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

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.