Genomic and phenotypic analyses of multidrug-resistant Acinetobacter baumannii NCCP 16007 isolated from a patient with a urinary tract infection

ABSTRACT Polymyxin B (PMB) is increasingly used as a last-line antibiotic; however, the emergence of PMB resistance is a serious threat to global health. Here, a total of 40 Acinetobacter baumannii clinical isolates were collected to screen for PMB-resistant strains. Several clinical isolates including NCCP 16007 were far more resistant to PMB (MIC: 128–256 μg/ml) than the ATCC 17978 strain (MIC: 2 μg/ml) and appeared to possess resistance to broad-spectrum antibiotics including meropenem and 12 others. Four highly PMB-resistant strains possessed point mutations in the histidine kinase PmrB, leading to an increased expression of pmrC encoding a phosphoethanolamine transferase. Whole-genome analyses revealed that the NCCP 16007 stain had acquired two additional copies of the pmrC gene with phage integrase and 13 antibiotic resistance genes (ARGs) from other pathogens, including Klebsiella pneumoniae and Pseudomonas aeruginosa. The GC ratios of the ARGs (50–60%) were higher than that of the chromosomal backbone (39.06%), further supporting the horizontal gene transfer of ARGs. Comparative genomics with other multidrug-resistant A. baumannii strains revealed that the NCCP 16007 strain has many additional ARGs and has lost several virulence factors including Csu pili and heme oxygenase but exhibited high pathogenicity in Galleria mellonella-infection models. The observation of condensed biofilm through confocal and scanning electron microscopy suggested that the NCCP 16007 strain may possess high adhesion capacity during urinary tract infection. Therefore, our genomic and phenotypic analyses suggested that the multidrug-resistant A. baumannii NCCP 16007 strain possesses high genome plasticity, natural transformation ability, and pathogenicity.


Introduction
Acinetobacter baumannii is a nosocomial pathogen often linked to catheter-related bloodstream infections and ventilator-associated pneumonia infections in immunocompromised patients [1,2]. Due to its ability to develop and spread antibiotic resistance genes (ARGs), the incidence of A. baumannii infection in clinical environments has significantly increased in recent years [3]. Multidrug-resistant (i.e. resistant to three or more antimicrobial agents) A. baumannii (MRAB) infection is linked to accelerated morbidity and mortality worldwide [4]. Carbapenems and broadspectrum β-lactam antimicrobial agents including meropenem, imipenem, and doripenem have been considered for the treatment of MRAB [5]. However, a nosocomial outbreak of carbapenem-resistant A. baumannii (CRAB) clonal complex 92 (international clone II) with blaOXA-23 encoding a carbapenemhydrolyzing oxacillinase has been reported in South Korea and many other countries [6][7][8][9]. Due to a lack of effective treatment and limited therapeutic options, conventional drugs such as polymyxin B (PMB) and fosfomycin are still used for MRAB and CRAB infections [10,11].
Polymyxin B (PMB) is a cationic and amphipathic therapeutic compound that consists of a cyclic polypeptide linked to a tripeptide linear chain with a fatty acid tail [12]. Although PMB has been linked to some adverse side effects such as neurotoxicity and nephrotoxicity, PMB is still used as a last-line treatment alone or in combination with carbapenem and vancomycin [13]. PMB binds to the lipid A of lipooligosaccharide (LOS) and lipopolysaccharide (LPS) in gram-negative bacteria, which modifies the charge of the negatively charged outer membrane (OM) [14]. LPS is then destabilized, thereby increasing the membrane's permeability, which causes leakage of components in the cytoplasm and ultimately leads to cell death [15]. Major bacterial PMB resistance occurs when lipid A is modified by the pmrC-encoded phosphoethanolamine (PetN) transferase and the arnT-encoded 4-amino-4-deoxy-L-arabinose (L-Ara4N) transferase. Both enzymes respectively add positively charged PetN and L-Ara4N to the lipid A, which reduces the binding of PMB to the OM [16,17]. In many PMB-resistant bacteria, several different point mutations found in the pmrB gene encoding a histidine kinase might prolong activation of the downstream transcriptional regulator PmrA by phosphorylation of an aspartate residue [18--18-20]. The phosphorylated PmrA binds to the promoter regions of the pmrCAB and arnBCADTEF operons, which increases the expression of the PmrC and ArnT enzymes, respectively [21,22]. Activation of the arnT operon (arnBCADTET, also called as the pmrHFIJKLM operon) adds LAra4N groups to lipid A only in several Gram-negative bacteria such as Escherichia coli, Yersinia pestis, Pseudomonas aeruginosa, and Ralstonia solanacearum, but not in Citrobacter rodentium and A. baumannii [23,24].
The plasmid-borne mcr-1 gene, which encodes a PetN transferase, was originally detected in Escherichia coli SHP45 isolated from a pig in China [25,26]. The mcr-1 gene product, which shares a 34% amino acid similarity with the PmrC of A. baumannii, might be transferred to other bacteria including A. baumannii and K. pneumoniae through horizontal gene transfer (HGT) via mobile genetic elements such as insertion sequences (IS), transposons, and conjugative plasmids [27]. IS elements can be classified into different families based on their transposase homologies, target specificity, composition, and length of their terminal inverted repeats [28]. A total of 59 different IS elements wave been identified. In many environmental and clinical A. baumannii strains (https://www-is.bio toul.fr/index.php), of which ISAbaI, belonging to the IS4 family located upstream of the genes encoding βlactamase and PetN transferase, appears to be crucial for ARG transfer [19,29]. In this study, the genome of multidrug-resistant A. baumannii NCCP 16007 isolated from a patient with a urinary tract infection was analyzed. We hypothesized that A. baumannii NCCP 16007 acquired ARGs and IS elements from other bacteria, resulting in variation of GC-content within those regions. Interestingly, two copies of pmrC genes with phage integrases were identified, in addition to 13 acquired ARGs. Our genomic analyses suggested that HGT events from other pathogens or plasmids including K. pneumoniae, P. aeruginosa, and Shigella flexneri were responsible for the multidrug-resistant (MDR) phenotype of the A. baumannii NCCP 16007 strain.

Bacterial strains and culture conditions
Forty A. baumannii clinical isolates were collected from two different facilities (National Culture Collection for Pathogens, Sungkyunkwan University, South Korea). The bacterial strains used in this study are listed in Table S1. These clinical strains were obtained from 2004 to 2013 in South Korea. The year, region, source, and genotypes of the clinical strains were provided by the National Culture Collection for Pathogens (see our Table 1). The genome of the Lab-WT ATCC 17978 reference strain is available in the NCBI database (Accession number: CP000521). All A. baumannii strains studied herein were grown at 37°C in Luria-Bertani (LB) broth with constant shaking at 220 rpm and aeration. All strains were cultured overnight (O/N) for 16 hours, after which each of the O/N cell cultures was diluted 1/100 and cultured for three additional hours to ensure that all experiments were conducted under the mid-exponential phase (optical density at 600 nm: 0.4).

Antimicrobial susceptibility testing
All antibiotics were purchased from Sigma-Aldrich, USA. A total of 14 antibiotics were selected to test the antibiotic resistance of Lab-WT and four PMB-resistant A. baumannii strains (NCCP 15995, NCCP 15996, NCCP 16007, and F-1629). The antimicrobial agents used in this study were polymyxin B, colistin, ampicillin, kanamycin, gentamicin, tetracycline, rifampicin,

Polymerase chain reaction (PCR) and sequencing of the pmrB gene
The primers used for PCR and sequencing of the pmrB gene are specified in

Quantitative reverse transcription-PCR (qRT-PCR)
Total RNA of A. baumannii was isolated from 5 ml of cell cultures in the mid-exponential growth phase (optical density at 600 nm: 0.4) using the RNeasy Mini Kit (QIAGEN, USA). The qRT-PCR primers used to determine the relative expression of the pmrC, ompA, bfmR, and pilA genes are listed in Table S2. All procedures were conducted in triplicate from at least three independent cultures. The relative expression levels were compared with the 16s rRNA expression level of each strain.

Galleria mellonella-infection model
Galleria mellonella larvae were obtained from SWORM, South Korea. Prior to injection, healthy G. mellonella larvae were starved to increase susceptibility to infection by suppressing the immune response for 4 h at 20°C. The larvae were then placed on ice and injected in the last left proleg with 10 µl of 106 CFU of either Lab-WT or NCCP 16007 strain with-/-without PMB (1/2 MIC; 1 μg/ml for Lab-WT, 128 μg/ml for NCCP 16007) using 31-gauge 6 mm syringes (BD, USA). A total of 15 G. mellonella larvae per condition were then tested after inoculation. The larvae were incubated in Petri dishes (SPL Life Sciences, South Korea) containing 100 mg of wheat bran powder (MG Natural, South Korea) at 37°C and inspected and scored every 24 h for death, failure to move in response to touch, and melanization.

Measurement of biofilm formation
Biofilms were grown in confocal dishes (SPL Life Sciences, South Korea) at 37°C for 24 h in LB broth. The biofilm cells were stained with FilmTracer™ SYPRO Ruby (Invitrogen, USA) for 30 min at room temperature, protected from light, and then washed with distilled water. The observed confocal laser scanning microscopy (CLSM; Carl Zeiss, Germany) images were analyzed and modified using the Zen 2.1 (Blue edition; Carl Zeiss, Germany) software.
LB broth in sterile 96-well microtiter plates (SPL Life Sciences, South Korea) was inoculated in triplicate with each overnight LB-grown culture and diluted 1:100 in LB broth. The volume of the cells was determined by converting the OD600 value of the O/N cells. Uninoculated LB broth was used as a negative control. The microtiter plates were then incubated at 37°C for 24 h. After removing planktonic cells, the biofilm biomass was stained with crystal violet and solubilized with 95% ethanol (v/v), after which its absorbance was measured at 550 nm.

Field-emission scanning electron microscopy (FE-SEM) analysis
Mid-exponential growth phase NCCP 16007 strain cells were used for SEM analyses. The cells were first fixed with low-strength Karnovsky's solution (2% paraformaldehyde, 2.5% glutaraldehyde, and 0.1 M phosphate buffer, final pH 7.2) for 4 h. Secondary fixation was done using 2% osmium tetroxide solution at 4°C for 2 h. These fixed samples were gradually dehydrated with ethanol (30%, 50%, 70%, 80%, 90%, 100%) for 10 min each and placed on an aluminum stub overnight to dry at room temperature. These samples were then coated with platinum and analyzed using a fieldemission scanning electron microscope (Quanta 250 FEG; FEI, USA).

Esterase activity assay using 4-nitrophenyl acetate
4-nitrophenyl acetate, the substrate used in esterase activity assay, was first dissolved in methanol (10 mg/ ml) (Sigma-Aldrich, USA). An aliquot (40 μl) of the cell cultures was then mixed with 120 μl phosphate buffer at pH 7.2 and 40 μl of the substrate within 96-well microtiter plates. After incubation at 37°C for 15 min, the mixtures were analyzed at 405 nm, following the hydrolysis of 4-nitrophenyl acetate. Distilled water was used as a negative control.
Modulation of the OM charge by the PmrA/B two-component system-activated PmrC appeared to confer PMB resistance to pathogenic A. baumannii strains [14]. It was speculated that a mutation in the pmrB gene leads to higher expression of the downstream pmrC gene, which might lead to higher PMB MICs in the four strains that exhibited this phenotype [18][19][20]. DNA analysis of PCR products with the pmrB-specific primer pairs indicated the occurrence of either point mutations or deletion in our tested strains (Figure 1(a) and Table S2). Only the NCCP 16007 strain had four amino acid deletions (Δ27-30) in the first transmembrane (TM) region. All four PMB-resistant strains exhibited the same point mutation in PmrB (A444V) and carried different point mutations in distinct regions (novel F26L in TM, NCCP 16007; novel A138T in the periplasmic domain, NCCP 15996; and P170L in HAMP domain, F-1629). Although the mechanisms by which these mutations affect PmrAphosphorylation remain unclear, our qRT-PCR analysis showed that the four tested strains had 20-to 150-fold higher pmrC expression levels compared to the Lab-WT strain (Figure 1(c)). Notably, given that the NCCP 16007 strain possesses the MDR phenotype, the highest PMB MIC, and the highest level of pmrC expression (150-fold increase), its genome should be investigated to dissect the detailed mechanisms of PMB resistance and MDR phenotypes.

Whole-genome analysis of multidrug-resistant A. baumannii NCCP 16007
The genome of NCCP 16007 was assembled into three contigs 3,884,786 bp in length with 3,618 coding regions, consisting of one chromosomal backbone (3,854,994 bp) and the pNC1 (8,031 bp) and pNC2 (21,761 bp) plasmids (Table S3). When compared to the average length of the 4,536 A. baumannii genomes (3,974,730 bp) listed in the NCBI database, the chromosomal length of NCCP 16007 is 120 kb shorter and its GC content (39%) is consistent with those of other A. baumannii strains. The NCCP 16007 strain whose genome size is shorter than the Lab-WT strain (ATCC 17978, 3,976,747 bp), lacked genes associated with cytosine-specific DNA methyltransferase (e.g. dcm), type IV secretion (e.g., trbL and tadA), and colicin V secretion (e.g. cvaA and cvaB), as well as other functionally unknown genes. The NCCP 16007 strain had the same 16S rRNA as ATCC 17978 but displayed a different average nucleotide identify (ANI) value (97.74%) from the Lab-WT strain, suggesting that many of its genes might have been horizontally transferred from other bacteria ( Figure S1). Unlike two of the plasmids present in the Lab-WT strain (pAB1: 13,408 bp; pAB2: 11,302 bp), the plasmids of the NCCP 16007 strain were distinctively characterized by their size and presence of genes encoding a TonB-dependent receptor and a phage integrase (Table S3).
Interestingly, the NCCP 16007 strain harbors a total of three copies of the pmrC gene that are 97% identical to each other, whereas the Lab-WT possesses only one pmrCAB operon. The pmrC and eptA genes have been reported as the same functional genes encoding PetN transferases [23,31]. The chromosome contains a pmrC gene in the pmrCAB operon and two pmrC gene copies are located next to the phage integrase genes in both the chromosome and pNC1 plasmid ( Table 2). The 150-fold increase in pmrC expression observed in this strain might be due to its location next to the IS element, which contains a strong promoter sequence [32,33] and 3 copies of the pmrC gene with 98% homology with the Lab-WT at the gene level (Figure 1(c) and Table S2). Our genome analyses therefore suggest that both the mutation of the pmrB gene and the presence of three pmrC gene copies resulted in high levels of pmrC expression and consequently high PMB MIC values among all tested strains (Table 1).

Acquired antibiotic resistance genes and insertion sequence elements
Our genome analyses demonstrated that the NCCP 16007 strain possessed a total of 32 ARGs linked to six different classes of antibiotics including carbapenem, β-lactam, gentamicin, and macrolide ( Table 2). Several ARGs in the genome of the NCCP 16007 strain could have contributed to the MDR phenotype against all 14 antibiotics tested ( Table 1). The GC ratios (50--60%) of many ARGs including sul1, aac (6ʹ)-lb3, aadA1, aph (3")-lb, and blaTEM-1D were much higher than that of the chromosomal backbone (39.06%), leading us to speculate that the ARGs may have been acquired from other bacterial species through HGT ( Figure 2). Therefore, we analyzed the homology of the ARGs and IS elements to determine whether the ARGs originated from other bacterial species using the Center for Genomic Epidemiology database and IS finder (see Materials and Methods section, Figure 2(b), Table 2, and Table S4). Fourteen of the 32 ARGs (including qacE, which encodes the efflux pump of Salmonella sp.) appeared to be originated from other pathogens, with gene homologies exceeding 98% identities ( Table  2). The similarity between the GC ratio of the original bacteria and that of each ARG further suggested that the NCCP 16007 strain acquired the genes from clinical environments (Table 2).
Additionally, a total of 19 different IS elements next to the ARGs might originate from many different pathogens including Proteus vulgaris and K. pneumoniae (Table S4). One IS91 family element was found to be identical (100%) to the ISVsa3 of Vibrio salmonicida, which is adjacent to the tet(R) and the aph(6)-ld genes (Figure 2(b-I) and Table 2). Several different IS elements (IS91, IS26, IS5, and Intl1) with high sequence identities (>99%) with those of other pathogenic bacteria were also present near the sul1, qacE, aadA1, catB8, and aac (6ʹ)-lb3 genes (Figure 2(b-II) and Table 2). The blaTEM-1D gene encoding a β-lactamase from E. coli was located between two IS6 family elements (Figure 2b-III). Unlike the single copy of the ISAbaI gene that was adjacent to the sul2 gene encoding a dihydropteroate synthases in the Lab-WT (Table S5), a total of 15 copies of the ISAbaI gene were scattered across the chromosome without the sul2 gene in the NCCP 16007. Several ISAbaI genes are located near the blaADC and blaOXA-23 genes (Table S4). Therefore, the extraordinary MDR capacity of the NCCP 16007 strain against the 14 antibiotics tested herein was due to the large numbers of ARGs and IS elements in its genome.
The genomes of the strains tested herein (Lab-WT and NCCP 16007) as well as 13 other well-known clinical isolates were investigated to identify ARGs using the Center for Genomic Epidemiology database and the Mauve software ( Figure 3 and Figure S2). Interestingly, the A. baumannii 1656-2 and ACICU strains, which were respectively isolated from the sputum of a hospitalized patient in South Korea and the cerebrospinal fluid of a patient in Italy, also exhibited multiple copies of the pmrC gene ( Figure S2). The AYE strain also possessed a total of 19 commonly acquired ARGs, and unlike the other studied strains, the AYE strain obtained the trimethoprim resistance genes dfrA1 and dfrA10 from E. coli (Figure 3 and Figure S2). In contrast to the uropathogenic UPAB1 strain, which does not possess acquired ARGs, the NCCP 16007 strain isolated from a urinary tract infection patient had a total of 13 ARGs (except for the qacE efflux pump gene), a large number compared to 13 other clinical strains (Figure 3).

High pathogenicity in Galleria mellonella-infection models
The virulence factor genes of the NCCP 16007 strain were compared with those of the Lab-WT, other MRAB, and antibiotic-sensitive A. baumannii strains ( Figure S3). The NCCP 16007 strain appeared to have  fewer virulence factors (76%, 44 out of 58 genes) and lacked several important genes that encode Csu pili and heme oxygenase, whereas the known MRAB strains A. baumannii TCDT-AB0715 and TYTH-1 exhibited many virulence-related genes (98%, 57 out of 58 genes) ( Figure S3). However, a Galleria mellonella-infection study revealed that the NCCP 16007 strain was highly pathogenic in vivo (Figure 4(a)). A total of 15 G. mellonella larvae were tested per condition after inoculation with 10 6 CFU of either the Lab-WT or NCCP 16007 strains with-/-without PMB (1/2 MIC; 1 μg/ml for Lab-WT, 128 μg/ml for NCCP 16007). The larvae exposed to the NCCP 16007 strain displayed a 90% mortality rate within five days, whereas only a 65% mortality rate was observed in the Lab-WTexposed larvae (Figure 4(a)). PMB treatment (1/2 MIC) reduced the larvae death rates from 90% to 45% in the NCCP 16007 infection samples; however, the PMB has no effect on infection outcome for the sensitive WT strain probably due to low in vivo PMB (1/2 MIC) efficacy against WT strain (Figure 4(a)). We assumed that although the NCCP 16007 strain has fewer virulence factors than Lab-WT and other clinical strains, the high mortality rate in the larvae infection study would be due to the high expression of pathogenicityassociated genes. The relative expressions of the outer membrane protein, ompA, the response regulator of biofilm formation, bfmR, and the main subunit of pili, pilA genes were respectively 1.3-fold, 1.5-fold, and fourfold higher than those in the Lab-WT, confirming the high virulence of the NCCP 16007 strain (Figure 4(b)). Additionally, virulence-associated biofilm formation was measured in the Lab-WT and NCCP 16007 strains ( Figure 5). The crystal violet assay for biofilm quantification indicated that the NCCP 16007 strain produced twofold more biofilm than the Lab-WT strain ( Figure  5(a)). Interestingly, unlike the Lab-WT strain, the NCCP 16007 strain formed thick, condensed, and pili-like biofilms  within 24 h ( Figure 5(b, c)). The PMB-treated NCCP 16007 strain at 1/2 MIC (128 μg/ml) produced longer pili-like biofilms than the untreated samples ( Figure 5(c)). Moreover, a twofold increase in esterase activity compared to the Lab-WT strain suggested that the NCCP 16007 strain might lead to more invasive infections and dissemination in the host cells ( Figure S4). Therefore, the high mortality and biofilm production of the NCCP 16007 strain may translate to more severe infectious diseases in clinical settings.

Discussion
Pathogenic bacteria possess a variety of molecular strategies to survive under antibiotic selective pressure. These strategies include random mutations, acquisition of ARGs through HGT, and activation of mobile DNA elements [34]. Acinetobacter species have a high genome plasticity and natural transformation ability, which could allow them to evolve by exchanging ARGs under antibiotic conditions [35]. Acinetobacter baylyi ADP1 has been reported to possess a remarkable natural competence ability, which is more than 100-fold higher than that of E. coli cells treated with CaCl2 [36]. Surprisingly, the MDR A. baumannii NCCP 16007 strain likely acquired many ARGs and IS elements through HGT, which resulted in high levels of resistance to most of the tested antibiotics (Table 2 and Table S4). The potential MDR mechanisms in the NCCP 16007 strain are summarized in Figure 6. The β-lactamases that inhibit β-lactam antibiotics (e.g. ampicillin and meropenem) can be divided into four types (classes A, B, C, and D) based on sequence similarities [37]. The blaTEM-1D gene product, which was first discovered in E. coli, belongs to the plasmidencoded constitutive broad-spectrum class A βlactamases (Table 2 and Figure 6) [38]. The NCCP 16007 strain possessed resistance to ampicillin (MIC > 512 μg/ml) and meropenem (MIC, 16 μg/ml, Table 1) not only due to a mutation in the oprD gene (Table 2 and Figure 6) but also due to the presence of several βlactamases, including the class D β-lactamase (blaOXA-23 and blaOXA-66 genes) and class A β-lactamase genes (GC ratio 48%) derived from E. coli (blaTEM-1D). Bacterial survival strategies against aminoglycoside antibiotics include i) inactivation of aminoglycosides by modifying enzymes, ii) enhanced production of efflux pumps, iii) reduction of membrane permeability, and iv) interference with aminoglycoside binding via modification of the 16S ribosomal RNA [39]. The NCCP 16007 strain possesses several HGT-mediated aminoglycoside-resistance genes from E. coli (aph(6)ld, GC ratio 53%), P. aeruginosa (aac(6ʹ)-lb3, aadA1, and aph(3")-lb, GC ratio 53%), and K. pneumoniae (armA, GC ratio 29%), which could modify aminoglycosides by adding phosphate, acetyl, adenyl, or methyl groups ( Figure 6 and Table 2). Macrolides with a macrocyclic lactone ring (e.g. erythromycin and clarithromycin) bind a 50S ribosome subunit with a specific target in the 23S rRNA and diverse ribosomal proteins [40]. Moreover, the msr(E) gene in the genome of the NCCP 16007 strain, which originated via HGT from P. multocida (GC ratio 39%), could protect ribosomes from macrolides [41] (Figure 6 and Table 2).
PmrAB-mediated lipid A modification participates in PMB resistance by changing membrane stability [42]. In the Enterobacteriaceae, P. aeruginosa, and A. baumannii, the PetN transferase PmrC, regulated by the PmrAB two-component system, reduces PMB affinity to LPS [19]. In our study, four PMB-resistant A. baumannii clinical isolates exhibited point mutations in the pmrB gene and high pmrC expression (Figure 1). It is not clear to what extent did the mutations in the pmrB affected the expression of pmrC and which PmrB domain was predominantly involved in PMB resistance. However, the pmrC gene expression of the four strains, 20-to 150fold higher than that of the Lab-WT strain, supported the hypothesis that lipid A modifications would prevent PMB from binding to the OM (Figure 1).
Many uropathogens initiate a urinary tract infection (UTI) using surface appendages, capsular polysaccharides, adhesins, and pili, which form biofilms by promoting bacteria-bacteria interactions [43,44]. A UTI is generally caused by contamination around the urethra by uropathogens in the intestine, followed by the colonization of the urethra and subsequent mobilization of the pathogens into the bladder, requiring appendages such as pili [45]. In this study, A. baumannii NCCP 16007 isolated from a patient with UTI also formed pili-like biofilms and the high expression of pilA encoding the type IV pili major subunit was determined (Figures 4 and Figures 5). Paradoxically, the NCCP 16007 strain does not possess the Csu pili-related genes (csuA, csuB, csuC, csuD, and csuE) needed to form biofilms, but the expression of the top regulator bfmR was 1.5-fold higher than that of the Lab-WT strain ( Figure S3 and Figure 4). These results indicate that Csu pili may not be essential for biofilm formation or that they could be replaced by other pili systems such as type IV pili [46].
In conclusion, four of the 40 strains of clinical strains isolated in South Korea were highly resistant to PMB, of which the genomic and phenotypic characteristics of NCCP 16007 with the highest pmrC expression were analyzed. We assumed that the NCCP 16007 strain has high resistance to all tested 14 antibiotics due to the HGT-mediated ARGs from other pathogen and plasmids. Interestingly, genomic analysis revealed that the NCCP 16007 uropathogenic strain acquired a total of 13 ARGs from other pathogens, a large number of ARGs compared with other clinical strains. Phenotypic analyses revealed that the NCCP 16007 strain exhibited an MDR phenotype, higher pathogenicity, and superior biofilm structure compared with the wild-type. Therefore, A. baumannii NCCP 16007 possesses a remarkable repertoire of MDR strategies for PMB resistance, including a large array of horizontally transmitted ARGs and extremely high levels of pmrC gene expression.