The S. maltophilia strains 142 and 156 carrying the novel aph(9)-Ic genes were collected from a tertiary hospital in Wenzhou, Zhejiang, China. The plasmid pMD19-T (Takara Bio, Inc., Dalian, China) was used as the vector for cloning the predicted resistance gene and Escherichia coli DH5α was used as the recipient for the recombinant. The pCold I vector was used for cold shock-induced expression of His6-tagged APH(9)-Ic and E. coli BL21 was used as the host. The bacterial strains were cultured overnight at 37°C in Luria-Bertani (LB) medium supplemented with the appropriate antimicrobial agents. The strains and plasmids used in this work are listed in Table 1 .

The whole-genomic DNA of S. maltophilia 142 and 156 was extracted using an AxyPrep Bacterial Genomic DNA Miniprep Kit (Axygen Biosciences, Union City, CA, USA). Whole-genome sequencing was achieved using the Illumina HiSeq 2500 and PacBio RS II platforms by Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China). Illumina short reads were assembled using SPAdes v3.14.1 (Bankevich et al., 2012), and the average length of short reads was 150 bp. The PacBio long reads of approximately 10 to 20 kb in length were initially assembled using Canu v1.8 software (Koren et al., 2017). The Illumina sequence reads were then mapped onto the primary assembly to correct possible misidentified bases using the Burrows−Wheeler Alignment v0.7.12 tool (Li and Durbin, 2009) and the Genome Analysis Toolkit (Mckenna et al., 2010). The open reading frames (ORFs) were predicted using Prokka v1.14.6 (Seemann, 2014) and further annotated by DIAMOND (Buchfink et al., 2015) against the UniProtKB/Swiss-Prot (http://web.expasy.org/docs/swiss-prot_guideline.html) and NCBI nonredundant (nr) protein databases (https://www.ncbi.nlm.nih.gov/refseq/about/nonredundantproteins/). Annotation of resistance genes was performed using ResFinder (Zankari et al., 2012) and Resistance Gene Identifier (RGI) from the Comprehensive Antibiotic Resistance Database (CARD) (Mcarthur et al., 2013). Multiple sequence alignment and a phylogenetic tree were generated by MEGAX (Kumar et al., 2018) using a maximum parsimony method with 1000 bootstrap replications. The multiple sequence alignment used for generating the phylogenetic tree is displayed in Supplementary Figure S1 . Analysis of conserved motifs in the APH(9)-Ic sequence was performed using the MEME Suite (http://meme-suite.org/). Gene organization diagrams were drawn with version 2.2.2 of Easyfig (Sullivan et al., 2011). FastANI v1.31 (Jain et al., 2018) was used to calculate the average nucleotide identity (ANI). ProtParam (https://web.expasy.org/protparam/) was used to predict the molecular weight and pI value. CGView Server (Petkau et al., 2010) was used to visualize the basic genomic features of chromosomes and to perform comparative genomic analysis. The promoter region of the aph(9)-Ic genes was predicted by BPROM (2016) (http://www.softberry.com/berry.phtml?topic=bprom&group=programs&subgroup=gfindb). The sequence type was analyzed according to multilocus sequence typing (MLST) for Stenotrophomonas maltophilia (https://pubmlst.org/bigsdb?db=pubmlst_smaltophilia_seqdef&page=sequenceQuery).

The minimum inhibitory concentrations (MICs) were determined by the agar dilution method with Mueller-Hinton (MH) agar plates. The susceptibility patterns were interpreted according to the Clinical and Laboratory Standards Institute (CLSI, 2022, Performance Standards for Antimicrobial Susceptibility Testing-Twenty-Eighth Edition: M100) guideline breakpoint criteria for S. maltophilia and Pseudomonas aeruginosa. As no CLSI breakpoint was available, the MIC result for spectinomycin was interpreted according to the publications by Hu et al. (Hu et al., 2017) and Jouybari et al. (Jouybari et al., 2021). E. coli ATCC 25922 was included in each test as a quality control standard. The MIC values were determined in triplicate on MH agar plates with twofold serial dilutions of the antimicrobials. The plates were incubated at 37°C for 16-20 hours before the results were analyzed.

Genomic DNA of S. maltophilia 142 and 156 was extracted as described above. The DNA fragments carrying the putative resistance genes with their promoter regions were amplified by PCR with the primers listed in Supplementary Table S1 . Each PCR product was ligated into the pMD19-T vector using a T4 DNA ligase cloning kit (Takara Bio, Inc., Dalian, China). The recombinant plasmids pMD19-T-pro-aph(9)-Ic (S. maltophilia 142) and pMD19-T-pro-aph(9)-Ic1 (S. maltophilia 156) were transformed into competent E. coli DH5α cells by the calcium chloride method, and the transformants were selected on LB agar plates supplemented with 100 μg/mL ampicillin. The cloned insert sequence in the recombinant plasmid of each transformant was further confirmed by both agarose gel electrophoresis and Sanger sequencing of the PCR products (Hangzhou Tsingke Biotechnology Co., Ltd., Hangzhou, China).

The ORF of the aph(9)-Ic gene was amplified with the primers listed in Supplementary Table S1 and cloned into the pCold I cold shock expression vector. The PCR product and the cloning vector pCold I were digested with both BamHI and HindIII (Takara Bio Inc., Dalian, China). Then, the resulting DNA fragments were ligated by T4 DNA ligase (Takara Bio Inc., Dalian, China). The resultant recombinant plasmid pCold I-aph(9)-Ic was introduced into E. coli BL21 competent cells by the calcium chloride method, and the transformant (pCold I-aph(9)-Ic/BL21) was selected on an LB agar plate containing 100 μg/mL ampicillin. The overnight culture of the recombinant strain (pCold I-aph(9)-Ic/BL21) was cultured in LB broth containing 100 μg/mL ampicillin at 37°C. When the OD₆₀₀ reached 0.6, isopropyl D-thiogalactopyranoside (IPTG) was added at a concentration of 1 mM to induce the expression of His6-tagged APH(9)-Ic, and cell cultivation was continued for 18 h at 16°C. Cells were harvested by centrifugation (5000 g, 10 min) at 4°C, resuspended in phosphate-buffered saline (pH = 7.4), and disrupted by sonication. The recombinant protein was purified using BeyoGold His-tag Purification Resin, first with nondenaturing wash buffer (50 mM NaH₂PO₄, 300 mM NaCl, 2 mM imidazole) and then eluted by the nondenaturing eluent (50 mM NaH₂PO₄, 300 mM NaCl, 50 mM imidazole) from the His-tag Protein Purification Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions ( Supplementary Figure S2 ). The His6-tag was removed by enterokinase (GenScript Inc., Nanjing, China) digestion at 4°C for 72 h ( Supplementary Figure S2 ). The purity of the protein was confirmed using sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) and subsequent staining with Coomassie Brilliant Blue. The protein concentration was determined spectrophotometrically using a BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, United States).

The kinetic assay used to monitor APH(9)-Ic activity was performed as reported previously (Lu et al., 2021). Briefly, kinetic parameters for APH(9)-Ic activity were determined by a continuous spectrophotometric assay, which coupled the production of ADP generated upon AG phosphorylation to the oxidation of NADH using the enzymes pyruvate kinase (PK) and lactate dehydrogenase (LD). The rate of ADP production was determined by monitoring the decrease in absorbance at 340 nm using a UV−VIS spectrophotometer (U-3900, Hitachi, Japan) at 37°C. Reactions were initiated by the addition of 250 nM APH(9)-Ic in a 250 μL reaction mixture that contained 100 mM HEPES (pH 7.0), 10 mM MgCl₂, 20 mM KCl, 2 mM phosphoenolpyruvate (Beijing Solarbio Science & Technology Co., Ltd.), 1 mM NADH (Roche 10107735001), a commercial mixture of PK and LD (Sigma P0294; 18-26 U/mL PK and 25-35 U/mL LD, final concentrations), 2 mM ATP, spectinomycin and streptomycin at various concentrations. The steady-state velocities were determined from the linear phase of the reaction progress curve and plotted as a function of the substrate concentration. Data were fit by nonlinear least squares methods with the Michaelis−Menten equation, v=(V _max[S])/(K _m+[S]), using GraphPad Prism 9 (GraphPad Software, Inc.) to calculate K _m and k _cat ( Supplementary Figure S3 ). In this equation, v is the steady-state velocity; V_max is the maximal velocity; [S] is the substrate concentration; K_m is the Michaelis constant for the substrate; and the turnover rate k _cat is calculated from the equation V _max = k _cat [E], where [E] is the enzyme concentration.

To analyze the expression of the aph(9)-Ic and aph(9)-Ic1, overnight cultures of S. maltophilia 142 and 156 were diluted in fresh LB broth at a ratio of 1:100 and incubated to OD₆₀₀ = 0.5, followed by the addition of 1/4 MIC spectinomycin (S. maltophilia 142, 4,096 μg/mL; S. maltophilia 156, 2,048 μg/mL) in the medium and further incubated for 1 h. Total RNA was extracted using RNAiso Plus (TaKaRa, Dalian, China) according to the manufacturer’s instructions. Total RNA was determined spectrophotometrically for RNA purity and concentration by an Implen NanoPhotometer (Implen GmbH, Munich, Germany). DNA-free RNA was confirmed by PCR amplification of the 16S rRNA gene of S. maltophilia. cDNA was synthesized using the Hiscript III All-in-one RT Super mix Perfect for qPCR Kit (Vazyme Biotech, Nanjing, China). RT Q-PCR was performed using SYBR qPCR Master Mix (Vazyme Biotech, Nanjing, China) in a CFX96TM Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, United States) to monitor the increase in fluorescence in real time. The primers used for quantitative RT−PCR (qRT−PCR) are listed in Supplementary Table S2 . The relative expression of the genes was calculated by the 2^-ΔΔCT method (Livak and Schmittgen, 2001) using 16S rRNA as the internal control (Wu et al., 2019). Expression levels were compared with and without antibiotic treatment using one-way ANOVA (LSD test). P ≤ 0.05 was considered significant.

The chromosomal nucleotide sequences of the S. maltophilia strains 142 and 156 reported in this study have been deposited in GenBank under accession numbers CP098483 and JAMQEE000000000, respectively. The accession numbers for the novel resistance genes and their corresponding proteins are aph(9)-Ic (ON693243), aph(9)-Ic1 (ON838098), APH(9)-Ic (USA18493.1), and APH(9)-Ic1 (MCM2524399.1).

The S. maltophilia isolates 142 and 156 carrying the novel AG resistance genes designated aph(9)-Ic and aph(9)-Ic1, respectively, were collected from a tertiary hospital in Wenzhou, Zhejiang, China. Initially, the two isolates identified in a clinical laboratory using a MALDI-TOF mass spectrometer (BioMérieux, Marcy l’Etoile, France) were all S. maltophilia. The ANI results showed that the genomes of the isolates 142 and 156 had the highest identities of 98.36% and 97.22% with that of S. maltophilia NCTC10258 (NZ_LS483377.1) and Pseudomonas hibiscicola ATCC 19867 (GCF_000382065.1), respectively. P. hibiscicola is a heterotypic synonym of S. maltophilia (van den Mooter and Swings, 1990) in the genus Stenotrophomonas (Anzai et al., 2000). Currently, P. hibiscicola is not recognized by the National Center for Biotechnology Information (NCBI), so we named this isolate S. maltophilia 156. In addition, the sequence type of S. maltophilia 142 was ST84, while S. maltophilia 156 cannot be assigned into any known sequence type.

When analyzing the resistance phenotypes and the genotypes of the two isolates, according to the working draft genome sequences obtained from Illumina sequencing, we found that there was no corresponding function-characterized resistance gene annotated from the genomes, even though the two isolates showed very high MIC levels against spectinomycin. According to the resistance gene annotation, several function-characterized aminoglycoside-modifying enzyme genes were found in the genomes of the two isolates, such as one (aph(3’)-IIc) in S. maltophilia 142, and two [aph(3’)-IIc and aac(6’)-Iz] in S. maltophilia 156, but none of them intermediated resistance to spectinomycin (Li et al., 2003; Okazaki and Avison, 2007).

Instead, a putative APH(9)-I gene (finally designated aph(9)-Ic in this work) was annotated in each of the two genomes. To verify whether this predicted gene was functional, we cloned the potential resistance genes, and their resistance functions were further confirmed. To better understand the genetic background of the novel spectinomycin resistance determinant, the complete genome of S. maltophilia 142 was further finished. The whole genome of S. maltophilia 142 consisted of a chromosome (without a plasmid), which was 4,830,983 bp in length and encoded 4,378 ORFs with an average GC content of 66.20% ( Table 2 and Supplementary Figure S4 ). The number of contigs and the average contig length of the S. maltophilia 156 draft genome were 114 and 38,836 bp, respectively.

The MICs of the tested antimicrobials for S. maltophilia strains 142 and 156 are shown in Table 3 , where MICs are defined as the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation (Andrews, 2001). The in vitro susceptibility test showed that S. maltophilia 142 exhibited resistance to 6 (among those whose breakpoints were available from CLSI or US FDA) of the 23 antimicrobials tested, while S. maltophilia 156 showed resistance to 7 of them. S. maltophilia strains 142 and 156 also showed high MIC levels for spectinomycin (16,384 and 8,192 μg/mL, respectively), streptomycin (>128 μg/mL), ribostamycin (1,024 and 64 μg/mL, respectively), paromomycin (256 and 128 μg/mL, respectively), kanamycin (64 μg/mL), neomycin (>16 μg/mL), cefoxitin (>64 μg/mL), and fosfomycin (>64 μg/mL), which have no established breakpoints. S. maltophilia 142 was susceptible to many other antimicrobials, including tobramycin, ceftazidime, levofloxacin and chloramphenicol ( Table 3 ).

Compared with the control (E. coli DH5α harboring the vector pMD19-T only), the two recombinants with the cloned aph(9)-Ic variants (pMD19-T-aph(9)-Ic/E. coli DH5α and pMD19-T-aph(9)-Ic1/E. coli DH5α) increased the MIC levels of spectinomycin by 8- and 4-fold, respectively, while no significant increase in MIC level was identified for the other AGs ( Table 3 ). Among the two recombinant clones, one from S. maltophilia 142 (aph(9)-Ic) showed a 2-fold greater increase in the MIC level of spectinomycin than that of the other one (aph(9)-Ic1).

The phosphotransferase activity and kinetic parameters of APH(9)-Ic were determined using purified APH(9)-Ic, and a variety of spectinomycin concentrations were used as substrates. The k _cat (1.09 ± 0.09) s^-1, K _m (31 ± 5) μM, and the catalytic efficiency (k _cat /K _m ratio) of (5.58 ± 0.31) × 10⁴ M⁻¹·s⁻¹ was observed for spectinomycin, while no significant catalytic efficiency was observed for streptomycin, which was consistent with the corresponding MIC level change of the recombinant strain (pMD19-T-aph(9)-Ic/DH5α) in the antimicrobial susceptibility test ( Table 3 ). The gene encoding APH(9)-Ia was first identified from Legionella pneumophila in 1997 (Suter et al., 1997). Further study revealed that APH(9)-Ia effectively phosphorylates spectinomycin with K _m = 21.5 μM, k _cat = 24.2 s^-1 and k _cat /K _m of 1.1 × 10⁶ M⁻¹·s⁻¹ and did not bind to other AGs, including kanamycin, amikacin, neomycin, butirosin, streptomycin and apramycin (Thompson et al., 1998). APH(9)-Ic showed some common functional characteristics with APH(9)-Ia and APH(9)-Ib, such as the narrow substrate spectrum and regiospecificity of the enzyme (Lyutzkanova et al., 1997; Ramirez and Tolmasky, 2010).

To investigate the effect of spectinomycin on the expression of the aph(9)-Ic and aph(9)-Ic1 genes, the mRNA levels of the two genes treated with or without spectinomycin were determined. The expression of the aph(9)-Ic and aph(9)-Ic1 genes increased significantly (P < 0.01) after induction with 1/4 MIC spectinomycin for 1 h ( Figure 1 ). Compared to the untreated control group, the expression of aph(9)-Ic in S. maltophilia 142 and aph(9)-Ic1 in S. maltophilia 156 increased 8.95- and 4.08-fold, respectively.

The nucleotide sequence length, number of encoded amino acids, predicted protein molecular mass and pI values of the aph(9)-Ic variants from S. maltophilia strains 142 and 156 are shown in Table 4 . The protein from S. maltophilia 142 is composed of 346 amino acids, while the protein from S. maltophilia 156 consists of 334 amino acids which is 12 amino acids shorter than that of S. maltophilia 142. It turned out that the amino acid residues at positions 335-346 in APH(9)-Ic are absent in APH(9)-Ic1 ( Figure 2 ). APH(9)-Ic1 (from S. maltophilia 156) shared 98.20% amino acid sequence identity with the corresponding amino acid sequence of APH(9)-Ic (from S. maltophilia 142). It could be concluded that the structural variation caused the MIC level difference between the two genes that the cloned aph(9)-Ic gene from S. maltophilia 142 showed a 2-fold MIC level to spectinomycin more than that of aph(9)-Ic1 from S. maltophilia 156.

A phylogenetic tree of APH(9)-Ic and the other function-characterized APH enzymes, including APH(3’), APH(3’’), APH(9), APH(7’), APH(2’’), APH(6) and APH(4), collected from the CARD (Mcarthur et al., 2013) and UniProtKB/Swiss-Prot (http://web.expasy.org/docs/swiss-prot_guideline.html) databases was constructed. The APH(9)-Ic proteins clustered closest to a branch composed of APH(9)-Ia ( Figure 3 ). Among the function-characterized AG resistance proteins, APH(9)-Ic shared the highest amino acid sequence identities of 33.75% and 28.04% with APH(9)-Ia (AAB58447.1) and APH(9)-Ib (AAB66655.1), respectively, while APH(9)-Ic1 shared 34.06% and 28.69% identities with APH(9)-Ia (AAB58447.1) and APH(9)-Ib (AAB66655.1), respectively. This finding indicates that the proteins identified in this work are novel AG O-phosphotransferases of the APH(9)-I family ( Figure 3 ). According to the nomenclature criteria recommended by Shaw (Shaw et al., 1993), the novel AG resistance protein from S. maltophilia 142 was thus designated APH(9)-Ic.

To explore the genetic environment of aph(9)-Ic, the structures of the approximately 10-kb flanking regions of aph(9)-Ic were analyzed and are shown in Figure 4 . Comparative analysis of the aph(9)-Ic gene-flanking regions of S. maltophilia strains 142 and 156 with the aph(9)-Ic gene-carrying fragments from other strains revealed that the regions adjacent to aph(9)-Ic were conserved in the corresponding parts of the chromosomal sequences of the S. maltophilia strains, with the exception that an IS481 was present next to aph(9)-Ic in the sequence from S. maltophilia 2013-SM24. Notably, several antimicrobial resistance-related genes, such as stp, orfA, mex-G like, orfD and aph(3’)-IIc, were present in the upstream regions of the aph(9)-Ic genes. The presence of aph(3’)-IIc may confer bacterial resistance to paromomycin, kanamycin and neomycin ( Table 3 ) (Okazaki and Avison, 2007). Due to the high degree of conservation of the genetic context of aph(9)-Ic and related genes in species of the genus Stenotrophomonas, aph(9)-Ic may be an intrinsic gene that is useful for resistance against spectinomycin produced naturally by Streptomyces (Gomes and Menawat, 1998) or introduced artificially.