Acinetobacter baumannii is a highly clinically relevant pathogen that can lead to fatal nosocomial infections affecting mainly compromised patients or patients with underlying diseases. Infections caused by A. baumannii include pneumonia due to mechanical ventilation, catheter associated infections, bacteremia, urinary tract and bone infections, skin and wound infections. A. baumannii has a propensity to cause outbreaks at Intensive Care Units (ICU), and is strongly linked to the multidrug resistance phenotype, particularly carbapenem-resistance. Its clinical success is mainly due to its high virulence and the ability to survive in adverse conditions such as inanimate surfaces, combined with its desiccation and biocide tolerance, but also its capacity to acquire antibiotic resistance genes that are often coded by mobile genetic elements facilitating their spread (Jeon et al., 2023; Lee et al., 2017). Especially worrying is resistance to last resort antibiotics such as carbapenems, that usually makes infections difficult to treat, prolonging the hospital stay and making it necessary the use of more harmful and expensive antibiotics, leading sometimes to the death of the patients (Castanheira et al., 2023; Hamidian and Nigro, 2019; Kyriakidis et al., 2021; Ramirez et al., 2020; Vijayakumar et al., 2019).

During the COVID-19 pandemic, carbapenem resistant A. baumannii (CRAB) infections increased due to the massive admission of patients into hospitals, globalization, migration and the inappropriate use of antimicrobials (Castanheira et al., 2023; Montrucchio et al., 2022; Müller et al., 2023). In 2017 the World Health Organization published a list of critical pathogens for which new antibiotics were needed, with CRAB as the number one critical priority pathogen, and is still heading the list in the updated list published in 2024 (Tacconelli et al., 2018; World Health Organization, 2024). These traits contribute to its dissemination and contribute to its propensity for nosocomial outbreaks, especially where there are gaps in the infection-prevention and control regimes making it a critical pathogen threatening global health (Castanheira et al., 2023; Hamidian and Nigro, 2019; Montrucchio et al., 2022; Rodríguez et al., 2018).

According to Rodríguez et al. analyzing data obtained from 2000 to 2013, only 60% of the Latin-American countries had reported CRAB, mainly in Argentina and Brazil (Rodríguez et al., 2018). Resistance to carbapenems (meropenem and imipenem) in Latin-America is especially alarming considering it is ranging from 80 to 90%. The data obtained after the COVID-19 pandemic raised concern about the health system since that trend was increased in several countries such as Peru and Argentina, but also in Paraguay, Guatemala, Bolivia, El Salvador, Nicaragua and Ecuador (Manobanda Nata and Jaramillo Ruales, 2023). Most of the carbapenemase-encoding genes circulating in Latin-America belong to the oxacillinase families bla _OXA-23, bla _OXA-58, bla _OXA-24/40 (bla _OXA-72), bla _OXA-143 (bla _OXA-253) and bla _OXA-214 (bla _OXA-215, bla _OXA-264, bla _OXA-265, bla _OXA-575) (Manobanda Nata and Jaramillo Ruales, 2023; Naas et al., 2017). However, less frequently, metallo-beta-lactamase genes such as bla _NDM, bla _VIM and bla _IMP are also reported (Manobanda Nata and Jaramillo Ruales, 2023). Although Paraguay is one of the Latin-American countries with limited accessible data regarding antibiotic resistance and epidemiology of the circulating A. baumannii isolates, the most recent reports showed that 70% of the isolates are carbapenem-resistant (Melgarejo-Touchet et al., 2021) usually carrying bla _OXA-23-like and bla _NDM-like genes, while coexistence of bla _NDM-like and bla _OXA-58-like genes was reported in a 2% of isolates (Melgarejo-Touchet et al., 2021). International clone (IC) 5 and IC7 are the most common lineages found in the country (Rodríguez et al., 2016).

The aim of this work was the analysis of the resistance to antibiotics during the years 2022, 2023 and 2024, and the molecular characterization by whole genome sequencing of a representative series of carbapenem-resistant A. baumannii isolates recovered from the National Hospital of Itaugua (NHI), Paraguay, in 2024.

Paraguay, like other Latin-American countries, has published little information on the incidence and epidemiology of A. baumannii. During 2022, an average of 743 microorganisms (bacteria and fungi) were recovered in the NHI, including isolates recovered upon hospital admission or and during hospitalization, of which 11% were identified as A. baumannii. Considering only the isolates recovered during hospitalization, A. baumannii is the most commonly isolated bacteria in the hospital. Thus, A. baumannii is of great concern to the healthcare community and it poses a challenge in the treatment of infections due to its high level of resistance to antibiotics (Rodríguez et al., 2018; Manobanda Nata and Jaramillo Ruales, 2023).

Carbapenem resistance rates of the A. baumannii isolates recovered in NHI in 2022 were above 80%. However, data obtained from the first two months of 2024 suggests that the carbapenem resistance rates are increasing up to 90% which is in accordance with data previously reported in the region (Melgarejo-Touchet et al., 2021; Manobanda Nata and Jaramillo Ruales, 2023). In the present study the majority of the A. baumannii isolates from the ICU patients were recovered from respiratory samples, which has also been reported in other studies, where isolates were recovered mainly from ICU patients that were suffering from pneumonia (Montrucchio et al., 2022; Brizuela Centurión et al., 2023; Castanheira et al., 2023).

Clonal relatedness analysis by MLST and cgMLST allowed us to identify the international clonal lineage to which the investigated isolates belong. A single isolate (PR7) was assigned to IC5 (ST79 Pasteur scheme), the so-called Pan-American clone that is characteristic and commonly described in CRAB isolates in Latin America, often along with clone IC7 (ST25) carrying the bla _OXA-23 gene, confirming its circulation in the area (Rodríguez et al., 2016; Nodari et al., 2020). It should be noted that the characteristics of this clone are consistent with those previously described by our group in strains from the neighboring country, Bolivia (Cerezales González, 2018). Most isolates were assigned to IC2, a lineage known for its global distribution, although its presence in Latin-American countries is rarely reported (Castillo-Ramirez, 2023; Morgado et al., 2023), as in Brazil in 2008 and 2015-2016 (Rodríguez et al., 2016; Camargo et al., 2022). Within the isolates belonging to IC2, ST2 (Pasteur scheme) was the most prevalent sub-lineage, which is in accordance with the reported by other authors worldwide (Hamidian and Nigro, 2019). The most recent region-wide study also including isolates from Paraguay between July 2013 and June 2014, showed that the circulating clones belonged to IC5 (ST79 Pasteur) and IC7 (ST25 Pasteur), but no IC2 isolates were detected (Rodríguez et al., 2016). The presence of IC2 isolates carrying the bla _OXA-23 gene in this study confirms the regional distribution of clone IC2, as described by other authors who identified this clone in South America (Levy-Blitchtein et al., 2018; Camargo et al., 2022; Martins-Gonçalves et al., 2024). During the COVID-19 pandemic, factors such as the massive influx of patients into ICUs and the increased use of antimicrobial agents intensified the selective pressure for multidrug-resistant pathogens. This led to outbreaks worldwide and also, in neighboring countries to Paraguay such as Brazil, where outbreaks caused by ST2 isolates, carrying bla _OXA-23, were identified between 2020 and 2021 (Camargo et al., 2022).

The detailed analysis of the antibiotic resistance genes showed the presence of two bla _OXA-51 variants, bla _OXA-66 and bla _OXA-65, with bla _OXA-66 as the characteristic variant of the IC2 clone and in line with its emergence at a regional level (Camargo et al., 2022). As previously mentioned, all the isolates were carrying the carbapenemase gene bla _OXA-23, a higher prevalence than the previously reported by Melgarejo et al (Melgarejo-Touchet et al., 2021). It is worth mentioning that IC2 isolates carried two copies of the gene, a frequent phenomenon among clinical A. baumannii isolates, according to the literature (Hua et al., 2016). These genes were located within transposons Tn2006 and Tn2008, a commonly described structure that plays a key role in the overexpression and mobilization of the gene (Hamidian and Nigro, 2019; Sánchez-Urtaza et al., 2023). Additionally, two different variants of the bla _ADC gene were identified: bla _ADC-73 and bla _ADC-5. The isolates belonging to IC2 were carrying bla _ADC-73 gene with ISAba1 upstream of it, a mechanism of overexpression that is commonly reported in clinical isolates (Nodari et al., 2020; Sánchez-Urtaza et al., 2023; Hamed et al., 2023). However, the bla _ADC-5 gene that was identified in the isolate belonging to IC5 was accompanied by IS3 upstream of it, which differs from the previously reported in other isolates from clone IC5 (Lopes et al., 2013).

Additional genes conferring resistance to other classes of antibiotics were also identified in isolates belonging to clone IC2: armA, aph(6)-Id, aph(3’’)-Ib, msr(E), mph(E), sul1, catA1 and tet(B); genes that have been reported recently in isolates that caused an outbreak in Brazil (Camargo et al., 2022). It is worth mentioning that in those countries where the IC2 isolates carrying the armA gene was detected and was predominant, they exhibited high resistance rates to aminoglycosides such as amikacin, gentamicin and tobramycin (Camargo et al., 2022; Martins-Gonçalves et al., 2024). In this study, the armA, mph(E) and msr(E) genes were identified in the chromosome within a Tn6180-derived fragment of the resistance island AbGRI3. This arrangement is globally spread and has been previously described in other CRAb isolates assigned to ST2 (Pasteur Scheme) and also ST195/1816 (Oxford Scheme), which is in accordance with the genetic contexts identified in our isolates (Wiradiputra et al., 2023; Wei et al., 2024). Although chromosomal integration may reduce the immediate risk of horizontal transfer compared to plasmid-borne elements, it can promote stable maintenance of these resistance genes within bacterial populations, and also, the presence of these genes in a Tn6180-derived structure suggests the potential for mobilization under selective pressure. This highlights the importance of monitoring chromosomal resistance islands as reservoirs of clinically significant resistance determinants. The aph genes (aph(6)-Id, aph(3’’)-Ib) together with tet(B) were found close to the carbapenemase gene bla _OXA-23 in an AbGRI1-like resistance island in all the isolates belonging to IC2. This island has been repeatedly documented in clinical IC2 isolates across different countries and contributes significantly to their multidrug-resistant phenotype (Cerezales et al., 2020; Vijayakumar et al., 2022; Wiradiputra et al., 2023; Naderi et al., 2023). All the studied isolates belonging to IC2 were also harboring sul1 and catA1 together with qacEΔ1 (antiseptic resistance) and merC (mercury resistance) genes in a complex structure of ~15 kb involving multiple mobile genetic elements such as IS26, IS6100 and Tn2-like elements, that closely resembles mosaic resistance regions previously described in Acinetobacter genomic islands (Hamidian and Hall, 2018b). Moreover, IC2 isolates have been shown to carry AbGRI variants with class 1 integron backbones containing sul1 and qacEΔ1 together with IS26-mediated rearrangements (Harmer et al., 2023). These findings support the hypothesis that this region represents a chromosomally integrated resistance island related to AbaR or AbGRI structures that emerged through IS26‐mediated horizontal gene capture.

The resistome of the IC5 isolate also included a variety of genes conferring resistance to non-β-lactam antibiotics such as aminoglycosides (armA, aph(6)-Id, aph(3’’)-Ib, aph(3’)-VIa, sat2 and aadA1), sulfonamides (sul2) and trimethoprim (dfrA1), which are commonly described in CRAb strains belonging to this clone (Nodari et al., 2020). In this isolate the aph(6)-Id, aph(3’’)-Ib and sul2 genes were identified within a non-canonical variant of the transposon Tn6250, characterized by the insertion of an ISAba125 element between the traA and traD genes. This transposon has been previously described by other authors (Brito et al., 2022), however, the presence of the ISAba125 was not previously reported. From a clinical perspective, such rearrangements may influence the mobility and transfer efficiency of resistance genes, potentially enhancing their spread across bacterial populations. The aph(3’)-VIa gene, is commonly associated with resistance to amikacin and is typically found within the TnAphA6 transposon, a composite element bracketed by ISAba125 insertion sequences (Khongfak et al., 2022; Brito et al., 2022). This transposon has been frequently identified in A. baumannii isolates, particularly those belonging to IC5, suggesting a strong association between this mobile element and the clonal lineage (Brito et al., 2022; Jouybari et al., 2021; Morgado et al., 2023). Moreover, TnAphA6 has been found both on conjugative plasmids and integrated into the chromosome, often in multidrug-resistant backgrounds (Khongfak et al., 2022; Brito et al., 2022). The mobility conferred by IS26 likely facilitates its dissemination not only through clonal expansion but also via horizontal gene transfer. The presence of aph(3’)-VIa within the TnAphA6 transposon in our IC5 isolate further supports its role in the global spread of aminoglycoside resistance within this lineage. In addition, dfrA1, sat2 and aadA1 genes were also identified together in isolate PR7 within a class 2 integron structure involving the bla _TEM-1 gene. However, the additional insertion of a Tn3 transposon harboring bla _TEM-1 within the Tn7 backbone, forming a Tn7::Tn3 composite transposon, suggests active modular recombination events. This unusual arrangement may enhance the mobility and plasticity of Tn7 by incorporating new resistance determinants. Similar nested transposition events have been reported for instance in ST79 A. baumannii isolates from Chile and Bolivia (Brito et al., 2022; Cerezales et al., 2020) but are relatively rare, and their presence highlights the role of Tn7 as a key element for the acquisition of multiple resistance genes, particularly in multidrug-resistant A. baumannii.

Regarding the virulome of the isolates, most of the genes were involved in biofilm production, which is key for survival and invasion of host’s cells (Kumar et al., 2021). All the isolates were harboring genes coding for Csu fimbriae, BfmRS system, Bap protein, extracellular polysaccharide PNAG, OmpA, Ata transporter and AbaI/AbaR proteins, which according to the literature, are linked with the ability to form biofilm (Ghasemi et al., 2018). Another important virulence factor is the capsule polysaccharide K locus (KL) and outer core OC locus (OCL) (Müller et al., 2023). Capsular polysaccharide genes vary between isolates within the same species, and recent studies associate certain types of capsular polysaccharides or their alterations with increased virulence and biofilm production (Cahill et al., 2022). In this work, different capsule types were identified: OCL1/KL3, OCL1/KL81 and OCL10/KL9. Isolates belonging to IC2 had the OCL1 gene cluster, the most predominant among IC1 and IC2 according to the literature (Kenyon et al., 2014). Among the IC2 isolates, it is common to find a wide variety of different K loci, with KL3 gene cluster predominant according to other authors (Müller et al., 2023; Camargo et al., 2022; Cahill et al., 2022), which is in accordance with our results, where 4 out of 7 IC2 isolates were KL3. The OCL10 gene cluster is predominantly found in isolates belonging to IC5, as well as K locus KL9 (Müller et al., 2023), which is in accordance with our findings. Despite sharing identical virulence gene content and OCL/KL loci as other biofilm producing strains, isolate PR6 exhibited significantly reduced biofilm formation. A possible explanation is that this phenotype results from differences in the expression of biofilm-related genes, which are regulated by the BfmRS two-component system and the csu operon—both essential for pili-mediated surface attachment. Disruption of either bfmR or bfmS can lead to reduced csu expression, thereby preventing biofilm formation (Gao et al., 2019).

Plasmid analysis showed the presence of a RepAci1 plasmid of 8.73 Kb in all the isolates belonging to IC2. This plasmid has been described previously in isolates belonging to IC1, IC2 and IC3, encoding a toxin-antitoxin system (brnT/brnA), septicolysin and TonB-dependent receptor ZnuD2 (Aranzamendi et al., 2024; Hamidian and Hall, 2018a), but unlike some of those isolates, the Paraguayan isolates were not harboring any carbapenemase gene in their plasmids. In fact, some variants of r3-T1 plasmids carrying a carbapenemase gene appear to be limited to North America and Europe (Lam and Hamidian, 2024). Replicon typing experiments by PCR were positive for replicase Aci4, however, typing using Acinetobacter plasmid typing database published by Lam et al. (2023) did not show the presence of that replicase, instead, r3-T14 was identified, which is very similar to the r3-T11 (Aci4) and may explain the positive result in the PCR experiment (Lam et al., 2023).

The results obtained in this study allowed us to update and get an approximate picture of the current antibiotic resistance rates, resistome, virulome, plasmid profiles and also the circulating clones of A. baumannii in Paraguay. This type of study is crucial not only for gaining knowledge about the characteristics of the clones circulating in the country, but also to design and implement control measures to prevent outbreaks.