Flow cytometry as a rapid test for detection of tetracycline resistance directly in bacterial cells in Micrococcus luteus

Elena Zayko,Mariya Grudistova,Anzhelika Makhova,Dagmara Bataeva,Yuliya Yushina

doi:10.5219/1354

Abstract

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 multidrug-resistant 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 Micrococcus luteus. Detection of cell viability and reliability of antibiotic resistance was carried out on the Guava EasyCyte flow cytometer (Merck Millipore, Germany) with SYBR Green and PI dyes. M. luteus was exposed to tetracycline (at 30, 90, 180 and 270 μg.mL-1) over 24 hours. Concentrations of live and dead cells were measured after 4 and 24 hours of incubation. The results revealed that the use of mixed dyes PI and SYBR Green allowed the division of cells into large subpopulations of live and dead cells and the DNA of destroyed cells. After 4 h exposure to tetracycline 30 μg.mL-1, the subpopulation of live cells decreased by 47% compared to the positive control. Tetracycline at 90 μg.mL-1 decreased the subpopulation of live cells by 59% compared to the 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. Incubation of M. luteus with 180 and 270 μg.mL-1 tetracycline decreased the subpopulation of live cells by 82% and 94%, respectively, in comparison with the positive control. After incubation with 30 μg of tetracycline over 24 h the number of living cells decreased by 70% in comparison with the positive control. Tetracycline treatment (90 μg.mL-1 for 24 h) killed 71% of cells. After exposure to 90 μg.mL-1 tetracycline 29% cells were viable. The viability of living cells was confirmed by a microbiological test.

Highlights

Antibiotics are one of the most beneficial discoveries in medicine and public health
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
There are reported experiences of antimicrobial susceptibility tests by flow cytometry (FC) (Woźniak-Kosek and Kawiak, 2005; Faria-Ramos et al, 2013), but despite the significant progress of clinically significant protocols of FC application, there is insufficient scientific information on its continuous use in microbiology. 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 Micrococcus luteus

Summary

Introduction

Antibiotics are one of the most beneficial discoveries in medicine and public health. We studied the speed and accuracy of flow cytometry for the detection of tetracycline resistance in the Gram-positive bacteria Micrococcus luteus. Detection of cell viability and reliability of antibiotic resistance was carried out on the Guava EasyCyte flow cytometer (Merck Millipore, Germany) with SYBR Green and PI dyes.

Results

Conclusion