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Identification of Escherichia coli clinical isolates producing macrolide 2'-phosphotransferase by a highly sensitive
K Taniguchi1, A Nakamura, K Tsurubuchi
1Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Abstract:
Macrolide is inactivated with ATP plus crude extract of Escherichia coli producing macrolide 2'-phosphotransferase (MPH(2')), but not by living cells. Therefore, a convenient method for detection of MPH(2') using intact cells is needed. In this report, we determine that the modified lysozyme-DNase-RNase (LDR) method (named ELDR method) is at least one hundred times more sensitive for the detection of MPH(2') activity than the LDR method and, in addition, highly sensitive for the detection of aminoglycoside-modifying enzymes. Therefore, three new MPH(2')-producing strains were found in clinically isolated E. coli in Japan in 1997 by this method. It suggests that MPH(2')-producing E. coli have been spread in Japanese clinical fields.
Insights
A new ELDR method enhances detection of macrolide 2'-phosphotransferase (MPH(2')) activity in Escherichia coli. This sensitive assay identified new MPH(2')-producing strains, indicating wider spread in clinical settings.
Area of Science:
- Microbiology
- Molecular Biology
- Enzymology
Background:
- Macrolide antibiotics are inactivated by macrolide 2 '-phosphotransferase (MPH(2 ')) produced by Escherichia coli.
- Detection of MPH(2 ') activity is crucial for understanding antibiotic resistance.
- Existing methods using crude extracts are less convenient than intact cell-based assays.
Purpose of the Study:
- To develop a sensitive and convenient method for detecting MPH(2 ') activity using intact Escherichia coli cells.
- To evaluate the efficacy of the modified lysozyme-DNase-RNase (ELDR) method for enzyme detection.
- To investigate the prevalence of MPH(2 ')-producing E. coli in Japanese clinical settings.
Main Methods:
- Development of the enhanced lysozyme-DNase-RNase (ELDR) method, a modification of the LDR assay.
- Testing the sensitivity of the ELDR method for MPH(2 ') activity detection compared to the LDR method.
- Screening clinically isolated E. coli strains from Japan for MPH(2 ') production using the ELDR method.
Main Results:
- The ELDR method demonstrated at least 100-fold higher sensitivity for MPH(2 ') detection than the LDR method.
- The ELDR method also proved highly sensitive for detecting aminoglycoside-modifying enzymes.
- Three novel MPH(2 ')-producing E. coli strains were identified among clinical isolates from Japan in 1997.
Conclusions:
- The ELDR method provides a sensitive and convenient approach for detecting MPH(2 ') activity in intact E. coli cells.
- The identification of new MPH(2 ')-producing strains suggests a significant spread of these resistant bacteria in Japanese clinical environments.
- This method facilitates the surveillance and understanding of macrolide and aminoglycoside resistance mechanisms in E. coli.