By Naomi Lubick

High levels of antibiotic resistance have been found in bacteria that live downstream from a waste-water treatment plant in Patancheru, near Hyderabad in India.

Two years ago, Joakim Larsson of the University of Gothenburg, Sweden, and his colleagues reported that the treatment plant released drugs in its effluent water at levels sometimes equivalent to the high doses that are given therapeutically. The antibiotic-containing water reaching the plant came from 90 bulk pharmaceutical manufacturers in the region, near Hyderabad, they determined. The researchers wondered what might be happening to bacteria in the environment exposed to these drugs.

Serendipitous resistance

Bacteria can trade bundles of drug-resistance genes in mobile "cassettes" carried, for example, on small circles of DNA called plasmids, which can replicate themselves independently of the bacterium's chromosome. To find these DNA snippets, Larsson and his colleagues used a DNA sequencing approach called "shotgun metagenomics," to analyze all the DNA present in the effluent, the river water and the river sediments they had gathered in the earlier study. Postdoctoral researcher Erik Kristiansson developed a bioinformatics method to parse the information and search for evidence of known antibiotic-resistance genes.

In three sites downstream of the plant, the resistance genes made up almost 2 percent of the DNA samples taken there, the researchers report in PLoS ONE. Because only one or two genes out of the typical genome of around 5,000 genes are necessary to protect the bacterium, that's a lot of genetic resistance, says Dave Ussery, a microbiologist at the Technical University of Denmark, who was not involved in the work.

The researchers found resistance genes for a wide range of antibiotics but the relationship to the antibiotics present was not straightforward. For example, the most frequent resistance genes found were for a class of antibiotics called sulfonamides, but the researchers found no evidence of the drugs themselves. They hypothesize that this may be an instance where resistance to one group of drugs could provide resistance to others.

And despite detecting high concentrations of fluoroquinolones, a chemical class that includes the heavy-hitting antibiotic ciprofloxacin, the team found less evidence of resistance to these drugs downstream than upstream from the plant. The researchers suggest that the levels of fluoroquinolones in the downstream effluent were so high that they overpowered even the resistant bugs.

Finding resistance amid so much exposure to active drug ingredients "is not surprising," comments David Graham at Newcastle University, UK, who has studied sites in Cuba, for example, exposed to lower levels of medical waste. "But in a way, it's sort of like a beaker experiment," he says, that tests the worst-case scenario, only this is "in a natural system. That's what makes it useful."

Round the world ticket?

The spread of antibiotic-resistance genes is complicated, says Björn Olsen, an infectious-disease specialist at Uppsala University in Sweden. Olsen says resistance hotspots like the one at Patancheru could end up behaving like a volcanic eruption: "the cloud is going to drop down somewhere else, not just around the sewage plant". His team recently documented multidrug-resistant Escherichia coli in the feces of birds that migrate to the Arctic.

The presence of high levels of antibiotics in the river and its sediments might not actually be the factor driving the genetic resistance, warns Sheridan Haack, a microbiologist at the U.S. Geological Survey (USGS) in Lansing, Mich. Resistance genes already present in bacteria from human waste, or developed by the bacteria used in the plant's treatment stages to break down the sludge, could be swept along with the effluent into the river.

Whatever the reason, the high rates of resistance found by Larsson and his team are interesting, says Haack, and the team seems to be the first to combine this metagenomic approach to environmental samples with bioinformatics. She says that Larsson's team should now use more traditional, specific searches for resistance genes and for the surviving bacterial species that are carrying those genes.

Ussery cautions that even if the bacteria found are not dangerous to humans or other animals in the area, they may transfer their resistance genes to bacteria that are. "They need to know who's there," says Ussery, to identify which species are surviving and which are the sources of genetic resistance.