Application of MS bacteriophages on contaminated trimmings reduces Escherichia coli O157 and non-O157 in ground beef
Introduction
Shiga-toxin E. coli (STEC) continue to be a major public health concern due to the severity of its foodborne infections that range from very mild to life-threatening (CDC, 2018). In, 2014, there were 4437 culture-confirmed STEC infections (43.2% due to O157, 56.5% due to nonO157) and costed more than $298 million in medical care costs in the United States (CDC, 2017; USDA-ERS, 2014). Animal products, especially ground beef, have demonstrated to be a common source of contamination of STEC (CDC, 2016). Previous research showed that STEC can be transferred to beef carcasses from hides during the harvesting process and persist in fresh beef after fabrication (Arthur et al., 2010; Bell, 1997; Byrne et al., 2000; Ferens & Hovde, 2011; McEvoy et al., 2000).
The USDA deemed STEC O157:H7 adulterant in ground beef in 1994 after a multistate outbreak from undercooked hamburger from a fast food chain. In 2012, six more serotypes including STEC O145, O121, O111, O103, O45, and O26 (the “Big Six”) were also deemed adulterant due to their association with foodborne outbreaks (USDA-FSIS, 2012). According to the USDA-FSIS, beef trim tested positive for STEC still can be used for human consumption, but it must be further processed into heat-treated products to eliminate bacteria (USDA-FSIS, 2014). Positive trim is sold at discounted price, leading to economic losses for the industry. Therefore, due to human health and economic impacts, regulatory agencies developed policies to control STEC contamination, whereas meat processors implemented food safety interventions to decrease the incidence of STEC in beef products. In the U.S., large beef processing facilities commonly use hot water carcass washing during harvest and applications of acidic solutions during harvest and fabrication as antimicrobial interventions (Wheeler et al., 2014). Although the effectiveness of acid solutions as antimicrobials against E. coli is supported by some research (Harris et al., 2006; Laury et al., 2009; Schmidt et al., 2014; Wolf et al., 2012; Zhao et al., 2009) several studies have shown that E. coli may become resistant to low pH after exposure to acid stress (Anderson & Marshall, 1989; Cheng et al., 2003; Kim et al., 2015; Leyer et al., 1995; Samelis et al., 2005; Smulders & Greer, 1998). Therefore, there is an immediate need for developing more efficient aids to decontaminate beef.
Bacteriophage (phage) solutions are known as bacteria-specific interventions that can be applied on food products (Atterbury et al., 2003; Endersen et al., 2014; Mahony et al., 2011). Recently, phage applications were reported to significantly decrease Salmonella contamination in ground meats (Grant et al., 2017; Jorquera et al., 2015; Sharma et al., 2015; Yeh et al., 2017). When in contact with a bacteria cell, phages attach to specific cell structures and introduce their genome into the bacteria. The lysis of the host is achieved by phage replication and release (Endersen et al., 2014; Montag et al., 1990; Rakhuba et al., 2010). In the United Sates, the Food Safety and Inspection Service (FSIS) Directive 7120.1 lists bacteriophage solutions as a processing aid to control only one STEC serotype (O157:H7) (USDA-FSIS, 2019). The objectives of this study were: i) to demonstrate that phages can also decrease “Big Six” contamination in contaminated beef; and ii) provide technical evidence to the United States Food and Drug Administration agency (FDA) to extend GRAS status for bacteriophages as processing aids against all adulterant STEC.
Section snippets
E. coli strains and inoculum preparation
Strains used in this study included E. coli O26 (ATCC® BAA-2196™), O45 (ATCC® BAA-2193™), O103 (ATCC® BAA-2215™), O111 (ATCC® BAA-2440™), O121 (ATCC® BAA-2219™), O145 (ATCC® BAA-2192™), and O157:H7 (ATCC® 35150™). Frozen stocks from each strain were briefly thawed and a loopfull of culture was aseptically streaked onto 1.6% agar LB agar plates in duplicate and incubated for 24 h at 37 °C to ensure cultures were live and viable. For each strain, a single isolated colony was transferred into
Results
The results of lysing efficacy of each bacteriophage against all STEC serotypes is demonstrated in Table 1. For each respective serotype (gray highlighted diagonal in the table), phages demonstrated at least 96.2% of lysing efficacy (MS1 103 against STEC O103). The highest lysing efficacy (99.9%) was observed for MS1 026 and MS1 157. Overall, when tested against their non-specific STEC serotypes, lysing efficacy of MS1 phages varied from 34.1% (MS1 103 against STEC O111) to 99.8% (MS1 157
Discussion
Bacteriophages MS1 exerted high lysing efficacy in vitro against their respective serotypes (above 1 log, Mirzaei et al., 2016). All MS1 bacteriophages were isolated from sewage as described by Mirzaei et al., 2014, Hudson et al. (2013), Carey-Smith et al., 2006, Hooton, Atterbury, and Connerton (2011), and Pereira et al. (2016) by using the same STEC serotypes that were used in this study to contaminate trim. The ability of each phage to lyse other non-specific STEC serotypes is possible
Conclusion
In this study, we demonstrated that bacteriophages are effective against the “Big Six” serotypes when treating beef trim under different temperatures. This study provides support documentation to biotechnology companies that bacteriophages may reduce all adulterant STEC, not only O157:H7. Therefore, future FDA requests to obtain GRAS status for phages targeting the “Big Six” must be considered. In addition, results of this research demonstrated that bacteriophage applications reduce STEC loads
Declaration of Competing Interest
None.
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