Plastic surgery patients were getting antibiotic-resistant infections, and doctors didn’t know why. It took a University of Connecticut microbiologist to unravel the mystery – turning up clues that connected the dots between sterile American hospitals, European leech farms, and poultry husbandry.

The research by Joerg Graf and colleagues, published today in mBio, provides proof that tiny levels of antibiotics found in the environment can result in antibiotic-resistant bacteria. Drug resistance is a major and growing concern in healthcare, given that it could lead to untreatable illnesses.

The Leech Lead

Graf, a professor in UConn’s Department of Molecular and Cell Biology, is intimately familiar with leech guts, where bacteria can live without making the animals sick. He’s examined the contents of thousands of leeches over the years – both those from the farm and those from the wild.

Graf thought he knew the bacteria in their digestive systems just as well. But in 2011, he got a surprise.

One of his graduate students, Sophie Colston, was having trouble growing a strain of Aeromonas bacteria in the leeches. They would feed the Aeromonas strain to a leech and later check how well it was growing. This time, the Aeromonas strain was having trouble.

At the same time, plastic surgeons began to report problems with patients getting infected with Aeromonas bacteria resistant to ciprofloxacin (Cipro), an important antibiotic. Normally they are easily treated with Cipro.

Graf’s interest was piqued because the plastic surgery patients had all been treated with a regular protocol of farm-raised leeches to improve blood flow at surgical sites. Leeches in medicine are raised on specialty farms, fed a controlled diet, and used just once on a single patient, so they seemed an unlikely source of contamination.

“Without ever having been in a hospital, without having seen a patient, these leeches contained Cipro-resistant bacteria,” Graf said, standing in front of a jar containing a mob of leeches – long flat ribbons, brown on top and green below, swimming, squirming, and climbing through the water, then extending weirdly into space, casting for prey. How could that happen, he wondered.

The Chicken Came First

Leeches are used in medicine because they are effective at increasing blood circulation and breaking up blood clots.

When they are being raised, leeches are hungry for blood. And they are not picky about where it comes from. At one point the source was cows but, when the mad cow disease outbreak made it undesirable to use cattle blood, a major medical leech farmer in Europe switched to feeding the leeches poultry blood instead. In Europe, the use of antibiotics has not been banned in poultry farms as it has in the U.S.

So, Graf and his team, including Colston and Lidia Beka, another graduate student, analyzed the gut contents of leeches from the farm that used poultry blood.

They found their answer: Traces of both ciprofloxacin and enrofloxacin, Cipro’s veterinary counterpart.

But the amount of antibiotic present was vanishingly low. Just around 0.01 micrograms per milliliter, four hundred times less than the concentration a bacteria must survive in order to be considered “resistant.” Could such trace levels of Cipro, hundredths of a microgram, really be causing antibiotic resistance?

Graf and Beka and their colleagues isolated strains of Aeromonas from leeches contaminated with antibiotics, and sequenced their genomes. Two colleagues, Matt Fullmer and Peter Gogarten, confirmed that they contained the three bits of DNA, two genes with mutations and a plasmid, necessary for resistance to Cipro.

Indeed, when the Cipro-resistant Aeromonas were grown alongside the test strain of Aeromonas in a clean lab medium or inside a leech, the test strain grew all over them. But if there was even a tiny bit of antibiotic added into the mix, the Cipro-resistant variety dominated.

“This was the first time such low levels of antibiotics were observed doing this in the natural environment,” Graf says.

This is worrisome, because ciprofloxacin and related drugs don’t break down very well in the environment. They persist. They’re found in hospital wastewater, in effluent from pharmaceutical manufacturers and farms, and even sometimes in sewage. And, apparently, in poultry blood.

The research of Graf and colleagues demonstrates that levels of the antibiotics circulating in the environment matter – even the tiniest of levels.

This research was funded through a grant from the National Institutes of Health’s National Institute of General Medical Sciences.