Method Article

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

DOI:

10.3791/53176

⸱

July 11th, 2016

 ,  ,  , 
1Institut für Medizinische Mikrobiologie und Hygiene, Medizinische Fakultät Carl Gustav Carus, TU Dresden, 2Institut für Virologie, Medizinische Fakultät Carl Gustav Carus, TU Dresden

In This Article

Summary

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Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

Abstract

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There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Introduction

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Secure identification of rare pathogens in routine diagnostics is hampered by the fact that classical cultural and biochemical methods are cumbersome and sometimes questionable. Furthermore, a diagnostic microbiology laboratory has to process a large number of pathogens, ranging from a few hundred to several thousands, daily, which requires the use of automated systems. In addition to the management of a high daily throughput, the precise identification of bacterial species is needed. This is warranted since they differ in their antimicrobial susceptibility pattern and therefore correct identification provides the clinician with essential information to choose appropriate antibiotics (e.g., Enterococcus spp., Acinetobacter spp.) 12,43.

Automated microbial identification systems (aMIS) apply standardized sets of enzymatic reactions to characterize the metabolic properties of bacterial isolates 13,15,16,26,27. Although the cartridges used in these systems utilize a large number of different biochemical reactions, e.g., 47 in the GN card of the aMIS used in this study 52, this strategy permits secure identification only for a limited set of bacteria. Furthermore, the database, an advanced expert system, is clearly focused on detection of relevant and highly relevant bacteria of medical importance 13,15,16,36. Two further systems, widely used in laboratories, also apply this biochemical approach for bacterial identification. Recent studies demonstrate a comparable identification accuracy between the aMIS used in this study and one of the competitors (93.7% and 93.0% respectively), while the 3rd aMIS has an identification accuracy of only 82.4% on species level 35. Such discrepancies may be explained by the quality of the underlying identification data references, the versions of kits and software, differences in metabolism and proficiency of technical personnel 35,36.

Two automated MALDI-TOF MS systems (MALDI-TOF microbial identification system, mMIS) are mainly used. These systems allow for detection of a large number of bacterial species based on their protein fingerprint mass spectra. For instance, the database of the mMIS used contains 6,000 reference spectra. Identification systems based on mass spectrometry offer fast and reliable detection of a great variety of microorganisms including rare pathogens 11,48,51. To date only a few direct comparisons are available between the mMIS used in this study and its competitor 19,33. According to Daek et al. both systems provide a similar high rate of identification accuracy, but the mMIS used in this study seems to be more reliable in species identification 19.

Similarly, molecular techniques addressing well conserved but also distinct genes (e.g., 16S rDNA or rpoB) permit a clear species identification 3,22,61. Among these, the 16S rDNA is the most widely used housekeeping gene because of its presence in all bacteria 34. Its function remains unchanged and finally, with roughly 1,500 bp, it is long enough to be suitable for bio-informatics 14,34. Many researchers regard 16S rRNA gene analysis as the "gold-standard" for bacterial identification 21. This is due to the fact that few laboratories use DNA-DNA hybridization techniques to date for identification of rare or new bacteria 14,34. Additionally, more and more databases are available which can be used for 16S rRNA gene analysis 50. However, it has to be taken into account that 16S rDNA based detection systems have a limited sensitivity compared to standard PCR protocols. Moreover, the molecular approach is sophisticated, time consuming and requires highly trained personnel as well as dedicated laboratory facilities and is, therefore, not easily implemented into routine diagnostics 55. Furthermore, it has been shown that the combination of at least two different methods of bacterial identification leads to highly accurate strain identification. The combination of MALDI-TOF MS and 16S rDNA sequencing permits the identification of large numbers of different bacterial species with high accuracy. Recently the combination of MALDI-TOF MS and 16S rRNA gene analysis was presented for bacterial identification studying epidemiological questions and rare pathogens 56.

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Protocol

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1. Extraction of Bacterial DNA

  1. Preparation of PBS Solution
    1. Weigh 1.65 g Na2HPO4 x 2H2O, 0.22 g NaH2PO4 x 2H2O and 8.80 g NaCl in a flask and fill with distilled water to a final volume of 1,000 ml. Adjust the pH to 7.4. For final use filter the solution through a bacteria-proof (0.22 µm) filter.
  2. DNA Extraction of Gram-negative Bacteria
    1. Streak the patient material on appropriate culture media (e.g., Columbia blood agar), identify and isolate potential pathogens.
    2. Prepare pure culture and perform Gram-staining in order to determine morphotype and to confirm purity and of the culture 49.
    3. Pick a single bacterial colony and transfer it with a 1 µl single use inoculation loop into a sterile 2.0 ml reaction tube which contains 1 ml PBS solution.
    4. Incubate this suspension in a thermomixer at 95 °C for 10 min. After incubation, let the bacterial extract (containing the dissolved DNA) cool to room temperature and either use it directly for amplification or store at -20 °C for further use (Figure 1a).
  3. DNA Extraction of Gram-positive Bacteria
    1. Streak the patient material on appropriate culture media (e.g., Columbia blood agar), identify and isolate potential pathogens. Perform Gram-staining in order to confirm morphotype of the culture.
    2. Prepare pure culture and perform Gram-staining in order to determine morphotype and to confirm purity of the culture.
    3. Pick a single bacterial colony with a sterile 1 µl inoculation loop and suspend it in 500 µl PBS solution in a 2.0 ml reaction tube.
    4. Add 500 µl glass-beads and transfer the tube to an oscillating homogenizer, operated at maximum frequency and amplitude (50 Hz), for 5 min. Use glass-beads of 1.0 mm diameter to disrupt the bacterial cell walls.
    5. Incubate this tube for 10 min at 95 °C and cool to room temperature. Use the extract either directly for the PCR reaction or store at -20 °C for further use.

2. 16S rDNA PCR

  1. Preparation of Amplification Primers
    1. Use the primers TPU1 (5´-AGA GTT TGA TCM TGG CTC AG-3' [M = A/C]) as forward primer and RTU4 (5´-TAC CAG GGT ATC TAA TCC TGT T-3´) as reverse primer 25. Prepare a primer stock solution with DNAse and RNAse free water, having a concentration of 100 pmol/µl (100 µM) and dilute it further to a working solution at 10 pmol/µl. Store the primer stock and working solution at -20 °C or use directly.
  2. Preparation of the PCR Reaction Mix
    1. Pipette 1 µl of each primer, 1 µl dNTP-mix (100 mM), 2.5 µl of the DNA extract, 5 µl 10x PCR buffer (containing 25 mM MgCl2), 0.25 µl Taq-polymerase (5 units/µl) and 39.25 µl DNAse- and RNAse free PCR water into a sterile 200 µl reaction tube.
  3. PCR Protocol
    1. Start the PCR program built as follows: First, an initial incubation step at 95 °C for 5 min which is necessary to activate the Taq-polymerase. Second, 35 amplification cycles, containing a 1 min. denaturation step at 95 °C, a 1 min. primer annealing step at 50 °C, and a 1.5 min primer extension step at 72 °C. Third, a final extension for 10 min at 72 °C. Finally, the PCR reaction is cooled to 4 °C.
      NOTE: Staphylococcus aureus and H2O serve as positive and negative control (Figure 1b).
    2. Place the tube containing the PCR reaction mix in the thermocycler and start the program.
  4. Purification of the PCR Product
    NOTE: In order to purify the PCR product, several protocols either enzyme based or with a silica adsorption matrix may be used. The single-step PCR cleanup used here utilizes two hydrolytic enzymatic reactions. While Exonuclease I (Exo I) removes single stranded DNA, shrimp alkaline phosphatase (SAP) hydrolyses unincorporated dNTPs. The second procedure is based on a silica membrane adsorption technique with a high salt fast binding - wash - low salt elution cycle.
    1. Add 1.5 µl of an enzymatic mix containing both Exo I and SAP to 25 µl PCR product, mix thoroughly and transfer to a thermocycler applying a simple protocol of first 15 min at 37 °C followed by 15 min at 80 °C. Store the purified PCR product either at -20 °C or use it directly as target for labeling PCR.

3. DNA Agarose Gel Electrophoresis

  1. Preparation of the Electrophoresis Buffer (TBE-buffer)
    1. Titrate 54.0 g (445 mM) TRIS base, 27.5 g (445 mM) boric acid and 20 ml of a 0.5 M EDTA (10 mM) solution at pH 8.0 with NaOH in a flask and fill it with distilled water to a total volume of 1,000 ml. Then dilute this 5x buffer 1:10 with distilled water (0.5x TBE) for electrophoresis.
  2. Preparation of the Loading Buffer
    1. Mix 250 mg bromophenol blue, 250 mg xylene cyanol FF and 15 g of polysucrose (Materials Table) into a flask. Then fill it with 0.5x TBE buffer to a total volume of 100 ml. Aliquot the loading dye to 1 ml portions and store at -20 °C for further usage.
  3. Casting of the Agarose Gel
    1. Weigh 6 g standard electrophoresis agarose to 300 ml 0.5x TBE buffer (see section 3.1.1) in a flask, to prepare a 2% (w/v) agarose gel. Then put the agarose mixture in a microwave oven, heat it near-boiling point until the agarose is completely dissolved and the solution appears clear.
    2. Let the molten agarose cool sufficiently and add 10 µl of a 1% (w/v) ethidium bromide stock solution. Pour the solution into a cast and place a gel comb (allowing for 30 wells) in the cast. Remove the comb when the gel is completely solidified and put the gel into the electrophoresis chamber containing 0.5x TBE buffer.
      NOTE: Ethidium bromide is toxic and mutagenic. Therefore it should be handled with caution. It is advisable to use nitrile gloves. There are alternative intercalating nucleic acid stains which can be used as a non-toxic alternative.
  4. Agarose Gel Electrophoresis of the PCR products
    NOTE: In order to verify the correct size of the PCR amplicons and to estimate the DNA concentration the purified PCR product is separated in an agarose gel.
    1. Pipette 2 µl of the loading buffer into a PCR reaction tube and add 8 µl of the PCR product. Pipette this mix into the wells of the gel. Add 8 µl of a molecular-weight size marker (DNA ladder) in a separate well to estimate the size of the PCR product.
    2. Apply a constant voltage at 10 V/cm and stop the electrophoresis as soon as the bromophenol blue marker has reached about ¾ of the total length of the gel. Visualize the separated PCR fragments using an UV-transilluminator (wavelength 302 nm) and document them using a gel-documentation system. Estimate the quantity of DNA by visual comparison with the DNA ladder. Use approximately 10 ng as target for the labeling PCR.

4. Sanger Sequencing

  1. Cycle Sequencing PCR
    NOTE: Perform the PCR labeling in a 10 µl reaction using a commercial kit (Materials Table).
    1. Add 2 µl of 5x reaction mastermix, 2 µl of the DNA preparation, 1.5 µl of TPU1 or RTU4 working solution (10 pmol/µl) and 4.5 µl of DNAse and RNAse free water.
    2. Put this sequencing reaction mix in a thermocycler and run another PCR. In total, process 25 cycles consisting of a denaturation step (96 °C, 10 sec), an annealing step (45-60 °C, 5 sec) and an extension step (60 °C, 2 min). Finally, cool the reaction to 4 °C.
  2. Purification of the Sequencing Reaction
    1. Purify the labeling reaction mix using commercial spin columns for PCR purification (Materials Table).
    2. Load 10 µl of the sequencing reaction onto the pre-hydrated gel-filtration matrix and perform a centrifugation step at 750 x g for 3 min.
      NOTE: By the use of this procedure unincorporated dye terminators are retained in the gel matrix.
    3. Then dry the aqueous eluate in a vacuum concentrator. Centrifuge at 2,500 x g for 20 to 30 min at 40 °C to complete dryness.
    4. Add 10 µl of highly deionized formamide then denature at 90 °C for 2 min and cool the mixture on ice. Pipette 10 µl of the denatured samples on a 96 well µl plate. Store the dried and cleaned PCR product at -20 °C if the analysis is to be carried out at a later time.
  3. 16S rRNA Gene Sequencing and Analysis of the DNA Sequence
    NOTE: Perform sequence analysis on an automated sequencer (Materials Table). The sequencer used here is an automated fluorescence-based capillary electrophoresis system that analyzes 4 samples simultaneously (50 cm 4-capillary array) with a liquid polymer 67 (Figure 1c).
    1. Place the microtiter plate in the sequencer. Write a sample sheet (plate record) as laid out in 2, save it and start the sequencing run/analysis following the steps given in 1.
      NOTE: Duration of a sequence run is 2 hr and provides data from 800 to 1,000 bp.
    2. Open the sequencing analysis software (Materials Table). Sequence data are given as a text file (.seq) and a file containing the electropherogram including quality values (QV) (.ab1).
      NOTE: Base calling is automatically performed by the sequencing analysis software and the underlying electropherogram is checked visually (e.g., peak height, peak separation) before the sequence is used for further applications.
    3. Compare the stored DNA sequence file to the NCBI nr database using BLAST algorithm (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide).

5. MALDI-TOF MS

NOTE: The mass spectrometer uses a 337 nm N2 laser and is operated by a specific control software (Materials Table). Spectra are recorded in linear mode and a mass range between 2,000 to 20,000 Da is covered. Data interpretation and allocation of scores to the samples is carried out in real time by an analysis software (Materials Table) (Figure 1d).

  1. Preparation of Bacteria for MALDI-TOF MS Analysis
    1. Grow the bacteria of interest (e.g., Myroides odoratimimus) on Columbia blood agar containing 5% horse blood at 37 °C for 18 to 24 hr, depending on the bacterial strain (Figure 2a). Perform Gram-staining to verify the morphotype of the organism under investigation as well as purity of the culture.
    2. Use a wooden toothpick for transferring a single bacterial colony to a well of a 96-well steel target (Figure 2b and 2c). Spot 1 µl of 70 % formic acid on top of the air dried organism on the steel target and leave to dry for approximately 2-3 min.
    3. Then, overlay the spot with 1 µl matrix solution, containing 50 mg/ml CHCA (α-Cyano-4-hydroxycinnamic acid) in an organic solvent (50% acetonitrile, 2.5% trifluoroacetic acid, 47.5% H2O).
      NOTE: The acid overlay method (on plate preparation method) may be used to improve the quality of the mass spectra. It has been shown that for Gram-positive bacteria such an "acid attack" increases the score values of the measured spectra 45.
      NOTE: Alternatively an automated sample preparation system (Materials Table) may be used in which contact free spots of 1 µl 70% formic acid solution and, after a first drying cycle, 1 µl matrix solution are applied on the bacterial smear. The samples are then dried under standardized conditions at 60% relative humidity and are ready to use for analysis.
    4. Let the smear with matrix overlaid air-dry again for about 5 min in a hood. This bacterial smear with the matrix applied is now stable for many hours.
      NOTE: Bacterial smears without matrix may not be stable due to protein degradation.
  2. Performing the MALDI-TOF MS Analysis
    1. Introduction of samples
      1. Open the control software of our mMIS (Materials Table).
      2. Aerate the sample loading port of the mass spectrometer by pressing the IN/OUT button of the instrument.
      3. Open the loading port after a click and insert the MALDI-TOF MS target plate with the dried bacterial smear plus matrix.
      4. Close the port and evacuate again. Observe vacuum and when it reaches 4.5 x 10-6 mbar start measurement.
    2. Sample Analysis
      1. Open the analysis software of our mMIS (Materials Table) and click the "File" menu. Select "New classification" and a new window "MALDI biotyper real time classification wizard" opens. Type the project name, for instance "Myroides measurement project_1, in the field "Project name" and press the button "New". Under the new dialog box named "New project" check project name and proceed by pressing the button "OK".
      2. In the "MALDI biotyper real time classification wizard" observe the "Analyte placement" window open. Select target positions (e.g., A1, A2, A3…), right-click any selected position (indicated by a blue square) and select "Add samples". In the automatically opened table view type in sample name, sample ID and optionally a comment. Click the button "Next" to proceed.
      3. In the "MALDI biotyper real time classification wizard" observe the "Selection of MALDI biotyper methods" window open. Select "Bruker Taxonomy" from "MSPs from taxonomy trees" and click "Next" to continue.
      4. In the "MALDI biotyper real time classification wizard" observe the "Project summary" window open. Check the inserted entries given above for the actual classification project. Start the classification run by pressing the button "Finish" (Figure 2d-f). Measurement starts automatically when the appropriate vacuum is reached.
      5. Follow the classification run until the measurement is successfully completed. View the results table in the "MALDI Biotyper Realtime Classification Project Myroides measurement project_1" open the menu "View" and click "Results".
      6. View the "Bruker Daltonics MALDI Biotyper Classification Results" as HTML file in the default web browser. Print and save the results.
    3. Measure a test standard daily prior to sample analysis to calibrate the instrument and perform instrument validation weekly (instrument self-test).
      NOTE: During measurement MALDI-TOF spectra are analyzed in real time by the analysis software (Materials Table). A list of ten species with their respective scores is given and is fed into the laboratory information system (LIS) for reporting.
    4. Critically evaluate the MALDI-TOF MS results (see representative results) and, if appropriate, include other tests, such as biochemical- and antibiotic susceptibility profiles or 16S rRNA gene sequencing and, if necessary, Gram-staining for assigning a bacterial species to the microbiological report.

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Results

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MALDI-TOF MS is a novel, fast and inexpensive method for microbiological routine diagnostics. Bacterial species identification by MALDI-TOF MS produces spectra mainly composed of ribosomal proteins but also other "very conserved proteins with house-keeping functions affected to a minimal extent by environmental conditions" 17.The database of this mMIS contains a large set of reference spectra and even bacteria which are rarely found in clinical isolates can be securely identifi...

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Discussion

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Both MALDI-TOF MS and 16S rRNA gene sequencing offer the possibility to identify large numbers of different bacteria. MALDI-TOF MS is a fast and inexpensive method, which is easy to handle and large databases of bacterial mass spectra are available. For this reason, MALDI-TOF MS is a rapid, cost effective and reliable method to conduct screening studies focused on rare bacterial pathogens 17,20,39,51. In a prospective study comparing MALDI-TOF MS with other phenotypic identification methods, Seng et al.

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Disclosures

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The authors declare that they have no competing financial interests.

Acknowledgements

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The authors would like to thank Prof. Enno Jacobs for his continuing support.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
CHROMASOLV, HPLC grade water, 1 LSigma-Aldrich Chemie, München, Germany270733
Tissue Lyser LTQiagen, Hilden, Germany85600Oscillating homogenizer
Glass-beads 1.0 mmVWR International, Darmstadt, Germany412-2917
Thermomixer 5436Eppendorf, Hamburg, Germany2050-100-05
QIAamp DNA Mini Kit (250)Qiagen, Hilden, Germany51306
Taq PCR Core Kit (1,000 U)Qiagen, Hilden, Germany201225
Forward Primer TPU1 (5´-AGA GTT TGA TCM TGG CTC AG-3’)biomers.net, Ulm, Germany -
Reverse Primer RTU4 (5´-TAC CAG GGT ATC TAA TCC TGT T-3´)biomers.net, Ulm, Germany -
Mastercycler Eppendorf, Hamburg, Germany- Thermocylcer
Reaction tube 1.5 mlSARSTEDT, Nümbrecht, Germany72,692
Reaction tube 2 mlSARSTEDT, Nümbrecht, Germany72,693,005
PCR 8er-CapStripsBiozym Scientific, Hessisch Oldendorf, Germany711040X
PCR 8er-SoftStripsBiozym Scientific, Hessisch Oldendorf, Germany711030X
Sharp R-ZV11 Sharp Electronics, Hamburg, Germany-Microwave
Titriplex III (EDTA Na2-salt dehydrate; 1 kg)Merck, Darmstadt, Germany1084211000
SeaKem LE AgaroseBiozym Scientific, Hessisch Oldendorf, Germany849006
SmartLadder SF - 100 to 1,000 bpEurogentec, Lüttich, BelgiumMW-1800-04
Bromophenol blue (25 g)Sigma-Aldrich Chemie, München, GermanyB0126
Xylene cyanol FF (10 g)Sigma-Aldrich Chemie, München, GermanyX4126
ComPhor L Maxi Biozym, Hessisch Oldendorf, Germany-
Ethidium bromide solution 1% (10 ml)Carl Roth, Karlsruhe, Germany2218.1
Gel Doc 2000Bio-Rad Laboratories, München, Germany-Gel-documentation system 
ExoSAP-IT (500 reactions)Affymetrix UK, Wooburn Green, High Wycombe, United Kingdom78201
Buffer (10x) with EDTA Life Technologies, Darmstadt, Germany402824
BigDye Terminator Kit v1.1Life Technologies, Darmstadt, Germany4337450
Hi-Di formamide (25 ml)Life Technologies, Darmstadt, Germany4311320
DyeEx 2.0 Spin Kit (250)Qiagen, Hilden, Germany63206
3130 Genetic AnalyzerLife Technologies, Darmstadt, Germany-Sequencer
MicroAmp optical 96-well reaction plate with barcodeLife Technologies, Darmstadt, Germany4306737
3130 Genetic Analyzer, plate base 96-wellLife Technologies, Darmstadt, Germany4317237
3130 Genetic Analyzer, plate retainer 96-wellLife Technologies, Darmstadt, Germany4317241
3130 Genetic Analyzer, well plate septaLife Technologies, Darmstadt, Germany4315933
3130 Genetic Analyzer, POP-7 Polymer, 7 mlLife Technologies, Darmstadt, Germany4352759
3130 Genetic Analyzer, 4-Capillary Array, 50 cmLife Technologies, Darmstadt, Germany4333466
Sequencing Analysis Software 5.4Life Technologies, Darmstadt, Germany-
microflexBruker Daltonik, Bremen, Germany-Our mMIS
VITEK MSbioMérieux, Nürtingen, Germany 2nd mMIS
flexControl 3.4Bruker Daltonik, Bremen, Germany-Device control software
Biotyper Realtime Classification 3.1 (RTC)Bruker Daltonik, Bremen, Germany-Analysis software
α-cyano-4-hydroxycinnamic acid, HCCA, 1 gBruker Daltonik, Bremen, Germany201344
Peptide Calibration Standard IIBruker Daltonik, Bremen, Germany222570
MSP 96 target polished steelBruker Daltonik, Bremen, Germany8224989
peqGREENpeqlab 37-5010
MALDI Biotyper Galaxy Bruker Daltonik, Bremen, GermanyPart No. 1836007 Automated sample preparation system
Vitek 2 bioMérieux, Nürtingen, Germany Our aMIS
MicroScan Beckman Coulter, Krefeld, Germany2nd aMIS
BD Phoenix™ Automated Microbiology SystemBD, Heidelberg, Germany3rd aMIS
Staphylococcus aureus subsp. aureus Rosenbach (ATCC® 25923™)American Type Culture Collection (ATCC), LGC Standards, Molsheim, FrancePositive control for PCR 

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16S rRNA Gene SequencingBacterial Pathogen IdentificationRare Bacteria DetectionMass Spectrometry AnalysisMolecular TechniquesDiagnostic MicrobiologyProteome Based ApproachBiochemical ProfilingSanger Sequencing

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