Rapid detection of cefotaxime-resistant Escherichia coli by LC–MS

Abstract

Antibiotic resistance is an unsolved healthcare problem with increasing impact on patient management in the last years. In particular, multidrug resistance among Gram-negative bacterial strains has become the most pressing challenge. In order to deliver the most efficacious antimicrobial therapy with minimum delay, rapid diagnostic tests are required in order to detect multidrug resistant pathogens early during infection. In line with these efforts, we have developed a mass spectrometry-based assay for the rapid determination of ampicillin and cefotaxime resistance. The assay quantifies beta-lactamase activities towards ampicillin and cefotaxime within a turnaround time of 150 min, which is substantially faster than classical susceptibility testing.

Introduction

In cases of sepsis, early knowledge-based de-escalation of the antimicrobial broad spectrum therapy reduces the spread of antimicrobial resistances (Fraser et al., 2006, Goldmann et al., 1996, McGowan, 1994) and is shown to reduce therapeutic costs (Beekmann et al., 2003, Coleman et al., 1991, Tumbarello et al., 2010). However, commercially available culture-based antibiotic susceptibility testing (AST) e.g. Vitek 2 requires 9.82 ± 2.32 h to generate a complete microbial report including antibiotic susceptibility results for Gram-negative rods (Gherardi et al., 2012). In cases of severe sepsis, a delay of antimicrobial therapy for only few hours is associated with high case fatality rates (Ferrer et al., 2014). Rapid susceptibility testing is thus urgently needed.

To accelerate microbial diagnostics, we have previously demonstrated a liquid chromatography-mass spectrometry (LC–MS) based assay to detect antibiotic susceptibilities and resistances (MAAST – mass spectrometry-based antibiotic susceptibility testing). The test detected resistance of Escherichia coli towards ampicillin within 90 min after microbial growth has been detected in blood cultures (Grundt et al., 2012).

MS-based assays for susceptibility testing rely upon monitoring of the microbial biotransformation of antibiotics (Hooff et al., 2012, Sparbier et al., 2012, Wimmer et al., 2012). This metabolism of antibiotics results in a mass shift of the antibiotics which can rapidly be detected by mass spectrometry.

In contrast to the qualitative MALDI-TOF MS approaches (Sparbier et al., 2012, Wimmer et al., 2012) MAAST is a combination of high performance liquid chromatography (HPLC) and mass spectrometry, which is capable to separate and quantify multiple compounds simultaneously. This setting allows the reproducible identification and quantification of compounds, such as antibiotics and their inactive metabolites. The compound concentration directly correlates with the signal intensity of the compound-specific mass at the respective separation time. Accordingly, LC–MS/MS can exactly quantify the native beta-lactam antibiotics as well as the hydrolysis products for susceptibility testing of beta-lactam antibiotics.