Insights into the Resistome and Phylogenomics of a ST195 Multidrug-Resistant Acinetobacter baumannii Clinical Isolate from the Czech Republic

Increasing antimicrobial resistance in nosocomial pathogens, such as Acinetobacter baumannii, is becoming a serious threat to public health. It is necessary to detect β-lactamase-producing microorganisms in clinical settings to be able to control the spread of carbapenem resistance. This study was conducted to evaluate the presence of β-lactamases in a selected clinical isolate of A. baumannii of ST2P/ST195Ox and to characterize possible enzymes, as well as its β-lactam resistome, using PCR and whole-genome sequencing analysis. PCR and sequencing confirmed that the isolate harbored five bla gene alleles, namely, blaADC-73, blaTEM-1, blaOXA-23, blaOXA-58 and blaOXA-66, as well as aminoglycosides, macrolides, sulfonamides and tetracyclines resistance determinants, which were either chromosomally and/or plasmid located. Furthermore, a gene order comparison using MAUVE alignment showed multiple changes compared with the clinical isolate of Malaysian A. baumannii AC30 genome and 76 regions with high homology. This study suggests that resistance to β-lactams in this A. baumannii isolate is mainly due to an overproduction of β-lactamases in combination with other resistance mechanism (efflux pump system).


Introduction
The emergence of carbapenem resistance in Enterobacteriaceae (e.g., Escherichia coli, Klebsiella pneumoniae) and Gram-negative non-fermenting (GNNF) bacteria (e.g., Acinetobacter baumannii, Pseudomonas aeruginosa) is a serious public health concern, mainly due to the production of carbapenemases, which ultimately limit therapeutic possibilities [1].
During evolution, bacteria have developed various genetic and biochemical mechanisms that allow them to resist the strong inhibitory effects of antimicrobials. At present, the most serious and feared mechanism leading to a rapid increase in resistance is considered to be the property of bacteria to exchange plasmids carrying genes encoding β-lactamases.
Several β-lactam resistance mechanisms have been described in bacteria, including enzymatic inactivation by β-lactamases, alterations in antibiotic binding sites, changes in cell permeability, modifications of metabolic pathways and an overexpression of efflux pumps [2]. Regarding the multi-drug resistance (MDR) phenotype in A. baumannii, many studies describe that it is related to the production of natural β-lactamases, reduced cell permeability and/or hyperexpression of efflux pumps [3]. Furthermore, it was found that the MDR phenotype in A. baumannii was significantly attributed to a resistance island carrying transposons, integrons and other mobile genetic elements, and also that efflux systems can provide resistance to a particular antibiotic to a lesser extent than the inactivating antibiotic enzymes [4].
According to the EARS-Net [5], the resistance of Enterobacteriaceae and GNNF bacteria to carbapenems was extremely different. For example, in 2019, Acinetobacter spp. showed a 31% resistance to carbapenems in the Czech Republic but reached more than 55% in Slovakia. Carbapenem resistance in Acinetobacter spp. is currently most critical in the following countries: Croatia (92%), Greece (92%) and Romania (88%) (available at: http: //atlas.ecdc.europa.eu/public/index.aspx; accessed on 31 March 2021).
Due to the increasing clinical importance of many β-lactamases, their detection is also essential in routine microbiological practice. This study was mainly focused on detecting genes encoding β-lactamases, as well as other resistance genes, and associated insertion sequences in a clinical A. baumannii strain 11069/A.

Bacterial Strain and Antimicrobial Susceptibility Testing
The A. baumannii strain 11069/A (imported from Hungary) was primarily isolated from the eye after a gunshot wound of a patient (man, 61-year-old) staying in the surgical intensive care unit of the University Hospital in Olomouc, Czech Republic.

Detection of β-Lactamase Genes by PCR
The presence of β-lactamase genes was tested using PCR with genomic DNA that was extracted from the isolate under previously reported conditions [7][8][9][10][11][12][13][14] and using primers shown in Table S1 in the Supplementary Materials.

Whole-Genome Sequencing, Annotation and Molecular Analysis
Genomic DNA from A. baumannii isolate 11069/A was extracted and purified using (NucleoSpin®Tissue, Macherey Nagel, Duerren, Germany). Fragment library was constructed using a Nextera XT kit followed by sequencing using Illumina HiSeq according to the manufacturer's instructions. The raw reads were assembled using a de novo method implemented in Geneious Prime [15] set to medium to high sensitivity of default setting (Biomatters, Auckland, New Zealand).
Open reading frames were captured using the Geneious software analysis tool Glimmer and sequences were searched against the public sequence databases using the BLASTn. Sequence features were annotated using the Geneious Prime annotation transfer tool.
Repeat regions, as well as insertion sequences (IS), were identified and analyzed using Geneious Prime. The predicted open reading frames (ORFs) located upstream and downstream of the ARGs were found using Glimmer and these ORFs were subsequently subjected to BLAST analysis. Predicted ORFs that showed significant sequence similarity to known IS were annotated as IS elements.

Genome Analysis and Phylogenetic Analysis
The genetic map of the assembled genome of A. baumannii 11069/A was visualized and compared with A. baumannii ST195 Ox strain AC30 using Mauve Genome Alignment (MCM algorithm) through the Geneious plugin with the default setting. An A. baumannii strain AC30 was used as the reference genome for sequence comparison.
Progressive Mauve with default setting in Geneious Prime was then used to compare the genome of the A. baumannii 11069/A strain with the genome sequences of other A. baumannii ST195 Ox strains downloaded from GenBank.
Further maximum likelihood phylogenetic tree was generated and visualized using PhyML implemented in Geneious with 100 bootstrap replicates.

Data Availability
Whole-genome assembly was deposited in GenBank under the accession number JAHBBB000000000.

Antimicrobial Susceptibility Testing
The isolate showed high resistance to specific antimicrobials and susceptibility was detected only for tigecycline and colistin (Table 1).

Whole-Genome Analysis of MDR A. baumannii 11069/A including Antibiotic Resistance Genes and Associated Genes
A total of 628,395 reads were assembled with the Geneious assembler. A. baumannii 11069/A draft genome sequence was assembled into 339 contigs (>201 bp long, base quality scores >Q50).
MLST revealed that the strain 11069/A belonged to the sequence type ST2 P /ST195 Ox (Table 1). Subsequently, genome sequence analysis confirmed that the A. baumannii isolate harbored five different β-lactamase genes, including bla ADC-73 , bla TEM-1 , bla OXA-23 , bla OXA-58 and bla OXA-66 . In all cases, a 100% nucleotide identity to the variants of the genes deposited in the BLDB database was confirmed (data not shown).
Our genome analyses demonstrated that the strain 11069/A possessed a total of 13 antibiotic resistance genes, including tet(B) (tetracycline resistance), sul2 (sulfonamide resistance), msr(E) and mph(E) (macrolide resistance), and four aminoglycoside resistance genes (aph(3 )-Ia, aph(3")-Ib, armA and aph(6)-Id) ( Table 2). The bla ADC-73 gene was found to be associated with an ISAba1 element ( Figure 1-I). Sequencing the data further indicated that bla TEM-1 was located between two inversely oriented copies of IS6-like elements of the IS26 family with perfect identical terminal inverted repeats of 14 bp on each side (Figure 1-II). This region showed a 100% nucleotide sequence identity to sequences from A. baumannii strain WM99c (GenBank accession number, GAN-CP031743). Nonetheless, CP031743 had a deletion of 12,201-bp AbGRI3 resistance region (GAN-KX011026) carrying three ARGs (armA, msr(E) and mph(E)), as well as protein involved in an initiation of plasmid replication (RepB) in the adjacent downstream region compared to the sequence of our isolate. Further, BLASTn was used to detect related plasmid sequences in GenBank. The right end of the contig showed a high level of similarity (56.7% coverage, 100% identity) to a 20,139-bp armA/msr(E)/mph(E)carrying plasmid (p2BJAB07104 (GAN-CP003907)) identified in a clinical isolate of A. baumannii ( Figure S2 in the Supplementary Materials). In addition, the BLAST results also showed a similar sequence identity (100%) with the (1) A. baumannii strain VB2107 chromosome (CP051474), (2) antibiotic resistance island AbGRI3 (KX011026) as well as transposon ∆Tn6279 chromosomally inserted in strain A072 (KT354507) (Figure 2).
The truncated insertion sequence ISAba1 was found upstream of the bla OXA-23 gene (Figure 1-III). ISAba3 family transposase, IS30 family transposase (both upstream) and an ISAba3-like were identified downstream of the bla OXA-58 gene (Figure 1-IV). BLASTn analysis showed a high level of similarity (9.5% coverage, 100% identity) to a 29,823-bp bla OXA-58 /msr(E)/mph(E)-carrying plasmid (pABIR (GAN-EU294228)) ( Figure S3   Other abbreviations symbols were Δ, genes that are truncated; IS, insertion sequence; TnpB, IS66 family insertion sequence element accessory protein TnpB. Promoter prediction was performed using Softberry BPROM webtool [19]. Putative gene annotations were assigned using a BLAST search. Other abbreviations symbols were ∆, genes that are truncated; IS, insertion sequence; TnpB, IS66 family insertion sequence element accessory protein TnpB. Promoter prediction was performed using Softberry BPROM webtool [19]. Putative gene annotations were assigned using a BLAST search.  Promoter prediction was performed using Softberry BPROM webtool [19]. Putative gene annotations were assigned using a BLAST search. Further, an investigation of the mutation in the carO gene and efflux pumps involved in carbapenem resistance in A. baumannii was performed. Sequencing revealed an altered nucleotide sequence by studying the carO outer-membrane protein gene, resulting in a 99.6% (change of one amino acid: glycine to aspartic acid at position 184) amino acid identity, respectively. No deletions or insertions were observed throughout the sequence. The adeABC efflux pump, as well as the presence of the adeRS genes, were not confirmed. On the contrary, we confirmed two additional resistance-nodulation cell division type efflux systems, namely, AdeIJK and AdeFGH. Further analysis revealed that the tested A. baumannii isolate had OprD (OprE3) porin, with no mutation over its coding frame and promoter sequences. In this study, the transcript level of efflux pumps and OprD was not studied. Genomic sequence analysis revealed the presence of the blc gene potentially involved in antibiotic resistance, but no β-lactamase genes were found in its vicinity.
A genomic comparison between A. baumannii strain AC30 (3.8 Mbp) and A. baumannii strain 11069/A (3.8 Mbp) genomes was conducted in order to identify genomic islands or regions of genomic plasticity. The Mauve alignment showed 76 regions with high homology named Local Colinear Blocks (LCBs), which encompassed 96.07 and 94.06% of their genomes, respectively (97.1% nucleotide sequence identity) (Figure 3). The region suggested to be inserted by the IS26 is marked with gray color. Genes and their transcriptional orientations are shown by horizontal arrows. Inverted repeat sequences are represented by an asterisk (*). Promoter prediction was performed using Softberry BPROM webtool [19]. Putative gene annotations were assigned using a BLAST search.
Further, an investigation of the mutation in the carO gene and efflux pumps involved in carbapenem resistance in A. baumannii was performed. Sequencing revealed an altered nucleotide sequence by studying the carO outer-membrane protein gene, resulting in a 99.6% (change of one amino acid: glycine to aspartic acid at position 184) amino acid identity, respectively. No deletions or insertions were observed throughout the sequence. The adeABC efflux pump, as well as the presence of the adeRS genes, were not confirmed. On the contrary, we confirmed two additional resistance-nodulation cell division type efflux systems, namely, AdeIJK and AdeFGH. Further analysis revealed that the tested A. baumannii isolate had OprD (OprE3) porin, with no mutation over its coding frame and promoter sequences. In this study, the transcript level of efflux pumps and OprD was not studied. Genomic sequence analysis revealed the presence of the blc gene potentially involved in antibiotic resistance, but no β-lactamase genes were found in its vicinity.
A genomic comparison between A. baumannii strain AC30 (3.8 Mbp) and A. baumannii strain 11069/A (3.8 Mbp) genomes was conducted in order to identify genomic islands or regions of genomic plasticity. The Mauve alignment showed 76 regions with high homology named Local Colinear Blocks (LCBs), which encompassed 96.07 and 94.06% of their genomes, respectively (97.1% nucleotide sequence identity) (Figure 3).

Phylogenetic Analysis of A. baumannii 11069/A
The phylogenetic tree diagram obtained using MAUVE and PhyML showed that the 11069/A strain is the most closely related strain to a clinical isolate of Malaysian A. baumannii AC30 (Figure 4).  Table S2-S4 in the Supplementary Materials.

Phylogenetic Analysis of A. Baumannii 11069/A.
The phylogenetic tree diagram obtained using MAUVE and PhyML showed that the 11069/A strain is the most closely related strain to a clinical isolate of Malaysian A. baumannii AC30 (Figure 4).    Table S5 in the Supplementary Materials.

Discussion
Pan-drug resistance is becoming an increasing threat worldwide [20]. This is mainly related to the fact that more and more carbapenemases are plasmid-encoded and harbored on integrons or transposons. In addition, there are recent concerns about the transmission of β-lactamase-producing bacteria in farm animals that have occurred in many European and Asian countries [21][22][23].
In the present study, a clinical isolate of A. baumannii with known phenotypic resistance was examined for the presence of most β-lactamase genes (mainly the largest and clinically significant groups), which were described only in GNNF bacteria. A PCR, performed using different primer combinations, confirmed the presence of different bla genes, specifically bla ADC , bla TEM , bla OXA-23-like , bla OXA-51-like and bla OXA-58-like . Homologous regions in nucleotide sequences of bla PER-like and bla SHV-like genes were used for designing primers with Primer3 (Geneious Prime). To detect some β-lactamase genes, a combination of two pairs of primers was used to cover all or almost all the allelic variants of these genes described in the BLDB database that also served to assess the sequence variant. The PER-F/R primers were aimed at detecting 11 subtypes of the bla PER genes but were unable to distinguish four allelic variants (PER-2/-6/-12/-14). The SHV-F/R primers can be used to detect 195 SHV subtypes, while not being able to distinguish five subtypes (SHV-39/-112/-202/-208/-210).
To identify the genes responsible for β-lactamase activity in the isolate, short-read WGS analysis was conducted. The analysis revealed that the isolate belonged to ST2 P /ST195 Ox , a high-risk clone that is known to be present in various (Eastern) Asian countries and was found to contain carbapenemases [24]. In addition, a carbapenem-resistant A. baumannii ST195 Ox had been isolated from a Pakistani male living in Denmark and from a Norwegian patient who was hospitalized abroad, suggesting the importation of this clinical strain [25,26]. Further, five bla genes were detected in the isolate of A. baumannii, including bla ADC-73 , bla TEM-1 , bla OXA-23 , bla OXA-58 and bla OXA-66 . These genes were found to be co-occurring with other resistance genes, such as tet(B), sul2, msr(E), mph(E), aph(3 )-Ia, aph(3")-Ib, armA and aph(6)-Id), together conferring an MDR profile to β-lactams, aminoglycosides, macrolides, sulfonamides and tetracyclines. Moreover, in our case, one type of AmpC (ampicillin chromosomal cephalosporinase) β-lactamase, bla ADC-73 , was found. Elsewhere, chromosomal-encoded AmpC β-lactamase has been reported to have very little activity against carbapenems, but may act synergistically with other resistance mechanisms, such as a diminished production of oprD, increased activity of ampC and increased expression of several efflux systems [27]. However, no novel β-lactamase genes were identified. All the above-mentioned ARGs have been identified in a chromosome and/or plasmid. However, due to some short contigs, such as contig 36 encompassing bla OXA-23 , we were unable to determine precisely whether the gene is chromosomal-or plasmid-encoded.
Truncated ISAba1 was detected upstream of the bla ADC-73 and bla OXA-23 genes, whereas no insertion sequence was observed upstream of bla OXA-66 . ISAba1 confers promoter sequences to the bla genes, resulting in β-lactamase overproduction in A. baumannii [28]. Concerning the bla OXA-58 gene, this gene was bracketed by insertion sequences (ISAba3-IS30-bla OXA-58 -ISAba3-like), both of which have been shown to provide promoter sequences enhancing the expression of bla OXA-58 [14]. In addition, the bla OXA-58 gene appears to be plasmid-borne. Poirel et al. reported that the bla OXA-58 gene is usually plasmid-borne and are associated with insertion sequences [29]. The analysis of the region surrounding the bla TEM-1 gene revealed two copies of IS26 in opposite orientations, including inverted repeats, which may play an essential role in transposition ( Figure 2). Furthermore, the aph(3 )-Ia gene, located between two copies of truncated IS26 with no duplicated target sequences, showed a 100% identity to sequences from A. baumannii strain TP3 (GAN-CP060013). However, this fragment likely resulted from a homologous recombination event. It has been reported that the IS26 element plays a role in the dissemination of chromosomal-or plasmid-localized resistance genes, including β-lactamases, by facilitating their mobilization between different genetic regions [30,31]. Taken together, these genes and the presence of insertion sequences such as ISAba1, ISAba3, ISAba3-like, IS26 and IS30 are important in the development of carbapenem-resistant A. baumannii.
Study of the β-lactam resistome in the isolate of interest confirmed the presence of the carO gene. A mutated version of this gene was observed. The type of mutation identified in the porin gene included only substitution (glycine to aspartic acid at position 184; G184D; numbering of amino acid position followed the GAN-CP059354), which probably has little effect on the function of the outer membrane protein. Zhu et al. reported that the CarO mutation is considered to be one of the molecular mechanisms that affect imipenem resistance in A. baumannii, indicating that the major differences were amino acid deletion at position 133 and insertion at 140-141 and 154-156 [32]. Furthermore, only one difference was observed at the 48th position (threonine → serine), which had little relation to antibiotic resistance.
Another major outer membrane protein associated with imipenem resistance is OprD [10]. OprD has been reported to be a key porin for the uptake of carbapenems into A. baumannii [33]. Our isolate was found to carry the oprD gene, whereas no modifications and mutations of this gene were found (the level of gene expression of OprD porin was not investigated). On the other hand, the abnormalities or loss of OprD porin represent one of the major mechanisms of carbapenem resistance in P. aeruginosa [34]. Estepa et al. have described many abnormalities in the oprD gene, including substitutions, insertions, deletions and premature stop codons.
Another important antimicrobial resistance mechanism present in A. baumannii is hyperexpression of various efflux pumps belonging to the resistance-nodulation cell division family (AdeABC, AdeFGH and AdeIJK) [35]. The adeABC operon was not confirmed in this strain. Further, two efflux systems were identified in the A. baumannii isolate, namely AdeFGH and AdeIJK, but the level of gene expression of efflux pumps was not investigated here. It has been reported that the efflux pumps AdeABC and AdeFGH are implicated in acquired resistance, while AdeIJK is important for intrinsic resistance [36][37][38]. According to the literature [39], strains containing the AdeIJK pump confer resistance to various antibiotics including carbapenems, whereas AdeFGH has been found to be important, for example, in resistance to chloramphenicol and fluoroquinolones. Therefore, in addition to the presence of ISAbal/bla OXA-23 , ISAbal/bla ADC-73, ISAba3-IS30-bla OXA-58 -ISAba3-like, bla OXA-66 and IS26/putative mobile element/bla TEM-1 /IS26, these pumps are other important mechanisms that occur in this strain.
In some enterobacteria, the blc gene (encoding an outer membrane lipoprotein) has been found to be in the vicinity of the bla AmpC gene that encodes a serine β-lactamase of chromosomal origin [40,41]. The isolates were characterized by carrying the transposonlike element (ISEcp1-bla CMY -blc-sugE) and composite transposon (IS26-ISEcp1-bla ACC -gdhA-IS26), wherein ISEcp1 represents an alternative promoter region regulating the overexpression of the bla gene. In our case, we were able to confirm only the presence of the blc gene, but there was no bla AmpC gene in its vicinity.
To date, the presence of β-lactamase genes in A. baumannii has been described in numerous papers. However, our findings provide more comprehensive information about the presence of various types of antimicrobial resistance genes, including β-lactamases, in a clinical isolate of A. baumannii, which may play a role in the development of antibiotic resistance at the clinical level and have important implications for the assessment of risks to human health. In addition, our results showed a match between PCR detection and WGS regarding the types of demonstrated β-lactamases, but WGS was able to detect other mechanisms of resistance to β-lactams unrelated to β-lactamases. Therefore, a PCR assay specifically aimed at detecting some β-lactamases in a selected group of microorganisms can be useful for laboratories that do not have next-generation sequencing platforms or do not have sufficient financial resources.
The increasing prevalence of carbapenem-resistant bacteria possessing different carbapenemase genes underscores the implementation of strict control measures to prevent their spread. Further, it is increasingly discussed in the literature that the emergence of resistance is associated with the uptake of resistance determinants throughout the genome. It has been found that the multi-drug resistance phenotype in A. baumannii is significantly attributed to the resistance island carrying transposons, integrons and other mobile genetic elements, while it has also been clarified that efflux systems may confer resistance to particular antibiotics to a lesser extent than drug-inactivating enzymes [4].

Conclusions
In the present study, five different β-lactamases and other resistance genes were detected together in an isolate of A. baumannii. The insertion sequences, ISAbal, ISAba3, ISAba3-like, IS26 and IS30, detected upstream and downstream of the β-lactamase genes, may be involved in their dissemination and expression processes. Altogether, these data indicate that the multi-drug resistance to β-lactams in this isolate may be a result of the overexpression of the β-lactamase genes and other resistance mechanisms (the efflux pump system). The high prevalence of β-lactamase-producing A. baumannii isolates is of great concern. Therefore, sustained investigation seems essential to monitor β-lactamaseproducing bacteria in patients with nosocomial and community-acquired infections.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/life11101079/s1, Table S1: Primers used to detect the β-lactamase genes using PCR; Table  S2: Distribution of locally colinear blocks (LCBs) of Acinetobacter baumannii AC30 relative to strain 11069/A; Table S3: The presence of gaps of the Acinetobacter strains studied; Table S4: The presence of mobile genetic elements including insertion sequences (ISs) identified on the gaps of the Acinetobacter strains studied; Table S5: Genomes of Acinetobacter baumannii ST195 Ox isolates used in this study; Figure S1: PCR products obtained by amplification of β-lactamase genes using specific primers in a clinical isolate 11069/A of Acinetobacter baumannii; Figure S2: Linear comparison of the complete nucleotide sequence of plasmid p2BJAB07104 from Acinetobacter baumannii BJAB07104 with putative plasmid found in this study; Figure S3: Linear comparison of the complete nucleotide sequence of plasmid pABIR from Acinetobacter baumannii with putative plasmid found in this study.