By spreading so rapidly and wreaking so much havoc, COVID-19 has revealed the susceptibility of global societies to both natural pathogens and intentionally released bioterror agents. The latter include bacteria that have been engineered for enhanced antibiotic resistance.1 Once pathogenic bacteria have been identified, they should be subjected without delay to an antibiotic susceptibility test (AST). Ideally, the AST should be fast and easy so that the appropriate treatment may be administered promptly. Unfortunately, traditional approaches for AST are time consuming and labor intensive.

Investigators at the Israel Institute for Biological Research have developed a rapid AST that can be performed directly on blood and environmental soil samples.2,3 In a recent study, the investigators used the AST on samples inoculated with Bacillus anthracis, Yersinia pestis, and Francisella tularensis. These bacteria—which cause anthrax, plague, and tularemia, respectively—are all Tier 1 bacteria. (According to the CDC, Tier 1 biological agents and toxins “present the greatest risk of deliberate misuse with significant potential for mass casualties or devastating effect to the economy, critical infrastructure, or public confidence, and pose a severe threat to public health and safety.)

Ronit Aloni-Grinstein, PhD, Ohad Shifman, PhD, and Shahar Rotem, PhD, all senior researchers in the department of biochemistry and molecular genetics, developed the micro agar PCR test (MAPt). MAPt is designed to improve on “gold standard” ASTs. These tests employ either a broth microdilution or a disk diffusion assay, and they take one to several days to complete.

“MAPt is applicable on a wide dynamic range of bacterial concentrations, has no need for isolation and purification steps, and provides results within hours to less than a day,” explains Aloni-Grinstein. “The method is based on the classical agar dilution test and a sensitive and specific PCR detection step. The latter can identify bacterial growth at lower bacterial concentrations and in shorter time frames than the usual visual examination. Moreover, by using target-specific PCR primers, only the specific pathogen is monitored within the heterogeneous sample.”

Aloni-Grinstein says the assay is sensitive and provides similar results to the traditional minimum inhibitory concentration (MIC),4 which identifies the susceptibility or resistance of bacterial strains to the applied antibiotic. The Clinical and Laboratory Standards Institute provides MIC guidelines and recommendations based on the pharmacokinetic (drug absorption, distribution, and elimination) and pharmacodynamic (biochemical and physiological) mechanisms of resistance.5

Coupling a micro agar dilution test with a quantitative PCR test provides the key steps in the process. “The sensitivity of the PCR method allows the detection of bacteria from concentrations as low as 5 × 102 colony forming units (cfu)/mL to concentrations as high as 109cfu/mL, which still enables a monolayer growth pattern, thus allowing an individual AST to each colony,” elaborates Aloni-Grinstein.

Besides enabling direct tests of whole blood, the AST can enable assessments of soil samples. Aloni-Grinstein says, “We have been dealing with the challenge of performing an antibiotic susceptibility test on environmental samples for quite a while. These samples harbor great amounts of various chemical (some are growth inhibitors) and biological (bacteria and fungus) environmental contaminants. Thus, determining an MIC value for a specific pathogen (bioterror agent) within such a sample traditionally requires time-consuming bacterial isolation, purification, and enrichment steps.”

She adds, “We found that with slow-growing bacteria, such as F. tularensis, one fast-growing contaminating bacteria in a culture of 106F. tularensis can take over the culture during a microdilution test, thus leading to an incorrect MIC value. To provide a clinically relevant test that can distinguish between antibiotic resistance and antibiotic susceptibility, we developed a rapid assay that may retain contaminants and yet provide an MIC value to specific bacteria within an heterogenic bacterial population.”

The agar-based MAPt supports bacterial growth even at low levels in the environmental sample. Further, the qPCR analysis enhances sensitivity and ensures specificity, allowing quantification of relatively lower amounts of bacteria from the heterogeneous environmental sample. As a result, the MIC can be determined in a much shorter timeframe.

“Our vision,” Aloni Grinstein declares, “is that MAPt will be an automated technique combining all steps, allowing high-throughput processing.” The group plans to expedite the automation of MAPt by establishing partnerships.

Aloni-Grinstein projects, “We are at the verge of starting a collaboration with a local hospital to examine the performance of MAPt side by side with the traditional assay on clinical samples derived from sepsis patients. The main drawback at the moment is the assay’s ‘cost per test’ when compared to the standard test. Automation of the process should be able to lead to higher throughput while decreasing manpower to lower the overall cost per test. This will place the MAPt as an affordable test in clinical scenarios.”

Moving forward, the researchers will be taking their technology into the clinic.



1. Rotem S, Steinberger-Levy I, Israeli O, Zahavy E, Aloni-Grinstein R. Beating the Bio-Terror Threat with Rapid Antimicrobial Susceptibility Testing. Microorganisms 2021; 9(7): 1535. DOI: 10.3390/microorganisms9071535.

2. Aloni-Grinstein R, Shifman O, Gur D, Aftalion M, Rotem S. MAPt: A Rapid Antibiotic Susceptibility Testing for Bacteria in Environmental Samples as a Means for Bioterror Preparedness. Front. Microbiol. 2020; 11: 592194. DOI: 10.3389/fmicb.2020.592194.

3. Rotem S, Shifman O, Aftalion M, Gur D, Aminov T, Aloni-Grinstein R. Rapid Antibiotic Susceptibility Testing of Tier-1 Agents Bacillus anthracis, Yersinia pestis, and Francisella tularensis Directly from Whole Blood Samples. Front. Microbiol. 2021; 12: 664041. DOI:10.3389/fmicb.2021.664041.

4. Kowalska-Krochmal B, Dudek-Wicher R. The Minimum Inhibitory Concentration of Antibiotics: Methods, Interpretation, Clinical Relevance. Pathogens 2021; 10(2): 165. DOI: 10.3390/pathogens10020165.

5. Khan ZA, Siddiqui MF, Park S. Current and Emerging Methods of Antibiotic Susceptibility Testing. Diagnostics (Basel). 2019; 9(2): 49. DOI: 10.3390/diagnostics9020049.


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