Automated Antimicrobial Susceptibility Testing

Disk diffusion testing was done according to the 2011 guidelines of the European Committee of Antimicrobial Susceptibility Testing (EUCAST) using standard antibiotic disks (i2a, Perols Cedex, France) and Mueller-Hinton agar plates (BD, Franklin Lakes, NJ). All measurements except those for investigator dependence were done by the same experienced laboratory technician to eliminate inter-person bias. In parallel, the disk diffusion Mueller-Hinton agar plates were measured with the Sirscan instrument (i2a, Perols Cedex, France) and manually using a standard calliper. Sirscan measurements were checked and corrected on-screen by the laboratory technician as recommended by the manufacturer. Standard deviations of zone diameter measurements were calculated from 19 independent and blinded readings by 19 experienced persons using antibiotic disk diffusion inhibition zones of S. aureus ATCC 29213, E. coli ATCC 25922, and P. aeruginosa ATCC 27853 (EUCAST quality control strains). Discrepancies of manual and Sirscan readings were categorised as follows: Discrepancies resulting in erratic assignment of bacterial isolates to adjacent interpretative categories (susceptible to intermediate, intermediate to susceptible, intermediate to resistant, resistant to intermediate) were referred to as “minor discrepancies”. Erroneous categorisation of true-susceptible isolates as resistant (considering the manual method as the gold standard) were referred to as “major discrepancies”. Categorisation of true-resistant isolates as susceptible (considering the manual method as the gold standard) were referred to as “very major discrepancies”.
The following parameters were used to test for the presence of individual resistance mechanisms using Sirscan readings: ESBL-screening was done using EUCAST clinical breakpoints for non-susceptibility to cefpodoxime, and/or ceftazidime, and/or cefotaxime, ceftriaxone, and/or cefepime, AmpC and MRSA-screening was done using EUCAST clinical breakpoints for non-susceptibility to cefoxitin, carbapenemase-screening was done using EUCAST clinical breakpoints for non-susceptibility to ertapenem, and/or meropenem, and/or imipenem, and VRE-screening was done using EUCAST clinical breakpoints for non-susceptibility to vancomycin [18 ].
All inhibition zone diameter results were recorded by the Sirweb software (i2a, Perols Cedex, France) and statistical parameters were calculated with the Microsoft Excel 2010 Software (Microsoft Corp., Redmond, WA).