Genomic Characterization of Mobile Genetic Elements Associated With Carbapenem Resistance of Acinetobacter baumannii From India

With the excessive genome plasticity, Acinetobacter baumannii can acquire and disseminate antimicrobial resistance (AMR) genes often associated with mobile genetic elements (MGEs). Analyzing the genetic environment of resistance genes often provides valuable information on the origin, emergence, evolution, and spread of resistance. Thus, we characterized the genomic features of some clinical isolates of carbapenem-resistant A. baumannii (CRAb) to understand the role of diverse MGEs and their genetic context responsible for disseminating carbapenem resistance genes. For this, 17 clinical isolates of A. baumannii obtained from multiple hospitals in India between 2018 and 2019 were analyzed. AMR determinants, the genetic context of resistance genes, and molecular epidemiology were studied using whole-genome sequencing. This study observed an increased prevalence of blaOXA–23 followed by dual carbapenemases, blaOXA–23, and blaNDM. This study identified three novel Oxford MLST sequence types. The majority of the isolates belonged to the dominant clone, IC2, followed by less prevalent clones such as IC7 and IC8. This study identified variations of AbaR4 and AbGRI belonging to the IC2 lineage. To the best of our knowledge, this is the first study that provides comprehensive profiling of resistance islands, their related MGEs, acquired AMR genes, and the distribution of clonal lineages of CRAb from India.


INTRODUCTION
Acinetobacter baumannii is a member of the ESKAPE group of pathogens and is considered to be one of the major global causes of hospital-acquired infections (HAIs) (Lee et al., 2017). A. baumannii is responsible for causing a wide range of infections, with pneumonia being the most commonly observed infection among critically ill patients (Dexter et al., 2015). This pathogen has a propensity to rapidly acquire antibiotic resistance genes and to develop resistance to multiple classes of antimicrobials (Lee et al., 2017). Carbapenems are one of the most commonly used antibiotics for the treatment of Acinetobacter infections. Carbapenem resistance in A. baumannii ranges between 70 and 85% in the Asia-Pacific region (O'Donnell et al., 2021). A study from SENTRY surveillance reported carbapenem resistance rates ranging from 55 to 90% in India (Gales et al., 2019). Both the Center for Disease Control (CDC) and the World Health Organization (WHO) categorized carbapenem-resistant A. baumannii (CRAb) under "Urgent Threat" and as Priority 1: Critical pathogen, respectively. Recently, the WHO Country Office for India developed the Indian Priority Pathogen List (IPPL) and categorized carbapenem-resistant, colistin-resistant A. baumannii under "Critical Priority." 1 With excessive genome plasticity, A. baumannii can acquire and disseminate antimicrobial resistance (AMR) genes that are often associated with various mobile genetic elements (MGEs) (Roca et al., 2012). Carbapenem resistance in A. baumannii is mainly due to genes encoding class D oxacillinases, bla OXA−23−like , bla OXA−51−like , and bla OXA−58−like (Poirel and Nordmann, 2006). The bla OXA−23 gene is the most predominant and is carried on many MGEs, including transposons, plasmids, and resistance islands (RIs) (Pagano et al., 2016). The association of insertion sequence (IS) elements with bla OXA−51like , bla OXA−23−like , bla NDM−like , and bla OXA−58−like genes was reported earlier (Poirel et al., 2005Turton et al., 2006a). Typically, bla OXA−23 is associated with transposons such as Tn2006, Tn2008, and Tn2009, while bla NDM −1 was mobilized by the Tn125-like composite transposon (Pagano et al., 2016). Recent studies have also indicated the role of conjugative plasmids as vehicles for disseminating resistance determinants such as bla OXA−23 in A. baumannii (Salto et al., 2018). Additionally and most importantly, the emergence of A. baumannii RIs carrying clusters of horizontally transferred genes is considered a significant contributor to the multidrug-resistant (MDR) phenotype of A. baumannii (Cameranesi et al., 2020). RIs in A. baumannii are made of transposons and are known to carry genes that confer resistance to multiple antibiotics and heavy metals (Hamidian and Hall, 2018). The AbaR3-type elements are confined to the International Clone 1 (IC1) and represented by ST1, ST19, ST20, and ST81. AbaR3 comprises a Tn6019 backbone and is consistently linked with Tn6018 or its components with multiple antimicrobial resistance regions (MARRs) (Hamidian and Hall, 2011). Similarly, studies have shown that Tn6022 can acquire bla OXA−23 transposon Tn2006 and form AbaR4 islands (Hamidian and Hall, 2017). Table 1 outlines the genomic and epidemiological features of different clones of CRAb.
Although the endemic burden of CRAb is a significant public health problem within Indian hospitals, the lack of genomic information makes it difficult to track its persistence (Mancilla-Rojano et al., 2019). Studying the genetic environment of resistance genes often provides valuable information on the 1 https://dbtindia.gov.in origin, emergence, evolution, and spread of resistance in bacterial populations (Hamidian and Nigro, 2019).
We aimed to characterize the prevalent genomic features of clinical isolates of CRAb in India. We also compared the structural configuration of RIs with the complete genetic information and observed structural variations within the genetic environment of resistance genes. We found that the backbone of MGEs and their associated AMR genes among this study isolates were similar to that of the global context.

Bacterial Isolates
A total of 17 clinical isolates of A. baumannii collected as a part of a surveillance study were used. Of the 17 isolates included in this study, 13 were from Christian Medical College (CMC), Vellore, three from All India Institute of Medical Sciences (AIIMS) Trauma Center, New Delhi, and one from Post Graduate Institute of Medical Education & Research (PGIMER), Chandigarh (Supplementary Figure 1). Among the isolates, ten isolates were from blood (B; n = 10), six from endotracheal aspirate (ETA; n = 6), and one from pus (P; n = 1). Phenotypic characterization of all the isolates as A. baumannii calcoaceticus (Acb) complex was determined using standard biochemical tests. Confirmation of the Acb complex at the species level was performed by Vitek-MS (Database v2.0, bioMerieux, France) as described earlier and by identifying chromosomally encoded bla OXA−51−like gene by PCR (Turton et al., 2006b).
For colistin, broth microdilution (BMD) was performed. Isolates identified as carbapenem-resistant by DD were further subjected to BMD to determine the minimum inhibitory concentration (MIC) for imipenem and meropenem. The susceptibility was interpreted as per the criteria defined by CLSI guidelines (Weinstein et al., 2018(Weinstein et al., , 2019. Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) were included in every batch for quality control (QC). For colistin susceptibility testing, in addition to QC strains, an mcr-1 positive E. coli isolate and two Klebsiella pneumoniae strains (BA38416 and BA25425) were also included for QC.

Genome Analysis
Further downstream analysis of the 17 complete genome sequences was performed using tools available at the Center for Genomic Epidemiology (CGE). 6 AMR genes were identified from the genome sequences using the BLASTn-based ABRicate (version 0.8.10) program 7 to query the ResFinder database. 8 The Capsular Polysaccharide loci (KL) and the Outer Core Lipooligosaccharide loci (OCL) types were identified using the Kaptive database. 9 The presence of ISs was identified using ISFinder. 10 Using BLAST analysis, the plasmid repAci types from the complete genomes were identified and characterized. The PHASTER server was used to determine the prophages. 11 The prophage regions identified were analyzed for the presence of any AMR genes using the ABRicate (version 0.8.10) program(see text footnote 7). A BLAST similarity search was performed on the individual genomes to identify the comM region that flanks AbaR type genomic islands. Based on the known AbaR sequences collected from published literature, the precise boundary of AbaRs and the respective backbone were curated manually. The sequence types were identified with MLST Finder 2.0 using Oxford MLST and Pasteur MLST schemes. 12

IC2-the Predominant and Endemic Lineage With Novel Structural Variations
Nine isolates belonged to IC2, and all had either XDR or PDR phenotypes. Six Oxford MLST STs (ST Oxf ) were identified, ST195 (2), ST451 (2), ST848 (2), ST208 (1), ST218 (1), and ST349 (1), but there was a single Pasteur MLST ST2 Pas . IC2 isolates predominantly carried either bla OXA−23 alone (7/9) or coproduced bla OXA−23 and bla NDM−1 (2/9). All had bla OXA−23 in Tn2006, an ISAba1-bounded composite transposon in the chromosome (Figure 1). Of the nine isolates, AB03, AB13, and AB16 carried the bla NDM−1 gene on the chromosome with two different structural variations in the genetic context. AB03 and AB16 were associated with the most commonly reported transposon, Tn125 (Figure 2A), while a truncated form of Tn125 (Tn125-like) was identified in AB13, where the genome harbors a single copy of ISAba125 and an incomplete transposase at the left-hand and right-hand extremities of Tn125, respectively ( Figure 2B). One to three plasmids were Frontiers in Microbiology | www.frontiersin.org   present among the nine genomes ( Table 3). The p1AB20 belongs to the plasmid family which encodes repAci6 and was found to carry the aphA6 gene within TnaphA6 that was bounded by two copies of ISAba125 in direct orientation. The most commonly observed prophage elements among the nine IC2 genomes include PHAGE_Acinet_Bphi_B1251_NC_019541, PHAGE_Psychr_Psymv2_NC_023734, and PHAGE_Acinet_ YMC11/11/R3177_NC_041866. Based on the genetic configurations, three different variants of RIs were identified among the nine genomes. Of which, AB03, AB16, AB19, and AB26 carried variants of AbGR1, which included the presence of a partial region of Tn6172 with the aminoglycoside resistance genes; aph(6) and aph(3)-I, mobilization gene; mobL, transposable element; CR2, phosphoglucosamine mutase; pgm, arsenic resistance encoding gene; arsR, tetracycline efflux protein; tet(B), and tetracycline resistance transcriptional repressor gene; tetR(B) along with Tn6022 and Tn2006 (Figure 3). AB18, AB20, and AB27 carried AbGRI variants with complex, diverse structures (Figure 4). AB18 and AB20 had a single copy of an ISAba1 element, sul2, rcr2, and a hypothetical protein inserted at the tniCA element on the Tn6172 backbone. Two copies of Tn2006 were observed in both the genomes but differed at the insertion site. In AB18, one copy of Tn2006 was inserted at orf4 while the second copy was inserted between orf BA and the tetracycline resistance transcriptional repressor gene, tetR(B). In AB20, one copy of Tn2006 was observed between the Tn6022 element and the plasmid linker, whereas another Tn2006 was inserted near orf BA. AB27 had two copies of ISAba1, one copy inserted at the tniE on the Tn6022 backbone and the second copy inserted at tniA of Tn6172. Two copies of Tn2006 were seen, one present on Tn6022 at orf4 with the second adjacent to orf BA on Tn6172. Insertion of sul2, rcr2, and hypothetical protein at the left inverted repeat of the Tn6172 element was also observed. Additionally, insertion of the arsenic resistance encoding gene, arsR, and the mobilization gene, mobL, was present on the Tn6172 element of all three genomes (Figure 4).
Interestingly, AB02 and AB13 carried the novel Tn6022derived, plasmid linker, and Tn6172-derived elements. Insertion of a single copy of Tn2006 and CR2-Tn10-MARR-like region in Tn6172 was observed in both the genomes (Figures 5A,B). However, one minor difference was identified between the genomes, where AB02 carried bla PER−7 within the class 1 integron of the Tn6172-derived element (Figure 5A), while Frontiers in Microbiology | www.frontiersin.org AB13 was found to carry a class 1 integron but was devoid of bla PER−7 (Figure 5B).

IC7 and IC8-the Emerging Lineage of CRAb Isolates in India
Two genomes, namely, AB14 and AB15, belonged to IC7 and were represented by ST1388 Oxf /ST25 Pas and ST691 Oxf /ST25 Pas , and both were XDR. AB14 and AB15 carried three and two plasmids, respectively. AB14 harbored bla OXA−23 in Tn2006 on the chromosome alone, whereas AB15 had it in both the chromosome (bla OXA−23 in Tn2006) and on an incomplete RepB family plasmid (bla OXA−23 in Tn2006). Both AB14 and AB15 showed the presence of two prophage regions, namely, PHAGE_Mannhe_vB_MhM_3927AP2_NC_028766 and PHAGE_Acinet_YMC11/11/R3177_NC_041866. AbaR4 that were mapped to Tn6022 backbone and Tn2006 linked bla OXA−23 locus was present in both the genomes (Figure 3).

IC1 Lineage With Tn6022-Derived Elements
AB28 had an XDR phenotype that corresponded to ST231 Oxf /ST1 Pas and belonged to IC1. Interestingly, in AB28, which carried a variant of bla OXA−51 (bla OXA−371 ), an insertion of ISAba16, TnpB, and an IS66 transposase, there was no upstream presence or insertional inactivation (Figure 6). AB28 carried Tn125 linked bla NDM−1 on the chromosome (Figure 2A). AB28 harbored six plasmids. Of which, p1AB28 carried bla OXA−23 on repAci6 family plasmid and several plasmid transfer (tra) genes. When we analyzed and compared the p1AB28 plasmid sequence with the reference plasmid, pA85-3 (accession number-KJ493819), we found the presence of a complete bla OXA−23 gene with one complete and an incomplete copy of an ISAba1 locus. However, some transposon-related genes such as uspA and sulP were intact. IS66 family transposase with its accessory protein, tnpB, was also encoded within the p1AB28 plasmid but was absent in the pA85-3 reference plasmid. The plasmid, p1AB28, also carried putative tra genes that are required for mating pair formation and trwC and trwB genes that are needed for plasmid mobilization (Figure 7). AB28 carried five different prophages as follows: PHAGE_Stx2_c_Stx2a_F451_NC_049924, PHAGE_Acinet_Bphi_B1251_NC_019541, PHAGE_Psychr_ pOW20_A_NC_020841, PHAGE_Escher_SH2026Stx1_NC_ 049919, and PHAGE_Acinet_vB_AbaS_TRS1_NC_031098. Notably, AB28 encoded Tn6022-derived elements in which the insertion of an IS256 family transposase, ISAba42, was observed between tniE and orf [Tn6022 (tniE-orf )::ISAba42] (Figure 8).

Pan-Susceptible Singleton and Pan-Drug Resistant CC862
The PSAB, AB01 represented as a singleton and belonged to the novel ST, ST2439 Oxf /ST285 Pas , while the PDRAB, AB06 belonged to CC862 and was represented by another novel ST, ST2440 Oxf /ST622 Pas . As expected, AB01 did not harbor any of the AMR determinants except bla ADC−25−like , bla OXA−337 . Three plasmids were present with no AMR genes. No intact prophage and RI were present.
AB06 carried dual carbapenemases, bla OXA−23 and bla NDM−1 , on the chromosome, and they were found to be carried on transposon, Tn2006, and Tn125, respectively. Two plasmids were observed with genes encoding resistance to β-lactamases, aminoglycosides, macrolides, and sulfonamides. Three intact prophages, PHAGE_Pseudo_phiCTX_NC_003278, PHAGE_Acinet_Bphi_B1251_NC_019541, and PHAGE_ FIGURE 4 | Structures of variants of AbGRI resistance island identified in this study. The typical AbGRI1 structure with an intact "Tn6022-linker-Tn6172" backbone is shown as a reference. The Tn6022 or Tn6022-derived part is shown in blue, the Tn6172 part or its partial segments are shown in orange, and the linker region is shown in green. The black arrows shown downward indicate the insertion of insertion sequence (IS) element or transposon or additional genes.

FIGURE 5 | (A)
Genetic backbone of AB13 carrying AbGRI variant identified in this study. The Tn6022 or Tn6022-derived part is shown in blue, the Tn6172 part or its partial segments are shown in orange, and the linker region is shown in green. The black arrows in the downward direction indicate the insertion of IS element, transposon, or additional genes. The pale yellow arrow indicates AMR genes, the light green arrow represents the tetracycline repressor gene, the gray arrow represents insertion elements, and the brown arrow indicates hypothetical protein. (B) Genetic backbone of AB02 carrying AbGRI variant identified in this study. The Tn6022 or Tn6022-derived part is shown in blue, the Tn6172 part or its partial segments are shown in orange, and the linker region is shown in green. The black arrows shown downward indicate the insertion of IS element or transposon or additional genes. The pale yellow arrow indicates AMR genes, the light green arrow represents the tetracycline repressor gene, the gray arrow represents insertion elements, and the brown arrow indicates hypothetical protein.
FIGURE 6 | Genetic backbone of bla OXA−51 . Two types of genetic structures were identified in this study. Sixteen isolates were identified with typical backbone, whereas one isolate with bla OXA−371 was identified with insertion sequence, ISAba16, TnpB, and IS66 family transposase.
FIGURE 7 | Circular representation of repAci6 plasmid (pink arrow), p1AB28, of Acinetobacter baumannii displayed using CG view server with the reference plasmid pA85-3 (accession number-KJ493819) (green-colored region). The two inner circles represent GC content and GC skew. The pink-colored arrow represents the presence of the OXA-23 gene along with the plasmid replication gene, repAci6, tra genes, and plasmid mobilization genes in p1AB28.

Phylogenetic Analysis of Core Genomes of CRAb
Analysis of core genomes of CRAb revealed the presence of multiple AMR genes among the IC2 isolates. Clone-specific OCL types such as OCL1 to IC2, OCL5 to IC7, and OCL2 to IC8 were observed. Diverse KL types were identified among the study isolates, and the tree showed the presence of STspecific KL types within a specific clonal lineage. AbaR4 was present among the IC1, IC7, and IC8 isolates, while AbaR4 and AbGRI variants were observed only among the IC2 isolates (Figure 9).

DISCUSSION
Acinetobacter baumannii has become an important hospitalacquired pathogen and is of major concern due to the rapid FIGURE 9 | Single nucleotide polymorphism (SNP)-based phylogenetic tree of carbapenem-resistant Acinetobacter baumannii sequenced in this study. The color-filled shape denoted presence, while the empty shape denoted the absence of the respective traits. The heat map represents the presence or absence of AMR genes; Dark red indicates the presence of the respective gene, while light red indicates the lack of the respective gene. The capsular types (KL), outer core lipopolysaccharide types (OCL), International clones/clonal complexes, and resistance/genomic islands of the CRAb isolates were represented by color-coded boxes as given in the legend. The Oxford and Pasteur scheme sequence types (STs) were given as text labels.
emergence of MDR, XDR, and PDR strains (Agoba et al., 2018;Havenga et al., 2019). Carbapenem resistance rates of more than 85% in A. baumannii have been reported from previous studies in India and are typically associated with isolates carrying either bla OXA−23 alone or both bla OXA−23 and bla NDM−1 , which concurs with this study (Vijayakumar et al., 2016;Vijayakumar et al., 2019;Vijayakumar et al., 2020). The majority of the isolates (13/17) in this study encoded more than one copy of the bla OXA−23 gene. However, we could not find any high-level carbapenem resistance genes in these isolates. Earlier, Hua et al. (2016) reported the presence of multiple copies of bla OXA−23 among CRAb as a common phenomenon without an increase in carbapenem resistance.
This study showed the endemicity of IC2 along with the emergence of sporadic clones, such as IC7 and IC8. Although previous studies from India reported the predominance of IC2, the presence of isolates that belongs to IC7 and IC8 indicates the dissemination of CRAb and reinforces the fact that the International clones of CRAb isolates are widespread among hospitals in India.
Several studies have reported that the bla OXA−23 gene has relocated to chromosomes and plasmids with the help of transposons (Hamidian and Nigro, 2019;Graña-Miraglia et al., 2020). Fourteen CRAb isolates were identified with Tn2006-linked bla OXA−23 in this study. Although experimental observations were not performed, carbapenem resistance in these isolates could be due to the ISAba1-mediated overexpression of the bla OXA−23 gene in Tn2006. Occasionally, carbapenem resistance in A. baumannii could happen due to the overexpression of bla OXA−51 variants by insertion of ISAba1 (Wong et al., 2019). In this study, the presence of ISAba16 was observed in one genome; however, insertional inactivation of bla OXA−51−like was not seen.
In A. baumannii, the bla NDM−1 gene can be encoded by either chromosomes or plasmids . However, this study observed A. baumannii isolates harboring bla NDM−1 only in chromosomes. Unlike Enterobacteriaceae, in which bla NDM−1 is often observed with a single copy of truncated ISAba125 on plasmids, the dissemination of bla NDM−1 in A. baumannii is always associated with a complete Tn125 Dortet et al., 2014). In contrast with the above statement, one genome in this study was identified with Tn125like linked bla NDM−1 , suggesting that it could have acquired bla NDM−1 from other species.
The presence of repAci6 harboring bla OXA−23 and belonging to IC1 was identified in the p1AB28 plasmid. Comparative analysis revealed that AB28 carries a plasmid closely related to the reference, as it harbors the bla OXA−23 gene in a different context (Hamidian et al., 2016). The p1AB28 plasmid is conjugative and can spread carbapenem resistance by disseminating the bla OXA−23 gene into diverse clones. However, further studies are warranted to confirm the same. Another genome, AB20, belonged to IC2 and carried a repAci6 conjugative plasmid. This plasmid harbors the aphA6 gene on the TnaphA6 transposon which encodes an aminoglycoside (3 ) phosphotransferase and confers resistance to amikacin. Previous studies from European and Asian countries have reported isolates of A. baumannii with large conjugative plasmid such as repAci6, carrying both the bla OXA−23 and aphA6 genes, which contribute to the dissemination of resistance to carbapenems and amikacin, respectively (Towner et al., 2011;Nigro and Hall, 2016). Earlier studies by Costa et al. (2018) reported the presence of AMR and virulence genes within the prophage regions of A. baumannii genomes. This study showed at least one prophage region in all the genomes except the PSAB. However, no prophages with AMR genes were detected.
Genomic analysis of AbaRs in this study unveiled novel genetic configurations specific to backbones, which involve either insertion of MGEs or structural modifications driven by known MGEs. For example, insertion of ISAba42 within the Tn6022 backbone leads to a truncated form of the tniE transposition gene, thereby forming the Tn6022 derived element. Furthermore, in this study, we identified an isolate (AB28) that belonged to IC1 but lacked an AbaR3 type island. Instead, it carried an IC2-specific Tn6022-derived backbone, which indicates the possibility of independent acquisition. Tn6022derived elements and AbaR4 and AbGRI variants are typically confined to IC2. In this study, we also found that none of the IC2 isolates carried AbaR4; instead, it was present among isolates belonging to other ICs such as IC7 and IC8. All the study isolates belonging to IC2 possessed either the AbGRI1 variant or the AbGRI variant with complex chimeric structures. Although the genetic events behind this process are unclear, such complex structural variation in the AbaR backbones might have resulted either due to the target sequences favorable for MGE insertion or due to the exposure of AbaRs with different MGEs in different clones. These findings indicate that AbaRs with diverse backbones might have evolved separately.

CONCLUSION
Overall, to the best of our knowledge, this study is the first that provides comprehensive profiling of RIs together with the MGEs, acquired AMR genes, and the distribution of clonal lineages among CRAb from India. Although this study provides a clear picture of the Indian scenario, further comparative analysis with an extensive collection of global isolates is required to understand the structural diversity and the evolution of these MGEs that drive the genome plasticity of A. baumannii.

DATA AVAILABILITY STATEMENT
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.

AUTHOR CONTRIBUTIONS
SV: laboratory methods, data analysis and interpretation, and manuscript writing. JJ: data analysis, interpretation, and manuscript writing. KV: hybrid genome assembly and other bioinformatics methods. PM, PR, SA, IB, and KW: manuscript correction. AN and AB: data analysis. BV: study design and supervising, manuscript writing, and manuscript correction. All authors contributed to the article and approved the submitted version.

ACKNOWLEDGMENTS
We would like to acknowledge Ms. Catherine Truman, Clinical Pharmacist, Christian Medical College and Hospital, India, for her valuable input in language editing.

869653/full#supplementary-material
Supplementary Figure 1 | A map of India showing the location of three hospitals from where the samples were collected as a part of the study. The red color represents PGIMER, Chandigarh, the green color represents AIIMS-Trauma, New Delhi, and the light red color represents CMC, Vellore. Map outline was created using mapchart.net. Republished from mapchart.net under a CC BY license, with permission from MapChart, original copyright 2021.