combination with boronic acid and EDTA in Entero- bacteriaceae that produced KPC or VIM enzymes.8 Isolates with MIC to plazomicin of 4 mg/ml were examined by PCR for the presence of 16S rRNA methylase genes (i.e., armA, rmtA, rmtB, rmtC, rmtD, and npmA) and the most common aminoglycoside- modifying enzymes in Gram-negative pathogens, using primers and conditions previously reported.9–11 In particular, the following genes were investigated: aac(69)-Ia, aac(69)-Ib, aac(69)-IIa, ant(29)-Ia, aac (3)-Ia, aac(3)-IIa, aac(3)-IVa and aph(39)-VIa. Results and Discussion The studied isolates included 241 K. pneumoniae, 33 E. coli and 26 Enterobacter spp. derived from blood (65.7%), pus (5.3%), bronchial secretions (1.7%), urine (8.0%), and fecal carriage (19.3%). Among the K. pneumoniae isolates, 138 (57.3%) were class A carbapenemase producers; 75 (31.1%) showed a positive EDTA-meropenem disc synergy test, which was suggestive of MBL production and 14 (5.8%) were designated both class A carbapenemase and MBL producers. PCR ampliﬁcation conﬁrmed the presence of blaKPC and blaVIM genes in all class A carbapenemase and MBL producers, respectively. ESBL production was conﬁrmed in 113 (81.9%) KPC producers, in 43 (57.3%) VIM producers and in 4 (28.6%) KPC and VIM producers. The remaining 14 K. pneumoniae isolates were all ESBL producers. Among the E. coli isolates, 15 (45.5%) were designated ESBL producers, whereas 9 (27.3%) were identiﬁed as KPC and 9 (27.3%) as VIM producers. Four of the VIM-positive E. coli isolates were also ESBL producers. Twenty-one (80.8%) Galani et al. Activity of Plazomicin against carbapenemase producers of the Enterobacter spp. isolates (19 E. cloacae and 7 E. aerogenes) harboured the blaVIM gene, while four (15.4%) harboured the blaKPC and one isolate possessed both blaVIM and blaKPC. The susceptibility results for all tested antimicrobials are shown in Table 1. Of the 300 isolates tested nine were pandrug-resistant, 157 were extensively drug- resistant and the remaining 134 were MDR according to deﬁnition given by ECDC.12 As shown, isolates were highly resistant not only to carbapenems (MIC50>8; MIC90.8 mg/ml) and piperacillin-tazobactam (MIC50. 64/4; MIC90.64/4 mg/ml) but also to ciproﬂoxacin (MIC50.2; MIC90.2 mg/ml). Approximately 78% of the strains were susceptible to colistin displaying MICs(2 mg/ml, while tigecycline’s MIC50 and MIC90 were 2 and 4 mg/ml, respectively (with 32% of the isolates being susceptible and 81% displaying MICs (2 mg/ml). Finally, fosfomycin demonstrated 56% susceptibility with an MIC50 of (16 and an MIC90 of 128 mg/ml. Isolates were highly resistant to tobramycin with only 6.7% of the strains being susceptible. Amikacin was active against 17.7% and gentamicin against 37.3% of the isolates. The vast majority (n5 242, 80.7%) of the isolates tested was non-susceptible to both amikacin and tobramycin, whereas 167 (55.7%) of them were resistant or intermediately susceptible also to gentamicin. Plazomicin had an MIC range of (0.25 to 4 mg/ ml, with an MIC50 of 1 and an MIC90 of 2 mg/ml that were substantially lower than those for comparator aminoglycosides. Only thirteen K. pneumoniae iso- lates (4.3%) exhibited MICs of 4 mg/ml. These Table 2 Activities of plazomicin against 300 MDR enterobacterial isolates with different resistance phenotypes Phenotype No. of isolates 0.25 8 16 Species Klebsiella pneumoniae Escherichia coli Enterobacter aerogenes Enterobacter cloacae KPC ESBL, KPC VIM ESBL, VIM KPC, VIM ESBL, KPC, VIM ESBL Total KPC VIM ESBL, VIM ESBL Total KPC ESBL, KPC VIM Total KPC VIM KPC, VIM ESBL, VIM Total 25 113 32 43 10 4 14 241 9 5 4 15 33 1 1 5 7 2 15 1 1 19 1 1 0.5 7 15 4 8 34 3 2 3 8 1 1 1 8 10 MIC (mg/ml) 4 1 3 4 4 1 13 1 12 69 14 22 7 3 9 136 4 3 3 8 18 1 4 5 1 6 1 8 2 5 26 10 9 3 1 4 58 2 4 6 1 1 1 1 Journal of Chemotherapy 2012 VOL. 24 NO. 4 193