MECHANISMS Characterization of ESBLs and Associated Quinolone Resistance in Escherichia coli and Klebsiella pneumoniae Isolates from an Urban Wastewater Treatment Plant in Algeria Souhila Alouache, 1,2 Vanesa Estepa, 3 Yamina Messai, 1 Elena Ruiz, 3 Carmen Torres, 3 and Rabah Bakour 1 The aim of the study was the characterization of extended spectrum beta-lactamases (ESBLs) and quinolone resistance in cefotaxime-resistant coliform isolates from a wastewater treatment plant (WWTP). ESBLs were detected in 19 out of 24 isolates (79%) from raw water and in 21 out of 24 isolates (87.5%) from treated water, identified as Klebsiella pneumoniae and Escherichia coli. Molecular characterization of ESBLs and quinolone re- sistance showed allele profiles CTX-M-15 (3), CTX-M-3 (5), CTX-M-15 + qnrB1 (1), CTX-M-3 + qnrB1 (1), CTX-M- 15 + aac-(6¢)-Ib-cr (4), and CTX-M-15 + qnrB1 + aac-(6¢)-Ib-cr (7). A double mutation S83L and D87N (GyrA) and a single mutation S80I (ParC) were detected in ciprofloxacin-resistant E. coli isolates. In K. pneumoniae, mutations S83I (GyrA) + S80I (ParC) or single S80I mutation were detected in ciprofloxacin-resistant isolates, and no mutation was observed in ciprofloxacin-susceptible isolates. bla CTX-M , qnrB1, and aac-(6¢)-Ib-cr were found, re- spectively, in these genetic environments: ISEcp1-bla CTX-M -orf477, orf1005-orf1-qnrB1, and Tn1721-IS26-aac-(6’)-Ib- cr-bla OXA-1 -catB4. bla CTX-M-15 was located on IncF plasmid in E. coli and bla CTX-M-3 on IncL/M plasmid in both species (E. coli and K. pneumoniae). E. coli isolates were affiliated to the phylogroups/MLST: D/ST405 (CC405), A/ST10 (CC10), A/ST617 (CC10), and B1/ST1431. K. pneumoniae isolates belonged to phylogroup KpI and to sequence types ST15, ST17, ST36, ST48, ST54, and ST147. The study showed a multi-drug resistance at the inflow and outflow of the WWTP, with ESBL production, plasmid-mediated quinolones resistance, and mutations in topoisomerases. The findings highlight the similarity of antibiotic resistance mechanisms in the clinical setting and the environment, and the role of the latter as a source of dissemination of resistance genes. Introduction T he indiscriminate use of antibiotics has contributed to their significant presence in the environment, promoting resistant microorganisms by selection, mutation, and re- combination. 4,12,30 Contaminated aquatic environments are instrumental in dissemination of resistant bacteria (resistance genes) that can reach humans through direct contact or food chains. 12,50 The assessment of antibiotic resistance and elu- cidation of its mechanisms in human-related environments are an invaluable complement to clinical studies, for the understanding of the development and spread of antimi- crobial resistance. The wastewater treatment plant (WWTP) is an important link in the recycling of water, it constitutes a site of conjunction and concentration of various types of pollution, and its effluents have a significant impact on sanitary quality of the environment. The analysis of micro- bial antibiotic resistance at the level of WWTP is interesting, because these data are representative of the evolution that might occur in the aquatic environment. Our study was fo- cused on the beta-lactams and quinolones resistance because of the major importance of these antibiotic classes in the anti- infective therapy. One of the most efficient mechanisms of resistance to broad-spectrum cephalosporins is the produc- tion of extended spectrum beta-lactamases (ESBL), which are plasmid-encoded clavulanate-susceptible enzymes confer- ring resistance to third- and fourth-generation cephalospo- rins and to monobactams. 21,37 ESBLs are a major public 1 Laboratory of Cellular and Molecular Biology, Faculty of Biological Sciences, University of Sciences and Technology Houari Boumediene, Algiers, Algeria. 2 High National School of Marine Sciences and Coastal Management, Campus Universitaire de Dely-Ibrahim, Algiers, Algeria. 3 A ´ rea de Bioquı ´mica y Biologı ´a Molecular, Universidad de La Rioja, Logrono, Spain. MICROBIAL DRUG RESISTANCE Volume 00, Number 00, 2013 ª Mary Ann Liebert, Inc. DOI: 10.1089/mdr.2012.0264 1