Rapid Identification and Antimicrobial Susceptibility Testing for Urinary Tract Pathogens by Direct Analysis of Urine Samples Using a MALDI-TOF MS-Based Combined Protocol.

Wei Li,Chen Li,Yi Zhang,Enhua Sun,Yong Li,Ying Wang,Xin Zhang,Lutao Du,Chuanxin Wang,Hongwei Pan

doi:10.3389/fmicb.2019.01182

Abstract

Usually, 18–48 h are needed for the identification of microbial pathogens causing urinary tract infections (UTIs) by urine culture. Moreover, antimicrobial susceptibility testing (AST) takes an additional 18–24 h. Rapid identification and AST of the pathogens allow fast and precise treatment. The objective of this study was to shorten the time of diagnosis of UTIs by combining pathogen screening through flow cytometry, microbial identification by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS), and AST using the VITEK 2 system for the direct analysis of urine samples. We analyzed 1,638 urine samples from patients with suspected UTIs submitted to the microbiology laboratory for culture. Each urine sample had an approximate volume of 30 mL and was divided into three aliquots. Urine processing included differential centrifugation and two washes to enrich the bacterial fraction for direct MALDI-TOF MS and direct AST. From a total of 1,638 urine samples, 307 were found to be positive through UF-1000i screening. Among them, 265 had significant growth of a single-microorganism. Direct identification was obtained in 229 (86.42%) out of these 265 samples, and no pathogens were misidentified. Moreover, species-level identification was obtained in 163 (88.59%) out of the 184 samples with Gram-negative bacteria, and 27 (38.03%) out of the 71 samples with Gram-positive bacteria. VITEK 2 AST was performed for 117 samples with a single-microorganism. Enterobacteriaceae data showed an agreement rate of antimicrobial categories of 94.83% (1,229/1,296), with minor, major, and very major error rates of 4.17% (54/1,296), 0.92% (12/1,296), and 0.08% (1/1,296), respectively. For Enterococcus spp., the overall categorical agreement was 92.94% (158/170), with a minor error rate of 2.94% (5/170) and major error rate of 4.12% (7/170). The turnaround time of this combined protocol to diagnose UTIs was 1 h for pathogen identification and 6–24 h for AST; noteworthily, only 6–8 h are needed for AST of Enterobacteriaceae using the VITEK 2 system. Overall, our findings show that the combination of flow cytometry, MALDI-TOF MS, and VITEK 2 provided a direct, rapid, and reliable identification and AST method for assessing urine samples, especially for Gram-negative bacterial infections.

Highlights

Urinary tract infections (UTIs) are among the most common community- and hospital-associated bacterial infections; affecting about 150 million persons worldwide each year (Flores-Mireles et al, 2015; McLellan and Hunstad, 2016)
We investigated a whole MALDI-TOF MSbased workflow to shorten the time of pathogenic diagnosis and effective treatment of UTIs by combining flow cytometry for screening, MALDI-TOF MS for microbial identification, and the VITEK 2 system for antimicrobial susceptibility testing (AST) of urine samples directly
We developed a fast and easy lysis-centrifugation-wash protocol to prepare bacterial pellets from blood cultures positive for the presence of microbial pathogens, that can be used for direct pathogen identification by MALDI-TOF MS and for direct AST using the VITEK 2 system (Pan et al, 2018)

Summary

Introduction

Urinary tract infections (UTIs) are among the most common community- and hospital-associated bacterial infections; affecting about 150 million persons worldwide each year (Flores-Mireles et al, 2015; McLellan and Hunstad, 2016). Other pathogens include Klebsiella pneumoniae, Proteus mirabilis, Staphylococcus saprophyticus, Enterococcus faecalis, group B Streptococcus (GBS), Pseudomonas aeruginosa, Staphylococcus aureus, and Candida spp., which are relevant as hospital-acquired and catheter-associated infectious agents (Flores-Mireles et al, 2015; McLellan and Hunstad, 2016). In conventional laboratory UTI diagnostics, identification of pathogens takes 18–48 h, and antimicrobial susceptibility testing (AST) needs an additional 18–24 h. A novel methodology is needed for both timely identification of pathogens and AST

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Conclusion