A Rapid Antimicrobial Susceptibility Test for Determining Yersinia pestis Susceptibility to Doxycycline by RT-PCR Quantification of RNA Markers.

Ohad Shifman,David Gur,Ronit Aloni-Grinstein,Emanuelle Mamroud,Raphael Ber,Moshe Aftalion,Izhar Ron,Ida Steinberger-Levy,Shahar Rotem

doi:10.3389/fmicb.2019.00754

Ohad Shifman, David Gur + Show 7 more

Open Access

https://doi.org/10.3389/fmicb.2019.00754

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Abstract

Great efforts are being made to develop new rapid antibiotic susceptibility tests to meet the demand for clinical relevance versus disease progression. This is important especially in diseases caused by bacteria such as Yersinia pestis, the causative agent of plague, which grows rapidly in vivo but relatively slow in vitro. This compromises the ability to use standard growth-based susceptibility tests to obtain rapid and proper antibiotic treatment guidance. Using our previously described platform of quantifying antibiotic-specific transcriptional changes, we developed a molecular test based on changes in expression levels of doxycycline response-dependent marker genes that we identified by transcriptomic analysis. This enabled us to determine the minimal inhibitory concentration of doxycycline within 7 h compared to the 24 h required by the standard CLSI test. This assay was validated with various Y. pestis strains. Moreover, we demonstrated the applicability of the molecular test, combined with a new rapid bacterial isolation step from blood cultures, and show its relevance as a rapid test in clinical settings.

Highlights

The development of rapid antimicrobial susceptibility tests (ASTs) is a major challenge in light of the worldwide escalation in antibiotic resistance
(Steinberger-Levy et al, 2016), we described a rapid molecular AST based on the Quantitative reverse-transcriptase PCR (qRT-PCR) quantitation of the changes in the expression of mRNA markers upon a short 2-h exposure of Y. pestis to ciprofloxacin and showed that this test can predict the standard MIC
We extend the applicability of our molecular AST approach by searching for doxycycline-responsive mRNA markers that can rapidly determine Y. pestis susceptibility to doxycycline

Summary

Introduction

The development of rapid antimicrobial susceptibility tests (ASTs) is a major challenge in light of the worldwide escalation in antibiotic resistance. Bacterial susceptibility is determined by standard ASTs, guided by the Clinical and Laboratory Standards Institute (CLSI) guidelines or the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (Matuschek et al, 2014; Clinical and Laboratory Standards Institute [CLSI], 2015, 2018). As these tests are growth dependent, they are time consuming and may delay proper treatment, especially upon infection with a pathogenic bacteria that grows rapidly in vivo but slowly in vitro. An example of such a bacterium is Yersinia pestis (Y. pestis), the causative agent of plague, which is

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