Bacteriophages LSA2308 and LSA2366 infecting drug-resistant Staphylococcus aureus: Isolation, characterization and potential application for milk safety
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.
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Strains
S. aureus ATCC25923, Salmonella Enteritidis ATCC13076, Salmonella Typhimurium ATCC14028, and Escherichia coli ATCC25922 were purchased from the American Type Culture Collection (ATCC, Virginia, USA). Listeria monocytogenes EGDe was a gift from Prof. Xiaohong Wang (College of Food Science and Technology, Huazhong Agricultural University). S. aureus SA027 was a mutant of S. aureus ATCC25923, which was obtained by adaptive evolution when subjected to repeated freeze/thaw cycles. Both S. aureus
Isolation and lytic spectrum of bacteriophages LSA2308 and LSA2366
Bacteriophages LSA2308 and LSA2366 were obtained from sewage using S. aureus ATCC25923 and S. aureus SA027 as the host strain, respectively. Both bacteriophages did not lyse Salmonella Enteritidis, Salmonella Typhimurium, Escherichia coli and Listeria monocytogenes. LSA2308 could lyse 17 of the 27 drug-resistant S. aureus strains isolated from food, while LSA2366 could lyse 18; notably, part of the lysed S. aureus were multidrug-resistant (Table 1). Additionally, all 11 strains isolated from
Conclusions
In this study, bacteriophages LSA2308 and LSA2366, which could infect a variety of drug-resistant foodborne S. aureus strains, were isolated and characterized. A combination of bacteriophage and ampicillin treatment showed improved bactericidal activity against foodborne drug-resistant S. aureus compared to treatment with either bacteriophage or ampicillin alone, which supported the possibility of combined application of bacteriophage and lower concentrations of antibiotics for treating
Declaration of competing interest
No conflict of interest declared.
Author declaration
We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us.
We confirm that we
CRediT authorship contribution statement
Fei Ma: Methodology, Writing – original draft. Yu Ning: Methodology, Writing – original draft. Qingqing Wan: Investigation, Methodology. Likou Zou: Investigation, Methodology. Yuntao Liu: Data curation, Writing – review & editing. Shujuan Chen: Data curation, Writing – review & editing. Jianlong Li: Data curation, Writing – review & editing. Zhen Zeng: Data curation, Writing – review & editing. Yong Yang: Formal analysis, Writing – review & editing. Hong Chen: Formal analysis, Writing – review
Acknowledgements
The authors are grateful for funding support from the National Natural Science Foundation of China (No. 31801629) and Sichuan Agricultural University, and help from Dr. Jinquan Li and Xiaohong Wang.
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