Extensively drug-resistant Acinetobacter baumannii: role of conjugative plasmids in transferring resistance

Bacterial Isolates and A. baumannii Identification

The five XDR A. baumannii clinical isolates included in the study were recovered from patients admitted to Jordanian hospitals in 2018. Two of these isolates were recovered from patients admitted to the ICUs; one isolated (ICU29) from sputum samples of an 84-year-old female, while the other (ICU40) was isolated from the cerebrospinal fluid sample of a 5-week-old male baby. The other three non-ICU isolates were recovered from a sputum sample of a 65-year-old male (Ab19), a urine sample of a 16-year male (Ab40), and a sputum sample of a 63-year female (Ab119). The isolates were identified biochemically using the VITEK©2. Molecular identification of A. baumannii was carried out by PCR amplification of internal fragments of the bla_OXA−51 gene (ying et al., 2015). This study was approved by the Jordanian Ministry of Health and the Institutional Review Board under the number MOH REC 180030.

Antibiotic susceptibility testing and minimal inhibitory concentration (MIC)

The antibiotic susceptibility testing was performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines following the method described previously (CLSI-2021) (Hudzicki, 2009). The antibiotics used in this study and their concentrations are listed in Table S1. MICs were determined for tigecycline, colistin, imipenem, and polymyxin B (SPL Life Science) drugs using the 96-well microdilution method (Wieg, Hilpert & Hancock, 2008). Polysorbate was added for colistin sulfate and polymyxin B to improve the delivery of antibiotics at a concentration of 0.002% in each well (Sader et al., 2012).

Next-generation sequencing and assembly

Whole-genome sequencing of the five isolates was done using two different sequencing platforms; isolate Ab119 was sequenced using Illumina and PacBio platforms. The other four isolates were sequenced using the Illumina platform only. DNA was extracted using Wizard genomic DNA purification kit (Promega, USA), and library preparation was done using the TruSeq Nano DNA Kit (Illumina) following the manufacturer’s instructions. The obtained 150bp short paired-end and long reads were assembled by SPAdes (version 3.14.0), and the genome of A. baumannii ATCC 19606 (NZ_CP045110) was used as a reference for the assembly (Bankevich et al., 2012). The quality of assembly was tested using the Sequencing Quality Assessment Tool (SQUAT) with the default settings for both (Yang et al., 2019).

Annotation, sequence typing, and phylogenetic classification

Assembled contigs were annotated using Prokka and the Rapid Annotation using Subsystem Technology (RAST) servers (Seemann, 2014; Aziz et al., 2008). The sequence types (ST) were determined as described by PubMLST (https://pubmlst.org) using the locus schemes Oxford and Pasteur separately and then order results by locus (Jolley, Bray & Maiden, 2018). Phylogenetic analysis was performed using RAxML-VI-HPC (randomized accelerated maximum likelihood for high-performance computing) tool provided by EDGE Bioinformatics (https://edgebioinformatics.org) to compare the five isolates against the A. baumannii database, and the Average Nucleotide Identity (ANI) was calculated as described previously (Philipson et al., 2017; Stamatakis, 2006; Goris et al., 2007).

Detection of antimicrobial resistance and virulence genes

Antimicrobial resistance genes were detected using ResFinder and Resistance Gene Identifier (CARD RGI) (https://card.mcmaster.ca). In the CARD RGI, only perfect and strict hits were chosen, while loose hits with less than 95% identity were excluded (Zankari et al., 2012; Alcock et al., 2020). Virulence genes were detected using the virulence factor database (VFDB) through the EDGE bioinformatics platform (Philipson et al., 2017; Chen et al., 2005).

Plasmid identification

In silico identification of plasmids was initially done using plasmidSPAdes and then confirmed using BlastN at NCBI (Antipov et al., 2016). PlasmidSeeker subsequently analyzed confirmed plasmid contigs to find the closest reference plasmid (Roosaare et al., 2018). RAST annotated the verified plasmids, and manual annotation for the replication (rep) genes was based on Bertini classification (Bertini et al., 2010).

Conjugation assay

Azide-resistance induction in A. baumannii recipient strains was performed as described previously (Leungtongkam et al., 2018). The donor and recipient cells were mixed at a 1:3 ratio in Luria-Bertini broth and incubated at 37 °C for 4 h. The selection was made on Mueller Hinton Agar supplemented with 300 µg/ml sodium azide and the appropriate antibiotic. Transconjugants were collected and tested against several antibiotics to detect the transfer of resistance genes by conjugation (Leungtongkam et al., 2018).

Plasmid curing

The curing agent acridine orange was used for plasmid curing as described previously (Saranathan et al., 2014). The curing broth was prepared by diluting acridine orange to the subinhibitory concentration using LB broth to a final volume of 10 ml. The tested isolates were inoculated into the curing broth, incubated for 48 h, and plated on LB agar. The antibiotic susceptibility testing was done for the isolates before and after curing to compare their antibiotic resistance profiles (Trevors, 1986).

Nucleotide sequences accession number

The complete sequence of the five isolates are deposited under Bio project number PRJNA739752. The five assembled genomes Ab40, Ab19, Ab119, ICU40, and ICU29 are assigned accession numbers GCA_019891135.1, GCA_019891055.1, GCA_019891085.1, GCA_019891095.1, and GCA_019891065.1, respectively.

Antimicrobial resistance

The five A. baumannii isolates investigated in this study were classified as XDR based on their antimicrobial resistance profiles (Table 1). Four isolates exhibited resistance against 18 out of the 21 tested antibiotics, but all were sensitive to tigecycline, polymyxin B, and colistin sulphate. Isolate Ab40 was susceptibility to trimethoprim-sulfamethoxazole and tobramycin and showed intermediate susceptibly to gentamicin. Additionally, isolate ICU40 showed intermediate susceptibility to trimethoprim-sulfamethoxazole.

Whole genome sequencing and sequence types

The reference-based assembly of the five Illumina sequenced isolates yielded an average length of 4,520,996 bp and an average G+C content of 38.76%. The number of detected open reading frames (ORFs) ranged between 3,754 and 4,324, with half of the ORFs coding for hypothetical proteins. Details of the WGSs results are listed in Table S2. Multiple locus sequence typing (MLST) showed that isolate Ab119 has a new ST according to the Oxford scheme and was not identified by the Pasteur scheme (Table 2). Details of MLST analysis can be found in Tables S3 and S4.

Antimicrobial resistance determinants

The isolates harbored an average of 27 different genes and gene mutations conferring resistance to antimicrobial agents through various mechanisms (Table 3). Isolate ICU40 contained the highest number of resistance determinants, whereas isolate Ab119 had the least. Genes that encode the resistance-nodulation-cell division (RND) efflux pumps were detected in all isolates. Tetracycline resistance MFS efflux pumps were less frequent; tetR and tetB were only found in isolates Ab19, ICU29, and ICU40. On the other hand, tet39 was only found in isolates ICU40 and Ab40. Two types of bla_OXA genes, bla_OXA−66 and bla_OXA−23, were found in most isolates, while bla_OXA−91 was only found in Ab40. These genes encode OXA-type beta-lactamases, conferring resistance to carbapenems, cephalosporins, and penams. Other carbapenem resistance genes detected include bla_ADC−30, bla_ADC−25, bla_ADC−81, and bla_ADC−73. The bla_NDM−1 gene encoding NDM-1 (zinc Metallo- β-lactamase “MBL”) was found in ICU40. In addition, the Extended-Spectrum β-Lactamase (ESBL) gene bla_TEM−1 was detected in three isolates. Aminoglycoside resistance genes were found in all isolates, with ant(2′)Ia, aph(3′)VI, and aph(3′)VIa being plasmid-borne. Two mutations in the fluoroquinolone-resistance determining regions (QRDRs) of gyrA and parC genes were detected in all isolates. Isolate Ab19 carries the disinfectant-resistance qacE/qacE Δ1 gene.

Virulence factors

An average of 60 virulence genes was detected in each isolate using the virulence factor database (VFDB). Those genes are involved in surface adherence, biofilm formation, virulence enzymes, immune evasion, iron uptake, quorum sensing, and serum resistance. Table 4 lists the virulence factors that were detected and their classification according to mechanisms of action.

Characterization of plasmids

The computational tools detected eight plasmids; four share the same structure with some differences. The ANI between these four plasmids ranged between 99.95% and 99.76%, and thus were named pAb19.1v(1-4). Additionally, two plasmids in both ICU isolates were found identical. Out of the eight plasmids, isolate Ab19 carried 3, ICU40 had 2, and ICU29 and Ab119 each carried one plasmid (Table 5). Replication genes were identified in six of the plasmids; four “pAb19.1v(1-4)” belong to Group 6 (GR6) of A. baumannii plasmids and carry genes for conjugation. In contrast, pAb-ICU29 found in both ICU isolates is a small cryptic plasmid (∼9 kb) that belonged to GR2 and didn’t carry any conjugation or resistance genes. Three resistance genes were found to be plasmid-borne; sul2, aph(3′)-VIa, and ant(2′)-Ia.

pAb19.1v1 (70,616 bp) is a large plasmid that carries the repAci6 replication gene and belongs to GR6 of A. baumannii plasmids. This plasmid carried 18 conjugation genes and is highly similar to the 70,100 bp plasmid pAb-G7-2 (accession number KF669606). pAb19.1v1 has two regions of conjugation genes; type IV secretion system tra genes and the other region harbors the trwB and trwC mobilization genes (Fig. 1). It also carries aminoglycoside resistance transposon TnaphA6, which consists of the aminoglycoside resistance gene aph(3′)-Via (aphA6) surrounded by two ISAba125. Plasmids very similar to pAb19.1v1 were detected in isolates Ab40, ICU40, and Ab119 (Fig. 2). Plasmid pAb19.1v2 (68,684 bp) from isolate Ab40 does not carry the resistance transposon TnaphA6, while pAb19.1v4 carries the transposon and is almost identical to pAb19.1v1 with a length of 70,183 bp. Although pAb19.1v3 is only 59,004 bp and does not have the second region of conjugative genes trwB and trwC, it does carry the aminoglycoside resistance transposon TnaphA6.

Plasmid pAb19.2 (32,796 bp) is harbored by Ab19 and seems a novel plasmid. Part of this plasmid is similar to plasmid pORAB01-2 (24,022 bp) from A. baumannii strain ORAB01 (accession number CP015485). The blast of pORAB01-2 as query and pAb19.2 as subject resulted in 99.86% percentage identity and 86% query coverage. Both plasmids carry the E. coli-generated sulfonamide resistance gene sul2 surrounded by the putative transposases TniA and TniB, and other transposases. Plasmid pAb19.2 also has two site-specific tyrosine recombinase XerC and six transposases, four of which are TniA putative transposase (Fig. 3A).

Plasmid pAb19.3 (6155 bp) harbors the aadB gene cassette, which confers resistance to tobramycin, gentamicin, and kanamycin. The plasmid also contains mobA and mobC mobilization genes. pAb19.3 is highly similar (97.84%) to pRAY* (AF003958) (Fig. 3B). Plasmid pAb-ICU29 (8,858 bp) found in ICU29 and ICU40 isolates is almost identical to pAC29a (CP008850). However, pAC29a (8,737 bp) has two rep genes, repA, and repB, while pAb-ICU29 carries only the repB gene (Lean et al., 2016). pAb-ICU29 belongs to GR2 as it carries RepB replication protein. AbkB/AbkA toxin-antitoxin (TA), Sel1 gene flanked by XerC/XerD recombination sites, and TonB receptor genes are encoded by both pAC29a and pAb-ICU29 (Fig. 3C).

Induction of azide-resistant Acinetobacter baumannii and conjugation

Of the two environmental A. baumannii isolates susceptible to all tested antibiotics, one isolate (Pk1) was able to grow in the presence of 300 µg/ml sodium azide and thus was used for conjugation assay. Out of the five isolates investigated in this study, transconjugants were generated from three donors only (Ab19, Ab40, and Ab119). After conjugation, transconjugants acquired resistance against amikacin, gentamicin, cefepime, and tetracycline. Also, transconjugants became less sensitive to other antibiotics even though the phenotype was not changed into resistance (Table 6).

Plasmid curing

The MIC of acridine orange differed between the five isolates, as Ab40, Ab119 were not able to grow on the minimum concentration (640 µg/ml) required for plasmid curing. Thus, the other three isolates (Ab19, ICU29, and ICU40) were cured successfully at 1,280 µg/ml sub-inhibitory concentration of acridine orange (Table 7). Isolate ICU40 did not show any change in the resistance profile, while Ab19 and ICU29 showed less resistance to a number of antibiotics. Still, the difference between the cured and non-cured cells did not change the resistance phenotype classification from resistant to sensitive for any of the tested antibiotics. Cured Ab19 was less resistant to amikacin, gentamicin, and meropenem, and its resistance to tobramycin became intermediate.

Phylogenetic and comparative analysis

Phylogenetic analysis was performed to examine the evolutionary relations between the five isolates (Fig. 4A) and other global A. baumannii strains (Fig. 4B). Isolates ICU29 and ICU40 were highly similar, with an ANI of 99.9%, belonged to the same sequence type of the Pasteur scheme, and contained the same plasmid (pAb-ICU29). The two non-ICU isolates, Ab119 and Ab19, showed considerable similarity (ANI: 99.21%). Also, Ab119, Ab19, and ICU40 have a shared plasmid (pAb19.1). Ab40 was the most distant on the phylogenetic tree compared to the other four isolates; ANI between Ab40 and ICU40 was 98.39%.