Alteration in the Morphological and Transcriptomic Profiles of Acinetobacter baumannii after Exposure to Colistin

Acinetobacter baumannii is often highly resistant to multiple antimicrobials, posing a risk of treatment failure, and colistin is a “last resort” for treatment of the bacterial infection. However, colistin resistance is easily developed when the bacteria are exposed to the drug, and a comprehensive analysis of colistin-mediated changes in colistin-susceptible and -resistant A. baumannii is needed. In this study, using an isogenic pair of colistin-susceptible and -resistant A. baumannii isolates, alterations in morphologic and transcriptomic characteristics associated with colistin resistance were revealed. Whole-genome sequencing showed that the resistant isolate harbored a PmrBL208F mutation conferring colistin resistance, and all other single-nucleotide alterations were located in intergenic regions. Using scanning electron microscopy, it was determined that the colistin-resistant mutant had a shorter cell length than the parental isolate, and filamented cells were found when both isolates were exposed to the inhibitory concentration of colistin. When the isolates were treated with inhibitory concentrations of colistin, more than 80% of the genes were upregulated, including genes associated with antioxidative stress response pathways. The results elucidate the morphological difference between the colistin-susceptible and -resistant isolates and different colistin-mediated responses in A. baumannii isolates depending on their susceptibility to this drug.


Introduction
Acinetobacter baumannii is a clinically important Gram-negative human pathogen that causes various infections, mainly in immunocompromised patients hospitalized in intensive care units [1].This bacterial species is successful in nosocomial environments due to its ability to persist on dry surfaces, to resist disinfectants, and to acquire genes for antimicrobial resistance.By displaying high rates of resistance to multiple drugs, including carbapenems, A. baumannii has become one of the largest threats to patients with bacterial infections by critically limiting their treatment options [1].The emergence of multidrug-resistant A. baumannii led to the resurgence of old drugs [2,3].
Polymyxins, including colistin, are cyclic lipopeptide antimicrobials produced by the soil bacterium Paenibacillus polymyxa [4].Although colistin showed significant activity against various Gram-negative pathogens, the use of the drug was stopped in the 1970s due to its nephro-and neurotoxicity [5], until it was recalled for use against multidrug-resistant bacteria.Amphiphilic colistin electrostatically interacts with polyanionic lipopolysaccharides (LPSs) in the bacterial membrane, displacing the divalent cations from the lipid A component of LPSs and thereby disrupting the outer membrane of Gram-negative bacteria [3].
Two main mechanisms of colistin resistance by alterations in the chromosomal gene have been characterized in A. baumannii through: (i) inactivation of the lipid A biosynthetic pathway-associated genes, resulting in complete loss of the LPSs [6], and (ii) overproduction of enzymes modifying the lipid A, such as phosphoethanolamine, decreasing the net charge of the outer membrane by adding a cationic group [7].Since the total loss of LPSs imposes a high biological cost upon the bacterial host [8], the former mechanism is rarely identified, but the latter one is common in clinical settings.Chromosomal mutations in the pmrA or pmrB genes, encoding the PmrAB two-component system, cause overproduction of PmrC phosphoethanolamine transferase, which is responsible for modifying lipid A and conferring colistin resistance upon A. baumannii.Some studies have reported amino acid alterations in PmrC, which also confers colistin resistance, but the actual role of the alterations was not clearly demonstrated [7,9].Supplementarily, possible involvement of the multidrug efflux pump overproduction, such as EmrAB, was proposed to be associated with colistin resistance [10].In addition, through a horizontal gene transfer, a plasmid-borne gene for colistin resistance, called mobilized colistin resistance (mcr), has been reported in Escherichia coli isolates in China [11].The mcr-1 gene-carrying plasmid could be introduced into A. baumannii on a bench, resulting in reduced susceptibility to colistin [12]; however, no studies have reported A. baumannii clinical isolates harboring the gene [13].
In this study, using an isogenic pair of colistin-susceptible and colistin-resistant isolates, new insights into the unique signature of colistin resistance and, especially, the effects of colistin treatment on both isogenic isolates were obtained.The physical, genomic, transcriptomic, and colistin-induced transcriptomic changes were evaluated in these isolates when exposed to subinhibitory or inhibitory concentrations of colistin.

Bacterial Isolates
A clinical A. baumannii isolate Z0317AB0082 was recovered from a transtracheal aspiration sample of a patient hospitalized in 2017 in South Korea.From the parental isolate, the isogenic mutant Z0317AB0082-R was obtained on Szybalski gradient media with 0-100 µg/mL colistin.Briefly, Szybalski gradient agar was prepared in a range between 0 and 100 µg/mL of colistin (Sigma-Aldrich, Saint Louis, MO, USA), and an overnight culture of the parental isolate was applied onto the gradient agar and cultured overnight at 37 • C to recover colonies of spontaneous mutants.

Antimicrobial Susceptibility Testing
Disk diffusion testing for drugs other than colistin was conducted using antimicrobial disks (Becton Dickinson, Sparks, MD, USA) on cationic-adjusted Mueller-Hinton (MH) agar (Difco Laboratories, Detroit, MI, USA) according to Clinical and Laboratory Standards Institute (CLSI) guidelines [14].Minimal inhibitory concentrations (MICs) of colistin were determined by the microdilution method using cationic-adjusted MH broth according to CLSI guidelines and interpreted through the criteria [15].Both E. coli ATCC 25922 and A. baumannii ATCC 19606 were used as quality control isolates for susceptibility testing, as well the mcr-1-positive E. coli CREC-527 strain harboring the mcr-1 gene [16].The colony forming units per milliliter in each well, containing a specific concentration of colistin, were determined, and for the spontaneous mutants, the heteroresistance phenotype was assessed using the manual of Sherman et al. [17].

Multilocus Sequence Typing
Multilocus sequence typing (MLST) was carried out according to the Oxford scheme [18].Briefly, fragments of seven housekeeping genes (gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD) were amplified by PCR, and the amplicons were Sanger-sequenced to determine the allele numbers.The sequence type of the isolate was determined using the PubMLST database (http://pubmlst.org,accessed on 27 March 2023).

Determination of Colistin-Resistance-Associated Gene Sequences
The pmrCAB operon and the genes for the lipid A biosynthesis system were amplified by PCR, and the nucleic acid sequences were determined by using Sanger sequencing.The primers used for the analysis were derived from Lesho et al. [19] for the pmrC, pmrA, and pmrB genes and Chen et al. [20] for the lpxA, lpxC, and lpxD genes.Total RNA from both A. baumannii isolates was extracted from cultures in the exponential growth phase in MH broth (optical density at 600 nm = 0.8) using the RNeasy Mini Kit (Qiagen Inc., Hilden, Germany).The expression of the pmrC, pmrA, and pmrB genes was quantified by RT-qPCR [19] and normalized to that of the rpoB gene [21].RT-qPCR reactions were performed on an Applied Biosystems™ 7500 Fast Dx Real-Time PCR instrument (Applied Biosystems, Waltham, MA, USA) using the One-Step Real-Time RT-PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA).Each experiment was performed in triplicates at least twice.

Whole-Genome Sequencing
Total DNAs of both the parental Z0317AB0082 and the colistin-resistant isogenic mutant Z0317AB0082-R A. baumannii isolates were prepared from bacterial cells grown overnight in MH broth, quality-checked using agarose gel electrophoresis, and quantified using the Qubit (Invitrogen, Waltham, MA, USA) system.The total DNA was subjected to library preparation and sequencing on PacBio single-molecule real-time (SMRT) cells (Pacific Biosciences, Menlo Park, CA, USA).The PacBio reads were retrieved from the raw data using PacBio's SMRT Analysis software (ver.2.3.0.) and quality control filters; a quality score of 85 or greater and a length of 13 kbp or greater were used to trim the set of reads to a manageable size of 300 Mbp.These reads were combined with available Illumina HiSeq data using Prooveread (ver.2.14) with default parameters.Using Bowtie 2 (ver.2.3.5.), mapped reads were obtained from the Illumina reads and the resulting file was applied to the variant calling software FreeBayes (ver.1.3.4).

Comparative Transcriptomics
Using TRIzol (Invitrogen), total RNAs were extracted from the parental A. baumannii Z0317AB0082 and its isogenic mutant Z0317AB0082-R isolates that had been induced for 30 min with either 2 µg/mL for both isolates or 64 µg/mL colistin for the isogenic mutant Z0317AB0082-R.Ribosomal RNA was eliminated using the NEB Nextra RNA Depletion Kit (New England Biolabs, Ipswich, MA, USA).The RNA integrity number was evaluated using the BioAnalyzer 2100 RNA Pico Chip (Agilent Technologies, Santa Clara, CA, USA), and the concentration was evaluated using Nanodrop.The RNA library was prepared with 2 µg total RNA using a TruSeq RNA Library Prep Kit (Illumina), and its quality was controlled using TapeStation HS D5000 Screening Tape (Agilent Technologies) and LightCycler qPCR.The sequencing data were 2 Gb per sample.Using the read-trimmed raw reads from Illumina HiSeq with a minimum length of 50 bp, Bowtie 2 (ver.2.3.5.) was employed for mapping, and the value of fragments per kilobase of transcript per million mapped reads was used to normalize the sequencing depth and transcript length.Differentially expressed gene analysis was conducted using the DEGseq package of R.

Scanning Electron Microscopy (SEM)
For SEM, bacteria were prefixed in 2% paraformaldehyde (Sigma-Aldrich) and 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) to prevent autolysis.To minimize chemical reactions between pre-and postfixation states of the sample, the specimens were washed thrice with the same buffer as that in the fixative solution and postfixed with 1% osmium tetroxide.After washing thrice with deionized water, ascending concentrations of ethanol (50%, 70%, 80%, 90%, 95%, and 100%) were used for dehydration, which was later substituted with hexamethyldisilazane (Sigma-Aldrich).The specimens were ion-coated with platinum and observed under a field-emission scanning electron microscope (JSM-7800F, JEOL, Japan) at an acceleration voltage of 15 kV and a working distance of 5-12 mm.

Statistical Analysis
Data are presented as means ± standard errors (SEs) for each group.Differences in quantitative measurement were assessed by Student's t test via Microsoft Excel, and a p value of <0.05 was considered significant.
Table 1.Antimicrobial susceptibility of the parental strain Z0317AB0082 and the colistin-resistant isogenic mutant Z0317AB0082-R.

Antimicrobial Agents
Antimicrobial Susceptibility Z0317AB0082 Z0317AB0082-R The colistin MIC for the colistin-susceptible parental isolate Z0317AB0082 was determined to be 2 µg/mL by the broth microdilution method, and that of the colistin-resistant isogenic mutant Z0317AB0082-R was 64 µg/mL.Both the parental isolate and the colistinresistant isogenic mutant had an identical antibiogram except for colistin (Table 1).The pmrC, pmrA, and pmrB genes of the pmrCAB operon and the lpxA, lpxC, and lpxD genes of the lipid A biosynthesis pathway were amplified by PCR, sequenced, and aligned against the nucleic acid sequences of the parental isolate.For all tested colonies, the only alteration noted was pmrB C625T , resulting in the PmrB L208F alteration.Only one of the mutants was chosen for further evaluation.

Genomic Features of the Z0317AB0082 Parental Isolate and the Colistin-Resistant Isogenic Mutant
The genome of the parental A. baumannii Z0317AB0082 isolate comprised a 4,012,716 bp long chromosome with two cryptic plasmids 100,260 bp and 8731 bp long.The isolate carried the intrinsic bla OXA-66 gene, encoding oxacillinase, and the bla ADC-25 -like gene, encoding cephalosporinase, in the chromosome, without any upstream insertion sequences.Another intrinsic ant(3 ′′ )-II gene, encoding the aminoglycoside-modifying enzyme [24], was also found in the chromosome.As an acquired resistance gene, two copies of the bla OXA-23 gene were found 2968 kbp apart in the chromosome, conferring high-level resistance to the carbapenems.In the quinolone-resistance-determining region, the mutations S83L in DNA gyrase subunit A and S80L in topoisomerase IV subunit A were identified, corresponding to the resistance phenotype to ciprofloxacin.
Compared with the nucleic acid sequence of the parental Z0317AB0082 isolate, the Z0317AB0082-R isolate harbored 13 single-nucleotide polymorphisms (SNPs) and one double T insertion (Table 2).Of the 13 SNPs, 9 were in the middle of six coding sequences (CDSs), and all except pmrB C625T were in the CDSs of transferases of insertion sequences.The other SNPs, including one in a plasmid, were in intergenic regions including the insertion case (Table 2).

Morphological Alterations Due to the PmrB Mutation
The parental and colistin-resistant isogenic mutant isolates were either untreated or treated for 30 min with subinhibitory or inhibitory concentrations of colistin, following which they were imaged using SEM (Figure 1).The untreated colistin-resistant A. baumannii isolate, with an average length of 0.976 µm, was 10% shorter than the untreated parental isolate (1.082 µm long).Both displayed a smooth and intact surface (Figure 1A,C).All colistin-treated groups, regardless of the concentration, showed shrunken cells and wrinkled cell surfaces along with cell debris (Figure 1B,D,E).The parental isolate treated with an inhibitory concentration of colistin 2 µg/mL presented a wrinkled cell surface along with bacterial filamentation.Cells were shrunken, and cell debris was observed (Figure 1B).The colistin-resistant isogenic isolate treated with the inhibitory concentration of colistin 64 µg/mL displayed a morphology similar to the correspondingly treated parental isolate: collocation of the filamented bacterial cells and shrunken cells with cell debris (Figure 1E), while that treated with 2 µg/mL of colistin had only shrunken cells (Figure 1D).

Effects of Colistin on the Transcriptomic Profiles of the Colistin-Susceptible and -Resistant Isolates
By RT-qPCR, the level of expression of pmrC, pmrA, and pmrB genes in the colistinresistant isogenic mutant were determined to be 1.3-, 1.7-, and 1.1-fold higher, respectively, than those in the parental isolate (Figure 2) and none of the differences were statistically significant.These three genes were overexpressed when cells were induced by inhibitory concentrations of colistin.A 30 min treatment with 2 µg/mL of colistin induced a 1.5-, 2.3-, and 2.3-fold increase in the expression of pmrC, pmrA, and pmrB in the parental isolate; however, no such overexpression was detected in the colistin-resistant isolate, for which 2 µg/mL was a subinhibitory concentration.At an inhibitory concentration of 64 mg/L of colistin, the colistin-resistant isolate showed a 2.1-, 1.7-, and 2.2-fold increase in expression of pmrC, pmrA, and pmrB, respectively.The transcriptional status of 3731 genes was determined, and fragments per kilobase of transcript per million mapped reads values were calculated and normalized using the average expression level of six 16S rDNAs.In parental A. baumannii treated with inhibitory concentrations of colistin, a total of 2435 genes (over 80% of the total genes) were upregulated (Table 3), and they were associated with various functions, including responses to different stresses, such as oxidation, ultraviolet radiation, chemicals, and heavy metals.In the colistin-resistant isolate, 2629 genes were upregulated upon exposure to inhibitory concentrations of colistin, while 2021 were upregulated upon exposure to subinhibitory concentrations (Table 3).Among these, 199 genes exhibited gradual upregulation as the colistin concentration increased (Figure 3).The relative transcriptional level of each gene is indicated in Table S1."Stay" indicates a relative level of transcription between 0.8 and 2.0, "Downregulation" indicates a relative level of transcription below 0.8, and "Upregulation" indicates a relative level of transcription over 2.0.* Among a total of 3731 genes, the genes presenting an uncountable level of expression, either in the testing group or in the reference group, were excluded, and numbers of the total genes for transcriptomic analysis are presented.The transcriptional status of 3731 genes was determined, and fragments per kilobase of transcript per million mapped reads values were calculated and normalized using the average expression level of six 16S rDNAs.In parental A. baumannii treated with inhibitory concentrations of colistin, a total of 2435 genes (over 80% of the total genes) were upregulated (Table 3), and they were associated with various functions, including responses to different stresses, such as oxidation, ultraviolet radiation, chemicals, and heavy metals.In the colistin-resistant isolate, 2629 genes were upregulated upon exposure to inhibitory concentrations of colistin, while 2021 were upregulated upon exposure to subinhibitory concentrations (Table 3).Among these, 199 genes exhibited gradual upregulation as the colistin concentration increased (Figure 3).The relative transcriptional level of each gene is indicated in Table S1."Stay" indicates a relative level of transcription between 0.8 and 2.0, "Downregulation" indicates a relative level of transcription below 0.8, and "Upregulation" indicates a relative level of transcription over 2.0.* Among a total of 3731 genes, the genes presenting an uncountable level of expression, either in the testing group or in the reference group, were excluded, and numbers of the total genes for transcriptomic analysis are presented.

Discussion
Colistin is the last resort against carbapenem-resistant A. baumannii.Its usage in clinical settings is ever-growing; thus, understanding colistin resistance and the impact of colistin exposure is important.Here, by comparing the colistin-resistant isogenic mutant with the parental isolate, the influence of colistin on the morphology and transcriptomics of A. baumannii were confirmed The mutation PmrBL208F, conferring colistin resistance, has been reported in A. baumannii clinical isolates from the period 2001 to 2008 in Spain and in the United States by the SENTRY Antimicrobial Surveillance Program [7].The isolate was recovered from bronchial aspirates of an Italian patient being treated with colistin [25].The leucine at 208 lies upstream of the histidine kinase domain (216-276) of the PmrB sensor kinase and its function is yet unknown.However, the previous study revealed that the mutation resulted in an overproduction of PmrC and conferred resistance to colistin by over-modifying the lipid A moiety with a MIC of 16 µg/mL.In addition, disruption of the pmrB coding sequence resulted in restoring the susceptibility to colistin with a MIC of 0.25 µg/mL by ceasing the over-modification [7].
In the SEM images, interesting changes in the cell length by the PmrBL208F alteration can be seen.In addition, bacterial filamentation and cell elongation were observed at inhibitory concentrations of colistin, similar to the findings of Otero et al., who developed a morphology-based rapid detection method for antimicrobial resistance in Gramnegative bacteria [26].Moreover, colistin-resistant A. baumannii was observed to have a shorter cell length.Filamented bacterial cells were observed when the A. baumannii strain were treated only by the inhibitory concentration of the colistin regardless of its susceptibility to the drug.Scatter plots showing the correlations between the normalized level of transcriptome of the parental colistin-susceptible Z0317AB0082 and the colistin-resistant isogenic mutant Z0317AB0082-R isolates after treatment with inhibitory concentrations of colistin (A) and inhibitory or subinhibitory concentrations of colistin (B).The transcriptional status of a total of 3731 genes was analyzed.Each dot represents a gene.The 10 dots located over 60 for the Z0317AB0082 isolate treated with inhibitory concentrations of colistin (65.6 to 280.7), which were paired with the 0.4 to 1.9 transcriptional level in the Z0317AB0082-R isolate treated with inhibitory concentrations of colistin, were omitted from graph A for clarity.

Discussion
Colistin is the last resort against carbapenem-resistant A. baumannii.Its usage in clinical settings is ever-growing; thus, understanding colistin resistance and the impact of colistin exposure is important.Here, by comparing the colistin-resistant isogenic mutant with the parental isolate, the influence of colistin on the morphology and transcriptomics of A. baumannii were confirmed The mutation PmrB L208F , conferring colistin resistance, has been reported in A. baumannii clinical isolates from the period 2001 to 2008 in Spain and in the United States by the SENTRY Antimicrobial Surveillance Program [7].The isolate was recovered from bronchial aspirates of an Italian patient being treated with colistin [25].The leucine at 208 lies upstream of the histidine kinase domain (216-276) of the PmrB sensor kinase and its function is yet unknown.However, the previous study revealed that the mutation resulted in an overproduction of PmrC and conferred resistance to colistin by over-modifying the lipid A moiety with a MIC of 16 µg/mL.In addition, disruption of the pmrB coding sequence resulted in restoring the susceptibility to colistin with a MIC of 0.25 µg/mL by ceasing the over-modification [7].
In the SEM images, interesting changes in the cell length by the PmrB L208F alteration can be seen.In addition, bacterial filamentation and cell elongation were observed at inhibitory concentrations of colistin, similar to the findings of Otero et al., who developed a morphology-based rapid detection method for antimicrobial resistance in Gram-negative bacteria [26].Moreover, colistin-resistant A. baumannii was observed to have a shorter cell length.Filamented bacterial cells were observed when the A. baumannii strain were treated only by the inhibitory concentration of the colistin regardless of its susceptibility to the drug.
Previously, a transcriptomic study was conducted on a colistin-resistant isogenic mutant and its parental isolate [27].In the current study, over five-fold overexpression was exhibited by seventeen genes in the mutant comparing with the parental isolate.Referring to the earlier results [27], six genes were overlapped: genes encoding poly-β-1,6-N-acetylglucosamine deacetylase, two putative membrane proteins, and an acetyl-CoA hydrolase and the pmrB and pmrA genes.The fold change in expression for pmrC was 1.8.In current study, the varying levels of expression could be due to either of the genetic alterations conferring colistin resistance, the genetic background, and possibly the different mRNA preparation methods.
Colistin resistance in clinical settings has been known to be associated mainly with the upregulation of the two-component regulatory system comprising pmrA and pmrB, which regulates the phosphoethanolamine transferase pmrC gene.This system was confirmed to be overexpressed after treatment with inhibitory or subinhibitory concentrations of colistin.Here, pmrC was moderately upregulated with a transcriptional level of 1.9.Besides the known genes associated with colistin resistance, many other genes were found to be overexpressed in the colistin-resistant isogenic isolate.Interestingly, the genes upregulated after exposure to an inhibitory concentration of colistin were different between the colistinsusceptible and the colistin-resistant isolate.
The expression profile of colistin-associated genes has been studied in Pseudomonas aeruginosa [28].Genes associated with the synthesis and modification of LPSs, oxidative stress response, and some components of the efflux pumping-out system, such as AdeABC, were found to be upregulated.Interestingly, the genes involved in antioxidative stress response pathways were overexpressed by exposure to the colistin.The upregulated genes are linked with the mechanism of action of colistin, concentrating the lethal hydroxyl radicals leading oxidative damage to DNA, lipids, and proteins bacteria like A. baumannii [29].In the current study, the oxidative stress response regulator soxR was 1.3-fold upregulated in colistin-susceptible isolates treated with inhibitory concentrations of colistin, and 2.0-and 2.6-fold upregulated in the colistin-resistant isolate treated with subinhibitory and inhibitory concentrations of colistin, respectively.Similarly, the iron homeostasisassociated fur gene was 1.6-fold upregulated in colistin-susceptible isolates treated with inhibitory concentrations of colistin and 1.2-and 2.9-fold upregulated in colistin-resistant isolates treated with subinhibitory and inhibitory concentrations of colistin, respectively.
Since colistin resistance in Gram-negative bacteria commonly develops when the bacterial host is exposed to colistin [7,21], understanding the transcriptomic changes triggered by colistin is important.A report using the Galleria mellonella invertebrate animal infection model showed that colistin resistance is associated with increased virulence in vivo [30].In addition, upregulation of the pgaB gene for the poly-beta-1,6-N-acetyl-D-glucosamine deacetylase was reported in colistin-resistant A. baumannii [27].The protein PgaB is associated with transportation of the surface polysaccharide and an adhesin for maintenance of biofilm structural stability [31].Correspondingly, the transcriptional level of the pmrB gene was three times higher in the colistin-resistant isolate than the colistin-susceptible strain in the current study.Moreover, the pgaB gene was 3.0-fold upregulated in colistin-susceptible isolates by colistin treatment with an inhibitory concentration and 1.5-and 1.7-fold upregulated in colistin-resistant isolates treated with subinhibitory and inhibitory concentrations of colistin, respectively.
Infections by colistin-resistant A. baumannii have very limited options for treatment, and trials exploring the possibility of synergistic antimicrobial activity of colistin with other drugs were carried out.Through the transcriptomic data in this study, upregulation of several genes was observed even in subinhibitory concentrations of colistin as those in the inhibitory concentration of colistin.And other drugs with different mechanisms of action could be combined with colistin to treat the infections of colistin-resistant A. baumannii.A study of the in vitro synergistic efficacy of colistin combined with tigecycline, amikacin, vancomycin, rifampin, ceftazidime, imipenem, or aztreonam was carried out, and all the combinations presented synergistic activity at least for partial group of colistin-resistant A. baumannii clinical isolates [30].Moreover, an in vivo study using mouse thigh and peritoneal infection models showed the combination of colistin and sulbactam eradicate better the infection-causative colistin-resistant A. baumannii from the lesion [31].While many studies present the possibilities of such treatment options for the infections by colistin-resistant A. baumannii [32,33], clinical usage of the combinatorial therapy needs more evidence because the results from clinical trials to date are not fully convincing.A colistin combination therapy with rifampin showed inconclusive achievement of treatment for pneumoniae caused by colistin-resistant A. baumannii [34], and therapy with colistin combined with meropenem presented higher 28-day mortality for the patients infected with colistin-resistant A. baumanii than colistin monotherapy [35].
This study has several limitations.First, RNAseq results were validated using RT-qPCR for only a limited number of genes because too many genes were found to be upregulated.Second, the 13 alterations, including twelve SNPs and one insertion, other than the pmrB C625T for the PmrBL208F mutation, were not deeply characterized for the reason that the alterations were located in the intergenic regions.Moreover, the results for the mutant with the pmrB C625T alteration may not be generalized to mutants with other mutations.And finally, colistin-mediated virulence both in vitro and in vivo was not examined and remains open for further study.However, to the best of our knowledge, this is the first trial involving a comparative assessment of the morphological and transcriptomic profiling by colistin treatment using the isogenic pair of colistin-susceptible and -resistant isolates.

Conclusions
In this study, using a colistin-susceptible clinical A. baumannii isolate and its colistinresistant isogenic mutant, colistin-mediated morphologic and transcriptomic characteristics were assessed after their exposure to colistin.The colistin-resistant mutant showed a shorter cell length than the parental isolate, and filamented cells were found when both isolates were exposed to the inhibitory concentration of colistin.Treatment with colistin led to upregulation of the genes associated with antioxidative stress response pathways, which is associated with the mechanism of antimicrobial action of colistin.In regard to the current study, future efforts are advocated to clarify the constraints of the study.

FIGURE 1 .
FIGURE 1. SEM micrographs of the parental Z0317AB0082 (A and B) and the colistin-resistant isogenic mutant Z0317AB0082-R (C, D, and E) Acinetobacter baumannii isolates cultured in media either without (A and C) or with colistin 2 g/mL (B and D) and 64 g/mL (E).Cell rupture and shrinkage (arrow head) were observed in colistin-treated groups and the filamentation (arrow) was observed in

Figure 1 .
Figure 1.SEM micrographs of the parental Z0317AB0082 (A,B) and the colistin-resistant isogenic mutant Z0317AB0082-R (C-E) Acinetobacter baumannii isolates cultured in media either without (A,C) or with colistin 2 µg/mL (B,D) and 64 µg/mL (E).Cell rupture and shrinkage (arrow head) were observed in colistin-treated groups, and the filamentation (arrow) was observed in the groups treated by inhibitory concentrations of colistin.The images were taken at ×5000 and ×20,000 magnifications and presented with the scale bar indicating 1 µm length.For each group, triplicate samples were used, and representative images are shown.

Microorganisms 2024 ,
12, x FOR PEER REVIEW 8 of 13 N/T, not treated

Figure 2 .
Figure 2. The transcriptional level of the pmrC, pmrA, and pmrB genes in the parental Z0317AB0082 and the colistin-resistant isogenic mutant Z0317AB0082-R isolates cultured in media either without (N/T) or with colistin 2 µg/mL and 64 µg/mL.The isolates were treated for 30 min either without or with colistin 2 µg/mL and 64 µg/mL, and the normalized transcriptional levels of pmrC (black), pmrA (gray), and pmrB (open) to that of the rpoB gene are indicated with error bars for standard error values out of biological triplicates.p values of <0.05 from Student's t test are indicated.

Figure 2 .
Figure 2. The transcriptional level of the pmrC, pmrA, and pmrB genes in the parental Z0317AB0082 and the colistin-resistant isogenic mutant Z0317AB0082-R isolates cultured in media either without (N/T) or with colistin 2 µg/mL and 64 µg/mL.The isolates were treated for 30 min either without or with colistin 2 µg/mL and 64 µg/mL, and the normalized transcriptional levels of pmrC (black), pmrA (gray), and pmrB (open) to that of the rpoB gene are indicated with error bars for standard error values out of biological triplicates.p values of <0.05 from Student's t test are indicated.

RFigure 3 .
Figure 3.Scatter plots showing the correlations between the normalized level of transcriptome of the parental colistin-susceptible Z0317AB0082 and the colistin-resistant isogenic mutant Z0317AB0082-R isolates after treatment with inhibitory concentrations of colistin (A) and inhibitory or subinhibitory concentrations of colistin (B).The transcriptional status of a total of 3731 genes was analyzed.Each dot represents a gene.The 10 dots located over 60 for the Z0317AB0082 isolate treated with inhibitory concentrations of colistin (65.6 to 280.7), which were paired with the 0.4 to 1.9 transcriptional level in the Z0317AB0082-R isolate treated with inhibitory concentrations of colistin, were omitted from graph A for clarity.

Figure 3 .
Figure 3.Scatter plots showing the correlations between the normalized level of transcriptome of the parental colistin-susceptible Z0317AB0082 and the colistin-resistant isogenic mutant Z0317AB0082-R isolates after treatment with inhibitory concentrations of colistin (A) and inhibitory or subinhibitory concentrations of colistin (B).The transcriptional status of a total of 3731 genes was analyzed.Each dot represents a gene.The 10 dots located over 60 for the Z0317AB0082 isolate treated with inhibitory concentrations of colistin (65.6 to 280.7), which were paired with the 0.4 to 1.9 transcriptional level in the Z0317AB0082-R isolate treated with inhibitory concentrations of colistin, were omitted from graph A for clarity.

Author
Contributions: E.-J.Y. conceived and designed the experiments, J.W.M., M.C.J. and J.-w.K. conducted the experiments, J.W.M., J.-w.K. and E.-J.Y. analyzed the data, J.W.M., J.-w.K. and E.-J.Y. wrote the manuscript, J.S.Y. and E.-J.Y. edited the manuscript.All authors contributed to the final version of the manuscript.All authors have read and agreed to the published version of the manuscript.Funding: This research was funded by the Korea Disease Control and Prevention Agency grant number 2019NI08302.

Table 2 .
Single-nucleotide polymorphisms and insert/deletions found in Z0317AB0082-R compared to the parental Z0317AB0082.

Table 3 .
Aspects of gene expression that were changed by colistin treatment.

Table 3 .
Aspects of gene expression that were changed by colistin treatment.