. As more strains of bacteria and other microbes evolve defenses against available drugs, more patients run the risk of contracting infections that defy treatment.

Now, 91Ě˝»¨ researchers offer new insights into measures currently used to control the spread of antibiotic resistant bacteria and other infectious agents in health care facilities.

in Environmental Science & Technology, the team studied the efficacy of nine common disinfectants used in health care facilities or households — such as ethanol, hydrogen peroxide, benzalkonium chloride and UV light — against three well-known strains of antibiotic-resistant bacteria. The researchers first evaluated how successfully each disinfectant killed (or more accurately “inactivated”) the bacteria.

Then the team went a step further. It also assessed the damage the disinfectants did to the root cause of the resistance: the bacterial genome itself. And while all the cleaners did a great job of stopping the spread of bacteria, the picture was very different when the team zeroed in on DNA.

“What we’re learning is that it’s not just the bacteria that we need to deal with in hospitals and elsewhere. It’s also the behavior of their DNA in these environments,” said lead author , who completed this research as a 91Ě˝»¨doctoral student in the civil and environmental engineering department and is now an assistant professor at Tongji University.

Within bacterial cells, the source of antibiotic resistance is specific genes — individual portions of DNA — that instruct a cell to protect itself against certain antibiotics. Modern disinfectants do an impressive job of stopping bacterial cells in their tracks, but a bacterium’s genes may survive even the death of the cell. And, thanks to a trick called “,” genes from one bacterium — even if that bacterium has been killed — can sometimes find their way into a new living bacterium, thus passing on antibiotic resistance.

In short, stopping the bacteria themselves isn’t always enough to prevent the creep of resistance.

“Increasingly, environmental engineers are thinking about and treating resistance genes as an emerging contaminant,” said He. “In public health settings such as hospitals, we might disinfect and sterilize an operating room to remove any bacterial contamination, but what if resistance genes survive? They could potentially reach other bacteria and contribute to more dangerous antibiotic resistant hospital-acquired infections.

“Our previous work has demonstrated resistance genes can stay active in horizontal transfer after water and wastewater disinfections, which led us to wonder whether similar things could occur in health care and personal-care disinfection practices.”

The experiment pitted the nine disinfectants against three kinds of antibiotic-resistant bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), the microbe responsible for life-threatening staph infections. Researchers placed samples of the bacteria in different environments, mostly as dried drops on stainless steel and nonstick surfaces that are common in hospitals and at home. They then applied the disinfectants and measured the effects on both the bacterial cells and the genes in question.

As expected, the disinfectants did a great job of stopping the bacteria. However, most had a negligible impact on the resistance-conferring genes. The DNA survived largely intact, and it was free to find its way into new bacteria.

There were some positive and negative standouts, though frequently not the cleaners the team expected.

“Chlorine, under the conditions we tested, seemed to be less effective against DNA than we originally anticipated, whereas another common cleaner called phenol, which we didn’t think would be effective, actually ended up working relatively well in some cases,” said senior author , a 91Ě˝»¨associate professor in the civil and environmental engineering department.

The winner in many of the experiments was UV light, which did significant damage to the offending genes — though ultimately less damage than the team anticipated.

“UV irradiation seems to be one of the more effective approaches to both inactivating bacteria and degrading their DNA,” Dodd said. “We know that UV light directly damages DNA, so we weren’t necessarily surprised to see it perform well here. But it was a welcome result nonetheless.”

The researchers were quick to point out that existing disinfection regimens in hospitals are still effective and critical for preventing the spread of disease.

This work can help researchers home in on the tools that offer the best one-two punch against problematic bacteria and their genes. Moving forward, the team wants to learn more about how best to optimize these cleaners’ effects, especially when new factors like ambient temperature, humidity and density of bacterial cells are taken into account. But, the results from this paper could already help hospitals refine their disinfection protocols.

“If you know you have a patient in a hospital or other health care facility who’s infected with an antibiotic-resistant pathogen, I think we do have enough evidence at this stage to suggest trying certain disinfectants over others when cleaning surfaces or instruments that the patient may have been in contact with,” said Dodd. “For example, UV light could be a good choice, whereas benzalkonium chloride might not be.”

Additional co-authors on this paper are , a former 91Ě˝»¨graduate researcher who is now a postdoctoral researcher at Gwangju Institute of Science and Technology; , a former 91Ě˝»¨research assistant who is now an associate professor at Gonzaga University; , a former 91Ě˝»¨graduate researcher who is now a medical resident at the 91Ě˝»¨School of Medicine; , a former 91Ě˝»¨guest researcher who is now a research professor at Gwangju Institute of Science and Technology; , a 91Ě˝»¨professor in the environmental and occupational health sciences department; , 91Ě˝»¨professor emeritus in the environmental and occupational health sciences department; and , a professor at the Gwangju Institute of Science and Technology.

This research was funded by the National Science Foundation, the National Natural Science Foundation of China, the Fundamental Research Funds for the Central Universities and the Allen & Inger Osberg Endowed Professorship.

For more information, contact Michael Dodd at doddm@uw.edu and Huan He at huanhe@tongji.edu.cn

Each year at least 2 million Americans are infected with bacteria that cannot be treated with antibiotics, and at least 23,000 of these people die, according to the

These bacteria can end up in our water, which is why we use disinfectants to kill or stop them from growing to treat both waste and drinking water.

But so far few researchers have looked at whether these treatments are effective in removing the genes that encode for the traits that make these bacteria resistant to antibiotics. Some researchers are concerned that, even after treatment, non-resistant bacteria could still become resistant by picking up intact genes left over from damaged antibiotic resistant bacteria.

Although it’s not clear if this is currently happening, researchers want to be prepared for this scenario. So a team at the 91Ě˝»¨ tested how well current water and wastewater disinfecting methods affect antibiotic resistance genes in bacterial DNA. While these methods work well to deter bacterial growth, they had varied success in either degrading or deactivating a representative antibiotic resistance gene.

The researchers recently in the journal Environmental Science & Technology and are developing a model for proper treatment of any antibiotic resistance gene.

“DNA is not in itself particularly toxic or harmful. But it’s important to consider its fate once it’s in the environment because it can potentially spread undesirable traits into bacterial communities,” said corresponding author , an associate professor in the UW’s civil and environmental engineering department. “We have been finding more and more medically relevant antibiotic resistance genes in the environment.

“The recognition that these genes are present in the environment isn’t new — other groups have already provided a great deal of information on their behavior as environmental contaminants. What’s unique about our work is that we’re focusing on really unraveling and characterizing how a variety of disinfection processes influence the fate of such genes, so we can better understand how these different treatments affect antibiotic resistant bacteria and their DNA in our water.”

Current water treatment plants use a variety of disinfecting methods. Most involve exposing water to UV light or to chlorine- or oxygen-containing compounds, such as chlorine by itself or ozone.

To determine how these methods affect both bacteria and antibiotic resistance genes, Dodd and his team used a model system: a harmless soil bacterium called Bacillus subtilis. The team worked with a strain of B. subtilis that overproduced a gene, called blt, which makes a protein that lets B. subtilis pump antibiotics out — making the bacterium resistant to a variety of common antibiotics.

The researchers exposed the bacteria to different disinfecting methods and then monitored two things: how well treated bacteria grew when exposed to antibiotics and whether the gene inside the bacteria was damaged.

“As we expected, all of the treatments we looked at were successful in disrupting bacterial viability,” said first author , a 91Ě˝»¨civil and environmental engineering doctoral student. “But we saw mixed results for DNA damage.”

At typical exposures used for water treatment, three methods showed greater than 90% degradation or deactivation of the gene: UV light, ozone and chlorine. The team determined that these three methods are largely successful in preventing the spread of antibiotic resistance by both deactivating the bacteria and damaging the resistance gene.

But two other disinfectants called chlorine dioxide and monochloramine showed barely any damage to the gene.

“We found that these two methods degrade DNA so slowly that almost nothing has happened during the amount of time water is exposed under typical treatment conditions,” said He. “In fact, we found that DNA from bacteria treated with chlorine dioxide and monochloramine retains the ability to transfer antibiotic resistance traits to non-resistant bacteria long after the original bacteria are killed.”

Currently the team knows how quickly these disinfecting methods affect the gene used in the study. Now the researchers are developing a model that would allow them to estimate how quickly any gene would be damaged.

“If we can predict how effectively each disinfectant method would deactivate or degrade a specific gene, then we can better evaluate effective treatment strategies for degrading any antibiotic resistance gene that presents a concern,” Dodd said. “Disinfection processes are very important tools for preventing the spread of antibiotic resistance. We’re trying to better understand them so we can more effectively design and operate them in the future.”

Other co-authors are and at the UW; at Corona Environmental Consulting who completed this research while at the UW; at Stanford University who completed this research while at the UW; and at the Gwangju Institute of Science and Technology in South Korea.

This research was funded by the National Science Foundation.

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For more information, contact Dodd at doddm@uw.edu.

Grant number: CBET-1254929