Prevalence and Characterization of Carbapenem-Hydrolyzing Class D β-Lactamase-Producing Acinetobacter Isolates From Ghana

Multidrug resistance, especially carbapenem resistance in Acinetobacter bacteria is a global healthcare concern. However, available data on the phenotypic and genotypic characteristics of Acinetobacter isolates from West Africa, including Ghana is scanty. Our aim was to investigate the antibiotic resistance profile and genotypic characteristics of Acinetobacter isolates from Ghana and to characterize carbapenemase producers using whole-genome sequencing (WGS). A total of 36 Acinetobacter isolates collected at three hospitals in Ghana between 2016 and 2017 were analyzed. MICs were determined by commercial antibiotic plates. Acinetobacter baumannii MLST was determined using the Pasteur scheme. WGS of OXA-carbapenemase producers was performed using short- and long-read sequencing strategies. The resistance rate was highest for trimethoprim/sulfamethoxazole (n = 22; 61%). Six (16.7%) and eight (22.2%) isolates were resistant to ceftazidime and colistin, respectively. Two (5.6%) isolates were resistant and one (2.8%) isolate had intermediate sensitivity to three carbapenems. Fifteen STs were identified in 24 A. baumannii isolates including six new STs (ST1467 ∼ ST1472). ST78 was the predominant (n = 6) followed by ST1469 (n = 3). Four carbapenemase-producing A. baumannii isolates also were identified. Isogenic ST103 isolates Ab-B004d-c and Ab-D10a-a harbored blaOXA–23 within Tn2007 on identical plasmids, pAb-B004d-c_3, and pAb-D10a-a_3. ST1472 isolate Ab-C102 and ST107 isolate Ab-C63 carried blaOXA–58 and blaOXA–420, a rare blaOXA–58 variant, respectively, within novel genetic contexts. Our results show that A. baumannii isolates of diverse and unique genotypes, including OXA-carbapenemase producers, are circulating in Ghana highlighting the need for a wider surveillance of antimicrobial resistance.


INTRODUCTION
Acinetobacter species are important pathogens, responsible for healthcare-related infections. Acinetobacter baumannii is the most clinically prevalent species, but the clinical significance of A. pittii and A. nosocomialis is also increasing (Adams et al., 2008;Chuang et al., 2011;Nemec et al., 2011;Doi et al., 2015). As Acinetobacter spp. have intrinsic resistance to several antibiotic classes and can acquire resistance determinants, carbapenems are an optimal treatment option for infections caused by these bacteria. However, carbapenem resistance is rising worldwide, leaving very limited antimicrobial treatment options (Dijkshoorn et al., 2007;Hamidian and Nigro, 2019). Therefore, carbapenemresistant Acinetobacter (CRA) is globally recognized as an urgent threat (World Health Organization, 2017; Centers for Disease Control and Prevention, 2019).
Carbapenem resistance in Acinetobacter is mainly caused by the production of carbapenem-hydrolyzing class D β-lactamases (CHDLs) including OXA-23-like, OXA-24-like and OXA-58like enzymes, or by the production of Ambler class B metalloβ-lactamases (MBLs) such as NDM-, IMP-and VIM-type enzymes (Potron et al., 2015). Until now, only NDM-1producing A. baumannii has been reported in Accra, the capital of Ghana (Codjoe et al., 2019). In A. baumannii, overexpression of the intrinsic bla OXA−51 -like gene mediated by the insertion sequence ISAba1 upstream also confers resistance to carbapenems (Wong et al., 2019). Changes in outer membrane proteins and overexpression of efflux pumps are also associated with carbapenem resistance (Poirel and Nordmann, 2006a;Coyne et al., 2011).
Surveillance of CRA prevalence, associated genotypes, and elucidating the mechanisms underlying resistance are crucial as they enable us to understand how these bacteria disseminate and how best to control their spread. However, due to the poor antimicrobial resistance surveillance in Ghana, data on CRA is limited. Moreover, no data is available yet for CHDL-producing Acinetobacter and their genotypic characteristics.
Our aim was to investigate the antibiotic resistance profile and the genotypic characteristics of clinical isolates of Acinetobacter spp. collected at three regional hospitals in Ghana. Furthermore, we also characterize OXA-carbapenemase producers using both short-and long-read whole-genome sequencing (WGS).
Acinetobacter baumannii complex (ABC) isolates were further characterized to species level using gyrB multiplex PCR method as previously described (Higgins et al., 2010).

Antibiotic Susceptibility Testing
The MICs of 15 antibiotics including meropenem, colistin, and minocycline were determined by broth microdilution using the commercial microplates DP34 and DP35 (Eiken Chemical Co., Tokyo, Japan). Results were interpreted according to the Clinical and Laboratory Standards Institute guideline document M100-S27 (Clinical and Laboratory Standards Institute, 2017).

Multilocus Sequence Typing
Multilocus sequence typing (MLST) was performed for all confirmed A. baumannii isolates using the Pasteur scheme. New allele types and new sequence types (STs) were submitted to PubMLST 1 and assigned new numbers. Genetic relatedness of STs was analyzed and visualized using PHYLOVIZ 2.0 goBURST Full MST analysis (Nascimento et al., 2017).

Whole-Genome Sequencing
Short-and long-read WGS was conducted in four CHDLproducing isolates to determine their complete chromosome and plasmid sequences with high accuracy. DNA was extracted with the NucleoSpin tissue kit (Macherey-Nagel, Düren, Germany) for short-read sequencing; in one of the isolates the DNA library was prepared with the Nextera DNA Flex library prep kit and sequenced with Illumina Miseq (Illumina, San Diego, CA, United States); GIEasy FS PCR-Free DNA Library Prep Set was used for the remaining three isolates and sequenced with MGI DNBSEQ (MGI Tech Co., Shenzhen, China). Nanopore MinION (Oxford Nanopore Technologies, Oxford, United Kingdom) was used for long-read sequencing; DNA was extracted with the MagAttract HMW DNA Kit (Qiagen, Hildon, Germany); multiplexed DNA libraries were prepared for all four isolates using the Native Barcoding Kit EXP-NBD104 and the ligation sequence Kit SQK-LSK109; and sequencing was performed on a single R9.4.1 flow cell. Low-quality reads (MinION Q < 10; DNBSEQ Q < 32; and MiSeq Q < 30) and short reads (MinION length <1,000 bp; DNBSEQ and Miseq length <10 bp) were filtered out.

Bioinformatics Analysis
Hybrid de novo genome assembly for WGS data was done with Unicycler v0.4.8 and Flye v2.6 using both short and long reads. Sequence annotation was performed using the online system RAST and BLAST searches. Whole-genome SNP typing was done for four CHDL producers using PathoBacTyper 2 . Resfinder v3.2 was used to detect acquired resistance genes (Zankari et al., 2012). Insertion sequences were identified using ISfinder 3 . Plasmid structures and genetic contexts of carbapenemase genes were compared and visualized using Easyfig v2.2.2 (Sullivan et al., 2011).

Phylogenetic Analysis
Forty-five bla OXA−58 plasmids and one bla OXA−420 plasmid in the repository of PLSDB v.2020_03_04 (Galata et al., 2019), as well as two plasmids from this study (pAbC102_1 and pAbC63_1) were used for phylogenetic analysis. Sequence alignment of plasmids was performed using CLC genomic workbench 20 (CLC bio, Aarhus, Denmark). A maximum parsimony tree with 1,000 bootstrap replicates was generated using MEGA X 10.1 (Kumar et al., 2018).

Accession Numbers
Complete chromosomal and plasmid sequences were deposited in GenBank under Bioproject PRJNA473419.

Phenotypic Characterization of Acinetobacter Isolates
Thirty-five out of the 36 isolates belonged to ABC, while the other isolate was identified as A. haemolyticus. Among all ABCs, 24 isolates (68.6%) were characterized as A. baumannii followed by seven A. nosocomialis (20%) and three A. pittii (8.6%). The species for the remaining one ABC isolate (identified using MALDI Biotyper) was not determined due to conflicting results from the gyrB multiplex PCR; this isolate was negative for the bla OXA−51 -like.
The allelic variant of bla OXA−51 was bla OXA−70 for both isolates. In addition, A. baumannii isolates, Ab-C102A and Ab-C63, belonging to the novel ST1472 and ST107 with bla OXA−51 variants of bla OXA−699 and bla OXA−51 respectively, were also identified and found to harbor CHDLs, bla OXA−58 and bla OXA−420 . All four isolates were collected at the Effia Nkwanta Regional Hospital. Other carbapenemase genes, such as MBL genes were not detected by conventional PCR.

Genetic Diversity of Acinetobacter baumannii Isolates
The genetic relatedness of the 24 A. baumannii isolates was assessed using the Pasteur MLST scheme. Isolates were assigned into 15 different STs, six of which were novel STs (ST1467, ST1468, ST1469, ST1470, ST1471, and ST1472). Minimum spanning tree analysis revealed that all STs had no genetic relationship with each other (Figure 1). ST78 (n = 6; 25%) was the most predominant, followed by the novel ST1467 (n = 3; 12.5%).

Genotypic Characterization of
Ab-B004d-c, Ab-D10a-a, and bla OXA−23 Genetic Context Two OXA-23-producing ST103 isolates, Ab-B004d-c, and Ab-D10a-a, were recovered from sputum and cerebrospinal fluid samples of different patients in January 2016 (Figure 2). Both isolates were resistant to most antibiotics, including carbapenems, but were fully susceptible to colistin ( Table 2). Over 4 Gb short-read sequences and about 1.8 Gb long-read sequences were obtained for each isolate after quality filtering (Supplementary Table 1). Using de novo hybrid assembly, two circular chromosomes with 4,091,477 and 4,100,469 bp were generated for Ab-B004d-c and Ab-D10a-a, respectively (Supplementary Table 2). Both isolates were isogenic, differing by only 10 SNPs (Figure 2). Four pairs of identical plasmids ranging in size from 2,697-48,239 bp also were generated for both isolates (Supplementary Table 2). Ab-D10a-a carried an extra 6,619 bp plasmid (Supplementary Table 2). The chromosomes of both isolates carried the aminoglycoside resistance gene aac(3)-IIa, the sulphonamide resistance gene sul1, the intrinsic β-lactamase gene bla OXA−70 , the extended-spectrum β-lactamase (ESBL) gene bla CTX−M−15 , and the intrinsic AmpC gene bla ADC−203 . No insertion sequences were detected upstream of bla OXA−70 and bla ADC−203 . The trimethoprim resistance gene dfrA1, the aminoglycoside resistance gene aac(3)-IId, the tetracycline resistance gene tet(39), and macrolide resistance genes msr(E) and mph(E) were found on two identical plasmids with 48,239 bp (pAb-B004d-c_1 and pAb-D10a-a_1) in both isolates.
The bla OXA−23 gene was present on two identical 8,215 bp plasmids (pAb-B004d-c_3 and pAb-D10a-a_3) within Tn2007, and flanked upstream by ISAba4 and downstream by a truncated ATPase gene ( Figure 3A). No other resistance genes were detected in these plasmids. Annotation results and BLAST searches for ORFs failed to identify any replication origin protein (Rep) gene in these plasmids. The linearized sequences of pAb-B004d-c_3 and pAb-D10a-a_3 were compared with Frontiers in Microbiology | www.frontiersin.org  the bla OXA−23 -containing region of pAB14 from A. baumannii (accession number: EF059914). Results showed that the genetic structure of Tn2007 was highly identical in pAb-B004d-c_3, pAb-D10a-a_3 and pAB14. In addition, a higBA toxin-antitoxin locus was found downstream of Tn2007.

Genotypic Characterization of Ab-C102 and bla OXA−58 Genetic Context
The OXA-58-producing ST1472 isolate Ab-C102 was recovered from the blood of a patient. Ab-C102 was susceptible to almost all antibiotics but displayed a low meropenem MIC (1 mg/L) ( Table 2). Similar to the isolates described before, more than 4 Gb of short-read and near 1 Gb of long-read sequences were obtained by WGS for the isolate Ab-C102, after quality filtering (Supplementary Table 1). A circular chromosome with 3,763,047 bp and three circular plasmids with 19,853, 67,097, and 90,089 bp were generated by hybrid de novo genome assembly (Supplementary Table 2). A novel variant of the intrinsic bla OXA−65 gene with three synonymous nucleotide substitutions, and an intrinsic bla ADC−32 gene were identified on the chromosome. No insertion sequences were detected upstream of these genes. The carbapenemase gene bla OXA−58 along with other resistance genes such as aph(6)-Id were identified on a 90,089 bp plasmid (pAb-C102_1; Table 2).
This plasmid encodes a RepB family plasmid Rep that share 100% identity with its counterpart in A. baumannii WB103 (accession number: AZM37906). Phylogenetic analysis showed that the genetically closest plasmid to pAb-C102_1 was pNDM-1_010045, with which it share 46% query cover and 97.25% identity (Figure 4). Interestingly, a BLAST search found that the full plasmid sequence of pAb-C102_1 had the highest homology with the draft WGS data of WB103, sharing 88% of query cover and 99.88% of identity.
The genetic environment of bla OXA−58 in pAb-C102_1 was comparable to that in pWA3 (accession number: JQ241791), with a similar genetic structure ( Figure 3B). In both plasmids, the  same ISAba3-like-araC1-lysE structure was found downstream of bla OXA−58 and the same three nucleotide substitutions were present in both ISAba3-like. The ISAba3-like-araC1-lysE was followed by one IS1006-like copy (one nucleotide substitution compared to IS1006) and the lysR gene. Upstream of bla OXA−58 , a 427 bp truncated form of ISAba3 was interrupted by a putative novel ISAjo2-like sequence. This contrasted with pWA3 in which a 121 bp truncated form of ISAba3 is interrupted by IS1008. A Re27-2 region, which is a pdif site targeted by the XerC/XerD-like site-specific recombinases, was found 13 bp upstream of the ISAjo2-like sequence. A second copy of ISAjo2-like and IS1006-like (three nucleotide substitutions compared with IS1006) was found upstream of the Re27-2 region. Neither a second copy of Re27-2 nor Re27-1 (single nucleotide variant of Re27-2) were found downstream of bla OXA−58 .

Genotypic Characterization of Ab-C63 and bla OXA−420 Genetic Context
Ab-C63 was an isolate recovered from a sputum sample that belonged to ST107 and produced OXA-420, a rare CHDL of the OXA-58 family. This isolate had intermediate susceptibility to carbapenems and was resistant to aminoglycosides, fluoroquinolones and colistin ( Table 2). A 3,873,864 bp circular chromosome and two plasmids with 81,353 and 10,662 bp were generated by hybrid assembly using quality-filtered ∼350 Mb short-read and ∼1.2 Gb longread sequences (Supplementary Tables 1 and 2). The intrinsic β-lactamase genes bla ADC−87 and bla OXA−51 , as well as the aminoglycoside resistance gene ant(3 )-IIa were found on the chromosome. The bla OXA−420 gene and other resistance genes such as mph(E) were located on a plasmid with 81,353 bp (pAb-C63_1; Table 2). This plasmid harbored a novel gene encoding a RepB family plasmid Rep which shared the highest similarity (99.7% identity) with proteins from Acinetobacter spp. and A. brisouii (accession numbers: WP_004761033, WP_151711075).
Phylogenetic analysis (Figure 4) showed pAB-C63_1 to be genetically distinct from the bla OXA−420 -containing A. baumannii ACN21 plasmid unnamed2 (NZ_CP038646), collected in India, but genetically close to the bla OXA−58containing pAI01 (NZ_CP044019), with which it shares 42% query cover and 95% identity. Plasmid backbone structure comparisons also revealed that pAb-C63_1 had a novel backbone compared with ACN21 plasmid unnamed2 ( Figure 3C). Moreover, a completely distinct genetic context was found surrounding bla OXA−420 in pAbC63_1, in which the gene was flanked by two copies of IS15DII, in contrast with ACN21 plasmid unnamed2 and A. baumannii strain IOMTU in which bla OXA−420 is flanked by ISAba3-like and truncated ISAba3-like elements ( Figure 3D). In addition, a third copy of IS15DII was found downstream, interrupting an ISAba3 sequence. The resistance genes ant(2 )-Ia and aph(3 )-Ia were flanked by the second and third copies of IS15DII. The Re27-2 sequence was detected 65 bp downstream of the truncated ISAba3. Moreover, a novel insertion sequence, a ISAlw27-like element (coverage = 1268/1282; identities = 1213/1268), was detected upstream of the first copy of IS15DII.

DISCUSSION
Carbapenem resistance in Acinetobacter spp. is a major health threat worldwide. Most of the studies on CRA in Africa were conducted in Northern or Southern regions (Manenzhe et al., 2015), while the current situation in sub-Saharan Africa may be underestimated given the limited laboratory and surveillance capacity. Here, we showed that Acinetobacter species, predominantly A. baumannii, are disseminating in Ghanaian healthcare facilities. A. nosocomialis and A. pittii (previously known as Acinetobacter genomics species 13UT and 3, respectively) were also prevalent. The proportion of these species is parallel to those causing nosocomial bloodstream infections in the United States (Wisplinghoff et al., 2012). Conventional identification methods can not distinguish between ABCs, resulting in misidentification of some species as A. baumannii (Wisplinghoff et al., 2012). Although, gyrB multiplex PCR has been reported as one of the most highly sensitive methods distinguishing species within ABCs (Lee et al., 2014), the conflicting result that we obtained for an ABC isolate using this method has proven the difficulty associated with species identification among ABCs. Multidrug resistance, including to carbapenems, was detected in the Acinetobacter isolates. However, carbapenem resistance was less prevalent (5.6%), in our study (primarily conducted in the Western region of Ghana) compared with a prior study conducted in the country's capital, Accra, in which seven out of nine A. baumannii isolates from burn wounds were resistant to meropenem (Forson et al., 2017). Higher carbapenem resistance, 51.5 and 10%, have been reported in the West African countries of Nigeria and Sierra Leone, respectively (Nsofor and Chekwube, 2017;Lakoh et al., 2020). Such discrepancy suggests that CRA dissemination varies by region, and may be attributed to the variability in antibiotic choice, infection control, and prevention practices. Moreover, the use of carbapenems in clinical setting in Ghana has been reported as low, and its unavailability as an essential drug on the Ghanaian national health insurance scheme may be associated with low prescription (Labi et al., 2018). We speculate this low carbapenems usage is correlated with low resistance selective pressure which may explain the observed lower carbapenem resistance prevalence in our study. The 22.2% prevalence of colistin resistance among all Acinetobacter isolates as well as 12.5% among A. baumannii isolates in this study are high in comparison with other countries where the resistance prevalence was mostly at single digit levels for A. baumannii (Pormohammad et al., 2020). Such high resistance is alarming especially since colistin has become one of the last-resort antibiotics used for multidrug-resistant gram-negative bacterial infections. The acquired resistance mediated by plasmid originated mcr gene resulted in the global spread of the colistin resistance among Enterobacteriaceae (Wang et al., 2018;Wise et al., 2018). In A. baumannii, colistin resistance is not associated with the mcr gene, rather caused by the overexpressed PetN FIGURE 4 | Maximum parsimony tree of OXA-58-and OXA-420-containing plasmids. Two plasmids from the current study (highlighted in bold), 45 OXA-58-producing plasmids and one OXA-420-producing plasmid listed in PLSDB database were included. Bootstrap values are shown at each node.
Frontiers in Microbiology | www.frontiersin.org transferases mediated by two pathways, overexpressed pmrC from mutations in pmrCAB operon and overexpressed pmrC homolog eptA from the insertion of ISAba1 on the upstream (Trebosc et al., 2019). Further investigation remains warranted to elucidate the resistance mechanisms involved in non-baumannii Acinetobacter isolates.
Several global clones (GC) of A. baumannii have emerged in the last decades with GC2, represented by ST2, being the most widespread, followed by GC1, represented by ST1. Multidrug resistance and carbapenemase production are mostly associated with the spread of these specific clones (Hamidian and Nigro, 2019). Here, we showed that A. baumannii isolates recovered in Ghana have high genetic diversity, associated with 15 STs among which six are novel. The predominant GC2 was absent and only a single isolate belonged to GC1. ST78, also known as the "Italian clone" (Carretto et al., 2011), was the predominant sequence type detected in Ghana. However, neither ST1 nor ST78 isolates were associated with carbapenemase production. The CHDL producers found belong to ST103, ST107 and ST1472; Both of ST103, and ST107 were uncommon, while ST1472 is a novel type. OXA-23 production is the leading carbapenem resistance mechanism in A. baumannii, mostly associated with GC2 and GC1 (Hamidian and Nigro, 2019). Although ST103 strains were associated with NDM-2-producing isolates in Egypt, UAE, and Palestine (Kaase et al., 2011;Ghazawi et al., 2012;Sjölander et al., 2014), and an OXA-235-producing strain was reported in Australia (Hamidian et al., 2017), to the best of our knowledge this is the first study to identify ST103 associated with OXA-23 production (isolates Ab-B004d-c and Ab-D10a-a). Based on the occurrence of OXA-58 carbapenemase in several Acinetobacter species and in Proteus mirabilis (Fu et al., 2014;Feng et al., 2016;Lange et al., 2017;Matos et al., 2019), it has been suggested that horizontal gene transfer rather than clonal spread is the main mechanism of bla OXA−58 dissemination. This may also explain the detection of a novel sequence type, ST1472 (isolate Ab-C102). ST107 isolate Ab-C63 carried bla OXA−420 , a rare variant of bla OXA−58 that was first described in A. baumannii ST623 and ST32 clinical isolates in Nepal (Shrestha et al., 2015). Ab-C63 was genetically distinct from the Nepalese isolates and other isolates identified in France and Brazil, which had the same ST and produced different CHDLs including OXA-24, OXA-143 and OXA-231 (Jeannot et al., 2014;Rodrigues-Costa et al., 2019).
We identified two isogenic OXA-23 producers, Ab-B004dc and Ab-D10a-a. These two isolates were recovered from different patients in the same hospital, indicating a possible nosocomial outbreak. The ESBL gene bla CTX−M−15 was also found on the chromosome of both isolates. Third-generation cephalosporin resistance in A. baumannii results mainly from the production of ESBLs of PER, GES and VEB types, or the overexpression of intrinsic AmpC β-lactamases owing to the upstream insertion of specific sequences such as ISAba1 (Hamidian and Hall, 2013;Potron et al., 2015). The ESBL gene bla CTX−M−15 is the main mechanism of resistance to third-generation cephalosporins in Enterobacteriaceae, but not a common one in Acinetobacter. There are only a few report of its occurrence in A. baumannii isolates from India, Haiti, and Brazil (Shakil and Khan, 2010;Potron et al., 2011;Zago et al., 2016). Therefore, by detecting bla CTX−M−15 in isolates from Ghana, our study provides evidence of its wider spread among Acinetobacter species.
The bla OXA−23 gene is the most common carbapenemase gene in A. baumannii and is usually mobilized by Tn2006, Tn2007, Tn2008 (including its variants), Tn2009, Tn6549, and AbaR4 into the chromosome and plasmids. Tn2006 is the most abundant of these mobile elements (Hamidian and Nigro, 2019). Tn2007, which was identified in plasmids pAb-B004d-c_3 and pAb-D10a-a_3 is relatively uncommon with no plasmids listed on PLSDB and only one in GenBank (pAB14) carrying it. However, its detection has been reported in isolates from Algeria, France, Spain, China, and Tanzania (Corvec et al., 2007;Guerrero-Lozano et al., 2015;Wang et al., 2015;Kumburu et al., 2019). The identification of Tn2007 in samples from Ghana, within a plasmid that share a high level of sequence identity with pAB14 (identified in Algeria and Tanzania) strongly supports the importance of plasmids in the spread of carbapenemase genes, especially in Africa.
The bla OXA−58 -containing plasmid pAb-C102_1 (isolate Ab-C102) share the same Rep as well as the highest query cover and sequence homology with the draft genome of A. baumannii WB103, an isolate recovered from hospital wastewaters in Singapore and that also carry bla OXA−58 . It was not possible to compare the genetic structure between the two isolates because WB103 plasmid sequences are not assembled, but the findings suggest that pAb-C102_1 likely derived from a plasmid from WB103. Diverse genetic environments are described for bla OXA−58 . The gene is commonly embedded in structures like ISAba2:ISAba3-bla OXA−58 -ISAba3 and IS6 family element-ISAba3-bla OXA−58 -ISAba3. Usually, these structures are accompanied downstream by araC1 and lysE (Poirel and Nordmann, 2006b;Fu et al., 2014;Cameranesi et al., 2018); the former is involved in bla OXA−58 regulation (Fondi et al., 2010). Mobilization and acquisition of bla OXA−58 is driven by site-specific recombination at XerC/D-like sites (including mediation by Re27) rather than through its surrounding IS elements, leading to diverse structure arrangements (Poirel and Nordmann, 2006b;Cameranesi et al., 2018). We observed a similar pattern in pAb-C102_1, in which an ISAba3 element interrupted by a novel ISAjo2-like sequence was followed by ISAba3-like-araC1-lysE, a structure that commonly surrounds bla OXA−58 . In addition, a Re27-2 site was found next to the novel ISAjo2-like element. These results indicate that bla OXA−58 was acquired via a similar XerC/D-like site-specific recombination in pAb-C102_1, and that the second copy of the gene created in the process may have been lost. Moreover, different arrangements in insertion sequences upstream of bla OXA−58 were associated with its overexpression; compared with ISAba3 this resulted in a stronger promoter leading to higher carbapenem resistance levels (Chen et al., 2008(Chen et al., , 2010Fu et al., 2014). It is unclear whether the low carbapenem MIC levels in Ab-C102 is a consequence of ISAjo2-like-ISAba3 arrangement and a relatively weak bla OXA−58 promoter. Further investigations are needed to clarify this issue.
The gene bla OXA−420 has three nucleotide substitutions and a single amino acid substitution compared with bla OXA−58 , and was first identified in 16 A. baumannii isolates from Nepal (Shrestha et al., 2015). Our BLAST search identified A. baumannii strain ACN21, isolated from India, that also carry bla OXA−420 , indicating that this specific variant is mostly spread in South Asia. To the best of our knowledge this study is the first to detect a bla OXA−420 -carrying isolate outside of Asia. Isolate Ab-C63 is not genetically related to earlier isolates from Nepal and India. Moreover, the genetic context of bla OXA−420 in pAb-C63_1 is novel while those from India and Nepal are similar. The presence of a Re27-2 sequence, commonly associated with bla OXA−58 acquisition, near bla OXA−420 , suggests that this resistance gene also was acquired by XerC/D-mediated sitespecific recombination in Ab-C63.
One limitation of our study is the limited sample size. Isolates were collected at three hospitals only, and the number of isolates obtained from two of the hospitals was extremely low. Hence, the results may not reflect the whole situation in Ghana.
In this study, we have demonstrated that the prevalence of carbapenem resistance in Ghana is low and that there is a high level of genetic diversity among clinical isolates of Acinetobacter. Furthermore, we characterized, for the first time in Ghana, OXA-23-, OXA-58-and OXA-420-producing A. baumannii isolated belonging to ST103, ST1472 and ST107, respectively. By combining short-read and long-read WGS we revealed that bla OXA−23 was within Tn2007 in two plasmids (pAb-B004d-c_3 and pAb-D10a-a_3) and identified novel genetic structures surrounding bla OXA−58 and bla OXA−420 (novel plasmids pAb-C102_1 and pAb-C63_1, respectively). Altogether, our results show that genetically diverse and unique A. baumannii isolates, including OXA-carbapenemase producers, are circulating in Ghana, measures to strengthen and expand surveillance of antimicrobial resistance worldwide remains essential.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the ethics committee of the Faculty of Medicine, Tokyo Medical and Dental University (M2017-208) and the ethics committee of Noguchi Memorial Institute for Medical Research, University of Ghana (FWA 00001824). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

AUTHOR CONTRIBUTIONS
AAy, AAb, and RS conceived and designed the experiments. AAy, AK, MS, WS, SM, IP, MM, and RS performed the experiments. AAy, MS, TH, TS, SI, AAb, and RS analyzed the data. MS, KM, TH, TS, SI, AAb, and RS contributed to reagents, materials, and analysis tools. AAy, MS, AAb, and RS contributed to the writing of the manuscript. All authors contributed to the article and approved the submitted version.

FUNDING
This work was supported by the Japan Agency for Medical Research and Development (AMED, URL: http://www.amed. go.jp/en/) under Grant Numbers JP19fm0108010 (TH, TS, SI, and RS) and JP20fk0108133 (MS). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.