Isolation, characterization and application of a polyvalent phage capable of controlling Salmonella and Escherichia coli O157:H7 in different food matrices

Highlights

• Salmonella and E. coli O157:H7 phages were isolated from chicken products.

• Polyvalent phage PS5 was characterized by host range, morphology, one-step growth, stability and genome sequencing.

• PS5 significantly reduced S. Enteritidis, S. Typhimurium and E. coli O157:H7 in in vitro and in foods.

Abstract

Salmonella Enteritidis, Salmonella Typhimurium, and Escherichia coli O157:H7 are the most important foodborne pathogens, causing serious food poisoning outbreaks worldwide. Bacteriophages are increasingly considered as novel antibacterial agents to control foodborne pathogens. In this study, 8 Salmonella phages and 10 E. coli O157:H7 phages were isolated from chicken products. A polyvalent phage PS5 capable of infecting S. Enteritidis, S. Typhimurium, and E. coli O157:H7 was further characterized and its efficacy in reducing these foodborne pathogens was evaluated in in vitro and in foods. Morphology, one-step growth, and stability assay showed that phage PS5 was a myovirus, with relatively short latent periods, large burst sizes, and high stability. Genome sequencing analysis revealed that the genome of PS5 does not contain any genes associated to antibiotic resistance, toxins, lysogeny, and virulence factors. In broth, phage PS5 significantly decreased the viable counts of all the three bacterial hosts by more than 1.3 log CFU/mL compared to controls after 2 h of incubation at 4 °C and 24 °C. In foods, treatment with PS5 also resulted in significant reductions of viable counts of all the three bacterial hosts compared to controls at temperatures tested. This is the first report on single phage capable of simultaneously controlling S. Enteritidis, S. Typhimurium and E. coli O157:H7 in both in vitro and in foods.

Introduction

Salmonella and E. coli O157:H7 are major causes of foodborne illness worldwide. Since Salmonella was first discovered in 1885, the bacterium has been associated with many foodborne outbreaks (Bell Kyriakides, 2002). Every year, Salmonella spp. is estimated to cause 1.0 million cases of gastroenteritis in the United States (Scallan et al., 2011). Although over 2500 serovars of Salmonella enterica have been identified, 2 serovars - S. Enteriditidis and S. Typhimurium are the most dangerous serovars responsible for more than 50% of reported salmonellosis cases (Fatica & Schneider, 2011). Typical symptoms of Salmonella infection can range from diarrhea to systemic typhoid fever (McGhie, Brawn, Hume, Humphreys, & Koronakis, 2009). After the first identification in 1983, E. coli O157:H7 has quickly become one of the most important foodborne bacteria due to the severe illness caused by this pathogen such as: mild diarrhea, haemorrhagic colitis, haemolytic uraemic syndrome and thrombotic thrombocytopenic purpura (O’Flynn, Ross, Fitzgerald, & Coffey, 2004). Annually, in the United States, there are approximately 73,000 illnesses, 2000 hospitalizations, and 60 deaths as the result of E. coli O157:H7 infection (Mead et al., 1999).

Although a variety of chemical, physical, and biological methods (irradiation, high-frequency heating, steam pasteurization, chlorine, organic acids, trisodium phosphate, ozone, plant extracts and essential oils) are being used to control Salmonella and E. coli O157:H7 in foods (Aymerich et al., 2008, Chen et al., 2012, Grant and Parveen, 2017, Petri et al., 2015), the decontamination of these pathogens in foods still presents real challenges due to the weakness of the conventional methods. Irradiation is one of the most effective physical means for controlling pathogenic bacteria such as E. coli, Salmonella, and Campylobacter in foods. However, it can lead to lipid oxidation, the inducing of unpleasant odor, the changes of color and texture, and nutrient loss (Ahn and Lee, 2012, Chen et al., 2012, Jo et al., 1999). Other limitations of irradiation are that this technology is not widely accepted by customers and requires complex operational facilities (Dincer and Baysal, 2004, Junqueira-Gonçalves et al., 2011, Maherani et al., 2016, Mittendorfer, 2016). Chlorine is recognized as the most commonly used chemical sanitizer. Washing with this chemical can reduce bacterial counts of foodborne pathogens (Sofos & Smith, 1998). However, chlorine poses health hazard since it can form carcinogenic compounds known as trihalomethanes in the presence of organic matter (Richardson, 2003). Therefore, the concentration of chlorine used in food industry is strictly limited (Byelashov & Sofos, 2009). In addition, chlorine can be rapidly counteracted by organic compounds present in foods (Huang & Nitin, 2019). Also, the extensive use of chlorine can result in the development of bacterial resistance to the antimicrobial compound, consequently reducing the efficacy of the sanitizer in further applications (Mokgatla et al., 1998, Mokgatla et al., 2002). Furthermore, this chemical agent may have negative impacts on not only food properties but also the environment (Ölmez & Kretzschmar, 2009). Therefore, novel antimicrobial agents are urgently needed for controlling foodborne pathogens (Yang, Huang, Yuan, Zhang, & Yousef, 2016).

Bacteriophages are increasingly recognized as potential antibacterial agents in food industry. They are bacterial viruses with many favorable features for bio-control of foodborne pathogens such as specific to target bacteria, self-replicating, harmless for human, and abundant in the environment (Hagens and Loessner, 2010, Loc-Carrillo and Abedon, 2011, Perera et al., 2015). Moreover, phages are able to kill antibiotic-resistant bacterial strains that pose substantial public health threat (Hoang Minh et al., 2016, Verraes et al., 2013). Due to these merits, a number of studies have focused on the bio-control of foodborne pathogens by lytic bacteriophages (Abuladze et al., 2008, Bao et al., 2015, Bigwood et al., 2008, Hooton et al., 2011, Hudson et al., 2013, Zinno et al., 2014). Most of phages are only capable of killing specific bacterial strains within a species (Hagens & Loessner, 2010). Phages lysing bacterial strains from different species or genera are very rare and called polyvalent phages (Hagens and Loessner, 2010, Hamdi et al., 2017). Although several polyvalent phages have been isolated (Goodridge et al., 2003, Hamdi et al., 2017, Kim and Ryu, 2011, Lopez-Cuevas et al., 2011, Santos et al., 2010, Yu et al., 2016), single phage infecting S. Enteritidis, S. Typhimurium and E. coli O157:H7 has not been reported. This study described the isolation, characterization and assessment of a polyvalent phage PS5 capable of controlling S. Enteritidis, S. Typhimurium and E. coli O157:H7 in in vitro and in foods.