Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

The overall goal of this procedure is to identify gram-negative organisms directly from blood culture broth using maloff technology to do this, five milliliters of a positive blood culture broth is transferred into a serum separating tube and centrifuged. Following the spin, the bacteria containing fluid layer is transferred into a micro centrifuge tube and then centrifuged at low speed. The supernatant is then transferred to a new micro centrifuge tube and centrifuged at high speed.

The resulting pellet is resuspended in formic acid and analyzed by maldi to to identify the bacteria species present. The main advantage of this technique over existing methods like traditional culture, is the earlier identification of gram-negative bacteria. The implications of this technique extend towards clinical microbiology because a diagnosis of bacteremia impacts on the choice of the antimicrobial prescribed by the clinician.

Demonstrating this procedure will be Leonardo Bascu, who's one of the technicians in our department who regularly performs this procedure. Aerobic or anaerobic blood culture bottles collected at the discretion of clinical staff, usually in the setting of a febrile illness, are incubated in automated machines that continuously monitor for the growth of bacteria. When there is evidence of growth in a blood culture bottle, the machine will identify the signaled bottle.

Begin this procedure by removing the signaled blood culture bottle from the incubator. Place it in a biological safety cabinet. Prepare a gram stain from the signaled blood culture broth according to local institutional protocols, and examine it by microscopy if gram-negative organisms are identified.

Process the culture broth following the method described here. If gram-positive organisms are identified, an alternative rapid molecular method may be used. Invert the blood culture bottle two to three times to mix.

Then in the biosafety cabinet. Attach a 10 milliliter syringe to a safety blood transfer device. Attach the blood transfer device to the blood culture bottle and withdraw five milliliters of the broth into the syringe transfer.

The aspirated blood culture broth into a serum separating tube centrifuge at 1, 250 times G for 15 minutes to remove red blood cells. After the spin, the blood culture from the aerobic bottles shows a clear separation of red blood cells to the bottom of the tube with the gel fluid interface appearing as an opaque white color. In contrast, the blood culture from the lytic anaerobic blood culture appears as a deep red color at the gel fluid interface.

Because the lysed red blood cell components remain suspended in the supernatant for either type aspirate and discard the supernatant using a sterile transfer pipette being careful to leave approximately one milliliter of the buffy coat immediately above the gel fluid interface. Next, using a sterile pipette, gently mix the last one milliliter of buffy coat fluid above the gel interface, and then transfer the entire volume into a micro centrifuge tube. Spin the new tube at 288 times G for 30 seconds using a plastic disposable one milliliter transfer pipette.

Transfer the supernatant into a new micro centrifuge tube and discard the tube with the pellet. It is critical in this step to avoid transferring the pellet. This is particularly important in anaerobic blood culture specimens because the supinate remains pigmented and the pellet is not always clearly seen.

Centrifuge the specimen at 18, 407 times G for one minute and then using a fine tip transfer pipette, aspirate, and discard the supernatant to leave as little residual liquid as possible without disrupting the pellet. Add one milliliter of sterile DNAs and RNA free water and pipette up and down to resuspend the residual pellet centrifuge. The resuspended solution at 18, 407 times G for one minute.

After the centrifugation, use a fine tip pipette to aspirate and discard the supernatant. Again, taking care to remove as much liquid as possible. Resuspend the pellet in 10 microliters of 70%formic acid.

For large pellets, it may be necessary to increase the formic acid volume by 50 to 100%Using the pipette mix well to ensure a homogenous solution if necessary, use the pipette tip to physically disrupt the pellet. The resulting solution contains concentrated bacteria isolated from the blood culture broth, which is relatively free of human cells and of blood culture Bottle resins Using a pipette inoculate, a clean maldi TOF target plate with two microliters of the prepared suspension per target spot. Prepare three target sites for each sample to overcome the occasional problem of failed reads.

Place the plate on the edge of the fume hood to maximize airflow across it so that it will quickly dry overlay each dried spot with one microliter of matrix solution. Allow the maldi to target plate to dry on the edge of the fume hood. As before, ensure a homogenous preparation fills each of the target spots on the plate.

Insert the target plate into the maloff mass spectrometer. Ensure the plate is sitting flush with the spring loaded plates. Do not insert the target plate until all of the target spots are dry.

Check the rubber seal to ensure that it is free of lint, dust and hair. This will allow the required vacuum conditions to be created. Next, close the maloff stage lid and then press the in-out button on the front of the instrument.

The lid will lock and a vacuum is created. Open the maldi typ and flex control software within the typ software. From the dropdown menu titled file, select new classification, name the project, and then select new.

Select next. To complete the setup within the flex control software window, identify the onscreen graphic of the target plate while holding down the left mouse button. Move the mouse over the inoculated spots and highlight the target spots that are in use.

Use the right mouse button to click anywhere on the selected target positions, and then select add analytes from the dropdown menu. For each of the target spots, add blood culture identification details to the ID column and click next. When complete, select the Commercial Taxonomy spectra database and select next laboratories may utilize additional in-house or commercial databases to increase the number of reference spectra, click finish and the MALDI TOF MS will begin generating spectra.

Occasionally higher maldi to scores may be achieved by utilizing the manual acquisition of maldi to spectra within the flex control software. Once the spectra are acquired and analysis is complete in the stereotyping software, select the plus button next to species identification. To expand the identification list for each target, study the top five identifications noting whether the top five are similar.

If the top five genre are discordant with scores of greater than or equal to 1.7 for the same target spot, a mix of species may be present. Note that OV technology has poor sensitivity for detection of mixed species. When a score of greater than or equal to 2.0 is noted for the highest match on a target spot report the species when the highest score on a target spot is greater than or equal to 1.7, but less than 2.0 report the gene.

Only if additional criteria of the top five identifications are concordant, then report to the species level. When all spectra are complete, press the in-out button on the MALDI to machine, open the target plate receptacle and remove the plate. If using a reusable maldi TOF target plate, use alcohol to clean all target spots and store the maldi to plate at room temperature once it is clean and dry to identify gram-negative bacteria from blood culture broth, spin wash cycles were employed to concentrate and isolate bacteria, which were then analyzed by MALDI TOF mass spectroscopy.

As described in this video article shown here is the MALDI TOF MS generated spectra for eureka coli with a logarithmic score of 1.86. One to the right are the first five matches identified by the typing software. Note that the species is consistently reported as re coli with the score of 1.861.

Since the top five matches are e coli, the identification is considered reliable to the species level. The MALDI to MS generated spectra for pseudomonas aerogen is shown here. The logarithmic score is 2.216 to the right are the first five matches identified by the typing software.

Note that the species is consistently reported as pseudomonas aerogen with a high score of 2.216. The identification is considered reliable to the species level. The MALDI to MS generated spectra for a mixed blood culture broth containing esia coli and SIO marsens is shown here to the right are the first five matches by the typing software, which reports both Reiki coli and SIO marsan.

In the first five matched spectra. This mixed gene is only identified in approximately one third of mixed broth cultures. This table summarizes the previously published results from the verification study of this method in which 91.8%of mono microbial broths achieved a maldi to score of less than 1.7 with 100%and 97%concordance to genius and species respectively.

After watching this video, you should have a good idea of how to prepare a signal blood culture bottle for identification using maldi toff. By following this method, we can use the same pellet prepared for bacterial identification to prepare a more standardized inocular for automated susceptibility testing. For example, This technique allows for laboratories to provide early diagnosis to clinicians with a potential to impact on management.

Don't forget that working with blood culture bottles can be hazardous and precautions, including a biological safety cabinet should always be used when manipulating blood culture bottles.