engleski

Identification of Escherichia coli, Klebsiella pneumoniae and Mycoplasma spp. directly from urine samples

Infections of the urinary tract are considered as one of the most prevalent bacterial infections in humans, with the highest rate of reoccurrence and antibiotic resistance. Therefore, rapid and accurate diagnosis is a key to a successful treatment and recovery. Urine is easily obtained, making itself a desirable sample for pathogen biomarker identification. Proteins found in urine are roughly divided on bacterial and human proteins. Proteome analysis of the named protein classes using mass spectrometry provides great sensitivity and selectivity for the detection of pathogen or human proteins. In the presented research, three urine samples: E. coli, K. pneumoniae and Mycoplasma spp. were analysed by standard clinical and mass spectrometry de novo sequencing method. E. coli and K. pneumoniae were confirmed by culture- based while Mycoplasma spp. was confirmed by PCR standardized clinical methods. On the other hand, mass spectrometry fast identification method includes urine proteome extraction protocol with bacterial cell lysis and protein isolation followed by Filter- Assisted Sample Preparation (FASP)1. After the protein lysis with trypsin and selective N- terminus peptide derivatization with CAF-/CAF+ reagent produced peptides are analysed in positive and negative ion mode. Tryptic peptide sequence identification in positive and negative MS/MS ion mode was carried out on MALDI-TOF/TOF mass spectrometer. E. coli and K. pneumoniae peptides were successfully identified after mass spectrometry analysis with in-house developed de novo sequencing Protein Reader software tool. Further on, analysis of small cell pathogen mycoplasma (1-2 m) was conducted on high resolution ESI mass spectrometry equipped with nano-LC separation system in order to distinguish human and pathogen peptides that were not separated after application of the same sample preparation protocol used on E. coli, K. pneumoniae urine samples. The results of later analysis showed a presence of mycoplasma in the sample suggesting the proposed protocol has potential for a routine identification of mycoplasma in human urine samples.

urinary pathogen identification ; mass spectrometry ; proteomics

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