Objectives: The aim of this study was to investigate the molecular epidemiology and the genetic support of carbapenem-resistant Enterobacteriaceae and Acinetobacter spp. isolates collected in the University Hospital of Ouargla, southern Algeria. Methods: A total of 99 Gram-negative bacteria (GNB) were collected from stool samples of colonised patients and from inanimate surfaces in the hospital environment between December 2014 and August 2015. Selected Enterobacteriaceae and Acinetobacter spp. isolates with reduced susceptibility to carbapenems were subjected to phenotypic study, including antibiotic susceptibility testing according to CA-SFM-EUCAST 2015 guidelines and modified Carba NP test. Genes encoding carbapenemases, extended-spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases were screened by PCR and sequencing. Clonal relatedness was determined by multilocus sequencing typing (MLST). Results: Of the 99 GNB isolates, 10 (10.1%) showed reduced susceptibility to carbapenems were studied further, including 7 Acinetobacter baumannii, 1 Acinetobacter nosocomialis, 1 Escherichia coli and 1 Klebsiella pneumoniae. PCR and sequencing showed that four A. baumannii isolates and the single A. nosocomialis isolate harboured blaNDM-1. In addition, blaOXA-23 was observed in three A. baumannii isolates, and blaOXA-48 was detected in the two Enterobacteriaceae isolates. MLST assigned the K. pneumoniae to ST999 and the E. coli to ST38. The seven A. baumannii isolates belonged to ST85 and ST2. Conclusions: This study describes the epidemiology of carbapenemases produced by Enterobacteriaceae and Acinetobacter spp. in southern Algeria and reports the first description of metallo-beta-lactamase NDM1-producing A. nosocomialis in Algeria. (C) 2016 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.