Discussion
Our findings indicated that Acinetobacter baumannii is the most common species found in clinical isolates from tracheal aspirates from ICU. Similarly, studies reported high rates of baumannii species in ICU samples elsewhere ( Qin et al., 2021 ; Bakhshi et al., 2022 ). Previous Ecuadorian studies reported high percentages of A. baumannii from respiratory tract samples, bronchial secretions, or tracheal aspirates ( Jiménez, 2013 ; Nuñez et al., 2016 ; Villacís et al., 2019 ). On the other hand, we found A. baumannii in the most polluted river environments, Machángara and Monjas rivers ( Yungán, 2010 ; Roldós, 2015 ). Similar, some studies found these bacteria in river water as well as sewage water ( Hrenovic et al., 2016 ; Kisková et al., 2023 ). Pathogens of clinical origin as A. baumannii can reach aquatic environments as a result of different human activities, such as sewage water discharge directly into the river without pretreatment ( Silva et al., 2016 ). Acinetobacter spp. survive in wastewater contributing to the spread clinical antibiotic resistance genes in aquatic systems ( Kisková et al., 2023 ; Tobin et al., 2024 ). Particularly, hospital sewage water is considered a major source of multidrug-resistant pathogenic bacteria and antibiotic residues ( Zhang et al., 2013 ). Contrary, we did not isolate any Acinetobacter spp. from the Guayllabamba River probably due to the distance (2 km), the collection points were far away from the wastewater reservoirs of urban areas and the bacterial concentrations decreases and distribution changes ( Zhang et al., 2009 ).

In our study, the clinical and environmental Acinetobacter isolates showed different susceptibility profiles. Clinical isolates exhibited a high rate of resistance to imipenem and meropenem; while, river isolates displayed a low resistance only to imipenem. In general, clinical isolates have high resistance to both antibiotics ( Cortivo et al., 2015 ). The percentage of resistance found in this study was higher compared with an Ecuadorian study ( Jiménez, 2013 ). River isolates are usually more sensitive compared to clinical isolates. The difference in the patterns of resistance found can be due to various factors such as, biological conditions, virulence factors, epidemiology, or place of origin ( Karmostaj, Najar & Salmanian, 2013 ). In general, it is expected that the antibiotic concentration is lower in aquatic environments than in clinical environments ( Zhang et al., 2009 ; Zhang et al., 2013 ). In clinical environments, antibiotics (in particular broad-spectrum antibiotics) are supplied continuously, promoting selective pressure in clinical isolates. Following this, the transmission of genetic determinants among clinical isolates could take place through horizontal gene transfer ( Zhang et al., 2009 ; Castanheira, Mendes & Gales, 2023 ). Nonetheless, the possibility of clonal dissemination as a mean of increasing antibiotic resistance, should not be discarded. This situation generates a faster antibiotic resistance evolution in clinical isolates than in environmental isolates ( Zhang et al., 2013 ). However, the susceptibility results of environmental samples should be treated with caution, as these profiles were based on clinical standards (CLSI) and a small sample size ( n = 10).

The high percentage of carbapenem-resistance found in clinical isolates may be increased due to the presence of bla OXA-51 , bla OXA-23 , bla OXA-72 , and bla OXA-143 genes. According to some studies, the association of bla OXA-23 and bla OXA-51 genes as the main cause for carbapenem resistance ( i.e ., Martínez & Mattar, 2012 ; Teixeira et al., 2013 ; Cortivo et al., 2015 ). The bla OXA-72 gene increases its resistance activity in the presence of imipenem and ampicillin/sulbactam ( Kuo et al., 2013 ). The bla OXA-143 and bla OXA-72 genes in association with intrinsic mechanisms as the alteration of the of the outer membrane proteins promotes a high resistance to meropenem ( Mostachio et al., 2012 ; Sen & Joshi, 2015 ). The low percentage of carbapenem-resistance found in environmental isolates may be explained by the absence of bla OXA genes. It is necessary the presence of insertion elements to promote overexpression of the bla OXA-51 gene since this gene it generates a weak resistance ( Higgins, Zander & Seifert, 2013 ; Teixeira et al., 2013 ; Sen & Joshi, 2015 ). The bla OXA-51 overexpression would be linked to the resistance to imipenem in A. baumannii river isolates ( Martínez & Mattar, 2012 ; Evans, Hamouda & Amyes, 2013 ). It would be important to know about possible IS Aba1 insertions upstream of the gene or alternative genes conferring resistance in the five isolates with resistance phenotype.

We found a high percentage of bla OXA-23 and bla OXA-51 genes in A. baumannii and A. pittii clinical isolates. The high percentage of bla OXA-23 gene found in A. baumannii , is related to its distribution worldwide since a single sequence was found in the study. The bla OXA-23 gene distribution has been associated with a clonal spread ( Higgins et al., 2010b ; Labarca et al., 2016 ). In the other hand, bla OXA-51 gene has been reported principally in baumannii species ( Lee et al., 2012 ; Teixeira et al., 2013 ; Périchon et al., 2014 ). Since bla OXA-51 gene can be found in the chromosome ( Poirel, Naas & Nordmann, 2010 ), some authors consider it as an intrinsic marker for A. baumannii identification ( Higgins et al., 2010b ; Périchon et al., 2014 ). However, the gene may be in transposons and plasmids ( Liu et al., 2015 ; Wang et al., 2015 ; Sennati et al., 2016 ). Its dissemination among other Acinetobacter species from different environments is possible, suggesting that the dissemination may not be by clonal strains (See Labarca et al., 2016 ).

Our findings detected two different bla OXA-51 variants OXA-65 and OXA-70 were circulating in the four hospitals; while, in rivers probably are OXA-65 and OXA-259, suggesting that the genes were under selective pressure. Similarly, previous studies OXA-65 was reported in A. baumannii clone ST79 from Guayaquil ( Rodríguez et al., 2016 ). In Latin America, is one of the variants found in A. baumannii in Brazil, Argentina, Paraguay, Mexico, and Honduras ( Rodríguez et al., 2016 ; Nodari et al., 2020 ; Graña-Miraglia et al., 2020 ). There is not report about the presence of OXA-70 neither OXA-259 variant in South America. The information about OXA-70 variant is scare worldwide. OXA-70 has been reported in Hong Kong, Malaysia, Ghana, and Honduras ( Alattraqchi et al., 2020 ; Ayibieke et al., 2020 ; Brown & Amyes, 2005 ; Galac et al., 2020 ). OXA-70 belong to the most diverse collection of class D carbapenemases, subgroup 3 that is the major mechanism of carbapenem resistance in A. baumannii ( Brown & Amyes, 2005 ). OXA-259 variant has been reported in A. baumannii clinical isolates from China, Tanzania, and Thailand ( Jia et al., 2019 ; Moyo et al., 2021 ; Chukamnerd et al., 2022 ) as well in bulk tank milk in Germani ( Sykes et al., 2023 ). The variant OXA-259 is located on the chromosome of A. baumannii and constitute an intrinsic oxacillinase genes with low-level carbapenemase activity that naturally occur ( Chukamnerd et al., 2022 ), due there is a not ISA balike elements ( Fedrigo et al., 2022 ). To the best of our knowledge, this is the first report of the OXA-70 and OXA-259 in A. baumannii in Ecuador.

Our findings showed the presence of bla OXA-366 gene in both species A. baumannii and A. pittii clinical isolates from Ecuador. The information about this variant is scare. GenBank previously reported a sequence of bla OXA-366 in A. baumannii from USA. Whereas the OXA-366 variant has been reported also in A. baumannii clinical isolates in Iran ( Bahador et al., 2015 ). To the best of our knowledge, this is the first report of the OXA-366 in A. baumannii and A. pittii in South America.

The bla OXA-72 gene co-harbored with bla OXA-23 and bla OXA-51 genes was identified in low levels in A. baumannii and A. pittii . This variant was documented previously in A. baumannii clinical isolates from Guayaquil ( Nuñez et al., 2016 ; Rodríguez et al., 2016 ) and from neighboring countries Colombia, Peru, and Brazil ( Saavedra et al., 2014 ; Saavedra et al., 2017 ; Werneck et al., 2011 ; de Sá Cavalcanti et al., 2013 ; Levy-Blitchtein et al., 2018 ; Nodari et al., 2020 ) and in A. pittii from Colombia ( Montealegre et al., 2012 ), Brazil ( Brasiliense et al., 2019 ), European and Asian countries ( Al Atrouni et al., 2016b ; Bonnin et al., 2014 ). The presence of this gene in two species may be due to the bla OXA-72 gene spreads through horizontal gene transfer mediated by plasmids ( Montealegre et al., 2012 ; Kuo et al., 2013 ; Nuñez et al., 2016 ; Rodríguez et al., 2016 ). To the best of our knowledge, this is the first report of the bla OXA-72 gene in A. pittii in Ecuador.

This is also the first report of the bla OXA-143 gene in A. pittii clinical isolates from Ecuador. In South America, this gene and its variants (OXA-231, OXA-253, and OXA-255) have been reported in A. baumannii clinical isolates in Brazil ( Higgins et al., 2009 ; Gionco et al., 2012 ; Girlich et al., 2014 ), Perú ( Levy-Blitchtein et al., 2018 ), and Colombia ( Saavedra et al., 2017 ). The variant found in this study is very different from OXA-143 variants reported in A. baumannii from Brazil, Honduras, and Korea ( Higgins et al., 2009 ; Zander et al., 2014 ; Kim et al., 2010 ). Interestingly, the sequence alignment showed a high similarity between our variant with two OXA-143 variants (OXA-499 and OXA-255) described to A. pittii in Korea ( D’Souza et al., 2017 ) and United States ( Zander et al., 2014 ). In particular, the difference between the variant reported in this study differs by a change in amino acid sequence with OXA-499 reported by D’Souza et al. (2017) . To the best of our knowledge, this is the first report of a variant similar to OXA-499 in A. pittii in South America.