Identification of a novel aminoglycoside nucleotidyltransferase ANT(3″)-Ic from Citrobacter telavivum S24

Abstract

Understanding the antibiotic resistance profile of the emerging pathogen Citrobacter telavivum S24 is important for clinical care, surveillance, and research. C. telavivum S24, a strain that exhibits resistance to both streptomycin and spectinomycin, was isolated from the soil at a broiler chicken farm. The strain was identified by whole-genome sequencing and average nucleotide identity analyses. Annotation of the resistance genes revealed that a novel aminoglycoside resistance gene, designated ant(3″)-Ic, is located on the chromosome of C. telavivum S24. This gene exhibits 52% similarity with the previously functionally characterized resistance gene aadA23. It distinguishes itself from ant(3″)-Ia and ant(3″)-Ib genes, and represents an independent branch. The ant(3″)-Ic gene is recognized as an inherent resistance gene of C. telavivum within a conserved genetic environment. The product of the ant(3″)-Ic gene is the aminoglycoside nucleotidyltransferase ANT(3″)-Ic, which has a theoretical pI of 5.05 and a molecular mass of 29 kDa. ANT(3”)-Ic inactivates streptomycin and spectinomycin with the K_m of 46.13 and 115.90 µM, respectively. The discovery of the novel antibiotic resistance gene ant(3″)-Ic and the functional characterization of ANT(3”)-Ic will facilitate more targeted surveillance and treatment strategies for preventing and controlling zoonotic pathogens of zoonotic diseases.

Introduction

The Citrobacter genus, which belongs to the Enterobacteriaceae family, comprises Gram-negative opportunistic pathogens that occasionally colonize the gastrointestinal tract of animals and humans. Citrobacter infections most commonly occur in immunocompromised and hospitalized patients with multiple comorbidities. Among infections caused by this genus, approximately half present as urinary tract infections [1]. The majority of Citrobacter infections are associated with Citrobacter koseri. To date, 15 species have been validated in the genus Citrobacter with the discovery of C. europaeus (2017) isolated from water and human fecal samples [2], C. portucalensis (2017) [3] from aquatic sample, and C. telavivum (2020) [4] from human rectal swab samples in recent years. Two C. telavivum strain, 6105T and 6106, which carry chromosomal mcr-9, were identified as a new species by Ribeiro et al. in 2020. These strains were isolated from two epidemiologically linked hospitalized patients in Israel [4]. This research highlighted that the emerging C. telavivum has the potential to acquire antimicrobial resistance genes and poses a risk for nosocomial transmission. However, the antimicrobial resistance phenotype and mechanisms of environmental C. telavivum S24 remain poorly understood and warrant further investigation

Aminoglycoside antibiotics play a crucial role in clinical treatment and agricultural breeding. However, in recent years, with the widespread use of antibiotics, the problem of bacterial resistance to aminoglycoside antibiotics has become increasingly serious, posing a huge threat to public health and the breeding industry [5, 6]. Therefore, in-depth research on newly emerging drug-resistant strains and the drug-resistant genes they carry has extremely practical significance. In the clinical setting, resistance to aminoglycosides is predominantly mediated by aminoglycoside-modifying enzymes (AMEs), which include aminoglycoside acetyltransferases (AACs), aminoglycoside nucleotidyltransferases (ANTs), and aminoglycoside phosphotransferases (APHs) [7]. ANTs inactivate aminoglycosides through adenylation, which transfers an AMP group from ATP to the aminoglycoside molecule, thereby obstructing its ability to bind with the bacterial ribosome [7].Based on their specificities for aminoglycoside modification, the ANT enzymes are divided into five classes: ANT(6), ANT(9), ANT(4'), ANT(2"), and ANT(3"), each targeting a different hydroxyl adenylation position. To date, ANT(3") enzymes are the most common type of ANT enzymes. They have two main subclasses, ANT(3")-Ia and ANT(3")-Ib, which confer resistance to spectinomycin and streptomycin.

In this study, we characterized a C. telavivum isolate from a broiler chicken farm. A novel gene conferring resistance to spectinomycin and streptomycin, designated ant(3″)-Ic, was identified on its chromosome. This study focused on understanding the characteristics of the novel resistance gene ant(3″)-Ic and its impact on aminoglycoside antibiotic resistance.

Materials and methods

Bacterial strains and plasmids

C. telavivum isolate S24 was retrieved from a soil sample sourced from a broiler chicken farm located at Wenzhou, Zhejiang, China (120°25′37.23″, 28°12′05.31″) in April 2015. The bacterial isolation was performed through a 10-fold serial dilution in sterile saline, followed by plating the dilutions on LB agar (Thermo Fisher Scientific, Waltham, USA) and incubation at 37 °C for 16 h. Single colonies were subsequently isolated and purified, from which S24 was selected for further study. Initial identification was performed by using a microorganism auto-analysis system (VITEK®2, BioMerieux, France), together with a comparative analysis of the 16 S rRNA gene sequence against the GenBank database, using the BLAST program (www.ncbi.nlm.nih.gov/BLAST/). To further determine the species status of S24, the average nucleotide identity (ANI) was calculated using a web-based ANI calculator [8] (http://www.ezbiocloud.net/tools/ani). This was based on 11 distinct phylogenetic groups within the genus Citrobacter [9]. The strains and plasmids employed in this work are detailed in Supplementary Table S1.

Whole-genome sequencing, assembly, annotation, and bioinformatic analysis

Genomic DNA from C. telavivum S24 was extracted using a Bacterial Genomic DNA Miniprep kit (Generay Biotech, Shanghai, China) according to the manufacturer’s instructions. The whole genome of C. telavivum S24 was sequenced using both the Illumina (HiSeq 2500, Illumina, California, USA) and PacBio (PacBio RS II, Pacific Biosciences, California, USA) platforms. The short reads from Illumina sequencing were assembled using the SKESA v2.4.0 [10]. The long reads from PacBio were assembled with Trycycler v0.5.1 [11] and Flye v2.9-b1768 [12]. The quality of the PacBio draft genome assembly was further improved with Pilon v1.24 by mapping the short reads from Illumina sequencing [13]. Open reading frames (ORFs) were predicted by Prokka v1.14.6 [14]. Antibiotic resistance genes were annotated by Resistance Gene Identifier v6.0.3, based on the comprehensive antibiotic resistance database (CARD) [15]. The promoter region of the antibiotic resistance gene was predicted using BPROM [16].

Antimicrobial susceptibility testing

The source information of the 23 antibiotics is shown in Table S2. The Minimal Inhibitory Concentrations (MICs) of antibiotics were determined using the standard agar dilution method on Mueller-Hinton (MH) agar (Hangzhou Binhe Microorganism Reagent Co., Ltd, Hangzhou, China) plates, following the guidelines of the Clinical and Laboratory Standards Institute (CLSI M100, 34th Edition, 2024, https://clsi.org/shop/standards/m100/) [17]. The interpretation of the susceptibility results adhered to the CLSI guidelines when applicable. In cases where these guidelines were unavailable, we referred to those provided by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Escherichia coli ATCC 25922 was used as the quality control strain.

Cloning experiments

To determine the gene function, the ant(3″)-Ic gene with its promoter region was amplified from the genomic DNA of C. telavivum S24 by PCR using designed primers (Table S3). The amplified fragment was inserted into the cloning vector pUCP20 digested with EcoRI (Takara, Shiga, Japan) and HindIII (Takara, Shiga, Japan). For expression and purification of ANT(3″)-Ic, the open reading frame (ORF) of the ant(3″)-Ic gene was PCR-amplified using primers designed with XhoI and EcoRI restriction sites and an enterokinase (EK) cleavage site (Table S3). The amplified product was subsequently cloned into the His-tagged expression vector pColdI. The recombinant plasmids pUCP20-ant(3″)-Ic and pColdI-ant(3″)-Ic were transformed into E. coli DH5α and E. coli BL21, respectively, and the resulting transformants E. coli DH5α/pUCP20-ant(3″)-Ic and E. coli BL21/pColdI-ant(3″)-Ic were obtained.

Overexpression and purification of ANT(3″)-Ic

The recombinant aminoglycoside nucleotidyltransferase ANT(3″)-Ic was overexpressed in E. coli BL21 after induction with 1 mM isopropyl-D-1-thiogalactopyranoside (IPTG) (Sigma, Missouri, USA) at 16 ℃ in LB medium (OXOID, Basingstoke, UK) for 16 h. The protein was further purified with BeyoGold™ His-tag Purification Resin (P2218, Beyotime, Shanghai, China), with a slight modification to the manufacturer’s instructions. Instead of using the standard elution buffer, different concentrations (50, 100, 200, 500 µM) of imidazole solution were used for ANT(3″)-Ic elution. An N-terminal His-tag was cleaved with enterokinase enzyme (Genscript, Nanjing, China) at 22 °C for different times (0, 2, 4, 8, 16 h) and removed by Ni-NTA affinity chromatography with BeyoGold™ His-tag Purification Resin. ANT(3″)-Ic was detected by SDS-PAGE and the quantity of the protein was determined by BCA protein assay Kit (Sangon, Shanghai, China) with spectrophotometer at 562 nm.

Enzyme activity and kinetic assays of ANT(3″)-Ic

ANT(3")-Ic activity was verified by the Kirby-Bauer disk susceptibility test with E. coli ATCC 25922, following the CLSI guidelines (M100, 34th Edition, 2024) [17]. Antimicrobial susceptibility test discs (Beijing Tiantan Biological Products Co., Ltd., Beijing, China) containing 10 µg of streptomycin, 10 µg of spectinomycin, 10 µg of gentamycin, and 30 µg of kanamycin, were placed on each MH plate. After incubating 15 µg of ATP with ANT(3″)-Ic of different contents (0, 8, 16, and 32 µg) for 30 min, the mixture was dropped onto the antimicrobial susceptibility test discs. All plates were cultured at 37℃ for 18 h. The inhibition zone diameters were measured using a vernier caliper. Each experiment was performed in triplicate independently, and the data were presented as the mean ± SD. Statistical analysis was performed using Student’s t-test with SPSS software version 23.0, with a significance level setting at p < 0.05.

For kinetic assays, the mixture for the ANT(3")-Ic enzymatic action incorporated 50 mM HEPES, 10 mM MgCl2, 0.2 mM UDP-glucose, 0.2 mM glucose 1,6-bisphosphate, 0.2 mM NADP, 0.2 mM dithiothreitol, 0.4 units/mL UDP-glucose pyrophosphorylase, 4 units phosphoglucomutase, 4 units glucose-6-phosphate dehydrogenase, 100 mM ATP, 1µg ANT(3")-Ic, and variable concentrations of aminoglycoside (0–800 µM) in a total volume of 0.2 mL [18]. The source information of the reagents is shown in Table S2. We monitored the generation of NADPH at a wavelength of 340 nm using a spectrophotometer (Synergy Neo2, BioTek, Vermont, USA).

Polypeptide analysis and phylogenetic evaluation of ANT(3″)-Ic protein

The molecular weight, pI, and instability index of ANT(3″)-Ic were predicted using ProtParam [19]. The putative signal peptide and cleavage site of ANT(3″)-Ic were predicted by SignalP 5.0 [20]. The phylogenetic tree was constructed using the MEGA 11 [21] with neighbor-joining method and bootstrap analysis, and visualized with Interactive Tree of Life (iTOL) v5 [22]. The percentage next to the branches of the tree was obtained from the bootstrap test with 1000 replicates. Multiple sequence alignment was conducted using the ESPript server [23].

Genetic environment, phylogenetic analysis, and distribution of the ant(3″)-Ic-like gene

Approximately 20-kb DNA fragments containing ant(3″)-Ic-like genes and their flanking regions were retrieved from the NCBI database and analyzed using BLAST + 2.12.0 [24] and visualized with Proksee [25]. The distribution of the ant(3″)-Ic-like genes was analyzed. Phylogenetic analysis based on ant(3″)-Ic-like genes was performed using MEGA 11 [21] with the neighbor-joining method.

Results and discussion

Phylogeny and genomic relatedness within C. telavivum S24

The isolate S24 is preliminarily identified as Citrobacter sp. based on 16 S rRNA gene (PQ669673) homology analysis, and further ANI analysis ultimately confirmed it to be C. telavivum, a species proposed by Ribeiro et al. in 2020 [4]. The ANI value of S24 when compared to the C. telavivum strain 6105T (CP045205.1) is 98.55% (Table 1), exceeding the boundary (95%) for the definition of prokaryotic species [26]. Conversely, the ANIs of C. telavivum S24, when compared with other species (such as C. farmeri GTC 1319 at 93.59%, C. rodentium ATCC 51459 at 84.65%, C. koseri TBCP-5362 at 83.54% and C. amalonaticus S1285 at 83.31%), were all below the species cut-off of 95% (Table 1) [9]. C. telavivum S24 has a higher genomic relatedness to C. farmeri, a clinically common pathogenic bacterium. These results clearly demonstrate the distinct genomic relationship of C. telavivum S24 with various Citrobacter species. The ANI values are presented in Table 1.

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C. telavivum S24 forms circular and smooth colonies on LB plates (Fig. 1A). The whole genome of C. telavivum S24 is a circular chromosome consisting of 5,099,022 bp and has an average GC content of 53.74%. The genome is predicted to have 4,688 coding sequences (CDSs), among which 3,638 are known proteins and 1,050 are hypothetical proteins. Additionally, there are 86 tRNA and 22 rRNA genes encoded in the genome (Fig. 1B). Comparative genomic analysis shows that the genome of C. telavivum 6105 exhibits the highest similarity to the genome of C. telavivum S24 (Fig. 1B). Unlike C. telavivum 6105 and 6106 carrying two plasmids, C. telavivum S24 is free of plasmid [4].

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Antimicrobial susceptibility profiles and genotypes of C. telavivum S24

A total of 35 antibiotic resistance genes were identified in the genome of C. telavivum S24 with mostly efflux pump-related resistance genes (28/35, 80%), including resistance-nodulation-division (RND) antibiotic efflux pumps (15/28), major facilitator superfamily (MFS) antibiotic efflux pumps (9/28), ATP-binding cassette (ABC) antibiotic efflux pumps (3/28), and a kdpE antibiotic efflux pump (1/28) (Fig. 1C). The details of these genes are listed in Table S4.

C. telavivum S24 is susceptible to a wide range of tested antibiotics, which include β-lactams, aminoglycosides, chloramphenicol derivatives, and tetracyclines, but it displays resistance to spectinomycin (MIC, 32 µg/mL), streptomycin (MIC, 64 µg/mL), fosfomycin (MIC, 64 µg/mL), and colistin (MIC, 256 µg/mL) (Table 2). The specific resistance patterns of C. telavivum S24 can be attributed to the presence of certain resistance genes. The fosfomycin and colistin resistance observed in C. telavivum S24 can presumably be attributed to the presence of the fosfomycin resistance genes glpT and uhpT [27], and the polymyxin resistance gene pmrF, respectively [28] (Fig. 1C). Interestingly, there is a high sequence similarity of 96% for glpT, 99.43% for uhpT, and 99.90% for pmrF between C. telavivum S24 and C. telavivum 6105. However, C. telavivum 6105 is susceptible to colistin and fosfomycin [4]. This phenomenon was also found in the putative β-lactamase gene blaSED. blaSED shares 99.10% identity with the Citrobacter telavivum strain 6105 class A beta-lactamase gene blaTEL (MN580933.1). Interestingly, blaSED confers resistance to various β-lactams, including ampicillin, temocillin, cefazolin, cefoxitin, cefotaxime, and ceftazidime to C. telavivum strain 6105. But C. telavivum S24 is susceptible to these antibiotics. It could be that certain mutations or modifications in the bacterial genome have disrupted the normal function of blaSED in conferring resistance.

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It should be noted that C. telavivum S24 is resistant to both streptomycin and spectinomycin. However, no aminoglycoside resistance gene with ≥ 80% amino acid (aa) identity with the functionally characterized resistance gene has been annotated from the whole genome. To uncover the resistance mechanism, a putative aminoglycoside nucleotidyltransferase gene ant(3″)-Ic with 52% similarity to aadA23 was identified. The construction of pUCP20-ant(3″)-Ic and colony PCR results were verified by 1% agarose gel (Fig. S1A, S1C). The recombinant strain E. coli DH5α/pUCP20-ant(3″)-Ic showed a high level of resistance to streptomycin and spectinomycin, with MICs of 64 and 512 µg/mL, respectively, indicating a 32-fold and 8-fold increase in resistance compared to E. coli DH5α/pUCP20. The high level of resistance exhibited by the recombinant E. coli DH5α/pUCP20-ant(3″)-Ic suggests a direct role of the ant(3″)-Ic gene in conferring resistance to streptomycin and spectinomycin. This could potentially be due to the specific enzymatic activity of the aminoglycoside nucleotidyltransferase ANT(3″)-Ic, which may modify the antibiotics and prevent their binding to target sites. Further studies are needed to elucidate the exact mechanism of action and determine whether other factors may also contribute to the observed resistance. In conclusion, we can infer that the ant(3″)-Ic gene confers resistance to streptomycin and spectinomycin in C. telavivum S24.

Putative aminoglycoside nucleotidyltransferase ANT(3″)-Ic is a novel member of the ANT(3″)-I family

ANT(3″)-Ic is 262 amino acids in length with a molecular weight of 29 KDa and a theoretical pI of 5.05, and it notably lacks a signal peptide (Fig. 2A). ANT(3”)-Ic is a stable protein with an instability index (II) of 38.84, exhibiting hydrophilic characteristics signified by a Grand Average of Hydropathicity (GRAVY) of −0.093 (Fig. 2A). To determine the relatives of the putative aminoglycoside nucleotidyltransferase, BLAST analysis of ANT(3”)-Ic was carried out on the CARD and 39 sequences with known functions were listed in Table S5. The comparison results show that the amino acid sequence similarity of these proteins ranges from 27 to 52%, with the closest relationship with ANT(3”)-Ic being AadA23 (ARO:3002620) [29] and AadA22 (ARO:3002619) [30] reported in Salmonella enterica (Table S5). ANT(3″)-Ic exhibits the highest similarity of 52% to AadA23 among the function-characterized resistance proteins. To further evaluate the phylogenetic relationship, the phylogenetic tree was constructed with 30 sequences of ANT(3″) enzymes, including 25 of ANT(3″)-Ia, 1 of ANT(3″)-Ib, 3 of ANT(3″)-II (ANT(3″)-IIa, ANT(3″)-IIb, ANT(3″)-IIc) and the one of this work. The result suggested that ANT(3″)-Ic could be a new branch of the ANT(3″)-I family (Fig. 2A), which inactivates streptomycin and spectinomycin by catalyzing the transfer of an AMP group from an ATP substrate [7]. ANT(3″)-I family currently includes ANT(3″)-Ia, ANT(3″)-Ib, and the newly discovered protein was thus designated ANT(3″)-Ic.

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Despite evolutionary distinctions, all members of the ANT(3″) family, including ANT(3″)-I and ANT(3″)-II, exhibit adenylation activity on streptomycin and spectinomycin [31]. By comparing the amino acid sequences of the ANT(3″)-I and ANT(3″)-II subfamilies, ANT(3″)-Ic maintains consistency at many highly conserved active amino acid sites (Fig. 2B). Notably, key amino acid sites such as S32, S42, D43, D45, E83, R188, K201, and Y227 are vital for ATP and ion binding; Q83, W108, and D178 are critical amino acid sites for binding with streptomycin and spectinomycin [31]. ANT(3″)-Ic exhibits high conservation at multiple active-site residues. Key sites S32, S42, D43, D45, E83, R188, K201, and Y227—critical for ATP and ion binding—are consistently preserved. Additionally, Q83, W108, and D178 represent essential residues for streptomycin and spectinomycin binding. Previous studies indicated that W169 and D174 are essential only for streptomycin resistance. The W169A mutant results in the 10-fold reduction in the MIC value for streptomycin. Multiple sequence alignment and the MIC result indicated that the W169I mutant may enhance the adenylation activity of ANT(3”) on streptomycin, for which evidence through varying MIC levels (ANT(3”)-Ic, 64 µg/mL; AadA (AAO49597.1), 128 µg/mL [32]; AadA36, (ON520657.1) 128 µg/mL [33]; AadA33 (ON520656), 256 µg/mL [34], ANT(3″)-IIa (EEX02086.1), > 1024 µg/mL [31]) has been recorded.

Aminoglycoside nucleotidyltransferase ANT(3″)-Ic mediated adenylation of streptomycin and spectinomycin

To elucidate its function in C. telavivum S24, we successfully expressed and purified the ANT(3″)-Ic using an E. coli expression system and Ni-NTA affinity chromatography (Fig. 3A Figure S1B, S1D). Standard inhibition zones were observed on E. coli ATCC 25922 when tested with streptomycin, spectinomycin, kanamycin and gentamicin (Fig. 3B). No inhibition zones were observed on the drug susceptibility discs of streptomycin and spectinomycin for C. telavivum S24 or E. coli DH5α/pUCP20-ant(3″)-Ic (Fig. 3B). This phenomenon might be attributed to the fact that the protein encoded by the ant(3″)-Ic gene confers streptomycin and spectinomycin resistance on C. telavivum S24 and E. coli DH5α. To further verify the hydrolytic activity of the ANT(3″)-Ic on various antibiotics, different contents of purified ANT(3″)-Ic were incubated with ATCC 25922. The results show that ANT(3″)-Ic can directly confer resistance to gentamicin and spectinomycin on E. coli ATCC 25922 in a concentration-dependent manner (Fig. 3D).

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To further explore the hydrolysis mechanism of the enzyme in depth, enzymatic kinetic parameters were evaluated. Spectinomycin and streptomycin were hydrolyzed by ANT(3″)-Ic with a Km of 115.90 and 46.13 µM, respectively (Table 3). The kinetic parameters indicate that the substrates of ANT(3″)-Ic are consistent with its MIC patterns.

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Genetic environment, phylogenetic analysis, and distribution of ant(3″)-Ic-like genes

Based on the whole genome sequence, the ant(3″)-Ic gene is located on the chromosome of C. telavivum S24 (Fig. 1). In order to understand the genetic environment of the ant(3″)-Ic-like gene, a total of 21 bacterial genomes carrying ant(3″)-Ic-like genes were retrieved from the NCBI nucleotide database. The approximately 20 kb flanking regions around the ant(3″)-Ic-like genes are highly conserved. The metal-binding protein gene zinT, universal stress protein gene uspF, transcriptional activator ampR, and a putative β-lactamase gene are located upstream of the ant(3″)-Ic-like genes (Fig. 4A). The minC, minD, and minE genes, which participate in cell division, are located downstream of the ant(3″)-Ic-like genes (Fig. 4A). No mobile genetic element was observed in the surrounding regions of ant(3″)-Ic-like genes (Fig. 4A). The phylogenetic tree demonstrates that ant(3″)-Ic-like gene sequences form distinct monophyletic clusters within each species, while exhibiting significant divergence across different species. (Fig. 4B). Based on the comparative and phylogenetic results, we speculate that the ant(3″)-Ic-like genes are a group of inherent resistance genes of the Citrobacter genus.

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The ant(3″)-Ic-like genes are exclusively distributed among different species of the genus Citrobacter, including C. amalonaticus (11/12), C. farmeri (3/3), C. freundii (1/1), C. telavivum (1/1) and Citrobacter sp. (5/8) (Fig. 4C) [4, 35,36,37]. The highest similarity (97.97%) of ant(3″)-Ic is with that of the strain C. telavivum 6105, followed by that of C. farmeri CCRI-24,236 (90.37%), while the similarities of the remaining ant(3″)-Ic-like genes were all around 88% (Fig. 4A). However, the adenylation activity of ant(3″)-Ic-like genes to streptomycin and spectinomycin remains uncharacterized in previous reports. A total of 21 complete genomes carrying ant(3″)-Ic-like genes have been documented across multiple geographic regions: the USA (5), United Kingdom (4), Germany (3), China (2), Switzerland (1), Canada (1), Australia (1), Israel (1), Thailand (1), and 2 strains with unrecorded origins (Fig. 4D). The ant(3″)-Ic-like genes are distributed in different parts of the world, raising concerns about the resulting resistance issues.

Conclusions

In this study, we first characterized a novel ANT resistance gene, ant(3″)-Ic, located in the genome of C. telavivum S24. The ant(3″)-Ic-encoded protein ANT(3″)-Ic can adenylate streptomycin and spectinomycin in a concentration-dependent manner. The ant(3″)-Ic gene has significant sequence differences from previously discovered enzymes with the same function and has the highest similarity to aadA23, with a similarity rate of 52%. The ant(3″)-Ic-like genes distribute among 6 species of Citrobacter, of which C. freundii is one of the predominant clinical pathogens. Based on its upstream and downstream structures, it can be speculated that both ant(3″)-Ic and ant(3″)-Ic-like genes are intrinsic resistance genes. Under the selective pressure of aminoglycoside antibiotics, Citrobacter strains harbouring the ant(3″)-Ic-like genes are positively selected and established in the antibiotic-rich environments.

This study systematically characterized the ant(3″)-Ic gene and established its fundamental functional properties. However, our preliminary investigations demonstrate substantial sequence and functional divergences among enzymes within the same functional family. For instance, AadA33 and AadA36, both belonging to the ANT(3″) family, display significant differences in catalytic efficiency and substrate specificity. Further explorations leveraging three-dimensional protein modeling, active site mutational analysis, and functional domain characterization could yield critical insights for rational drug design and therapeutic development.

Data availability

The nucleotide sequences data reported in this study have been deposited to the NCBI database and the GenBank accession numbers for the sequences are as follows: complete chromosome of C. telavivum S24, CP175802; ant(3″)-Ic gene, PQ679945; and 16 s rDNA of C. telavivum S24, PQ669673.

Abbreviations

AMEs:

Aminoglycoside modifying enzymes

AACs:

Aminoglycoside acetyltransferases

ANTs:

Aminoglycoside nucleotidyltransferases

APHs:

Aminoglycoside phosphotransferases

MICs:

Minimal inhibitory concentrations

CLSI:

Clinical and Laboratory Standards Institute

ANI:

Average nucleotide identity

ORFs:

Open reading frames

CDSs:

Coding sequences

References

Ranjan KP, Ranjan N. Citrobacter: an emerging health care associated urinary pathogen. Urol Ann. 2013;5(4):313–4. PMID: 24311922IF: 0.8 Q4 B4.

Ribeiro TG, Clermont D, Branquinho R, Machado E, Peixe L, Brisse S. Citrobacter Europaeus sp. nov., isolated from water and human faecal samples. Int J Syst Evol Microbiol. 2017;67(1):170–3. https://doi.org/10.1099/ijsem.0.001606.

Ribeiro TG, Goncalves BR, Silva MS, Novais A, Machado E, Carrico JA, Peixe L. Citrobacter portucalensis sp. nov., isolated from an aquatic sample. Int J Syst Evol Microbiol. 2017;67(9):3513–7. https://doi.org/10.1099/ijsem.0.002154.

Ribeiro TG, Izdebski R, Urbanowicz P, Carmeli Y, Gniadkowski M, Peixe L. Citrobacter telavivum sp. Nov. With chromosomal mcr-9 from hospitalized patients. Eur J Clin Microbiol Infect Dis. 2021;40(1):123–31. https://doi.org/10.1007/s10096-020-04003-6.

Zhao X, Li W, Hou S, Wang Y, Wang S, Gao J, Zhang R, Jiang S, Zhu Y. Epidemiological investigation on drug resistance of Salmonella isolates from Duck breeding farms in Shandong Province and surrounding areas. China Poult Sci. 2022;101(8):101961. https://doi.org/10.1016/j.psj.2022.101961.

Liu C, Wang P, Dai Y, Liu Y, Song Y, Yu L, Feng C, Liu M, Xie Z, Shang Y, et al. Longitudinal monitoring of multidrug resistance in Escherichia coli on broiler chicken fattening farms in shandong, China. Poult Sci. 2021;100(3):100887. https://doi.org/10.1016/j.psj.2020.11.064.

Ramirez MS, Tolmasky ME. Aminoglycoside modifying enzymes. Drug Resist Updat. 2010;13(6):151–71. https://doi.org/10.1016/j.drup.2010.08.003.

Yoon SH, Ha SM, Lim J, Kwon S, Chun J. A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie Van Leeuwenhoek. 2017;110(10):1281–6. https://doi.org/10.1007/s10482-017-0844-4.

Yuan C, Yin ZQ, Wang JY, Qian CQ, Wei Y, Zhang S, Jiang LY, Liu B. Comparative genomic analysis of citrobacter and key genes essential for the pathogenicity of citrobacter Koseri. Front Microbiol. 2019;10:2774. https://doi.org/10.3389/fmicb.2019.02774.

Souvorov A, Agarwala R, Lipman DJ. SKESA: strategic k-mer extension for scrupulous assemblies. Genome Biol. 2018;19(1):153. https://doi.org/10.1186/s13059-018-1540-z.

Wick RR, Judd LM, Cerdeira LT, Hawkey J, Meric G, Vezina B, Wyres KL, Holt KE. Trycycler: consensus long-read assemblies for bacterial genomes. Genome Biol. 2021;22(1):266. https://doi.org/10.1186/s13059-021-02483-z.

Kolmogorov M, Yuan J, Lin Y, Pevzner PA. Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol. 2019;37(5):540–6. https://doi.org/10.1038/s41587-019-0072-8.

Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Cuomo CA, Zeng Q, Wortman J, Young SK, Earl AM. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS ONE. 2014;9(11):e112963. https://doi.org/10.1371/journal.pone.0112963.

Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30(14):2068–9. https://doi.org/10.1093/bioinformatics/btu153.

Alcock BP, Huynh W, Chalil R, Smith KW, Raphenya AR, Wlodarski MA, Edalatmand A, Petkau A, Syed SA, Tsang KK, Baker SJC, Dave M, McCarthy MC, Mukiri KM, Nasir JA, Golbon B, Imtiaz H, Jiang X, Kaur K, Kwong M, Liang ZC, Niu KC, Shan P, Yang JYJ, Gray KL, Hoad GR, Jia B, Bhando T, Carfrae LA, Farha MA, French S, Gordzevich R, Rachwalski K, Tu MM, Bordeleau E, Dooley D, Griffiths E, Zubyk HL, Brown ED, Maguire F, Beiko RG, Hsiao WWL, Brinkman FSL, Domselaar GV, McArthur AG. CARD 2023: expanded curation, support for machine learning, and resistome prediction at the comprehensive antibiotic resistance database. Nucleic Acids Res. 2023;51(D1):690–9. https://doi.org/10.1093/nar/gkac920.

Solovyev V, Salamov A. Automatic annotation of microbial genomes and metagenomic sequences. In Chapter 4: Li RW, editor. metagenomics and its applications in agriculture, biomedicine and environmental studies. Nova Science; 2011. p. 61–78. https://novapublishers.com/shop/metagenomics-and-its-applications-in-agriculture-biomedicine-and-environmental-studies/.

Lewis IIJS, Mathers AJ, Bobenchik AM, Bryson AL, Campeau S, Cullen SK, Dingle T, Galas MF, Humphries RM, Kirn TJ, Limbago B, Pierce VM, Richter SS, Satlin M, Schuetz AN, Sharp S, Simner PJ, Tamma PD, Weinstein MP. CLSI M100™ Performance Standards for Antimicrobial Susceptibility Testing. 34th ed. CLSI standard M100. Clinical and Laboratory Standards Institute. 2024. https://clsi.org/shop/standards/m100/.

Kim C, Hesek D, Zajicek J, Vakulenko SB, Mobashery S. Characterization of the bifunctional aminoglycoside-modifying enzyme ANT(3’’)-Ii/AAC(6’)-IId from Serratia marcescens. Biochemistry. 2006;45(27):8368–77. https://doi.org/10.1021/bi060723g.

Wilkins MR, Gasteiger E, Bairoch A, Sanchez JC, Williams KL, Appel RD, Hochstrasser DF. Protein identification and analysis tools in the expasy server. Methods Mol Biol. 1999;112:531–52. https://doi.org/10.1385/1-59259-584-7:531.

Armenteros JJA, Tsirigos KD, Sonderby CK, Petersen TN, Winther O, Brunak S, von Heijne G, Nielsen H. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat Biotechnol. 2019;37(4):420–3. https://doi.org/10.1038/s41587-019-0036-z.

Tamura K, Stecher G, Kumar S. MEGA11: molecular evolutionary genetics analysis version 11. Mol Biol Evol. 2021;38(7):3022–7. https://doi.org/10.1093/molbev/msab120.

Letunic I, Bork P. Interactive tree of life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 2021;49(W1):293–6. https://doi.org/10.1093/nar/gkab301.

Gouet P, Robert X, Courcelle E. ESPript/ENDscript: extracting and rendering sequence and 3D information from atomic structures of proteins. Nucleic Acids Res. 2003;31(13):3320–3. https://doi.org/10.1093/nar/gkg556.

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215(3):403–10. https://doi.org/10.1016/S0022-2836(05)80360-2.

Grant JR, Enns E, Marinier E, Mandal A, Herman EK, Chen CY, Graham M, Domselaar GV, Stothard P. Proksee: in-depth characterization and visualization of bacterial genomes. Nucleic Acids Res. 2023;51(W1):484–92. https://doi.org/10.1093/nar/gkad326.

Richter M, Rossello-Mora R. Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci USA. 2009;106(45):19126–31. https://doi.org/10.1073/pnas.0906412106.

Kurabayashi K, Tanimoto K, Fueki S, Tomita H, Hirakawa H. Elevated expression of GlpT and UhpT via FNR activation contributes to increased fosfomycin susceptibility in Escherichia coli under anaerobic conditions. Antimicrob Agents Chemother. 2015;59(10):6352–60. https://doi.org/10.1128/AAC.01176-15.

Joo H, Eom H, Cho Y, Rho M, Song WJ. Discovery and characterization of polymyxin-resistance genes PmrE and PmrF from sediment and seawater Microbiome. Microbiol Spectr. 2023;11(1):e0273622. https://doi.org/10.1128/spectrum.02736-22.

Michael GB, Cardoso M, Schwarz S. Class 1 integron-associated gene cassettes in Salmonella Enterica subsp. Enterica serovar agona isolated from pig carcasses in Brazil. J Antimicrob Chemother. 2005;55(5):776–9. https://doi.org/10.1093/jac/dki081.

Herrero A, Rodicio MR, Echeita MA, Mendoza MC. Salmonella enterica serotype typhimurium carrying hybrid virulence-resistance plasmids (pUO-StVR): a new multidrug-resistant group endemic in Spain. Int J Med Microbiol. 2008;298(3–4):253–61. https://doi.org/10.1016/j.ijmm.2007.04.008.

Zhang G, Leclercq SO, Tian J, Wang C, Yahara K, Ai G, Liu S, Feng J. A new subclass of intrinsic aminoglycoside nucleotidyltransferases, ANT(3)-II, is horizontally transferred among acinetobacter spp. By homologous recombination. PLoS Genet. 2017;13(2):e1006602. https://doi.org/10.1371/journal.pgen.1006602.

Stern AL, Van der Verren SEV, Kanchugal SP, Nasvall J, Gutierrez-de-Teran H, Selmer M. Structural mechanism of aada, a dual-specificity aminoglycoside adenylyltransferase from Salmonella enterica. J Biol Chem. 2018;293(29):11481–90. https://doi.org/10.1074/jbc.RA118.003989.

Gao MD, Feng CL, Ji YA, Shi YK, Shi WN, Zhang L, Liu S, Li AQ, Zhang XY, Li QL, Lu JW, Bao QY, Zhang HL. AadA36, a novel chromosomal aminoglycoside nucleotidyltransferase from a clinical isolate of Providencia stuartii. Front Microbiol. 2022;13:1035651. https://doi.org/10.3389/fmicb.2022.1035651.

Feng CL, Gao MD, Jiang WY, Shi WN, Li AQ, Liu S, Zhang L, Zhang XY, Li QL, Lin HL, Lu JW, Li KW, Zhang HL, Hu YL, Bao QY, Lin X. Identification of a novel aminoglycoside O-nucleotidyltransferase AadA33 in Providencia vermicola. Front Microbiol. 2022;13:990739. https://doi.org/10.3389/fmicb.2022.990739.

Abed JY, Deraspe M, Berube E, D’Iorio M, Dewar K, Boissinot M, Corbeil J, Bergeron MG, Roy PH. Complete genome sequences of Klebsiella Michiganensis and citrobacter farmeri, KPC-2-producers serially isolated from a single patient. Antibiot (Basel). 2021;10(11):1408. https://doi.org/10.3390/antibiotics10111408.

AbuOun M, Jones H, Stubberfield E, Gilson D, Shaw LP, Hubbard ATM, Chau KK, Sebra R, Peto TEA, Crook DW, Read DS, Gweon HS, Walker AS, Stoesser N, Smith RP, Anjum MF, on behalf of the REHAB consortium. A genomic epidemiological study shows that prevalence of antimicrobial resistance in enterobacterales is associated with the livestock host, as well as antimicrobial usage. Microb Genom. 2021;7(10):000630. https://doi.org/10.1099/mgen.0.000630.

Romero R, Zarzycka A, Preussner M, Fischer F, Hain T, Herrmann JP, Roth K, Keber CU, Suryamohan K, Raifer H, Luu M, Leister H, Bertrams W, Klein M, Shams-Eldin H, Jacob R, Mollenkopf HJ, Rajalingam K, Visekruna A, Steinhoff U. Selected commensals educate the intestinal vascular and immune system for immunocompetence. Microbiome. 2022;10(1):158. https://doi.org/10.1186/s40168-022-01353-5.