Direct Microbial Identification using An Automated Microbial Identification System to Facilitate the EUCAST RAST Method Without Mass Spectrometry

This research aims to evaluate the diagnostic accuracy of Gram negatives directly from positively flat blood culture bottles by automated microbial identification system. The purpose is to utilize it as a tool for early clinical reporting by EUCAST rapid anti microbial susceptibility testing method. EUCAST RAST method was recently introduced in 2018 to enable reporting of anti-microbial susceptibility testing results within four to eight hours of positive flagging of automated blood culture bottles.

It involves performing Kirby-Bauer disc diffusion method directly from positively flagged bottle and reading results at either four, six, or eight hours. For clinical reporting by EUCAST RAST method, the microbial identification is a prerequisite for determining the interpretative category of a AST results. The major challenge in implementing this method is early identification of the microbes within eight hours, especially in resource poor settings, which lacks mass spectrometry.

We found that the direct inoculum protocol was around 94%accurate in identifying a RAST reportable gram negative, which includes Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii complex. This approach of identifying gram negative directly was around four times faster than the standard approach of identification after overnight incubation. We'll now focus on assessing the impact of this approach of clinical reporting in gram-negative sepsis on patient outcomes in terms of length of hospitalization and mortality rate.

Furthermore, it can also be used as a tool to reduce antimicrobial usage to facilitate antimicrobial torture. To begin swab the septum of an automated blood culture bottle containing monomorphic gram-negative organisms with 70%isopropyl alcohol inside a Class two a biosafety cabinet. Then draw one milliliter of blood broth mixture into a sterile syringe equipped with a 21 gauge needle.

Dispense a big drop of the mixture onto the surface of a plated media. After streaking the plates, incubate the cultures at 37 degrees Celsius for 18 to 24 hours. Examine the plates for isolated colonies inside the biosafety cabinet.

Next, dispense three milliliters of sterile saline into an aMIAST tube with a dispenser bottle. Now pick up three to five morphologically similar colonies with a sterile straight inoculation wire. Transfer the inoculum into the first tube, use saline and a densitometer to adjust the turbidity of the suspension between 0.47 and 0.63 McFarland.

Then place a capillary attachment of the gram-negative aMIAST identification card in the first tube. Position the selected cards on the cassette. Now load the cassette into its position in the filler chamber with the sample barcode facing inward.

Close the door and press fill on the user interface screen. When the filling cycle is complete, the blue indicator light on the system will flash. Now transfer the cassette from the filling chamber into the loading chamber.

Remove the cassette waste when loading is completed. Subculture the remaining suspension in the tubes on CLED agar to check the purity of the isolates. The standard inoculum protocol identified 105 out of 204 tested isolates as RAST reportable gram negatives with Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumanni complex being the primary organisms identified.

To begin swab the septum of an automated blood culture bottle having monomorphic gram-negative organisms with 70%isopropyl alcohol inside a Class two A biosafety cabinet. Draw five milliliters of blood broth mixture into a sterile syringe equipped with a 21 gauge needle. Next, disinfect the rubber septum of a rubber separator tube.

Transfer the blood broth mixture into the tube centrifuge the sample at 160 G for 10 minutes. Now carefully pipette out the supernatant inside a biosafety cabinet. Transfer the supernatant to a new plain blood collection vial.

Place the capped vial in a centrifuge at 2000 G for 10 minutes. Now aspirate the supernatant with a sterile pipette. Then dispense three milliliters of sterile saline into an aMIAST tube with a dispenser bottle.

Using a sterile inoculation loop, pick the bacterial pellet from the bottom of the vial and inoculate it into the aMIAST tube. Use sterile saline and a densitometer to adjust the turbidity of the suspension between 0.47 and 0.63. Then place a capillary attachment of the gram-negative aMIAST identification card in the first tube.

Position the selected cards appropriately on the cassette. Now load the cassette into its position in the filler chamber with the sample barcode facing inward, close the door and press fill on the user interface screen. When the filling cycle is complete, the blue indicator light on the system will flash.

Now transfer the cassette from the filling chamber into the loading chamber. Remove the cassette waste when loading is completed. Subculture the remaining suspension in the tubes on CLED agar to check the purity of the isolates.

The results of seven positive blood culture bottles identified using the direct inoculum protocol were discordant until this species level. Of the 105 RAST reportable gram negatives identified by the standard inoculum protocol 98 were correctly identified by the direct inoculum protocol. Among 99 non RAST reportable gram negatives, A RAST reportable gram negative was identified in four positive blood culture bottles.

To begin place two un inoculated 90 millimeter Mueller into agar plates in a biosafety cabinet. With gloves on clean the septum of an automated blood culture bottle containing monomorphic gram-negative organisms with 70%isopropyl alcohol. Use a sterile syringe to aspirate 100 to 150 microliters of undiluted blood broth mixture from the blood culture bottle.

Add the sample to the center of a Mueller into an agar plate. Now use a sterile cotton swab to gently spread the broth over the plates in three directions. Then apply about six or fewer antimicrobial discs on each plate.

Transfer the plates to an aerobic incubator at 35 degrees Celsius for eight hours. When inoculation is complete, observe the purity of the isolate. Read the inhibition zones within eight hours, give or take five minutes.

Use the RAST breakpoint table for short incubation to interpret the results. After checking the bacterial identification results in the aMIAST. For the weekly quality control testing of the RAST method.

Place four sterile glass tubes on a tube stand. Dispense sterile saline into the tubes. Next, transfer the QC strain from the isolated colonies of an overnight culture into the first tube to create a 0.5 McFarland suspension with a sterile pipette and tip.

Transfer 10 microliters of the suspension from the first tube to the second tube. Once mixed transfer 10 microliters of the suspension from the second to the third tube, and then to the last tube. Next, take one milliliter of the inoculum from the last tube with a sterile syringe.

Transfer the inoculum into an automated blood culture bottle after swabbing the septum with 70%isopropyl alcohol. Simultaneously with a separate sterile needle add five milliliters of sterile sheet blood into the same blood culture bottle. Incubate it in a continuous blood culture monitoring system until it flags positive.