In-house method for direct bacterial identification in positive blood culture broths using microfiltration, bead beating, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Highlights

• Highly accurate method for direct bacterial identification in positive BC broths.

• Microfiltration and mechanical disruption steps were used to remove blood cells.

• Rapid, low-cost and chemical-free method using MALDI-TOF MS.

• Performance of the IH method in the identification of clinical organisms was comparable to that of Sepsityper method.

Abstract

Rapid identification of bacterial pathogens facilitates earlier optimization of antibiotic treatment and reduces morbidity and mortality in sepsis patients. The aim of this research was to design an in-house chemical-free method for direct bacterial identification in positive blood culture (BC) broths and to compare the performance of this method with that of the commercial Sepsityper® kit. The overall species identification rates for the in-house and Sepsityper methods were 88.4% and 85.8%, respectively (n = 190). Among 146 facultative anaerobes, 92.5% and 95.9% were identified to the species level using the in-house and Sepsityper methods, respectively. For 32 anaerobic bacteria, the in-house method showed a higher species identification rate (75.0%) than the Sepsityper method (53.1%). The in-house method correctly identified more Bacteroides species (100.0%) than the Sepsityper method (18.2%). Our novel in-house method and the Sepsityper method showed a high accuracy for direct bacterial identification in positive BC broths using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Introduction

Bloodstream infections are a major cause of morbidity and mortality in hospitalized patients (Goto and Al-Hasan, 2013). The survival rate of patients with bacteremia depends on the initial administration of an appropriate empirical antimicrobial therapy, which underscores the importance of rapid identification of microorganisms (Kollef, 2003; Lodise et al., 2003). The mortality rate of patients with anaerobic bacteremia who received adequate empirical treatment was observed to be significantly lower than that of patients who received inadequate empirical treatment (Kim et al., 2016). Hence, earlier and accurate species identification of the causative pathogen is important for selecting appropriate antibiotics before obtaining the results of antimicrobial susceptibility testing (Seifert, 2009; Torres et al., 2020).

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which enables the rapid identification of bacteria from fresh colonies grown on agar plates, is a routine method in clinical microbiology laboratories (Van Belkum et al., 2017). In addition, many laboratories have developed preparation methods for identifying pathogens directly from positive blood culture (BC) broths, which have shown diverse performances (Di Gaudio et al., 2018; Tanner et al., 2017; Van Belkum et al., 2017).

Furthermore, a commercial preparation kit (Sepsityper®; Bruker Daltonics, Bremen, Germany) has been developed to simplify the processing steps required for the purification and extraction of bacterial components from positive BC broths (Jamal et al., 2013; Morgenthaler and Kostrzewa, 2015). A previous study has shown that the Sepsityper method could identify 67.3–95.1% of bacteria to the species level (Morgenthaler and Kostrzewa, 2015). We established an in-house (IH) preparation method, which involves the use of filters and beads for cell lysis, without requiring any chemical agents such as saponin. In this study, we compared the performances of the Sepsityper and IH methods for the direct identification of bacteria in positive BC broths.