Rapid Phenotypic Detection of Microbial Resistance in Gram-Positive Bacteria by a Real-Time Laser Scattering Method†

Abstract

We developed a methodology for antimicrobial susceptibility testing (AST) based on the BacterioScanTM216R laser scattering technology, using methicillin resistance in Staphylococcus aureus and vancomycin resistance in enterococci as exemplar for important resistance phenotypes. Fifty methicillin-resistant (MRSA) and 50 methicillin-susceptible (MSSA) S. aureus, as well as 50 vancomycin-resistant enterococci (VRE) and 50 vancomycin-susceptible enterococci (VSE) isolates were used for the study. Optimal test conditions were derived by investigating the effects of inoculum size, medium, incubation temperature and broth filtration. We proposed four different statistical approaches for rapid discrimination between resistant and susceptible bacteria. The statistical approach based on raw measurements of bacterial concentrations delivered sensitivity of 100% and specificity of 94% for discrimination between MRSA and MSSA already after 3 hours of incubation. Categorical agreement of ≥90% was achieved after 140 min with this approach. Differentiation between VRE and VSE was possible with 98% sensitivity and 92% specificity after 3 hours, using a sophisticated statistical approach based on concentration slopes derived from the raw concentration measurements. This approach provided categorical agreement of ≥90% after 165 min. The sensitivity and specificity estimates were confirmed by leave-one-out cross validation. In conclusion, the phenotypic AST methods developed in this study are promising for rapid detection of MRSA and VRE. The development and application of this technology would allow early detection of the resistant pathogens, thus facilitating swift change to the targeted antimicrobial treatment as well as timely initiation of appropriate infection control measures. Further studies are warranted to validate this approach for the detection of other resistance phenotypes, including direct testing from clinical specimens.

Innovative and rapid antimicrobial susceptibility testing systems

“…Recently, it was shown that sur face labelling of bacteria substantially shortened the microscopic analysis of AST results in 96well formats using only a standard microplate reader 42 . To improve the current systems, various prototype AST methods with different approaches have been reported, includ ing the use of mobile phones 37 , laser scatter technol ogy 43 , sensing of bacterial vibrations using phase noise measurements on resonant piezo electric substrates 44 , protein adsorbed magnetic nanoparticle mediated proto cols 45 , field effect enzymatic detection 46 , microfluid ics [47][48][49] , glucose metabolization 50 , optical screening and diffusometry 51 , nanometre scale bacterial deformation measurement 52 , mass spectrometry 53 , nanowire sen sors 54 , electrochemical sensing 55 , real time laser scatter ing 56 , atomic force microscopy 57 , fluorescence activated cell sorting (FACS) 58 , Raman and infrared spectroscopy at the single cell level 59,60 , nanotube assisted micro wave electroporation 61 , hydrodynamic trapping 62 and video microscopy 63 . In this Review, we do not describe these methods in detail, due to the vast differences in the protocols.…”

Recent Advances in the Race to Design a Rapid Diagnostic Test for Antimicrobial Resistance

“…The most basic method of detecting bacteria growth is by absorbance measurements as a function of optical density. Recent studies have employed a commercially available Forward Laser Light Scatter System (BacterioScan), which is a sensitive optical method for measuring light scattered by bacteria suspended in a liquid sample. − By measuring laser light scattered at specific angles, information such as cell shape and size can be included with the density of the bacterial suspension to confirm antibiotic susceptibility with higher sensitivity than typical optical density (transmittance) measurements . Others have improved popular fluorescence sensing modalities into microarray systems capable of scanning thousands of microscopic biofilms in one readout .…”

Rapid phenotypic methods to improve the diagnosis of bacterial bloodstream infections: meeting the challenge to reduce the time to result

“…Recently, this method was applied to artificial BC pellets inoculated with Escherichia coli and resulted in complete AST in <3 h [78]. Flow cytometry performed on positive BC produced AST results in 2 h with a 98% agreement with the reference microdilution [79], while the Real-Time Laser Scattering Method correctly detected methicillin resistance in S. aureus and vancomycin resistance in enterococci in <3 h [80]. Finally, a direct-on-target microdroplet growth assay coupled with MALDI-TOF MS accurately detected antimicrobial resistance in 5 h [81].…”