Comparison of selected methods in bacteriological diagnostics and their use in bacterial typingBenulič, Katarina (Avtor) Cerar Kišek, Tjaša (Mentor) 16S rDNA PCRreal-time PCRbacteriasyndromic testingcultivationventilator-associated pneumoniameningitisarthritisKlebsiella pneumoniaewhole-genome sequencingWGSNGSresistancecarbapenemaseOXA-48NDMoutbreakObjectives The aim of the study was comparison of PCR-based molecular methods to cultivation and evaluation of their role in bacterial diagnostics of different syndromes. We wanted to further investigate the first hospital outbreak of carbapenemase-producing Klebsiella pneumoniae and other isolates circulating in the population. We also wanted to evaluate the role of whole-genome sequencing in clinical samples. Methods Syndrome-specific real-time PCR panels and 16S rDNA eubacterial PCR were compared to cultivation for diagnostics of ventilator-associated pneumonia, bacterial meningitis and septic arthritis. Klebsiella pneumoniae isolates were analysed with whole-genome sequencing for determination of antimicrobial resistance and virulence genes, plasmid content and typing. Whole-genome sequencing was also used for detection of Klebsiella pneumoniae in spiked blood samples. Results Concordance between molecular methods and cultivation was low in bronchoalveolar lavage samples. Eubacterial PCR detected the most pathogens in case of bacterial meningitis and it also detected many pathogens in synovial fluid samples, in which cultivation was negative. Specific PCR detected all pathogens included in the panels in samples where other methods were positive, except in one synovial fluid sample. We were able to determine additional outbreak isolate and new potential outbreak with whole- genome sequencing. Identical plasmids carrying blaOXA-48 were found in outbreak isolates. Detection and to some extent typing of Klebsiella pneumoniae was possible in blood samples. Conclusions Eubacterial PCR proved to be very useful for diagnostics of bacterial meningitis and it also seems to be a great complementary tool to cultivation in synovial fluid samples. However, its role is questionable in ventilator-associated pneumonia. Specific PCR panels are limited with the range of pathogens included in the assay. Whole-genome sequencing was successfully used for investigation of the outbreak and clones circulating in the population, although its use for detection of pathogens directly in clinical samples in the near future does not seem probable.20202020-10-22 07:15:55Doktorsko delo/naloga121666VisID: 14092COBISS_ID: 33926659sl