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Biology

Hurtig antimikrobiel følsomhedstest ved stimuleret Raman-spredningsbilleddannelse af deuteriuminkorporering i en enkelt bakterie

Published: February 14, 2022
doi:
10.3791/62398
, ,
1Department of Electrical and Computer Engineering,Boston University, 2Boston University Photonics Center,Boston University, 3Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine,Virginia Polytechnic Institute and State University, 4Department of Biomedical Engineering,Boston University, 5Department of Chemistry,Boston University

Summary

Denne protokol præsenterer hurtig antimikrobiel følsomhedstest (AST) assay inden for 2,5 timer ved enkeltcellestimuleret Raman-spredningsbilleddannelse af D2O-metabolisme. Denne metode gælder for bakterier i urin- eller fuldblodsmiljøet, hvilket er transformerende for hurtig enkeltcellet fænotypisk AST i klinikken.

Abstract

For at bremse og forhindre spredning af antimikrobielt resistente infektioner er der et presserende behov for hurtig antimikrobiel følsomhedstest (ASAT) for kvantitativt at bestemme de antimikrobielle virkninger på patogener. Det tager typisk dage at gennemføre AST ved konventionelle metoder baseret på langtidskulturen, og de fungerer ikke direkte for kliniske prøver. Her rapporterer vi en hurtig AST-metode muliggjort af stimuleret Raman-spredning (SRS) billeddannelse af deuteriumoxid (D2O) metabolisk inkorporering. Metabolisk inkorporering afD2O i biomasse og metabolisk aktivitetshæmning ved eksponering for antibiotika på enkeltbakterieniveau overvåges ved SRS-billeddannelse. Enkeltcellemetabolismeinaktiveringskoncentrationen (SC-MIC) af bakterier ved eksponering for antibiotika kan opnås efter i alt 2,5 timers prøveforberedelse og påvisning. Desuden er denne hurtige AST-metode direkte anvendelig på bakterieprøver i komplekse biologiske miljøer, såsom urin eller fuldblod. SRS metabolisk billeddannelse af deuteriuminkorporering er transformativ for hurtig enkeltcellet fænotypisk AST i klinikken.

Introduction

Antimikrobiel resistens (AMR) er en voksende global trussel mod effektiv behandling af infektionssygdomme1. Det forudsiges, at AMR vil forårsage yderligere 10 millioner dødsfald om året og et globalt BNP-tab på 100 billioner dollars inden 2050, hvis der ikke træffes foranstaltninger til bekæmpelse af antibiotikaresistente bakterier 1,2. Dette understreger det presserende behov for hurtige og innovative diagnostiske metoder til test af antibiotikafølsomhed (AST) af infektiøse bakterier for at bremse fremkomsten af antibiotikaresistente bakterier og reducere den relaterede dødelighed3. For at sikre det bedst mulige kliniske resultat er det afgørende at indføre effektiv terapi inden for 24 timer. Imidlertid kræver den nuværende guldstandardmetode, som diskdiffusion eller bouillonfortyndingsmetode, normalt mindst 24 timer til præinkubationsproceduren for kliniske prøver og yderligere 16-24 timer for at opnå resultaterne af den minimale hæmmende koncentration (MIC). Samlet set er disse metoder for tidskrævende til at styre en øjeblikkelig beslutning om behandling af infektionssygdomme i klinikken, hvilket fører til fremkomsten og spredningen af antimikrobiel resistens4.

Genotypiske AST-metoder, såsom polymerasekædereaktion (PCR)-baserede teknikker5, er blevet udviklet til hurtig påvisning. Sådanne teknikker måler de specifikke resistensgenetiske sekvenser for at give hurtige AST-resultater. De er ikke afhængige af tidskrævende cellekultur; Det er dog kun specifikke kendte genetiske sekvenser med resistens, der testes. Derfor er dens anvendelse begrænset til forskellige bakteriearter eller forskellige resistensmekanismer. De kan heller ikke give MIC-resultater for terapibeslutninger 6,7. Desuden er nye fænotypiske metoder til hurtig AST under udvikling for at overvinde disse begrænsninger8, herunder mikrofluidiske enheder 9,10,11,12,13, optiske enheder14,15,16, fænotypiske AST-kvantificering af nukleinsyrerne kopinummer17,18 og Raman-spektroskopiske metoder 19, 20,21,22,23,24. Disse metoder reducerer tiden til at guide AST-resultater, men de fleste af dem gælder kun for bakterieisolater, ikke direkte til kliniske prøver, og kræver stadig langvarig præinkubation.

I dette arbejde præsenterer vi en metode til hurtig bestemmelse af følsomheden af bakterier i urinen og fuldblod via overvågning af den cellulære metaboliske aktivitet ved SRS-billeddannelse. Vand (H2O) deltager i langt de fleste essentielle biomolekylære synteseprocesser i levende celler. Som en isotopologue af vand gennem enzymkatalyseret H/D-udvekslingsreaktion mellem det redoxaktive hydrogenatom i NADPH og D-atomet iD2O kan deuterium inkorporeres i biomasse inde i en celle25,26. En deutereret fedtsyresyntesereaktion medieres af deuterium mærket NADPH. D2O-inkorporeringen i reaktioner af aminosyrer (AA’er) resulterer i den deutererede proteinproduktion26 (figur 1). På denne måde kan de nyligt syntetiserede CD-bindingsholdige biomolekyler i enkelte mikrobielle celler anvendes som en generel metabolisk aktivitetsmarkør, der skal detekteres. For at aflæse de novo syntetiserede CD-bindinger anvendes Raman-spektroskopi, et alsidigt analytisk værktøj, der giver specifik og kvantitativ kemisk information om biomolekyler, i vid udstrækning til at bestemme antimikrobiel modtagelighed og reducere testtiden betydeligt til et par timer27,28,29,30 . På grund af den iboende lave effektivitet af Raman-spredningsprocessen er den spontane Raman-spektroskopi imidlertid af lav detektionsfølsomhed. Derfor er det udfordrende at opnå billedresultater i realtid ved hjælp af spontan Raman-spektroskopi. Sammenhængende Raman-spredning (CRS), herunder sammenhængende anti-Stokes Raman-spredning (CARS) og stimuleret Raman-spredning (SRS), har nået høj detektionsfølsomhed på grund af det sammenhængende lysfelt for at generere størrelsesordener større end spontan Raman-spektroskopi, hvorved højhastigheds-, specifik og kvantitativ kemisk billeddannelse på enkeltcelleniveau 31,32,33,34,35 ,36,37,38,39.

Her, baseret på vores seneste arbejde40, præsenterer vi en protokol til hurtig bestemmelse af den metaboliske aktivitet og antimikrobielle modtagelighed ved femtosekund SRS C-D-billeddannelse af D2O-inkorporering af bakterier i det normale medium, urin og fuldblodsmiljø på enkeltcelleniveau. Femtosekund SRS-billeddannelse letter overvågning af enkeltcellemetabolismeinaktiveringskoncentration (SC-MIC) mod antibiotika på enkeltbakterieniveau inden for 2,5 timer. SC-MIC-resultaterne valideres ved standard MIC-test via bouillonmikrofortynding. Vores metode er anvendelig til bestemmelse af antimikrobiel modtagelighed af bakterier urinvejsinfektion (UTI) og blodbaneinfektion (BSI) patogener med en meget reduceret analysetid sammenlignet med den konventionelle metode, hvilket åbner mulighed for hurtig fænotypisk AST i klinikken på enkeltcelleniveau.

Protocol

Brugen af humane blodprøver er i overensstemmelse med retningslinjerne fra IRB fra Boston University og National Institutes of Health (NIH). Specifikt er prøverne fra en bank og er fuldstændigt afidentificeret. Disse prøver anses ikke for at være menneskelige emner af institutional review board (IRB) kontor ved Boston University. 1. Fremstilling af bakterier og antibiotika lageropløsning Antibiotika (gentamicinsulfat eller amoxicillin) stamopløsning fremstilles i en koncentrat…

Representative Results

Effekten af inkubationstid på deuteriuminkorporering måles ved spontan Raman-mikrospektroskopi ved C-D (2070 til 2250 cm-1) og C-H (2.800 til 3.100 cm-1) regionen (figur 4a). Time-lapse enkeltcellede Raman-spektre af P. aeruginosa dyrket i 70O indeholdende medium viser stigende CD / CH-intensitet over inkubationstid fra 0 til 180 min. (figur 4b) Den stigende C-D-overflod i enkelte mikrobielle celler afslører, at D<…

Discussion

Hurtig AST kan opnås ved at vurdere responsen af bakteriel metabolisk aktivitet på antibiotikabehandling ved hjælp af enkeltcellet SRS metabolisk billeddannelse inden for 2,5 timer fra prøven til SC-MIC-resultater. Responsen af bakteriel metabolisk aktivitet og antimikrobiel modtagelighed kan detekteres ved at overvåge den metaboliske inkorporeringaf D2O til biomolekylesyntese ved hjælp af SRS-billeddannelse af CD-bindinger. Da vand er allestedsnærværende anvendt i levende celler, giver SRS metabolisk …

Disclosures

The authors have nothing to disclose.

Acknowledgements

Dette arbejde blev støttet af NIH R01AI141439 til J.-X.C og MS og R35GM136223 til J.-X.C.

Materials

Acousto-optic modulation Gooch&Housego R15180-1.06-LTD Modulating stokes laser beam
Amoxicillin Sigma Aldrich A8523-5G
Bandpass filter Chroma HQ825/150m Block the stokes laser beam before the photodiode
Calcium chloride Sigma Aldrich C1016-100G Cation adjustment
Cation-adjusted Mueller-Hinton Broth Fisher Scientific B12322 Antimicrobial susceptibility testing of microorganisms by broth dilution methods
Centrifuge Thermo Scientific 75002542
Cover Glasses VWR 16004-318
Culture tube with snap cap Fisher brand 149569B
Daptomycin Acros A0386346
Deuterium oxide 151882 Organic solvent to dissolve antibiotics
Deuterium oxide-d6 Sigma Aldrich 156914 Organic solvent as a standard to calibrate SRS imaging system
Escherichia coli BW 25113 The Coli Genetic Stock Center 7636
Eppendorf polypropylene microcentrifuge tubes 1.5 mL Fisher brand 05-408-129
Gentamicin sulfate Sigma Aldrich G4918
Hydrophilic Polyvinylidene Fluoride filters Millipore-Sigma SLSV025NB pore size 5 µm
ImageJ software NIH Version: 2.0.0-rc-69/1.52t Image processing and analysis
Incubating orbital shaker set at 37 °C VWR 97009-890
Inoculation loop Sigma BR452201-1000EA
InSight DeepSee femtosecond pulsed laser Spectra-Physics Model: insight X3 Tunable laser source and fixed laser source at 1045 nm for SRS imaging
Lock-in amplifier Zurich Instrument HF2LI Demodulate the SRS signals
Oil condenser Olympus U-AAC NA 1.4
Pseudomonas aeruginosa ATCC 47085 (PAO1) American Type Culture Collection ATCC 47085
Photodiode Hamamatsu S3994-01 Detector
Polypropylene conical tube 15 mL Falcon 14-959-53A
Polypropylene filters Thermo Scientific 726-2520 pore size 0.2 µm
Sterile petri dishes Corning 07-202-031
Syringe 10 mL Fisher brand 14955459
UV/Vis Spectrophotometer Beckman Coulter Model: DU 530 Measuring optical density at wavelength of 600 nm
Vortex mixer VWR 97043-562
Water objective Olympus UPLANAPO/IR 60×, NA 1.2
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Tags

Keywords: Rapid Antimicrobial Susceptibility TestingStimulated Raman ScatteringDeuterium IncorporationSingle BacteriumBacterial ConcentrationOptical DensityColony Forming UnitsAntibioticMHB MediumSerial DilutionPositive ControlNegative ControlIncubationHomogenization
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Zhang, M., Seleem, M. N., Cheng, J. Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium. J. Vis. Exp. (180), e62398, doi:10.3791/62398 (2022).
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