Identification of pathogens from native urine samples by MALDI-TOF/TOF tandem mass spectrometry

Abstract

Background: Reliable high-throughput microbial pathogen identification in human urine samples is crucial for patients with cystitis symptoms. Currently employed methods are time-consuming and could lead to unnecessary or inadequate antibiotic treatment. Purpose of this study was to assess the potential of mass spectrometry for uropathogen identification from a native urine sample. Methods: In total, 16 urine samples having more than 10 5 CFU/mL were collected from clinical outpatients. These samples were analysed using standard urine culture methods, followed by 16S rRNA gene sequencing serving as control and here described culture-independent MALDI-TOF/TOF MS method being tested. Results: Here we present advantages and disadvantages of bottom-up proteomics, using MALDI-TOF/TOF tandem mass spectrometry, for culture-independent identification of uropathogens (e.g. directly from urine samples). The direct approach provided reliable identification of bacteria at the genus level in monobacterial samples. Taxonomic identifications obtained by proteomics were compared both to standard urine culture test used in clinics and genomic test based on 16S rRNA sequencing. Conclusions: Our findings indicate that mass spectrometry has great potential as a reliable high-throughput tool for microbial pathogen identification in human urine samples. In this case, the MALDI-TOF/TOF, was used as an analytical tool for the determination of bacteria in urine samples, and the results obtained emphasize high importance of storage conditions and sample preparation method impacting reliability of MS2 data analysis. The proposed method is simple enough to be utilized in existing clinical settings and is highly suitable for suspected single organism infectious etiologies. Further research is required in order to identify pathogens in polymicrobial urine samples.

CD5L as an Extracellular Vesicle-Derived Biomarker for Liquid Biopsy of Lung Cancer

“…Spectra were collected from 350 shots per spectrum over the m/z range of 600–3000 and calibrated by two-point internal calibration using trypsin autodigestion peaks ( m / z 842.5099, 2211.1046). The mass list of PMF was analyzed using Mascot (version 2.1, Matrix Science, UK) to search for matching proteins in the NCBI NR database (human; 276,468 entries) [ 16 ]. The following parameters were used in the database search: trypsin as the cleaving enzyme, a maximum of one missed cleavage, iodoacetamide as a complete modification of cysteine, oxidation of methionine as a partial modification, monoisotopic masses, and a mass tolerance of 0.1 Da.…”

Pathobionts: mechanisms of survival, expansion, and interaction with host with a focus on Clostridioides difficile

“…MALDI-TOF MS or ESI-TOF (Electrospray Ionization Time-of-Flight) technology that depends upon the reference database of enriched peptide mass fingerprints database of the type strains can be used to identify pathogens from prepared clinical samples, i.e., stools and urine, to overcome the limitations of the cultivation methods. 128 Microbial identification with the help of meta-proteomic data is based on the analysis of unique, strain-specific peptide biomarkers, which are also examples of noninvasive monitoring of pathobionts at the genus and species levels. 128 REIMS is a high-throughput MS-based method that is used in the metabolomics and metabolite profiling of a wide range of human samples including cell culture, and fecal microbiota.…”

“… 128 Microbial identification with the help of meta-proteomic data is based on the analysis of unique, strain-specific peptide biomarkers, which are also examples of noninvasive monitoring of pathobionts at the genus and species levels. 128 REIMS is a high-throughput MS-based method that is used in the metabolomics and metabolite profiling of a wide range of human samples including cell culture, and fecal microbiota. 129 During a C. difficile outbreak, S-layer proteins (30–50 kDa region) were diagnosed using the MALDI-TOF MS method.…”

New Microbiological Techniques for the Diagnosis of Bacterial Infections and Sepsis in ICU Including Point of Care

“…However, due to the limited panel of pathogens and resistance genes detected, its clinical benefit remains uncertain [ 75 ]. MALDI-TOF has been used directly on clinical samples including blood, urine [ 76 ] and CSF [ 77 ]. PCR/ESI-MS combines pathogen-specific PCR with mass spectrometry based on electrospray ionisation (ESI-MS): the IRIDICA system (Abbott) could detect 780 microorganisms and 4 resistance genes from various samples.…”