Multicenter retrospective genomic characterization of carbapenemase-producing Acinetobacter baumannii isolates from Jiangxi patients 2021–2022: identification of a novel international clone, IC11

ABSTRACT This study aimed to characterize carbapenem-resistant Acinetobacter baumannii (CRAB) isolates from Jiangxi patients using whole-genome sequencing (WGS). We subjected 100 clinical CRAB strains isolated from the three local largest teaching hospitals to WGS and antimicrobial susceptibility testing. Molecular epidemiology was investigated using multilocus sequence typing, core genome multilocus typing, core genome single-nucleotide polymorphism phylogeny, and pulsed-field gel electrophoresis. The most prevalent acquired carbapenemase was blaOXA-23, predominant in all isolates (100%). Isolates belonging to the dominating international clone IC2 accounted for 92% of all isolates. International IC11 (ST164Pas/ST1418Ox) clone was found in an additional 8% (eight isolates), with seven isolates (87.5%) carrying an acquired additional blaNDM-1 carbapenemase. The oxa23-associated Tn2009, either alone or in a tandem repeat structure containing four copies of blaOXA-23, was discovered in 62% (57 isolates) of IC2. The oxa23-associated Tn2006 was identified in 38% (35 isolates) of IC2 and all IC11 isolates. A putative conjugative RP-T1 (formerly RepAci6) plasmid with blaOXA-23 in Tn2006 within AbaR4, designated pSRM1.1, was found in IC2 A. baumannii strain SRM1. The blaNDM-1 gene found in seven IC11 isolates was located on a novel Tn6924-like transposon, a first-time report in IC11. These findings underscore the significant importance of real-time surveillance to prevent the further spread of CRAB. IMPORTANCE Carbapenem-resistant Acinetobacter baumannii (CRAB) is notorious for causing difficult-to-treat infections. To elucidate the molecular and clinical epidemiology of CRAB in Jiangxi, clinical CRAB isolates were collected and underwent whole-genome sequencing and antibiotic susceptibility phenotyping. Key findings included the predominance of OXA-23-producing IC2 A. baumannii, marked by the emergence of OXA-23 and NDM-1-producing IC11 strains.

Multilocus sequence typing (MLST) schemes are still widely used to investigate the molecular epidemiology and outbreaks of A. baumannii (5,6).The clonal complexes of clustered MLST sequence types (STs) are now corresponding to the international clones (ICs) (7).The assignment to ICs has become an established criterion for classifying A. baumannii isolates in an epidemiological context.To date, IC1-11 A. baumannii isolates have been described, the most widespread of which is IC2 commonly harboring the acquired carbapenemase OXA-23 (8).
Since the advent of relatively cheap whole-genome sequencing (WGS), this technique has become the ultimate approach to studying the dissemination of many bacterial pathogens.WGS has proven to be extremely useful, offering a higher resolution than the two MLST schemes in the case of A. baumannii, where the very dynamic nature of its genome renders MLST typing faulty (7,9).Although the use of single-nucleotide polymorphism (SNP) typing gives the highest resolution, core genome multilocus typing (cgMLST) provides a more stable nomenclature and results that are easier to share with others, especially considering the very closely related isolates from the same place or strains at very short periods of time (10).
WGS has been used to study the spread of clones of A. baumannii at a national level and even the continental level.Over the last years, in some countries, such as the USA, Brazil, Lebanon, Malaysia, United Kingdom, Vietnam, and some parts of China, WGS has been used to analyze clone diversity within and between hospital settings (3,7,(11)(12)(13).However, there is a significant lack of epidemiological and genomic data about different lineages causing CRAB infections in a single hospital setting in Jiangxi province mainly due to limited research infrastructure and resources.
The goal of our study was to elucidate the contemporary population structure of clinical CRAB isolated from patients at the three largest tertiary teaching hospitals in Jiangxi province and the mechanisms of carbapenem resistance.WGS was applied to extract ST OX , ST Pas , cgMLST, acquired carbapenemase genes and their mobile genetic elements, and capsule locus K as well as outer core locus (OCL) from CRAB isolates (2021-2022), along with epidemiological data and antimicrobial susceptibility testing.

Clinical characteristics and antimicrobial susceptibility of clinical CRAB isolates
A total of 100 non-duplicate CRAB strains were collected, with one lacking clinical ward information and two missing specimen origin data.Out of 98 isolates with specified sources, 82.5% originated from the respiratory tract, followed by 7.1% from blood (Table 1).Of the 99 isolates with known origins, 60.6% were derived from ICUs (Table S2).Rates of resistance to all the 14 tested antimicrobials were high among the isolates (Fig. 1; Table S3).Among the 100 tested isolates, 79.0% were classified as extensively drug-resistant Acinetobacter baumannii (XDRAB), defined as exhibiting non-susceptibility to at least one agent in all but two or fewer antimicrobial categories (14)(15)(16).Rates of tigecycline and polymyxin B resistance were relatively low, at 7.0% (7/100) and 5% (5/100), respectively.Furthermore, 11.0% (11/100) of the isolates showed intermediate resistance to tigecycline.

Dominance of IC2 and emergence of IC11 within CRAB population across multiple hospitals
According to the recently established international clones (IC1-11), a total of 100 isolates could be assigned to the two ICs, IC2 and IC11.A whole-genome phylogeny based on SNPs in the core genomes of all isolates is shown in Fig. 2A.We defined a SNP cutoff based on the observed SNP distribution to cluster study isolates into clearly separated clonal lineages and sublineages, which were then compared to established ICs.A cutoff of 46,000 SNPs differentiated CRAB lineages belonging to IC2 and IC11.A cutoff of 220 SNPs further defined major sublineages within the ICs (Table S1).Three sublineages     within IC2 with various degrees of heterogeneity are showed in Fig. 2A and presented in Table S1.The geographical distribution of CRAB isolates by study hospital, culture source distribution, and ward distribution is illustrated in Fig. 2; Tables 1 and 2; Table S2.IC2 constituted the largest and most widely spread clonal lineage with isolates (92%) distributed over all participating hospitals.Novel IC11 was as widespread over all study sites as IC2, but with a much lower prevalence, accounting for only eight isolates (8%) (Fig. 2; Table 2).
cgMLST sequence types (cgSTs) were determined by CGE cgMLSTFinder based on the pubMLST database with 2,133 alleles.Seven different cgSTs were assigned to the 100 available isolates (Fig. 2B2).IC2 sublineage A (IC2A) comprised two CGE cgSTs, including cgST596 and cgST668 (Tables 1 and 2; Tables S1 and S2).IC2 sublineage B (IC2B) corresponded to cgS906.The majority of IC2 sublineage C (IC2C) isolates belonged to cgST923, in addition to cgST909 and cgST661 (Table 2).IC11 belonged to cgST1101.The cgST distributions of CRAB isolates including study hospital, culture sources, and isolation ward are depicted in Tables 1 and 2; Table S2; Fig. 2B1.cgST596, cgST906, and cgST923 are the three major dominant lineages widely distributed across all three hospitals, but IC11/cgST1101 is widespread with low prevalence (Table 2; Fig. 2B1).The body site distribution of cgSTs was similar to one another; most cgSTs are characterized by the predominance in the respiratory tract, with the exception of cgST596 and cgST906 distributed in a variety of isolation sources (Table 1).All cgSTs were primarily found in hospital ICUs, but cgST596, cgST906, and cgST923 were also present in different clinical wards (Table S2).

Characterization of the resistance transposons, genomic resistance islands, and plasmids among clinical CRAB population
An acquired carbapenemase gene was identified in all tested isolates across all study sites (Fig. 2A and 3).The most frequently encountered acquired carbapenemase was bla OXA-23 , present in all tested isolates.Seven isolates (7%) harbored an additional bla NDM-1 ; interestingly, bla NDM-1 was restricted to IC11 isolates (87.5%).Two oxa23-asso ciated transposons, Tn2006 and Tn2009, were identified (Fig. 3).oxa23-containing Tn2006 was confined to IC2B and IC11, and oxa23-containing Tn2009 was present only in IC2A and IC2C isolates.A novel transposon associated with bla NDM-1 , designated Tn6924-like, was found in IC11 isolates (100%).To characterize the genetic context of the acquired bla OXA-23 and bla NDM-1 genes in each sublineage, high-quality complete genomes of eight representatives with distinct cgSTs from each sublineage were analyzed (Table S4; Fig. 4).The genetic environment of bla OXA-23 is illustrated in (Fig. 4A1 through A3).In IC2A and IC2C, bla OXA-23 -containing Tn2009 was either found alone or in a tandem repeat structure with four copies of bla OXA-23 , which upon integration into the chromosome generated diverse 9 bp target site duplications (TSDs).In IC2B, bla OXA-23 -containing Tn2006 was commonly present within AbaR4 or its derivatives, targeting the chromosomal comM gene or disrupting a repAci6 plasmid backbone.The AbGRI1 variant harboring AbaR4 also targeted the chromosomal comM gene.In the IC11 strain (SRM3), two copies of bla OXA-23 -containing Tn2006 transposed into two distinct chromosomal locations, producing different 9 bp TSDs.One Tn2006 was located near an intact prophage region, generating a TSD of AAAATTGAG, while the other Tn2006 integrated into a gene encoding an efflux RND transporter permease, with a TSD of AAAATATCG.The genetic structure of the NDM-1associated Tn6924-like transposon is depicted in Fig. 4B.This transposon, a 31.5 kb member of the Tn7 superfamily, confers resistance to carbapenem and amikacin.It comprises multiple insertion sequences, a truncated Tn125 transposon carrying a copy of the bla NDM-1 gene, an aphA6 TnaphA6-like transposon, and a type I restriction methyla tion (R1M1S1) system.Additionally, it generates a 5 bp TSD upon insertion into the 3′ end of chromosomal glmS (17)(18)(19).

DISCUSSION
Due to the limited availability of detailed data on clinical CRAB populations in Jiangxi province, we conducted a genome-based survey on CRAB strains obtained from the three largest tertiary teaching hospitals in this region.As expected, the majority of strains analyzed (60.6%) were collected from hospital ICUs, and they commonly (82.5%) occurred in the patients' respiratory tracts (1,3,11).Consistent with previous reports, most strains (80%) were phenotypically XDRAB (3,15).However, of great concern, the CRAB strains analyzed in this study exhibited higher resistance rates to polymyxin B (5%) and tigecycline (7%) compared to the results reported in China's ICUs in 2020 (3).Investigating the underlying resistance mechanisms for polymyxin B and tigecycline is beyond the scope of this work; further research on these aspects will be conducted in the future.
IC2 was by far the most frequent clonal lineage and was spread worldwide, although new lineages have become common in some parts of the world and are often associated with the dissemination of bla OXA-23 gene among CRAB (9).In the current study, IC2 predominated in our CRAB population, accounting for 92%, consistent with previous findings, and was followed by IC11 (8%).
ST164 Pas A. baumannii has spread globally over the past decade, though with low prevalence.It has, thus, been recognized as an international clone, recently proposed to be named IC11 (8,(23)(24)(25)(26)(27).However, to date, we still lack sufficient IC11 data in China.In this study, the sporadic occurrence of international clone IC11 provides an update on the molecular epidemiology of CRAB.More importantly, the emergence of NDM-1 and OXA-23-producing IC11 isolates is of great concern for healthcare settings, requiring real-time and effective surveillance to prevent further dissemination.NDM-1-producing A. baumannii isolates are uncommon.Many studies found that NDM-1-positive CRAB are IC9 (ST85 Pas ) from the Middle East and sporadically in Germany and Denmark (8,27).A recent study by Zafer et al. described an NDM-1-producing IC11 (ST164 Pas /ST1418 Ox ) A. baumannii from Cairo, Egypt (27).Although OXA-23-/NDM-1-producing A. baumannii isolates are relatively rare, Miltgen et al. (28) reported an outbreak of such IC1 A. baumannii without special phenotype in the Southwest Indian Ocean Area.Similarly, Hansen et al. ( 8) identified six similar isolates in Denmark, comprising five IC2 and one IC1.Notably, Zhao et al. (23) first reported an IC11 (ST164 Pas /ST1418 Ox ) strain collected from an ICU patient in Hangzhou, marked by three copies of OXA-23 and one copy of NDM-1 inserted into the chromosome via different composite transposons.In accord ance with previous studies, our comprehensive genomic analysis suggests that the IC11 (ST164 Pas /ST1418 Ox ) A. baumannii strain has spread worldwide over the last decade (8,24,27), but it remains at low prevalence and requires further surveillance due to the limited number of available genomes.Also, in the present analysis, we identified two distinct IC11 clades, one cluster characterized by OXA-23-producing strains primarily from Thailand and another comprising OXA-23-/NDM-1-producing strains originating from China.This provides important evidence for empirical clinical treatment of CRAB infections in healthcare settings in China.
Recent studies have revealed that traditional seven-loci schemes (Oxford and Pasteur) and pulsed-field gel electrophoresis typing (our data also showed in Fig. S1) fail to accurately depict the relationships among A. baumannii, primarily due to the high levels of recombination events (29)(30)(31).In our study, we achieved finer resolution in CRAB population analysis using core genome single-nucleotide polymorphisms and cgMLST typing, thereby uncovering detailed genetic patterns.Variations in geographic distribution, antimicrobial susceptibility, and mobile genetic elements (RIs, plasmids, and transposons) were observed between IC2 (with its distinct sublineages) and IC11.Previous findings indicated that cgSTs displayed a close association with transposon types among IC2 isolates (3,11).In this study, Tn2006 was present in IC2B, correspond ing to cgST906, and in IC11, classified under cgST1101.The RP-T1 (formerly RepAci6) pSRM1.1 found in IC2B isolates was indicative of playing a great role in the dissemination of bla OXA-23 between species, given that this plasmid contained a copy of bla OXA-23 in Tn2006 within AbaR4 inserted into the plasmid maintenance region, but with a complete conjugative region.RepAci6 plasmids were known to facilitate the spread of oxa23 gene in IC1 and IC2 (4).Some RepAci6 plasmids have been reported to be conjugative and include a complete set of genes for conjugation and could be a helper plasmid to facilitate a bla OXA-23 -carrying RepAci1 plasmid mobilization (4,32).Additionally, Tn2009, frequently found in IC2A and IC2C strains, could be represented in a tandem repeat structure with four copies of bla OXA-23 , which has been poorly reported (4,9,33,34).Few studies demonstrated that the formation of a Tn2009 tandem repeat circular intermediate, assisted by the ISAba1 element, suggests a novel mechanism for bla OXA-23 translocation, warranting further investigation (9).The acquisition of bla NDM-1 by a novel Tn6924-like was firstly reported in IC11 (ST164 Pas /ST1418 Ox ) A. baumannii, and its contribution to the spread of bla NDM1 shall be more concerning (17,19,23).
Our study has several limitations.This analysis did not encompass all hospitals in this region, suggesting that the clinical and genome epidemiology of CRAB may differ.Another limitation of this study was that we did not determine the impact of bla OXA-23 copy number on phenotypic susceptibility.
In summary, IC2 has predominated the OXA-23-producing CRAB population in this region, marked by the emergence of the rare IC11 (ST164 Pas /ST1418 OX ) lineage co-harbor ing bla OXA-23 and bla NDM-1 .This discovery highlights the critical importance of ongoing surveillance to prevent further dissemination.

Strain collection
A total of 100 non-duplicate CRAB strains were collected by three clinical laboratories at the three local distinct largest tertiary teaching hospitals in Jiangxi province.All strains were recovered from patients admitted to the hospitals between February 2021 and February 2022.The study was approved by the Ethics Committee of the Second Affiliated Hospital of Nanchang University.As we conducted a retrospective study, it was exempt from the requirement for informed consent.

Species identification and antimicrobial susceptibility testing
Acinetobacter strains were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (Bruker, Bremen, and Germany).MICs of different antimi crobial agents were determined by the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI 2021) guidelines with the reference strain Escherichia coli ATCC 25922 being used as the quality control (35).The MIC interpretive breakpoints of tigecycline for A. baumannii were determined based on the FDA recommendations for Enterobacteriaceae: susceptible (≤2 µg/mL), intermediate (4 µg/mL), and resistant (≥8 µg/mL) (3,16,36).

Whole-genome sequencing and analysis
Genomic DNA of all isolates was extracted using the TIANamp Bacteria DNA kit (Tiangen Biochemical Technology, Beijing) following the manufacturer's instructions and was subjected to WGS described below.A total of 100 CRAB isolates were sequenced using a HiSeq X Ten Sequencer (Illumina, San Diego, CA, USA), with 150 bp paired-end short reads and 200× coverage (Table S6).Of the 100 isolates, eight isolates were also subjected to long-read sequencing using a MinION Sequencer (Nanopore; Oxford, United Kingdom).The obtained paired-end Illumina reads were assembled de novo with the use of the SPAdes v3.13 (37).Both short and long reads of the additional eight isolates were utilized to generate a de novo hybrid assembly using Canu v1.8 and Pilon v1.24 (38).Genomes were annotated using Prokka v.12-beta and NCBI Prokaryotic Genome Annotation Pipeline (39,40).Short reads were mapped to reference genomes of respective international clones using Snippy v.4.6.0 (IC2, A9844, CP102580.1;IC11, XH1935, CP088894.1).Recombination was identified and removed by Gubbins v.3.3.0 (41), and the resulting alignment was used for tree estimation using RAxML v8.2.12 with GTRGAMMA model and 100 rapid bootstrap replicates (42).In order to understand the epidemiology of IC11 (ST164 Pas /ST1418 OX ) clone from a global perspective, we analyzed the IC11 (ST164 Pas /ST1418 OX ) sequences by investigating 28 assemblies available through Pathogenwatch (https://pathogen.watch/) and only two genomes deposited in the NCBI database, from a variety of countries (Table S5).Assemblies and genomes were downloaded in December 2023 through their websites and included with our own assemblies.SNP distances were calculated from the Gubbins-filtered polymorphic sites file using SNP-dists v0.6.3.Visualization of trees was carried out using Figtree v1.4.4 and iTOL v6 (11).

FIG 2
FIG 2 Phylogeny of A. baumannii with distribution of isolates and international clones.(A) The midpoint rooted phylogeny was constructed from SNPs in the core genomes of all isolates using RAxML.The phylogeny is annotated based on the study site of isolation, IC, Oxford ST, core genome MLST (determined by cgMLSTFinder v1.2), acquired carbapenemase, capsular polysaccharide K locus (KL), and lipooligosaccharide OCL.Branches are shaded by lineages and sublineages described in the text.EFY, the Second Affiliated Hospital of Nanchang University; YFY, the First Affiliated Hospital of Nanchang University; SRM, Jiangxi Provincial People's Hospital.(B1) Geographical distribution of cgMLST of the 100 CRAB isolates.The maps were drawn using the R package mapchina (https://github.com/xmc811/mapchina),and the data source was derived from https://www.openstreetmap.org.(B2) Minimum spanning tree constructed on the basis of cgMLST allelic genes of 100 clinical CRAB isolates.Each circle depicts an allelic profile based on sequence analysis of 2,133 cgMLST genes.Colors of the circles denote different cgMLST types.Closely related genotypes (less than 10 alleles difference) are shaded in the same node.The length of the connecting lines represents the number of target genes with different alleles.

FIG 3
FIG 3 Genetic characteristics of OXA-23-producing IC2 and IC11 clinical CRAB originated from Jiangxi region.The phylogeny is annotated based on Pasteur ST, Oxford ST, cgMLST, and study hospital of isolation.The presence of plasmids and plasmid types is shown in black, and their absence is in white; pSRM1.2 and pYFY24.2have identical genome sequences, represented by pSRM1.2;pYFY21.1 and pYFY24.1 have nearly identical genome sequences illustrated by pYFY21.1;pYFY3.1 and pSRM25.1 contain identical genome sequences, depicted by pSRM25.1.The presence of genomic resistance islands (RIs) and resistance transposons is shown in gray, and their absence is in white.

FIG 4
FIG 4 Genetic context of acquired bla OXA-23 and bla NDM-1 .The orientation and extent of genes are indicated by horizontal arrows.Inverted repeats and TSDs are shown as black and violaceous vertical bars, respectively.(A1) Structure comparison of Tn2009 and Tn2006 carrying the bla OXA-23 gene.*2 or *3 indicates that Tn2009 or Tn2006 move independently into two or three chromosomal locations.(A2) Genetic structure of a Tn2009 tandem repeat structure with four copies of bla OXA-23 (located in the chromosomes of isolates IC2A_cgST668/YFY3, IC2C_cgST661/SRM21, and IC2C_cgST923/YFY21). (A3) Tn2006 within AbaR4 and AbaR4 derivative as part of AbGRI1 variant in the chromosomal comM of strain IC2B_cgST906/YFY24.Orange, dark blue, and purple denote Tn6022, Tn6172, and the linker elements, respectively.(B) bla NDM-1 -containing Tn6924-like transposon structure identified in strain IC11_cgST1101/SRM3 in this study.Comparison of Tn6924 found in Cl300 (GenBank accession no.CP082952) and 11W359501 (GenBank accession no.CP041035).Tn7 transposition genes are colored purple, antibiotic resistance genes red, and insertion sequence green.

FIG 5 A
FIG 5 A global view of IC11 (ST164 Pas /ST1418 Ox ) Acinetobacter baumannii based on WGS.Core genome phylogeny is linked with country, collection date, study center, capsular polysaccharide KL, lipooligosaccharide OCL, and antimicrobial resistance genes.The highlighted boxes denote the substructure within ST1418 Ox (Thailand subclade and China subclade).The presence of plasmid types is shown in black, and absence is in white; the presence of resistance transposons is shown in gray, and absence is in white.Abbreviations: NA, not applicable; Tpns, transposon.

TABLE 1
Culture source distribution of CRAB isolates a

TABLE 2
Study site distribution of CRAB isolates