Results
Acinetobacter spp. clinical isolates originated mainly proceeded from pediatric population (77%; 40/52), followed by the adult population (19.2%; 10/52). Isolates derived principally from patients admitted in UCI 32.69% (17/52), followed by unidentified units 15.38% (8/52) and general surgery 13.46% (7/52) ( Figure 2A ). According to the clinical specimens, the isolates came from tracheal aspirates 28.85% (15/52), followed by catheters 11.54% (6/52) and abscesses 9.62% (5/52) ( Figure 2B ). The majority of clinical isolates were identified as A. baumannii (96.15% 50/52) and A. pittii (3.85%; 2/52) according rpoB gene. Only the identity of A. pittii (3.85%; 2/52) was confirmed with both markers, rpoB and gyrB genes. A. baumannii was found in all type of clinical specimen from all hospital units while A. pittii was found only in tracheal aspirates from UCI. The biochemical and molecular identification of each isolate is detailed in Tables S2 and S3 . The nucleotide and amino sequences of partial rpoB gene are detailed in Informations S1 and S2 . We submitted all rpoB sequences to GenBank with assigned accession numbers OP796738 – OP796781 .

A total of 27 water samples were collected from five rivers. Twenty-two samples presented suggestive Acinetobacter isolates, four samples (RMO3, RS5, RP4, and RG3) showed no growth, and one sample (RMO4) had isolates not related to Acinetobacter spp. We recovered twenty-six suggestive isolates from San Pedro (2), Machángara (4), Pita (1), and Monjas (3) rivers, of which only 38.5% (10/26) were identified as Acinetobacter spp. based on VITEK 2 compact® (Biomerieux, Marcy-I’Étoile, France) and as A. baumannii based on gyrB gene amplification. The remaining corresponded to other genera ( Pseudomonas aeruginosa : 34.6%, Stenotrophomonas maltophila : 19.3%, Burkkolderia cepacia : 3.8%, and Moraxella group: 3.8%). The biochemical and molecular identification of each isolate is detailed in Tables S2 and S3 .

Clinical isolates presented the higher resistance (100%) to the three antibiotics: imipenem, meropenem, and piperacillin/tazobactam. Forty-eight percent were susceptible to tobramycin ( Figure 3A ). The river isolates displayed an antimicrobial profile that varied between resistant and intermediate to eight antibiotics tested. Nevertheless, the main resistance was to piperacillin/tazobactam (100%). All of them were susceptible to tobramycin and ampicillin/sulbactam (100%) ( Figure 3B ). According to the synergistic inhibition, both river and clinical isolates did not present metallo-β-lactamases enzymes. The antimicrobial susceptibility profile of the clinical and river isolates is detailed in Table S2 .

According the screening of carbapenem-resistance genes, clinical isolates presented the following genes: bla SIM , bla SPM , bla GES , bla OXA-23 , bla OXA-24 , bla OXA-51 , and bla OXA-143 ; whereas, seven river isolates showed bla OXA-51 gene ( Table S3 ). The bla SIM gene, co-harbored with the bla OXA-72 and bla OXA-143 genes, was the least frequently detected (1.9%; 1/52; 15-1175) in A. pittii . The bla SPM gene, co-harbored with the bla OXA-51 and bla OXA-23 genes, was detected in 13.46% (7/52; 15-0122, 15-0591, 15-690, 15-780, 15-795, 15-853, 15-1,138) in A. baumannii . The bla GES gene, co-harbored with the bla OXA-51 , bla OXA-23 , bla OXA-72 , bla OXA-143 , and bla SIM genes was present in 11.53% (6/52) in A. baumannii (15-0669, 15-690, 15-853, 15-900) and A. pittii (15-691, 15-1,175). The bla OXA-23 gene, co-harbored with the bla OXA-51 gene, was found in 98.07% (51/52) present in A. baumannii . The bla OXA-24 gene, co-harbored with both bla OXA-23 and bla OXA-51 genes, was present in 9.6% (5/52) in A. baumannii (15-0659, 15-0985, 15-1064, and 15-1367) and in A. pittii (15-0691). The bla OXA-143 gene was the least frequently detected (1.9%; 1/52; 15-1,175); this gene was co-harbored with the bla OXA-24 gene in A. pittii .

We reported 47 bla OXA-51 sequences: 42 complete sequences of 813 bp and five partial sequences of 340 bp from A. baumannii clinical isolates. Of bla OXA-51 sequences, we identified two genes encoding OXA-65 ( n = 44), OXA-70 ( n = 2) and one unidentified ( MF594746 ). Forty complete sequences ( MF594725 – MF594728 , MF594730 – MF594737 , MF594735 , MF594739 , MF594741 – MF594745 , OP554146 – OP554168 ) and four partial sequences ( MF594729 , MF594734 , MF594736 , and MF594737 ) shared 100% of nucleotide identity with bla OXA-65 of A. baumannii (GenBank accession number CP033869.1 ); whereas two entire sequences ( MF594740 and MF594747 ) shared 100% nucleotide identity with bla OXA-70 of A. baumannii (GenBank accession number NG_049811.1 ).

We found 48 bla OXA-23 sequences: 42 complete sequences of 820 bp and six partial sequences of 471 bp from A. baumannii clinical isolates. Of bla OXA-23 sequences, we identified two genes encoding OXA-23 ( n = 38) and OXA-366 ( n = 10). Thirty-two entire sequences ( MF594756 – MF594759 , MF594762 – MF594767 , MF594769 , MF594772 – MF594777 , OP554169 , OP554170 , OP554172 – OP554178 , OP554180 – OP554182 , OP554185 – OP554187 , OP554189 , OP554191 – OP554193 ) and six partial sequences ( MF594755 , MF594760 , MF594761 , MF594768 , MF594770 , and MF594771 ) shared 100% of nucleotide identity with bla OXA-23 of A. baumannii (GenBank accession number CP110468.1 ). Ten complete sequences ( MF594757 , MF59474 – MF59476 , OP554171 , OP554179 , OP554183 , OP554184 , OP554188 , and OP554190 ) shared 100% nucleotide identity with bla OXA-366 of A. baumannii (GenBank accession number NG_049659.1 ).

We reported five bla OXA-24/40 entire sequences of 817 bp ( MF594778 , MF594779 , MF594781 , MF594782 , and MF594783 ) and one partial sequence of 204 bp ( MF594780 ). Of bla OXA-24/40 sequences, we identified one gene encoding OXA-72 present in three A. baumannii and two A. pitti clinical isolates that shared 100% of nucleotide identity with bla OXA-72 of A. baumannii (GenBank accession number MN495626 ) and also A. pittii (GenBank accession number MN481287.1 ).

Sequencing of the bla OXA-143 PCR product obtained from A. pitti clinical isolates identified gene encoding OXA-499. Our bla OXA-143 sequence ( MF594724 ) shared 99% nucleotide and amino acid identity with bla OXA-499 of A. pittii (GenBank accession numbers NG_049775.1 and ALM96709.1 ). The nucleotide sequence presented one specific change in nucleotide 280, first position of the codon (GTG) (ATG), resulting in a valine (V) to methionine (M) change in position 94 ( Figure 4 ).

Sequencing of the bla OXA-51 PCR products obtained from A. baumannii river isolates identified two genes encoding OXA-65 and OXA-259. Four partial sequences ( MF594748 – MF594751 ) from three rivers (Pita, Machángara, and San Pedro) shared 100% of nucleotide identity with bla OXA-65 of A. baumannii (GenBank accession number OL961415.1 ) and three partial sequences ( MF594752 – MF594754 ) from Machángara River shared 100% of nucleotide identity with bla OXA-259 of A. baumannii (GenBank accession number CP053098.1 ).

The nucleotide and amino sequences of bla OXA genes are detailed in the Informations S3 to S10 . We submitted all sequences to GenBank with assigned accession numbers MF594724 – MF594783 , OP554146 – OP554193 .