Chromosomal dif sites and associated modules identified in Acinetobacter sp. drive the horizontal transfer of antibiotic resistance

Introduction: Modules containing antibiotic resistance genes (ARGs) flanked by Xer site-specific recombination sites have been identified in Acinetobacter plasmids and are considered mobile genetic elements (MGEs) that facilitate horizontal gene transfer via the XerCD site-specific recombination (XerCD SSR) system. Although additional dif-like sites have been identified on the Acinetobacter chromosome beyond the main locus, it remains unclear whether these sites are associated with chromosomal dif modules.

Methods: MacConkey agar plates supplemented with meropenem were used to isolate the resistant strain. Whole-genome sequencing (WGS) was performed on the Oxford Nanopore platform, and the bacterial species was identified using Average Nucleotide Identity (ANI) and digital DNA-DNA hybridization (dDDH). Antimicrobial susceptibility testing was performed against 18 antibiotics. Identification of dif and pdif sites was performed using BLAST tools.

Results: This study identified numerous Xer modules containing resistance genes, IS elements, and other functional genes within the chromosome and plasmid of strain M10 (Acinetobacter sp.) isolated from a farmer at a cattle farm in Guangxi, China. Genome analysis and antimicrobial susceptibility testing confirm the association between these modules carrying resistance genes and resistant phenotypes. Chromosomal dif sites and associated dif modules in the strain were highly similar (sequence identity >99%) to plasmid-carried pdif sites and associated pdif modules in the public database. These suggest that additional chromosomal dif-like sites facilitate dif module formation, and that gene flow occurs between the chromosomes and plasmids of Acinetobacter. Furthermore, most Xer sites clustered to form a linear multi-module array, termed chromosomal dif module island and plasmid-borne pdif module island. Similar configurations were frequently observed in public Acinetobacter plasmid genomes.

Discussion: Additional dif-like sites are present in Acinetobacter chromosomes, which are unlikely to play a function in chromosomal dimer resolution, and the modules they form are functionally similar to pdif modules, both of which play an important role in horizontal gene transfer.

Introduction

Acinetobacter is known for its ability to spread through food production, processing, and storage; through the hands of healthcare personnel; and through cross-contamination of medical devices, making it a significant contributor to nosocomial infections (Rathinavelu et al., 2003; De Amorim and Nascimento, 2017). The rise of antimicrobial resistance in Acinetobacter is a critical medical challenge. Although research on the transfer of resistance genes via canonical MGEs (such as plasmids, ICEs, and transposons) in Acinetobacter remains limited (Pérals et al., 2000; Wang et al., 2025), an untypical class of mobile genetic elements whose transposition likely depends on the action of the XerCD site-specific recombination (XerCD SSR) system was recently discovered in Acinetobacter. This system consists of the homologous recombinases XerC/XerD (tyrosine recombinase family) (Lin et al., 2020; Balalovski and Grainge, 2020), which catalyze two sequential pairs of DNA strand cuts and exchanges at a defined locus termed dif, located in the terminus region of the chromosome (Balalovski and Grainge, 2020; Grosso et al., 2012; Poirel and Nordmann, 2006; Midonet and Barre, 2014). Furthermore, this system is critical for stabilizing plasmids by resolving multimers (Colloms et al., 1998; Summers et al., 1993). Typically, the dif site is a 28-bp site consisting of two inverted repeat 11-bp Xer-binding motifs (the left and right regions) separated by a 6-bp interval called the central region (Lin et al., 2020; Balalovski and Grainge, 2020). A monomer of XerC and XerD each binds to a 11-bp semi-binding site (Balalovski and Grainge, 2020; Shao et al., 2023). The dif sites located in Acinetobacter plasmids are called pdif sites and appear multiple times in a plasmid (Shao et al., 2023). Modules containing ARGs flanked by pdif sites have been identified. Substantial copies of these modules are widely distributed across plasmid genomes within the Acinetobacter genus (Grosso et al., 2012; Poirel and Nordmann, 2006; Shao et al., 2023; Cameranesi et al., 2018; Merino et al., 2010; Blackwell and Hall, 2017; Tran et al., 2012), suggesting horizontal gene transfer mediated by pdif modules. In addition to ARGs, genes encoding other functional proteins and proteins of unknown function are carried by pdif modules, such as pdif-ser, pdif-ISAjo2, pdif-higA-higB, pdif-chrAB, pdif-kup, pdif-terC, pdif-add, pdif-sulP, and pdif-ohr (Blackwell and Hall, 2017; Harmer et al., 2023; Mindlin et al., 2019). Meanwhile, the site-specific recombination reaction can be observed between the two sites in different plasmids (Cameranesi et al., 2018; Mindlin et al., 2019), further suggesting that the pdif module is involved in horizontal genetic transfer. However, modules containing various genes flanked by chromosomal dif sites are rarely observed. Usually, a single dif site (the main dif site) is involved in dimeric chromosome resolution (Mindlin et al., 2019; Carnoy and Roten, 2009). This site is considered not to participate in dif module formation, as its involvement would impair the dimer resolution process (Mindlin et al., 2019). Thus, in addition to the main dif site, additional dif-like sites may exist that could contribute to dif module formation. This study found numerous Xer sites in the chromosome and plasmid of strain M10. The presence of multiple chromosomal dif-like sites suggests that some may serve functions beyond canonical dimer resolution.

Results

Source

In September 2023, we isolated a meropenem-unsusceptible strain, M10, from the feces of a farmer at a commercial bovine farm in Guangxi, China. PCR amplification and Sanger sequencing confirmed that this strain carried the bla_NDM gene.

Identification of bacterial species

This strain was classified as Acinetobacter genus using MALDI-TOF-MS and 16S rDNA. Subsequently, this strain was sequenced using whole-genome sequencing (WGS) on the Oxford Nanopore platform (long-read sequencing technology). The analysis confirmed 98.56% completeness and 0.83% contamination in this genome assembly (Acinetobacter towneri genome assembly ASM4857284v1 – NCBI – NLM). To confirm the species of this strain, the average nucleotide identity (ANI) match was performed using the NCBI annotation service on the NCBI database. This result showed that the strain best matched Acinetobacter towneri DSM 14962 = CIP 107472 (GCA_000368785.1), with an ANI value of 93.82%. Subsequently, these strains were submitted to the genome-to-genome distance calculator (GGDC) on the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulture) platform to calculate the digital DNA–DNA hybridization (dDDH) value. This result demonstrated a dDDH value of 53.3%. Current standards require ANI (>95% for the same species) or digital DNA–DNA hybridization (dDDH) (>70%) values to assign a novel strain to a species robustly (Riesco and Trujillo, 2024; Meier-Kolthoff et al., 2013). These results indicate that strain M10 falls below the standard ANI (>95%) and dDDH (>70%) thresholds for assignment to an existing species. In view of this, this strain was submitted to the JSpeciesWS and DSMZ platforms using the Tetra Correlation Search (TCS) and Type (Strain) Genome Server (TYGS) for further analysis. These results were similar to those in the NCBI annotation service. Thus, genome M10 should be considered a potential novel species within the Acinetobacter genus.

Phylogenetic analysis

We retrieved 33 genomes closely related to strain M10 from the JSpesiesWS (TCS) and DSMZ platforms (TYGS). Subsequently, all genomes were used to build an ML phylogenetic tree (Figure 1). Phylogenetic analysis revealed that the tree diverged into three branches, highlighting three distinct lineages. Branch two included strain M10, A. towneri (DSM 14962 CIP 107472 and DSM 14962 CIP 107472 DSM 14962), A. kanungonis (PS-1), A. tibetensis (Y-23), and A. tandoii (DSM 14970 CIP 107469 and DSM 14970 CIP 107469 DSM14970). Among these, strain M10 and A. towneri belonged to a clade, showing their highest genetic similarity. Branch lengths in phylogenetic trees serve as important indicators for measuring genetic differences among strains. The genetic difference in strain M10 significantly exceeds that in A. towneri [branch length: 0.029 (M10) > 0.0077 (A. towneri)], indicating genetic similarity between them, though they belong to different lineages.

Figure 1

Figure 1. Phylogenetic tree. Bootstrap values are shown on the branch of this tree as circles. Branch lengths are displayed as numbers on each branch of this tree.

Antibiotic resistance phenotype

The antimicrobial susceptibility testing showed that strain M10 was resistant to meropenem (16 mg/L), ampicillin (128 mg/L), ceftazidime (>1,024 mg/L), cefepime (128 mg/L), gentamicin (16 mg/L), tetracycline (16 mg/L), imipenem (16 mg/L), and ertapenem (64 mg/L) (Table 1). However, it was susceptible to aztreonam, chloramphenicol, colistin, kanamycin, fosfomycin, ciprofloxacin, sulfamethoxazole, azithromycin, tigecycline, and amikacin (Table 1).

Table 1

Table 1. MIC and ARGs of the strain M10.

Genetic diversity analysis

ONT long-read sequencing revealed that strain M10 carried a chromosome (chromosome-NDM-M10) and a plasmid (pM10), with sizes of 2,896,975 bp and 162,635 bp (Table 2). The chromosome-NDM-M10 harbored multiple ARGs, including aadA2b, aac(3)-IId, tmrB, aph(3′)-VIa, bla_NDM-1, ble_MBL, bla_OXA-58, msr(E), mph(E), and tet(39) (Table 2). In addition, the chromosome carried metal resistance genes (MRGs) against arsenic, mercury, and copper, as well as genes encoding virulence factors (VFs) related to bacterial outer membrane protein, type IV pili, phospholipase D, LPS, two-component system, and serum resistance (Table 2). The pM10 carried three ARGs, including msr(E), mph(E), and tet(39) (Table 2). The analysis using MOB-Typer revealed that pM10 could be typed as rep_cluster_1656 and predicted to be mobilizable.

Table 2

Table 2. Genomic characteristics of the strain M10.

Chromosomal dif sites

A total of 22 dif sites were identified in the chromosome of strain M10. Among these, one dif site was identified as the main dif site (1,434,250–1,434,277 bp). The remaining dif sites likely facilitate the formation of dif modules. Notably, ten dif sites clustered in a region of 36,106 bp (2,529,048–2,565,154 bp) that contained all identified chromosomal ARGs, except for aadA2b. This region also included genes encoding IS elements, other functional proteins, proteins of unknown function, and the toxin–antitoxin system (Figures 2A,B). These clustered dif sites formed nine dif modules, and these modules in a row built an island designated as the “dif module island” (Figures 2A,B).

Figure 2

Figure 2. The show of dif and pdif modules and the islands they build. (A) The locations of Xer module islands are shown in the circular genome of the chromosome and plasmid. (B) Linear environment of the dif and pdif module islands. (C) Linear skeletons of dif modules. (D) Linear skeletons of pdif modules. Vertical bars indicate dif or pdif (C/D and D/C) sites; the direction of each site is shown.

Plasmid-borne pdif sites

We found 19 pdif sites in the pM10 of strain M10. Like the configuration of the dif module island in the chromosome, 15 pdif sites clustered in the region with a size of 25,749 bp (43,812–69,561 bp) that contained all plasmid-borne ARGs and various functional genes (Figures 2A,B). This region was divided into 14 pdif modules. Here, we called this region a “pdif module island” (Figures 2A,B).

Xer-ARG modules

In addition to aadA2b, the remaining ARGs in the chromosome and plasmid of strain M10 were located within seven Xer modules, including five dif-ARG modules (dif-bla_OXA-58, dif-aac(3)-IId, dif-tet(39), dif-msr(E)-mph(E), and dif-bla_NDM-1) in the dif module island (Figure 2C) and two pdif-ARG modules (pdif-tet(39) and pdif-msr(E)-mph(E)) in the pdif module island (Figure 2D). The linear alignment in Figure 3A was built based on genome analyses of the dif-bla_NDM-1, dif-bla_OXA-58, and dif-aac(3)-IId modules. The linear alignment in Figure 3B was constructed based on genome analyses of the Xer-tet(39) and Xer-msr(E)-mph(E) modules.

Figure 3

Figure 3. Linear comparisons of the Xer-ARG modules. (A) Linear comparisons were built based on the dif-blaNDM-1, dif-blaOXA-58, and dif-aac(3)-IId modules. (B) Linear comparisons were built based on the Xer-tet(39) and Xer-msr(E)-mph(E) modules. The C/D and D/C sites of dif-ARG and pdif-ARG modules are shown in Table 3. The color-marked genome names indicate the species under Acinetobacter spp. Vertical bars indicate dif or pdif (C/D and D/C) sites; the direction of each site is shown above.

Genome analyses

Dif-bla_NDM-1 module

The Dif-bla_NDM-1 module, carrying multiple resistance elements (bla_NDM-1, aph(3′)-Via, and ble_MBL) and insertion sequences (ISAba23, ISAba14, and ISAba125), was located within the right-hand region of the dif module island (Figure 2B). This module, spanning 11,245 bp in size (Figure 2C), was considerably longer than the previous reported (Poirel and Nordmann, 2006; Shao et al., 2023; Merino et al., 2010; Blackwell and Hall, 2017) pdif-bla_OXA-72, pdif-bla_OXA-24, pdif-bla_OXA-58, pdif-sul2, pdif-tet(39) and pdif-msr(E)-mph(E) modules. Notably, the dif-bla_NDM-1 module carried the bla_NDM-1 genetic context, ISAba125-bla_NDM-1-ble_MBL-trpF-dsbD. BLASTn analysis revealed that the dif-bla_NDM-1 module exhibited >99% identity and coverage to a segment containing bla_NDM-1 in the pNDM_SCLZS30 (Figure 3A). However, this segment could not be identified as a pdif module due to the absence of the right and center regions in the D/C site (Table 3A). Furthermore, the region ISAba125-bla_NDM-1-ble_MBL-trpF-dsbD in Tn125 (DANMEL) is highly similar (99.95% identity and 100% coverage) to that in the dif-bla_NDM-1 module, but the remaining region of Tn125, cutA-groES-groEL-insE-ISAba125, is missing in the dif-bla_NDM-1 module. No other ISAba125 could be identified in the downstream region of the bla_NDM-1 genetic context.

Table 3

Table 3. The C/D and D/C sites of dif-ARG and pdif-ARG modules in Figure 3.

Dif-bla_OXA-58 module

Two inverted bla_OXA-58 genes were identified within the different modules in the dif module island (Figure 2B). These bla_OXA-58 genes were flanked by two inverted ISAba3 elements, with sizes 427 bp and 774 bp, respectively. These IS elements were shorter than the full-length ISAba3 element (794 bp, ISFinder). The right-hand dif-bla_OXA-58 module (10,747 bp) shared the D/C and C/D sites of the dif-lysE and dif-bla_NDM-1 modules. The analysis in GenBank databases found that no segment matches this module. The left-hand dif-bla_OXA-58 module (2,318 bp) shared the D/C site with the dif-aac(3)-IId module. BLASTn analysis revealed multiple segments of Acinetobacter plasmids exhibiting >99% identity and coverage with this module. Thus, this study focuses on the left-hand dif-bla_OXA-58 module. All segments containing bla_OXA-58 in Figure 3A could be identified as pdif-bla_OXA-58 modules. These modules were highly conserved (>99% identity and coverage) with the chromosomal dif-bla_OXA-58 module. Four variant bases emerged in the center and right regions of the C/D site within pdif sites compared to dif sites (Table 3A).

dif-aac(3)-IId module

The dif-aac(3)-IId module of 3,736 bp carrying aac(3)-IId, tmrB, and IS15 (Figure 2C) was positioned between the dif-bla_OXA-58 and dif-tet(39) modules, sharing their C/D and D/C sites (Figure 2B). BLASTn analysis revealed two plasmid-borne fragments (pNDM_SCLZS30 and pNDM_SCLZS86) exhibiting >99% identity and coverage with the dif-aac(3)-IId module (Figure 3A). In pNDM_SCLZS86, the downstream region of the segment containing aac(3)-IId was truncated by a 24,500-bp region, leading to the absence of D/C site (Table 3A). Pdif-aac(3)-IId module in pNDM_SCLZS30 could be identified and was highly conserved (>99% identity and coverage) with the chromosomal dif-aac(3)-IId module. Only one variant base emerged in the left region of the D/C site within pdif sites compared to dif sites (Table 3A).

Xer-tet(39) modules

Xer-tet(39) modules were identified in the dif and pdif module islands, respectively (Figures 2B–D). These modules exhibited >99.5% identity and coverage (2056/2057 bp) (Figure 3B). BLASTn analysis revealed they showed high similarity (>99% identity and coverage) to numerous segments of the chromosomes and plasmids of Acinetobacter in the public database. Eight pdif-tet(39) modules could be identified from public plasmid genomes (Figure 3), two of which were identified in pSP19M058–4. Notably, the pdif-tet(39) module in pS30-1 has been previously reported (Blackwell and Hall, 2017). It differed from the chromosomal dif-tet(39) module in the center regions of the C/D and D/C sites (Table 3B). The remaining modules were highly conserved (>99% identity and coverage) to chromosomal dif-tet(39) module (Figure 3). Only one variant base emerged in the center region of C/D site within pdif sites compared to dif sites (Tables 3A,B).

Xer-msr(E)-mph(E) modules

Xer-msr(E)-mph(E) modules were located in the dif and pdif module islands, respectively (Figures 2B–D). These modules exhibited >99.5% identity and coverage (3,004/3006 bp). Like the dif-tet(39) module, they were also highly similar (>99% identity and coverage) to numerous segments of the chromosomes and plasmids of Acinetobacter in the public database. In addition to the segment in p19110F47, nine pdif-msr(E)-mph(E) modules could be identified from public plasmid genomes (Figure 3). Interestingly, the pdif-msr(E)-mph(E) modules in pOXA58_010055 and pAS74-1 additionally carried IS481 and ISAba26, respectively. Apart from these modules, the remaining modules were highly similar (>99% identity and coverage) to the chromosomal dif-msr(E)-mph(E) module (Figure 3). Compared to dif sites, two variant bases or one inserted base emerged in the right region of the D/C site within pdif sites (Tables 3A,B).

Chromosomal dif modules containing other genes

In addition to ARGs, three dif modules containing other genes have been identified in the dif module island. The dif-ser module (2,537,075-2,537,939 bp) was located between the dif-tet(39) and dif-msr(E)-mph(E) modules, sharing their D/C and C/D sites (Figure 2B). This gene encoded a helix-turn-helix domain-containing protein (UniProt, 203 aa), which is a small serine recombinase and belongs to the site-specific recombinase resolvase family. The copy of this gene could be identified in the plasmid-carried pdif-ser-ISAba17 module in pM10. Interestingly, the Xer-ser module could be frequently identified in public plasmid genomes and was typically located in the downstream region of mph(E) (Figure 3). The dif-higA-higB module (2,540,890-2,541,651 bp) was located between dif-msr(E)-mph(E) and dif-lysE modules and shared their D/C and C/D sites (Figure 2B). The higB encoded a stable toxin protein (106 aa) and the higA encoded its labile antitoxin (90 aa). These genes belong to the type II toxin–antitoxin system (a critical genetic regulatory module in bacterial plasmids), which maintains plasmid stability by inhibiting growth when the plasmid is lost, and is widely distributed across bacterial plasmids and chromosomes. This module was highly similar (>99% identity and coverage) to the pdif-higA-higB module (58,052–58,809 bp) identified in the pdif module island (Figure 2B) and public plasmid genomes (Figure 3). Furthermore, the dif module (2,541,624–2,543,218 bp, Figure 2B), harboring a similar gene (99.5% identity) to lysE family translocators (201 aa, bacterial transmembrane transporters specialized in amino acid efflux to maintain intracellular homeostasis), was located between dif-higA-higB and dif-bla_OXA-58 modules and shared their C/D and D/C sites (Figure 2B). This module was undetectable in pM10 and public plasmid genomes (Figure 3).

Plasmid-borne pdif modules containing other genes

In addition to ARGs, multiple pdif modules carrying other genes were detected in the pdif module island. A similar gene (99.5% identity) to STAS domain-containing proteins (485 aa, a conserved structural domain ubiquitously present in prokaryotic and eukaryotic organisms) was detected in two dif modules (43,812–45,969 and 50,114–52,850 bp), respectively (Figure 2B). The STAS domain is functionally implicated in diverse cellular processes, particularly mediating ion transport and signal transduction. The pdif module (56,834–57,585 bp) contains a functionally uncharacterized gene and a gene encoding RelE/ParE family toxin (100 aa) of the Type II toxin–antitoxin system (Figure 2B). Adjacent to this module, a pdif module (57,558–58,080) carried a gene encoding a SMI1/KNR4 family protein (139 aa), a functionally unknown protein in bacterial systems (Figure 2B). Notably, the above two modules were commonly present across public plasmid genomes (Figure 3B). In addition, three pdif modules, including 48,072–49,400 bp, 49,373–50,141 bp, and 52,823–53,701 bp, were identified in the pdif module island and carried multiple uncharacterized genes (Figure 2B).

pdif-ISAjo2 module

In the pdif module island, two highly similar IS elements were identified in the pdif modules of 45,942–48,099 bp and 67,207–69,561 bp and were 1,482 bp (left-hand module) and 1,475 bp (right-hand module) (Figure 2B). They showed 97% identity to the ISAjo2 reference sequence, 96% identity to ISAso2, and 90% identity to ISApi2 (ISFinder). Given its highest similarity to ISAjo2 (an ISAba32 group member of the IS1202 family), this element is designated as ISAjo2 (ISfinder). Notably, the ISAjo2 in the left-hand module was five bp from the C/D site. This characteristic was similarly observed in previous reports (Blackwell and Hall, 2017; Harmer et al., 2023). However, two additional genes, relB (81 aa) and relE (83 aa), were identified downstream of ISAjo2 in this module (Figure 2B). These genes encode functional components of the family addiction module antitoxins and constitute essential elements within bacterial toxin–antitoxin systems. The ISAjo2 in the right-hand module was 27 bp from the C/D site. Two genes encoding a BrnT-family toxin (103 aa) and a cytoplasmic protein (104 aa) were identified downstream of the ISAjo2 (Figure 2B). However, the characteristics of this module were different from the previous reports (Blackwell and Hall, 2017; Harmer et al., 2023).

pdif-ISAba25 module

A previously unreported IS in the Xer module was identified in the pdif module (53,674–56,861 bp, Figure 2B) of the pdif module island. Sequence analysis found that this element exhibited 95% identity to the ISAba25 reference sequence and 93% identity to ISAlw34. Based on its best match, it was designated as ISAba25 (ISfinder), a member of the IS66 family. Furthermore, a gene encoding an uncharacterized protein (170 aa) was detected downstream of this IS.

pdif-ISAba17 module

Another previously unreported IS in the Xer module was identified in the pdif module (61,760–65,205 bp, Figure 2B) of the pdif module island. This element exhibited 95% identity to the reference sequence ISAba17 (ISfinder), a member of the IS66 family. A gene that was similar to the ser gene in the dif module (2,537,075–2,537,939 bp) was detected upstream of this IS. However, its 102 bases at the 3′ end were replaced by ISAba17 (Figure 2B).

The configuration of the dif and pdif module islands

Xer-msr(E)-mph(E) module was closely adjacent to Xer-tet(39) module in the dif and pdif module islands (Figure 2B). This characteristic was also frequently observed in public plasmid genomes (Figure 3). Similarly, dif-bla_OXA-58 module was closely adjacent to dif-aac(3)-IId module, and this characteristic also appeared in some public plasmid genomes (Figure 3A). The dif and pdif module islands were formed by multiple dif/pdif modules arranged in a row. This configuration inevitably leads to sharing the internal C/D or D/C sites to form two types of dif modules: one flanked by a C/D and a D/C site and another flanked by a D/C and a C/D site (Figure 2B). A similar configuration was also frequently observed in public plasmid genomes (Figure 3).

Discussion

Acinetobacter, a heterogeneous bacterial genus, is widely distributed across human and animal communities and exhibits robust environmental adaptability and survival capabilities (De Amorim and Nascimento, 2017). Many of them, primarily A. baumannii, but also A. nosocomialis, A. pittii, A. lwoffii, and others, are listed as clinically relevant pathogens (Wang et al., 2025; Touchon et al., 2014; Peleg et al., 2012). The rising antimicrobial resistance, particularly resistance to last-resort antibiotics, among this genus has become an urgent medical challenge, with numerous mobile genetic elements contributing to its dissemination. A presumptive pdif-bla_OXA-24 module was initially identified in the A. baumannii plasmid pABVA01a (D’Andrea et al., 2009). This module was also found in different contexts within another Acinetobacter plasmid, suggesting its transfer (D’Andrea et al., 2009). Subsequent studies revealed that Acinetobacter plasmids carry pdif modules with a wide variety of resistance genes, metal resistance genes, IS elements, and other genes (Wang et al., 2025; Grosso et al., 2012; Midonet and Barre, 2014; Shao et al., 2023; Cameranesi et al., 2018; Merino et al., 2010; Blackwell and Hall, 2017; Tran et al., 2012; Harmer et al., 2023; Mindlin et al., 2019). Thus, these modules were previously considered to be associated with Acinetobacter plasmids. Although additional dif-like sites have been identified on the chromosome of Acinetobacter beyond the main locus (Mindlin et al., 2019), it remains to be determined whether these sites are associated with the dif module. This study revealed that multiple Xer sites are present in the chromosome and plasmid of Acinetobacter sp. M10, mediating the formation of simple Xer modules carrying one to several genes or complex Xer modules containing more than ten genes. Genome analysis confirmed that the dif-tet(39) and dif-msr(E)-mph(E) modules in the chromosome were highly similar (sequence identity >99%) to those in the plasmid of this strain. Multiple chromosomal dif modules in this strain showed high sequence identity (>99%) to plasmid-carried pdif modules in public databases. Genome analysis and antimicrobial susceptibility testing confirm the association between these modules and the resistant phenotype. These suggest that Acinetobacter chromosomes also function as a vehicle for dif modules, and various genes (such as genes encoding antibiotic resistance, IS elements, toxin–antitoxin systems, and other functional proteins) flow via Xer modules between the chromosomes and plasmids of Acinetobacter. These modules, arranged one after another in the chromosome and plasmid, form special configurations: a chromosomal dif module island and a plasmid-borne pdif module island, suggesting their presence either as single modules or linear multi-module arrays. Similar configurations were frequently observed in public Acinetobacter plasmid genomes. The exploration of the chromosomal dif modules and the plasmid-carried pdif modules provides a novel perspective for understanding the dissemination of antibiotic-resistant Acinetobacter worldwide.

The recurrent appearance of Xer modules on the chromosomes and plasmids of Acinetobacter demonstrates their ability to spread. Like other MGEs, Xer modules mediate the horizontal transfer of genes, accelerating bacterial genome evolution. The Xer module itself functions as a “gene cassette” that can be integrated into larger genetic contexts, such as plasmids and chromosomes (Shao et al., 2023). When such a plasmid transfers among bacterial populations via conjugation, the Xer module “hitchhikes” along, reflecting the synergistic mechanism among MGEs. The transfer mechanism of Xer modules shares common characteristics with other MGEs while exhibiting specific characteristics. Like the transfer of other canonical MGEs (such as plasmids, ICEs, and transposons) (Wang et al., 2025; Correa et al., 2024; Bi et al., 2012; Brovedan et al., 2020), which depend on various elements (such as oriT, relaxase, Rep, and transposases), the Xer module relies on the XerCD site-specific recombination system. However, unlike other MGEs, the Xer module encodes only Xer sites and does not encode other self-transfer elements (Shao et al., 2023; Blackwell and Hall, 2017). Its mobilization usually depends on the mechanism provided by the host cell. These features give the Xer module a more compact structure and potentially reduce the burden on the host bacteria. As a novel gene dissemination system, Xer modules facilitate the spread of antibiotic resistance within the Acinetobacter genus, together with other MGEs, forming a complex and dynamic network of gene flow.

Previous studies (Mindlin et al., 2019; Blakely et al., 1993; Blakely et al., 1991; Kuempel et al., 1991) found that a single main dif site in the terminus region of the chromosome is required for the resolution of chromosome dimers by the XerC/XerD system. If it is transferred, the dimer resolution process is impaired (Balalovski and Grainge, 2020; Kuempel et al., 1991). Numerous dif sites identified in the chromosome of Acinetobacter sp. M10 suggest that additional dif-like sites are present in the chromosome of Acinetobacter beyond the main locus and unlikely to play the function of dimer resolution. Genome analysis revealed that dif sites and associated dif modules were highly similar (sequence identity >99%) to pdif sites and associated pdif modules, suggesting that additional chromosomal dif-like sites facilitate dif module formation. The results of this study and previous studies (Blackwell and Hall, 2017; Mindlin et al., 2019; D’Andrea et al., 2009; Feng et al., 2016) collectively highlight the common presence of the Xer site and associated Xer module across plasmids and chromosomes within the Acinetobacter genus. The XerCD site-specific recombination system, initially functioning to stabilize chromosomes and plasmids (Balalovski and Grainge, 2020; Carnoy and Roten, 2009; Castillo et al., 2017), has subsequently played an additional role in driving horizontal gene transfer, underscoring that bacterial evolution never ceases. This study identified a chromosomal dif module island and a plasmid-borne pdif module island. These islands consist of multiple dif/pdif modules arranged one after another, leading to modules flanked by a C/D and a D/C sites or by a D/C and a C/D sites. This characteristic raises the question of whether the horizontal mobilization of the dif/pdif modules occurs as an individual module or as a linearly contiguous multi-module array.

The bla_NDM gene encodes New Delhi Metallo-β-lactamase (NDM), which hydrolyzes nearly all β-lactam antibiotics, including carbapenems, the last-resort therapeutic agents for multidrug-resistant bacterial infections (Kikuchi et al., 2022; Wang et al., 2017; Acman et al., 2022; Chatterjee et al., 2016; Partridge and Iredell, 2012). The bla_NDM-positive strains were first retrospectively identified in 2005 from A. baumannii in an Indian hospital (Kikuchi et al., 2022; Acman et al., 2022; Partridge and Iredell, 2012). In early isolates, bla_NDM is located within the intact Tn125 transposon, leading to the hypothesis that Tn125 is the ancestral transposon of bla_NDM (Acman et al., 2022). The upstream region of Tn125 carries ISs from families such as IS5/IS30, and frequent recombination among these IS elements has generated diverse genetic backgrounds (Kikuchi et al., 2022; Acman et al., 2022). ISAba125, a member of the IS30 family, is typically located upstream of bla_NDM and forms the structure ISAba125-bla_NDM-ble_MBL-trpF-dsbD (Acman et al., 2022). This structure was frequently detected in Acinetobacter, Klebsiella, and Escherichia (Acman et al., 2022). It has been widely accepted that bla_NDM-1 is regulated by a hybrid promoter located between bla_NDM-1 and ISAba125, and ISAba125 is commonly present in some form within bla_NDM-positive isolates (Kikuchi et al., 2022). This study identified an intact bla_NDM-1 genetic backbone carried by the chromosomal dif module, suggesting that, for effective expression of carbapenem resistance, bla_NDM-1, closely associated elements, and the hybrid promoter are integrally preserved in the dif module.

This study identified multiple Xer modules carrying genes encoding toxin–antitoxin systems, IS elements, helix-turn-helix domain-containing proteins, and proteins of uncharacterized function. Genome analyses confirmed that copies of these modules were widely distributed in Acinetobacter genomes. Given that genes in Xer modules are frequently associated with toxin–antitoxin systems, antibiotic resistance, and metal resistance, the Xer modules may contribute to the persistence of their resident vehicles, such as plasmids or chromosomes, by enhancing adaptability and tolerance. Various Xer modules were detected in the chromosome and plasmid of Acinetobacter sp. M10, which may suggest the presence of numerous undiscovered Xer modules within the Acinetobacter population. Furthermore, numerous dif and pdif modules detected in a novel species of the Acinetobacter genus underscore the potential for widespread dissemination of ARGs and mobile elements in this genus.

Conclusion

Additional chromosomal dif-like sites are functionally similar to plasmid-carried pdif sites. Modules flanked by Xer sites, as a novel mobile genetic element, form a close interaction network with their vehicles, such as plasmids and chromosomes, and play a vital role in disseminating resistance genes and other functional elements.

Materials and methods

Sampling and microbial identification

Strain M10 was collected from the feces of a farmer at a commercial bovine farm in Guangxi, China. Human-derived specimens were obtained following written informed consent procedures. This strain was isolated from MacConkey (Huan Kai Microbial, China) agar plates supplemented with 0.5 mg/mL meropenem and cultured at 37 °C for 16 h. As described previously (Wang et al., 2017), PCR amplification and Sanger sequencing were used to detect the bla_NDM gene.

Whole-genome sequencing

Briefly, the genome was sequenced using the long reads on the Oxford Nanopore platform (Beijing Genomics Institution, China) and the short reads on the DNBSEQ platform (Beijing Genomics Institution, China). The corrected reads were obtained by hybrid assembly of DNBSEQ and Oxford Nanopore. The assembled genome was checked for completeness and contamination using CheckM (v1.2.3) of the NCBI annotation service.

Bacterial-species identification

Bacterial genus identification was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Singhal et al., 2015) (MALDI-TOF MS; Bruker Daltonik, Germany) and 16 s rDNA (Kim et al., 2010). Bacterial species identification was carried out on the NCBI database using the ANI match of the NCBI annotation service, on the JSpeciesWS platform (JSpeciesWS – Taxonomic Thresholds) using TCS, and on the DSMZ platform (Type Strain Genome Server) using TYGS. The dDDH value was calculated using GGDC on the DSMZ platform (Leibniz Institute DSMZ: Welcome to the Leibniz Institute DSMZ).

Phylogenetic construction

Core genomes were extracted using Roary (Roary). Recombination was filtered using Gubbins (Gubbins). Filtered polymorphic sites were employed to build a tree on the PhyML platform (PhyML). The iTOL (Interactive Tree Of Life) was used to visualize the tree based on each genome’s features.

Antimicrobial susceptibility testing

Minimum inhibitory concentrations (MICs) were determined for 18 antibiotics, listed in Table 1, using the broth microdilution method in Mueller–Hinton medium (HuanKai Microbial, China). Interpretive criteria of Clinical and Laboratory Standards Institute (CLSI) guidelines (M100-S34, 2024) were applied. The CLSI breakpoints for Enterobacteriaceae were used for aztreonam, chloramphenicol, kanamycin, fosfomycin, azithromycin, and ertapenem, as criteria for Acinetobacter were unavailable. The resistance breakpoints for Enterobacterales set by the U. S. Food and Drug Administration (FDA) were used to interpret tigecycline (R ≥ 8 mg L⁻¹) as the CLSI criteria for this antimicrobial, and the FDA criteria for Acinetobacter were absent.

Bioinformatics analysis

The MOB-typer on the Galaxy platform (Galaxy | China) was used to analyze plasmid characteristics. The VFanalyzer on the VFDB platform (VFDB: Virulence Factors of Bacterial Pathogens) was used to search for genes encoding VFs. Genome annotation was performed using the RAST genome annotation service (RAST Server – RAST Annotation Server), and further manual correction was performed using ORFfinder (ORFfinder Home-NCBI), UniProt (UniProt), and ISFinder (ISfinder). The linear alignment was created using Easyfig (v2.2.5).

The identification of dif and pdif sites

Identification of dif and pdif sites was performed using BLAST tools. pdif sites were identified according to the pdif sites of A. baumannii and A. lwoffii in the previous report (Mindlin et al., 2019). dif sites were identified based on the chromosomal additional dif-like sites of Acinetobacter in the previous report (Mindlin et al., 2019). Only sites that were at least 85% identical to the reference sequences were taken into account.

Data availability statement

The assembled genome of strain M10 has been deposited in the NCBI database under accession no. GCA_048572845.1.

Ethics statement

All animal studies were performed according to the US National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Lanzhou Institute of Husbandry and Pharmaceutical Science of CAAS.

Author contributions

QW: Supervision, Formal analysis, Methodology, Data curation, Writing – original draft, Writing – review & editing, Resources, Investigation, Visualization, Validation, Software. WW: Investigation, Visualization, Project administration, Writing – review & editing. YQ: Writing – original draft, Software, Investigation, Validation. GD: Writing – original draft, Visualization, Validation. BL: Methodology, Writing – original draft, Data curation. YZ: Investigation, Writing – original draft, Data curation. JZ: Writing – review & editing, Funding acquisition, Project administration. YB: Investigation, Software, Writing – original draft.

Funding

The author(s) declared that financial support was received for this work and/or its publication. This study was supported by the National Natural Science Foundation of China (grant No. 32273068), the earmarked fund for the China Agriculture Research System (CARS) (grant No. CARS-37), and the National Key Research and Development Program of China (grant No. 2022YFD1602201).

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that Generative AI was not used in the creation of this manuscript.

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