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Microbiol Spectr
Microbiol Spectr
spectrum
Microbiology Spectrum
2165-0497
American Society for Microbiology 1752 N St., N.W., Washington, DC

39101706
spectrum00501-24
10.1128/spectrum.00501-24
spectrum.00501-24
Research Article
clinical-microbiologyClinical MicrobiologyWhole genome sequencing insight into carbapenem-resistant and multidrug-resistant Acinetobacter baumannii harboring chromosome-borne blaOXA-23
Wang Wei 1 2 Methodology Software Writing – original draft
Weng Jiahui 3 Methodology Writing – original draft
Wei Jie 4 Methodology Software Writing – original draft
Zhang Qinghuan 4 Formal analysis Methodology
Zhou Yu 5 Resources
He Yanju 1 Investigation Methodology
Zhang Limei 6 Data curation Formal analysis
Li Wenting 6 Formal analysis Resources
https://orcid.org/0000-0002-9905-7888
Zhang Yi 1 Formal analysis Visualization Writing – review and editing 386947136@qq.com

https://orcid.org/0000-0001-5327-8901
Zhang Zhiren 6 Supervision Writing – review and editing 610225801@qq.com

https://orcid.org/0000-0002-8645-8332
Li Xiaobin 6 Conceptualization Funding acquisition Methodology Supervision Writing – original draft Writing – review and editing xiaobinli@alumni.sjtu.edu.cn

1 Department of Pulmonary and Critical Care Medicine, Zhuhai People’s Hospital (Zhuhai Clinical Medical College of Jinan University) , Zhuhai, China
2 Department of Critical Care Medicine, Zhuhai People’s Hospital (Zhuhai Clinical Medical College of Jinan University) , Zhuhai, China
3 School of Basic Medical Sciences, Guangzhou University of Chinese Medicine , Guangzhou, China
4 Department of Clinical Laboratory, Zhuhai People’s Hospital (Zhuhai Clinical Medical College of Jinan University) , Zhuhai, China
5 Department of Anesthesiology, Zhuhai People’s Hospital (Zhuhai Clinical Medical College of Jinan University) , Zhuhai, China
6 Guangdong Provincial Key Laboratory of Tumor Interventional Diagnosis and Treatment, Zhuhai People’s Hospital (Zhuhai Clinical Medical College of Jinan University) , Zhuhai, China
Editor Chow Siu-Kei MultiCare Health System , Tacoma, Washington, USA

Address correspondence to Xiaobin Li, xiaobinli@alumni.sjtu.edu.cn
Address correspondence to Zhiren Zhang, 610225801@qq.com
Address correspondence to Yi Zhang, 386947136@qq.com
Wei Wang, Jiahui Weng, and Jie Wei contributed equally to this article. The author order was determined by drawing straws.

The authors declare no conflict of interest.

9 2024
05 8 2024
05 8 2024
12 9 e00501-2423 2 2024
26 6 2024
Copyright © 2024 Wang et al.
2024
Wang et al.
https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

ABSTRACT

Carbapenem-resistant Acinetobacter baumannii (CRAB) poses a significant threat to hospitalized patients as effective therapeutic options are scarce. Based on the genomic characteristics of the CRAB strain AB2877 harboring chromosome-borne blaOXA-23, which was isolated from the bronchoalveolar lavage fluid (BALF) of a patient in a respiratory intensive care unit (RICU), we systematically analyzed antibiotic resistance genes (ARGs) and the genetic context associated with ARGs carried by CRAB strains harboring chromosome-borne blaOXA-23 worldwide. Besides blaOXA-23, other ARGs were detected on the chromosome of the CRAB strain AB2877 belonging to ST208/1806 (Oxford MLST scheme). Several key genetic contexts associated with the ARGs were identified on the chromosome of the CRAB strain AB2877, including (1) the MDR region associated with blaOXA-23, tet(B)-tetR(B), aph(3'')-Ib, and aph(6)-Id (2); the resistance island AbGRI3 harboring armA and mph(E)-msr(E) (3); the Tn3-like composite transposon containing blaTEM-1D and aph(3')-Ia; and (4) the structure “ISAba1-blaADC-25.” The first two genetic contexts were most common in ST195/1816, followed by ST208/1806. The last two genetic contexts were found most frequently in ST208/1806, followed by ST195/1816.

IMPORTANCE

The blaOXA-23 gene can be carried by plasmid or chromosome, facilitating horizontal genetic transfer and increasing carbapenem resistance in healthcare settings. In this study, we focused on the genomic characteristics of CRAB strains harboring the chromosome-borne blaOXA-23 gene, and the important genetic contexts associated with blaOXA-23 and other ARGs were identified, and their prevalent clones worldwide were determined. Notably, although the predominant clonal CRAB lineages worldwide containing the MDR region associated with blaOXA-23, tet(B)-tetR(B), aph(3'')-Ib, and aph (6)-Id was ST195/1816, followed by ST208/1806, the CRAB strain AB2877 in our study belonged to ST208/1806. Our findings contribute to the knowledge regarding the dissemination of CRAB strains and the control of nosocomial infection.

KEYWORDS

carbapenem-resistant Acinetobacter baumannii
chromosome-borne blaOXA-23
ST
genetic context
comparative genomic analysis
MOST | National Natural Science Foundation of China (NSFC) 82002170 Li Xiaobin Zhuhai City Science and Technology Plan Project ZH22036201210092PWC Wang Wei Xiangshan Talent Project of Zhuhai People&apos;s Hospital 2020XSYC-02 Li Xiaobin cover-dateSeptember 2024
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pmcINTRODUCTION

Acinetobacter baumannii, a ubiquitous, strictly aerobic, non-fermentative Gram-negative bacillus, has become an important opportunistic pathogen in nosocomial infections, which commonly occur in patients with high-risk factors, including immunocompromised status, old age, heavy use of antibiotics, indwelling catheters, and length of hospital and/or intensive care unit (ICU) stay (1, 2). The mortality rate of nosocomial blood infections caused by A. baumannii has reached 34% and up to 43% of blood infections in patients in the ICU (3). The success of A. baumannii as a nosocomial pathogen is likely attributable to its high antibiotic resistance (4).

The emergence of multidrug-resistant (MDR) and extremely drug-resistant (XDR) A. baumannii has made it one of the most troublesome nosocomial pathogens worldwide in recent years (5). Data from the China Antimicrobial Surveillance Network (CHINET) indicate that the resistance levels of A. baumannii isolates to imipenem and meropenem will remain relatively stable (accounting for more than 75%) from 2018 to 2022 (6). Indeed, in 2017, the World Health Organization listed CRAB as a priority one critical pathogen, with an urgent need for the development of new treatments (7).

Horizontal gene transfer and mutational changes in chromosomal structures are the main causes of multidrug-resistant (MDR) and extreme drug-resistant (XDR) A. baumannii (8). The spread of antibiotic resistance genes (ARGs) is facilitated by mobile genetic elements, such as plasmids, insertion sequences (ISs), transposons, and integrons (9). To better understand the characteristics of ARGs and the genetic environment associated with ARGs carried by the A. baumannii strain AB2877, which was isolated from a patient in a tertiary hospital, we performed in silico typing and comparative analysis of the A. baumannii strain AB2877 with other A. baumannii strains available in the NCBI database. This study highlights the important relationship between ARGs and mobile genetic elements in A. baumannii that harbor chromosome-borne blaOXA-23.

RESULTS

Antibiotic resistance profiles of A. baumannii strain AB2877

Antimicrobial susceptibility testing showed that the A. baumannii strain AB2877 was resistant to cephalosporins (ceftazidime and cefepime), carbapenems (imipenem and meropenem), quinolones (ciprofloxacin and levofloxacin), aminoglycosides (amikacin and tobramycin) and β-lactam/β-lactamase inhibitor combinations (piperacillin/tazobactam, ampicillin/sulbactam, cefoperazone/sulbactam, and ticarcillin/clavulanate) (Table 1). It was also resistant to doxycycline and sulfamethoxazole (Table 1). Additionally, the A. baumannii strain AB2877 showed intermediate-level resistance to tigecycline and minocycline (Table 1). Notably, for the antibiotics tested, the A. baumannii strain AB2877 was only susceptible to colistin and cefiderocol (Table 1).

TABLE 1 Minimum inhibitory concentration (MIC) values of CRAB AB2877b

Antibiotics	MIC(μg/mL)	R or S	
Categories	Name	
Cephalosporins	Ceftazidime	≥ 64	R	
	Cefepime	≥ 32	R	
	Cefiderocola	-	S	
Carbapenems	Imipenem	≥ 16	R	
	Meropenem	≥ 16	R	
Quinolones	Ciprofloxacin	≥ 4	R	
	Levofloxacin	≥ 8	R	
Tetracyclines	Doxycycline	≥ 16	R	
	Minocycline	8	I	
	Tigecycline	4	I	
Aminoglycosides	Amikacin	≥ 64	R	
	Tobramycin	≥ 16	R	
Sulfonamides	Sulfamethoxazole	≥ 320	R	
Polymyxins	Colistin	≤ 0.5	S	
β-lactam/β-lactamase inhibitor combinations	Piperacillin/tazobactam	≥ 128/4	R	
Ampicillin/sulbactam	≥ 32/16		
Cefoperazone/sulbactam	≥ 64/32	R	
Ticarcillin/clavulanate	≥ 128/2	R	
a Cefiderocol, KB = 24.

b S, Susceptible; R, Resistant; I, Intermediate.

Genomic analysis of the A. baumannii strain AB2877

Genome analysis revealed that the A. baumannii strain AB2877 comprised a 3.89 Mb chromosome and a plasmid (pAB2877) of 8731 bp size. MLST analysis revealed that the A. baumannii strain AB2877 belonged to ST208/1806 (Oxford MLST scheme) and ST2 (Pasteur MLST scheme). The chromosome of the A. baumannii strain AB2877 harbored 13 acquired ARGs, including beta-lactam resistance genes (blaADC-73, blaOXA-23, blaOXA-66, blaTEM-1D), sulphonamide resistance gene (sul2), tetracycline resistance (tet(B) and tet(R)), aminoglycoside resistance (aph(3')-Ia, aph(3'')-Ib, aph (6)-Id, and armA), and macrolide resistance (mph(E) and msr(E)). The genome also contains several insertion sequence (IS) elements, most of which belong to the IS4, IS5, IS6, IS66, and IS91 families. However, no ARGs or virulence genes were found in plasmid pAB2877. Notably, ARGs carried by the chromosome were categorized into five multi-drug resistant (MDR) regions.

MDR region associated with blaOXA-23, tet(B)-tetR(B), aph(3'')-Ib, and aph(6)-Id

The genes encoding beta-lactam resistance (blaOXA-23), tetracycline resistance (tet(B)-tetR(B)), and aminoglycoside resistance (aph(3'')-Ib and aph (6)-Id) were located in the ~13 kb MDR region (Fig. 1A). The blaOXA-23 gene, together with the DEAD/DEAH box helicase-like and ATPase genes, were flanked by two copies of ISAba1 in different orientations, thus constituting the composite transposon Tn2006 (Fig. 1A). In this MDR region, the insertion sequence ISVsa3 is located upstream and downstream of the aminoglycoside (aph(3'')-Ib and aph (6)-Id) and the tetracycline (tet(B)-tetR(B)) resistance genes, respectively (Fig. 1A).

Fig 1 (A) Genetic structures of the MDR region associated with blaOXA-23, tet(B), tetR(B), aph(3'')-Ib, and aph (6)-Id. Genome sequences used to draw the diagrams from GenBank (A. baumannii VB31459 plasmid, A. baumannii AB34299 chromosome, A. baumannii CUVET-MIC596 chromosome, A. baumannii AB2877 chromosome, K. pneumoniae KP59 chromosome, A. baumannii RBH2 plasmid, A. baumannii VB82 plasmid). Genes and ORFs are indicated by arrows, and the direction of transcription is indicated by arrowheads. The resistance and transposase genes are shown in red and blue, respectively. (B) The unrooted tree shows the Top 5 most prevalent MLST from 135 strains of A. baumannii. The phylogenetic tree was created using the kSNP v3.1 based on the whole genomes of 135 strains of A. baumannii, which harbored the MDR region associated with blaOXA-23, tet(B), tetR(B), aph(3'')-Ib, and aph (6)-Id. The five most common MLST genotypes are ST195/1816 (red), ST208/1806 (green), ST451/1809 (blue), ST218/2164 (purple), and ST357! (orange).

BLAST analysis using the GenBank nr database based on the 13-kb MDR region carried by the A. baumannii strain AB2877 was conducted, and the results indicated that the most common species carrying the 13-kb MDR region was A. baumannii (135 fully sequenced genomes of A. baumannii; coverage ≥99% and identity ≥99%). In addition, we found that the 13-kb MDR region was also present on the chromosome of Klebsiella pneumoniae (GenBank accession CP076322, 100.00% coverage with 99.97% identity; Fig. 1A). Of the 135 A. baumannii strains encoding the 13-kb MDR region, the Top 5 prevalent Oxford STs (in descending order) were ST195/1816 (42 strains), ST208/1806 (17 strains), ST451/1809 (11 strains), ST218/2164 (10 strains), and ST357! (nine strains) (Fig. 1B).

Genetic context associated with armA and mph(E)-msr(E)

armA and mph(E)-msr(E) were located on a ~11.5-kb Tn6180-derived fragment of the resistance island AbGRI3, which was bracketed by ISEc28 and IS26 (Fig. 2A). ISEc28 was inserted upstream of the armA gene. For the macrolide resistance genes (mph(E)-msr(E)), an IS4-like element ISEc29 was inserted upstream of the mph(E)-msr(E), and an IS66-like element ISAba24 was inserted downstream of the mph(E)-msr(E) (Fig. 2A).

Fig 2 (A) Genetic contexts associated with armA and mph(E)-msr(E). The genome sequences used to draw the diagrams from GenBank (A. baumannii ABCR01 chromosome, A. baumannii MDR-CQ chromosome, A. baumannii AB2877 chromosome, A. baumannii BJAB0868 plasmid, A. baumannii AC30 plasmid, E. hormaechei ECL-14–60 plasmid, K. pneumoniae A2293 plasmid, Providencia sp. 1709051003 chromosome, and P. rettgeri CHS4.1 plasmid). Genes and ORFs are shown as arrows and the direction of transcription is indicated by arrowheads. The resistance and transposase genes are shown in red and blue, respectively. (B) The unrooted tree shows the TOP5 most prevalent MLST from 154 strains of A. baumannii. The phylogenetic tree was created using the kSNP v3.1 based on the whole genomes of 154 strains of A. baumannii, which harbored the genetic contexts associated with armA and mph(E)-msr(E). The five most common MLST genotypes are ST195/1816 (red), ST208/1806 (green), ST451/1809 (blue), ST368/1962 (purple), and ST191! (orange).

Based on the BLAST analysis using the GenBank nr database, with a minimum coverage of 99% and minimum identity of 99%, the resistance island AbGRI3 harboring armA and mph(E)-msr(E) carried by the A. baumannii strain AB2877 was present not only in the chromosomes of A. baumannii (154 fully sequenced genomes of A. baumannii) but also in A. baumannii (e.g. A. baumannii BJAB0868 plasmid p3BJAB0868). The resistance island AbGRI3 harboring armA and mph(E)-msr(E) carried by the A. baumannii strain AB2877 was also identified in other species, including Enterobacter hormaechei (plasmid, GenBank accession MZ836805), K. pneumoniae (plasmid, GenBank accession MN310378), Providencia (both chromosomes [GenBank accession CP042861] and plasmid [GenBank accession OL908906]). Of the 154 A. baumannii strains that carried the 11.5-kb resistance island AbGRI3 harboring armA and mph(E)-msr(E), the dominant Oxford STs of TOP5 (in descending order) were ST195/1816 (39 strains), ST208/1806 (34 strains), ST451/1809 (9 strains), ST368/1962 (9 strains), and ST191! (nine strains) (Fig. 2B).

Genetic context associated with blaTEM-1D and aph(3')-Ia

blaTEM-1D and aph(3')-Ia were located on a 9.4-kb Tn3-like composite transposon, which was bracketed by two copies of IS26 in the same orientation (Fig. 3A). Additionally, four copies of IS26 in the same orientation were found in the 9.4-kb Tn3-like composite transposon. The blaTEM-1D gene was flanked by two direct repeats of IS26, and the aph(3')-Ia gene was also flanked by two direct repeats of IS26 (Fig. 3A).

Fig 3 (A) Genetic structures associated with blaTEM-1D and aph(3')-Ia. Genome sequences used to draw the diagrams from GenBank (A. baumannii F11, A. baumaannii AB329 chromosome, A. baumannii AB2877 chromosome, A. baumannii X4-300 plasmid, and Acinetobacter sp. FDAARGOS_560 chromosome). Genes and ORFs are shown as arrows, and the direction of transcription is indicated by arrowheads. The resistance and transposase genes are shown in red and blue, respectively. (B) The unrooted tree shows the Top 5 most prevalent MLST from 156 strains of A. baumannii. The phylogenetic tree was created using the kSNP v3.1 based on the whole genomes of 156 strains of A. baumannii, which harbored the genetic structures associated with blaTEM-1D and aph(3')-Ia. The five most common MLST genotypes are ST208/1806 (red), ST195/1816 (green), ST350! (blue), ST451/1809 (purple), and ST381! (orange).

Based on the BLAST analysis hit from the GenBank nr database, with a minimum coverage of 99% and a minimum identity of 99%, the 9.4-kb Tn3-like composite transposon containing blaTEM-1D and aph(3')-Ia was widely present on the chromosomes of A. baumannii (156 fully sequenced genomes of A. baumannii; coverage ≥99% and identity ≥99%). The top 5 predominant Oxford STs among the 156 strains of A. baumannii (in descending order) were ST208/1806 (65 strains), ST195/1816 (17 strains), ST350! (nine strains), ST451/1809 (eight strains), and ST381! (eight strains) (Fig. 3B).

Genetic context of blaADC-25

In the A. baumannii strain AB2877, ISAba1 was located downstream of blaADC-25 (Fig. 4A). The structure “ISAba1-blaADC-25” was widely present on the chromosomes of A. baumannii (461 fully sequenced genomes of A. baumannii; coverage ≥99% and identity ≥99%). Of the 461 A. baumannii strains carrying the structure “ISAba1-blaADC-25,” the five most prevalent Oxford STs (in descending order) were ST208/1806 (114 strains), ST195/1816 (44 strains), ST191! (20 strains), ST345/1857 (16 strains), and ST368/1962(15 strains) (Fig. 4B).

Fig 4 (A) Genetic structure of blaADC-25. Genome sequences used to draw the diagrams from GenBank (A. baumannii MDR-CQ chromosome, A. baumannii VB723 chromosome, A. baumannii AB2877 chromosome, A. baumannii J9 plasmid). Genes and ORFs are shown as arrowheads, and the direction of transcription is indicated by arrowheads. The resistance and transposase genes are shown in red and blue, respectively. (B) The unrooted tree shows the most prevalent TOP5 MLST from 461 strains of A. baumannii. The phylogenetic tree was created using the kSNP v3.1 based on the whole genomes of 461 strains of A. baumannii, which harbored the genetic structure of blaADC-25. The five most common MLST genotypes are ST208/1806 (red), ST195/1816 (green), and ST191! (blue), ST345/1857 (purple), and ST368/1962 (orange), respectively.

DISCUSSION

In this study, we describe a CRAB strain, AB2877, belonging to ST208/1806 (Oxford MLST scheme) or ST2 (Pasteur MLST scheme), which was isolated from the bronchoalveolar lavage fluid (BALF) of a 66-year-old patient in the respiratory intensive care unit (RICU). Among the antibiotics tested, the CRAB strain AB2877 was only susceptible to colistin and cefiderocol, indicating their great clinical value in the treatment of CRAB-causing infections. The CRAB strain AB2877 carries chromosomal blaOXA-23 gene, which is a key determinant of antibiotic resistance found in Acinetobacter species, which encodes a class D β-lactamase enzyme that confers resistance to carbapenem antibiotics (10). The blaOXA-23-like gene is prevalent in China and is present in 97% of carbapenem-resistant isolates (11). ISAba1 is a strong promoter sequence that influences the expression of neighboring genes, including those encoding antibiotic resistance determinants (12). Upstream of resistance genes, ISAba1, can significantly increase their expression, leading to increased resistance to antibiotics (13).

In CRAB AB2877, the blaOXA-23 gene was embedded in Tn2006, which is bracketed with two copies of ISAba1 in different orientations. The transposon Tn2006 is one of the most common transposons worldwide (14) and may be one of the main reasons for the global dissemination of carbapenem resistance in A. baumannii (15). In particular, besides transposon Tn2006, transposons Tn2008 and Tn2009 also appeared to contribute significantly to the dissemination of blaOXA-23 in China (16–18). Furthermore, we found that Tn2006 harboring blaOXA-23 was present not only on the chromosomes but also on the plasmids of A. baumannii, as well as other pathogens, such as K. pneumoniae and Proteus mirabilis (19), suggesting that several species may be reservoirs or scatterers for this class D carbapenemase gene. In addition to the blaOXA-23 gene, other beta-lactamase genes (blaOXA-66, blaADC-25, and blaTEM-1D) were also detected in this study. This finding is consistent with those of previous reports. For example, an A. baumannii isolate has been reported to harbor blaOXA-23, blaOXA-66, and blaADC-25 in south China (20). Several coexisting multi-beta-lactam resistance genes contribute significantly to the extensive drug resistance observed in A. baumannii strains, which poses significant challenges for infection control and clinical management.

In this study, the CRAB strain AB2877 belonged to ST208/1806 (Oxford MLST scheme). Analysis of the genetic context associated with detected ARGs indicated that the genetic context associated with blaTEM-1D and that of blaADC-25 were easily detected in ST208/1806, followed by ST195/1816. The MDR regions associated with blaOXA-23, tet(B)-tetR(B), aph(3'')-Ib, aph (6)-Id, and the resistance island AbGRI3 harboring armA and mph(E)-msr(E) were commonly detected in ST195/1816, followed by ST208/1806. ST208 is widely recognized as a predominant lineage of A. baumannii GC2 worldwide, and some researchers believe that ST208 may have originated in North America and evolved into two clades (21). Reports of carbapenem-resistant A. baumannii ST195 in China have increased gradually in recent years (22, 23).

The blaOXA-23-like gene is almost always found within transposons and is commonly associated with an antibiotic-resistance genomic island (AbGRI) (14). The ribosomal RNA methyltransferase gene armA, an aminoglycoside resistance gene located on the resistance island AbGRI3, has been widely reported in A. baumannii (24). In the AB2877 isolate, the genetic contexts associated with armA and mph(E)-msr(E) were similar to those of Tn6180. Tn6180-borne armA or AbGRI3 has spread worldwide, especially in Japan and East Asia (25, 26).

In this study, we describe the genomic characteristics of the multidrug-resistant CRAB strain AB2877 belonging to ST208/1806 (Oxford MLST scheme) harboring chromosome-borne blaOXA-23, which was isolated from the BALF of a patient in the RICU in China. Several key genetic contexts associated with blaOXA-23 and other ARGs were found on the chromosome of the CRAB strain AB2877. Based on the genomes of A. baumannii available in the GenBank database, we explored the predominant clonal lineages of A. baumannii worldwide, including different genetic contexts of the CRAB strain AB2877. The blaOXA-23 gene was located in the MDR region associated with blaOXA-23, tet(B)-tetR(B), aph(3'')-Ib, and aph (6)-Id, which was most commonly found in ST195/1816, followed by ST208/1806. Furthermore, the resistance island AbGRI3 that harbors armA and mph(E)-msr(E) carried by strain AB2877 was most frequently found in CRAB ST195/1816, followed by CRAB ST208/1806. The CRAB strain AB2877 also carried one Tn3-like composite transposon bracketed by two copies of IS26 containing blaTEM-1D and aph(3')-Ia and one structure “ISAba1-blaADC-25,” which were most commonly found in ST208/1806, followed by ST195/1816.

MATERIALS AND METHODS

Isolation, identification, and antimicrobial susceptibility testing

The strain AB2877 was isolated from a BALF sample of a patient in the RICU who stayed for more than 14 days and received antibiotic treatment with carbapenem in the hospital. Bacterial species were identified using a fully automatic VITEK-2 Compact system (bioMérieux, France) and by sequencing the 16S rRNA gene. Antimicrobial susceptibility was measured using the VITEK-2 Compact system, which used the following antimicrobial agents: cephalosporins (ceftazidime, cefepime, and cefiderocol), carbapenems (imipenem and meropenem), quinolones (ciprofloxacin and levofloxacin), tetracyclines (doxycycline, minocycline, tigecycline), aminoglycosides (amikacin and tobramycin), sulfonamides (sulfamethoxazole), polymyxins (colistin), and β-lactam/β-lactamase inhibitor combinations (piperacillin/tazobactam, ampicillin/sulbactam, cefoperazone/sulbactam, and ticarcillin/clavulanate). The minimum inhibitory concentration (MIC) breakpoints for cefoperazone–sulbactam were those for A. baumannii: S, ≤16/8 mg/L; I, 32/16 mg/L; R, ≤64/32 mg/L. Colistin resistance was confirmed by broth microdilution test (Mikrolatest; Erba Lachema, Brno, Czech Republic) as suggested by EUCAST. Susceptibility testing for cefiderocol was determined by Kirby–Bauer’s disk diffusion (KB) method. The results of other antimicrobial agents were interpreted according to the Institute of Clinical and Laboratory Standards (CLSI M100–S33) (CLSI, 2023).

Whole genome sequencing, assembly, and annotation

Whole-genome sequencing of the A. baumannii strain AB2877 was performed by GENEWIZ Co., Ltd. (Suzhou, China) using paired-end sequencing with Novaseq 6000 (150-bp paired-end reads) and long sequencing with PacBio Sequel. Hybrid assembly with long and short reads was used to produce the complete bacterial genome of the A. baumannii strain AB2877. First, PacBio long reads were assembled using HGAP (v.4.0)/Falcon (v.0.3) of WGS-Assembler 8.2 (27), and assembly polishing was performed with Pilon (version 1.22) (28) using Illumina short reads. The assembled genome of the A. baumannii strain AB2877 was submitted to the NCBI GenBank database (29) and annotated using the NCBI Prokaryotic Annotation Pipeline (PGAP) (30).

Bioinformatics analysis for the genome of the A. baumannii strain AB2877

The multilocus sequence typing (MLST) of the A. baumannii strain AB2877 was performed using MLST software (v.2.0; https://cge.food.dtu.dk/services/MLST/) (31). ResFinder (32) v.4.5 (http://genepi.food.dtu.dk/resfinder) identified several acquired antimicrobial resistance genes. Insertion sequence (IS) elements were identified using ISfinder (https://www-is.biotoul.fr/blast.php) (33). A sequence similarity search was conducted against the GenBank non-redundant (nr) database using MegaBLAST (34). Sequence comparisons were visualized using Easyfig v.2.2.5 (35). Genome-wide single-nucleotide polymorphism (SNP) calling and phylogenetic analysis were performed by using kSNP v3.1 (36), and the tree was displayed with iTOL (37).

ACKNOWLEDGMENTS

This work was supported financially by grants from the National Natural Science Foundation of China (Grant No. 82002170), the Zhuhai City Science and Technology Plan Project (Grant No. ZH22036201210092PWC), and the Xiangshan Talent Project of Zhuhai People’s Hospital (Grant No. 2020XSYC-02).

DATA AVAILABILITY

The complete sequences of the chromosome and plasmid of A. baumannii strain AB2877 were submitted to GenBank database, under accession numbers CP092485-CP092486.

ETHICAL APPROVAL

This study has been approved by the Ethics Committee of Zhuhai People’s Hospital (Permission Number: [2020] No. 28).
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