Antibiotic factors in crocodile and alligator blood - Part 1

In response to the discovery this week by Dr Mark Merchant (McNeese State University) that American alligators have powerful antibiotics in their blood, I've decided to write a short history of our research into crocodile blood and the discovery of the antibiotic "crocodillin" back in 1998. It's an eventful tale, so in the nature of all good cliffhangers I'm going to split it into at least two parts. Or perhaps that's because I don't have time to write the whole thing at once!

So, let's travel in time back to 1998 (don't worry, we're not staying forever). I was involved in a research project that I thought would have major repercussions around the world, because what we discovered was potentially ground-breaking. I was approached by BBC Science to suggest an interesting research project that they could fund, and film. Did I have an interesting project, oh boy!

I had long been intrigued by the immune system of crocodiles, having seen numerous examples where they generously gave each other some pretty horrific physical injuries - slashes and gashes exposing muscle and bone, ripping off legs, and biting off tails. Just a typical night down the swamp. And yet I couldn't recall ever seeing such wounds become infected. Put yourself in the same position: you've just had your arm bitten, you're lying in filthy water full of bacteria, and you just know it's going to get infected and you might even lose your arm. Not so with crocodiles.

What was it about crocodiles, I wondered, that gave them such potent immune systems? And was it something we could use in human medicine to treat our own disease?

The BBC Science producer, Jill Fullerton-Smith, wasn't particularly interested in this at first. Perhaps it didn't feature enough heads being ripped off wildebeests (the usual staple of TV documentaries about crocodiles)? Or perhaps she needed time to think about it? Yes, that was it. In fact, she apparently woke up one night a couple of weeks later, sat bolt upright in bed, and realised what a great question it was!

So a few months later with a BBC camera crew in tow, we started catching both captive and wild crocodiles to get a few ml of blood. We invited a few "celebrities" along to make the show more exciting (as if a British guy catching crocs in northern Australia wasn't exciting enough!) and sent the blood across to Dr Gill Diamond in New Jersey. I remember suggesting Gill because I'd recently read about his work looking at Komodo Dragon blood, investigating why those lizards with particularly unsavoury saliva didn't infect each other during fighting. He'd developed a technique to fractionate serum into its constituents for amplification and analysis. In short, he seemed like the ideal person for the job of looking at our crocodile blood. After preparing the samples and sending Gill red blood cells, white blood cells (leucocytes) and serum, we crossed our fingers and waited. We actually had to send the samples a second time because the courier company screwed up and let everything defrost and rot. Good job we only sent half the samples in case of such an event!

A few weeks later I was sitting in my office and the phone rang, it was the BBC producer Jill. I could tell she was excited. Gill Diamond had isolated the active constituent in the blood and tested it against a range of bacteria - it killed them all. He then thought he'd try for the jackpot and tested it against MRSA (methycillin-resistant Staphylococcus aureus) otherwise known as Golden Staph - resistant to all known antibiotics.

The constituent in the crocodile blood killed the MRSA bacteria.

I don't think it hit me straight away just how exciting it all was. I think Jill was expecting me to dance around the room punching my fists into the air, but perhaps it was my combined British reserve and scientific scepticism that kept it in check. For a while at least.

The results of our exploits were shown in the BBC documentary "Secret Life of Crocodiles" (also known as "Crocodile Secrets" on the Discovery Channel).

Gill's team and I were able to present these findings at the IUCN-SSC Crocodile Specialist Group meeting in Florida in 2002, and the abstract appeared in the proceedings:

Britton, A.R.C., Diamond, G., Laube, D. and Kaiser, V., 2002. Antimicrobial activity in the blood of the saltwater crocodile (Crocodylus porosus). [abstract presented by G. Diamond at the 16th Working Meeting of the IUCN/SSC Crocodile Specialist Group, Florida, USA, October 2002]

The saltwater crocodile shows a low incidence of infection from serious injuries sustained during intraspecific aggression, in spite of the microbe-laden environment in which it lives. This suggests a well-developed innate immune system, which provides a rapid, non-specific first line of host defense. In other aquatic species such as amphibians and fish, this defense is found in the mucous skin secretions as antimicrobial peptides. Due to the anatomy of the crocodile, we reasoned that a homologous defense would be found in the circulatory system, either as soluble factors or as agents expressed in phagocytic cells. To address the first hypothesis, we extracted serum from wild saltwater crocodiles to isolate naturally occurring antibiotics. The serum was maintained at -80C until fractionation. Serum was fractionated by Reverse-phase HPLC on a C-18 column with a 0-60% acetonitrile gradient, and fractions were assayed for antibiotic activity against E. coli in a modified radial diffusion assay. Preliminary results indicated strong antibiotic activity in several fractions. We have taken a single fraction, eluting at 13% acetonitrile, for further characterization. Based on our initial observations, we predict that the crocodile exhibits both peptide and non-peptide based antimicrobial activity in its blood.

In our next exciting episode, we travel forward in time to 2002 to meet Supercroc!