2087 Journal o f Food Protection, Vol. 76, No. 12, 2013, Pages 2087-2092 doi: 10.4315/0362-028X. JFP-13-143 Copyright © , International Association for Food Protection
Research Note
Transfer of Methicillin-Resistant Staphylococcus aureus from Retail Pork Products onto Food Contact Surfaces and the Potential for Consumer Exposure HEATHER L. SNYDER, STEVEN E. NIEBUHR,
and
JAMES S. DICKSON*
Department o f Animal Science, Food Safety Research Laboratory, Iowa State University, 2292 Kildee H alf Ames, Iowa 50010, USA MS 13-143: Received 8 April 2013/Accepted 5 August 2013
ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogen that has developed resistance to beta-lactam antibiotics and has been isolated at low population numbers in retail meat products. The objectives of this study were to estimate the potential transfer of MRSA from contaminated retail pork products to food contact surfaces and to estimate the potential for human exposure to MRSA by contact with those contaminated surfaces. Pork loins, bacon, and fresh pork sausage were inoculated with a four-strain mixed MRSA culture over a range of populations from approximately 4 to 8 log, vacuum packaged, and stored for 2 weeks at 5°C to simulate normal packaging and distribution. Primary transfer was determined by placing inoculated products on knife blades, cutting boards, and a human skin model (pork skin) for 5 min. Secondary transfer was determined by placing an inoculated product on the contact surface, removing it, and then placing the secondary contact surface on the initial contact surface. A pork skin model was used to simulate transfer to human skin by placing it into contact with the contact surface. The percentages of transfer for primary transfer from the inoculated products to the cutting board ranged from 39 to 49%, while the percentages of transfer to the knife ranged from 17 to 42%. The percentages of transfer from the inoculated products to the pork skin ranged from 26 to 36%. The secondary transfer percentages ranged from 2.2 to 5.2% across all products and contact surfaces. Statistical analysis showed no significant differences in the amounts of transfer between transfer surfaces and across cell concentrations.
Methicillin-resistant Staphylococcus aureus (MRSA) strain are strains that have acquired resistance to beta-lactam antibiotics. They first gained notice as the agents respon sible for nosocomial infections (hospital-acquired MRSA) and have since been increasingly isolated from infections in the community (community-acquired MRSA) among those with no history of long-term hospitalization or surgical procedures (3). Within the last decade, livestock-acquired MRSA strains, known as ST398 (sequence type 398), have been isolated from healthy livestock, primarily swine. This sequence type has also been isolated from those who work in close contact with swine, and some human cases have also resulted in infections (18, 19). There have also been cases in those with no previous animal contact (22). Recently, concerns have been raised from a food safety standpoint, as ST398 strains and other MRSA strains have been isolated from raw retail meats (5, 9, 20). Though there have not been any documented cases of human infection with ST398 strains due to raw meats (14), MRSA strains are present in retail raw meat and may serve as a possible source of bacterial infections of food preparers in the food industry (21) and hands of consumers in the home.
* Author for correspondence. Tel: 515-294-4733; Fax: 515-294-5066; E-mail: [email protected].
MRSA may commonly be found on animals at harvest, and the animals themselves may be the source. Research has shown MRSA to be present on carcasses and to carry through to the final products (2), and ST398 has been isolated as a source of contamination of raw meat (5,10,11, 20). Other findings point to humans as the source. On occasion, the MRSA contamination of meat was found to be USA 100 or USA 300, common human strains (9). However, ST398 strains have been shown to transfer between animals and humans more readily than USA 100 or USA 300 (17). MRSA can survive a range of pHs, temperatures, and salt levels and could survive on raw meat that reaches the consumer. MRSA is able to be carried through the stages of a production facility (2); it may also be carried around the kitchen. Several studies have reported that knives and cutting boards are major sources of cross-contamination with bacteria during food preparation and that bacteria on these surfaces from raw meat may in turn contaminate hands (6-8,12,14). Research suggests that bacteria will transfer readily to food contact surfaces and to skin (4,16). Despite these findings, a dose-response level is still not known, so it is difficult to make a quantitative risk assessment regarding the consumer (1). The objectives of this study were to estimate the percentages of transfer of MRSA from pork to knives, cutting boards, and skin and to estimate the potential
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for human exposure from surfaces which have become contaminated with MRSA. M A TER IA LS A N D M E TH O D S Pork skin was used as a model for human skin (17), to represent the potential transfer from pork products or contaminated surfaces to humans. Primary exposure was evaluated after storing the inoculated product at 4°C for 14 days and involved laying the inoculated product on the contact surface for 5 min with no additional pressure. Secondary exposure involved laying the inoculated product on the contact surface, placing a 500-g lead donut on top of the inoculated product, and sliding the product back and forth on a 10-cm path on the transfer surface for 20 complete transits, after which the inoculated product was removed and pork skin was applied to the contact surface to simulate transfer from a contaminated surface to pork skin. All contact surfaces were at ambient temperature (ca. 22°C) during the experiments. Bacterial cultures. Four MRSA isolates were used in this experiment: an ST398 isolate from ground pork, an ST398 isolate from pork chops (Dr. Catherine Logue, Iowa State University College of Veterinary Medicine, Ames), an ST398 isolate from a 24-week-old hog (Dr. Tara Smith, University of Iowa, College of Public Health Department of Epidemiology, Center for Emerging Infectious Diseases, Iowa City), and strain ATCC BAA-44, which is resistant to multiple antibiotics, including methicillin. The isolates were streaked onto Baird-Parker (BP) agar (Difco, BD, Sparks, MD) and Spectra MRSA agar (Remel, Lenexa, KS) and incubated at 37°C for 24 h. Black colonies on BP agar were indicative of S. aureus, and denim-colored colonies on Spectra MRSA agar were indicative of MRSA. The colonies isolated were also tested using the Staphylase test (Oxoid Ltd., Basingstoke, Hants, UK) and the PBP2 latex agglutination test (Oxoid Ltd.) and were MRSA positive. All strains were maintained in tryptic soy broth (Difco, BD). The day before experimentation, the cultures were transferred to tryptic soy broth, grown aerobically at 37°C for 24 h, and then combined to form a mixed culture with a cell concentration of approximately 109 CFU/ml. Inoculation procedure. Fresh boneless pork loins were purchased from a local source and held at 4°C until used for the experiments. Five loins were separately inoculated with 10 ml of five different decimal dilutions to achieve a range of populations on the pork surfaces from ca. 104 to 108 CFU/cm2. One loin was used as a negative control; 100 cm2 was swabbed with a SpeciSponge (Whirl-Pak Speci-Sponge bags, Nasco, Fort Atkinson, WI) hydrated with 10 ml of 0.01% buffered peptone water (BPW; Difco, BD), and 0.1-ml amounts of the swab sample were surface plated onto BP agar with egg yolk tellurite supplement (BP-EYTA; Difco, BD) and Spectra MRSA agar in duplicate. No growth was observed for the negative controls after 24 h of incubation at 37°C. After inoculation, inoculated loins were held at 4°C for 30 min to allow attachment of the bacteria. An area 5 by 5 cm of each loin was swabbed with a Speci-Sponge hydrated with 10 ml of BPW. The loins were then each vacuum packaged and stored for 2 weeks at 5°C to simulate typical packaging and distribution. Swabs were taken to determine bacterial counts after inoculation but before storage. The swabs were hand massaged for 1 min, serially diluted in BPW, surface plated on BP-EYTA in duplicate, and incubated at 37°C for 24 h. Bacon was purchased from a local source and held at 4°C until used for the experiments. Each of the replications involved four strips of bacon. All inoculated bacon was stored at 4°C for
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30 min to allow attachment of the bacteria. The samples were swabbed and the bacterial populations enumerated as described above. Fresh pork sausage was purchased from a local source and held at 4°C until used. For each replication, 30 g of sausage was inoculated and then divided into four 10-g portions and formed into 5-mm-thick strips measuring 2 by 10 cm, using a mold (Department of Engineering, Iowa State University, Ames). The surface (2 by 10 cm) was swabbed with a Speci-Sponge, and the sausage strips were then vacuum packaged and stored for 2 weeks at 4°C to simulate typical packaging and distribution. The samples were swabbed and the bacterial populations enumerated as described above. Primary transfer. For each replication, five contact surfaces were used to correspond to the five cell concentrations used in inoculation. The pork skin was sanitized with a UV light in a biosafety cabinet for 15 min, and the cutting boards and knives were sterilized by autoclaving. Loins inoculated with different cell concentrations were aseptically removed from the vacuum package and divided into three sections, one section for each contact surface. The individual sections were placed on the contact surfaces for 5 min without any movement or additional pressure. After 5 min, the loins were moved, and an area 5 by 5 cm of each pork loin was swabbed with a Speci-Sponge rehydrated with 10 ml of BPW. An area 5 by 5 cm of each cutting board and each pork skin section were also swabbed. The entire surface of the knife blade (14 by 1.5 cm) of each knife was swabbed. All swabs were hand massaged for 1 min, serially diluted in BPW, plated on BPEYTA, and incubated at 37°C for 24 h. This procedure was followed for the bacon and fresh pork sausage, except that four slices of bacon were used to achieve a surface area of 5 by 5 cm that could be swabbed. Sampling for secondary transfer. After the 2-week storage period, the vacuum-packaged loins were placed on the contact surfaces as described above. A 500-g lead donut was then placed on the loin, and the loin with the weight was moved back and forth on a 10-cm path on the transfer surface for 20 complete transits. After the surfaces were exposed to the inoculated loins, they remained at ambient temperature for 5 min. The pork skin was applied to the surface with the 500-g lead donut on top, and the skin was passed along the same 10-cm path for 20 back-and-forth transits. The surfaces and the pork skin were then immediately swabbed with a Speci-Sponge, and after swab sampling, the pork skin sample was diluted 1:10 (wt/vol) with BPW. Sample analysis. BP-EYTA was prepared according to the manufacturer’s instructions. This medium was chosen because it is commonly used for the isolation and enumeration of S. aureus (8). Since the identity of the MRSA strains in this experiment had been determined with ancillary tests, BP-EYTA was used for these experiments. Statistical analysis. The numeric populations of MRSA were reported as CFU per square centimeter of the surface area swabbed. The percentage of transfer of MRSA from the pork products to the surfaces was calculated by dividing the population of MRSA on the contact surface after contact by the initial population present on the product either 30 min or 2 weeks (with storage at 4°C) after inoculation and multiplying the result by 100 (13). Three independent replicate experiments were performed. The percent ages of transfer for each product and surface were analyzed with SAS (Statistical Analysis System, version 9.2, SAS Institute, Inc.,
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TABLE 1. Primary transfer o f MRSA from pork products to contact surfaces after initial inoculation Least squares mean % transfer (SD) from product to contact surface Product Pork loin
Avg population (log CFU/g) on product
Cutting board
4.12 4.94 5.83 6.99 8.04 Avg"
33.33 37.69 54.0 24.33 85.58 46.09
(1.88) (19.8) (20.2) (14.85) (26.67)
Bacon
4.35 5.09 6.37 7.11 8.34 Avg
Sausage
4.68 5.89 7.04 8.16 9.25 Avg
16.99 22.32 26.04 26.99 53.61 48.97 15.3 5.6 29.6 70.6 75.0 38.96
(6.25) (23.24) (16.45) (16.45) (18.03) (53.81) (18.2) (2.3) (15.4) (38.6) (29.2) (29.97)
Knife 7.14 24.96 48.52 40.45 28.63 33.45
(23.47) (32.71) (37.2) (19.27) (26.71)
7.09 11.49 9.1 33.15 22.12 16.95
(4.4) (8.73) (4.56) (41.9) (15.73) (20.69)
26.3 51.0 39.9 36.4 80.5 42.19
(15.9) (5.7) (9.2) (22.7) (43.1) (40.69)
Pork skin 60.0 12.5 19.69 11.08 80.62 36.91
(2.8) (13.7) (7.42) (20.75) (32.84)
17.97 23.29 44.33 21.15 27.68 27.54 11.8 56.9 11.2 38.4 27.8 26.54
(11.26) (14.79) (35.43) (8.37) (11.58) (18.99) (2.1) (32.5) (1.6) (12.3) (6.4) (38.99)
a Average percentage of transfer across all inoculation levels. Cary, NC) with a general linear model using least squares means and quantile-quantile (Q-Q) plots (which indicated that the data were normally distributed). The least squares means test was performed to determine if there was no significant difference in the ability of the bacteria to transfer among the surfaces—the polyethylene cutting board, the knife, and the pork skin. Unless otherwise noted, statistical significance was determined as a P value of 0.10) in the percentage of transfer of MRSA between either the initial population on the pork loin or the contact surface. A similar trend was observed with bacon (Table 1), although the percentage of transfer to the knife contact surface was statistically lower (P = 0.07) than the percentage of transfer to either the pork skin or cutting board surface. The population on the inoculated surface of pork sausage significantly (P < 0.05) affected the percentage of transfer to the contact surfaces, with the lower inoculation levels resulting in lower percentages of transfer to all three surfaces (Table 1). When the data from
all of the initial inoculated populations on each product type were pooled, there was no significant difference between the different contact surfaces, but there was a trend (P = 0.06) toward a lower percentage of transfer from bacon than from pork loin or sausage. Primary transfer after 2 weeks of storage. When the percentages of transfer of MRSA after 2 weeks of storage were analyzed, there was no significant difference (P > 0.10) between inoculum level or contact suiface type (Table 2). However, when the data for each product was pooled across the different inoculum levels and contact surfaces, the percentage of transfer from bacon was significantly less (P < 0.05) than from either pork loin or sausage. Pork loin and fresh pork sausage were not significantly different from each other. Secondary transfer. The objective of these experi ments was to evaluate the potential for common consumer kitchen contact surfaces to transfer MRSA from contami nated pork products to human skin. When compared within product type, there was no significant difference in transfer between the initial inoculation on the product and the final transfer from the contact surface to the pork skin model (Table 3). When combined across all product types and contact surfaces, there was no significant difference in secondary transfer between any of the inoculated products or either of the contact surfaces. Effect of contact pressure on transfer. Comparisons were made between the percentages of transfer of MRSA from the inoculated products to the cutting board. This comparison was between the data which resulted from the primary transfer experiments (product weight only) and the secondary transfer experiments (product weight plus 500 g
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TABLE 2. Primary transfer of MRSA from pork products to contact surfaces after 2 weeks of storage at 4°C Least squares mean % transfer0 (SD) to indicated contact surface Product
squares mean log CFU/cm2 (SD)]
Pork loin
4.85 (0.73) 5.34 (1.32) 6.57 (0.73) 7.69 (0.81) 8.63 (0.85) Avgc 5.34(0.11) 6.07 (0.19) 7.33 (0.22) 7.97 (0.41) 9.09 (0.70) Avg 5.66 (0.16) 6.88 (0.11) 8.03 (0.09) 8.69 (0.17) 9.73 (0.11) Avg
Bacon
Sausage
Cutting board 6.07 12.45 15.04 4.26 13.66 10.82 4.12 8.64 4.19 23.85 4.00 8.96 19.75 8.24 10.91 16.35 18.64 14.78
Knife
(0.00)* (10.68) (14.91) (4.62) (10.19) (9.74) (2.29) (5.69) (1.02) (30.22) (1.99) (14.12) (26.28) (8.18) (11.34) (7.86) (11.59) (13.27)
0.36 18.15 8.24 3.31 3.92 7.79 1.42 4.34 1.74 12.21 4.15 4.77 4.13 8.32 8.99 21.54 13.77 11.35
(0.00) (25.57) (9.80) (3.06) (5.04) (13.02) (1.03) (2.99) (1.11) (7.24) (3.31) (5.2) (1.15) (5.50) (4.57) (1.88) (10.17) (10.52)
Pork skin 8.57 8.52 18.88 1.98 25.47 13.72 3.98 7.19 6.59 9.92 3.04 6.14 11.87 4.27 12.89 15.75 13.89 11.73
(0.00) (2.10) (24.14) (0.85) (29.18) (18.74) (2.63) (0.68) (2.89) (8.52) (0.97) (4.38) (9.06) (2.71) (6.10) (13.68) (11.65) (8.99)
° (MRSA population on contact surface/MRSA population on product) x 100. * For the pork loin at the lowest inoculum level, the populations on the contact surfaces were below the detectable limit of the assay for two of the three replications. c Average across all initial inoculum levels. and movement). For inoculated pork loin, there was a significant difference (P < 0.05) in the percentages of transfer with light and heavy pressure, with fewer bacteria transferred (lower percentage of transfer) with the heavy pressure (Table 4). There was no significant difference in the percentages of transfer with light and heavy pressure for bacon or sausage.
DISCUSSION MRSA was shown to be able to survive refrigeration temperatures on vacuum-packaged fresh pork for at least 2 weeks, a common shipping and distribution time. This suggests that retail pork products contaminated with MRSA could reach the consumer and present a source of possible
TABLE 3. Secondary transfer of MRSA from inoculated pork to contact surface to pork skin Least squares mean % transfer* (SD) to pork skin from contact surface Inoculated product Pork loin
Bacon
Pork sausage
Population on inoculated product [least squares mean log CFU/cm2 (SD)]° 4.29 (0.35) 4.86 (0.26) 6.50 (0.42) 6.92 (0.12) 8.08 (0.30) Avgc 4.60 (0.30) 5.40 (0.14) 6.66 (0.18) 7.52 (0.09) 8.61 (0.11) Avg 4.58 (0.30) 5.53 (0.19) 6.57 (0.03) 7.45 (0.17) 8.46 (0.25) Avg
“ Population of MRSA on inoculated loin prior to contact with surface. * (Population on pork skin/population on pork loin) x 100. c Average transfer across all inoculation levels.
Cutting board 5.47 3.59 3.94 3.78 1.98 3.63 4.22 3.40 0.73 8.23 2.27 3.77 2.90 2.68 2.46 5.09 6.39 3.90
(6.07) (2.56) (3.75) (3.01) (0.37) (2.92) (2.89) (3.65) (0.65) (9.12) (1.87) (4.72) (1.94) (1.13) (1.22) (4.03) (2.32 (2.57)
Knife 2.68 3.32 5.90 2.95 3.54 3.68 4.22 0.73 1.61 1.39 4.05 2.26 5.35 2.57 6.01 5.58 6.39 5.18
(1.99) (2.38) (4.22) (1.15) (2.55) (2.54) (1.11) (0.36) (1.19) (1.40) (5.02) (2.58) (1.05) (2.36) (4.77) (4.68) (2.64) (3.21)
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TABLE 4. Comparison o f the effects o f light and heavy pressure on the percentage o f transfer o f MRSA from inoculated product to cutting board Inoculated surface and approx population (log CFU/cm2) on surface Pork loin 4 5 6 7 8 Avgc
Least squares mean % transfer (SD) to contact surface with indicated pressure" Light
Heavy
33.33fc 37.69 (1.88) 54.0 (19.8) 24.33 (20.2) 85.58 (14.85) 46.08 (26.66)
6.02 29.69 13.53 21.01 11.22 16.64
(1.85) (37.19) (5.98) (10.78) (7.18) (17.25)
16.99 22.32 26.04 26.99 53.61 29.96
(6.25) (23.24) (16.45) (16.45) (18.03) (18.87)
45.10 18.3 45.5 44.07 33.7 38.83
(20.3) (13.1) (27.9) (40.28) (18.3) (23.33)
15.3 5.6 29.6 70.6 75.0 38.96
(18.2) (2.3) (15.4) (38.6) (29.2) (29.97)
18.31 8.23 20.31 34.31 12.13 19.19
(11.0) (7.63) (11.31) (22.11) (2.19) (14.79)
Bacon 4 5 6 7 8 Avg Sausage 4 5 6 7 8 Avg
“ Light pressure was the weight of the product, and heavy pressure was a 500-g lead doughnut and back-and-forth movement. * At an inoculum level of 4.12 CFU/cm2, two of the three replications had no recoverable populations of MRSA on the contact surfaces. c Average transfer across all inoculation levels.
contamination or infection. Viable cells remain on the retail pork products and are capable of contaminating food contact surfaces, as has been previously demonstrated (4, 6, 13). The initial objective was to determine the extent to which MRSA could be transferred from inoculated pork to contact surfaces, including a pork skin model which represented human skin. This study did not find a significant difference in the percentages of transfer from inoculated pork products to any of the three contact surfaces, which is consistent with a finding in earlier research (4). However, other research has found a relationship between the texture of the surface and bacterial transfer (14). The cutting boards used in the experiments described in the current research were new, smooth polyethylene cutting boards that had not been previously used and had no cut marks, damage, or grooves on the surface. The lack of transfer of cells observed with the lowest inoculation levels for the pork loins may be attributable either to the inoculum preparation or simply to limitations of the recovery methods. Although the results of this study show that the percentages of transfer during primary transfer increased numerically as the populations on the product increased, this observed increase was not significantly different (P > 0.10). While this was not unexpected, other studies have described
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an inverse relationship in which a high initial bacterial load leads to a lower total amount transferred and lower initial bacterial counts will lead to higher percentages of transfer due to bacterial interactions on the meat surface (6, 13). However, one of these studies used raw poultry that was naturally contaminated (6), while the research presented here used inoculated products. Pork product surfaces vary among type (loin, bacon, and sausage) and may be quite different from the surfaces of raw poultry. Therefore, bacterial interactions on inoculated pork and naturally contaminated poultry cannot be easily compared due to the differences in their surface types. The secondary transfer experiments (Table 3) show that there was not a significant difference (P > 0.10) in transfer to the pork skin from the contact surfaces among the products and across the cell populations. It was expected that the cell transfer would be higher if pressure was applied to the product while it was being moved across a surface. One study reported that applied pressure would facilitate bacterial removal from one surface to another, resulting in higher transfer rates (13). It was also expected that subsequent weighted skin contact on that surface would result in high transfer. The observed percentages of transfer (Table 4) are lower than those from the primary transfer when the contaminated product was simply placed on the pork skin for 5 min. However, a direct comparison cannot be made because the secondary transfer was to determine if a contaminated surface could transfer bacteria to skin, whereas the primary transfer showed that direct skin contact on the contaminated product could transfer the bacteria. Still, the results show that transfer occurs across all levels of initial contamination, which indicates that risk of consumer contact and possible colonization or infection exists. In order for a pathogen to present a risk to the consumer, it must be able to survive on meat surfaces and on surfaces used in home food preparation, such as cutting boards and knives (15). These experiments quantified the percentages of transfer of MRSA on retail pork products to food contact surfaces at the consumer level. As the doseresponse of MRSA is still not known, these data may allow the construction of a model to determine what MRSA bacterial cell range places the consumer at the most risk of becoming colonized with MRSA or developing a MRSA infection due to the preparation of a contaminated retail product in the home kitchen. ACKNOWLEDGMENTS The authors thank Dr. Byron Brehm-Stecher and Dr. Joe Sebranek for their editorial comments and Karl Pazdemik for his assistance with statistical analysis. For this research, funding, wholly or in part, was provided by the National Pork Board.
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Pu, S., F. Han, and B. Ge. 2009. Isolation and characterization of methicillin-resistant Staphylococcus aureus strains from Louisiana retail meats. Appl. Environ. Microbiol. 75:265-267. Rodriquez-Perez, F., A. Valero, E. Carrasco, R. M. Garcia, and G. Zurera. 2008. Understanding and modelling bacterial transfer to foods: a review. Trends Food Sci. Technol. 19:131-144. Tang, J. Y. H., M. Nishibuchi, Y. Nakaguchi, F. M.Ghazali, A. A. Saleha, and R. Son. 2011. Transfer of Campylobacter jejuni from raw to cooked chicken via wood and plastic cutting boards. Lett. Appl. Microbiol. 52:581-588. Todd, E. C. D., J. D. Grieg, C. A. Bartleson, and B. S. Michaels. 2009. Outbreaks where food workers have been implicated in the spread of foodbome disease. Part 6. Transmission and survival of pathogens in the food processing and preparation environment. J. Food Prot. 72:202-219. Todd, E. C. D., B. S. Michaels, J. D. Grieg, D. Smith, J. Holah, and C. A. Bartleson. 2010. Outbreaks where food workers have been implicated in the spread of foodbome disease. Part 7. Barriers to reduce contamination of food by workers. J. Food Prot. 73:15521565. Vanderhaeghen, W., K. Hermans, F. Haesebrouck, and P. Butaye. 2010. Methicillin-resistant Staphylococcus aureus (MRSA) in food production animals. Epidemiol. Infect. 138:606-625. van Loo, I., X. Huijsdens, E. Tiemersma, A. de Neeling, N. van de Sande-Bruinsma, D. Beaujean, A. Voss, and J. Kluytmans. 2007. Emergence of methicillin-resistant Staphylococcus aureus of animal origin in humans. Emerg. Infect. Dis. 13:1834-1839. Voss, A., F. Loeffen, J. Bakker, C. Klaassen, and M. Wulf. 2005. Methicillin-resistant Staphylococcus aureus in pig farming. Emerg. Infect. Dis. 11:1965-1966. Weese, J. S., B. Avery, and R. Reid-Smith. 2010. Detection of methicillin-resistant Staphylococcus aureus (MRSA) clones in retail meat products. Lett. Appl. Microbiol. 51:338-342. Weese, J. S., R. Reid-Smith, J. Rousseau, and B. Avery. 2010. Methicillin-resistant Staphylococcus aureus contamination of retail pork. Can. Vet. J. 51:749-752. Welinder-Olsson, C., K. Floren-Johansson, L. Larsson, S. Oberg, L. Karlsson, and C. Ahren. 2008. Infection with Panton-Valentine leukocidin-positive methicillin-resistant Staphylococcus aureus t034. Emerg. Infect. Dis. 14:1271-1272.
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Research Note
Transfer of Methicillin-Resistant Staphylococcus aureus from Retail Pork Products onto Food Contact Surfaces and the Potential for Consumer Exposure HEATHER L. SNYDER, STEVEN E. NIEBUHR,
and
JAMES S. DICKSON*
Department o f Animal Science, Food Safety Research Laboratory, Iowa State University, 2292 Kildee H alf Ames, Iowa 50010, USA MS 13-143: Received 8 April 2013/Accepted 5 August 2013
ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogen that has developed resistance to beta-lactam antibiotics and has been isolated at low population numbers in retail meat products. The objectives of this study were to estimate the potential transfer of MRSA from contaminated retail pork products to food contact surfaces and to estimate the potential for human exposure to MRSA by contact with those contaminated surfaces. Pork loins, bacon, and fresh pork sausage were inoculated with a four-strain mixed MRSA culture over a range of populations from approximately 4 to 8 log, vacuum packaged, and stored for 2 weeks at 5°C to simulate normal packaging and distribution. Primary transfer was determined by placing inoculated products on knife blades, cutting boards, and a human skin model (pork skin) for 5 min. Secondary transfer was determined by placing an inoculated product on the contact surface, removing it, and then placing the secondary contact surface on the initial contact surface. A pork skin model was used to simulate transfer to human skin by placing it into contact with the contact surface. The percentages of transfer for primary transfer from the inoculated products to the cutting board ranged from 39 to 49%, while the percentages of transfer to the knife ranged from 17 to 42%. The percentages of transfer from the inoculated products to the pork skin ranged from 26 to 36%. The secondary transfer percentages ranged from 2.2 to 5.2% across all products and contact surfaces. Statistical analysis showed no significant differences in the amounts of transfer between transfer surfaces and across cell concentrations.
Methicillin-resistant Staphylococcus aureus (MRSA) strain are strains that have acquired resistance to beta-lactam antibiotics. They first gained notice as the agents respon sible for nosocomial infections (hospital-acquired MRSA) and have since been increasingly isolated from infections in the community (community-acquired MRSA) among those with no history of long-term hospitalization or surgical procedures (3). Within the last decade, livestock-acquired MRSA strains, known as ST398 (sequence type 398), have been isolated from healthy livestock, primarily swine. This sequence type has also been isolated from those who work in close contact with swine, and some human cases have also resulted in infections (18, 19). There have also been cases in those with no previous animal contact (22). Recently, concerns have been raised from a food safety standpoint, as ST398 strains and other MRSA strains have been isolated from raw retail meats (5, 9, 20). Though there have not been any documented cases of human infection with ST398 strains due to raw meats (14), MRSA strains are present in retail raw meat and may serve as a possible source of bacterial infections of food preparers in the food industry (21) and hands of consumers in the home.
* Author for correspondence. Tel: 515-294-4733; Fax: 515-294-5066; E-mail: [email protected].
MRSA may commonly be found on animals at harvest, and the animals themselves may be the source. Research has shown MRSA to be present on carcasses and to carry through to the final products (2), and ST398 has been isolated as a source of contamination of raw meat (5,10,11, 20). Other findings point to humans as the source. On occasion, the MRSA contamination of meat was found to be USA 100 or USA 300, common human strains (9). However, ST398 strains have been shown to transfer between animals and humans more readily than USA 100 or USA 300 (17). MRSA can survive a range of pHs, temperatures, and salt levels and could survive on raw meat that reaches the consumer. MRSA is able to be carried through the stages of a production facility (2); it may also be carried around the kitchen. Several studies have reported that knives and cutting boards are major sources of cross-contamination with bacteria during food preparation and that bacteria on these surfaces from raw meat may in turn contaminate hands (6-8,12,14). Research suggests that bacteria will transfer readily to food contact surfaces and to skin (4,16). Despite these findings, a dose-response level is still not known, so it is difficult to make a quantitative risk assessment regarding the consumer (1). The objectives of this study were to estimate the percentages of transfer of MRSA from pork to knives, cutting boards, and skin and to estimate the potential
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for human exposure from surfaces which have become contaminated with MRSA. M A TER IA LS A N D M E TH O D S Pork skin was used as a model for human skin (17), to represent the potential transfer from pork products or contaminated surfaces to humans. Primary exposure was evaluated after storing the inoculated product at 4°C for 14 days and involved laying the inoculated product on the contact surface for 5 min with no additional pressure. Secondary exposure involved laying the inoculated product on the contact surface, placing a 500-g lead donut on top of the inoculated product, and sliding the product back and forth on a 10-cm path on the transfer surface for 20 complete transits, after which the inoculated product was removed and pork skin was applied to the contact surface to simulate transfer from a contaminated surface to pork skin. All contact surfaces were at ambient temperature (ca. 22°C) during the experiments. Bacterial cultures. Four MRSA isolates were used in this experiment: an ST398 isolate from ground pork, an ST398 isolate from pork chops (Dr. Catherine Logue, Iowa State University College of Veterinary Medicine, Ames), an ST398 isolate from a 24-week-old hog (Dr. Tara Smith, University of Iowa, College of Public Health Department of Epidemiology, Center for Emerging Infectious Diseases, Iowa City), and strain ATCC BAA-44, which is resistant to multiple antibiotics, including methicillin. The isolates were streaked onto Baird-Parker (BP) agar (Difco, BD, Sparks, MD) and Spectra MRSA agar (Remel, Lenexa, KS) and incubated at 37°C for 24 h. Black colonies on BP agar were indicative of S. aureus, and denim-colored colonies on Spectra MRSA agar were indicative of MRSA. The colonies isolated were also tested using the Staphylase test (Oxoid Ltd., Basingstoke, Hants, UK) and the PBP2 latex agglutination test (Oxoid Ltd.) and were MRSA positive. All strains were maintained in tryptic soy broth (Difco, BD). The day before experimentation, the cultures were transferred to tryptic soy broth, grown aerobically at 37°C for 24 h, and then combined to form a mixed culture with a cell concentration of approximately 109 CFU/ml. Inoculation procedure. Fresh boneless pork loins were purchased from a local source and held at 4°C until used for the experiments. Five loins were separately inoculated with 10 ml of five different decimal dilutions to achieve a range of populations on the pork surfaces from ca. 104 to 108 CFU/cm2. One loin was used as a negative control; 100 cm2 was swabbed with a SpeciSponge (Whirl-Pak Speci-Sponge bags, Nasco, Fort Atkinson, WI) hydrated with 10 ml of 0.01% buffered peptone water (BPW; Difco, BD), and 0.1-ml amounts of the swab sample were surface plated onto BP agar with egg yolk tellurite supplement (BP-EYTA; Difco, BD) and Spectra MRSA agar in duplicate. No growth was observed for the negative controls after 24 h of incubation at 37°C. After inoculation, inoculated loins were held at 4°C for 30 min to allow attachment of the bacteria. An area 5 by 5 cm of each loin was swabbed with a Speci-Sponge hydrated with 10 ml of BPW. The loins were then each vacuum packaged and stored for 2 weeks at 5°C to simulate typical packaging and distribution. Swabs were taken to determine bacterial counts after inoculation but before storage. The swabs were hand massaged for 1 min, serially diluted in BPW, surface plated on BP-EYTA in duplicate, and incubated at 37°C for 24 h. Bacon was purchased from a local source and held at 4°C until used for the experiments. Each of the replications involved four strips of bacon. All inoculated bacon was stored at 4°C for
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30 min to allow attachment of the bacteria. The samples were swabbed and the bacterial populations enumerated as described above. Fresh pork sausage was purchased from a local source and held at 4°C until used. For each replication, 30 g of sausage was inoculated and then divided into four 10-g portions and formed into 5-mm-thick strips measuring 2 by 10 cm, using a mold (Department of Engineering, Iowa State University, Ames). The surface (2 by 10 cm) was swabbed with a Speci-Sponge, and the sausage strips were then vacuum packaged and stored for 2 weeks at 4°C to simulate typical packaging and distribution. The samples were swabbed and the bacterial populations enumerated as described above. Primary transfer. For each replication, five contact surfaces were used to correspond to the five cell concentrations used in inoculation. The pork skin was sanitized with a UV light in a biosafety cabinet for 15 min, and the cutting boards and knives were sterilized by autoclaving. Loins inoculated with different cell concentrations were aseptically removed from the vacuum package and divided into three sections, one section for each contact surface. The individual sections were placed on the contact surfaces for 5 min without any movement or additional pressure. After 5 min, the loins were moved, and an area 5 by 5 cm of each pork loin was swabbed with a Speci-Sponge rehydrated with 10 ml of BPW. An area 5 by 5 cm of each cutting board and each pork skin section were also swabbed. The entire surface of the knife blade (14 by 1.5 cm) of each knife was swabbed. All swabs were hand massaged for 1 min, serially diluted in BPW, plated on BPEYTA, and incubated at 37°C for 24 h. This procedure was followed for the bacon and fresh pork sausage, except that four slices of bacon were used to achieve a surface area of 5 by 5 cm that could be swabbed. Sampling for secondary transfer. After the 2-week storage period, the vacuum-packaged loins were placed on the contact surfaces as described above. A 500-g lead donut was then placed on the loin, and the loin with the weight was moved back and forth on a 10-cm path on the transfer surface for 20 complete transits. After the surfaces were exposed to the inoculated loins, they remained at ambient temperature for 5 min. The pork skin was applied to the surface with the 500-g lead donut on top, and the skin was passed along the same 10-cm path for 20 back-and-forth transits. The surfaces and the pork skin were then immediately swabbed with a Speci-Sponge, and after swab sampling, the pork skin sample was diluted 1:10 (wt/vol) with BPW. Sample analysis. BP-EYTA was prepared according to the manufacturer’s instructions. This medium was chosen because it is commonly used for the isolation and enumeration of S. aureus (8). Since the identity of the MRSA strains in this experiment had been determined with ancillary tests, BP-EYTA was used for these experiments. Statistical analysis. The numeric populations of MRSA were reported as CFU per square centimeter of the surface area swabbed. The percentage of transfer of MRSA from the pork products to the surfaces was calculated by dividing the population of MRSA on the contact surface after contact by the initial population present on the product either 30 min or 2 weeks (with storage at 4°C) after inoculation and multiplying the result by 100 (13). Three independent replicate experiments were performed. The percent ages of transfer for each product and surface were analyzed with SAS (Statistical Analysis System, version 9.2, SAS Institute, Inc.,
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TABLE 1. Primary transfer o f MRSA from pork products to contact surfaces after initial inoculation Least squares mean % transfer (SD) from product to contact surface Product Pork loin
Avg population (log CFU/g) on product
Cutting board
4.12 4.94 5.83 6.99 8.04 Avg"
33.33 37.69 54.0 24.33 85.58 46.09
(1.88) (19.8) (20.2) (14.85) (26.67)
Bacon
4.35 5.09 6.37 7.11 8.34 Avg
Sausage
4.68 5.89 7.04 8.16 9.25 Avg
16.99 22.32 26.04 26.99 53.61 48.97 15.3 5.6 29.6 70.6 75.0 38.96
(6.25) (23.24) (16.45) (16.45) (18.03) (53.81) (18.2) (2.3) (15.4) (38.6) (29.2) (29.97)
Knife 7.14 24.96 48.52 40.45 28.63 33.45
(23.47) (32.71) (37.2) (19.27) (26.71)
7.09 11.49 9.1 33.15 22.12 16.95
(4.4) (8.73) (4.56) (41.9) (15.73) (20.69)
26.3 51.0 39.9 36.4 80.5 42.19
(15.9) (5.7) (9.2) (22.7) (43.1) (40.69)
Pork skin 60.0 12.5 19.69 11.08 80.62 36.91
(2.8) (13.7) (7.42) (20.75) (32.84)
17.97 23.29 44.33 21.15 27.68 27.54 11.8 56.9 11.2 38.4 27.8 26.54
(11.26) (14.79) (35.43) (8.37) (11.58) (18.99) (2.1) (32.5) (1.6) (12.3) (6.4) (38.99)
a Average percentage of transfer across all inoculation levels. Cary, NC) with a general linear model using least squares means and quantile-quantile (Q-Q) plots (which indicated that the data were normally distributed). The least squares means test was performed to determine if there was no significant difference in the ability of the bacteria to transfer among the surfaces—the polyethylene cutting board, the knife, and the pork skin. Unless otherwise noted, statistical significance was determined as a P value of 0.10) in the percentage of transfer of MRSA between either the initial population on the pork loin or the contact surface. A similar trend was observed with bacon (Table 1), although the percentage of transfer to the knife contact surface was statistically lower (P = 0.07) than the percentage of transfer to either the pork skin or cutting board surface. The population on the inoculated surface of pork sausage significantly (P < 0.05) affected the percentage of transfer to the contact surfaces, with the lower inoculation levels resulting in lower percentages of transfer to all three surfaces (Table 1). When the data from
all of the initial inoculated populations on each product type were pooled, there was no significant difference between the different contact surfaces, but there was a trend (P = 0.06) toward a lower percentage of transfer from bacon than from pork loin or sausage. Primary transfer after 2 weeks of storage. When the percentages of transfer of MRSA after 2 weeks of storage were analyzed, there was no significant difference (P > 0.10) between inoculum level or contact suiface type (Table 2). However, when the data for each product was pooled across the different inoculum levels and contact surfaces, the percentage of transfer from bacon was significantly less (P < 0.05) than from either pork loin or sausage. Pork loin and fresh pork sausage were not significantly different from each other. Secondary transfer. The objective of these experi ments was to evaluate the potential for common consumer kitchen contact surfaces to transfer MRSA from contami nated pork products to human skin. When compared within product type, there was no significant difference in transfer between the initial inoculation on the product and the final transfer from the contact surface to the pork skin model (Table 3). When combined across all product types and contact surfaces, there was no significant difference in secondary transfer between any of the inoculated products or either of the contact surfaces. Effect of contact pressure on transfer. Comparisons were made between the percentages of transfer of MRSA from the inoculated products to the cutting board. This comparison was between the data which resulted from the primary transfer experiments (product weight only) and the secondary transfer experiments (product weight plus 500 g
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TABLE 2. Primary transfer of MRSA from pork products to contact surfaces after 2 weeks of storage at 4°C Least squares mean % transfer0 (SD) to indicated contact surface Product
squares mean log CFU/cm2 (SD)]
Pork loin
4.85 (0.73) 5.34 (1.32) 6.57 (0.73) 7.69 (0.81) 8.63 (0.85) Avgc 5.34(0.11) 6.07 (0.19) 7.33 (0.22) 7.97 (0.41) 9.09 (0.70) Avg 5.66 (0.16) 6.88 (0.11) 8.03 (0.09) 8.69 (0.17) 9.73 (0.11) Avg
Bacon
Sausage
Cutting board 6.07 12.45 15.04 4.26 13.66 10.82 4.12 8.64 4.19 23.85 4.00 8.96 19.75 8.24 10.91 16.35 18.64 14.78
Knife
(0.00)* (10.68) (14.91) (4.62) (10.19) (9.74) (2.29) (5.69) (1.02) (30.22) (1.99) (14.12) (26.28) (8.18) (11.34) (7.86) (11.59) (13.27)
0.36 18.15 8.24 3.31 3.92 7.79 1.42 4.34 1.74 12.21 4.15 4.77 4.13 8.32 8.99 21.54 13.77 11.35
(0.00) (25.57) (9.80) (3.06) (5.04) (13.02) (1.03) (2.99) (1.11) (7.24) (3.31) (5.2) (1.15) (5.50) (4.57) (1.88) (10.17) (10.52)
Pork skin 8.57 8.52 18.88 1.98 25.47 13.72 3.98 7.19 6.59 9.92 3.04 6.14 11.87 4.27 12.89 15.75 13.89 11.73
(0.00) (2.10) (24.14) (0.85) (29.18) (18.74) (2.63) (0.68) (2.89) (8.52) (0.97) (4.38) (9.06) (2.71) (6.10) (13.68) (11.65) (8.99)
° (MRSA population on contact surface/MRSA population on product) x 100. * For the pork loin at the lowest inoculum level, the populations on the contact surfaces were below the detectable limit of the assay for two of the three replications. c Average across all initial inoculum levels. and movement). For inoculated pork loin, there was a significant difference (P < 0.05) in the percentages of transfer with light and heavy pressure, with fewer bacteria transferred (lower percentage of transfer) with the heavy pressure (Table 4). There was no significant difference in the percentages of transfer with light and heavy pressure for bacon or sausage.
DISCUSSION MRSA was shown to be able to survive refrigeration temperatures on vacuum-packaged fresh pork for at least 2 weeks, a common shipping and distribution time. This suggests that retail pork products contaminated with MRSA could reach the consumer and present a source of possible
TABLE 3. Secondary transfer of MRSA from inoculated pork to contact surface to pork skin Least squares mean % transfer* (SD) to pork skin from contact surface Inoculated product Pork loin
Bacon
Pork sausage
Population on inoculated product [least squares mean log CFU/cm2 (SD)]° 4.29 (0.35) 4.86 (0.26) 6.50 (0.42) 6.92 (0.12) 8.08 (0.30) Avgc 4.60 (0.30) 5.40 (0.14) 6.66 (0.18) 7.52 (0.09) 8.61 (0.11) Avg 4.58 (0.30) 5.53 (0.19) 6.57 (0.03) 7.45 (0.17) 8.46 (0.25) Avg
“ Population of MRSA on inoculated loin prior to contact with surface. * (Population on pork skin/population on pork loin) x 100. c Average transfer across all inoculation levels.
Cutting board 5.47 3.59 3.94 3.78 1.98 3.63 4.22 3.40 0.73 8.23 2.27 3.77 2.90 2.68 2.46 5.09 6.39 3.90
(6.07) (2.56) (3.75) (3.01) (0.37) (2.92) (2.89) (3.65) (0.65) (9.12) (1.87) (4.72) (1.94) (1.13) (1.22) (4.03) (2.32 (2.57)
Knife 2.68 3.32 5.90 2.95 3.54 3.68 4.22 0.73 1.61 1.39 4.05 2.26 5.35 2.57 6.01 5.58 6.39 5.18
(1.99) (2.38) (4.22) (1.15) (2.55) (2.54) (1.11) (0.36) (1.19) (1.40) (5.02) (2.58) (1.05) (2.36) (4.77) (4.68) (2.64) (3.21)
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TABLE 4. Comparison o f the effects o f light and heavy pressure on the percentage o f transfer o f MRSA from inoculated product to cutting board Inoculated surface and approx population (log CFU/cm2) on surface Pork loin 4 5 6 7 8 Avgc
Least squares mean % transfer (SD) to contact surface with indicated pressure" Light
Heavy
33.33fc 37.69 (1.88) 54.0 (19.8) 24.33 (20.2) 85.58 (14.85) 46.08 (26.66)
6.02 29.69 13.53 21.01 11.22 16.64
(1.85) (37.19) (5.98) (10.78) (7.18) (17.25)
16.99 22.32 26.04 26.99 53.61 29.96
(6.25) (23.24) (16.45) (16.45) (18.03) (18.87)
45.10 18.3 45.5 44.07 33.7 38.83
(20.3) (13.1) (27.9) (40.28) (18.3) (23.33)
15.3 5.6 29.6 70.6 75.0 38.96
(18.2) (2.3) (15.4) (38.6) (29.2) (29.97)
18.31 8.23 20.31 34.31 12.13 19.19
(11.0) (7.63) (11.31) (22.11) (2.19) (14.79)
Bacon 4 5 6 7 8 Avg Sausage 4 5 6 7 8 Avg
“ Light pressure was the weight of the product, and heavy pressure was a 500-g lead doughnut and back-and-forth movement. * At an inoculum level of 4.12 CFU/cm2, two of the three replications had no recoverable populations of MRSA on the contact surfaces. c Average transfer across all inoculation levels.
contamination or infection. Viable cells remain on the retail pork products and are capable of contaminating food contact surfaces, as has been previously demonstrated (4, 6, 13). The initial objective was to determine the extent to which MRSA could be transferred from inoculated pork to contact surfaces, including a pork skin model which represented human skin. This study did not find a significant difference in the percentages of transfer from inoculated pork products to any of the three contact surfaces, which is consistent with a finding in earlier research (4). However, other research has found a relationship between the texture of the surface and bacterial transfer (14). The cutting boards used in the experiments described in the current research were new, smooth polyethylene cutting boards that had not been previously used and had no cut marks, damage, or grooves on the surface. The lack of transfer of cells observed with the lowest inoculation levels for the pork loins may be attributable either to the inoculum preparation or simply to limitations of the recovery methods. Although the results of this study show that the percentages of transfer during primary transfer increased numerically as the populations on the product increased, this observed increase was not significantly different (P > 0.10). While this was not unexpected, other studies have described
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an inverse relationship in which a high initial bacterial load leads to a lower total amount transferred and lower initial bacterial counts will lead to higher percentages of transfer due to bacterial interactions on the meat surface (6, 13). However, one of these studies used raw poultry that was naturally contaminated (6), while the research presented here used inoculated products. Pork product surfaces vary among type (loin, bacon, and sausage) and may be quite different from the surfaces of raw poultry. Therefore, bacterial interactions on inoculated pork and naturally contaminated poultry cannot be easily compared due to the differences in their surface types. The secondary transfer experiments (Table 3) show that there was not a significant difference (P > 0.10) in transfer to the pork skin from the contact surfaces among the products and across the cell populations. It was expected that the cell transfer would be higher if pressure was applied to the product while it was being moved across a surface. One study reported that applied pressure would facilitate bacterial removal from one surface to another, resulting in higher transfer rates (13). It was also expected that subsequent weighted skin contact on that surface would result in high transfer. The observed percentages of transfer (Table 4) are lower than those from the primary transfer when the contaminated product was simply placed on the pork skin for 5 min. However, a direct comparison cannot be made because the secondary transfer was to determine if a contaminated surface could transfer bacteria to skin, whereas the primary transfer showed that direct skin contact on the contaminated product could transfer the bacteria. Still, the results show that transfer occurs across all levels of initial contamination, which indicates that risk of consumer contact and possible colonization or infection exists. In order for a pathogen to present a risk to the consumer, it must be able to survive on meat surfaces and on surfaces used in home food preparation, such as cutting boards and knives (15). These experiments quantified the percentages of transfer of MRSA on retail pork products to food contact surfaces at the consumer level. As the doseresponse of MRSA is still not known, these data may allow the construction of a model to determine what MRSA bacterial cell range places the consumer at the most risk of becoming colonized with MRSA or developing a MRSA infection due to the preparation of a contaminated retail product in the home kitchen. ACKNOWLEDGMENTS The authors thank Dr. Byron Brehm-Stecher and Dr. Joe Sebranek for their editorial comments and Karl Pazdemik for his assistance with statistical analysis. For this research, funding, wholly or in part, was provided by the National Pork Board.
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