Identification of<i>Escherichia coli</i>clinical isolates producing macrolide 2â²-phosphotransferase by a highly sensitive detection method

Macrolide 2′-phosphotransferase [MPH(2′)] transfers the γ phosphate of ATP to the 2′-OH group of macrolide antibiotics. The role of aspartic acids in the putative ATP-binding site of MPH(2′)II was investigated through the substitution of alanine for aspartate by site-directed mutagenesis. D200A, D209A, D219A, and D231A mutant strains were unable to inactivate the substrate oleandomycin, while a D227A mutant retained 7% of the activity of the original enzyme.

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Identification of Functional Amino Acids in the Macrolide 2′-Phosphotransferase II

Taniguchi

Nakamura

Tsurubuchi

et al. 1999

Antimicrob Agents Chemother

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Assay for Evaluating the Abundance of Vibrio cholerae and Its O1 Serogroup Subpopulation from Water without DNA Extraction

Nasreen

Hussain

et al. 2022

Pathogens

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Cholera is a severe diarrheal disease caused by Vibrio cholerae, a natural inhabitant of brackish water. Effective control of cholera outbreaks depends on prompt detection of the pathogen from clinical specimens and tracking its source in the environment. Although the epidemiology of cholera is well studied, rapid detection of V. cholerae remains a challenge, and data on its abundance in environmental sources are limited. Here, we describe a sensitive molecular quantification assay by qPCR, which can be used on-site in low-resource settings on water without the need for DNA extraction. This newly optimized method exhibited 100% specificity for total V. cholerae as well as V. cholerae O1 and allowed detection of as few as three target CFU per reaction. The limit of detection is as low as 5 × 103 CFU/L of water after concentrating biomass from the sample. The ability to perform qPCR on water samples without DNA extraction, portable features of the equipment, stability of the reagents at 4 °C and user-friendly online software facilitate fast quantitative analysis of V. cholerae. These characteristics make this assay extremely useful for field research in resource-poor settings and could support continuous monitoring in cholera-endemic areas.

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Macrolide Esterase-producing Escherichia coli Clinically Isolated in Japan.

et al. 2000

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Current Japanese clinical practice involves the usage of large amounts of new macrolides such as clarithromycin and roxithromycin for the treatment of diffuse panbronchiolitis, Helicobacter pylori and Mycobacterium avium complex infections. In this study, the phenotypes, genotypes, and macrolide resistance mechanisms of macrolide-inactivating Escherichia coll recovered in Japan from 1996 to 1997, were investigated.The isolation rate of erythromycin A highly-resistant E. coli (MIC > 1600 /xg/ml) in Japan slightly increased from 0.5% in 1986 to 1.2% in 1997. In six macrolide-resistant strains, recovered from the strains collected for this study during 1996 to 1997, the inactivation of macrolide could be detected with or without added ATPin the assay system. The appearance of erythromycin A-inactivating enzyme independent of ATPwas novel from Japanese isolates, and the ]H NMRspectra of oleandomycin hydrolyzed by the three ATP-independent isolates were examined. It was clearly shown that the lactone ring at the position of C-13 was cleaved as 13-H signal in aglycon of oleandomycin upper shifted. These results suggested the first detection of macrolide-lactone ring-hydrolase from clinical isolates in Japan. These results suggested the first detection of an ATP-independent macrolide-hydrolyzing enzyme from Japanese clinical isolates. Substrate specificity of the macrolide-hydrolyzing enzyme was determined with twelve macrolides including the newer membersof this group and it was found that not only erythromycin A but also the new macrolides, such as clarithromycin, roxithromycin, and azithromycin were inactivated. The NMRdata, broad spectrum of activity, and independence of co-enzymesupported our namingof the enzymeas a macrolide esterase. PCRmethodology was employed to detect an ereB-like gene from the 3 isolates producing macrolide esterase, and one of these was subsequently shown to contain both ereB-like and ermB-Wksgenes. It was also clearly shown that the other three isolates, which inactivated macrolide in the presence of ATP, had an mphA-liko gene.

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Identification ofEscherichia coliclinical isolates producing macrolide 2â²-phosphotransferase by a highly sensitive detection method

Cited by 3 publications

References 16 publications

Identification of Functional Amino Acids in the Macrolide 2′-Phosphotransferase II

Identification of Functional Amino Acids in the Macrolide 2′-Phosphotransferase II

Assay for Evaluating the Abundance of Vibrio cholerae and Its O1 Serogroup Subpopulation from Water without DNA Extraction

Macrolide Esterase-producing Escherichia coli Clinically Isolated in Japan.

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Identification ofEscherichia coliclinical isolates producing macrolide 2â²-phosphotransferase by a highly sensitive detection method

Cited by 3 publications

References 16 publications

Identification of Functional Amino Acids in the Macrolide 2′-Phosphotransferase II

Identification of Functional Amino Acids in the Macrolide 2′-Phosphotransferase II

Assay for Evaluating the Abundance of Vibrio cholerae and Its O1 Serogroup Subpopulation from Water without DNA Extraction

Macrolide Esterase-producing Escherichia coli Clinically Isolated in Japan.

Contact Info

Product

Resources

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Identification ofEscherichia coliclinical isolates producing macrolide 2â²-phosphotransferase by a highly sensitive detection method