Genomic and phenotypic characterization of Acinetobacter colistiniresistens isolated from the feces of a healthy member of the community

Acinetobacter species are widely known opportunistic pathogens causing severe community and healthcare-associated infections. One such emerging pathogen, Acinetobacter colistiniresistens, is known to exhibit intrinsic resistance to colistin. We investigated the molecular characteristics of A. colistiniresistens strain C-214, isolated from the fecal sample of a healthy community member, as part of a cohort study being conducted in Segamat, Malaysia. Comparison of the whole genome sequence of C-214 with other A. colistiniresistens sequences retrieved from the NCBI database showed 95% sequence identity or more with many of the genome sequences representing that species. Use of the Galleria mellonella killing assay showed that C-214 was pathogenic in this model infection system. The strain C-214 had a colistin and polymyxin B MIC of 32 and 16 mg/L, respectively. Besides, it was resistant to cefotaxime, amikacin, and tetracycline and showed moderate biofilm-producing ability. Different genes associated with virulence or resistance to major classes of antibiotics were detected. We observed mutations in lpxA/C/D in C-214 and other A. colistiniresistens strains as probable causes of colistin resistance, but the biological effects of these mutations require further investigation. This study provides genomic insights into A. colistiniresistens, a potentially pathogenic bacterium isolated from a community member and notes the public health threat it may pose.


Sample collection and Isolation of Acinetobacter spp.
A single colistin-resistant Acinetobacter isolate was obtained after screening 233 fecal samples from Segamat for bacterial and fungal isolates in 2018.The isolate formed part of a larger cohort of Acinetobacter spp.isolates identified during a community research project that involved the isolation and investigation of ESKAPE pathogens from individuals living in the Segamat District 19 .
The sample collection and processing steps have been described earlier 20 .The samples were plated on Leeds Acinetobacter Agar (HiMedia, India) and MacConkey Agar (Oxoid, UK) and subsequently incubated at 37 °C for 24 h.Colony morphology and nature of the strains were observed and recorded.Three colonies with Acinetobacter morphology were selected from each sample and identified by standard biochemical methods (Gram stain, Catalase test and Oxidase reactions).
PCR amplification of a 16S rRNA gene fragment and subsequent sequencing was performed to confirm the Acinetobacter spp.The 16S rRNA gene was targeted using the universal primers described in previous studies 21 .Bacterial DNA extraction for PCR was carried out by the boiling extraction method described by Dashti et al. 22 .
Colistin and other antimicrobial susceptibility testing.Antimicrobial susceptibility testing and interpretation were performed using the standard disk diffusion method for 12 different antibiotics on Mueller Hinton agar (Oxoid, UK) according to the Clinical Laboratory Standards Institute (CLSI) guidelines 23 .The antibiotic disks used in this study were piperacillin, piperacillin-tazobactam, ceftazidime, cefepime, aztreonam, imipenem, meropenem, gentamicin, amikacin, ciprofloxacin and tetracycline.
However, broth microdilution was used for colistin and polymyxin B, the only method CLSI recommends.The minimum inhibitory concentration (MIC) for colistin and polymyxin B was performed following the CLSI, 2015 guidelines and observed breakpoints (≤ 2 mg/L, susceptible; > 4 mg/L, resistant).Acinetobacter baumannii ATCC BAA 1605 and E. coli ATCC 2325 were used as controls with known antibiotic resistance patterns.
Biofilm production and quantification assay.Biofilm production and quantification assays were performed according to Huet et al. 20 with slight modifications.In brief, a total of 100 μl Tryptone Soya Broth (TSB) medium (Oxoid, UK) supplemented with 0.2% glucose was added to each well.Using an overnight bacterial culture, the cell suspension was adjusted to 0.5 McFarland standard in TSB supplemented with 0.2% glucose and 100 μl of each suspension was inoculated into each well.Two wells were left uninoculated and used as negative controls.The plates were incubated at 37 °C for 24 h for biofilm production.Following the biofilm production assay, biofilm quantification was carried out using Crystal violet (CV) and XTT assays.
DNA extraction and whole genome sequencing.To achieve the complete genome sequence, hybrid short and long-read based whole genome sequencing (WGS) was performed.Total genomic DNA was extracted using the phenol-chloroform phase-separation method, according to Sambrook & Russell 24 .Extracted DNA quality and concentration was assessed using a Nanodrop bioanalyzer spectrophotometer (Thermo Scientific, Delaware, USA).
The short-read sequencing data was generated with a Nextera XT library preparation kit (Illumina, San Diego, CA, USA) and sequencing was performed using an Illumina MiSeq sequencer with the MiSeq Reagent Kit v3 as per the manufacturer's protocol (2 × 250 bp paired-end read setting).Additionally, for long-read sequencing, DNA libraries were prepared according to the Ligation Sequencing Kit protocol (SQK-LSK109).The long-read sequencing data was then generated using a MinION FLO-MIN106 flow cell and a MinION MK1B sequencing device (Oxford Nanopore Technology).
Precise species identification.Species identification was carried out through the average nucleotide identity (ANI) based on BLAST and in silico DNA-DNA hybridization (isDDH) using the online server tool JSpeciesWS 33 and genome-to-genome distance calculator 34 , respectively, with default parameters.An ANI value of more than 95% and isDDH values ≥ 70.0% were used as a cut-off to define bacterial species precisely.A phylogenomic analysis of closely related Acinetobacter spp.whole genome sequences was carried out using GToTree program v.1.7.05 35 .These sequences were retrieved from the National Center for Biotechnology Information (NCBI) based on the presence of single copy genes in each genome, including our isolate C-214.
An ampC gene was detected in the genome of the C214 isolate.It resembles the UniRef90_N9PW73 cluster (UniRef50_A0A0N1I997 cluster at 50% cutoff), whose protein sequences belonged exclusively to A. colistiniresistens.The AmpC protein sequence from C214 was compiled together with protein sequences of the UniRef50_ A0A0N1I997 cluster and Ambler class C beta-lactamases from the BLDB database 41 to build a phylogenetic tree using FastTree 42 .The ampC gene tree was visualized using iTol v6 43 .

In vivo Galleria mellonella killing assay.
To determine the virulent nature of A. colistiniresistens, an in vivo killing assay was performed on the greater wax moth, Galleria mellonella.The G. mellonella larvae were purchased from Carolina Biological, US.Larvae showing symptoms of melanization or deformation were omitted from the assay to eliminate the potential for bias.Each larva was weighed and those meeting the criteria of 250 ± 50 mg were used in the study.Killing assay experiments were performed by injecting 10 µl of two different bacterial solutions with 10 7 and 10 6 colony-forming units per larva (CFU/larva), respectively, into the last left proleg using a Hamilton syringe.To check for death caused by physical damage, one group of larvae was injected with 10 µl of PBS as a negative control.Another control group did not receive an injection.The larvae were incubated for seven days at 37 °C and checked for symptoms of death every 24 h.Larva that did not respond to tactile stimulation or had a blackish discolouration were reported dead.A. baumannii C-98 and E. coli OP50 were selected as high and low-pathogenicity reference strains, respectively.The experiments were repeated three times, with the average reading taken into account.

Statistical data analysis.
All analyses were performed using three separate experiments using GraphPad Prism software 6.01.The significance of differences was determined at p ≤ 0.05.The killing of G. mellonella by A. colistiniresistens was analyzed using the Kaplan-Meier method.Log-rank test was performed.

Results
Characteristics of the isolate.A study of A. baumannii from fecal samples from the community in Segamat district, Johor, Malaysia led to the isolation of a single colistin-resistant Acinetobacter spp.designated as C-214, on selective agar plates.The carrier was a 34-year-old female housewife from the indigenous Orang Asli Jakun community.For preliminary species identification, PCR was done with the universal primers 27F and 1492R followed by Sanger sequencing to get the nearly complete 16S rRNA gene sequence of the strain 36 .The use of BLAST for the 16S rRNA sequence against the NCBI database revealed that the isolate belonged to the genus Acinetobacter and is a member of the species colistiniresistens.
FE-SEM imaging was carried out, and the colony morphology was compared to explore any differences in bacterial cell morphology between A. baumannii and A. colistiniresistens.No significant difference was observed in their cell membrane and colony formation.Both were found to have coccobacillus phenotypes.On selective Leeds Acinetobacter agar media, they produced identical colonies and colours (data not shown).
Antibiotic resistance profile and biofilm-forming ability.The isolate, C-214, had a colistin and polymyxin B MIC of 32 and 8 µg/ml, respectively (Table 1).In addition to colistin resistance, this isolate was phenotypically resistant to cefotaxime, amikacin and tetracycline but susceptible to cefepime, ceftazidime, ciprofloxacin, gentamicin, piperacillin/tazobactam and carbapenems based on the disk diffusion antibiotic susceptibility testing (Table 1).
The biofilm-forming ability of C-214 was also assessed.Based on XTT and CV assays, isolate C-214 showed moderate biofilm-forming capability (Table 1).
Genomic features of the strain C-214.C-214 was sequenced using both short-reads (Illumina MiSeq) and long-read (Oxford Nanopore) sequencing technologies.Hybrid genome assembly revealed that the Acinetobacter strain C-214 contained one circular chromosome of 3,865,171 bp (GC content 41.33%) (Fig. 1).The GC content is almost identical to that reported for the A. colistiniresistens sequence represented by GCF_003227755.1 and the genome size is typical for this strain.Besides, three circular plasmids were also detected with sizes of 10,411 bp (p214-1), 5509 bp (p214-2), 8305 bp (p214-3) and GC content of 35.4,30.79 and 33.65%, respectively (Supplementary B).The average genome size is similar to A. baumannii, whose genomes range between 3.6 and 4 Mbp in size with a GC content of around 39% 44,45 .
The genome annotation analysis using Prokka detected 3844 genes with 3,705 coding sequences, 75 tRNA sequences, 18 rRNA sequences, 1 tmRNA sequence and 45 misc RNA sequences.
Precise species detection.ANI and in silico DNA-DNA hybridization analyses of strain C-214 were conducted against 79 different Acinetobacter spp along with 19 A. colistiniresistens strains (Supplementary B).The highest ANI (98.08%) and DDH (71.04) values were found against A. colistiniresistens strain NR1165 (Supplementary B).A phylogenetic tree was constructed on the basis of 20 A. colistiniresistens, three A. baumannii, one A. gyllenbergii and one A. proteolyticus genomes, and it is clear that C-214 is a genomovar within the A. colistiniresistens group which is itself composed of two sub-groups (Fig. 2).

Presence of antibiotic resistance genes in C-214 compared with other A. colistiniresistens strains.
The distinct patterns of antibiotic resistance phenotypes observed in strain C-214 prompted us to investigate the known genes linked to resistance in the sequenced genome and compare them with other available A. colistiniresistens genomes.In addition, we also compared the presence of AMR genes in three A. baumannii, one A. gyllenbergii and one A. proteolyticus genomes.Detailed results are summarized in Fig. 2. The AMR gene analysis using the CARD database detected five antibiotic resistance genes in the genome of strain C-214, where one tetracycline resistance gene (tet39) was found within a plasmid (p214-1).Besides, one beta-lactam resistance gene bla OXA302 , two aminoglycoside resistance genes (ANT(3'')-IIc, AAC(6')-Ij) and multidrug efflux pump gene adeB were also present in the isolate.Detection of these genes also supported our AST phenotypic data (Table 1), where the strain C-214 showed resistance against beta-lactams (CTX-30), tetracycline (TE30) and aminoglycoside (AK30).However, even though the isolate is highly resistant against colistin and polymyxin B, the gene responsible for resistance could not be identified.Studies have found that the plasmid-mediated gene mcr is responsible for colistin resistance in Acinetobacter baumannii 46 .We could not find any mcr gene in any A. colistiniresistens isolates.After comparison of the putative resistance genes in C-214 with 19 other A. colistiniresistens strains, it was discovered that most of the isolates carried a similar collection of resistance genes (n ≤ 5).However, the tetracycline resistance A gene (tet39) was only detected in the strain, C-214, described in this paper.An insertion sequence ISaba26 was detected in both the chromosome and plasmid (p214-1).In addition, when Acinetobacter derived cephalosporinases (ADCs) were compared between A. colistiniresistens isolates and A. baumannii, considerable differences were observed.Both A. baumannii and the A. colistiniresistens isolates carried the class C intrinsic beta-lactamase gene.However, whereas the A. baumannii isolates analyzed in this study carried an ADC-1 type Figure 1.Genome map of Acinetobacter colistiniresistens strain C-214 chromosome (CP102099) plotted using BLAST ring image generator (BRIG).The outer coloured circle denotes the GC skew of genomic sequences (purple: negative; green: positive), followed by distributions of antimicrobial resistance genes (red), virulence genes (blue) and CRISPR-Cas system loci (grey). of gene, the A. colistiniresistens carried an ADC-8 type gene (Fig. 3).The amino acid sequence similarity of these two types is about 50%.Efflux pump related genes were commonly seen in both A. baumannii strains, but only two genes were found in the A. colistiniresistens isolates.

Virulence factor related genes observed in A. colistiniresistens. Different virulence factor related
genes were analyzed in the C-214 strain, along with 19 other A. colistiniresistens, three A. baumannii, one A. gyllenbergii and one A. proteolyticus genomes derived from NCBI, where two A. baumannii genomes from the same project.The results are summarized in Fig. 4. The outer membrane protein gene ompA, which promotes bacterial biofilm formation, eukaryotic cell infection, antibiotic resistance, and immunomodulation, was found in all A. colistiniresistens isolates, including C-214 47 .Genes related to lipopolysaccharide (LPS) production, such as lpxA, B, C, D and lpxL, were present in all A. colistiniresistens genomes.The lpxA, lpxC, and lpxD genes are primarily involved in the initial stages of lipid A production and the hydrophobic anchor of LPS 48 .It has been found that mutations in lpxA, lpxC and lpxD may play a role in the development of colistin resistance 48 .We compared these lpx genes acquired from WGS of all A. colistiniresistens and three A. baumannii colistin-sensitive strains (A.baumannii ATCC19606 type strain, A. baumannii H-10112, A. baumannii C-98) (Supplementary A, Fig. S1).We found similar polymorphisms in lpxA/C/D and lpxL genes in all A. colistiniresistens strains suggesting that alterations in LPS metabolism could be the reason for colistin resistance observed in these strains.We also found other virulence factor related genes including; type VI secretion system, stress adaptation, antiphagocytosis, two-component regulatory system (bfmR, bfmS), serum resistance, iron uptake and adherence genes.While most isolates shared similar virulence-related genes (Fig. 4), certain differences were noted between A. colistiniresistens and A. baumannii isolates.Although both hospital and community A. baumannii isolates possessed a complete set of type VI secretion system genes, only one to three genes encoding this system were found in six A. colistiniresistens isolates, including C-214.Type VI secretion system (T6SS) genes are well recognized as a crucial virulence factor in A. baumannii and toxins produced by T6SS genes could kill other bacteria as well as eukaryotic cells 49 .

Pangenome analysis.
Extensive pan-genome analysis investigations can aid in understanding a bacterial species' functional adaptability 50 .To get insight into the pan-genome information of A. colistiniresistens, we created different plots to visualize the number of total genes, core accessory genes, and unique genes as a function of the sequenced genomes.
Acinetobacter colistiniresistens is classified into two genomovars.To explore the genomic diversity, we performed a pan-genome analysis using the Anvi' o pan-genome workflow 40 (Fig. 5).The pan-genome consisted of a total of 6825 genes, with 2601 core genes shared among all strains and 2179 core genes occurring only once.Furthermore, we investigated the enrichment between the two genomovars 51 .The enrichment was defined as an enrichment score greater than 15 and an adjusted q-value below 0.01.Functional enrichment analysis was conducted based on COG20 annotation (Supplementary B).Our strain, C-214, was found in clade 2, which exhibited enrichment in 17 genes, while clade 1 showed enrichment in eight genes.The genomes were arranged according to the phylogenomic tree (Fig. 5).Isolate pathogenicity.The pathogenicity of A. colistiniresistens C-214 was tested in the G. mellonella model and compared using a virulent A. baumannii strain C-98 (unpublished) and a non-virulent E. coli OP-50.Figure 6 highlights the variability of pathogenicity in two different bacterial inoculums.We administered parenteral injections of two different concentrations (10 7 and 10 6 CFU) of A. colistiniresistens strain C-214 to examine the impact on larval pathogenicity.The infected larvae exhibited distinct symptoms, including nodulation, blackening of the cuticle, and eventual mortality.Notably, the degree of melanization increased significantly with higher inoculum doses, indicating that the initial infectious inoculum size plays a crucial role in the progression of the infection.To analyze the survival outcomes, we employed Kaplan-Meier survival distributions for each bacterial inoculum and conducted a log-rank (Mantel-Cox) test, which revealed significant differences (p < 0.001).The survival probability of the larvae depended on the number of CFU injected.For larvae injected with an inoculum size of 10 7 CFU/larvae, the survival rate after 24 h was 40% for A. colistiniresistens C-214, 0% for A. baumannii C-98, and 100% for the non-virulent E. coli strain OP50.However, the survival rate for C-214-treated larvae decreased to 0% after 120 h.In comparison, larvae injected with 10 6 CFU/ larva exhibited a 90% survival rate for C-214 after 24 h, 20% for C-98, and 100% for OP-50.Within the observation period of 168 h post-inoculation, 60% of the C-214-treated larvae survived.Further dilution (10 5 CFU/ larva) resulted in 100% survival but showed melanization in 30% of the population after 168 h of observation.

Figure 2 .Figure 3 .
Figure 2. The phylogenomic tree of Acinetobacter colistiniresistens, annotated with ANI values and percentage identity matrix against antimicrobial resistance genes from CARD.Note that the type strain NIPH2036 is represented by the genome assembly GCF_000413935.1, the 13BJ/14TU isolate by GCF_003227755.1 and three TUM isolates by GCF_009013115.1, GCF_9013295.1 and GCF_9013055.1.

Figure 4 .
Figure 4. Presence of genes involved in virulence in the strain C-214 and 19 other A. colistiniresistens, three A. baumannii, one A. gyllenbergii and one A. proteolyticus genomes.The presence of genes in an isolate is specified by a colored rectangle, colored according to sequence similarity to curated VFDB sequences.The absence of genes is shown as blank spaces with no color.

Figure 5 . 2 .
Figure5.Anvi' o pangenome display of 20 A. colistiniresistens genomes.Layers were coloured according to the two proposed A. colistiniresistens genomovars.Genomes were sorted based on the phylogenomic tree from Fig.2.Gene clusters were sorted based on the presence and absence of genes.Gene clusters that fall under the single copy genes and the core genome were indicated.Barplots representing singleton gene clusters per each genome were also shown.