Klebsiella aerogenes, previously identified as Enterobacter aerogenes, is a Gram-negative bacterium belonging to the Enterobacteriaceae family. It can be found in various environments including water, soil, air, and the human digestive system as a commensal organism. However, this bacterium is also a significant opportunistic pathogen that has been associated with hospital-acquired diseases such as pneumonia, meningitis, skin, soft tissue and urinary tract infections (Chen et al., 2015).

β-lactam antibiotics are one of the most commonly prescribed drug classes with numerous clinical uses. These antibiotics are sub-classed as penicillins (these being the most commonly prescribed), cephalosporins, cephamycins, monobactams, and carbapenems (Sta Ana et al., 2021). Klebsiella aerogenes is intrinsically resistant to ampicillin, amoxicillin, first-generation cephalosporins, and cefoxitin due to the expression of a constitutive AmpC β-lactamase. AmpC type lactamases are also known as extended spectrum β-lactamases (ESBLs) and these provide resistance against the majority of β-lactam antibiotics, such as extended-spectrum cephalosporins and monobactams, with the exception of carbapenems and cephamycins (Davin-Regli and Pages, 2015; Castanheira et al., 2021).

The presence of ESBLs in K. aerogenes strains has been well-documented, leading to the use of carbapenems as a last-resort treatment for serious infections caused by these pathogens. However, previous studies have shown a high prevalence of antibiotic resistance to cephalosporins and carbapenems in clinically relevant K. aerogenes strains worldwide (Ma et al., 2020). These infections pose a significant public health challenge due to the limited treatment options available and their elevated mortality rates (Mulani et al., 2019; Denissen et al., 2022).

The main resistant mechanism of carbapenemase-producing K. aerogenes is the production of carbapenemases, although other mechanisms have been proposed, including overproduction of β-lactamases, efflux pumps, porin deficiency, and changes in penicillin-binding proteins (Pan et al., 2021). Carbapenem resistance in clinical isolates of Carbapenem Resistant Enterobacterales (CRE) is predominantly caused by the presence of these carbapenemases, especially Klebsiella pneumoniae Carbapenamase (KPC) and New Delhi Metallo-β-lactamase (NDM). bla_KPC is commonly found on various plasmids like IncF-, IncI-, IncA/C-, IncX-, and IncR-type plasmids, while bla_NDM is mostly associated with IncX3-type plasmids. These plasmids are easily transferable and can promote the dissemination of bla_KPC and bla_NDM through horizontal gene transfer among diverse bacterial populations spreading antibiotic resistance (Yuan et al., 2023).

Although carbapenemases such as KPC, NDM, and Imipenemase (IMP) have been detected in K. aerogenes, there are limited studies demonstrating the simultaneous presence of bla_KPC and bla_NDM genes. Here, we conducted an in-depth analysis of genes related to the antimicrobial resistance and mobile genetic elements in carbapenemase-producing K. aerogenes, found in Brazilian hospitals, to understand its genomic diversity. To the best of our knowledge, this paper is the first to report the simultaneous presence of both bla_KPC and bla_NDM in K. aerogenes isolated from clinical samples in Brazil.

Klebsiella aerogenes is known as an opportunistic pathogen of patients admitted to the intensive care unit and is frequently associated with multidrug resistance (MDR) (Azevedo et al., 2018). It has been detected in various hospital sources and is known for its ability to adapt to this setting. This study found that K. aerogenes was mainly detected in rectal swabs and urine samples, followed by tracheal aspirate, blood, and catheter tips. Previous studies have highlighted that K. aerogenes is commonly present in human specimens such as urinary, gastrointestinal, respiratory, blood, abscesses, and cutaneous samples (Davin-Regli et al., 2019). Additionally, rectal swabs are employed for active surveillance of asymptomatic carriers. These findings are also consistent with prior studies demonstrating the presence of E. aerogenes in surveillance rectal swabs (Vrioni et al., 2012). But the small number of K. aerogenes isolates is one of the major limitations of this study.

Our study found that all K. aerogenes isolates displayed some level of resistance to the β-lactams tested, including carbapenems, and 50% of K. aerogenes isolates exhibited a MDR profile (Table 1). Carbapenems have traditionally been a preferred treatment for infections caused by MDR Gram-negative strains (Bouza, 2021). Therefore, the emergence of carbapenem-resistant K. aerogenes strains present a new challenge in the treatment of these infections and pose a public health threat worldwide (Kamio and Espinoza, 2022).

The presence of resistance genes was verified using polymerase chain reactions. Out of the 10 isolates, we discovered that two only contained the bla_KPC-2 gene and three only contained the bla_NDM-1 gene while, interestingly, five strains carried both the bla_KPC-2 and bla_NDM-1 genes. Previous studies have reported the presence of bla_KPC or bla_NDM genes in clinical K. aerogenes isolates from various countries (Pulcrano et al., 2016; Franolić et al., 2019; Ma et al., 2020), including Brazil (Bispo Beltrão et al., 2020; Soares et al., 2021). However, our literature review indicated that there has been only one study of K. aerogenes co-harboring both bla_KPC and bla_NDM genes, which was observed in China (Zhang et al., 2017). Therefore, to the best of our knowledge, this is the first report describing clinical samples of K. aerogenes isolated from Brazil with simultaneous carriage of bla_KPC and bla_NDM genes. It is noteworthy that we found a significant number of K. aerogenes isolates exhibiting a bla_KPC-2 or bla_NDM-1 carbapenemase in addition to ESBLs genes, including members of the bla_CTX-M-1; bla_{OXA1, 4, 30}; bla_TEM; and bla_SHVvariants. Our findings are in line with studies that reported the concomitant presence of carbapenemase and ESBLs genes in K. aerogenes strains (Ma et al., 2020; Pan et al., 2021).

Although most of the CRKA strains were susceptible to aminoglycosides (gentamicin and/or amikacin), a high percentage of the isolates were found to contain the aph(3’)-VI gene, a plasmid-encoded aminoglycoside phosphotransferase that confers resistance to amikacin. Studies have shown that Enterobacter aerogenes can carry both aph(3’)-VI and bla_KPC (Firmo et al., 2019), and also aph(3’)-VI with bla_NDM (Chen et al., 2015). However, to the best of our knowledge, this is the first report of Klebsiella aerogenes concomitant harboring aph(3’)-VI, bla_KPC-2, and bla_NDM-1 genes, as can be seen in the CRKA315 strain. Most of the CRKA isolates also harbored the aac(6’)-Ib gene, responsible for encoding an aminoglycoside 6’-N-acetyltransferase type Ib, which provides resistance to amikacin (Machuca et al., 2016). Interestingly, amikacin susceptible isolates harboring the aac(6′)-Ib gene have been reported in K. pneumoniae strains (Almaghrabi et al., 2014; Haldorsen et al., 2014; Galani et al., 2019). Despite half of the CRKA strains being susceptible to ciprofloxacin, 80% of the isolates harbored the qnrS (qnrS1 and/or S2) gene. The qnrS genes have been detected in a variety of microorganisms and environments, where they can be located in both the chromosome and in plasmids (Xu et al., 2023). Studies have indicated an association between qnrS1 and a Tn3-like-blaTEM-1-containing transposon, leading to enhanced recombination and insertion effectiveness (Guan et al., 2013). The expression of the qnrS1 gene is enhanced by quinolones, in contrast to certain other qnr genes (Monárrez et al., 2018). QnrS2, associated with quinolone resistance which demonstrates a 92% similarity in amino acid composition with qnrS1, is frequently identified in IncQ, IncU, and ColE-type plasmids as part of a mobile insertion cassette element bracketing inverted repeats but lacking a transposase (Picão et al., 2008; Han et al., 2012; Dobiasova et al., 2016; Wen et al., 2016). Notably, a newly identified surrounding genetic structure of qnrS2 flanked by IS26 elements was observed in E. coli strains from China (Tao et al., 2020). This finding highlights the important role of IS26 in facilitating the horizontal spread of quinolone resistance genes. Therefore, our data suggests that the variance in results between phenotyping and genotyping may be linked to the presence of multiple concurrent resistance mechanisms (Galani et al., 2019). However, we should mention that the incomplete sequencing of K. aerogenes CRKA317 may undermine the confidence level of this speculation.

Efflux pumps are important membrane proteins that play a crucial role as defense mechanisms by actively exporting harmful substances, such as antibiotics, detergents, and heavy metals (Jang, 2023). In our study, all of the isolates studied harbored acrA, encoding a subunit that functions as an adapter protein linked to AcrB, and TolC outer membrane channel proteins. Although we did not identify acrB in our analysis, together these proteins form the AcrAB-TolC stable efflux complex, known to contribute to multidrug resistance in nosocomial pathogens (Chen et al., 2022). These genes have been identified in K. aerogenes clinical isolates and are responsible for expelling various compounds, including antimicrobial agents like quinolones, tetracyclines, and chloramphenicol (Pradel and Pagès, 2002; Masi et al., 2007; Chevalier et al., 2008). Although the expression levels of acrAB and tolC genes were not determined in our K. aerogenes isolates, it was observed that all strains carried both genes which might be involved in the development of MDR carbapenem-resistant profile of K. aerogenes. The mdtK gene encodes an efflux pump that has the ability to expel acriflavine, doxorubicin, norfloxacin, and dipeptides (Hayashi et al., 2010; Andersen et al., 2015). This gene was found in a majority of our CRKA strains. Previous studies have reported the presence of the mdtK gene in K. aerogenes isolated from river sediment (Iyer et al., 2017).

WGS is a valuable tool for identifying and characterizing disease-associated bacteria in clinical settings. Thus, we conducted a comprehensive analysis of the entire genome of K. aerogenes CRKA317 to gain deeper insights into its genomic diversity, and methods of resistance.

The draft genome of K. aerogenes CRKA317 comprised of a single circular chromosome with a length similar to most K. aerogenes genomes in NCBI GenBank and harbored various essential genes for bacterial cellular processes. Additionally, the results of the RAST and eggNOG analyses showed that our K. aerogenes CRKA317 carried genes linked to drug resistance.

Next, we explored the phylogenetic affiliation of K. aerogenes CRKA317. The16S rRNA gene sequence analysis showed that the K. aerogenes CRKA317 was not closely related to K. aerogenes as a specie. Although, 16S rRNA gene is used extensively in bacterial phylogenetics, the limitations of using 16S rRNA gene relatedness to classify bacteria have been extensively documented (Rossi-Tamisier et al., 2015; Thorell et al., 2019; Soares et al., 2023). Therefore, a combination of ANI, dDDH, and TYGS technologies were employed to determine the phylogenetic position of the K. aerogenes CRKA317, which predicted a close phylogenetic relationship with K. aerogenes.

In our investigation of the genetic relationship between K. aerogenes CRKA317 and another 26 K. aerogenes strains, we found three strains (K. aerogenes 57, K. aerogenes CAVI1320, and K. aerogenes EA46506) that were closely related to K. aerogenes CRKA317. When analyzing the distribution of shared gene families among four strains, we found that our K. aerogenes CRKA317 had the highest number of singleton genes compared to its closest three relatives. Some of these singleton genes were related to antibiotic resistance. Singleton genes are typically acquired through horizontal gene transfer (HGT) or mutations in pre-existing genes. These genes are often associated with specific metabolic pathways, virulence, antibiotic resistance mechanisms, or other environmental adaptations (Costa et al., 2020). Furthermore, the KEGG pathway analysis revealed that most of the genes were in the core genome and were related to metabolic pathways. As the analyses were based on small number of K. aerogenes, we cautiously speculated that K. aerogenes might have genomic plasticity, that may contribute to antibiotic resistance and environmental adaptation.

Our comprehensive analysis of the K. aerogenes CRKA317 using WGS confirmed the correlation between the genotype and the phenotype to its antimicrobial resistance. Furthermore, our analysis found a large number of antibiotic resistance-associated genes such as porin and efflux pump-encoding genes giving resistance to both previously tested and untested antibiotics, suggesting a wide-ranging antibiotic resistance profile (Tables 2, 3). AmpC β-lactamases are usually encoded within the chromosome or found as ampC genes on a plasmid. Our K. aerogenes CRKA317 harbored two copies of bla_AmpC gene. Several Gram-negative organisms, including E. aerogenes, Enterobacter cloacae, Serratia marcescens, Providencia stuartii, Pseudomonas aeruginosa, Hafnia alvei, and Morganella morganii, have presented AmpC in their genomes (Jacoby, 2009; Ghanavati et al., 2018; Tamma et al., 2019). This enzyme provides resistance against aminopenicillins, cephalosporins, oxyimino-cephalosporins (e.g., ceftriaxone, cefotaxime, and ceftazidime), cephamycins (e.g., cefoxitin and cefotetan), and monobactams (aztreonam) (Jacoby, 2009). The gene sul-2 implicated in sulphonamide resistance due to inducing high levels of dihydropteroate synthase was found in our strain (Teichmann et al., 2014). This gene has been widely studied and its association with sulfamethoxazole resistance has been demonstrated in numerous studies worldwide (Teichmann et al., 2014; Shin et al., 2015), including in an E. aerogenes from Brazil (Grazziotin et al., 2016). The K. aerogenes CRKA317 contained the fosA gene, which is commonly found in the genomes of K. pneumoniae, K. oxytoca, E. cloacae, E. aerogenes, S. marcescens, M. morganii, P. stuartii, and P. aeruginosa. All these species haboring fosA gene presented intrinsic resistance or reduced susceptibility to fosfomycin (Ito et al., 2017). The ermC gene, known for its role in conferring erythromycin resistance in S. aureus and other Staphylococci (Jamrozy et al., 2017) was also detected in the K. aerogenes CRKA317. The K. aerogenes CRKA317 also contained the catA-like gene, responsible for producing a chloramphenicol acetyltransferase that catalyzes chloramphenicol (Huang et al., 2017). This gene is commonly present on transposons and plasmids, and it is widespread among a range of organisms such as Acinetobacter spp., Bacillus methylotrophicus and Chryseobacterium indologenes (Obayiuwana and Ibekwe, 2020; Damas et al., 2022).

We also detected three alterations in amino acids in marA in our K. aerogenes CRKA317 (Ser3Asn, Val96Ile, Gly103Glu) and one in soxS (Ala2Glu). These findings partially corroborate the results of Maneewannakul and Levy (1996), where they identified three mutations in marA (Ser3Asn, Val96Glu, Gly103Ser) leading to resistance to fluoroquinolones in E. coli. Moreover, Aly et al. (2015) demonstrated that a mutation in soxS (Ala12Ser) contributed to resistance against ciprofloxacin, enrofloxacin, chloramphenicol, and doxycycline in E. coli strains. We suggest these same mechanisms gave rise to antimicrobial resistance in our K. aerogenes CRKA317 due to the similar mutations noted.

Additionally, our K. aerogenes CRKA317 presented several MGEs harboring resistance genes (Table 4). MGEs are important tools for acquiring resistance genes through horizontal gene transfer. ISs, for example, are transposable DNA segments that have been previously linked to resistance genes and can be transferred horizontally by plasmids or by bacteriophages (Siguier et al., 2014). The bla_OXA-9, bla_TEM-1 genes encoding for resistance to β-lactams, and aac(6’)-Ib, aadA1 encoding resistance aminoglycosides were related to an IS110 family transposase (IS4321/IS5075 and ISKpn25 insertion elements). These findings are partially supported by previous studies that have shown ESBL-encoding genes (bla_TEM-1B) and genes related to aminoglycoside resistance (aph-Id, aph-Ib) are located near to an IS5075 insert in K. pneumoniae (Pajand et al., 2023). The bla_CTX-M-15 gene was in close proximity to ISEcp1, which is a member of the IS1380 family (Poirel et al., 2008), and has been identified as one of several elements responsible for facilitating the transfer of bla_CTX-M genes across various species of Enterobacteriaceae (Shawa et al., 2021; Wang et al., 2023). The bla_NDM-1 and ble_MBL genes (a class B3 β-lactamases with carbapenemase activity) were close to the insertion sequence ISAba125 (IS30 family). Studies have proposed that the ISAba125–ble_NDM combination occurred initially in Acinetobacter spp. and later transferred to other Gram-negative bacteria (Castanheira et al., 2023). Additionally, the ISAba125–bla_NDM-1–ble_MBL combination has been found in a structure referred to as NDM-GE-U.S, first observed in a K. pneumoniae strain from the United States and subsequently detected in various strains worldwide (Hudson et al., 2014; Peirano et al., 2018).

The qnrS1 the aph(3”)-VI genes, were found in close proximity to the ISKpn19 insertion sequence of the ISKra4 family in our isolate. Studies have shown that qnrS1 gene, which confers resistance to ciprofloxacin, is related to ISKpn19 in various bacteria, such as Leclercia adecarboxylata, Salmonella corvallis; K. pneumoniae, E. coli, and S. marcescens (Xu et al., 2020; Chen et al., 2023; Sano et al., 2023; Zheng et al., 2024). The aph(3”)-VI gene, responsible for amikacin modification, was not found to be flanked by the ISKpn19 insertion sequence in our literature review. It is worth noting that K. aerogenes CRKA317 presents a large number of singleton genes, including bla_NDM-1, aadA/ant(3”)-Ia, aph(3’)-VI, and qnrS1 and its presence indicates the variability and diversity within the genome of our strain. It is possible that these genes were obtained from other lineages through horizontal gene transfer, leading to the development of new functions, such as resistance to antibiotics. This assumption can be reinforced by the presence of a genomic island that carries most of the resistance genes (bla_OXA-9, bla_TEM-1, bla_NDM-1, ble_MBL, bla_CTX-M-15, aac(6’)-Ib, aadA/ant(3”)-Ia, aph(3’)-VI, and qnrS1) found in our genome. Genomic islands are distinct regions in the bacterial genome with genes related to each other and often associated with specific functions. These islands are often acquired through horizontal gene transfer events and are associated with the widespread distribution of antimicrobial resistance factors among bacteria (Juhas et al., 2009). The composition of genomic islands is conducive to the acquisition of new antibiotic resistance genes, as they include several MGEs, which facilitate the incorporation of new genes, but also their own transfer, for example, using tRNA genes as recombination sites into the chromosome (da Silva Filho et al., 2018).

Our K. aerogenes CRKA317 displayed many genes of interested linked to antibiotic resistance (Table 3). The ramA, acrR, acrA and acrB genes, and MFS_MMR_MDR-like gene were found to be associated with MITEEc. MITEEc belongs to the IS630 family and was found in an extensively drug-resistant (XDR) Escherichia coli isolate (Jain et al., 2021). RamA belongs to the AraC/XylS protein family and shows a close association with the marA and soxS proteins (Rosenblum et al., 2011). Elevated expression of ramA is associated with the activation of the acrAB efflux pump, which confers multidrug resistance in various bacterial species such as E. aerogenes, K. pneumoniae, and E. cloacae (Chollet et al., 2004; Keeney et al., 2007; Ruzin et al., 2008). The acrR gene regulates the multidrug efflux pump AcrAB-TolC (Subhadra et al., 2018). The MFS_MMR_MDR-like gene is linked to methylenomycin resistance, and the antibiotic Methylenomycin A is produced naturally by Streptomyces coelicolor A3, a model organism for streptomycetes (Bentley et al., 2002; Bowyer et al., 2017). The acrAB-tolC system where the acrAB fusion protein, members of the RND-type efflux family, function with another antibiotic efflux pump, tolC, to pump out various compounds such as SDS, novobiocin, deoxycholate, aminoglycosides, and dianionic β-lactams including carbenicillin, oxacillin, nafcillin, and aztreonam (Guérin et al., 2023). We also found many other RND antibiotic efflux pumps, the first pair being eefA and eefB which are part of the eefABC locus known to encode a tripartite efflux pump that gives rise to resistance to erythromycin and other antibiotics in E. aerogenes (Pradel and Pagès, 2002; Masi et al., 2007). The oqxA and oqxB operon has also been described to increase antibiotic resistance in E. aerogenes, this time to quinolones as they combine to make the oqxAB efflux pump (Wong et al., 2015; Moosavian et al., 2021). The HAE1 family, also contained in our isolate, contains large number of identified RND transporters (Nikaido, 2018). These pumps are commonly found in Gram-negative bacteria, typically existing as trimers, and are involved in the transportation of drugs and other hydrophobic substances (Nikaido, 2018). In addition, we found members of the acrB/acrD/acrF family, specifically acrD, mdtA and mdtB. The mdtABC operon is transcriptionally activated by baeR and leads to the formation of the mdtABC tripartite complex, which provides resistance to novobiocin and deoxycholate in E. coli (Nagakubo et al., 2002). Related to this complex, it was found mdtH and mdtK both of which give rise to proteins that function has multidrug efflux pumps that contribute to resistance against quinolone antibiotics such as norfloxacin and enoxacin in E. coli strains (Nishino and Yamaguchi, 2001; Yu et al., 2020). The final RND antibiotic efflux pump sequenced in our isolate is oprM which is part of MexX-MexY-OprM efflux systems that mediate intrinsic antibiotic resistance to aminoglycosides and erythromycin in bacteria such as P. aeruginosa (Wong et al., 2001), Brevundimonas brasiliensis sp. nov and Burkholderia vietnamiensis (Shinoy et al., 2013; Soares et al., 2023).

We also found putative ICE with T4SS just harboring sul-2 gene, encoding for resistance to sulfonamide, and sox gene, a key component of a central regulatory system present in all Enterobacteriaceae, which detects and reacts to internal chemical stressors like antibiotics (Chubiz, 2023). ICEs are mobile elements integrated into the chromosomes that can be excised and transferred horizontally to other bacteria and, therefore, have been associated with antimicrobial resistance genes (Johnson and Grossman, 2015). Despite this, due to the finding of only one resistance gene in the ICEs studied here (sul-2), we can assume that this structure is not the main source of resistance gene acquisition in K. aerogenes CRKA317.

Genes associated with porins were the second category identified in our isolate (Table 3). Porins belong to a category of transmembrane proteins called omps, which form small channels in the membrane and facilitate the passive movement of hydrophilic compounds. They regulate cellular permeability and can either enhance or reduce resistance to antibiotics. In our strain, we specifically found the outer membrane protein encoding genes oprD, ompC, ompA, ompX and ompW which have been found to have clinical significance. For instance, a reduction or absence of OmpC in clinical E. aerogenes isolates has been linked to a slight increase in imipenem MIC (Lavigne et al., 2012). Meanwhile, overexpressing ompX in E. aerogenes results in elevated resistance to β-lactam antibiotics, possibly due to significant reduction in the Omp36 porin (Hejair et al., 2017). OmpW expression in A. baumannii isolates was found to increase when exposed to ciprofloxacin and decrease when exposed to imipenem (Gurpinar et al., 2022). Conversely, A. baumannii strains with mutations in ompA exhibited reduced permeability for cephalothin/cephaloridine and lower minimum inhibitory concentrations for a range of antibiotics including imipenem, colistin, meropenem, chloramphenicol, aztreonam, and nalidixic acid (Smani et al., 2014; Tsai et al., 2020). Finally, in P. aeruginosa, the porin oprD plays a significant role in the uptake of basic amino acids and carbapenems (Wong et al., 2001).

Other genes of interest included: a superoxide response transcriptional regulator (soxS), a multiple antibiotic resistance transcriptional regulator (marA), and a major facilitator superfamily member (kdeA),

Prophages play a role in the survival mechanisms of their hosts and contribute to the enhancement of genetic diversity within the host genome (Kondo et al., 2021). In our study, we found two intact regions which were associated with the presence of a prophage highly similar to the Salmonella phage SEN34 (National Center for Biotechnology Information reference sequence NC_028699.1), and Escherichia phage vB_EcoM_ECO1230-10 (NC_027995.1). Prophage regions of Salmonella phage SEN34 (NC_028699.1) has been identified in Salmonella salamae (Hounmanou et al., 2022), and Salmonella enterica serovar Paratyphi B (Castellanos et al., 2020) and have been linked to drug resistance.

There are limitations to our study that need to be acknowledged. We encountered difficulties in assembling complete plasmid sequences, primarily due to the short reads generated by high-throughput sequencer. This can result in antimicrobial resistance genes being located on incomplete contigs, leading to uncertainty about whether they are situated on a plasmid or within the chromosome (Orlek et al., 2017; Berbers et al., 2020). Nonetheless, it is important to highlight those studies conducted in China have shown that clinical isolates of carbapenem-resistant K. aerogenes carried bla_NDM-1 gene on plasmids of the IncFIIAs type. In another study, Shen et al. (2019) identified a plasmid (p1564) containing genes for plasmid replication (IncA/C repA), antibiotic resistance (bla_NDM-1, rmtC, aacA4, ble_MBL, bla_CMY-6 and sul-1), and conjugation (tra clusters). In K. aerogenes, there is still no consensus on the location of the bla_KPC-2 gene in the plasmid, the transposon variants capable of carrying this gene, and which incompatibility (Inc) groups carry the bla_KPC gene (Bispo Beltrão et al., 2020). However, a study conducted by Bispo Beltrão et al. (2020) in Brazil has described a non-Tn4401 element (NTEKPC-IId) that carries the bla_KPC-2 and aph(3’)-VII genes in IncQ1 plasmids in K. aerogenes. To date, the IncQ1 bla_KPC-2-positive plasmids have been found in different strains such as E. coli, K. pneumoniae of CG258, Klebsiella quasipneumoniae, and P. aeruginosa (de Oliveira Santos et al., 2018).

Although K. aerogens has not been reported to carry the plasmids found in our sequencing study, it is important to note genes such as bla_KPC and bla_NDM are commonly associated with the plasmid fragments found in K. aerogenes CRKA317. Takei et al. (2022) found nine isolates of K. pneumoniae carrying bla_NDM-1 and bla_CTX-M-15 on the IncFIB (pQil) plasmid and another five isolates carrying bla_NDM-1 on the IncC plasmid. Similar data were also observed in the results of Zeng et al. (2022), who identified the bla_NDM-1 gene in IncC plasmids from 21 K. pneumoniae isolates. Finally, the fragmented INCFIIK plasmid observed in our genome has already been noted to carry genes such as bla_KPC-2, bla_CTX-M-15, bla_TEM-1 and, less commonly, bla_NDM-1 (Chen et al., 2013; Bi et al., 2018). This suggests an emerging mechanism, using Inc. groups, that plays a role in the dissemination of carbapenem resistance in clinically important bacteria.

In conclusion, our current research has uncovered a concerning scenario involving K. aerogenes demonstrating resistance to commonly utilized drugs for treating infections, including those considered as last-resort options for life-threatening infections in ICU patients. Moreover, the presence of mobile genetic elements highlights the alarming potential for the transmission of various resistance genes such as bla_NDM-1 and bla_KPC-2 within hospital settings to susceptible populations. This scenario poses significant challenges for managing infectious diseases and underscores the necessity of early detection of such genetic features or mutations.

Our study did not involve human genetic material or biological samples. The strains were obtained from the collection of the Central Laboratory of Public Health, a leading diagnostic center in Tocantins, Brazil. This was a retrospective study and epidemiological data were obtained from a database at LACEN-TO in accordance with Resolution 466/12 of the National Health Council (Conselho Nacional de Saúde/Ministério da Saúde, 2012). Informed consent was not required as per Resolution 466/12 regarding research involving humans by the National Health Council. The study received approval from the Committee of Ethics in Human Research at the Federal University of São Carlos (no. 1.088.936), and permissions to conduct it were obtained from the State Department of Health in Tocantins and LACEN/TO.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

The studies involving humans were approved by Committee of Ethics in Human Research at the Federal University of São Carlos (no. 1.088.936). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and institutional requirements.

SR: Formal analysis, Investigation, Methodology, Writing – review & editing. GN: Formal analysis, Investigation, Methodology, Writing – review & editing. GS: Formal analysis, Investigation, Methodology, Writing – review & editing. MD: Formal analysis, Investigation, Methodology, Writing – review & editing. RF: Formal analysis, Investigation, Methodology, Writing – review & editing. PL: Formal analysis, Investigation, Methodology, Writing – review & editing. RS: Formal analysis, Methodology, Writing – review & editing. LC: Methodology, Writing – review & editing. AC: Formal analysis, Writing – review & editing. IM: Visualization, Writing – review & editing. AC: Visualization, Writing – review & editing. M-CP: Conceptualization, Funding acquisition, Project administration, Supervision, Writing – original draft, Writing – review & editing.