High efficacy of a characterized lytic bacteriophage in combination with thyme essential oil against multidrug-resistant Staphylococcus aureus in chicken products

Abstract

Background: The emergence and spread of methicillin-resistant Staphylococcus aureus (MRSA) in the food industry have led to using alternative natural bioagents for controlling S. aureus in food.

Aims: The current work aimed to isolate and characterize a lytic phage specific to S. aureus and evaluate its efficacy with thyme essential oil for controlling S. aureus growth in chicken fillets.

Methods: Twenty S. aureus strains previously isolated from ready-to-eat chicken products were tested for antimicrobial susceptibility and used for phage isolation.

Results: All S. aureus strains were multidrug-resistant (MDR). The isolated phage (vB_SauM_CP9) belonged to the family Myoviridae and maintained its stability at pH (4-9) and temperature (30-70°C). The phage showed lytic activity on ten S. aureus strains and had a burst size (228 PFU/infected cell), latent period (45 min), and rise period (15 min). A combination of S. aureus phage multiplicity of infection (MOI) 10 + thyme oil 1% caused a higher significant reduction in S. aureus growth (87.22%) in artificially inoculated chicken fillets than individual treatment with bacteriophage or thyme essential oil.

Conclusion: To our knowledge, this is the first report to evaluate the efficacy of bacteriophage and thyme oil for controlling the growth of MDR S. aureus in chicken products and recommending application of S. aureus phage and thyme oil combination in the food industry to achieve food safety goals and consumer protection as well as mitigate the antimicrobial resistance crisis.

Keywords: Bacteriophage; Biocontrol; Chicken products; Staphylococcus aureus; Thyme essential oil.

Fig. 1

Morphological characterization and restriction enzyme analysis of vB_SauM_CP9 phage. A: The arrow shows phage particles under TEM. The virions were negatively stained with uranyl acetate (scale bar, 200 nm), and B: Restriction enzyme digestion analysis of phage DNA. Lane M: DNA marker (New England Biolabs), Lane 1: Undigested DNA, Lane 2: The phage DNA was digested with Hind III, and Lane 3: No digestion with EcoRI

Fig. 2

The effect of different pH (3-10), and temperature (30-90 C) on the stability of S. aureus vB_SauM_CP9 phage. The data were presented for three independent experiments. A: The phage titer was stable at pH range 4-9 with maximum stability at pH = 6-7, and B: The phage titer was stable at temperature range 30-70 C

Fig. 3

The activity of vB_SauM_CP9 phage (MOI 1 and 10) on the growth of S. aureus in an infected culture at 37°C compared with control. The growth was measured at OD₆₀₀. The data were presented for three independent experiments

Fig. 4

One-step growth curve of vB_SauM_CP9 phage. The plaque forming units (PFU) per infected cell were shown at different times post infection. The samples were taken every 15 min up to 90 min

Fig. 5

The effect of thyme oil (0.5 and 1%), phage (MOI 1, MOI 10), and thyme oil 1% + phage MOI 10 on the growth of MDR S. aureus in chicken fillets after 30, 60, and 120 min. The log₁₀ CFU/g was presented for three independent experiments. The higher reduction in CFU/g was observed after treatment with a combination of phage MOI 10 and thyme 1% (P<0.05)