2.1 Bacterial strains and growth conditions

A. veronii CTe-01 and E. coli K-12 BW25113 were grown in LB medium at 37 °C for 24 h. Strain stocks were stored at −20 °C in LB medium with 30 % glycerol.

2.2 Molecular identification

The identity of A. veronii CTe-01 was confirmed through sequence analysis of the 16S rRNA, rpoD, and gyrB genes from the draft genome (VATZ00000000.2), compared with the GenBank database using BLASTn for nucleotide collection and highly similar sequences (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST_SPEC=GeoBlast&PAGE_TYPE=BlastSearch). The sequences are available under accession codes MK876839.1, VATZ01000092.1:14350–16215, and VATZ01000001.1:16817–19228, respectively.

2.3 Biochemical and physiological characterization

In addition to biochemical characteristics assessed with the VITEK® 2 Compact system (Tataje-Lavanda et al., 2019), further tests included citrate utilization; fermentation of glucose, lactose, and sucrose; catalase and oxidase tests; lysine decarboxylation and deamination; acetoin production; urease, gelatinase, and ornithine decarboxylase activities; indole production; motility; and esculin hydrolysis.

2.4 Determination of minimal inhibitory concentration (MIC) for heavy metals and antibiotics

The MICs (µg/mL) of various metal(loid) salts for A. veronii CTe-01 were determined as follows: HgCl₂, AgNO₃, K₂Cr₂O₇, and K₂CrO₄ (0–100); K₂TeO₃ (0–200); CdCl₂ and ZnSO₄·7H₂O (0–300); CoCl₂·6H₂O (0–400); NiSO₄·6H₂O and CuSO₄ (0–600). Triplicate assays were conducted at 37 °C in 2 mL LB medium, without agitation. Antibiotic susceptibility and MICs were assessed using the MicroScan WalkAway 96 Plus system (Siemens).

2.5 Molecular characterization of plasmid pCTe-01

The plasmid pCTe-01 was purified with the QIAprep Spin Miniprep kit (QIAGEN) and its size estimated by agarose gel electrophoresis (1 %) in TAE buffer, using a Supercoiled DNA ladder (New England Biolabs) as a standard. The plasmid was then digested with BamHI, BsTNI, MboI, and PstI, according to the manufacturer's instructions (New England Biolabs). Fragment sizes were determined by 1.5 % agarose gel electrophoresis using a 1 kb Plus DNA ladder (Thermo Fisher Scientific) as a standard.

2.6 Sequencing plasmid pCTe-01 and improving the draft genome of A. veronii CTe-01

Draft genome of A. veronii CTe-01 [VATZ00000000.1], and plasmid pCTe-01 were additionally sequencing at Macrogen, Inc. (South Korea) using the Illumina platform with 101-bp paired-end reads (TruSeq Nano DNA Kit) [BioSample: SAMN11620977]. Quality control included FastQC v0.11.9 and Trimmomatic v0.39 (Bolger et al., 2014) for the reads of three libraries. De novo genome assembly using SPAdes v3.13.1 improved metrics compared to the previous version (N50: 103,789; L50: 12). Contig identity was verified with KmerFinder v3.2, showing similarity to A. veronii strain X12 (NZ_CP024933.1). Contiguator2 (Galardini et al., 2008) organized the contigs into 60 mapped and 809 unmapped contigs. Plasmid pCTe-01 was manually assembled and circularized, incorporating unmapped contigs with coverage over 10 000 bp. The updated draft genome has been deposited in GenBank for annotation with NCBI PGAP v5.1 after automatically removing small contigs and contaminants (VATZ00000000.2).

2.7 In silico predictions

Resistome analysis was performed using ARIBA v.2.146 with default parameters and databases including ARG-ANNOT (Gupta et al., 2014), CARD (McArthur et al., 2013), MEGARes (Doster et al., 2020), ResFinder (Zankari et al., 2012), and VFDB (Chen et al., 2016), using unfiltered reads, and KmerFinder software. The RASTtk (Aziz et al., 2008), KmerResistance v2.2, and Phaster (Arndt et al., 2016) were used to identify antimicrobial and heavy metal resistance genes, and phage and prophage sequences in contigs, using default parameters. Prophage DNA sequences were classified as intact (>90), questionable (70–90), or incomplete (<70). The pCTe-01 sequence was analyzed in silico with PGAP v5.1 and RASTtk, and subjected to virtual digestion using SnapGene 1.1.3. We also examined A. veronii CTe-01 genome for the presence of aer, hlyA, alt, and ast virulence genes.

3.1 Characterization of A. veronii CTe-01

The identity of A. veronii was confirmed by 16S rRNA, rpoD, and gyrB sequence similarity. This strain is motile, non-lactose fermenting, catalase-negative, oxidase-negative, and indole-positive. It utilizes citrate and succinate, and efficiently ferments D-glucose, D-maltose, D-mannitol, D-mannose, and D-trehalose, with less efficiency for sucrose. Additionally, A. veronii CTe-01 exhibits L-proline-arylamidase, tyrosine-arylamidase, β-N-acetyl-glucosaminidase, and lysine decarboxylase activities.

This bacterium harbors the hemolytic aer gene, which is 1467 bp long and located between nucleotides 14 313 and 15 779, on NODE 18 (VATZ00000000.2). The Aer protein is predicted to be a 54 kDa polypeptide (https://web.expasy.org/compute_pi/). No similar sequences to other hemolytic genes as hlyA, alt, and ast genes were found on the gDNA sequence.

3.2 Characterization of plasmid pCTe-01

We identified a 9059 bp plasmid in A. veronii CTe-01 with a GC content of 54.8 %, confirmed by electrophoresis and in silico analysis (Fig. 1, Fig. 2). Gel electrophoresis fragment sizes (Fig. 1), matched predictions from in silico digestion using SnapGene v1.1.3. Digestion with BamHI and PstI enzymes yielded fragments of approximately 5586, 2752, 721 bp; and 2813, 2216, 1941, 1696, 393 bp, respectively. BsTNI produced a 2000 bp fragment and several smaller than 700 bp, while MboI yielded no fragments.

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Fig. 1

Agarose gel electrophoresis (1.5 % in TAE buffer) of plasmid pCTe-01 after restriction endonuclease digestion. Lane M, molecular weight standard (1 kb plus); lane 1, undigested pCTe-01; lanes 2–5, digestions carried out using BamHI, BsTNI, MboI, and PstI enzymes, respectively.

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Fig. 2

Circular diagram of plasmid pCTe-01. Colored arrows indicate coding sequences (CDS): green, ParAB and RepAB; blue, Rel; yellow, antitoxins; red, Sel1 family proteins; orange, integrase domain proteins; and gray, hypotheticals.

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In silico predictions indicated that plasmid pCTe-01 replicates with RepA/RepB proteins and segregates using ParA-like and ParB proteins. Stabilization proteins identified include a type II toxin-antitoxin (TA) system (RelE/ParE, RelB/StbD, and RelB/DinJ) (Table 1). Additionally, a secretory immunoglobulin A-binding protein, EsiB, and five hypothetical proteins also were detected.

3.3 Heavy metal and antibiotic resistance of A. veronii CTe-01

A. veronii CTe-01 demonstrated resistance to various toxic metal (loid) salts, including K₂TeO₃, HgCl₂, AgNO₃, K₂CrO₄, CdCl₂, ZnSO₄·7H₂O, and NiSO₄·6H₂O In silico analysis revealed potential heavy metal (loid) resistance genes on the chromosome (Table 2). Antibiotic MIC results showed intrinsic resistance to penicillin, ampicillin, ampicillin/sulbactam, amoxicillin/clavulanic acid, erythromycin, and cefazolin (Table 3). Both strains tested were resistant to clindamycin, oxacillin, linezolid, daptomycin, and synercid; showed intermediate resistance to piperacillin/tazobactam and rifampicin; and were susceptible to cefepime, ceftazidime, gentamycin, levofloxacin, moxifloxacin, ertapenem, nitrofurantoin, ticarcillin/clavulanic acid, trimethoprim/sulfamethoxazole, and tobramycin.

3.4 Improved genomic annotation and identification of resistance-associated genes, pathogenicity factors, and phage-related sequences on A. veronii CTe-01 genome

PGAP annotations of the A. veronii CTe-01 draft genome revealed significant improvements: contigs decreased from 272 to 200, pseudogenes from 162 to 145, rRNAs increased from 4 to 14, and tRNAs from 40 to 107.

Analysis of the raw data revealed several resistance genes, including β-lactamases like ampH/ampS, bla_OXA12, bla_TEM, and cphA4, among others. Additionally, type III secretion systems (acr, asc, exs) and motility genes, key pathogenicity factors, were predicted (Table 4). A search for heavy metal resistance genes identified candidates for Cu, Co, Zn, Cd, As, Ni, Hg, and Te (Table 5). The draft genome of A. veronii CTe-01 showed four regions with phage-associated genes related to Aliivibrio fischeri, Bacillus cereus, Acinetobacter baumannii, and E. coli (Table 6). Predicted phage and prophage proteins, included recombinase/integrase sequences and proteins related to phage DNA synthesis and structure, were found. Components of the psp operon, such as pspABC, were also detected using RASTtk (Table 7).

4.1 Characterization of A. veronii CTe-01

A. veronii CTe-01 was isolated from wastewater containing domestic, hospital, and industrial effluents, rich in various microorganisms and chemicals (Tataje-Lavanda et al., 2019). This bacterium can utilize various carbohydrate sources and shows diverse enzymatic activities, which could contribute to its survival in the complex environment. Its β-hemolytic property may be related to aerolysin (Aer), encoded by the aer gene, which was the only gene detected among those investigated. This property may contribute to degrade animal or organic compounds in the environment, similar to processes in domestic wastewater (Tataje-Lavanda et al., 2019). β-hemolysis from aerolysin is a common feature in Aeromonas isolates from fish, clinical, and food samples (Janda and Abbott, 2010). In contrast, other strains have additional hemolytic genes like act, hly, and ast (Sun et al., 2021).

4.2 Characterization of plasmid pCTe-01

The plasmid pCTe-01 is about 9 kb in size. Analysis of the intact plasmid and fragments from BamHI, BsTNI, and PstI digestion matched in silico predictions using SnapGene v1.1.3. The MboI enzyme did not cut the DNA, likely due to absent recognition sites or methylation (Fig. 1). These results confirm the plasmid size and Illumina sequence, though the plasmid sequence remains incomplete.

In silico analysis predicted that pCTe-01 replicates using repA and repB genes, which encode for DNA replication initiation and transcriptional regulation. These genes, part of the repABC operon, are found in plasmids with low copy number, as seen in pCTe-01. They belong to several incompatibility groups (Pérez-Oseguera and Cevallos, 2013). Additionally, also predicted it use ParAB partitioning proteins for segregation, and type II toxin-antitoxins (TA) for stabilization (Kamruzzaman et al., 2021), but not antibiotic resistance genes (Table 1). The absence of resistance genes on pCTe-01 suggests that A. veronii CTe-01 carries these genes on its chromosome, and the observed resistance genes in this bacterium may have been acquired through phages or other mobile elements integrated into the genome (Table 6), rather than plasmids, as no mobilization genes were detected. The plasmid pCTe-01 also encodes EsiB, a protein that interacts with secretory immunoglobulin A, potentially aiding the bacterium in evading the neutrophil response during infections in fish and aquatic animals (Pastorello et al., 2013). Additionally, also contains a gene for an integrase domain-containing protein (Table 1), which phages use to integrate into the bacterial genome, suggesting exposure to MGEs similar to those in A. veronii C198 (Hatrongjit et al., 2020). Several hypothetical proteins were also identified and warrant further investigation.

4.3 Heavy metals and antibiotic MICs

A. veronii CTe-01 showed resistance to various heavy metals (loids) and the in silico analysis suggest genes related (Table 2). This is the first demonstration of tellurite resistance and the terABD operon in A. veronii, suggesting a possible link. However, similar traits and genes have been reported in A. caviae (Arenas et al., 2014). The bacterium forms black colonies, indicating tellurite reduction, but the mechanism in this species is unknown. In A. caviae, the dihydrolipoamide dehydrogenase enzyme (LpdA) reduces tellurite to elemental tellurium (Arenas et al., 2014). The copA and czcA genes, involved in copper and cadmium-zinc resistance, respectively, were not found in A. veronii CTe-01. However, they have been detected in 25 % and 61 % of 36 Aeromonas spp. isolates from shellfish Ruditapes philippinarum, respectively (Dahanayake et al., 2019). Similar genes for Cu, Pb, Cr, Hg, and Cd resistance have been observed in other Aeromonas species, supporting the idea of natural bioremediation of heavy metals (De Silva et al., 2018).

A. veronii CTe-01 showed resistance to β-lactams like penicillin, ampicillin, and cefazolin, as well as to the macrolide erythromycin, while remaining sensitive to several other antibiotics (Table 3). These resistances may be related to the presence of β-lactamases and extended-spectrum β-lactamases (Table 4), which are common in these bacteria (De Silva et al., 2018). Additionally, Aeromonads are known for resistance to tetracyclines, quinolones, and cephalosporins (Zhou et al., 2019). The presence of multiple antibiotic resistance genes in environmental bacteria represents a global public health concern, particularly with the rising prevalence of resistances.

4.4 Genome analysis of A. veronii CTe-01: Identification of antibiotic and heavy metal resistance, virulence genes, potential phages, and prophages through in silico analysis

The resistome analysis suggest that resistance to penicillin and ampicillin (Table 3) may be associated with extended-spectrum β-lactamase genes such as bla_TEM-10, bla_TEM-101, bla_OXA-12, and others (Table 4). Some of these genes, such as blacphA3 and bla_OXA-12, have been observed in A. veronii C198 (Hatrongjit et al., 2020), and bla_OXA-12 and cphA3, in A. veronii XhG1.2 (Das et al., 2021). These genes, known as mobile, are widely disseminated among enteric bacteria. They can be acquired horizontally through MGEs (Piotrowska and Popowska, 2015). The resistance to cefazolin displayed could be related to bla_OXA-12 (Table 4), an antibiotic-inactivation gene detected in Aeromonas (Hatrongjit et al., 2020). The resistance to erythromycin is likely associated with the presence of the macrolide-specific efflux protein MacA (Table 5).

The heavy metal resistances observed in A. veronii CTe-01 could be associated with various genes (Table 2, Table 5), similar to other members of its genus (De Silva et al., 2018). However, further investigation is needed to explore its potential as a bioremediator.

Several genes related to the asc family type III secretion system (T3SS) have been identified on the chromosome of A. veronii CTe-01 (Table 4). This virulence system is also found in other bacteria such as Salmonella enterica, Shigella spp., Citrobacter rodentium, and pathogenic E. coli (Sanchez-Garrido et al., 2022). In A. salmonicida, the T3SS is recognized as the primary virulence system (Frey and Origgi, 2016). A comparison between the T3SS components of A. veronii CTe-01 and A. salmonicida reveals significant similarity, indicating that these genes might contribute to the pathogenic traits and resistance profile in A. veronii. However, no previous reports were found on the presence of T3SS or the RelBE toxin-antitoxin system in this species. Other pathogenicity factors detected were the fim and fli genes, related to fimbria and the flagellar motor (Table 4), which may contribute to virulence, by aiding cell surface adherence (Fernández-Bravo and Figueras, 2020).

Phage and prophage-related sequences on A. veronii CTe-01 gDNA (Table 6) suggest these MGEs originate from water-associated species, such as A. fischeri, B. cereus, A. baumannii, and E. coli. Additionally, several components, including integrase, phage DNA synthesis elements, and the phage shock pspABC operon, have been identified (Table 7). This operon is critical in phage stress response, PspA protein helps maintain membrane integrity where damage is perceived by PspBC, as reported in Yersinia enterocolitica (Flores-Kim and Darwin, 2016).

The specific compounds produced by A. veronii during heavy metal activity −such as volatile mercury, reduced tellurium, and nanodots- have not yet been identified. Consequently, the functional roles of genes and proteins remain undemonstrated. Future research will focus on completing the A. veronii CTe-01 genome using PacBio or nanopore technologies, exploring its evolutionary relationships with other bacteria and plasmids, analyzing its transcriptome in the presence of specific heavy metals, and identifying the resulting compounds for potential applications.