MICs of TGC (MICTGC) were interpreted as per the US Food and Drug Administration’s breakpoint recommendations for Enterobacteriaceae. MICs of RIF (MICRIF) and AZT (MICAZT) were interpreted using CLSI breakpoints for Haemophilus influenzae (Clinical & Laboratory Standards Institute, 2009). The test was conducted in triplicate. Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 strains were used as controls. Of the 33 strains from the susceptibility testing, 13 clone A strains and seven clone click here B strains were randomly chosen for the in vitro testing of the following combinations using Etest: IMP–RIF, IMP–COL,

IMP–DOX, IMP–AN, IMP–AZT, COL–RIF, COL–DOX, COL–AN, COL–TGC, TGC–AN, and TGC–AZT. Two strips, one containing antibiotic A and another containing antibiotic B, were aligned at 90° at the respective MIC (mg L−1) of two antibiotics, MICA and MICB, as previously described (White et al., 1996). To evaluate interactions between antibiotics, the fractional inhibitory concentration (FIC) and FIC index were calculated as previously described (White et al., 1996; Tascini et al., 1998; Timurkaynak et al., 2006; Tan et al., 2007). Antibiotic combinations were evaluated based on the FIC index, which ranges as follows: synergistic if ≤ 0.5; additive Sirolimus in vivo if > 0.5 but < 1; indifferent if ≥ 1 but < 4; and antagonistic

if ≥ 4. The effect of adding RIF on the mean MICs of IMP (MICIMP) and of COL (MICCOL) for the same 20 strains was analyzed using two-tailed paired t-test with 95% confidence interval. The tests were performed in duplicate, with additional tests performed until two identical results were selected as confirmed. No fifth test was required. Three clone A strains and two clone B strains from the in vitro testing of antimicrobial combinations were analyzed using analytical Nintedanib (BIBF 1120) isoelectric focusing (aIEF) as previously described (Paterson et al., 2001). These strains

had different MICs of IMP, AN, AZT, COL, DOX, RIF, and TGC. Using PCR we screened for β-lactamase genes (blaTEM, blaSHV, blaPER, blaADC, blaIMP, blaVIM, blaOXA-23,blaOXA-Ab, and blaOXA-58) and genes encoding AMEs (aphA6, aadA1, aadB, aacC1, and aacC2). Additional determinants included integrase genes (intII, intI2 and intI3); disruptions in the outer membrane protein gene, carO; and the presence of adeR. We also examined the mutations in QRDRs of gyrA and parC by direct sequencing of the PCR products. All determinants were PCR-amplified using established primers and controls previously described (Hujer et al., 2006). According to our medical record review, of 121 MDR A. baumannii strains, 102 were hospital-acquired. Based on rep-PCR, 76 belonged to a major clone A and eight to a minor clone B. The study strains’ clonality, sources, and antimicrobial susceptibility data based on VITEK 2 are summarized in Supporting Information, Table S1.