Direct identification of disease-causing microorganisms in clinical samples using genomics and proteomics

Currently, our research is focusing on identifying peptide biomarkers for pathogens in several infectious diseases, including respiratory tract infections (RTI), blood-stream infections (BSI) and urinary tract infections (UTI), using genomics and mass spectrometry (MS)-based proteomics (proteotyping), with the overarching goal of improving infectious disease diagnostics. The foundation for the proteotyping workflow has been developed during the last 10 years starting with exploring the proteome of various strains of bacteria (Karlsson et al. 2012) and in particular through an FP7 Health-Innovation project (Tailored Treatment), where attention was given to detect and identify RTI directly in clinical samples (Van Houten et al. 2018, 2019).

We have shown that peptide biomarkers have the power to differentiate bacterial species (Karlsson et al. 2018), as well as strains within the same species (Karlsson et al. 2012). This proteotyping approach (Karlsson et al. 2012, 2015, 2018, 2020) can also be used for differentiating taxonomically-close species, such as the pathogen S. pneumoniae from commensal species, S. pseudopneumoniae and S. mitis of the Mitis Group of the genus Streptococcus (Karlsson et al. 2018). Having identified peptide biomarkers for species- and strain-level identification (Karlsson et al. 2012, 2020), as well as peptide biomarkers for antibiotic resistance and virulence, the next logical step is the exploration of how to implement the use of the peptide biomarkers in a clinical routine laboratory setting.

The novelty of this proposed project lies in the departure from the traditional diagnostic methodologies that rely upon microbial isolation and phenotypic analyses of antibiotic sensitivity. These methods require time, usually measured in days, while patients often need to be hospitalized and medical costs rise. An important factor of the new diagnostic approach is the fact that analyses of the proteome detect the expression of the genotypic template. Bacterial strains may be observed to carry antimicrobial resistance genes without any discernible resistance being noted for the microorganism. Such false positives may cause confusion in developing a treatment plan by physicians. Our peptide biomarker approach on the other hand allows for detection of an expressed phenotypic antimicrobial resistance and traits of virulence (not only genetic potential).

The proteotyping approach provides the potential for highly sensitive and rapid diagnoses of infectious bacteria, i.e., without the time-consuming cultivation steps, including identification at the species level, and adding comprehensive antimicrobial resistance and virulence assessments, directly from clinical samples. Thus, the physician treating a patient could receive the results of analyses on the same day as the clinical sample was collected from the patient, identifying the infectious agent, the strain-type of any species detected and confirmation of the presence and types of virulence and antimicrobial resistance factors. Such an approach provides the possibility of routine ‘bed-to-lab-to-physician’ diagnostics within hours’ time, rather than days’ time. Such innovation would have a significant impact on the overall treatment of infectious diseases.

Our research group is part of both Clinical Microbiology at the Sahlgrenska University hospital and Department of infectious diseases, University of Gothenburg (UGOT), with the overarching goal of improving infectious disease diagnostics. We are also part of Center for Antibiotic Resistance Research (CARe) at UGOT, a cross-disciplinary collaboration with a vision to limit mortality, morbidity and socioeconomic costs related to antibiotic resistance on a global scale through research.

To understand the molecular mechanisms of antibiotic resistance and to discover novel targets for treatment, we apply tandem mass spectrometry (MS/MS) for elucidating protein biomarkers (‘proteotyping’) associated with microbial antibiotic multi-resistance, with focus on Extended-Spectrum β-Lactamase (ESBL)-producing, carbapenemase-producing and colistin-resistance in clinically-relevant bacteria. We employ quantitative proteomics to enable determination of differential expression of resistance factors during exposure to antibiotics. We also use targeted gene ‘knock-outs’ (generated by homologous recombination) to elucidate the impacts of individual resistance factors on overall bacterial phenotypic antimicrobial resistance and apply quantitative proteomics for identification of expressed known and novel protein biomarkers associated with antimicrobial resistance.

Furthermore, we are involved in projects focused on genome-based natural product discovery and characterization of bioactive compounds in marine Actinobacteria, employing methods of microbiology, molecular biology, analytical chemistry, bioinformatics and organic chemistry. Isolates possessing particular genes together with antimicrobial activity are analyzed for the production and characterization of antimicrobial bioactive compounds with the aim to identify new compounds with novel mode of action.

Projects
2022- : VGR Regional samverkan FoU. Direkt, odlingsoberoende analys av kliniska prov för identifiering av art och stam av sjukdomsframkallande bakterier samt deras virulens- och antibiotikaresistensprofiler VGFOUREG-969330

2020- : Pioneering Strategies Against Bacterial Infections - PEST-BIN - is a Marie Skłodowska-Curie Action (MSCA) Innovation Training Network (ITN), funded by the European Commission, Horizon 2020 Program.

2018-2021, 2022-2024: VGR ALF-LUA Project; “Proteomics and Genomics Diagnostics of Infection, Virulence and Antimicrobial Resistance: Rapid Applications for Septicemia and Sepsis” (Project No. ALFGBG-720761).

2018-2020: University of Gothenburg Centre for Antibiotic Resistance Research (CARe) Project; “Rapid, Reliable Proteomics-Based Diagnostics of Antibiotic Multi-Resistance in Infectious Disease” (Project Nr. 5311_205314021).

2018-2020: Vetenskapsrådet (Joint-Programme Initiative – Anti-Microbial Resistance – JPIAMR) Project: “Predicting cell-cell horizontal transmission of antibiotics resistance from genome and phenome (TRANSPRED)” (Project No. VR-2016-06504).
2017-2020: VGR Regional samverkan FoU. Snabb diagnostik av sepsis - användning av biomarkörer för invasiva patogena bakterier baserad på proteomik och masspektrometri VGFOUREG-665141

2013-2017: EC FP7-HEALTH-2013-INNOVATION-1 Project, “Development of Tailored Anti-Microbial Treatment Regimens and Novel Host-Pathogen Insights for Respiratory Tract Infections and Sepsis (TAILORED-Treatment)” (Project No. HEALTH-F3-602860-2013).