Abstract
Bacteriophage lytic enzymes have recently attracted considerable interest as novel antimicrobials against Gram-positive bacteria. In this work, antimicrobial activity in milk of HydH5 [a virion-associated peptidoglycan hydrolase (VAPGH) encoded by the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88], and three different fusion proteins created between HydH5 and lysostaphin has been assessed. The lytic activity of the five proteins (HydH5, HydH5Lyso, HydH5SH3b, CHAPSH3b and lysostaphin) was confirmed using commercial whole extended shelf-life milk (ESL) in challenge assays with 104 CFU/mL of the strain S. aureus Sa9. HydH5, HydH5Lyso and HydH5SH3b (3.5 µM) kept the staphylococcal viable counts below the control cultures for 6 h at 37°C. The effect is apparent just 15 minutes after the addition of the lytic enzyme. Of note, lysostaphin and CHAPSH3b showed the highest staphylolytic protection as they were able to eradicate the initial staphylococcal challenge immediately or 15 min after addition, respectively, at lower concentration (1 µM) at 37°C. CHAPSH3b showed the same antistaphyloccal effect at room temperature (1.65 µM). No re-growth was observed for the remainder of the experiment (up to 6 h). CHAPSH3b activity (1.65 µM) was also assayed in raw (whole and skim) and pasteurized (whole and skim) milk. Pasteurization of milk clearly enhanced CHAPSH3b staphylolytic activity in both whole and skim milk at both temperatures. This effect was most dramatic at room temperature as this protein was able to reduce S. aureus viable counts to undetectable levels immediately after addition with no re-growth detected for the duration of the experiment (360 min). Furthermore, CHAPSH3b protein is known to be heat tolerant and retained some lytic activity after pasteurization treatment and after storage at 4°C for 3 days. These results might facilitate the use of the peptidoglycan hydrolase HydH5 and its derivative fusions, particularly CHAPSH3b, as biocontrol agents for controlling undesirable bacteria in dairy products.
Highlights
Staphylococcus aureus is a bacterial pathogen responsible for a wide range of human and animal infections, including food poisoning caused by the ingestion of enterotoxins produced in food by enterotoxigenic strains [1,2]
We have described the virion-associated peptidoglycan hydrolase (VAPGH) HydH5 encoded by the S. aureus phage vB_SauS-phiIPLA88
Total viable bacterial counts were below the detection limit (,10 CFU/ml) in extended shelf-life milk (ESL) milk, whereas about 7.086104 CFU/ml and 3.896101 CFU/ml were detected in raw and pasteurized milk, respectively
Summary
Staphylococcus aureus is a bacterial pathogen responsible for a wide range of human and animal infections, including food poisoning caused by the ingestion of enterotoxins produced in food by enterotoxigenic strains [1,2]. It has been shown that antimicrobial synergy exists in vitro between some phage endolysins and antibiotics or antimicrobial peptides of bacterial origin against S. aureus [18,23,24] In this regard, the in vitro synergy observed between phage lytic proteins and lysostaphin was recently expanded to include the in vivo protection of murine mammary glands from an S. aureus challenge [25]. There is a largely untapped group of phage lytic proteins the virion-associated peptidoglycan hydrolases (VAPGHs) that are involved in local cell-wall degradation to facilitate the injection of phage DNA into the cell cytoplasm [27] These PGHs have been reported to be encoded by phages infecting S. aureus [28,29] and other bacterial species [30,31,32,33]. We have assessed the antimicrobial ability of HydH5 and its derivative fusion proteins in milk, in order to explore new biopreservation strategies to effectively inhibit S. aureus growth in dairy products
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