Carbapenemase genes distribution in clonal lineages of Acinetobacter baumannii: a comprehensive study on plasmids and chromosomes

Background The global spread of plasmids carrying carbapenemase genes within carbapenem resistant Acinetobacter baumannii (CRAB) strains poses a worldwide public health issue. In this study, we conducted a comprehensive genetic analysis of plasmids and chromosomes harboring the major carbapenemase genes (bla NDM, bla KPC, bla VIM, bla IMP, bla GES, bla OXA-58-like, bla OXA-24/40-like, bla OXA-143-like, and bla OXA-23-like) in CRAB strains using bioinformatic tools. Methods We retrieved plasmids and chromosomes carrying the major carbapenemase genes from GenBank. The size, replicon type, and conjugal apparatus of the plasmids were also determined. Furthermore, allele types, co-existence of other antimicrobial resistance genes alongside carbapenemases in plasmids or chromosomes, co-occurrence of carbapenemase genes, gene repetition, and sequence types (ST) of whole genomes were characterized. Results The database contained 113 plasmids and 38 chromosomes harboring carbapenemase genes. This investigation revealed that bla NDM and bla OXA-58-like were the predominant allele types in both the plasmids and chromosomes. Nine (7.96%) plasmids with bla NDM-1 were potentially conjugative. The most common replicon types of the plasmids were R3-T1, R3-T8, R3-T2, R3-T23, and RP-T1. The analysis revealed that bla NDM-1 and bla OXA-58-like genes possessed the highest variety of co-existence with other antibiotic resistance genes. The co-occurrence of dual carbapenemases was identified in 12 plasmids and 19 chromosomes. Carbapenemase gene repetitions were identified in 10 plasmids and one chromosome. Circular alignment revealed that the plasmids carrying the co-occurrence of bla NDM-1 and bla OXA-58 were more homogeneous. However, there was heterogeneity in certain regions of these plasmids. According to the minimum spanning tree (MST) results, the majority of the plasmids belonged to the genomes of ST2Pas, ST1Pas, ST422Pas, ST622Pas, and ST85Pas. Conclusion A. baumannii appears to have a strong ability for genome plasticity to incorporate carbapenemase genes on its plasmids and chromosomes to develop resistance against carbapenems. Mobilizable plasmids harboring carbapenemases significantly contribute to the dissemination of these genes. The genetic structure of the plasmids revealed a strong associations of class I integrons, ISAba-like structures, Tn4401 elements, and aac (6′)-Ib with carbapenemases. Furthermore, gene repetition may also be associated with carbapenem heteroresistance.


Introduction
Acinetobacter baumannii is a member of the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. baumannii, Pseudomonas aeruginosa, and Enterobacter spp.). A. baumannii is an opportunistic pathogen that has been identified as an important cause of nosocomial infections, including ventilator-associated pneumonia in intensive care units, urinary tract, and bloodstream infections (Flores-Paredes et al., 2021;Nocera et al., 2021).These infections are associated with significant mortality rates owing to high levels of antimicrobial resistance (Alrahmany et al., 2022).Carbapenems are used as a last resort to treat severe infections caused by multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains (Zalegh et al., 2023).In addition, carbapenem-resistant A. baumannii (CRAB) isolates have been categorized by the World Health Organization (WHO) as one of the 12 top priority resistant bacteria that pose the greatest threat to public health (Hsieh et al., 2020).The main cause of carbapenem resistance in A. baumannii isolates is the acquisition of metallo-beta-lactamases (MBLs), class A carbapenemases (for example, KPC), and carbapenem-hydrolyzing class D betalactamase (CHDLs) enzymes (Kyriakidis et al., 2021).
Treatment of antibiotic-resistant A. baumannii isolates is becoming increasingly difficult due to the co-existence of carbapenemase-encoding and other antibiotic-resistance genes, including aminoglycoside-modifying genes and plasmid-mediated quinolone resistance (Wasfi et al., 2021).However, these resistance genes are often located in mobile genetic elements (MGEs), including conjugative plasmids, transposons, integrons, and insertion sequences.They can be easily expanded and transferred between various bacterial species (Horne et al., 2023).Conjugative plasmids, which include the origin of the transfer (oriT) region, relaxase enzyme, type IV coupling protein (T4CP), and a gene cluster for the bacterial type IV secretion system (T4SS) apparatus, are essential for the horizontal gene transfer and dissemination of carbapenemase genes (Li et al., 2018).Lam et al. (2022) classified A. baumannii plasmids into three categories based on the Pfam domains of the replication initiation protein including RP, R1, and R3.Plasmids that contain conjugation genes and are often associated with various acquired antibiotic resistance genes belong to the four most common types: RP-T1, R3-T1, R3-T2, and R3-T3 (Lam et al., 2023).Plasmids carrying resistance genes are strongly related to specific sequence types (STs), including ST2, ST1, and ST3, which can cause nosocomial infections and outbreaks (Frenk et al., 2022).Therefore, the genetic characterization of these plasmids in successful international clones is important for understanding the carbapenemase-harboring bacteria and for effective decision-making in infection prevention strategies.
In this study, we conducted an in silico analysis and comparative assessment of carbapenemase harboring plasmids (CHPs) and chromosomes (CHCs) in A. baumannii.The primary objective was to identify the genetic structures of various allele types of the major carbapenemase genes (bla IMP , bla VIM , bla GES , bla NDM , bla OXA-58-like, bla OXA24/40 -like, bla OXA-143 -like, and bla OXA-23 -like) using bioinformatic tools.Additionally, this study characterized the genetic properties of both CHPs and CHCs, encompassing replicon types, conjugation ability, co-existence (linkage of carbapenemases with other antimicrobial resistance genes), co-occurrence (presence of at least two carbapenemase genes in one strain), gene repetition, and phylogenetic relatedness.

Allele types and genetic environment of carbapenemases
The allele types of the mentioned carbapenemase genes were determined (cut-off was 100% identity and 100% coverage) using the beta-lactamase database (http://bldb.eu/)(Naas et al., 2017).Additionally, the prevalence of each allele type was determined.Moreover, the genetic contexts of nine carbapenemase genes present on both plasmids and chromosomes were characterized.The most prevalent of genetic structure for each gene was depicted.

Detection of other AMR genes on the retrieved DNAs
The Comprehensive Antibiotic Resistance Database (CARD) (https://card.mcmaster.ca/home)was used to identify the presence of various antimicrobial resistance genes against betalactams, carbapenems, macrolides, aminoglycosides, and other antibiotics (Islam et al., 2023).The co-existence (gene linkage) of A. baumannii plasmids and chromosomes harboring major carbapenemase genes with various antimicrobial resistanceassociated genes was investigated.Co-occurrence was determined based on the presence of at least two major carbapenemase genes in an isolate.

Genetic characterization and replicon typing of plasmids carrying carbapenemases
The conjugation apparatus of the plasmids was identified using oriTfinder (https://bioinfo-mml.sjtu.edu.cn/oriTfinder/)(Li et al., 2018).The presence or absence of conjugation elements, including oriT, relaxase-encoding genes, T4CP, and T4SS in the CHPs was determined using this web tool.The replicon type for each plasmid was determined using local BLASTn.In addition, carbapenemase gene repetition in the plasmids was performed.Moreover, information on the geographical regions, isolation sources, collection date, host disease, and hosts of all isolates harboring crabapenemase genes were extracted.

Clonal relatedness of strains harboring carbapenemase genes
The distribution of major carbapenemase genes among the different STs was assessed.For each plasmid, ST of the related chromosome was determined using seven housekeeping genes (cpn60, fusA, gltA, pyrG, recA, rplB, and rpoB) from the PubMLST database (https://pubmlst.org).The clonal relatedness of STs was characterized using PHYLOViZ version 2.0 (http:// www.phyloviz.net)to generate a minimum spanning tree (MST) for all STs (Nascimento et al., 2017).

The co-occurrence of plasmids and chromosomes harboring carbapenemase genes
Analysis of the data retrieved from the GenBank database revealed the co-occurrence of predominant allele types of carbapenemase genes in the plasmids and chromosomes.This cooccurrence was observed in 12 plasmids, among which 10 harbored both bla NDM-1 and bla OXA-58 -like genes, one contained both bla OXA- 58 -like and bla IMP genes, and one harbored both bla GES and bla OXA- 23 -like genes.Notably, there was no co-occurrence of bla OXA-24 -like and other carbapenemase genes in the plasmids dataset (Table 1 and Figure 3).Furthermore, co-occurrence was found in 19 chromosomes, which can be categorized as follows:17 chromosomes carried both bla NDM-1 and bla OXA-23 -like genes, one chromosome contained both bla NDM-1 and bla OXA-58 -like genes, and one chromosome exhibited co-occurrence of bla OXA-23 -like and bla OXA-10 genes.Notably, there was no co-occurrence of bla KPC-3 with other carbapenemase genes in the chromosomes (Table 2).

Genetic environment of carbapenemase genes
Two copies of ISAba125 flanked bla NDM-1 .The bla NDM cluster was sequentially embedded in the ISAba125-IS30bla NDM -ble MBLtat-cutA structure.bla OXA-58 is located in the ISAba3-bla OXA-58 -ISAba3-like structure.This was followed by araC1 (a putative transcriptional regulator) and lysE (a putative threonine efflux protein).The bla KPC gene was flanked by two copies of Tn4401.This transposon also harbors the ISKpn6, ISKpn7, transposase, and resolvase genes.The bla GES gene is located in a class 1 integron.The bla GES gene cassette is downstream of aacA4 gene, which encodes AAC (6′)-Ib aminoglycoside acetyltransferase.This was followed by the dfrA7 gene cassette, trimethoprim resistance gene, and qacEdelta1.The bla IMP gene was found in the bla IMP -qacG2-aacA4-catB3-sul cassette array and was the most abundant gene in the class 1 integrons.This array contained genes that confer resistance to quaternary ammonium compounds (qacG), aminoglycosides (aacA4), and chloramphenicol (catB3).XerC/ XerD-like binding sites flanked the bla OXA-24/40 -like genes.In addition, bla OXA-23 was flanked by two copies of ISAba1.This arrangement was followed by the ATPase and helicase genes (Figure 4).

Discussion
Carbapenem resistant A. baumannii poses a significant challenge because it is a leading cause of community-acquired and nosocomial infections, contributing to elevated morbidity and mortality rates (Mirzaei et al., 2020).Carbapenems are last-line antibiotics against these infections.However, MDR and XDR A. baumannii isolates complicate treatment (Katip et al., 2022).
carbapenemase genes on conjugative plasmids contribute to higher dissemination rates of these genes between bacteria (Benson et al., 2017).In the current study, bioinformatic tools were used to present more information on the genetic characteristics of A. baumannii plasmids and chromosomes harboring carbapenemase genes.
According to the results of this study, bla NDM and bla OXA-58 were the most prevalent carbapenemase genes in the plasmids and chromosomes.Consistent with the current study, Monnheimer et al. showed that the most abundant carbapenemases in A. baumannii were belonged to these genes (Monnheimer et al., 2021).
Our study revealed significant findings regarding the coexistence of various aminoglycoside resistance genes, such as aac  2014) have previously reported similar observations regarding the co-existence of carbapenemases and aminoglycoside resistance genes (Nowak et al., 2014).
Any action to combat the spread of antibiotic resistance genes requires the identification of the potential sources and genetic environment of these genes.The study of MGEs associated with antibiotic resistance genes could provide valuable epidemiological information to identify potential sources.The genetic environment of the carbapenemase genes is consistent with the results of many studies conducted on this topic.Among the MBLs, bla KPC and bla NDM were associated with Tn4401/non-Tn4401 elements and ISAba125/Tn125, respectively.Whereas, the bla VIM and bla IMP were associated with class 1 integron (Reyes et al., 2020;Wang et al., 2023).Nguye et al. showed that the bla OXA-58 is located in an ISAba3-bla OXA-58 -ISAba3-like structure (Nguyen et al., 2020).
In parallel with our study findings, Lasarte-Monterrubio et al. revealed that the bla OXA-24/40 -like was associated with the XerC/ XerD-like binding site, while bla OXA-23 was flanked by ISAba (Lasarte-Monterrubio et al., 2022).Transposons and integrons, found in different plasmids and bacterial clones, are indicative of their transmissibility and mobilization within various genetic elements (Reyes et al., 2020).MGEs, including conjugative plasmids, transposons, integrons, and bacteriophages, act as carriers for the acquisition and transfer of antibiotic resistance genes.They play an important role in transferring resistance genes among bacteria (Nadella et al., 2022).In the current study, the majority of conjugative plasmids were linked to bla NDM , which may be the primary reason for their high prevalence co-existence with other antibiotic-resistance genes.In the present study, the most prevalent replicon types were R3-T1 and R3-T2.Previous studies have shown that bla NDM-1 in A. baumannii isolates may have chromosomal or plasmid origin (Sańchez-Urtaza et al., 2023).According to the results of current study, Chen et al., demonstrated the co-existence of bla OXA-58 -like with bla NDM , bla OXA-58 -like with bla IMP , and bla GES with bla OXA-23 -like.This phenomenon is related to the fact that these genes are located on the same conjugated plasmid (Chen et al., 2019).In a recent study, bla OXA-24/40 -like genes were not found to co-occur with other carbapenemase genes.Moreover, CHCs analysis revealed the co-occurrence of bla NDM-1, bla OXA-23 , and bla OXA-58 .This finding was consistent with the results of Ramoul et al. (2016).Additionally, we identified the co-occurrence of bla OXA-565 and bla OXA-10 .
Carbapenemase gene repetition is a significant evolutionary process that affects environmental adaptation of bacteria.Repetition of antimicrobial genes is a prevalent metabolic factor that gradually changes during evolution, and plays an important role in antimicrobial resistance.Specifically, we found carbapenemase gene repetitions in bla OXA-24 and bla OXA-58 , which were located on both the plasmids and chromosomes.These duplications might have contributed to the development of carbapenems heteroresistance.
In the current study, ST2 Pas , ST1 Pas , ST422 Pas , ST622 Pas , and ST85 Pas were the most prevalent sequence types among the A. baumannii isolates.This finding is consistent with the finding of Khuntayaporn et al., study indicated that ST2 is the predominant ST in Thailand (Khuntayaporn et al., 2021).Several studies have The multiple circular sequence alignment and genetic environment of plasmids harboring bla NDM-1 /bla OXA-58 in A. baumannii.Inner ring: size of plasmids with co-occurrence of bla NDM-1 and bla OXA-58 , and outer ring: the reference strain.Color spectrum: identity percent of the plasmids with the reference strain.
consistently shown that the majority of CRAB isolates are associated with the international clone ST2, which has been reported in various studies in Mediterranean countries According to reports from various countries, the clonal diversity of A. baumannii, according to the Pasteur scheme of MLST, shows that ST2 pas , ST1 pas , and ST3 pas are the predominant carbapenemresistant clones worldwide.In ST studies, ST2 was the most dominant (Abhari et al., 2019;Khuntayaporn et al., 2021).Between 1999 and 2009, a study conducted in four Mediterranean countries (Greece, Italy, Lebanon, and Turkey) revealed that A. baumannii outbreaks were predominantly driven by the dissemination of ST2, with fewer contributions to ST1, ST25, ST78, and ST20 (Cherubini et al., 2022).These clones were found to carry bla OXA-58 , bla OXA-23 , and bla OXA-72 (Nawfal Dagher et al., 2019;Li et al., 2023).In Greece, ST2 is the most common clone circulating in hospitals (Pournaras et al., 2017).In addition, the international clone ST2 is widely distributed in Lebanon (Nawfal Dagher et al., 2019).In a study by Thadtapong et al., ST2 was most frequently identified among colistin-and carbapenem resistant A. baumannii isolates.This observation aligns with the findings of the current study, indicating a consistent prevalence of ST2 pas in these isolates (Thadtapong et al., 2021).Khorshid et al. investigated the prevalence of various STs associated with genes encoding aminoglycoside-modifying enzymes.Their findings revealed that ST2 was the most prevalent among these STs.Remarkably, ST2, identified as an international clone, exhibits substantial genetic capacity for acquiring antimicrobial resistance genes within its genome (Khurshid et al., 2020).
However, an increased resistance to carbapenems has been reported worldwide.MBLs and CHDL-producing A. baumannii isolates, which are responsible for outbreaks, have been reported in different regions worldwide (Reyes et al., 2023).Although OXA-like enzymes weakly hydrolyze carbapenems, they can confer high resistance to carbapenems when associated with ISAba1 and ISAba125 (Li et al., 2019).MBLs and CHDLs located on MGEs spread rapidly to clonal lineages of A. baumannii (Shropshire et al., 2022).
Conjugative plasmids are involved in the rapid spread of CRAB (Chen et al., 2017).Therefore, these strains carrying plasmids and chromosomes harboring different carbapenemases and other antimicrobial resistant genes could pose a major threat to the healthcare system.Therefore, genetic characterization of these plasmids and chromosomes plays an important role in the control of bacteria carrying carbapenemases.
genes in CRAB.Nevertheless, the study has several limitations.First, the initial dataset and completeness relied on GenBank submissions and annotations.Furthermore, sampling bias exists because the plasmids and isolates studied may not encompass the full spectrum of CRAB strains and carbapenemase gene variations worldwide.In summary, the limitations of this study stem from data source dependence and sampling bias, highlighting the need for caution when interpreting the findings.

Conclusion
Characterization of the genetic structures revealed that carbapenemase genes appear not only in plasmids but also in the chromosomes of CRAB.The co-existence of plasmids encoding carbapenemases with other antibiotic resistance genes, cooccurrence, and gene repetition of carbapenemases in plasmids and chromosomes were notable findings.Conjugative plasmids containing bla NDM-1 and bla OXA-58 pose a threat to the expansion of carbapenem resistance.On the other hand, plasmids harboring bla NDM are widespread.A. baumannii employ different genetic strategies such as gene repetition and various genetic elements (transposons, integrons, and insertion sequences) to develop efficient resistance against carbapenems.Gene repetition, association of resistance gene cassettes with mobile genetic elements, acquisition of conjugative plasmids, high capacity to acquire carbapenemase genes on plasmids and chromosomes, and expansion of carbapenemases through successful international clones (ST2 Pas , ST1 Pas , ST422 Pas , ST622 Pas , and ST85 Pas ) play major roles in the development of resistance to carbapenems in A. baumannii worldwide.The high consumption of antibiotics in clinical settings exacerbates antimicrobial resistance worldwide.Therefore, a global campaign is necessary to combat against CRAB infection.

TABLE 1
The data on the geographical regions, isolation sources, years, hosts, genetic characteristics and Rep types of the plasmids harboring carbapenemases isolated from A. baumannii strains.

TABLE 1 Continued
The data on the geographical regions, isolation sources, years, hosts, and clonal relatedness of the chromosomes of A. baumannii strains harboring carbapenemases.

TABLE 2 Continued
*ST, Sequence type.N.D, Not determined.