Feasibility and potential significance of rapid in vitro qualitative phenotypic antimicrobial susceptibility testing of gram-negative bacilli with the ProMax system.

Jade Chen,Dakai Liu,Amorina Cruz,Vincent Gau,William H Rodgers,Matthew L Faron,Michael Tomasek,Supram Hosuru Subramanya

doi:10.1371/journal.pone.0249203

Abstract

The emergence and evolution of antibiotic resistance has been accelerated due to the widespread use of antibiotics and a lack of timely diagnostic tests that guide therapeutic treatment with adequate sensitivity, specificity, and antimicrobial susceptibility testing (AST) accuracy. Automated AST instruments are extensively used in clinical microbiology labs and provide a streamlined workflow, simplifying susceptibility testing for pathogenic bacteria isolated from clinical samples. Although currently used commercial systems such as the Vitek2 and BD Phoenix can deliver results in substantially less time than conventional methods, their dependence on traditional AST inoculum concentrations and optical detection limit their speed somewhat. Herein, we describe the GeneFluidics ProMax lab automation system intended for a rapid 3.5-hour molecular AST from clinical isolates. The detection method described utilizes a higher starting inoculum concentration and automated molecular quantification of species-specific 16S rRNA through the use of an electrochemical sensor to assess microbiological responses to antibiotic exposure. A panel of clinical isolates consisting of species of gram-negative rods from the CDC AR bank and two hospitals, New York-Presbyterian Queens and Medical College of Wisconsin, were evaluated against ciprofloxacin, gentamicin, and meropenem in a series of reproducibility and clinical studies. The categorical agreement and reproducibility for Citrobacter freundii, Enterobacter cloacae, Escherichia coli, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae, and Pseudomonas aeruginosa were 100% and 100% for ciprofloxacin, 98.7% and 100% for gentamicin and 98.5% and 98.5% for meropenem, respectively.

Highlights

growth control (GC) ratio reporting ranges differed among organism-antibiotic combinations with a majority of species having a range of 0.6 to 0.8 for both ciprofloxacin and meropenem and 0.3 to 0.4 for gentamicin
A total of 43 strains (39 CDC, 3 ATCC, 1 International Health Management Associates (IHMA)) covering all ten target species were tested across three separate reproducibility studies to report a total 346 susceptibility results compared to the disk diffusion reference method
Repeated testing and validation of optimization efforts including higher antimicrobial susceptibility testing (AST) inoculum and antibiotic concentrations led to a final overall categorical agreement and reproducibility of 94.0% and 96.4% by the end of Reproducibility Study 3

Summary

Introduction

The widespread—and often inappropriate—use of antimicrobial agents partially due to the lack of rapid, evidence-based identification (ID)/antimicrobial susceptibility testing (AST). Feasibility of rapid automated AST of gram-negative bacilli. The NIAID website may be accessed here: https:// www.niaid.nih.gov/. The NICHD website may be accessed here: https://www.nichd.nih.gov/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. As a non-academic, commercial company, the employer and funder provided support in the form of salaries for authors [J.C., M.T.,V.G.], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section

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