Genomic Landscape Reveals Chromosomally-Mediated Antimicrobial Resistome and Virulome of a High-Risk International Clone II Acinetobacter baumannii AB073 from Thailand

This study utilized integrative bioinformatics' tools together with phenotypic assays to understand the whole-genome features of a carbapenem-resistant international clone II Acinetobacter baumannii AB073. Overall, we found the isolate to be resistant to seven antibiotic classes, penicillins, β-lactam/β-lactamase inhibitor combinations, cephalosporins, carbapenems, aminoglycosides, fluoroquinolones, and folate pathway antagonists. These resistance phenotypes are related to various chromosomal-located antibiotic resistance determinants involved in different mechanisms such as reduced permeability, antibiotic target protection, antibiotic target alteration, antibiotic inactivation, and antibiotic efflux. IC2 A. baumannii AB073 could not transfer antibiotic resistance by conjugation experiments. Likewise, mobilome analysis found that AB073 did not carry genetic determinants involving horizontal gene transfer. Moreover, this isolate also carried multiple genes associated with the ability of iron uptake, biofilm formation, immune invasion, virulence regulations, and serum resistance. In addition, the genomic epidemiological study showed that AB073-like strains were successful pathogens widespread in various geographic locations and clinical sources. In conclusion, the comprehensive analysis demonstrated that AB073 contained multiple genomic determinants which were important characteristics to classify this isolate as a successful international clone II obtained from Thailand.


Introduction
One of the problematic pathogens belonging to the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) is A. baumannii due to its ability to "escape" killing by antibiotics and its high burden of healthcare-associated infections (HAIs) [1,2].According to the WHO priority pathogens list, carbapenem-resistant A. baumannii (CRAB) is also designated as a critical priority pathogen for encouraging the research and development of new antimicrobials [3,4].HAIs caused by A. baumannii include ventilator-associated pneumonia, bloodstream, skin, and urinary tract infections, and secondary meningitis [5].
Molecular epidemiology investigations have classifed nine international clonal lineages (IC1-9) of A. baumannii, which represent genetically distinct populations spreading in various geographic locations [6][7][8].Te ICs distributed worldwide comprise three major clones (IC I, II, and III) also known as global clones (GC I, II, and III) [6,7].Like in European countries, IC2 was reported as a dominant clone that successfully spread in many regions of Tailand [7,9,10].A. baumannii is an important carrier of antibiotic resistance genes located on both chromosomal or plasmid DNA, which can be exchanged by horizontal transfer or multiplied by clonal expansions [7].Te majority of virulence factors found in A. baumannii are capsular polysaccharides, outer membrane proteins (OMPs), protein secretion systems, lipopolysaccharides (LPS), phospholipases, proteases, and iron-acquisition systems [11].
Whole-genome sequencing (WGS) technologies can be applied to monitor public health surveillance and molecular epidemiology along with putative mechanisms involving virulence and drug resistance [12].In this study, we aimed to use the whole-genome sequence to understand genomicbased epidemiology, and identify genes associated with antimicrobial resistance and pathogenicity of an IC2 A. baumannii AB073 isolated from Tailand.

Whole-Genome Sequencing and General Genome
Analysis.Te total genomic DNA sample of A. baumannii strain AB073 was extracted and purifed using a Wizard ® Genomic DNA Purifcation Kit (Promega, USA).Te purifed genomic DNA was quantifed and qualifed using an Agilent ® 2100 Bioanalyzer, (Agilent Technologies, Inc., Santa Clara, CA, United States).Te DNA library was generated by following the Nextera XT DNA Library Prep Kit Reference Guide prior to paired-end sequencing on a MiSeq sequencer, according to the manufacturer's instructions (Illumina).Te raw reads were subjected to quality trimming, de novo assembling, and assembledcontig correcting by using bioinformatics software, Trim Galore v0.6.7 [21], Unicycler v0.4.8 [22], and Pilon v1.23 [23], respectively.To identify the relative genomes of AB073, a similar genome fnder was conducted [24].Te resulting contigs were ordered in the Multi-CSAR web server [25] by using the relative genomes of AB073 as references.

Global Health, Epidemiology and Genomics
Likewise, many genes associated with bioflm formation were found such as genes that contributed to AdeFGH efux pump (adeFGH), bioflm-associated protein (Bap), Csu pili (csuABCDE), and PNG (pgaABCD) (Table 2).Tere were seven known virulence genes (lpsB and lpxABCDLM) related to LPS synthesis which were responsible for immune invasion.AB073 was predicted to harbor a few genes encoded by phospholipase C and D (plc1, plc2, and plcD) and an outer membrane adhesin such as OmpA.Te two pairs of genes including abaIR and bfmRS were identifed and classifed as virulence regulon genes in AB073.A single gene, pbpG related to serum resistance was also found to be located on the AB073 chromosome (Table 2).
On the other hand, according to the immunogenic polysaccharide typing, AB073 carried 2 loci conferring the biogenesis of lipooligosaccharide outer core (OCL1) and capsular polysaccharide (KL3) (Figure 1).We also predicted biosynthetic gene clusters and found 6 putative clusters of AB073 (cluster I to VI) related to known biosynthetic gene clusters with sizes ranging from 18.4 to 77.8 kb (Figure 1 and Table S2).Among them, cluster I was related to a redoxcofactor content, clusters II and VI appeared to be associated with siderophore productions, cluster III was found to be a beta-lactone synthesis element, and the last two clusters (IV and V) were classifed as aryl polyene biogenesis operons (Table S2).
Calling core genes among AB073 and other 2,647 genomes, retrieved from the PubMLST database, revealed that AB073 was found to be an international clone II (IC II) as represented in Figure 3.We also performed wholegenome SNP calculations to track the close isolates of AB073 against both complete genome and draft genome databases as shown in Tables 1 and S3, respectively.Tese results demonstrated that AB073-like strains could be isolated from various clinical samples such as blood, sputum, urine, and nasopharynx and are obtained from many countries such as Malaysia, China, and Belgium (Tables 1  and S3).
3.6.In Silico Survey of AB073 Mobilome.By using Mobi-leElementFinder, only three insertion sequences were identifed in AB073 (ISAba24, IS26, and ISAba26).While, searching for bacterial mobile genetic elements in oriTfnder revealed that AB073 did not carry any genes responsible for relaxase, type 4 secretion system, and type IV coupling protein.
To confrm these fndings, we conducted the conjugational transfer assay and failed to detect the transfer of resistance to imipenem, kanamycin, and ticarcillin between AB073 and antibiotic-sensitive recipients.

Discussion and Conclusions
Multidrug-resistant Acinetobacter baumannii and carbapenem-resistant Acinetobacter baumannii (MDRAB and CRAB) are known as successful pathogens responsible for nosocomial infections, especially for ventilatorassociated pneumonia [5].In this study, we used WGS together with phenotypic assays to retrospectively investigate the epidemiology and genomic landscape related to drug resistance and virulence factors of A. baumannii AB073 obtained from a patient with ventilator-associated pneumonia.Our fndings corroborate the results of previous studies which reported that the most common global or international clone of carbapenemase-producing A. baumannii belongs to IC2 (CCoxf92/CCpas 2) in Africa, Europe, and other neighboring Asian countries such as Malaysia, China, and Vietnam [7][8][9][10]42].Trough indepth analysis, we found that many strains closely related to AB073 (SNP diferences of less than 100) could be isolated from various clinical sources, countries, and years which tended to be successful pathogens.By comparing AB073 with a set of 10 CRAB isolates revealed that ST2international clone 2 was the most dominant clone distributed in Tailand.On the other hand, many AB073-like strains carried two copies of gdhB detected by in silico analysis which was the barrier for assignation of the MLST Oxford scheme of A. baumannii [43].
In this work, we found that AB073 harbored many genes that contributed to multiple resistance mechanisms located on the chromosome.Major resistance mechanisms of AB073 included reduced permeability, antibiotic target protection, antibiotic target alteration, antibiotic inactivation, and antibiotic efux.Among fve putative resistance mechanisms, an antibiotic inactivation by hydrolyzing enzymes was the common pathway related to CRAB phenotype as documented previously [7].Mobile genetic elements (MGEs), including insertion sequences (ISs), transposons (Tn), and conjugative plasmids (T4SS and T4CP) play an essential role in transferring antimicrobial resistance gene among different kinds of pathogens or between the chromosome and plasmids in a strain [44].In contrast, we did not detect antimicrobial resistance genes embedded in MGEs of AB073.Tis in silico analysis was consistent with conjugation experiments, whereas AB073 does not transfer any antibiotic resistance to receipts.Moreover, in silico plasmids typing also identifed only a small GR2 plasmid which is known to be an LN_1 plasmid lineage.Tis plasmid lineage was reported to be smaller than 10 kb in size distributed in diverse STs' A. baumannii strains and is nonmobilizable [33,34].Together, these results suggested the genetic stability of AB073 resistome.
Te pathogenicity of the genome sequenced in this work and 10 genomes retrieved from the NCBI database was exacerbated by the presence of diverse genes involving a quorum sensing system, bioflm formation, iron uptake, immune invasion, toxic enzyme production, and serum resistance.Nutrient iron is known to be a key factor for A. baumannii growth and persistence within infection sites [45,46].Likewise, the strain studied in this work contained two gene clusters encoding acinetoferrins, and many genes involved in acinetobactin and heme synthesis which are important for AB073 to utilize iron.In addition, bacterial persistent invasion of the human immune system is associated with capsules, cell-surface polysaccharides, and the bioflm [47].
In conclusion, many resistance genes identifed on the chromosome of AB073 could not transfer and resulted in genome stablity.However, AB073-like strains were found in diferent sources and countries which illustrated the ability to survive, distribute, and to be successful pathogenic strains.In support of these fndings, AB073 virulome revealed the diverse key components involved in pathogenicity and successful colonizer.

Figure 1 :
Figure 1: Te genetic arrangement of the essential genes on the chromosome of A. baumannii AB073.Acquired antibiotic resistance genes, prophage genomes, locus of cell-surface polysaccharide (KL and OCL), and secondary metabolite gene clusters found in various regions on the chromosome of AB073 are labeled with various colors.

Figure 2 :
Figure 2: Te comparative-genomic analysis of AB073 compared to additional CRAB obtained from Tailand.(a) A WGS-based phylogeny presented with a complete antimicrobial resistome and metadata of AB073 versus selected 10 CRAB isolated from Tailand.(b) A phylogenetic tree based on WGS sequence together with a complete virulome of AB073 compared to selected 10 CRAB collected from Tailand.Gray boxes represent several resistance mechanisms, virulence classes, and metadata.

Figure 3 :
Figure 3: A GrapeTree clustering based on A. baumannii cgMLST of AB073 and other genomes deposited in the PubMLST database.Nodes difering by fewer than 7 alleles were collapsed together.Each node corresponds to a single ST oxf obtained from the MLST Oxford scheme, with the diameter scaled to the number of strains and number of samples contained in them.A dashed-line circle represents the AB073 characterized in this study.A gray box illustrates the ST oxf with a number of isolates.

Table 1 :
General genome properties of A. baumannii AB073 and its relative strains.
ND, not detected; STpas, MLST Pasteur scheme; SToxf, MLST Oxford scheme; CC, clonal complex.* Two copies of gdhB were detected by in silico determination based on the MLST Oxford scheme.* *

Table 2 :
Computational identifcation of genes encoding for virulence factors of AB073.