Laboratories currently use plate culturing methods to detect pathogens, but the process is lengthy and may not deliver results in a useful time period. The researchers' goal is to develop a simple, inexpensive test that could provide results in a matter of a few hours and be used as a tool by growers to make timely risk assessments.
"This will give growers a better idea that something might be a problem so they can take action on it," Nugen said.
Collecting water for pathogen testing is fairly straight forward, but the challenge has been sampling the produce itself.
In order to conduct an assay, the bacteria must first be separated from the plant material.
"How can we pretreat samples if we have something like produce, which might have internalized pathogens or biofilms?" Nugen said.
To do that, the researchers harnessed enzymes that digest the leafy plant material. Nugen said this process results in recovering twice as many Salmonella organisms compared to just washing plant leaves and collecting the rinse water.
Users simply place a leaf sample in a small plastic bag that contains enzymes and incubate it for about 1.5 hours.
Users would then squeeze a small liquid sample through a filter and place it in a tube with bacteriophages - viruses that are harmless to humans but infect specific bacterium, such as Salmonella or E. coli. Some phages are so specific they will only infect one bacterium serotype while others will infect a broader range of serotypes within an individual species.
Phages also will only infect and replicate in viable bacteria, ensuring that non-viable organisms are not detected. This distinction is useful if prior mitigation steps, such as chlorination, have already been used.
The phages used in the test were engineered to insert a particular gene into the bacteria. Nugen credited Dr. David Sela, an assistant professor of microbiology at the University of Massachusetts, Amherst, for his expertise with the genetic engineering of the phages.
Ideally, Nugen said, the test would use a cocktail of about 10 to 15 phages, with some having a very specific host range and some very broad.
The engineered phages seek out and bind to target organisms while at the same time initiating infection and replication. When the process is completed, the bacteria cell wall lyses or breaks down, releasing the replicated engineered phages - a process known as amplification. The phages are engineered with a reporter enzyme, which can be detected in the last step of the process.
This final step involves putting a few drops of reagent in the tube, waiting a few minutes and inserting a dip stick similar to those used in over-the-counter pregnancy tests.
Known as a lateral flow assay, the dipstick can quantify the reporter enzyme expressed by bacteria during the infection with the engineered phages. By using multiple phages/enzymes, several bacteria can be targeted simultaneously.
If the reactive portion of the dipstick remains white, it means that a very low number or no target organisms were detected.
Nugen was quick to point out that just because a user receives a negative result doesn't mean pathogens aren't present. The test reflects one sample collected from one location at a single point in time. Instead, he said the tests should be viewed as another tool to help growers make more informed decisions.
"They might be deciding about using irrigation water from a river and are worried about a gross contamination that might have occurred upstream," he said. "Or they're routinely using produce rinse water, and they might want to check to see if there are different time periods when Salmonella is showing up."
The researchers have put their rapid assay to real-world tests on samples of rinse water collected from four Massachusetts commercial produce operations by Dr. Amanda Kinchla, a University of Massachusetts Extension food safety specialist and project collaborator.
"We wanted to know the highs and lows in salinity and pH to make sure our phages would bind and work," Nugen said.
Project abstract may be found on the Center for Produce Safety website: