Insights into mobile genetic elements and the role of conjugative plasmid in transferring aminoglycoside resistance in extensively drug-resistant Acinetobacter baumannii AB329

Bacterial strains and antibiotic susceptibility testing (AST)

A. baumannii AB329, which is a phage-susceptible XDRAB strain, was isolated from patient sputum obtained from our previous study (Leungtongkam et al., 2018a). This strain is representative of the outbreak clone obtained from a hospital in east Thailand, and it was collected from November 2013 to February 2015 (Leungtongkam et al., 2018a). Antimicrobial susceptibility testing (AST) was performed as previously described (Kongthai et al., 2021). The AST results were interpreted according to the Clinical Laboratory Standard Institute (CLSI, 2020). The protocol was approved by the Naresuan University Institutional Biosafety Committee, and the project number was NUIBC MI 63-07-21.

Polymerase chain reaction (PCR)-based replicon typing method

The AB-PBRT method was used to detect plasmid groups (GRs) with primers specific to each GR from GR1 to GR19, which were described by a previous study (Bertini et al., 2010).

DNA extraction, genome sequencing and assembly

Genomic DNA of A. baumannii AB329 was extracted using the Real Genomics DNA Extraction Kit (RBC Biosciences, Taiwan), and it was quantified using a Qubit^® DNA Assay Kit in a Qubit^® 2.0 Fluorometer (Life Technologies, CA, USA) prior to sequencing with both short-read (Illumina paired-end) and long-read (PacBio, Menlo Park, CA, USA) sequencing systems. For short-read sequencing, paired-end sequencing libraries (2 × 250 bp) were constructed using the Nextera XT sample preparation kit following the manufacturer’s suggestions, and they were sequenced using the Illumina MiSeq platform. The Illumina reads were trimmed with Sickle v1.33 using the default parameters (Joshi & Fass, 2011). For long-read sequencing, a large insert library (10 kb) was constructed and sequenced on the PacBio RS platform (Pacific Biosciences, Menlo Park, CA, USA). A hybrid assembly was conducted with the Illumina trimmed reads and PacBio reads using Unicycler v0.4.8.0 with the default settings (Wick et al., 2017). Unicycler automatically identified and trimmed the overlapping ends, and circular sequences were rotated to dnaA.

Genome annotation and bioinformatic analysis

The assembled circular chromosome and plasmids were functionally annotated using Prokka v1.12 with the default options. MLST types were determined in silico using the MLST database (https://pubmlst.org/organisms/acinetobacter-baumannii). The complete genomes of 292 A. baumannii strains retrieved from the NCBI database in December 2021 were used to identify the core genome (Table S1). A single-nucleotide polymorphism (SNPs) phylogenetic tree of the core genome was reconstructed using CSI Phylogeny with the default settings (Kaas et al., 2014). This tree was visualized and edited using Interactive Tree of Life (iTOL) (https://itol.embl.de/). A pangenomic analysis was executed using Roary v.3.13.0, which compared the five closest related genomes, including A. baumannii NIPH17_00019 (AP024415.1), A. baumannii XH856 (CP014541.1), A. baumannii KAB02 (CP017644.1), A. baumannii KAB06 (CP017652.1) and A. baumannii KAB05 (CP017650.1). Then, the output was illustrated using R studio as described in https://github.com/IamIamI/pADAP_project/tree/master/Roary_stats. The average nucleotide identity (ANI) was calculated using FastANI v.1.3 to estimate the whole-genome similarity among the two XDRAB strains, which were obtained from hospitalized patients in Thailand (Kongthai et al., 2021), and the five closest related genomes identified from the pan-genome analysis. Antimicrobial resistance and virulence genes were retrieved using the Comprehensive Antibiotic Resistance Database (CARD) and VFanalyzer (Liu et al., 2019), respectively. The large-scale BLAST score ratio (LS-BSR) pipeline was utilized to compare the virulence and drug resistance genes with 292 A. baumannii genomes (Table S1). A BSR value of 0.4 and above was interpreted as the presence of genes, and a BSR value below 0.4 was inferred as gene absence (Sahl et al., 2014; Yakkala et al., 2019). Then, these BSR values were used to build a hierarchical clustering heatmap using the R packages pheatmap and tidyverse. MGEs were detected using MobileElementFinder (Johansson et al., 2021). The presence of prophage sequences in the genome of A. baumannii AB329 was analyzed using the PHAge Search Tool Enhanced Release (PHASTER) online server (Arndt et al., 2016). Prophage open reading frames (ORFs) were examined as described in a previous study (Fu et al., 2017). The prediction of genomic islands (GIs) and AbaR was conducted by running BLASTn. A set of nucleotide queries of GIs identified in A. baumannii ACICU was used for BLASTn searching (Di Nocera et al., 2011; Thummeepak et al., 2020). Plasmid comparison and the identification of the AbaR structure were accomplished using Easyfig version 2.1 (Sullivan, Petty & Beatson, 2011). The complete genome was deposited in the NCBI GenBank database under the accession numbers CP091452 (chromosome), CP091453 (pAB329a), and CP091454 (pAB329b).

Conjugation experiment

A broth-mating conjugation assay was performed according to a previously published protocol with minor modifications (Leungtongkam et al., 2018b). Overnight cultures of the donor (A. baumannii AB329) and the sodium azide resistance recipient (A. baumannii NU13R) were adjusted in 0.85% NaCl until the cell suspensions reached a turbidity equal to a McFarland value of 0.5, which was measured using a densitometer (SiaBiosan, Riga, Latvia). Equal volumes (250 µl) of adjusted cell suspensions of the donor and the recipient were mixed in 500 µl of 2 × Luria-Bertani (LB) broth and incubated for 4 h at 37 °C. Transconjugants were selected on three LB agar plates containing 250 µg/ml sodium azide (negative control), 20 µg/ml kanamycin (negative control), and 250 µg/ml sodium azide plus 20 µg/ml kanamycin.

The conjugation frequency (CF) was calculated as previously described (Leungtongkam et al., 2018b). PCR to detect plasmid groups (Bertini et al., 2010), aminoglycoside resistance genes (Kongthai et al., 2021) and traU genes (Kongthai et al., 2021) was performed using cell lysates from donor (AB329), recipient (NU13R), and transconjugants (NU13R-pAB329b) as templates.

Antibiotic susceptibility testing (AST)

Antibiotic susceptibility testing of A. baumannii AB329 using the disk diffusion method revealed that it was resistant to imipenem, meropenem, amikacin, ciprofloxacin, gentamicin, trimethoprim/sulfamethoxazole, cefoperazone/sulbactam, colistin, and tigecycline (Table S2). The minimum inhibitory concentrations (MICs) of imipenem, colistin, and tigecycline were 32, 1 and 0.25 ug/mL, respectively (Table S2).

Complete genome sequence of A. baumannii AB329

The complete genome sequence of A. baumannii AB329 generated by short- and long-read sequencing revealed a circular chromosome 3,948,038 bp in length with 39% GC content, and it contained two plasmids (pAB329a and pAB329b) (Table 1). pAB329a is a small circular plasmid of 8,731 bp, and pAB329b is a megaplasmid of 82,120 bp. The Prokka prokaryotic genome annotation system identified 18 rRNAs, 72 tRNAs, 3,837 ORFs, and a total of 3,747 protein-coding genes on the main chromosome of AB329 (Table S3). Genes for tRNAs and rRNAs were detected only in the chromosome (Table 1). pAB329a contained 12 ORFs, while pAB329b contained 113 ORFs. AB329 was assigned to MLST type 1166/98 (Oxford/Pasteur) and was found to belong to the IC2 lineage.

Phylogenomic and comparative genomic analyses of A. baumannii AB329

Phylogenomic analysis was performed using the core genome of A. baumannii AB329 and 292 additional A. baumannii strains deposited in the NCBI database. As shown in Fig. 1A, phylogenetic analysis of A. baumannii AB329 presented in the same cluster with the A. baumannii strains NIPH17_00019 (AP024415.1), XH856 (CP014541.1), KAB02 (CP017644.1), KAB06 (CP017652.1), and KAB05 (CP017650.1) (Fig. 1B and Table S4). We also compared the genome of A. baumannii AB329 with two XDRAB isolates from two different hospitals in Thailand as previously described (Kongthai et al., 2021). The ANI (%) values of A. baumannii AB329 with A. baumannii AB140 and A. baumannii AB053 were 99.72% and 99.27%, respectively (Table S4). A pangenome of A. baumannii AB329 consisting of 4,628 genes represented the core, shell, and cloud genomes (Fig. 1B). The core genome represented a pool of conserved genes that were present in all genomes and included 3,238 genes. The accessory genes, which included shell genes (genes present in two or more strains) and cloud genes (genes only found in a single strain), constituted a total of 1,390 genes.

Virulence genes, antibiotic resistance genes, and mobile genetic elements of A. baumannii AB329

Analysis of the virulence genes of A. baumannii AB329 revealed that genes were involved in iron uptake (hemO, barA, barB, basA, basB, basC, basD, basF, basG, bash, basI, basJ, bauA, bauB, bauC, bauD, bauE, bauF, entE), serum resistance (pbpG), stress adaptation (katG, katE), gene regulation (bfmR, bfmS), immune evasion (lpsB, lpxA, lpxB, lpxC, lpxD, lpxL, lpxM), enzyme phospholipase (plcC, plcD), biofilm formation (adeF, adeG, adeH, csuA, csuB, csuC, csuD, csuE, pgaA, pgaB, pgaC, pgaD), and host cell adherence (ompA) (Fig. 2A). Resistome analysis detected a number of resistance mechanisms in the chromosome, including beta-lactam-inactivating enzymes, aminoglycoside-modifying enzymes, efflux pumps, permeability defects, and target site modifications. As shown in Fig. 2B, genes conferring drug resistance, including beta-lactam resistance (bla_OXA-51, bla_ADC-25, bla_OXA-23, bla_TEM−1D), aminoglycoside resistance (aph(3′)-Ia, aph (3″)-Ib, aph (6)-Id, arm A), tetracycline resistance (tet (B), tet (R)), and macrolide resistance (mph (E), msr (E)), were detected on the chromosome. Genes encoding resistance-nodulation-cell division (RND) (Ade ABC, Ade IJK, and Ade FGH), multidrug and toxic efflux (MATE) (Abe M), major facilitator superfamily (MFS) (tet (A) and tet (B)), and small multidrug resistance (SMR) efflux systems (Abe S) were found. In silico analysis of the pattern of ARGs was conducted, and the 292 selected A. baumannii genomes worldwide were grouped into three clusters (A, B, and C) (Fig. 2B). We found that A. baumannii AB329 was grouped into Cluster C and was closely related to A. baumannii VB2181 (CP050401.1) and AC29 (CP007535.2), which were isolated from India and Malaysia. The MGEs of A. baumannii AB329 included two plasmids, three prophages, 19 GIs, and 33 ISs. The two plasmids were pAB329a and pAB329b. pAB329a is a small circular plasmid of 8,731 bp, and pAB329b is a megaplasmid of 82,120 bp. pAB329b carries conjugation-gene clusters required for autonomous conjugative transfer, which involves F pilus (traD, traE, traK, traB, traV, traC, traW, traN, traF, traH, traG, traW), plasmid replication (repA), and a recombinase (recD2). A few hypothetical proteins and one copy of the aminoglycoside resistance gene aph (3′)-VIa were detected in pAB329b (Fig. 3A). The genomic features of plasmid pAB329b aligned with 11 closely related plasmids deposited in the GenBank database are represented in Fig. 3A. pAB329a was closely related to plasmid p2AB5075 (CP008708.1) in lineage_2(LN_2), and pAB329b was similar to pACICU2 (NC_010606) in LN-1 (Table S5). Compared to pACICU2, we found that both plasmids had the same backbone regions; however, pAB329b harbored the aph(3′)- VIa gene, while it the ARG was absent from pACICU2 (Fig. 3B). We found three prophages, one intact and two incomplete, in the genome of A. baumannii AB329. Our bioinformatic analysis revealed that the intact prophage sequence contained 69 ORFs involved in DNA processing, drug resistance, host lysis, integrase, metabolism process, and phage proteins (Fig. 4A). Interestingly, the MFS transporter was detected in the genome of this prophage, which was homologous to Acinetobacter phage YMC11/11/R3177 (KP861230.1), with 57.98% ANI. The genome of A. baumannii AB329 was examined for GIs, and 19 were identified (Table S6). In addition, one resistance island, AbaR4, which harbored tetracycline and aminoglycoside resistance genes, was detected in the genome of A. baumannii AB329 (Fig. 4B). Transposable elements such as transposons and ISs were investigated, and 34 ISs were detected in the chromosome, except for ISAba 1, which was detected in the chromosome and the plasmid (Table 1). ISAba125 was detected only in the plasmid, and it bracketed the aph (3′)-VIa gene (Fig. 3A). The ARGs in the chromosome located near the ISs were bla_OXA-23 (ISAba1), bla_TEM (ISAba33), and aph(3′)-VIa ((IS6).

Conjugative transfer of the aminoglycoside resistance gene of plasmid pAB329b

To investigate the role of pAB329b in the transfer of ARG, we performed a conjugation assay to study the plasmid’s ability to transfer the aminoglycoside resistance gene to sodium azide-resistant A. baumannii NU13R (recipient). As shown in Table 2, aminoglycoside resistance could be transferred from the donors (A. baumannii AB329) to the recipient (A. baumannii NU13R). The conjugation frequency was approximately 1.2 × 10⁻⁷. The resistance genes, plasmid typing results, and antibiotic susceptibility of the donors, the recipient, and the transconjugants are shown in Table 2. The transconjugant MIC of kanamycin was > 64 µg/ml, which is approximately 32-fold higher than the MIC of kanamycin on the recipient strains. PCR amplification of aminoglycoside resistance genes (aph(3′)- VIa) and traU revealed their presence only in donors and transconjugants (Table 2). The plasmid GR6 was detected in the transconjugant NU13R-pAB329b.