Antibacterial properties of serum from the American alligator (Alligator mississippiensis)

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Abstract

Treatment of alligator (Alligator mississippiensis) and human serum samples with Escherichia coli resulted in a time- and concentration-dependent inhibition of bacterial proliferation. When inoculated with E. coli, alligator serum exhibited 10-fold lower bacterial survival rates after 1 h than human serum. In addition, the antibacterial spectrum of alligator serum was shown to be much broader than that of human serum, with growth inhibition occurring in 100% of bacterial strains tested (compared to only 35% for human serum). Additional results showed that the antimicrobial activities of alligator serum could be completely inhibited by preincubation with proteases, indicating the proteinaceous nature of the antimicrobial activities. Furthermore, incubation of alligator serum at 56 °C for 30 min (classical human serum complement inactivation conditions) obliterated all antimicrobial properties of the alligator serum. The antibacterial activities occurred relatively quickly in vitro, with significant activity occurring within 5 min of inoculation with E. coli and maximal activity at 20 min. Also, the antimicrobial activity exhibited temperature dependence, with a substantial decrease in activity below 15 °C. These data suggest that the antimicrobial properties of alligator serum may be due to an active serum complement system.

Introduction

Eukaryotic organisms must continuously defend themselves against infiltration and colonization by microorgamisms. Host defense occurs via complex mechanisms and can be divided into two distinct, but interrelated, types of responses: acquired immunity and humoral (innate) immunity. Acquired immunity requires prior exposure to a specific antigen before a full immunological assault can be established by the host organism. In addition, the acquired immune response is complex and can often take several days to become fully activated. The humoral immune response comprises a significant portion of the immune system and acts as an initial defense mechanism against microbial growth shortly after infection occurs. These innate defense responses are activated shortly after exposure and act to slow or stop an infection in the initial stages so that the acquired immune response can be initiated. Also, the innate immune system functions in the activation of the acquired immune system by generating chemotactic factors and producing cytokines that initiate the development and maturation of specific T-cell and B-cell populations.

Anecdotal evidence suggests that alligators are resistant to microbial infections. These animals often sustain serious injuries, including open wounds, due to interspecies fighting and collisions with boat propellers. However, despite the fact that they live in marsh environments, which may harbor potentially infectious microorganisms, alligators often heal without signs of infection. Despite the propensity of alligators to resist microbial infection, the mechanisms of immunity are not well characterized. The primary goal of this study was to investigate and characterize the antibacterial properties of the serum of the American alligator in vitro.

Section snippets

Chemicals and biochemicals

Nutrient broth and nutrient agar were purchased from ISC Bioexpress (Kaysville, Utah). Lyophilized ATCC bacterial strains were purchased from Chrisope Technologies (Lake Charles, LA). The following ATCC-registered strains were used: Klebsiella oxytoca (49131), Providencia stuartii (33672), Escherichia coliform (25922), Proteus mirabilis (43607), Enterobacter aerogenes (49469), Salmonella typhimurium (14028), Pseudomonas aeruginosa (27853), Citrobacter freundii (C109820), Shigella sonnei

Results

Inoculation of nutrient broth with approximately 106 CFUs of E. coli resulted in a time-dependent increase in bacterial proliferation as measured by spectrophotometry at 430 nm (Fig. 1a,b). Inclusion of different concentrations of human or alligator serum in the broth produced a concentration-dependent decrease in bacterial growth. Inoculation of 10% alligator serum reduced bacterial growth by 84% at 3 h, compared to only a 47% reduction by human serum (Fig. 1a,b). The addition of 10, 25, 50,

Discussion

Exposure of eukaryotes to pathogenic microorganisms results in stimulation of complex host defense mechanisms. The spectrum of protective immunological mechanisms includes the induction of innate immune mechanisms. Innate immunity is non-specific in nature and requires no previous exposure to a specific antigen (Hoffman et al., 1999).

The immune systems of crocodilians have not been well-characterized. However, several reports have described the presence of cellular components of the immune

Acknowledgements

The authors wish to acknowledge the contributions of Dr William Taylor of the Department of Biological and Environmental Sciences at McNeese State University. We also wish to thank Damon Thibodeaux, Katerina Cabello, Brett Young and Robert Van Gossen for their technical assistance. In addition, we wish to acknowledge the technical support of Phillip ‘Scooter’ Trosclair with the Louisiana Department of Wildlife and Fisheries. This research project was supported by the Shearman Research

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