Usage of 150 mm Mueller – Hinton Agar for the EUCAST rapid antimicrobial susceptibility test (RAST) directly from positive blood culture bottles

In this study, we evaluated the performance of modified rapid antimicrobial susceptibility test (mRAST) with 150 mm Mueller Hinton Agar (MHA) plates which was earlier standardized for 90 mm MHA by EUCAST. Blood culture bottles spiked with ATCC quality control strains were prepared. For quality control Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, and Enterococcus faecalis ATCC 29212 strains were used. By calculating and proportioning the surface areas of the plates comparing with 90 mm plates, 350 ± 50 µL undiluted blood culture samples were inoculated in 150 mm MHA, and 12 disks were placed. This process was repeated independently for three days and three times on each day for reproducibility. The mRAST test was performed on 50 samples with positive signals and gram-negative bacilli on Gram-stained samples (20 Klebsiella pneumoniae, 15 E. coli, 10 Acinetobacter baumannii, and five P. aeruginosa).Comparison of 90 mm MHA and 150 mm MHA showed that the categorical agreement of ATCC strains and 50 gram negative isolates was 100% and >95%, respectively, for all antibiotics. For K. pneumoniae, only 0.4 major error (ME) was detected at 4 h. For E. coli, 3.2, 1.6, and 1.5 ME were detected at 4, 8, and 20 h, respectively, whereas 1.6 very major error (VME) was detected at 4 h and 1.0 VME was detected at both 8, and 20 h, respectively. No errors were detected for P. aeruginosa or A. baumannii.These results indicated that 350 ± 50 µL of undiluted blood culture in 150 mm MHA was suitable for the mRAST test in vitro.