MIBIE Summer School Molecular diagnostics of UTI & STI by using PCR, DHPLC and NGS

Transcription

1 MIBIE Summer School 2014 Molecular diagnostics of UTI & STI by using PCR, DHPLC and NGS Eugen Domann Institute for Medical Microbiology Justus-Liebig University Gießen

2 Human microbiome 14

3 Superorganism man: Symbiosis between man and microbes Homo sapiens ca cells ca kg bacterial colonization ca cells ca. 1-2 kg ca. 10x bacteria with ca. 100x genes! Our Self-Portrait: the Human Microbiome (Nature, )

4 Superorganism man: Symbiosis between man and microbes Homo sapiens ca cells ca kg 100 % human Birth bacterial colonization ca cells ca. 1-2 kg ca. 10x bacteria with ca. 100x genes! 10 % human 90 % microbial amount of microbes Death

5 ca bacteria per gram stool Major source for UTI: intestinal microbiota

6 Anatomy of the urogenital tract of female and male

7 Quantification and identification calibrated loops > bacteria/ml 1 µl 10 µl a b b C.L.E.D. agar plate a (Bromthymolblue-Lactose-Cystin according to SANDY, 1960) bacteria/ml

8 Identification of bacteria Pseudomonas aeruginosa

9 MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry transfer parts of a colony

10 MALDI-TOF MS Prinzip der MALDI-TOF-MS-basierten Identifikation von Proteinen und Peptiden. Die ribosomalen Proteine der Bakterien- oder Pilzkultur werden mit der Matrix nach Beschuss mit dem Laser desorbiert, ionisiert und im elektrischen Feld beschleunigt. Dabei trennen sie sich im Flugrohr entsprechend ihrer Ladung und Masse auf, und speziesspezifische Massenspektren werden erzeugt.

11 MALDI-TOF MS Massenspektrum einer Pseudomonas aeruginosa-probe. Das Muster charakteristischer Massenspitzen (Peaks) des Spektrums wird mit entsprechenden Datenbankeinträgen verglichen

12 Frequently found bacteria able to cause UTI Proteus sp. Morganella morganii Escherichia coli Enterococci Enterobacter ssp. Staphylococci Aerococcus sp. Providencia sp. Klebsiella sp. Pseudomonas aeruginosa Corynebacterium urealyticum Staphylococcus saprophyticus Frequent problem: Patient has disorders, leukozyte esterase or nitrit is positive but the microbiological report is negative (no culture)!

13 Overview about standard and molecular diagnostics of UTI, STD molecular Sample standard nucleic acid extraction (Maxwell 16, Kits) broad-range PCR 16S rdna DHPLC analysis (WAVE) PCR positive: collection of peaks (fragment collector) sequencing of the peaks identification of bacteria via bioinformatics specific real-time PCR STD-Panel and others qualitative and quantitative detection microbiome, metagenome sequencing of selected samples; next-generation sequencing (NGS) (PGM, MiSeq) culture of microorganisms identification of isolates (biochemistry, MALDI-TOF MS, sequencing and bioinformatics) storage of isolates (strain collection) antibiotic susceptibility, biofilm formation, pathogenicity/virulence (Galleria mellonella infection model),

14 Quantitative real-time PCR analysis: Specific detection of microorganisms StepOne Plus (Applied Biosystems) A) Sample B) Chlamydia trachomatis Amplex Biosystems GmbH MW C) Mycoplasma sp. STD Panel: Neisseria gonorrhoeae Chlamydia trachomatis Mycoplasma, Ureaplasma Treponema pallidum Haemophilus ducreyi

15 PCR and sequencing of the 16S rdna bioinformatics, data base analysis sequencing machine sequences covering the variable regions sequencing PCR product primer pairs 16S rdna of an unknown bacterium (isolate or original patient sample)

16 Analysis of microbial communities (polymicrobial infections) polymicrobial infection (4) total DNA extraction 16S rdna PCR gel electrophoresis: all amplicons are of the same size but are of different sequences gel electrophoresis: amplicons with similar size question: how to separate and to identify the different amplicons?

17 DHPLC-based microbiome analysis Escherichia coli Denaturing High-Performance Liquid Chromatography E. faecalis E. coli UR695 Staph. aureus E. faecalis K. pneumoniae Bacterial mixture E. coli P. aeruginosa

18 DHPLC analysis: Standard curve for quantification Absorbance (mv) Absorbance (mv) e+0 1e+1 1e+2 1e+3 1e+4 1e+5 1e+6 1e+7 1e+8 Amount of of bacteria per ml ml Detection limit: ~ 5-10 bacteria/ml

19 16S rdna amplicon sequencing (example PGM, Ion Torrent) PCR of Amplicon V5-V6 785F 1061R 275 bp amplicon analyse data MG-RAST ligate barcode adapters amplify the library run sequence apply the Ion Sphere particles to the Ion chip emulsion PCR

20 Analysis of ca urine samples Miscellanous bacteria (by culture and by PCR) (23,03 %) E. coli (35,15 %) E. faecalis (19,39 %) Fastidious bacteria (only by PCR) 22,43 % correct identification facilitates the choice of the appropriate weapon Experiences regarding polymicrobial UTI (based on ca samples): 67% one bug, 33% two or three bugs (more is a rare event)

21 Where do the bugs come from? human microbiome sexual acts environment e.g. Gonococci Treponema pallidum e.g. - Rat urine in drinking water: Leptospira - Mouse urine and feces as aerosol or dust: Hanta virus - Smear infection: Chlamydia trachomatis