Research and new technologies are striving to minimize the impacts of the relentless lurker that is Listeria.
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Investigators at North Carolina State University (NCSU), Raleigh, have completed a proof-of-concept study in which they identified several compounds that may be effective in minimizing Listeria’s virulence.
Simply stated, a proof-of-concept study demonstrates that a particular theory has the potential for practical real-world application.
In this case the process started with the key understanding that inhibiting a particular enzyme of Listeria, glucose-1-phosphate uridylyltransferase (GalU), leads to rather dramatic modifications of the bacterial cell surface, according to Paul Orndorff, PhD, professor emeritus of microbiology.
“From this point we determined that these chemical modifications in turn rendered Listeria much less virulent than it normally is, and thus less able to cause illness,” Dr. Orndorff says. The work was published in Molecular Informatics in March 2018.
Dr. Orndorff and his collaborators in the NCSU Department of Chemistry, postdoctoral researcher Melaine Kuenemann, PhD, and Denis Fourches, PhD, an assistant professor of computational chemistry, embraced the task of identifying potential compounds that could inhibit the function of GalU. Using computers and cheminformatics methods, Dr. Fourches and Dr. Kuenemann characterized, analyzed, and virtually screened more than 88,000 drug-like compounds using a technique called 3D molecular docking.
All those computations, which were based on the three-dimensional structure of the GalU protein and virtual representations of each compound, took several weeks to accomplish, Dr. Fourches mentions.
“Through computer modeling, we prioritized 37 compounds predicted to bind the GalU active site and thus looked promising enough to be tested in vitro,” he relates. “Of the 37, three compounds showed good experimental activity and were deemed effective enough to warrant further study. This is a great result, considering we had no idea what type of chemical could actually bind the GalU pocket.”
Moreover, all those compounds, including the less active ones, yielded important information about how their chemical structures relate to their activity in inhibiting GalU’s function, Dr. Fourches notes. “We were able to derive several predictive structure-activity relationships based on those 37 compounds, and these relationships will help us design even more effective GalU inhibiting compounds,” he elaborates. “This study shows that one can develop small molecules to shut down the activity of one specific bacterial enzyme, leading to the suppression of virulence. This is a completely new avenue, especially for fighting antibiotic-resistant bacteria.”
This is true research at the interface of several complimentary disciplines, Dr. Fourches emphasizes. “The synergistic use of artificial intelligence, molecular modeling techniques, and in vitro confirmation is a game-changer in the way we rationally design chemicals,” he points out.
The next step, Dr. Fourches says, would be using computers to virtually generate thousands of new analogues, virtually screening them, and then selecting another batch of 50 molecules to be tested experimentally to determine their impact on GalU. “Unfortunately, this project is not funded at the moment, even though direct commercial applications from this study are reachable,” he mentions. “We need more support from both federal agencies and commercial partners to get this research moving forward.”
The NCSU research also determined that inhibiting GalU made Listeria more vulnerable to cefotaxime, an antibiotic to which it has a natural resistance.
“This antibiotic susceptibility suggests the possibility of viable therapies that could combine a GalU inhibitor and a known antibiotic such as cefotaxime,” Dr. Orndorff says. “However, we believe if the GalU inhibitor is effective enough, the host, whether human or animal, should be able to eliminate the Listeria population without antibiotics. This holds promise to be a great solution for farmers striving for antibiotic-free livestock and poultry operations.”
More Robust Risk Assessment
To benefit the dairy processing industry, the Midwest Dairy Foods Research Center and the South Dakota Agricultural Experiment Station are funding researchers in the South Dakota State University (SDSU), Brookings, Dairy and Food Science Department, who, in a cooperative effort with a commercial ice cream and frozen desserts manufacturer, are developing models to more accurately predict the risk from Listeria.
To that end, SDSU food microbiology professor Sanjeev Anand, PhD, and doctoral student Neha Neha, MS, are focusing specifically on the recovery potential of any Listeria cells injured in a variety of ways.
Collaborating on the project is Gemechis Djira, PhD, an associate professor in the SDSU Department of Mathematics and Statistics, who is creating regression models.
“Our risk assessment models use both product matrix parameters and environmental considerations, such as storage temperature, duration, pH, and water activity, in addition to the potential levels of cross-contamination from the environment,” Dr. Anand relates.
This is important, Dr. Anand says, because recent cases of Listeria in frozen foods that have resulted in recalls, ranging from frozen vegetables to ice cream bars, have reinforced the need for better methods of gauging the risk of foodborne pathogen contamination in processing plants.
“Listeria contamination has been recently traced to niches in the food processing environment that harbor the bacteria,” he points out. “For example, Listeria contamination in one commercial ice cream plant was traced to bacteria on the spout of an ice cream freezer. This is not surprising, since Listeria is a cold-loving microorganism. Pasteurization and cooking kill this organism, but the bacteria can grow at temperatures 40 degrees Fahrenheit and above in refrigerators and can even survive freezing.”
Neha says that, although injured Listeria cells are not known to cause illness, they may have the ability to recover and repair themselves.
To better understand the risk from injured cells, she looked at the organism’s behavior in different types of ice cream mixes with total solid levels ranging from 36 percent to 45 percent. She spiked the samples with three levels of a nonpathogenic Listeria strain before pasteurization.
“Results showed that injured Listeria cells did not recover in the ice cream mix itself under the normal conditions of mix handling,” she reports. “Studies are currently underway to evaluate the influence of any handling abuse on the recovery potential of injured cells.”
To address the issue of cross-contamination in the manufacturing environment, the next step is to determine how Listeria builds up in the environment, what characteristics make this possible and how it resists cleanup. This phase of the study starts later in 2018, Neha notes.
“We want to understand what characteristics make it possible for Listeria to persist and recover,” Neha relates.
She is planning to do whole genome sequencing of the bacteria, with the goal of understanding the gene expression that leads to colonization. “This should be beneficial in comparing any resident strain of Listeria, which can form resilient biofilms in the harborage sites and is difficult to eradicate,” Neha says.
“If we gain more knowledge about injured cells and integrate that information among the variables for the product and the environmental site, we should be able to design more robust risk-assessment models,” Neha predicts. “Additionally, examining injured Listeria at a molecular level will help scientists worldwide design novel cleaning techniques that can eliminate the bacteria in the manufacturing environment and prevent their persistence.”
New Lab Assay
“Solus One Listeria is an assay offering next-day detection of Listeria species in environmental samples,” says Ray Wakefield, CEO of Solus. “This assay provides a negative or a presumptive positive result from a single enrichment step in less than 25 hours.”
The whole process is a selective enrichment followed by an immunoassay, Wakefield explains. “If you break this down, the enrichment incubation is 22 hours and the immunoassay is 2 hours and 45 minutes, hence a total of 24 hours and 45 minutes,” he elaborates.
According to Wakefield, benefits of Solus One Listeria, which is AOAC certified (PTM No. 051802), include a significant reduction of technician hands-on time. “High sample throughput can be achieved with a single instrument, giving the laboratory the ability to cope with fluctuating sample volumes, and also improving capacity to grow,” Wakefield relates.
Same-Shift Listeria Results
CERTUS, Chicago, Ill., expects to make commercially available on Sept. 15, 2018 a new in-house pathogen testing system that offers real-time detection and same shift results. To start, the CERTUS System is focusing on environmental Listeria detection, according to John Coomes, company president.
“The CERTUS System utilizes surface-enhanced Raman spectroscopy nanoparticle technology,” Coomes relates. “As part of its path to AOAC validation, which is expected in August this year, the CERTUS System recently underwent an intensive battery of performance-based tests proving detection of Listeria species at 1 x 104 colony forming units (CFUs) during a standard eight-hour work shift. Results also show that 1 CFU of Listeria monocytogenes can be detected in as little as 18 hours from swab to result.”
Coomes says tests demonstrate the CERTUS System’s ability to detect Listeria in produce wash samples known to have high bioburden. “With a range of matrices that include stainless steel, ceramic, plastic, and concrete sample sites, the CERTUS system provides 98 percent accuracy, which is equivalent to other commercial pathogen detection systems that are much more complex, have longer prep times and require expensive laboratories and technicians,” he points out. “Moreover, inclusivity and exclusivity test results have demonstrated that all challenging strains of Listeria are accurately detected with the CERTUS System, and interfering bacteria are
excluded from results.”
The CERTUS System is comprised of a detection unit, consumable detection kits featuring trademarked Bio-Lock sampling swabs, and the control pad. “The CERTUS control pad includes complimentary facility management software that enables plant test scheduling, test site result mapping, instant notifications, and remediation management, while automatically recording critical data points that support Food Safety Modernization Act compliance and storing data for instant access to reporting,” Coomes notes.
Coomes believes a major benefit of the CERTUS System’s combination of in-house capability and real-time detection is that food processors can have test results in-hand in less time than it takes for a third-party lab to receive samples by mail.