Increased prevalence of aminoglycoside resistance in clinical isolates of Escherichia coli and Klebsiella spp. in Norway is associated with the acquisition of AAC(3)-II and AAC(6′)-Ib Bjørg C. Haldorsen a , Gunnar Skov Simonsen a, b , Arnfinn Sundsfjord a, b , Ørjan Samuelsen a, ⁎ Norwegian Study Group on Aminoglycoside Resistance 1 a Reference Centre for Detection of Antimicrobial Resistance, Department of Microbiology and Infection Control, University Hospital of North Norway, Tromsø, Norway b Research Group for Host-Microbe Interactions, Department of Medical Biology, Faculty of Health Sciences, University of Tromsø, Tromsø, Norway abstract article info Article history: Received 3 April 2013 Received in revised form 17 September 2013 Accepted 2 October 2013 Available online 14 October 2013 Keywords: RmtB AAC(6′)-Ib AAC(3)-II ESBL CTX-M In this study, we show that the increasing prevalence of aminoglycoside resistance observed in Norway among clinical Escherichia coli and Klebsiella spp. isolates is mainly due to the presence of the aminoglycoside- modifying enzymes AAC(3)-II and AAC(6′)-Ib. A frequent co-association of aminoglycoside resistance with Cefotaximase-München group 1 extended-spectrum β-lactamases was also observed. © 2014 Elsevier Inc. All rights reserved. Aminoglycosides are an important group of antibiotics often used together with β-lactams in the treatment of severe infections caused by both Gram-negative and Gram-positive bacteria. Increasing aminoglycoside resistance among Gram-negatives is now threatening the clinical efficacy of these antibiotics. Aminoglycoside resistance in Gram-negatives is mainly conferred by production of aminoglycoside- modifying enzymes (AMEs) and 16S rRNA methylases (Ramirez and Tolmasky, 2010; Wachino and Arakawa, 2012). Genes encoding AMEs and 16S rRNA methylases are located on mobile genetic elements along with other resistance determinants such as extended-spectrum β- lactamases (ESBLs) and carbapenemases resulting in multi-drug re- sistant isolates (Ramirez and Tolmasky, 2010; Wachino and Arakawa, 2012). In Norway, gentamicin resistance has steadily increased during the last decade and has now reached ~5% among Escherichia coli and ~4% among Klebsiella spp. blood culture isolates in 2011, threatening the current national standard empirical treatment regimen for septicemia that includes gentamicin and a β-lactam (NORM/NORM-VET, 2012). Further, the prevalence of ESBLs among Gram-negative bacteria has also increased to similar levels (NORM/NORM-VET 2012). In this study, we have performed a molecular characterization of 2 nationwide strain collections to examine the molecular basis of aminoglycoside-resistant invasive E. coli and Klebsiella spp. and the potential association to the increasing prevalence of Cefotaximase-München (CTX-M)-type ESBLs. Two strain collections were retrieved through the Norwegian surveillance program for antimicrobial resistance (NORM) from diag- nostic microbiology laboratories (Table 1): i) the NORM-AMG collec- tion consisting of all clinical isolates of E. coli (n = 105), Klebsiella pneumoniae (n = 31), and Klebsiella oxytoca (n = 1) from blood and urine samples, reported as resistant or intermediate susceptible to gentamicin and/or tobramycin among E. coli (n = 2510) and Klebsiella spp. (n = 1578) isolates included in the national surveillance program in 2009 (NORM/NORM-VET, 2010) and ii) the NORM-ESBL collection consisting of all ESBL-positive E. coli (n = 60) and K. pneumoniae (n = 8) clinical isolates from blood and urine in 2007–2008 (NORM/NORM-VET, 2008; NORM/NORM-VET, 2009). Species identification was performed using VITEK2 (bioMérieux, Marcy l’Etoile, France). All isolates were subjected to antimicrobial susceptibility testing using gentamicin, tobramycin, and amikacin E-tests according to the manufacturer’s instructions (bioMérieux). The results were interpreted according to the clinical breakpoints of the European Committee on Antimicrobial Susceptibility Testing (www.eucast.org). Screening for genes encoding AMEs (aac(6′)-Ib, aac(3)-IIa/c, aac(3)-Ia, ant(2″)-Ia, and ant(4′)-IIb) and 16S rRNA methylases (armA, rmtA, rmtB, rmtC, rmtD, rmtE, and npmA) was performed by PCR on isolates with reduced susceptibility to aminogly- cosides (Table 2). The presence of CTX-M-ESBL genes was examined as previously described (Tofteland et al., 2007), and PCR products were Diagnostic Microbiology and Infectious Disease 78 (2014) 66–69 ⁎ Corresponding author. Tel.: +47-77627043. E-mail address: orjan.samuelsen@unn.no (Ø. Samuelsen). 1 Members of the Norwegian Study Group on Aminoglycoside Resistance are listed in the Acknowledgement. 0732-8893/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.diagmicrobio.2013.10.001 Contents lists available at ScienceDirect Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio