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

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 ongoing 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.

Assessing the microbiomes of scalder and chiller tank waters throughout a typical commercial poultry processing day

“…While Anoxybacillus sequences were the most abundant Firmicutes OTU for the Mid and End samples using the filtrate method, they accounted for only ∼10% of the overall relative abundance in those samples -well below the massive dominance they exhibit in the comparable direct method samples. Overall bacterial concentrations were previously shown to be 5 to 6 logs higher in the direct final scalder samples compared to the filtrate samples (Rothrock et al, 2013), and the microbiomic analysis indicates that Anoxybacillus spp. are responsible for this shift both qualitatively and quantitatively.…”

“…The richness, and to a lesser degree diversity, of the final scalder bacterial communities increased once carcasses began to be processed, and as was observed with the scalder water analyses (Table 1), these values plateaued halfway through the processing day (Mid). Considering that the carcasses introduce not only organic particulates to the scalder tank (Bryan and Doyle, 1995), but also a variety of carcass-and fecal-associated bacteria that increase bacterial contamination of scalder tanks throughout a processing d (Whyte et al, 2004;Rothrock et al, 2013), these increases were not unexpected. In terms of the chiller tank, richness decreased over the course of the processing d, most likely due to the fact that chlorination within these tanks is used to reduce bacterial loads (Stopforth et al, 2007;Finstad et al, 2012).…”

“…Salmonella, Campylobacter, Escherichia) (Stopforth et al, 2007;Finstad et al, 2012), this large increase in Proteobacteria was unexpected, but it should be noted that these are relative abundance values, not absolute abundance values. Previously published data on these samples showed that total bacterial concentrations in the chiller water tank samples did not significantly change throughout the processing d and remained quite low (∼10 2 copies/mL; (Rothrock et al, 2013)). Overall, by the end of the processing d, the relative abundances of the major phyla (Proteobacteria, Firmicutes, Actinobacteria, Bacteriodetes) were similar to what was observed prior to carcasses being introduced (Start), supporting the efficacy of the chlorination to resist major changes at the phylum level within the chiller tank microbiome during the processing d, even though significant changes were observed physiochemically (Table 1).…”

“…While the filtrate and direct methods resulted in similar microbiomic profiles for the chiller tank samples (circles and squares, respectively), the direct sampling method (triangles) produced unique microbiomic profiles as compared to the filtrate method (diamonds) for the final scalder tank water samples. In a previously published work from this study, significant differences also were observed qualitatively between the filtrate and direct sampling methods for the final scalder tank using both qPCR and droplet digital PCR (Rothrock et al, 2013). Considering the water sampling method affected only the microbiomic profiles of the final scalder tank, only these samples were further analyzed.…”

“…Scalder and chiller waters used for this study contained a variety of organic particulates such as feathers, fecal material, blood, fats, and oils that can impair bacterial recovery, since bacterial loads can be intimately associated with these organic materials within processing waters (Rothrock et al, 2013). Therefore, to account for bacteria potentially associated with these organic particulates (which were removed during the filtrate processing method used above), direct nonfiltered samples were concurrently analyzed from homogenized final scalder and chiller tank water.…”