Bacteriophages LSA2308 and LSA2366 infecting drug-resistant Staphylococcus aureus: Isolation, characterization and potential application for milk safety

Highlights

• Phages LSA2308 and LSA2366 with lytic activity against drug-resistant foodborne S. aureus were isolated.

• Combination of phage and ampicillin achieved better biocontrol than treatment with either phage or ampicillin alone.

• Phages LSA2308 and LSA2366 showed application potentials in milk production chain.

Abstract

Bacteriophages are considered a promising candidate for controlling food pathogens. In this study, we isolated and characterized S. aureus-infecting bacteriophages, and investigated their potential for antibiotic substitute and bacteriophage cocktail effect in Luria-Bertani (LB) broth and skim milk. Bacteriophages LSA2308 and LSA2366 possessing lytic activity against drug-resistant S. aureus were isolated. The latent period of LSA2308 and LSA2366 was approximately 20 and 10 min, with burst size of 407 and 258 PFU per infected cell, respectively. A combination of LSA2308 (or LSA2366) and 1/2 MIC ampicillin achieved better clearance of S. aureus CR003 in LB broth than treatment with either bacteriophage or ampicillin alone. Biocontrol with 10⁵–10⁷ PFU/mL LSA2308 or LSA2308/LSA2366 cocktail was effective in LB broth, whereas it was less active in skim milk. Compared with treatment using 10⁵ or 10⁶ PFU/mL bacteriophages, the bactericidal efficacy of 10⁷ PFU/mL bacteriophages on single or mixed S. aureus strains in skim milk was the best at 4 and 25 °C, revealing a dose-dependent mode. Our study further expands the repertoire of bacteriophages infecting drug-resistant S. aureus, and provides insights into the control of S. aureus in milk production chain.

Introduction

Milk and milk products are widely consumed worldwide, but raw milk may be a risk for public health if contaminated with zoonotic foodborne pathogens (Artursson, Schelin, Thisted, Hansson, & Olsson, 2018). Staphylococcus aureus (S. aureus) is an important pathogen responsible for clinical and subclinical mastitis in dairy cows and contaminating raw milk during production (Ruegg, 2017). Antibiotics are often used to treat and prevent diseases caused by a variety of bacteria that affect dairy cows. However, the development and spread of antibiotic resistance pose a public health threat to consumers (Seow, Mahyudin, Amin-Nordin, Radu, & Abdul-Mutalib, 2021). S. aureus contamination may also occur in milk during processing, handling, and transportation, etc. (Yoon, Wei, & Oh, 2018). Uses of antibiotics in food are not permitted, whereas chemical agents may have adverse effects because of chemical residue; consequently, alternatives for control of S. aureus are urgently needed.

Bacteriophages, which are viruses that parasitize bacteria, can effectively lyse bacterial cells. In recent years, bacteriophages have been considered a potential alternative to antimicrobials in the treatment or control of foodborne pathogens (Pollock et al., 2020), and there are some commercial bacteriophage products available in the market (Duc, Son, Yi, et al., 2020). Generally, the approach of using bacteriophage products is based on adding an appropriate concentration of bacteriophages targeting the pathogenic bacteria contaminating the foods. Compared with synthetic antibiotics or other antimicrobials, bacteriophages are natural antimicrobials that exist widely in the environment, and have strong host specificity. However, bacteria have bacteriophage resistance mechanisms, and can evolve a series of barriers to prevent bacteriophages adsorption, thus developing resistance against bacteriophages (Labrie, Samson, & Moineau, 2010). Consequently, isolation of novel S. aureus bacteriophages with different lysis spectrum and high lytic ability is essential. Meanwhile, many researchers try to utilize bacteriophages in combination with other antimicrobials, or combine different bacteriophages into a cocktail to extend their scope of application. For example, simultaneous treatment with bacteriophage Sb-1 and rifampicin or daptomycin resulted in successful eradication of S. aureus biofilms (Tkhilaishvili, Lombardi, Klatt, Trampuz, & Di Luca, 2018). Yuan et al. (2019) studied the effect of bacteriophage cocktail therapy to control the development of bacteriophage resistance in multidrug-resistant Acinetobacter baumannii, and showed that cocktail therapy significantly reduced the emergence of bacteriophage-resistant mutants.

Milk products have been considered as preferred foods for testing biocontrol potential of bacteriophages (Ali Gharieb, Saad, Mohamed, & Tartor, 2020; Luo et al., 2021) since the dairy industry is familiar with destructive effect of bacteriophages on the beneficial starter cultures used for fermented dairy products (El Haddad et al., 2016). In this study, we isolated two bacteriophages against S. aureus, and studied their phenotypes, genotypes, and biological characteristics. Additionally, the potential of bacteriophages for antibiotic substitute and bacteriophage cocktail effect in Luria-Bertani (LB) broth and skim milk were evaluated.