Optimization of several parameters in order to reduce time in antibiotic susceptibility testing in a clinical laboratory

• Background - When sepsis or bacteraemia is suspected, patient blood samples are cultivated in blood culture bottles and then further incubated for identification of the organism and antimicrobial susceptibility testing. These methods are slow, identifying causative pathogens in a couple of hours, and antibiotic susceptibility results within 18-36 hours.Here we present optimization of several parameters in order to evaluate if the MBT ASTRA™ method can be a rapid tool, used for routine antibiotics susceptibility testing, in a clinical laboratory.Methods – MALDI-TOF MS measurements were performed with a Microflex LT/SH bench-top mass spectrometer (Bruker) with standard settings. The resulting spectra were uploaded in the MBT-ASTRA™ software, which normalizes the peaks and determines the AUC and RG values for each setup.Results - The bacterial preparation steps generated a new protocol, which reduced time with 30-60 minutes.The antibiotics susceptibility test was optimized for 90 minutes incubation time. 200 µl McFarland 0.5 bacterial suspension in broth were incubated in broth at 37°C, with and without 32 µg/ml Cefotaxime, 16 µg/ml Meropenem and 4 µg/ml Ciprofloxacin.The suspensions were transferred to 0.45 µm pore size filter membraned 96 well plate. They were centrifuged; washed; fixated and eluted; put on a MALDI-target, and covered by matrix solution. All could be automated with robot, which reduced time with 60 minutes.Conclusion – Rapid susceptibility testing becomes more requested with the increase of resistance bacteria causing infections. Our study can be a valuable tool for clinical laboratories striving for reduction in time handling of antibiotic susceptibility testing.

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