Here, we report the complete genome sequences of four clinical isolates of extensively drug-resistant Acinetobacter baumannii (XDRAB), isolated in Thailand. These results revealed multiple antimicrobial-resistant genes, each involving two sequence type 16 (ST16) isolates, ST2, and a novel sequence type isolate, ST1479.
ABSTRACT
Here, we report the complete genome sequences of four clinical isolates of extensively drug-resistant Acinetobacter baumannii (XDRAB), isolated in Thailand. These results revealed multiple antimicrobial-resistant genes, each involving two sequence type 16 (ST16) isolates, ST2, and a novel sequence type isolate, ST1479.
ANNOUNCEMENT
Acinetobacter baumannii is one of the multidrug-resistant bacteria listed as a priority by the World Health Organization, as it exhibits resistance to most commercially available antibiotics and causes hospital-acquired infections (1–3). In particular, carbapenem-resistant Acinetobacter baumannii (CRAB) infections present limited therapeutic options and are associated with high morbidity and mortality as well as longer hospitalization (3). Recently, incidences of extensively drug-resistant A. baumannii (XDRAB) infections have been reported worldwide, including in Thailand (4, 5). Four strains were isolated from individual sputum (n = 3) and bile (n = 1) samples by a tertiary hospital in northeastern Thailand between 2017 and 2018 (6). All isolates were cultured on sheep blood agar for DNA extraction. They were identified to the species level using gyrB multiplex PCR (7). These strains were resistant to 11 representative antimicrobial agents of all drug classes (piperacillin, piperacillin-tazobactam, ceftazidime, cefepime, imipenem, meropenem, ciprofloxacin, gentamicin, amikacin, trimethoprim-sulfamethoxazole, and tetracycline), whereas they had intermediate resistance to colistin, according to the Clinical and Laboratory Standards Institute (CLSI, 2020) guidelines (8). The four XDRAB strains were grouped into international clone II (n = 2) and no clonal group (n = 2) based on a multiplex PCR assay (9).
Bacterial genomic DNA samples extracted using ZymoBIOMICS DNA kits (Zymo Research, USA) were sequenced using the Oxford Nanopore Technologies (ONT) and Illumina platforms. Library preparation for ONT sequencing followed the rapid barcoding DNA sequencing protocol with the SQK-RBK004 kit without DNA size selection, to preserve the plasmid DNA, and the libraries were sequenced using a single R9.4.1/FLO-MIN106 flow cell on a MinION Mk1B sequencer. We base called and demultiplexed the raw data using Guppy v3.4.5 (ONT), specifying the high-accuracy model (-c dna_r9.4.1_450bps_hac.cfg). The ONT adapters were trimmed using Porechop v0.2.4 (https://github.com/rrwick/Porechop). Quality control of ONT reads was undertaken using NanoPlot v1.28.1 (https://github.com/wdecoster/NanoPlot). For the Illumina platform, the sequencing library was generated using the NEBNext Ultra II DNA library prep kit for Illumina (New England Biolabs, USA) following the manufacturer’s recommendations. We applied Fastp v0.19.5 (10) for quality filtering of the Illumina reads. Adapters were trimmed using Skewer v0.2.2 (11) (https://github.com/relipmoc/skewer). Quality checking of the Illumina reads was performed using FastQC v0.11.8 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Hybrid assemblies with the ONT and Illumina data were performed using Unicycler v0.4.8 (12) (https://github.com/rrwick/Unicycler/releases), and the genome sequences were checked for quality using QUAST v5.0.2 (13) (http://quast.sourceforge.net/). Complete circular DNA structures of the bacterial chromosomes and plasmids were automatically produced using Unicycler software. The genome sequences were submitted to the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v4.12 for annotation. Default parameters were used for all software unless otherwise specified.
All isolates contained plasmids, and we detected seven plasmids in one isolate (Table 1). From the whole-genome sequencing, the following antimicrobial resistance determinants were ascertained using ResFinder (14) (https://cge.cbs.dtu.dk/services/ResFinder/) and CARD (15) (https://card.mcmaster.ca/): blaNDM-1, blaOXA-23, blaOXA-66, blaTEM1D, blaVEB-1, blaIMP-14, blaADC-25, sul1, sul2, cmlA1, mphE, msrE, ARR2, tetB, tet(39), aac(3)-IId, aadA1, ant(2″)-Ia, aph(3′)-Ia, aph(3″)-Ib, aph(3′)-VI, aph(6)-Id, and armA. The multilocus sequence type (MLST) findings using the PubMLST database indicated that two isolates were ST16 (or ST355), and the other isolates were ST2 (or ST195) and ST1479 (new sequence type), according to the Pasteur MLST scheme (with the Oxford MLST scheme in parentheses) (16, 17) (https://pubmlst.org/abaumannii/).
TABLE 1.
Assembly metrics and accession numbers of four clinically extensively drug-resistant Acinetobacter baumannii strains
| Strain | Sequence type | Total no. of bases (millions) | No. of reads (thousands) | N50 (bp) | BioSample accession no. | SRA accession no. for: |
GenBank accession no. | Total no. of contigs | Type of contig | Total length (bp) | GC content (%) | No. of ORFsa | No. of rRNAs | No. of tRNAs | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ONT reads | Illumina reads | ||||||||||||||
| A217 | ST16 | 1,194 | 173 | 12,710 | SAMN15596636 | SRR12517409 | SRR12517413 | JACEIH000000000 | 5 | Chromosome | 3,897,448 | 39.05 | 3,606 | 18 | 75 |
| Plasmid | 118,657 | 41.06 | 127 | 0 | 1 | ||||||||||
| Plasmid | 78,040 | 43.88 | 82 | 0 | 0 | ||||||||||
| 1,266 | 8,400 | 150 | |||||||||||||
| Plasmid | 43,239 | 38.81 | 62 | 0 | 0 | ||||||||||
| Plasmid | 12,366 | 34.46 | 14 | 0 | 0 | ||||||||||
| A466 | ST16 | 1,440 | 248 | 10,073 | SAMN15596638 | SRR12517407 | SRR12517411 | JACEIF000000000 | 2 | Chromosome | 3,944,195 | 39.02 | 3,683 | 18 | 74 |
| 1,208 | 8,100 | 150 | Plasmid | 8,731 | 34.37 | 10 | 0 | 0 | |||||||
| A522 | ST2 | 1,481 | 222 | 12,765 | SAMN15596639 | SRR12517406 | SRR12517410 | JACEIJ000000000 | 4 | Chromosome | 3,897,674 | 39.05 | 3,621 | 18 | 75 |
| Plasmid | 118,652 | 41.05 | 127 | 0 | 1 | ||||||||||
| Plasmid | 66,679 | 43.99 | 69 | 0 | 0 | ||||||||||
| 684 | 2,800 | 150 | |||||||||||||
| Plasmid | 12,366 | 34.46 | 14 | 0 | 0 | ||||||||||
| A423 | ST1479 | 1,407 | 256 | 10,240 | SAMN15596637 | SRR12517408 | SRR12517412 | JACEIG000000000 | 8 | Chromosome | 3,806,348 | 39.00 | 3,517 | 18 | 75 |
| Plasmid | 108,874 | 43.13 | 116 | 0 | 0 | ||||||||||
| Plasmid | 95,364 | 41.49 | 104 | 0 | 1 | ||||||||||
| Plasmid | 6,078 | 39.17 | 5 | 0 | 0 | ||||||||||
| Plasmid | 4,554 | 41.26 | 4 | 0 | 0 | ||||||||||
| 1,310 | 8,700 | 150 | |||||||||||||
| Plasmid | 2,924 | 37.82 | 4 | 0 | 0 | ||||||||||
| Plasmid | 2,816 | 38.74 | 4 | 0 | 0 | ||||||||||
| Plasmid | 2,309 | 38.54 | 2 | 0 | 0 | ||||||||||
ORFs, open reading frames.
Data availability.
These sequences have been submitted to GenBank under BioProject accession number PRJNA647677. The accession numbers and assembly statistics are provided in Table 1.
ACKNOWLEDGMENTS
The Kasetsart University Research and Development Institute (KURDI), Bangkok, Thailand, provided a grant and English-editing assistance.
This study was reviewed and approved by the Human Research Ethics Committee of Roi Et Hospital (ERB). The ERB waived the requirement for informed consent as the study satisfied the conditions of the policy statement on ethical conduct for research involving humans. This study was conducted according to the principles of the Declaration of Helsinki.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
These sequences have been submitted to GenBank under BioProject accession number PRJNA647677. The accession numbers and assembly statistics are provided in Table 1.