Background

Beef carcass contamination during processing has been shown to occur principally from hides. During the hide removal process, pathogens such as E. coli O157:H7 and Salmonella are transferred from the hide, where they are high in prevalence, to the carcass. Numerous carcass interventions have been implemented into beef processing plants to remove carcass microbial contamination. However, occasional process failures occur that result in greater contamination than can be effectively removed with current carcass interventions. Furthermore, it would be more effective to prevent carcass contamination rather than rely on decontamination procedures.  

We have recently reported that if bacterial contamination of the hide were eliminated or greatly reduced before hide removal, the prevalence of Escherichia coli O157:H7 on the preevisceration carcass was nearly eliminated (Nou et al., 2003, submitted). The strategy evaluated by Nou et al. was chemical dehairing, a process not widely in use due to a variety of difficulties in process application. However, their results imply that processes that effectively clean the hides before hide removal may be effective interventions for preventing carcass microbial contamination. The common oral antimicrobial compound cetylpyridinium chloride (CPC) may have potential for use as a similar hide intervention process. CPC has been used in numerous studies to reduce carcass contamination of poultry, and Cutter et al. have demonstrated the effectiveness of CPC for reducing microbial counts on beef carcasses as well. However, before CPC could be tested as a hide intervention several protocol questions needed to be addressed, and a number of conditions optimized.

The stated objectives for this work were: The objective of this study was to determine the experimental and application parameters necessary to develop protocols to adequately assess the efficacy of cetylpyridinium chloride (CPC) as a beef carcass hide intervention treatment; and to then apply those protocols to determine the of effects of a CPC hide intervention on the levels of E. coli O157:H7, Enterobacteriaceae and Aerobic Plate Count Bacteria in a processing plant environment.

Methodology

Sampling of hides and carcasses. All hide samples were collected using sample sponge bags containing 25mL 2xDE. Samples were collected from 500cm2 area on hides with 10 bi-directional strokes of sponge, turned over half way through the process. The site of sampling varied by experiment, as some experiments required numerous samples to be collected from the same hide. In these cases the sample sites were defined and rotated among hides to control for potential variability. Carcass samples were collected using sponge bags containing BPW. Samples were collected from an 8000cm2 area that covered the anal hock and brisket.

E. coli O157:H 7 isolation. Isolation of E. coli O157:H7 was carried out according to established procedures. Briefly, 75mL TSB was added to each sample bag containing a wetted sampling sponge. This was followed by incubated for enrichment at 2 hours at 25°C, 6 hours at 42°C, and finally held at 4°C until a 1mL aliquot was removed for immunomagnetic separation (IMS) of O157. Following the IMS procedure, half of each preparation was plated to ctSMAC, and nRainbow agar plates. The plates were incubated at 37°C for 16 hours and suspect colonies were confirmed to be O157 through latex agglutination.

APC and EB enumeration. The sample bags were thoroughly massaged and 1.5mL aliquots were taken for serial dilution.  Hide samples underwent a centrifugation and resuspention step to remove potential CPC residue in the supernatant prior to serial dilution. One milliliter of the subsequent serial dilutions was plated to 3M APC and EB PetriFilm. PetriFilm were incubated according to manufacturer’s specifications and counted manually or using an automated imagining system.

Findings

Two experiments were conducted to determine the need for and methods of CPC neutralization during sampling and enrichment in order to obtain valid measurement of the effects of CPC on microbial populations. Residual CPC in hide sponge samples prevented bacterial growth when buffered peptone water was used for sample collection. Dey-Engley neutralization broth at 2X concentration was required to effectively neutralize up to 0.1% CPC at a broth to CPC ratio of 10:1. However, neutralization was not complete when used with 1% CPC. An additional step to prevent the antimicrobial effects of residual CPC in the samples was tested by centrifugal removal of the CPC containing supernatant and resuspending the bacteria in DE. APC and EB counts were not affected by this additional step, and it was implemented into the protocol to provide additional assurance that artifactual data were not obtained from incomplete CPC neutralization.

Two experiments were conducted to determine the effect of CPC concentration and dwell time after treatment on microbial populations (tables 1 and 2). All dwell times after 1% CPC application to cattle hides resulted in lower APC and EB counts than controls (Table 1). However, for APC, 30 sec dwell time had the lowest counts and 30 min, 2 h, and 4 h were not different from one another. For EB, 30 min dwell time had the highest counts and 30 sec, 2 h, and 4 h were not different from one another. For CPC concentration (Table 2), APCs were not different among 0.5, 1, and 3% CPC after either 30 sec or 4 h dwell time, but 4 h counts were lower than 30 sec counts. For EB counts, 3% CPC was lower for control, 30 sec, and 4 h samples than was 0.5 or 1%. An experiment was conducted to determine the effect of spray pressure and two-spray combinations of CPC and water on hide microbial populations (Table 3). Two-spray combinations resulted in lower APC and EB counts as spray pressure increased. High pressure water followed by high pressure water resulted in slightly lower APC and EB counts that for controls. High pressure CPC followed by high pressure CPC had the lowest APC and EB counts. Low pressure CPC followed by low pressure CPC was the least effective CPC treatment. Medium pressure CPC followed by medium pressure CPC was slightly less effective than high/high CPC.

These results indicate that under the proper conditions, CPC treatment can be effective for reducing microbial populations on cattle hides and warranted further study on whether this effect will result in reduced contamination of the carcass by the hide during processing. To do this 149 control cattle and 139 treated cattle were compared. Hides and preevisceration carcasses of each set were sampled. Although, the levels of APC and EB on CPC treated hides were not significantly affected (Table 4) the carcasses of treated cattle showed 1.5 and 1.0 log decreases of APC and EB respectively. Moreover, when the prevalence of E. coli O157:H7 was compared between treated and untreated groups notable decreases were observed (Table 5). CPC treatment of hides reduced O157:H7 prevalence by an average of 22%.  Carcass contamination was significantly reduced to less than 3% from control levels of 23%. One individual sampling day demonstrated a reduction of carcass contamination from 43% to 0%.

Analysis of the post wash carcasses showed that there was no detectable CPC residue on the carcasses. Also, grade and yield data showed no significant differences between CPC sprayed cattle and controls.

Implications

These studies solved problems in measuring the effectiveness of CPC on treated hides containing residual CPC; developed an effective application protocol for reducing hide contamination; and determined the efficacy of CPC treatment to reduce carcass contamination under optimal controlled conditions. Dey-Engley neutralizing broth used at a 2x concentration combined with a centrifugal step to remove CPC contaminated supernatant was necessary to obtain valid measurement of CPC efficacy. Immediately prior to slaughter, cattle were washed with high-pressure water spray to remove organic matter, and then hides were saturated with a 1% CPC spray. This CPC treatment reduced pre-evisceration carcass levels of APC by 1.5log/100cm², and EB by 1log/100cm². Moreover, carcass contamination by E. coli O157:H7 was reduced to less than 3%, whereas levels as high as 42% were observed. The results suggest that the use of CPC as a hide intervention holds significant promise for reducing pathogen contamination during beef processing.