 9, 2000, pp. 1184-1188.
[24]	J. D. Stopforth, R. O’Connor, M. Lopes, W. E. Hill and M. Samadpour, “Validation of Individual and Multiple Sequential Interventions for Reduction of Microbial Populations during Processing of Poultry Carcasses and Parts,” Journal of Food Protection, Vol. 70, No. 6, 2007, pp. 1393-1401.
[25]	K. C. Tamblyn, D. E. Conner and S. F. Bilgili, “Utilization of the Skin Attachment Model (SAM) to Determine the Antibacterial Activity of Potential Carcass Treatments,” Poultry Science, Vol. 76, No. 9, 1997, pp. 1318-1323.
[26]	C. Voidarou, D. Vassos, T. Kegos, A. Koutsotoli, A. Tsiotsias, J. Skoufos, et al., “Aerobic and Anaerobic Microbiology of the Immersion Chilling Procedure during Poultry Processing,” Poultry Science, Vol. 86, No. 6, 2007, pp. 1218-1222.
[27]	J. K. Northcutt, D. Smith, R. I. Huezo and K. D. Ingram, “Microbiology of Broiler Carcasses and Chemistry of Chiller Water as Affected by Water Reuse,” Poultry Science, Vol. 87, No. 7, 2008, pp. 1458-1463. doi:10.3382/ps.2007-00480
[28]	G. Harms, A. C. Layton, H. M. Dionisi, I. R. Gregory, V. M. Garrett, S. A. Hawkins, et al., “Real-Time PCR Quantification of Nitrifying Bacteria in a Municipal Wastewater Treatment Plant,” Environmental Science and Technology, Vol. 37, No. 2, 2003, pp. 343-351. doi:10.1021/es0257164
[29]	B. Malorny, E. Paccassoni, P. Fach, C. Bunge, A. Martin and R. Helmuth, “Diagnostic Real-Time PCR for Detection of Salmonella in Food,” Applied Environmental Microbiology, Vol. 70, No. 12, 2004, pp. 7046-7052. doi:10.1128/AEM.70.12.7046-7052.2004
[30]	Y. He, X. Yao, N. W. Gunther IV, Y. Xie, S.-I. Tu and X. Shi, “Simultaneous Detection and Differentiation of Campylobacter jejuni, C. coli, and C. lari in Chickens Using a Multiplex Real-Time PCR Assay,” Food Analytical Methods, Vol. 3, No. 4, 2010, pp. 321-329.
[31]	B. Suo, Y. He, S.-I. Tu and X. Shi, “A Multiplex Real-Time Polymerase Chain Reaction for Simultaneous Detection of Salmonells spp., Escherichia coli O157, and Listeria monocytogenes in Meat Products,” Foodborne Pathogens & Disease, Vol. 7, No. 6, 2010, pp. 619-628. doi:10.1089/fpd.2009.0430