|Rodriguez-Rubio, Lorena -|
|Martinez, Beatriz -|
|Garcia, Pilar -|
|Rodriguez, Ana -|
Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 17, 2013
Publication Date: January 24, 2013
Citation: Rodriguez-Rubio, L., Martinez, B., Donovan, D.M., Garcia, P., Rodriguez, A. 2013. Potential of the virion-associated peptidoglycan hydrolase HydH5 and its derivative fusion proteins in milk biopreservation. Applied Microbiology and Biotechnology. 8(1):e54828. Interpretive Summary: Problem-- There is a need for novel antimicrobials to fight pathogens that infect dairy cattle mammary glands (mastitis). Treatment of mastitis and the resultant reductions in both milk yield and quality cost the US dairy industry up to $2 billion in losses annually. The same antimicrobials would be helpful if they were effective in raw or pasteurized milk to enhance the food safety aspects of dairy products. Resistance development in milk pathogens is common, so novel antimicrobials are sorely needed. Accomplishment— This manuscript describes the use of thermostable phage (viruses that infect and kill bacteria) proteins to reduce or eliminate contaminating bacteria in both raw and pasteurized milk. Contribution of Accomplishment to Solving the Problem-- Currently antibiotic treatments of mammary gland infections (mastitis) in cattle are less than 50% effective. The first step in identifying novel antimicrobials for treating mastitis infections is to show that they are active in milk. This work describes a novel group of proteins from viruses that infect bacteria that are effective at eradicating or reducing the pathogen Staphylococcus aureus in milk. These proteins will likely be useful for both mastitis treatment but due to the heat and cold tolerance, they will also likely be useful in food safety uses. This is the first time that these proteins have been shown to be useful in milk, so could potentially be of great impact not just for staphylococcal infections, but other pathogens as well.
Technical 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 hass been assessed. The lytic activity of the four proteins (HydH5, HydH5Lyso, HydH5SH3b and CHAPSH3b) was confirmed in challenge assays using commercial whole extended shelf-life milk (ESL) contaminated challenged 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 throughout 6 h time at 37ºC. The effect is apparent just 15 minutes after the addition of the lytic enzyme, being the viable counts significantly different from 15 min after protein addition onwards. Of note, CHAPSH3b showed the highest staphylolytic activityprotection as it was able to eradicate kill the initial staphylococcalchallenge population in 15 min at lower concentrations at both 37ºC (0.99 µM) and room temperature (1.65 µM). No regrowth was observed, and not re-growth occurred thereafter for the remainder of the experiment (up to 6 hrs). CHAPSH3b activity (1.65 µM) was also assayed in raw (whole and skimmed) and pasteurized (whole and skimmed) milk. Pasteurization of milk clearly enhanced CHAPSH3b staphylolytic activity in both whole and skimmed milk at both temperatures. This effect was most dramatic, but particularly at room temperature as this protein was able to reduce S. aureus viable counts to undetectable levels immediately after addition with no and not re-growth was detected for the duration of the experiment (360 min) thereafter. Furthermore, CHAPSH3b protein is known to be heat tolerant and retained some lytic activity after pasteurization treatment and after storage at 4ºC 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.