Materials and Methods
The National Reference Laboratory for Antimicrobial Resistance provided us a total of 52 Acinetobacter spp. clinical isolates recovered through the surveillance network from January to December 2015. As part of the standard procedure, the Reference Laboratory get back the Acinetobacter isolates from four different hospitals: Hospital Pediátrico Baca Ortiz (H1, n = 42), Hospital Pablo Arturo Suárez (H2, n = 5), Hospital del Sur Enrique Garcés (H3, n = 3), and Hospital San Francisco de Quito (H4, n = 2) in Quito ( Figure 1 ). The Reference Laboratory isolated Acinetobacter spp. from the following clinical samples: pleural fluid, cerebrospinal fluid, blood, urine, catheters, secretions, wounds, skin, abscesses, sputum, tracheal aspirates, and unidentified samples recovered from eleven units (ICU, general surgery, traumatology, burn unit, external consultation, neonatal unit, infectious diseases, pediatric unit, internal medicine, emergency, cardiology, and unidentified units). The data from each isolate are detailed in Table S1 . The Reference Laboratory previously identified all isolates as A. baumannii based on VITEK 2 compact system® (Biomerieux, Marcy-I’Étoile, France).

We inoculated the frozen clinical isolates in sheep blood and MacConkey agars and incubated at 37 °C for 24 h. We used Gram staining, oxidase test, and catalase test to re-confirmed the identity of the isolates. We performed DNA extraction by boiling according to the protocol described by Cuaical et al. (2012) ; briefly, we boiled an overnight culture diluted in sterile water (500 μl) at 100 °C for 15 min, following centrifugation at 10,000 g for 2 min. We quantified the supernatant by spectrophotometry using the Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA) and stored at −20 °C for subsequent amplification. We confirmed species identification by the amplification of partial RNA polymerase β ( rpoB ) gene and DNA gyrase subunit B ( gyrB ) gene according to La Scola et al. (2006) and Higgins et al. (2007 , 2010a) , respectively. The primers selected are detailed in Table 1 .

Note:

The primer name, the sequences of primer forward and reverse, the expected size, and reference where the sequences were obtained.

We collected the water samples from five principal rivers: Monjas (MO, n = 6), Machángara (MA, n = 6), San Pedro (S, n = 6), Pita (P, n = 6), and Guayllabamba (G, n = 3) in March and April 2016 ( Figure 1 and Table S1 ). We chose those rivers for two reasons: (i) the four rivers are the main ones that run through Quito, all of them flow into the Guayllabamba River, and (ii) sewage is released into the city’s rivers without prior treatment. In addition, we chose the number of water collection points based on the accessibility and proximity to the urban center. We collected 100 ml of water samples at each river point and transported to the laboratory with ice packs according to Zhang et al. (2013) with few modifications. Subsequently, we centrifuged the water samples at 1,000 g for 10 min at room temperature, resuspended the pellets with 5 ml of fluid thioglycollate medium, and incubated at 37 °C overnight. We inoculated thirty microliters of cultured sample onto CHROMagar Acinetobacter plates with antimicrobial selective supplement (CR 202; CHROMagar Company, Paris, France) and incubated at 37 °C overnight. We selected all colonies with red pigmentation and identified through morphological and conventional biochemical tests used for the clinical isolates. We identified the positive potential isolates using VITEK-2 compact system® (Biomerieux, Marcy-I’Étoile, France). We confirmed the species identification by the amplification of DNA gyrase subunit B ( gyrB) gene according to Higgins et al. (2007 , 2010a) .

We use the disc diffusion method and the EDTA sinergy test to determinate the antimicrobial susceptibility and the production of metallo β-lactamases of clinical and river Acinetobacter spp. isolates ( Clinical Laboratory Standards Institute, 2022 ). We spread a concentration corresponding to 0.5 of the McFarland scale of each isolate on Muller Hinton agar plates and incubated at 37 °C overnight. We included the following eleven antimicrobial susceptibility test discs (OXOID, Waltham, MA, USA): ampicillin/sulbactam (20 μg), ceftazidime (30 μg), ciprofloxacin (10 μg), imipenem (10 μg), meropenem (10 μg), gentamicin (10 μg), amikacin (30 μg), cefepime (30 μg), trimethoprim/sulfamethoxazole (25 μg), piperacillin/tazobactam (110 μg), and tobramycin (30 μg). We interpreted the resistance based on the susceptibility breakpoints of the Clinical Laboratory Standards Institute (2022) . To assess the synergistic inhibition, we included an ethylenediaminetetraacetic (EDTA) disc (5 mg) with two carbapenem discs, imipenem (10 mg) and meropenem (10 mg), located at 2 cm from the center of the EDTA disc.

We performed two different multiplex PCRs to screen the presence of carbapenems-resistant genes including six genes encoding enzymes type serine β-lactamases ( bla GES , bla OXA-23 , bla OXA-24 , bla OXA-51 , bla OXA-58 , and bla OXA-143 ) and three genes encoding metallo-β-lactamases ( bla SPM , bla SIM , and bla GIM ) using specific primers previously described ( Ellington et al., 2007 ; Woodford et al., 2006 ) ( Table 1 ). We achieved a PCR amplification in a final volume of 25 μl containing 1X of Master Mix Green GoTaq® (12.5 μl) (Promega, Madison, WI, USA), 10 μM of forward and reverse primers (0.5 μl), and 50 ng DNA (1 μl). To each PCR assay, we put positive, internal, and negative controls. Both multiplex PCR reaction conditions are detailed in Table 2 . We identified all PCR products through electrophoresis in a 2% agarose gel stained with GelstarTM Nucleic Acid Gel Stain 10000 X staining (Lonza, Rockland, ME, USA) and visualized using Gel Doc XR system (Bio-Rad, Hercules, CA, USA).

Note:

The different thermal cycling conditions for various molecular analyses in this study.

We performed a conventional PCR to amplified the entire sequence of the following genes bla OXA-23 , bla OXA-24 , bla OXA-51 , and bla OXA-143 genes using primers previously reported ( Heritier, Poirel & Nordmann, 2006 ; Afzal-Shah, Woodford & Livermore, 2001 ; Jeon et al., 2005 ; Woodford et al., 2006 ) ( Table 1 ), following the conditions detailed in Table 2 . We cleaned the PCR products using exonuclease I and shrimp alkaline phosphatase (ExoSAP-IT®). We send both DNA strands of all positive bla OXA genes for sequencing to Macrogen (Seoul, Korea). We aligned manually both forward and reverse sequences for editing and cleaning to obtain a single consensus sequence with the Clustal W program implemented in the MEGA 7 software ( Kumar, Stecher & Tamura, 2016 ). We performed the identification of sequences obtained using a basic local alignment search tool (BLAST) ( Altschul et al., 1997 ). We determined the number of sequences of bla OXA-51 and bla OXA-23 genes with DnaSP v.5.10 software ( Rozas et al., 2010 ).