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Robin Kelleher Davis, Pablo Argueso; Antimicrobial Activity Detected in Ocular and Salivary Secretions from Marine Mammals. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4163.
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© ARVO (1962-2015); The Authors (2016-present)
Marine mammal species’ first ancestors evolved from the land to the sea more than 30 million years before man’s ancestors started walking upright, a mere 3-6 million years ago. We hypothesize that the marine mammal tear film has evolved unique mechanisms to cope with the harsh environment of the sea. We undertook this study to determine whether mucosal secretions, specifically tears and saliva, from marine mammals have antimicrobial activity.
Tear and saliva samples from bottlenose dolphins, manatees, and humans were collected using IRB and ACUC approved protocols. Samples were lyophilized and reconstituted in sterile water (vehicle). Antimicrobial activity was tested against strains of E. coli (DH5α, New England Biolabs) and P. aeruginosa 6294 (generous gift of Dr. Mihaela Gadjeva, Brigham and Women's Hospital, Boston, MA). Bacterial cultures were grown in LB (Luria-Bertani) media overnight at 37oC, diluted to an optical density at 600 nm (OD600) of 0.2, and aliquoted to 96-well plates containing vehicle, gentamicin (0.1 mg/ml), or sample in triplicate, in a total volume of 120 ul per well. Final protein concentrations in the assay were 12-39 ug/ml for dolphin tear (n=3), 115-370 ug/ml for dolphin saliva (n=5), 30-51 ug/ml for manatee tear (n=3), and 108 ug/ml for human tear (n=3), respectively. Plates were incubated with shaking in a water bath at 37oC and bacterial growth was monitored by a standard turbidity assay with OD600 measurements taken at hourly intervals.
Within two hours of incubation, growth of E. coli was substantially inhibited (p<0.05) by samples of dolphin tear (OD600 = 0.460 +/- 0.025), dolphin mouth mucus (OD600 = .478 +/- 0.230), manatee tear (OD600 = 0.549 +/- 0.313), and human tear (OD 600 = 0.543 +/- 0.051) as compared to vehicle alone (OD600 = 1.012 +/- 0.141). The antibiotic, gentamicin, also inhibited bacterial growth (OD600 = 0.445 +/- 0.086). P. aeruginosa growth was slower and was not affected significantly at 2 hours by any of the samples tested. At 22.5 hours, 2 of the 5 dolphin saliva samples inhibited P. aeruginosa growth by 76 and 79% respectively, however none of the other agents had an effect.
It appears that under the conditions of this study, marine mammal tears and saliva do exhibit antimicrobial activity. Future studies will focus on determining which factors in these secretions are responsible for inhibiting bacterial growth.
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