Multi-excitation Raman Spectroscopy Complements Whole Genome Sequencing for Rapid Detection of Bacterial Infection and Resistance in WHO Priority Pathogens

Abstract

Current methods for diagnosing acute and complex infections mostly rely on culture-based methods and, for biofilms, fluorescence in-situ hybridization. These techniques are labor-intensive and can take 2-4 days to return a test result, especially considering an extra culturing step required for the antibiotic susceptibility testing (AST). This places a significant burden on healthcare providers, delaying treatment and leading to adverse patient outcomes. Here, we report the complementary use of our newly developed multi-excitation Raman spectroscopy (ME-RS) method with whole-genome sequencing (WGS). Four WHO priority pathogens are AST phenotyped and their antimicrobial resistance (AMR) profile determined by WGS. On application of ME-RS method we find high correlation with the WGS characterization. Highly accurate classification based on the species (98.93%), wild-type/non-wild type (99.45%), and presence or absence of thick peptidoglycan layers in cell walls (100%), as well as at the individual strain level (99.29%). These results clearly demonstrate the potential of ME-RS as a rapid and first-stage tool for species, resistance and strain-level classification which can be followed up by WGS for confirmation. Such a workflow can facilitate efficient antimicrobial stewardship to handle and prevent the spread of AMR.

Highly sensitive quantitative phase microscopy and deep learning complement whole genome sequencing for rapid detection of infection and antimicrobial resistance

“…The wild-type/sensitive and phenotypically resistant/non-wild-type strains of Gram-negative Acinetobacter baumannii (n=6), Escherichia coli (n=7), Klebsiella pneumoniae (n=5), and Grampositive Staphylococcus aureus (n=2), Bacillus subtilis (n=1) were used for this study. Phenotypic information on E. coli, K. pneumoniae, and S. aureus strains was previously reported in [6,16,36]. Minimum inhibitory concentration (MIC) values of Acinetobacter strains were as described in [37].…”

“…Culture-independent diagnostic tests can also detect dead bacteria when antibiotic therapy has been administered before sampling [12]. Label-free optical techniques such as quantitative phase microscopy [13] and Raman spectroscopy [14, 15] have recently been shown to measure phenotypic and molecular signatures in pathogens at very low concentrations. We have recently reported the multi-excitation Raman spectroscopy (ME-RS) method for the species, resistance, and strain-level classification of pathogens [16].…”

“…Real-time sequencing, for example, has been successfully implemented in lower respiratory tract infections, urine infections, cerebral spinal fluid, surgical site infections, and orthopedic devices with up to 100 % sensitivity and specificity in pathogen detection. Label-free optical techniques such as quantitative phase microscopy [13] and Raman spectroscopy [14, 15] have recently been shown to measure phenotypic and molecular signatures in pathogens at very low concentrations. We have recently reported the multi-excitation Raman spectroscopy (ME-RS) method for the species, resistance, and strain-level classification of pathogens [16].…”

“…Label-free optical techniques such as quantitative phase microscopy [13] and Raman spectroscopy [14, 15] have recently been shown to measure phenotypic and molecular signatures in pathogens at very low concentrations. We have recently reported the multi-excitation Raman spectroscopy (ME-RS) method for the species, resistance, and strain-level classification of pathogens [16]. In combination with downstream machine learning-based analysis, such methods may open doors to developing new culture-free identification of bacteria strains and their clinically relevant properties, such as wild-type (WT) and non-wild type (NWT) bacteria and provide insight into the AMR profile.…”

Highly sensitive quantitative phase microscopy and deep learning aided with whole genome sequencing for rapid detection of infection and antimicrobial resistance

“…Real-time sequencing, for example, has been successfully implemented in lower respiratory tract infections, urine infections, cerebral spinal fluid, surgical site infections, and orthopedic devices with up to 100% sensitivity and specificity in the pathogen detection ( Wang et al, 2020 ; Whittle et al, 2022 ). Label-free optical techniques such as quantitative phase microscopy ( Kim et al, 2022 ) and Raman spectroscopy ( Ho et al, 2019 ; Lister et al, 2022 ) have recently been shown to measure phenotypic and molecular signatures in pathogens at very low concentrations. Oh et al (2020) utilized optical diffraction tomography to quantitatively analyze the response of Escherichia coli and Bacillus subtilis to varying concentrations of ampicillin ( Oh et al, 2020 ).…”