Abstract

The first ancestors of marine mammals evolved from land to the sea more than 30 million years before man's ancestors began walking upright, which was a mere 3–6 million years ago. We hypothesize that marine mammal mucosal surfaces have 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 37°C, diluted to an optical density measured 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 µl per well. Final protein concentrations in the assay were 12–39 µg/ml for dolphin tear (n = 3), 115–370 µg/ml for dolphin saliva (n = 5), 30–51 µg/ml for manatee tear (n = 3), and 108 µg/ml for human tear (n = 3), respectively. Plates were incubated with shaking in a 37°C water bath, and bacterial growth was monitored using 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 reconstituted samples of dolphin tear and saliva, manatee tear, and human tear as compared to vehicle. P. aeruginosa growth was slower and was not affected significantly by any of the samples at 2 hours; however, 2 of the 5 dolphin saliva samples inhibited P. aeruginosa growth by 76 and 79% respectively at 22.5 hours. The antibiotic, gentamicin, also inhibited bacterial growth.

Under the conditions of this study, marine mammal tears and saliva exhibited antimicrobial activity. In future studies, we will focus on determining which factors in these secretions are responsible for inhibiting bacterial growth.

Acknowledgments

We thank Dolphin Quest Oahu, Oahu, HI; Gulfworld Marine Park, Panama City Beach, FL; University of Florida, FL; Aquarium of Niagara, Niagara Falls, NY; and New England Aquarium, Boston, MA, for their very generous donations of marine mammal samples for this project; and David A. Sullivan, Schepens Eye Research Institute and Massachusetts Eye and Ear Infirmary, Boston, MA, for advice. Grant Support: NIH EY05612; Arey's Pond Boat Yard, S. Orleans, MA.