Correct effective doses of antibiotics are important in the treatment of infectious diseases. The most frequently used methods for determination of the antibiotic susceptibility of bacterial pathogens are slow. The detection of multidrugresistant bacteria currently relies on primary isolation followed by phenotypic detection of antibiotic resistance by measuring bacterial growth in the presence of the antibiotic being tested. The basic requirements for methods of detection of resistance to antibiotics include speed and accuracy. We studied the speed and accuracy of flow cytometry for the detection of tetracycline resistance in the Gram-positive bacteria
Antibiotics are one of the most beneficial discoveries in medicine and public health. However, the use, overuse and misuse of these drugs have led to increases in antibioticresistant bacterial infections. Antimicrobial resistance (AMR) poses a serious global threat of growing concern to human, animal and environmental health. This is due to the emergence, spread, and persistence of multidrugresistant bacteria (
Fast, accurate antibiotic susceptibility testing is a critical need in addressing escalating antibiotic resistance, since delays in identifying multidrug-resistant organisms increase mortality (
Standard methods of detection of antibiotic sensitivity are labor- and time-consuming. Detection of multidrugresistant bacteria currently relies on primary isolation followed by the phenotypic detection of antibiotic resistance by measuring bacterial growth in the presence of the antibiotic being tested. These conventional methods take a minimum of 24 hours to obtain results after the pure culture is isolated (the analysis typically lasts up to 72 hours) (
One of these directions is the use of flow cytometry (FC) for the detection of microorganism viability and resistance. Flow cytometry was adopted for microbiological purposes almost 40 years ago, and the usefulness of this method for the identification of microbial pathogens directly in clinical samples or the detection of specific antibodies in serum has been well studied (
The quantitative assessment of prokaryotic viability is essential, especially for the confirmation of the activity of novel antimicrobial substances (
This manuscript reports the results of the use of FC with the dyes SYBR Green and PI for rapid assessment of cell viability and antibiotic resistance of the Gram-positive bacteria
There is a considerable need for new techniques that enable quick and specific diagnosis of pathogens resistant to antibiotics to guide correct treatment and to slow the development of resistance. Flow cytometry can provide quick essential information about the resistance to antibiotics of pathogenic microorganisms.
To obtain negative control, 1 mL suspension was heated at 100 °C for 10 min. To prepare mixed samples, 200 μL of positive control (live cells) and 200 μL of a negative control (dead cells killed by heating) were used.
Cultures were grown to early exponential phase, at which time tetracycline (Sigma-Aldrich, USA) was added in various concentrations (30 μg.mL-1, 90 μg.mL-1, 180 μg.mL-1 and 240 μg.mL-1). Incubation was carried out at 37 °C for 24 h. Concentrations of live and dead cells were measured after 4 and 24 h of incubation with the antibiotic.
SYBR Green stock solution was prepared by dissolving 5 μL of SYBR TM Green I Nucleic Acid Gel Stain, 10,000 X concentrate in DMSO (Thermo, USA) in 495 μL of deionized water. A PI working solution was prepared by dissolving 1 mg PI (AppliChem, Germany) in 1 mL of deionized water immediately before the study.
A volume of 975 μL of 1X pH 7.4 PBS (Santa Cruz, USA) was mixed with 10 μL of SYBR Green (Thermo, USA) and 5 μL PI (AppliChem, Germany), shaken vigorously, then 10 μL of
Verification of cell viability was obtained by microbiological test by transferring 100 μL of each concentration onto a nutrient agar plate (TSA, Liofilchem). All plates were incubated for 3 – 5 days at 30 °C. The growth on nutrient agar was evaluated by the presence of viable cells in the sample.
Measurements were repeated three times. STATISTICA 10.0 software was used for statistical analyses. The results were calculated as mean ± standard deviation (M ±
Routine techniques for the detection of resistance to antibiotics are based on a phenotypic study in which microbial growth is observed in the presence of different antibiotics. They yield results in not less than 24 h. In the last two decades, faster AST methods, such as PCR-based tests (
Figure
Result of flow cytometry analysis of positive, negative controls and mixed sample of
After 4 h exposure of
Results of flow cytometry after exposure of
In Figure
Results of the FC analysis of
Results of flow cytometry after exposure of
After 4 h exposure to tetracycline at 30 μg.mL-1, the subpopulation live cells decreased by 47.0% compared to the positive control (Table
The results of measuring the number of cells by flow cytometry.
Time incubation | K «+» | Tetracycline | |||||||||||||
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30 μg.mL-1 | 90 μg.mL-1 | 180 μg.mL-1 | 270 μg.mL-1 | ||||||||||||
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*107 cells.mL-1 | |||||||||||||||
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total | live | dead | total | live | dead | total | live | dead | total | live | dead | total | live | dead | |
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5.34a | 5.20 | 0.14 | 3.07b,c | 2.80 | 0.27 | 2.84b | 2.10 | 0.74 | 2.40b | 0.90 | 1.5 | 1.50b,d | 0.30 | 1.20 | |
22.28a | 22.00 | 0.28 | 3.30b,c | 1.60 | 1.70 | 3.40b | 1.50 | 1.90 | 2.58b | 0.58 | 2.0 | 2.06b,d | 0.26 | 1.80 |
Note: * – significant differences of experimental (tetracycline) doses compared with positive control (
A continued increase in concentration caused a shift in the population and an increase in dead cells, indicating damage to the cells of the microorganism. So, incubation of
This completely coincided with the results of the cytometric analysis. In analogical research, FC was used for detecting resistant
Cytometry analysis showed a decline in the numbers of viable bacteria after 24 h incubation of
The viability of living cells was confirmed by a microbiological test. Incubation on the plate was increased to 5 days. A study by (
After 24 h exposure to tetracycline (180 μg.mL-1 and 270 μg.mL-1) the majority of the cells in the population had received antibiotic-induced damage. The number of stained living cells decreased by 89% and 98.9%, respectively, in comparison with the positive control (
Dormancy is a protective state that enables bacteria to survive antibiotics, starvation and the immune system. Dormancy comprises different states, including persistent and viable but nonculturable (VBNC) states that contribute to the spread of bacterial infections (
Food products are a source of antibiotic-resistant pathogenic bacteria, a way for the transmission of antibiotic-resistant ‘food’ pathogens through the food chain to humans (
Fast and accurate antibiotic susceptibility tests can significantly reduce mortality rates and reduce financial costs (
As to future work, the same strategy may usefully be applied to other microorganisms (including pathogens in difficult food matrixes and other antibiotics. However, the present work shows that one may indeed expect to be able to determine antibiotic susceptibility by FC methods. This could be a very useful tool in the fight against antimicrobial resistance.
The current conventional methods for the determination of minimal inhibitory concentration (MIC) rely on the growth of the test organism in the presence of the antibiotic, which can be time-consuming depending on the growth speed of the organism, ranging from days for fastgrowing bacteria to weeks for slow-growing bacteria. We reported a rapid and novel antibiotic susceptibility testing methodology using FC. This study to demonstrate the feasibility of FC used SYBR Green/PI dyes to assay rapidly the viability of cells and to detect reliably the antibiotic resistance of the Gram-positive bacteria
The authors wish to thank Elena Kotenkova (Experimental Clinic–Laboratory of Biologically Active Substances in V. M. Gorbatov Federal Research Center for Food Systems of RAS) for performing the FC.
The work was performed as part of state assignment Federal Research Center for Food Systems after VM Gorbatov of Russian Academy of Sciences No. 0585-2019-0009.