An electrode instead of a Petri dish

The time of the analysis for antibiotic resistance was reduced to one and a half hours

Natalia Kondratenko, N+1

Bioengineers from Washington State University has developed an electrochemical method that will quickly determine the resistance of bacteria to antibiotics. With the help of a system that consists of an electrode and a mediator substance and measures the effectiveness of cellular respiration, the test can be carried out in an hour and a half.

The article was published in the journal Biosensors and Bioelectronics (Tibbits et al., Rapid differentiation of antibiotic-susceptible and -resistant bacteria through mediated extracellular electron transfer).

In the treatment of bacterial infections, situations may arise when it is necessary to assess the sensitivity of the pathogen to the drug — for example, if the patient is prescribed a repeated course of antibiotics. When conducting a resistance test, the growth rate of a bacterial culture under the influence of an antibiotic is usually assessed. Such a study takes two to three days, during which the patient receives probably ineffective medication. This creates favorable conditions for the selection of individual antibiotic-resistant bacteria, which leads to the emergence of new resistant strains.

To speed up the analysis, an alternative method based on measuring the effectiveness of cellular respiration is used. During respiration, electrons are transferred between the proteins of the respiratory chain located on the membrane of the bacterial cell — and these electrons are able to create an electric current that can be registered using an electrode. This method takes less time than growing bacteria in Petri dishes, but it is not used much, since not all bacteria are able to transfer electrons directly to the electrode.

A group of bioengineers from Washington State University, led by Gretchen Tibbits, presented an improved electrochemical method for determining antibiotic resistance. The problem with the existing method was that electrons can be transferred to the electrode only in the presence of a mediator substance capable of being reduced and then oxidized, and most bacteria cannot release such substances into the external environment. Bioengineers solved this problem by adding phenazine methosulfate to the system, which is used to conduct a cell viability test (MTS test). In this study, it was used not for the MTS test, but as an electron carrier. The improved method was called READAS (Rapid Electrochemical Assay for Detecting Antibiotic Susceptibility).

To determine antibiotic resistance, scientists took different — sensitive and antibiotic— resistant strains of four types of pathogenic bacteria: Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae. The first two species cause a wide range of human diseases, from skin inflammation to meningitis. E. coli is part of the normal intestinal microflora, but some pathogenic strains can cause intestinal infections. K. pneumoniae is the causative agent of pneumonia, as well as some urogenital infections. The researchers tested four antibiotics with different mechanisms of action on these bacteria: tobramycin (inhibits protein synthesis), ciprofloxacin (disrupts transcription), as well as imipenem and oxacillin (inhibit cell wall synthesis).

When exposed to an antibiotic (gray curve) on sensitive strains, the recorded current decreased — this indicates a decrease in respiratory efficiency. In the case of resistant strains, the curve hardly changed. Figure from the article by Tibbitts et al.

Cell viability was assessed by the current strength, which decreases if the bacterium is sensitive to an antibiotic, and does not change or changes slightly if the strain is resistant. None of the measurements took more than 90 minutes. To classify the test results, the scientists introduced a value called the antibiotic sensitivity index. If the index is greater than 0.4, then the strain is considered sensitive to the antibiotic, and if less — resistant.

Bioengineers hope that their improved method will soon be used in medical research. It is unlikely that it will be possible to completely move away from the usual scenario with the cultivation of bacteria, but shortening the period of testing for resistance is an undoubted advantage, since this will allow patients to receive effective treatment earlier, as well as weaken the positive selection of antibiotic—resistant individuals.

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