Draft Genome Sequences of Two Extensively Drug-Resistant Strains of Acinetobacter baumannii Isolated from Clinical Samples in Pakistan.

Infections in immunocompromised patients that are caused by extensively drug-resistant (XDR) Acinetobacter baumannii strains have been increasingly reported worldwide. In particular, carbapenem-resistant A. baumannii strains are a prominent cause of health care-associated infections. Here, we report draft genome assemblies for two clinical XDR A. baumannii isolates obtained from hospitalized patients in Pakistan.

ANNOUNCEMENT

Acinetobacter baumannii, a Gram-negative opportunistic pathogen of the Moraxellaceae family, can carry multiple antimicrobial resistance (AMR) determinants. Infections and outbreaks caused by multidrug-resistant (MDR) or extensively drug-resistant (XDR) A. baumannii strains in immunocompromised patients have been increasingly reported worldwide (1), with isolates often belonging to international clone 2/sequence type 2 (ST2) (2). Carbapenem-resistant A. baumannii (CRAB) strains are a significant cause of health care-associated infections in Pakistan, with various prevalence rates (62% to 100%) (3). The most common β-lactamases in CRAB strains are acquired (e.g., blaOXA-23, blaOXA-40, blaOXA-58, blaOXA-143, and blaOXA-235) and intrinsic (e.g., blaOXA-51 and blaOXA-69) carbapenem-hydrolyzing oxacillinases (4).

Draft genomes are reported here for two clinical XDR A. baumannii isolates obtained from urine (CFSAN059604, isolated in 2004) and throat (CFSAN059618, isolated in 1998) specimens from hospitalized patients in Pakistan. Patient samples were inoculated onto nonselective (e.g., blood agar) and differential (e.g., MacConkey agar) plates and incubated for 24 to 48 h at 37°C. API 20E and Vitek 2 (bioMérieux) systems were used for species identification and confirmation, respectively. Susceptibility testing against clinically relevant antimicrobials was performed by conventional broth microdilution (5) following CLSI and EUCAST guidelines and breakpoints (5–7). The two isolates were resistant to 17 antibiotics with the same MICs, as follows: ≥128 μg/ml for piperacillin-tazobactam; ≥64 μg/ml for cefotaxime and aztreonam; ≥32 μg/ml for ampicillin, ceftriaxone, and tetracycline; ≥16 μg/ml for cefoxitin, ceftazidime-avibactam, gentamicin, and chloramphenicol; and ≥8 μg/ml for cefazolin, doripenem, meropenem, ertapenem, ciprofloxacin, levofloxacin, and trimethoprim-sulfamethoxazole. CFSAN059604 and CFSAN059618 were also resistant, but with different MICs, to ampicillin-sulbactam (≥32 and ≥16 μg/ml, respectively), ceftazidime (≥128 and ≥16 μg/ml, respectively), cefepime (≥32 and ≥16 μg/ml, respectively), and imipenem (≥8 and ≥16 μg/ml, respectively). Both isolates were susceptible to colistin (≤2 μg/ml) and minocycline (≤4 μg/ml). Finally, CFSAN059604 and CFSAN059618 had markedly different resistance profiles for amikacin (≤2 and ≥64 μg/ml, respectively) and tobramycin (≤1 and ≥16 μg/ml, respectively).

Isolates were grown overnight in lysogeny broth (Lennox), and DNA was extracted using the DNeasy blood and tissue kit (Qiagen). Libraries were prepared using the Nextera XT DNA library preparation kit and sequenced on a MiSeq (CFSAN059618) or NextSeq (CFSAN059604) sequencer (Illumina), with paired-end sequencing technology (2 × 250-bp and 2 × 150-bp sequencing, respectively). Minimum sequence quality was represented by average coverage greater than 50× and Q scores for reads 1 and 2 greater than 30 (8). Absence of contamination was confirmed with Kraken (9). Default parameters were used unless otherwise noted. De novo assemblies were obtained with Shovill v0.9 (https://github.com/tseemann/shovill), available in the GalaxyTrakr pipeline (10). The “trim reads” option was selected, and 500 bp was set as the minimum contig length. Draft genomes were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (11). Table 1 lists the number of reads, number of contigs per assembly, genome size, and GC content for each isolate. The isolates were assigned to ST2 (CFSAN059604) and ST23 (CFSAN059618), based on the multilocus sequence typing Pasteur scheme (https://pubmlst.org/abaumannii). NCBI Pathogen Detection (PD) (https://www.ncbi.nlm.nih.gov/pathogens) was used to identify 14 (CFSAN059604) and 8 (CFSAN059618) AMR genes. NCBI PD uses AMRFinderPlus to assign the most specific AMR protein by using a hierarchy of gene families/symbols/names (https://www.ncbi.nlm.nih.gov/pathogens/antimicrobial-resistance/AMRFinder). Results are available in Table 1 and from the NCBI PD website (CFSAN059604 and CFSAN059618).

TABLE 1

Assembly statistics, accession numbers, and AMR genes for the XDR Acinetobacter baumannii isolates examined in this work

Samplea	BioSample accession no.	Yr isolated	SRA accession no.	GenBank accession no.	No. of contigs	Total length (bp)	N50 (bp)	GC content (%)	Genome coverage (×)	Avg read quality score for:		No. of reads	No. of AMR genes
										Read 1	Read 2
CFSAN059604	SAMN10086814	2004	SRR8837010	SSMO00000000	48	3,944,772	261,641	38.9	712	32b	30b	21,896,362	14c
CFSAN059618	SAMN10086821	1998	SRR8837150	SSMN00000000	79	3,907,254	116,856	38.8	93	35	33	1,792,448	8d

The two isolates belong to NCBI BioProject PRJNA342326.

Average of four lanes.

Genes aac(3)-I, aadA1, adeC, ant(3ʺ)-IIa, aph(3ʺ)-Ib, aph(3′)-Ia, aph(6)-Id, blaADC, blaOXA-254, blaTEM-1, qacEΔ1, sul1, sul2, and tet(B).

Genes ant(2ʺ)-Ia, ant(3ʺ)-IIa, aph(3′)-VIa, blaADC-76, blaOXA-235, blaOXA-68, sul2, and tet(39).

The described draft genomes will be useful in comparative genomic analyses of A. baumannii strains from different regions and clinical settings. These data can also provide phylogenetic insights into the emergence of XDR A. baumannii strains and support epidemiological investigations of outbreaks.

Data availability.

The complete genome sequences of A. baumannii CFSAN059604 (SRA number SRR8837010) and CFSAN059618 (SRA number SRR8837150) are available in GenBank under accession numbers SSMO00000000 and SSMN00000000, respectively (first versions).