Comparison of selected methods in bacteriological diagnostics and their use in bacterial typing

Benulič, Katarina (author), Cerar Kišek, Tjaša (mentor)

Objectives 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.

• Language: English
• Keywords: 16S rDNA PCR, real-time PCR, bacteria, syndromic testing, cultivation, ventilator-associated pneumonia, meningitis, arthritis, Klebsiella pneumoniae, whole-genome sequencing, WGS, NGS, resistance, carbapenemase, OXA-48, NDM, outbreak
• Work type: Doctoral dissertation
• Organization: MF - Faculty of Medicine
• Year: 2020
• PID: 20.500.12556/RUL-121666
• COBISS.SI-ID: 33926659
• Publication date in RUL: 22.10.2020

Secondary language

• Language: Slovenian
• Title: Primerjava izbranih molekularnih metod v bakteriološki diagnostiki in njihova uporaba za tipizacijo bakterij

Abstract

Namen Namen dela je bila primerjava molekularnih metod na osnovi PCR s kultivacijo in ocena njihove uporabnosti v bakterijski diagnostiki različnih sindromov. Želeli smo dodatno raziskati prvi izbruh z odporno bakterijo Klebsiella pneumoniae in druge izolate, ki krožijo v populaciji. Namen je bil tudi ovrednotenje vloge sekvenciranja celotnega genoma v klinični diagnostiki. Metode Za sindrom specifične PCR in 16S evbakterijski PCR smo primerjali s kultivacijo za diagnostiko pljučnice, povezane z mehanskim predihavanjem, bakterijskega meningitisa in septičnega artritisa. Izolate Klebsiella pneumoniae iz zbirke smo analizirali z metodo sekvenciranja nove generacije za določitev genov za virulenčne dejavnike in rezistenco, za določitev plazmidov in za tipizacijo. Sekvenciranje naslednje generacije smo uporabili tudi za detekcijo Klebsiella pneumoniae iz krvi. Rezultati Ujemanje molekularnih metod in kultivacije je bilo slabo v vzorcih bronhoalveolarne lavaže. Z evbakterijskim PCR smo zaznali največ patogenov v primeru meningitisa, prav tako smo detektirali mnogo patogenov v vzorcih sklepne tekočine, kjer je bila kultivacija negativna. S specifičnimi PCR smo zaznali vse patogene, ki so bili vključeni v panel v vzorcih, ki so bili pozitivni tudi z drugimi metodami, razen v enem vzorcu sklepne tekočine. S sekvenciranjem celotnega genoma smo določili dodaten izolat, ki spada k znanemu izbruhu z odpornimi enterobakterijami in tudi nov dodatni izbruh. Identični plazmid z zapisom za OXA-48 smo dokazali v izolatih iz izbruha. V vzorcih krvi smo uspeli detektirati bakterijo Klebsiella pneumoniae in jo do neke mere tipizirati. Sklepi Evbakterijski PCR se je izkazal kot koristen za diagnostiko bakterijskega meningitisa, uporaben je tudi za dopolnitev diagnostike artritisa, je pa njegova vloga vprašljiva pri vzorcih bronhoalveolarne lavaže. Specifični PCR so omejeni z naborom tarč, ki so vključene v test. Sekvenciranje naslednje generacije je koristno pri opredelitvi izbruhov in populacijske strukture, v bližnji prihodnost pa njegova vloga za rutinsko detekcijo patogenov direktno v kužnini ni verjetna.

• Keywords: 16S rDNK PCR, PCR v realnem času, bakterije, sindromsko testiranje, kultivacija, pljučnica, mehansko predihavanje, meningitis, artritis, Klebsiella pneumoniae, sekvenciranje celotnega genoma, WGS, NGS, odpornost, karbapenemaze, OXA-48, NDM, izbruh