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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Biosciences & Biotechnology Laboratory » Research » Publications at this Location » Publication #322519
Title: Citrus-derived oils inhibit Satphylococcus aureus growth and alter its interaction with bovine mammary cells

Author
item FEDERMAN, C - University Of Maryland
item ALMARIO, J - University Of Maryland
item SALAHEEN, S - University Of Maryland
item MOYES, K - University Of Maryland
item Elsasser, Theodore
item BISWAS, D - University Of Maryland

Submitted to: Infection and Immunity
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/21/2015
Publication Date: 5/5/2016
Citation: Federman, C., Almario, J.A., Salaheen, S., Moyes, K.M., Elsasser, T.H., Biswas, D. 2016. Citrus-derived oils inhibit Satphylococcus aureus growth and alter its interaction with bovine mammary cells. Infection and Immunity. J Dairy Sci. 2016 May;99(5):3667-74. doi: 10.3168/jds.2015-10538.

Interpretive Summary: While traditional antibiotic therapy has long been the standard treatment for bacterial infections, recent developments in the emergence of bacteria that are resistant to the killing effects of antibiotics have raised concerns over what constitutes prudent use of these valuable drugs. The wide spread use of antibiotics in food animals in particular has come under tremendous scrutiny and with this concern, the White House has placed a high priority on research that can develop alternatives to traditional antibiotics for prevention and treatment of common bacterial diseases in livestock. The present research was undertaken to look into the potential for a natural citrus oil, known for its ability to clean surfaces of contamination, to have potential as an alternative to antibiotics in the prevention of attachment of bacteria to epithelial cells and thus be of benefit in applications in food animal agriculture. When tested at several relatively low concentrations, citrus oil was effective in preventing both the growth of a serious test bacteria Staphylococcus aureus as well as the attachment and invasion of skin and organ epithelial cells. The citrus oil had no detrimental effects on the growth and health of the cells at the doses used that were effective in the capacity to neutralize the bacteria. We conclude that the naturally-derived components of citrus oil may be affective in many uses in animal agriculture to limit bacterial disease spread and save the use of antibiotic drugs for the most important purposes.

Technical Abstract: This experiment examined the effects of cold-pressed, terpeneless citrus oil (CDO) on growth of Staphylococcus aureus, which a major cause of contagious bovine mastitis, and invasion of epithelial cells as modeled with bovine mammary cells (MAC-T). The broth dilution method (Muthaiyan et al., 2012) was implemented to determine in vitro minimum inhibitory concentration, using CDO concentrations ranging from 0.0125% to 0.4% with half step dilutions. Growth inhibition was examined by adding 0.00%, 0.05%, 0.025%, 0.0125%, and 0.00625% CDO to 100,000 CFU per ml S. aureus in nutrient broth, and diluting and plating on to nutrient agar to count colonies. Effects of CDO on epithelial cell attachment and invasion was assessed by agar plating of supernats of lysed cells following incubation with bacteria. The effects of CDO on pre-formed biofilm stability was assessed in a 96 well plate format; 10 million CFU per ml S. aureus was allowed to form a biofilm and then treated with 0.00625% CDO, or 0.0125% CDO in 1% dimethysulfoxide (DMSO). Biofilm integrity was calculated on the basis of residual crystal violet absorption at 540 nm. Lastly, cytotoxic effects of CDO on epithelial cells was examined at various concentrations of CDO using the MTT assay. We observed that the minimum inhibitory concentration of CDO to inhibit the growth of S. aureus in vitro, was 0.025% CDO. A time kill curve for CDO’s action on S. aureus over 4 h was generated. CDO was capable of inhibiting all growth of S. aureus after 3 h incubation at concentration of 0.25% or after 2 h incubation at concentrations of 0.05%. It was also observed that CDO had no effect on pre-formed biofilms except at a concentration of 0.05%, in which there was a modest reduction in the measured absorbance. In addition, CDO was capable of inhibiting association to and invasion of MAC-T cells by S. aureus after a 1 h treatment with CDO. CDO had no cytotoxic effects on cells at the concentrations tested and increased proliferation of epithelial cells at concentrations up 0.05%. In conclusion, CDO should be considered for further research as a preventative measure against or treatment of bovine mastitis.