By Deborah Borfitz
January 11, 2021 | Energy-generating bacteria, only in recent years leveraged to power biobatteries, have now been deployed for paper-based biosensing of antibiotic effectiveness. The innovative twist here is a high-throughput antimicrobial susceptibility testing device that eliminates the need for lab culturing and provides actionable results within five hours, according to Seokheun "Sean" Choi, associate professor at Binghamton University's Thomas J. Watson College of Engineering and Applied Science.
In a recent article appearing in Biosensors and Bioelectronics (DOI: 10.1016/j.bios.2020.112518), Choi and his colleagues describe the scientific feat in experiments with the prototype device using the electricity-producing pathogen Pseudomonas aeruginosa, one of the most common causes of infections in humans, and the first-line antibiotic gentamicin. The team is now working with chemical compounds that can prompt extracellular electron transfer from non-electricity-producing microorganisms, such as E. coli, to make the technique more “generalizable to all bacteria cells,” says Choi.
If successful, the device could serve as an important point-of-care diagnostic tool, especially in resource-limited regions of the world where paper-based biosensors are in high demand, he adds. Choi was the first to develop single-use, paper-based biobatteries powered by electrogenic bacteria from human sweat, providing the developing world with an eco-friendly and cost-effective alternative to commercial batteries for disposable devices—potentially including, he speculates, biosensors for detecting COVID-19.
As a Ph.D. student, Choi developed biosensors for finding cancer. Work on the antimicrobial susceptibility testing device has allowed him to capitalize on his twin interests in diagnostics and energy, he says.
His motivation is the high and growing rate of bacterial infections worldwide, including infections secondary to COVID-19. The problem is that antibiotics are “carelessly and excessively used” as treatments, leading to the rapid emergence and spread of antibiotic-resistant bacteria posing a serious threat to public health and the economy, says Choi. The Centers for Disease Control and Prevention reports 2.8 million antibiotic-resistant infections in the U.S. each year, with more than 35,000 associated deaths.
"To effectively treat infections, we need to select the right antibiotics with the exact dose for the appropriate duration," he says. "So, there's an urgent need to develop an antibiotic susceptibility testing method and offer effective [treatment] guidelines."
Antimicrobial susceptibility testing typically involves probing for resistant or nonresistant phenotypes of pathogens, says Choi. Getting to a result can take anywhere from two days to a week from the time of taking the sample and, during the wait, clinicians will often prescribe a broad-spectrum antibiotic in large doses to ensure its efficacy on the target pathogen.
The alternative, genotypic-based method is fast and simple, he adds, but of limited utility because the “[genetic] mechanisms of antibiotic resistance are not fully understood and only a handful of resistance genes are available for detection.” A genotypic assay would not be useful if the culprit bacteria develops new antibiotic-resistant genes, which can theoretically happen.
“Generally, the phenotypic method is more reliable and effective,” continues Choi. But, in addition to the relatively long time it takes to monitor bacteria growth, the approach requires central laboratories equipped with a benchtop imaging system.
Signal Detection
Extracellular electron transfer is “a remarkable biochemical process that certain microorganisms use for growth, overall cell maintenance and information exchange with surrounding microorganisms,” says Choi. Most microorganisms use respiration to convert stored chemical energy into electrical energy, using a soluble compound such as oxygen, nitrate, or sulfate as the electron acceptor in the process.
Many pathogens can natively transfer electrons across the cell membrane to an external electrode, he continues, and the mechanism plays an essential role in determining power generation and energy efficiency. Bacteria might make the transfer directly or using nanowires or chemical compounds they produce or are introduced.
The paper-based antimicrobial susceptibility testing device is an eight-well sensing array that characterizes bacteria electrical response in different concentrations of antibiotics, explains Choi. The first step is to incubate bacteria samples in the antibiotic mixtures for a few hours to induce antibiotic effects. Next, the samples would be loaded into the array for “tens of minutes” to extract the corresponding electrical signatures.
Within five hours, the device will generate minimum inhibitory concentration values of the antibiotics needed to inhibit pathogen growth. Test results come in the form of an electrical readout, with current generation of the bacteria measured against a baseline level without antibiotics, says Choi.
The hypothesis was that “antibiotic exposures would cause sufficient inhibition to the bacterial metabolisms, and the readout… would be sensitive enough to show even small variations in electric output caused by changes in antibiotic effectiveness,” says Choi.
The prototype could be extended to 64 or 96 sensors to build other tests into the device, says Choi, who recently had a hand in developing a high-throughput, rapid-screening platform of extracellular electron transfer in microbial fuel cells that was described in a separate article published in Biosensors and Bioelectronics early last year (DOI: 10.1016/j.bios.2020.112259). The paper substrate used for the antimicrobial susceptibility test could be easily patterned with hydrophobic or hydrophilic areas to create boundaries between individual test fields on the microfluidics-based platform.
“The beauty of our paper-based [antibiotic effectiveness] biosensor is that you don’t need a pumping system to move the liquid because the paper itself uses capillary force … [so] it just flows through a channel to the desired sensing area,” says Choi. In experiments soon to get underway, the research team plans to preload chemicals on the array and let it dry before dropping various non-electrogenic bacteria cells into antibiotics of differing concentrations.