A novel chromosomal aminoglycoside 6′-N-acetyltransferase, AAC(6′)-Io, confers resistance to multiple aminoglycosides identified from Bacillus cereus

A novel chromosomal aminoglycoside 6′-N-acetyltransferase, AAC(6′)-Io, confers resistance to multiple aminoglycosides identified from Bacillus cereus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A novel chromosomal aminoglycoside 6′-N-acetyltransferase, AAC(6′)-Io, confers resistance to multiple aminoglycosides identified from Bacillus cereus Weina Shi, Lei Zhang, Chunlin Feng, Hongqiang Lou, Junwan Lu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6041341/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract Background : The emergence of various resistance determinants in microbes is a growing concern for the clinical application of antimicrobial agents to treat bacterial infections. Research on the aminoglycoside resistance mechanism may help us to determine the complexity of bacterial resistance mechanisms and effective treatment of infectious diseases. Methods : Bacteria were isolated from environmental samples via the plate streak method. The minimum inhibitory concentration (MIC) of the antibiotics was determined using the agar dilution method. Gene cloning and antibiotic susceptibility testing were conducted to confirm the function of the new resistance gene. The kinetic parameters of the enzyme were determined after the protein AAC(6’)-Io was expressed in E. coli . Whole-genome sequencing and bioinformatic analysis were subsequently conducted to analyze the structure and evolution of the resistance gene-related sequences. Results : A novel aminoglycoside resistance gene, aac(6')-Io, which was identified in the chromosome of B. cereus DW444, confers resistance to tobramycin, kanamycin, amikacin, netilmicin, sisomicin and ribostamycin. Of the aminoglycoside substrates tested, AAC(6')-Io demonstrated the highest catalytic efficiency for netilmicin ( k cat / K m , 2.11 × 10 2 M −1 ·s −1 ). Among the functionally characterized antimicrobial resistance proteins, AAC(6')-Io demonstrated the highest amino acid (aa) sequence similarity (47.51%) to AAC(6')-34, and it had the functional essential residues or domains of the AAC(6’)-I proteins, including F 100 -G 102 and G 112 T 113 , which are Ac-CoA binding sites, and L 115 , which is the key site for the acetylation of amikacin. Conclusion : The new aminoglycoside resistance gene aac(6')-Io was described in this study, along with its molecular characteristics. Elucidating the antibiotic resistance mechanism of this pathogen will benefit the clinical application of aminoglycosides to treat infections caused by bacteria carrying its homogous genes. aminoglycoside acetyltransferase AAC(6’) antimicrobial resistance Bacillus cereus kinetic parameter Figures Figure 1 Figure 2 Background Aminoglycosides are broad-spectrum antibacterial compounds that typically contain one aminocyclitol ring (most commonly 2-deoxystreptamine) linked to one or more amino sugars by a glycosidic bond ( 1 ). Since the 1940s, aminoglycosides have been primarily applied clinically to treat a variety of bacterial infections ( 2 , 3 ). The primary mechanism of action of these antibiotics is the binding of aminoglycosides to the 16S rRNA of the 30S ribosomal fragment, leading to the misreading of mRNA and thus to the biosynthesis of nonfunctional misfolded proteins ( 4 ). Currently, the use of aminoglycosides as treatments has become more conservative because of their toxicity, bacterial resistance, and the complexity associated with their chemical syntheses ( 3 ). However, in addition to the use of aminoglycosides for bactericidal therapy, modern medicine has found other uses for these agents, including treatments for various human genetic diseases and Meniere’s disease ( 5 , 6 , 7 , 8 ). Moreover, aminoglycosides are being researched as multifunctional agents for the treatment and prevention of HIV-1 infection ( 3 ). Unfortunately, due to the overuse and selective pressure of antibiotics, bacteria have developed several resistance mechanisms that threaten the use of aminoglycosides ( 9 ). The main resistance mechanism to these antibiotics is the enzymatic modification of aminoglycosides by bacteria encoding aminoglycoside-modifying enzymes (AMEs) ( 10 , 11 ). These enzymes act to inactivate aminoglycoside antibiotics by modifying them through acetylation (aminoglycoside acetyltransferase, AAC), phosphorylation (aminoglycoside phosphotransferase, APH), and adenylation (aminoglycoside nucleotidyltransferase, ANT) ( 4 , 12 ). The aminoglycoside acetyltransferase class comprises four subtypes: AAC(2'), AAC(6'), AAC( 1 ), and AAC( 3 ) ( 13 ). The ability of aminoglycoside 6’- N -acetyltransferases [AAC(6′)s] to inactivate several clinically significant aminoglycosides makes them quite intriguing. The AAC(6’)-I enzymes confer resistance to amikacin, tobramycin, sisomicin, kanamycin, isepamicin, dibekacin, and netilmicin. However, the AAC(6’)-II type acetylates gentamicin, tobramycin, netilmicin, and sisomicin but not amikacin ( 14 , 15 , 16 ). Bacillus cereus is a gram-positive, aerobic-to-facultative, spore-forming rod-shaped bacterium widely found in nature ( 17 ). The ability of this bacterium to form endospores that are resistant to a range of physical stressors and endure time and harsh conditions is an essential trait of its ability to cause foodborne illness ( 18 , 19 ). B. cereus can cause various infections, including gastrointestinal, eye, bloodstream, skin, and central nervous system infections. In addition, this bacterium can coinfect other bacteria, such as Bacillus anthracis and Clostridium perfringens , resulting in anthrax-like pulmonary infections and gas gangrene-like cutaneous infections, respectively ( 17 ). In terms of antibiotic resistance, B. cereus constantly exhibits resistance to β-lactams, fluoroquinolones, tetracyclines and trimethoprim, while susceptibility to clindamycin, erythromycin, chloramphenicol, vancomycin, aminoglycosides and tetracyclines is usually observed ( 17 , 20 , 21 ). In this work, the environmental isolate B. cereus DW444 was found to chromosomally harbor a novel aminoglycoside 6’- N -acetyltransferase gene, designated aac(6’)-Io , and the molecular characteristics of this gene and the protein it encodes were characterized. Materials and methods Bacterial strains and plasmids An environmental isolate, B. cereus DW444, was obtained from a soil sample from an animal farm in Wenzhou, southeastern China, in 2020. Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 29213 were utilized as quality controls for antimicrobial susceptibility tests of nonfastidious gram-positive bacteria (22). Escherichia coli DH5α and E. coli BL21 (DE3) were used as the recipients for the recombinant plasmids aac(6’)-Io -pMD19-T (cloning of aac(6’)-Io with its promoter region) and aac(6’)-Io -pCold I (cloning of ORF of aac(6’)-Io ), respectively. The bacteria and plasmids utilized in this study are listed in Table 1. Table 1 Bacteria and plasmids used in this work. Bacterium/plasmid Relevant characteristic(s) Source Bacterium Bacillus cereus DW444 DW444, the wild-type isolate This study Escherichia coli DH5α DH5α, a recipient for cloning of aac(6’)-Io Our laboratory collection Escherichia coli BL21(DE3) BL21, a recipient for the AAC(6’)-Io expression Our laboratory collection Staphylococcus aureus ATCC 29213 ATCC 29213, a quality control for antimicrobial susceptibility test Our laboratory collection Enterococcus faecalis ATCC 29212 ATCC 29212, a quality control for antimicrobial susceptibility test Our laboratory collection pMD19-T- aac(6’)-Io /DH5α DH5α harboring pMD19-T- aac(6’)-Io (a recombinant plasmid) This study pCold I- aac(6’)-Io /BL21 BL21 harboring pCold I- aac(6’)-Io (a recombinant plasmid) This study Plasmid pMD19-T a vector for cloning the aac(6’)-Io gene with its promoter region, ampicillin resistance Takara Bio, Inc., Dalian, China pCold I/BL 21(DE3) a vector for expressing AAC(6’)-Io, ampicillin resistance Our laboratory collection In vitro antibiotic susceptibility tests The minimum inhibitory concentration (MIC) was determined using Mueller-Hinton (MH) solid medium and the agar dilution method in accordance with the suggested guidelines of the Clinical and Laboratory Standards Institute (CLSI) (22). Genome sequencing and annotation The methods used for genome sequencing and annotation were described in a previous publication (23). Briefly, the genome sequence data from the PacBio RS II sequencing platform were initially assembled using Canu v1.8 (24), and the sequence quality was further corrected by mapping the Illumina sequencing data to the initial assembly using Pilon software (25). The coding sequences (CDSs) were predicted by Prokka v1.14.6 (26). The deduced proteins were annotated via DIAMOND v2.0.11 (27) and NCBI nonredundant protein databases. The antimicrobial resistance genes were predicted based on the comprehensive antibiotic resistance database (CARD) (28) by Resistance Gene Identifier v5.2.0 (https://github.com/arpcard/rgi). The average nucleotide identity (ANI) was computed by FastANI v1.31 (29). The promoter region of a gene was predicted using BPROM (2016) (http://www.softberry.com/berry.phtml?topic=bprom&group=programs&subgroup=gfindb). The genomic features of the genome map were visualized by the CGView Server (30). Multiple sequence comparison and phylogenetic tree reconstruction The methods for multiple sequence alignment, phylogenetic tree reconstruction, and visualization were described in a previous publication (23). The molecular weight and pI of the protein were analyzed using ProtParam (https://web.ExPASy.org/protparam/). The conserved domains of the proteins were analyzed by CD-search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The program clinker v0.0.24 (31) was applied to visualize the genetic context of the genes. Cloning of the aac(6 ’ )-Io gene Using B. cereus DW444 genomic DNA as a template, the open reading frame (ORF) of aac(6’)-Io with its upstream promoter region was amplified by polymerase chain reaction (PCR) with a pair of primers (forward primer pro-aac-F, 5’-AGGTGCAATGAAAGATGTAACAGCAAG-3’; reverse primer pro-aac-R, 5’-CACACATCCCCCTTTGTTTGTATAAGC-3’). To clone the PCR products, the vectors pMD19-T and T4 DNA ligase (Takara Bio, Inc., Dalian, China) were used. Using the calcium chloride method, the recombinant plasmid was transformed into competent E. coli DH5α cells. Subsequently, the transformant was selected on an LB agar plate containing 100 μg/mL ampicillin, and the inserted DNA fragment was verified by Sanger sequencing (23). Recombinant protein AAC(6 ’ )-Io expression and purification The ORF of aac(6’)-Io from B. cereus DW444 was amplified by PCR using the primer set Bam HI- enterokinase- aac -F (5’- CGGGATCCGACGACGACGACAAGATGCTTTTTCAAAAAGAAGGTTTATTGATT-3’) and Hind III- aa c-R (5’- CCAAGCTTTTATTGTTTATATTCCATCATCCAA-3’). According to previous methods (23), the PCR products digested with restriction enzymes ( Bam HI and Hind III) (Takara Bio Inc., Dalian, China) were inserted into the pCold I expression vector, and the resulting plasmid, pCold I-enterokinase- aac(6’)-Io , was transformed into E. coli BL21(DE3) competent cells. The cloned fragment of the transformant was verified by Sanger sequencing (Hangzhou Tsingke Biotechnology Co., Ltd., Hangzhou, China) and was then used for recombinant protein expression. The transformant pCold I-enterokinase- aac(6’)-Io/ BL21 was cultivated at 37 °C in LB medium supplemented with 100 μg/mL ampicillin until the optical density (OD) at 600 nm reached 0.5. To promote the expression of His6-tagged AAC(6')-Io, 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) was added, and the bacterial cells were continuously incubated for 18 hours at 16 °C. After obtaining the bacterial culture mixture, the bacterial cells were sonicated, and the soluble fraction containing His6-tagged AAC(6’)-Io was obtained by centrifugation (5000 g, 10 min, 4 °C). The supernatant and the precipitate from the soluble fraction were further separated by centrifugation (22,000 g, 30 min, 4 °C). Following the manufacturer's instructions, the Histag Protein Purification Kit (Beyotime, Shanghai, China) was used to extract His6-tagged AAC(6')-Io from the supernatant. Enterokinase (GenScript Inc., Nanjing, China) was utilized to digest the His6-tag for 24 hours at 16 °C. Sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) and Coomassie Brilliant Blue staining were used to validate the purity of AAC(6')-Io. The protein content was measured spectrophotometrically with a BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, United States). Kinetic studies of AAC(6 ’ )-Io Specifically, AAC(6’)-Io catalyzes the transfer of an acetyl group of acetyl coenzyme A to position 6’ of the 6-aminohexose ring of the substrate (14). The free sulfhydryl group of CoASH reacts with the 5,5'-dithiobis of (2-nitrobenzoic acid) (DTNB) to generate 5-thio-2-nitrobenzoic acid (TNB), whose absorbance at 412 nm can be monitored spectrophotometrically (32, 33). For a total of ten minutes, the reaction was continually observed by a Synergy Neo2 Multi-Mode Microplate Reader (Biotek, VT, USA). The kinetic assay was conducted in a total volume of 200 μL that contained 2 mM DTNB, 80 μM acetyl-CoA, 50 mM HEPES (pH 7.0), 1 mM EDTA, 25 mM 2-(N-morpholino) ethanesulfonic acid (pH 6.0), and different quantities of aminoglycosides (5–150 μM). The assays were started by adding 4.2 μM of purified enzyme(34). With GraphPad Prism 9 (GraphPad Software, San Jose, CA, United States), the initial rates were plotted against substrate concentration and fitted by nonlinear regression to the Michaelis‒Menten equation to calculate K m and k cat . Nucleotide sequence accession number The following genomic (protein) sequences of B. cereus DW444 are available in the NCBI databases: CP130335 (chromosome), CP130336 (pDW444-290265), CP130337 (pDW444-67560), CP130338 (pDW444-77364), OR282771 ( aac(6’)-Io ) and WKT62500 (AAC(6’)-Io). Results and discussion Identification of the novel aminoglycoside resistance gene aac(6 ’ )-Io in B. cereus More than 500 isolates, including DW444, were obtained from animal anal swabs and sewage samples collected from rural animal farms in Wenzhou, southern China, via the plate streak method (35). In addition to the in vivo antibiotic susceptibility test for the isolates, the whole-genome sequences of numerous isolates were also sequenced by the Illumina NovaSeq platform. An analysis of the relationships between the bacterial resistance phenotypes and genotypes revealed that one soil bacterium, DW444, had high MICs against nearly all 35 antimicrobials of the 8 classes tested (Table 2), and even though it exhibited higher MICs (> 2048 μg/mL) for aminoglycosides such as kanamycin, amikacin, spectinomycin and ribostamycin, no gene with > 80% identity to the functionally characterized genes responsible for aminoglycoside resistance could be predicted from the genome sequence. This may suggest the presence of a novel resistance mechanism concerning the aminoglycoside resistance phenotype of the isolate. To investigate the potential resistance mechanism, the genome annotation results were examined, and a few hypothetical aminoglycoside resistance proteins, such as an AAC(6’)-34-like protein sharing 47.51% amino acid similarity with AAC(6’)-34 (APB03223.1), an ANT(6)-Ia-like protein sharing 42.0% amino acid similarity with ANT(6)-Ia (AHE40557.1) and an AAC(3)-VIIa-like protein with 30.0% amino acid similarity with AAC(3)-VIIa (AAA88552.1), were found (Table S1). Among the three detected genes, we selected the one with the highest similarity to known genes, that is, the aac(6')-34 homologous gene (designated aac(6’)-Io in this study), and further studied and confirmed that it has acetyltransferase activity. Table 2 Minimum inhibitory concentrations of antimicrobials for B. cereus DW444 and the recombinant and control strains (μg/mL). Class Antibiotic B. cereus DW444 pMD19-T- aac(6')-Io /DH5α pMD19-T/DH5α DH5α E. faecalis ATCC 29212 S. aureus ATCC 29213 Aminoglycosides Amikacin >2,048 8 1 1 64 512 1 0.5 0.25 16 0.125 Kanamycin >2,048 256 8 8 512 32 1 0.125 0.125 0.5 4,096 512 <2 <2 64 256 8 <1 2,048 8 8 8 64 64 Streptomycin 1024 2 2 2 64 4 Tobramycin 512 16 0.5 0.5 32 0.125 β-Lactams Ampicillin >2,048 >2,048 >2,048 8 8 2 Amoxicillin 2,048 >2,048 >2,048 4 <1 16 0.125 0.25 0.06 >16 >16 Cefazolin 256 8 16 1 32 1 Cefepime >16 0.0625 0.0625 0.016 >16 2 Cefotaxime >16 0.125 0.125 0.06 >16 2 Cefoxitin 128 2 4 4 256 2 Ceftazidime >256 0.5 1 1 >256 8 Ceftriaxone >128 0.0625 0.125 8 >128 8 Cefuroxime >1,024 16 16 16 128 4 cephalothin 512 32 64 <4 32 2,048 2,048 2048 16 32 32 Piperacillin >128 128 >128 8 16 8 Carbapenems Meropenem >32 <0.004 <0.004 64 0.125 0.25 0.125 1 32 <0.5 <0.5 <0.5 <0.5 <0.5 Nalidixic acid 1,024 64 64 32 128 128 Macrolides Azithromycin 512 <1 <1 <1 2 1,024 32 32 32 2 8 Tetracyclines Tetracycline 128 2 1 2 16 32 <0.0625 <0.0625 <0.0625 <0.0625 1,024 4 4 8 128 2 Amphenicols Chloramphenicol >1,024 16 16 16 16 16 Florfenicol 512 8 4 8 <2 <2 General c haracteristics of the B. cereus DW444 genome To investigate the structure of the novel resistance gene aac(6’)-Io , the whole genome of DW444 was sequenced. The genome consisted of a chromosome and three plasmids (Table 3). The chromosome was 5,548,786 bp in length and encoded 5,717 CDSs with a GC content of 35.24%. The three plasmids pDW444-290265, pDW444-67560, and pDW444-77364 encoded 313, 109 and 88 CDSs, respectively. Eight genes encoding proteins sharing aa sequence similarities of ≥ 80% with proteins encoded by the antimicrobial resistance genes in the CARD were found to be encoded in the chromosome, which included fosB (fosfomycin thiol transferase), bcl (class A B. cereus Bc beta-lactamase), rpoB (rifamycin-resistant beta-subunit of RNA polymerase), bcII (subclass B1 B. cereus Bc beta-lactamase), bcIII (class A B. cereus Bc beta-lactamase), satA (streptothricin acetyltransferase), Enterococcus faecium EF-Tu mutants (elfamycin resistant EF-Tu), and isaB (lsa-type ABC-F protein) (Table S2). No putative antibiotic resistance gene was identified to be encoded on any of the three plasmids. Table 3 General features of the B. cereus DW444 genome. Chromosome pDW444-67560 pDW444-77364 pDW444-290265 Size (bp) 5,548,786 67,560 77,364 290,265 GC content (%) 35.24 33.41 30.29 32.83 Predicted coding sequences (CDSs) 5,717 88 109 313 Known proteins 4,249 (74.32%) 8 (9.09%) 10 (9.17%) 86 (27.48%) Hypothetical proteins 1,468 (25.68%) 80 (90.91%) 99 (90.83%) 227 (72.52%) Protein coding (%) 95.97 100.00 98.20 88.92 Average ORF length (bp) 810.60 655.10 599.80 675.60 Average protein length (aa) 269.10 217.40 201.50 230.40 tRNAs 106 0 0 0 rRNA operons (16S-23S-5S) *14 0 0 0 Species identification of the isolate revealed that the genome sequence of DW444 had the highest percentage (72.3%) of in silico DNA–DNA hybridization ( is DDH) with B. cereus ATCC 14579 (GCF_006094295.1), followed by Bacillus thuringiensis ATCC 10792 (GCF_002119445.1, 68.3%). The ANI results demonstrated that the whole genome of DW444 shared the highest identity (99.88%) with B. thuringiensis (GCF_024712805.1), and the identity with B. cereus ATCC 14579 (GCF_006094295.1) was 96.84% (Table S3). According to the threshold (cutoff scores ≥70% for is DDH and ≥95% for ANI) (29, 36) to specify a bacterium to a certain species, there was only one strain, B. cereus ATCC 14579, in the public database, sharing both higher is DDH and ANI with DW444 than the thresholds. The isolate DW444 was thus tentatively classified as the species B. cereus and was therefore designated B. cereus DW444. The resistance phenotype and distribution of aac(6’)-Io (-like) genes The aac(6’)-Io gene is 537 bp in length and encodes a predicted peptide of 179 amino acids with a predicted molecular mass of 20.72 kDa and a pI value of 5.12. The aac(6’)-Io gene with its promoter region was PCR-amplified and cloned and inserted into the pMD19-T vector. Compared with the control (pMD19-T/DH5α), the recombinant (pMD19-T- aac(6’)-Io /DH5α) strains showed resistance to tobramycin, kanamycin, amikacin, netilmicin, sisomicin and ribostamycin, with increased MICs of 32-, 32-, 8-, 8-, >8-, and >256-fold, respectively, for the 6 antimicrobial agents (Table S4). Analysis of the resistance profiles of aac(6’)-Io with its close relatives aac(6’)-34 , aac(6’)-Ian , aac(6’)-Ie , and aac(6’)-Im revealed that among the aminoglycoside antibiotics they tested, they exhibited similar resistance characteristics, although the MICs of the aminoglycosides may vary (37, 38, 39) (Table S4). To date, 33 functionally characterized resistance genes sharing amino acid similarities between 47.51% (AAC(6’)-34) and 22.73% (AAC(6’)-Ib-cr3) with AAC(6’)-Io are present in the CARD, and all of them are AAC(6’)-I proteins. According to the phylogenetic analysis of these proteins, AAC(6')-Io formed a new branch and was closer to AAC(6')-34, AAC(6')-Ian, AAC(6')-Ie, and AAC(6')-Im. This suggested that AAC(6’)-Io was a novel lineage of the AAC(6’)-I subfamily (Figure 1). To analyze the distribution of AAC(6')-Io-like proteins, AAC(6')-Io was used as a query to search for homologous proteins in the NCBI nr database. A total of 517 sequences with both coverage and identity >80% were retrieved, and all of them were from the genus Bacillus . The closest relative of AAC(6’)-Io was a functionally uncharacterized protein (WP_000898772.1) of GCN5-related N -acetyltransferases (GNATs) from Bacillus spp., with an aa sequence identity of 100.0% and coverage of 100% for AAC(6’)-Io. Kinetic parameters and structural characteristics of AAC(6’)-Io and its relatives The enzymatic activity analysis of AAC(6’)-Io revealed that tobramycin, kanamycin, amikacin, isepamicin and netilmicin were acetylated by AAC(6’)-Io, while no acetylation was detected when using spectinomycin or streptomycin as a substrate (Table 4). To analyze the differences in the enzymatic activities of the functionally characterized AAC(6’) proteins with closer phylogenetic relationships (Figure 1) and greater amino acid similarity (> 40%) to AAC(6’)-Io, the kinetic parameters of four AAC(6’) proteins [AAC(6’)-34, AAC(6’)-Ian, AAC(6’)-Ie, and AAC(6’)-Im] were compared. The substrates of AAC(6’)-Io fall within the range of substrates of the subfamily AAC(6’)-I. Analyzing the substrate specificity of these 5 proteins (including AAC(6’)-Io of this work) revealed that among the 9 antimicrobial agents analyzed with AAC(6’)-Io, except for two (spectinomycin and streptomycin) with AAC(6’)-Io alone and both with a negative result, the other 7 antimicrobials were analyzed with the other 2-4 enzymes, and they all showed positive results. Of the other four enzymes, (AAC(6')-Ian and AAC(6')-Ie) were examined with all 7 substrates. AAC(6’)-Im was used to test 6 compounds, while AAC(6')-34 was used to analyze only two compounds (Table S5). Table 4 Steady-state kinetic parameters for AAC(6’)-Io. Substrate k cat (s -1 ) K m (μM) a k cat / K m (M −1 · s −1 ) AAC(6')-Io AAC(6')-34 AAC(6')-Ie AAC(6')-Io AAC(6')-34 AAC(6')-Ie AAC(6')-Io AAC(6')-34 AAC(6')-Ie Amikacin (1.17 ± 0.13) × 10 -3 ND a 2.11 ± 0.35 7.6 ± 0.5 ND a 20.9 ± 5.3 1.54 × 10 2 ND a 1.01 × 10 5 Fortimycin ND a ND a 0.19 ± 0.01 ND a ND a 2.2 ± 0.4 ND a ND a 1.01 × 10 5 Isepamicin (3.28 ± 0.65) × 10 -3 ND a 1.66 ± 0.50 ND a ND a 18.7 ± 9.0 0.19 × 10 2 ND a 8.87 × 10 4 Kanamycin (2.21 ± 0.86) × 10 -3 ND a 0.31 ± 0.05 ND a ND a 1.4 ± 0.5 1.50 × 10 2 ND a 2.21 × 10 5 Netilmicin (2.67 ± 0.22) × 10 -3 ND a 0.87 ± 0.04 12.6 ± 3.8 ND a 3.2 ± 0.5 2.11 × 10 2 ND a 2.27 × 10 5 Sisomicin ND a 0.65 ± 0.06 2.92 ± 0.31 ND a 4.7 ± 0.4 6.5 ± 2.1 ND a 1.4 × 10 5 4.5 × 10 5 Tobramycin (3.26 ± 0.37) × 10 -3 ND a 0.68 ± 0.1 58.6 ± 9.9 ND a 2.3 ± 0.9 0.56 × 10 2 ND a 3.0 × 10 5 Spectinomycin NA b ND a ND a NA b ND a ND a NA b ND a ND a Streptomycin NA b ND a ND a NA b ND a ND a NA b ND a ND a a ND, not detected. b NA, no acyl transfer activity was detected. Among the other four AAC(6’) proteins, two (AAC(6')-Ian and AAC(6')-Im) were free of a specific digital kinetic parameter. The enzymatic activity of AAC(6')-Ian was determined by using thin-layer chromatography (38), while the activity of AAC(6')-Im, due to substrate inhibition, was monitored through changes in absorbance (39). The kinetic parameters of one substrate, sisomicin, were measured for AAC(6')-34 (37), which was not analyzed by AAC(6')-Io in this work. AAC(6')-Ie had 1.0 × 10 3 - to 1.0 × 10 4 -fold greater catalytic efficiency ( k cat / K m ) than did AAC(6')-Io for the five substrates (tobramycin, kanamycin, amikacin, isepamicin and netilmicin), which were analyzed with both enzymes (39, 40) (Table 4). The lower catalytic efficiency of AAC(6’)-Io than that of AAC(6’)-Ie could be attributed to the low turnover rate ( k cat ) of AAC(6’)-Io (Table 4). To analyze the functional essential residues or domains of the AAC(6’)-Io protein and its relatives, alignments of the deduced amino acid sequence of AAC(6’)-Io with seven other closely related AAC(6’)-I proteins were performed (Figure 2). The residues related to the function of the AAC(6)-I enzymes are conserved in these proteins, including those in this work. These residues include F 100 -G 102 and G 112 T 113 , which are Ac-CoA binding sites predicted by CDD/SPARCLE (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml.) (41), L 115 is the key site for the acetylation of amikacin (42), and the conserved motif (K 109 -Y 122 ) for AAC(6)-I enzymes (42). Conclusions A novel chromosomal aminoglycoside 6'- N -acetyltransferase gene, aac(6’)-Io , was identified in B. cereus DW444. AAC(6’)-Io shares the highest aa sequence similarity (47.51%) with the functionally characterized AAC(6’)-34 from Paenibacillus sp. LC231 and confers resistance to aminoglycosides, including tobramycin, kanamycin, amikacin, isepamicin, and netilmicin. Deciphering new antibacterial resistance mechanisms in bacteria from different sources would help clinics treat infections caused by bacteria harboring the same resistance genes effectively. Abbreviations MIC : Minimum inhibitory concentration aa : Amino acid AMEs: Aminoglycoside-modifying enzymes AAC: Aminoglycoside acetyltransferase APH: Aminoglycoside phosphotransferase ANT: Aminoglycoside nucleotidyltransferase MH: Mueller-Hinton CLSI: Clinical and Laboratory Standards Institute CDSs: Coding sequences CARD: Comprehensive antibiotic resistance database ANI: Average nucleotide identity ORF: Open reading frame PCR: Polymerase chain reaction IPTG: Isopropyl-β-d-thiogalactopyranoside OD: Optical density NCBI: National center for biotechnology information SDS‒PAGE : Sodium dodecyl sulfate‒polyacrylamide gel electrophoresis DTNB: 5,5'-dithiobis of (2-nitrobenzoic acid) TNB: 5-thio-2-nitrobenzoic acid isDDH : in silico DNA–DNA hybridization GNATs: GCN5-related N-acetyltransferases Declarations Ethics approval and consent to participate This study used strains isolated from the environment and animals in animal farms in Wenzhou, China. The owners of the farms were informed in writing of the study and provided approval for the sampling of animals. The studies involving human participants and animals were reviewed and approved by the Animal Welfare and Ethics Committee of Wenzhou Medical University, Zhejiang Province, China (protocol number: wydw2021-0323). All authors have reviewed and approved the manuscript for publication. As our study is not a clinical trial, no clinical trial registration or approval is required. Consent for publication Not applicable. Availability of data and materials The datasets generated during the current study are available in the NCBI repository, CP130335 (chromosome), CP130336 (pDW444-290265), CP130337 (pDW444-67560), CP130338 (pDW444-77364), OR282771 ( aac(6’)-Io ), and WKT62500 (AAC(6’)-Io). The data is publicly available. Competing Interests No potential conflicts of interest are reported by the authors. There are no conflicts of interest between the coauthors. Funding This study was supported by the Science & Technology Project of Jinhua City, China (2024-4-030, 2022-2-013), and the Zhejiang Provincial Natural Science Foundation of China (LTGY24H190003, LGD22C040006), and the Science & Technology Project of Wenzhou City, China (N20210001). Author’s contributions Conceived and designed the experiments: QB, CC and JL; Performed the experiments: WS, LZ, CF, HL and JWL; Data analysis and interpretation: WS, CF and JWL; Drafting of the manuscript: WS, QB, CC and JL. Acknowledgments The authors would like to acknowledge the teachers and scientists of the Science and Technology Platforms of Wenzhou Medical University who helped with the analysis of the enzyme kinetic parameters. References Favrot L, Blanchard JS, Vergnolle O. Bacterial GCN5-Related N-Acetyltransferases: From Resistance to Regulation. Biochemistry. 2016;55(7):989-1002. Serio AW, Magalhães ML, Blanchard JS, Connolly LE. Aminoglycosides: Mechanisms of action and resistance. Antimicrobial Drug Resistance: Mechanisms of Drug Resistance, Volume 1. 2017:213-29. Houghton JL, Green KD, Chen W, Garneau-Tsodikova S. The future of aminoglycosides: the end or renaissance? Chembiochem. 2010;11(7):880-902. Azucena E, Mobashery S. Aminoglycoside-modifying enzymes: mechanisms of catalytic processes and inhibition. Drug Resist Updat. 2001;4(2):106-17. Pullens B, van Benthem PP. Intratympanic gentamicin for Meniere's disease or syndrome. Cochrane Database Syst Rev. 2011(3):CD008234. Hainrichson M, Nudelman I, Baasov T. Designer aminoglycosides: the race to develop improved antibiotics and compounds for the treatment of human genetic diseases. Org Biomol Chem. 2008;6(2):227-39. Pokrovskaya V, Nudelman I, Kandasamy J, Baasov T. Aminoglycosides redesign strategies for improved antibiotics and compounds for treatment of human genetic diseases. Methods Enzymol. 2010;478:437-62. Hermann T. Aminoglycoside antibiotics: old drugs and new therapeutic approaches. Cell Mol Life Sci. 2007;64(14):1841-52. Guidry CA, Davies SW, Metzger R, Swenson BR, Sawyer RG. Whence Resistance? Surg Infect (Larchmt). 2015;16(6):716-20. Wright GD. Aminoglycoside-modifying enzymes. Curr Opin Microbiol. 1999;2(5):499-503. Ahmadian L, Norouzi Bazgir Z, Ahanjan M, Valadan R, Goli HR. Role of Aminoglycoside-Modifying Enzymes (AMEs) in Resistance to Aminoglycosides among Clinical Isolates of Pseudomonas aeruginosa in the North of Iran. Biomed Res Int. 2021;2021:7077344. Ramirez MS, Tolmasky ME. Aminoglycoside modifying enzymes. Drug Resist Updat. 2010;13(6):151-71. Ramirez MS, Tolmasky ME. Amikacin: Uses, Resistance, and Prospects for Inhibition. Molecules. 2017;22(12). Vakulenko SB, Mobashery S. Versatility of aminoglycosides and prospects for their future. Clin Microbiol Rev. 2003;16(3):430-50. Tada T, Miyoshi-Akiyama T, Shimada K, Shimojima M, Kirikae T. novel 6'-n-aminoglycoside acetyltransferase AAC(6')-Iaj from a clinical isolate of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2013;57(1):96-100. Tada T, Miyoshi-Akiyama T, Shimada K, Dahal RK, Mishra SK, Ohara H, et al. A Novel 6'-N-Aminoglycoside Acetyltransferase, AAC(6')-Ial, from a Clinical Isolate of Serratia marcescens. Microb Drug Resist. 2016;22(2):103-8. Bottone EJ. Bacillus cereus, a volatile human pathogen. Clin Microbiol Rev. 2010;23(2):382-98. Stenfors Arnesen LP, Fagerlund A, Granum PE. From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiol Rev. 2008;32(4):579-606. Pitt TL, McClure J, Parker MD, Amezquita A, McClure PJ. Bacillus cereus in personal care products: risk to consumers. Int J Cosmet Sci. 2015;37(2):165-74. Jung J, Jin H, Seo S, Jeong M, Kim B, Ryu K, et al. Short Communication: Enterotoxin Genes and Antibiotic Susceptibility of Bacillus cereus Isolated from Garlic Chives and Agricultural Environment. Int J Environ Res Public Health. 2022;19(19). Mbhele ZN, Shobo CO, Amoako DG, Zishiri OT, Bester LA. Occurrence, Antibiotic Resistance, Virulence Factors, and Genetic Diversity of Bacillus spp. from Public Hospital Environments in South Africa. Microb Drug Resist. 2021;27(12):1692-704. CLSI. Performance Standards for Antimicrobial Susceptibility Testing—Twenty-Eighth Informational Supplement: M100-S28.2020. Shi W, Lu J, Feng C, Gao M, Li A, Liu S, et al. Functional characterization of a novel aminoglycoside phosphotransferase, APH(9)-Ic, and its variant from Stenotrophomonas maltophilia. Front Cell Infect Microbiol. 2022;12:1097561. Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 2017;27(5):722-36. Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One. 2014;9(11):e112963. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30(14):2068-9. Buchfink B, Reuter K, Drost HG. Sensitive protein alignments at tree-of-life scale using DIAMOND. Nat Methods. 2021;18(4):366-8. McArthur AG, Waglechner N, Nizam F, Yan A, Azad MA, Baylay AJ, et al. The comprehensive antibiotic resistance database. Antimicrob Agents Chemother. 2013;57(7):3348-57. Jain C, Rodriguez RL, Phillippy AM, Konstantinidis KT, Aluru S. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat Commun. 2018;9(1):5114. Petkau A, Stuart-Edwards M, Stothard P, Van Domselaar G. Interactive microbial genome visualization with GView. Bioinformatics. 2010;26(24):3125-6. Gilchrist CLM, Chooi YH. clinker & clustermap.js: automatic generation of gene cluster comparison figures. Bioinformatics. 2021;37(16):2473-5. Magnet S, Lambert T, Courvalin P, Blanchard JS. Kinetic and mutagenic characterization of the chromosomally encoded Salmonella enterica AAC(6')-Iy aminoglycoside N-acetyltransferase. Biochemistry. 2001;40(12):3700-9. Galimand M, Fishovitz J, Lambert T, Barbe V, Zajicek J, Mobashery S, et al. AAC(3)-XI, a new aminoglycoside 3-N-acetyltransferase from Corynebacterium striatum. Antimicrob Agents Chemother. 2015;59(9):5647-53. Zhou K, Liang J, Dong X, Zhang P, Feng C, Shi W, et al. Identification and Characterization of a Novel Chromosomal Aminoglycoside 2'-N-Acetyltransferase, AAC(2')-If, From an Isolate of a Novel Providencia Species, Providencia wenzhouensis R33. Front Microbiol. 2021;12:711037. Figueroa-Bossi N, Balbontin R, Bossi L. Basic Bacteriological Routines. Cold Spring Harb Protoc. 2022;2022(10):Pdb prot107849. Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM. DNA-DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol. 2007;57(Pt 1):81-91. Pawlowski AC, Wang W, Koteva K, Barton HA, McArthur AG, Wright GD. A diverse intrinsic antibiotic resistome from a cave bacterium. Nat Commun. 2016;7:13803. Jin W, Wachino J, Kimura K, Yamada K, Arakawa Y. New plasmid-mediated aminoglycoside 6'-N-acetyltransferase, AAC(6')-Ian, and ESBL, TLA-3, from a Serratia marcescens clinical isolate. J Antimicrob Chemother. 2015;70(5):1331-7. Smith CA, Bhattacharya M, Toth M, Stewart NK, Vakulenko SB. Aminoglycoside resistance profile and structural architecture of the aminoglycoside acetyltransferase AAC(6')-Im. Microb Cell. 2017;4(12):402-10. Daigle DM, Hughes DW, Wright GD. Prodigious substrate specificity of AAC(6')-APH(2"), an aminoglycoside antibiotic resistance determinant in enterococci and staphylococci. Chem Biol. 1999;6(2):99-110. Lu S, Wang J, Chitsaz F, Derbyshire MK, Geer RC, Gonzales NR, et al. CDD/SPARCLE: the conserved domain database in 2020. Nucleic Acids Res. 2020;48(D1):D265-D8. Shaw KJ, Rather PN, Hare RS, Miller GH. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbiol Rev. 1993;57(1):138-63. Additional Declarations No competing interests reported. Supplementary Files TABLES1.docx Table S1 | The aminoglycoside resistance-associated putative resistance genes identified in the B. cereus DW444 genome. TableS2.docx Table S2 | The antibiotic resistance-associated putative resistance genes with ≥ 80% similarities with the function-characterized resistance genes identified in B. cereus DW444. TABLES3.xlsx Table S3 | The result of the type strains of Bacillus species with ≥ 95% ANI with DW444 TableS4.docx Table S4 | Comparation of MIC results of aminoglycosides for the recombinants carrying aac(6')-Io or its close relatives (μg/mL). TableS5.docx Table S5 | Corresponding substates of the antibiotic-modifying enzymes. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 03 Mar, 2026 Reviews received at journal 27 Feb, 2026 Reviewers agreed at journal 18 Feb, 2026 Reviews received at journal 13 May, 2025 Reviews received at journal 23 Apr, 2025 Reviewers agreed at journal 16 Apr, 2025 Reviewers agreed at journal 16 Apr, 2025 Reviewers invited by journal 14 Apr, 2025 Editor assigned by journal 04 Apr, 2025 Editor invited by journal 31 Mar, 2025 Submission checks completed at journal 28 Mar, 2025 First submitted to journal 28 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6041341","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":442882025,"identity":"a08e1049-deb3-42a7-a469-fa8999f0583c","order_by":0,"name":"Weina Shi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYDACZgiVwMbeAKQKGBgkiNfCc4CB4YABMVqgIIFBIoFILQbHmY89+LinLo9P8vEx6Q8GNnKSDcwPH93Ap+UwW7rhjGeHi9mk09IkDhikGUszsBkb5+DRYnaYx0ya58CBxDbpHDOglsOJ8xh42KTxa+H/Jv3nQF1im+QZorUAFTAcYE5sk+CBaJlNSIv9YTYzyZ4DhxPbeNKSLc4A/SLZTMAvkv2Hn0n8ADpsfvvhgzcqKmzkJI43P3yMTwsWwEya8lEwCkbBKBgFWAAAXQJHIKfTrAoAAAAASUVORK5CYII=","orcid":"","institution":"Lishui University","correspondingAuthor":true,"prefix":"","firstName":"Weina","middleName":"","lastName":"Shi","suffix":""},{"id":442882026,"identity":"7619f443-0dba-4132-9185-2a844505032c","order_by":1,"name":"Lei Zhang","email":"","orcid":"","institution":"Central Hospital Affiliated to Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Zhang","suffix":""},{"id":442882027,"identity":"ad4036b1-1003-4ebc-9e56-87dca6498ec6","order_by":2,"name":"Chunlin Feng","email":"","orcid":"","institution":"Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chunlin","middleName":"","lastName":"Feng","suffix":""},{"id":442882032,"identity":"b25aa8e2-b38c-42c2-877f-b5e4f557b29e","order_by":3,"name":"Hongqiang Lou","email":"","orcid":"","institution":"Jinhua University of Vocational Technology","correspondingAuthor":false,"prefix":"","firstName":"Hongqiang","middleName":"","lastName":"Lou","suffix":""},{"id":442882037,"identity":"d9005ed5-4f71-40d0-a2ff-37f34b13abde","order_by":4,"name":"Junwan Lu","email":"","orcid":"","institution":"Jinhua University of Vocational Technology","correspondingAuthor":false,"prefix":"","firstName":"Junwan","middleName":"","lastName":"Lu","suffix":""},{"id":442882041,"identity":"6a8cec15-0997-472f-b2f5-214d0ff73c43","order_by":5,"name":"Qiyu Bao","email":"","orcid":"","institution":"Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qiyu","middleName":"","lastName":"Bao","suffix":""},{"id":442882043,"identity":"a9d33dd5-2fd6-4e16-bf32-7dbb94c2e13a","order_by":6,"name":"Cong Cheng","email":"","orcid":"","institution":"Lishui University","correspondingAuthor":false,"prefix":"","firstName":"Cong","middleName":"","lastName":"Cheng","suffix":""},{"id":442882045,"identity":"ca69958b-75fc-4109-8b78-94b6a2673ff5","order_by":7,"name":"Jun Lu","email":"","orcid":"","institution":"Quzhou People’s Hospital, The Quzhou Affiliated Hospital of Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Lu","suffix":""}],"badges":[],"createdAt":"2025-02-16 13:23:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6041341/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6041341/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80728759,"identity":"75dd047e-b899-42a5-bf51-440d6fb7ac16","added_by":"auto","created_at":"2025-04-16 12:12:44","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":122744,"visible":true,"origin":"","legend":"\u003cp\u003eA phylogenetic tree illustrating the relationship between AAC(6’)-Io and other members within the AAC(6’)-I subfamily. AAC(6’)-Io is highlighted in red. The three adjacent columns provide additional information, including the protein names, accession numbers, and percentages of amino acid similarities with AAC(6’)-Io.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6041341/v1/05db4461dce85ee02247b08f.jpeg"},{"id":80728761,"identity":"56d5d392-db69-4752-b408-1241db9fd28a","added_by":"auto","created_at":"2025-04-16 12:12:44","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":247011,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple sequence alignment of AAC(6’)-Io with its close relatives. Symbols such as “exclamations” denote residues that are completely conserved, while asterisks indicate strongly similar residues. Hyphens are used to represent gaps in the sequence alignment. The numbers on the right side of the alignment indicate the length of each sequence. Functional residues are highlighted within frames, and a specific motif is mentioned. The AAC(6’) proteins and their accession numbers are AAC(6’)-Io (WKT62500), AAC(6’)-34 (APB03223.1), AAC(6’)-Ian (BAQ22025.1), and AAC(6’)-Im (AAK63041.1).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6041341/v1/0ddcf726d036f49405c22030.jpeg"},{"id":80730444,"identity":"2baefc5f-80b1-474a-b711-2dffc954395b","added_by":"auto","created_at":"2025-04-16 12:28:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1510160,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6041341/v1/d63b9e38-44c9-46f7-b5d0-4b12e18f1065.pdf"},{"id":80728762,"identity":"f4a8a1d9-55cf-4d3b-b206-d0312531ec9f","added_by":"auto","created_at":"2025-04-16 12:12:44","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19555,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1 | The aminoglycoside resistance-associated putative resistance genes identified in the \u003cem\u003eB. cereus\u003c/em\u003e DW444 genome.\u003c/p\u003e","description":"","filename":"TABLES1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6041341/v1/d2b0063f322447747557c47d.docx"},{"id":80729423,"identity":"33125fb8-cab3-4760-880c-5e4af7d73997","added_by":"auto","created_at":"2025-04-16 12:20:44","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15386,"visible":true,"origin":"","legend":"\u003cp\u003eTable S2 | The antibiotic resistance-associated putative resistance genes with ≥ 80% similarities with the function-characterized resistance genes identified in \u003cem\u003eB. cereus \u003c/em\u003eDW444.\u003c/p\u003e","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6041341/v1/a06a2534db217b39e97a0b50.docx"},{"id":80728772,"identity":"e4f09fbc-97a2-4614-9583-3862a4f59310","added_by":"auto","created_at":"2025-04-16 12:12:44","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":384850,"visible":true,"origin":"","legend":"\u003cp\u003eTable S3 | The result of the type strains of Bacillus species with ≥ 95% ANI with DW444\u003c/p\u003e","description":"","filename":"TABLES3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6041341/v1/8114e3fd28ead0967bb3742e.xlsx"},{"id":80728763,"identity":"c3a75fdc-2375-49ea-9d9e-4e7e7fc0b0b0","added_by":"auto","created_at":"2025-04-16 12:12:44","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":18197,"visible":true,"origin":"","legend":"\u003cp\u003eTable S4 | Comparation of MIC results of aminoglycosides for the recombinants carrying \u003cem\u003eaac(6')-Io\u003c/em\u003e or its close relatives (μg/mL).\u003c/p\u003e","description":"","filename":"TableS4.docx","url":"https://assets-eu.researchsquare.com/files/rs-6041341/v1/f4de80f914e27ccc3ba45913.docx"},{"id":80729424,"identity":"a3e1a6c5-28e6-4663-894c-558e0fe62f6a","added_by":"auto","created_at":"2025-04-16 12:20:44","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":14706,"visible":true,"origin":"","legend":"\u003cp\u003eTable S5 | Corresponding substates of the antibiotic-modifying enzymes.\u003c/p\u003e","description":"","filename":"TableS5.docx","url":"https://assets-eu.researchsquare.com/files/rs-6041341/v1/93fc0386d451f1aef3cc7455.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A novel chromosomal aminoglycoside 6′-N-acetyltransferase, AAC(6′)-Io, confers resistance to multiple aminoglycosides identified from Bacillus cereus","fulltext":[{"header":"Background","content":"\u003cp\u003eAminoglycosides are broad-spectrum antibacterial compounds that typically contain one aminocyclitol ring (most commonly 2-deoxystreptamine) linked to one or more amino sugars by a glycosidic bond (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Since the 1940s, aminoglycosides have been primarily applied clinically to treat a variety of bacterial infections (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The primary mechanism of action of these antibiotics is the binding of aminoglycosides to the 16S rRNA of the 30S ribosomal fragment, leading to the misreading of mRNA and thus to the biosynthesis of nonfunctional misfolded proteins (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCurrently, the use of aminoglycosides as treatments has become more conservative because of their toxicity, bacterial resistance, and the complexity associated with their chemical syntheses (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). However, in addition to the use of aminoglycosides for bactericidal therapy, modern medicine has found other uses for these agents, including treatments for various human genetic diseases and Meniere\u0026rsquo;s disease (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Moreover, aminoglycosides are being researched as multifunctional agents for the treatment and prevention of HIV-1 infection (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUnfortunately, due to the overuse and selective pressure of antibiotics, bacteria have developed several resistance mechanisms that threaten the use of aminoglycosides (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). The main resistance mechanism to these antibiotics is the enzymatic modification of aminoglycosides by bacteria encoding aminoglycoside-modifying enzymes (AMEs) (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). These enzymes act to inactivate aminoglycoside antibiotics by modifying them through acetylation (aminoglycoside acetyltransferase, AAC), phosphorylation (aminoglycoside phosphotransferase, APH), and adenylation (aminoglycoside nucleotidyltransferase, ANT) (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). The aminoglycoside acetyltransferase class comprises four subtypes: AAC(2'), AAC(6'), AAC(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), and AAC(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). The ability of aminoglycoside 6\u0026rsquo;-\u003cem\u003eN\u003c/em\u003e-acetyltransferases [AAC(6\u0026prime;)s] to inactivate several clinically significant aminoglycosides makes them quite intriguing. The AAC(6\u0026rsquo;)-I enzymes confer resistance to amikacin, tobramycin, sisomicin, kanamycin, isepamicin, dibekacin, and netilmicin. However, the AAC(6\u0026rsquo;)-II type acetylates gentamicin, tobramycin, netilmicin, and sisomicin but not amikacin (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eBacillus cereus\u003c/em\u003e is a gram-positive, aerobic-to-facultative, spore-forming rod-shaped bacterium widely found in nature (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). The ability of this bacterium to form endospores that are resistant to a range of physical stressors and endure time and harsh conditions is an essential trait of its ability to cause foodborne illness (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). \u003cem\u003eB. cereus\u003c/em\u003e can cause various infections, including gastrointestinal, eye, bloodstream, skin, and central nervous system infections. In addition, this bacterium can coinfect other bacteria, such as \u003cem\u003eBacillus anthracis\u003c/em\u003e and \u003cem\u003eClostridium perfringens\u003c/em\u003e, resulting in anthrax-like pulmonary infections and gas gangrene-like cutaneous infections, respectively (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). In terms of antibiotic resistance, \u003cem\u003eB. cereus\u003c/em\u003e constantly exhibits resistance to β-lactams, fluoroquinolones, tetracyclines and trimethoprim, while susceptibility to clindamycin, erythromycin, chloramphenicol, vancomycin, aminoglycosides and tetracyclines is usually observed (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this work, the environmental isolate \u003cem\u003eB. cereus\u003c/em\u003e DW444 was found to chromosomally harbor a novel aminoglycoside 6\u0026rsquo;-\u003cem\u003eN\u003c/em\u003e-acetyltransferase gene, designated \u003cem\u003eaac(6\u0026rsquo;)-Io\u003c/em\u003e, and the molecular characteristics of this gene and the protein it encodes were characterized.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eBacterial strains and plasmids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn environmental isolate, \u003cem\u003eB. cereus\u003c/em\u003e DW444, was obtained from a soil sample from an animal farm in Wenzhou, southeastern China, in 2020. \u003cem\u003eEnterococcus faecalis\u003c/em\u003e ATCC 29212 and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 29213 were utilized as quality controls for antimicrobial susceptibility tests of nonfastidious gram-positive bacteria (22). \u003cem\u003eEscherichia coli\u003c/em\u003e DH5\u0026alpha; and \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) were used as the recipients for the recombinant plasmids \u003cem\u003eaac(6\u0026rsquo;)-Io\u003c/em\u003e-pMD19-T (cloning of \u003cem\u003eaac(6\u0026rsquo;)-Io\u0026nbsp;\u003c/em\u003ewith its promoter region) and \u003cem\u003eaac(6\u0026rsquo;)-Io\u003c/em\u003e-pCold I (cloning of ORF of \u003cem\u003eaac(6\u0026rsquo;)-Io\u003c/em\u003e), respectively. The bacteria and plasmids utilized in this study are listed in Table 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 1 Bacteria and plasmids used in this work.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eBacterium/plasmid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 267px;\"\u003e\n \u003cp\u003eRelevant characteristic(s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003eSource\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eBacterium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 267px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003e\u003cem\u003eBacillus cereus\u0026nbsp;\u003c/em\u003eDW444\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 267px;\"\u003e\n \u003cp\u003eDW444, the wild-type isolate\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003e\u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003eDH5\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 267px;\"\u003e\n \u003cp\u003eDH5\u0026alpha;, a recipient for cloning of \u003cem\u003eaac(6\u0026rsquo;)-Io\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003eOur laboratory collection\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003e\u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003eBL21(DE3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 267px;\"\u003e\n \u003cp\u003eBL21, a recipient for the AAC(6\u0026rsquo;)-Io expression\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003eOur laboratory collection\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u0026nbsp;\u003c/em\u003eATCC 29213\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 267px;\"\u003e\n \u003cp\u003eATCC 29213, a quality control for antimicrobial susceptibility test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003eOur laboratory collection\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003e\u003cem\u003eEnterococcus faecalis\u003c/em\u003e ATCC 29212\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 267px;\"\u003e\n \u003cp\u003eATCC 29212, a quality control for antimicrobial susceptibility test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003eOur laboratory collection\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003epMD19-T-\u003cem\u003eaac(6\u0026rsquo;)-Io\u003c/em\u003e/DH5\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 267px;\"\u003e\n \u003cp\u003eDH5\u0026alpha; harboring pMD19-T-\u003cem\u003eaac(6\u0026rsquo;)-Io\u0026nbsp;\u003c/em\u003e(a recombinant plasmid)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003epCold I-\u003cem\u003e\u0026nbsp;aac(6\u0026rsquo;)-Io\u003c/em\u003e/BL21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 267px;\"\u003e\n \u003cp\u003eBL21 harboring pCold I-\u003cem\u003eaac(6\u0026rsquo;)-Io\u0026nbsp;\u003c/em\u003e(a recombinant plasmid)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003ePlasmid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 267px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003epMD19-T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 267px;\"\u003e\n \u003cp\u003ea vector for cloning the \u003cem\u003eaac(6\u0026rsquo;)-Io\u003c/em\u003e gene with its promoter region, ampicillin resistance\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003eTakara Bio, Inc., Dalian, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003epCold I/BL 21(DE3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 267px;\"\u003e\n \u003cp\u003ea vector for expressing AAC(6\u0026rsquo;)-Io, ampicillin resistance\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 137px;\"\u003e\n \u003cp\u003eOur laboratory collection\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eantibiotic susceptibility tests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe minimum inhibitory concentration (MIC) was determined using Mueller-Hinton (MH) solid medium and the agar dilution method in accordance with the suggested guidelines of the Clinical and Laboratory Standards Institute (CLSI) (22).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenome sequencing and annotation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe methods used for genome sequencing and annotation were described in a previous publication (23). Briefly, the genome sequence data from the PacBio RS II sequencing platform were initially assembled using Canu v1.8 (24), and the sequence quality was further corrected by mapping the Illumina sequencing data to the initial assembly using Pilon software (25). The coding sequences (CDSs) were predicted by Prokka v1.14.6 (26). The deduced proteins were annotated via DIAMOND v2.0.11 (27) and NCBI nonredundant protein databases. The antimicrobial resistance genes were predicted based on the comprehensive antibiotic resistance database (CARD) (28) by Resistance Gene Identifier v5.2.0 (https://github.com/arpcard/rgi). The average nucleotide identity (ANI) was computed by FastANI v1.31 (29). The promoter region of a gene was predicted using BPROM (2016) (http://www.softberry.com/berry.phtml?topic=bprom\u0026amp;group=programs\u0026amp;subgroup=gfindb). The genomic features of the genome map were visualized by the CGView Server (30).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMultiple\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003esequence comparison\u0026nbsp;and phylogenetic tree reconstruction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe methods for multiple sequence alignment, phylogenetic tree reconstruction, and visualization were described in a previous publication (23). The molecular weight and pI of the protein were analyzed using ProtParam (https://web.ExPASy.org/protparam/). The conserved domains of the proteins were analyzed by CD-search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The program clinker v0.0.24 (31) was applied to visualize the genetic context of the genes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCloning of the \u003cem\u003eaac(6\u003c/em\u003e\u003c/strong\u003e\u0026rsquo;\u003cstrong\u003e\u003cem\u003e)-Io\u0026nbsp;\u003c/em\u003egene\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing \u003cem\u003eB. cereus\u003c/em\u003e DW444 genomic DNA as a template, the open reading frame (ORF) of \u003cem\u003eaac(6\u0026rsquo;)-Io\u003c/em\u003e with its upstream promoter region was amplified by polymerase chain reaction (PCR) with a pair of primers (forward primer pro-aac-F, 5\u0026rsquo;-AGGTGCAATGAAAGATGTAACAGCAAG-3\u0026rsquo;; reverse primer pro-aac-R, 5\u0026rsquo;-CACACATCCCCCTTTGTTTGTATAAGC-3\u0026rsquo;). To clone the PCR products, the vectors pMD19-T and T4 DNA ligase (Takara Bio, Inc., Dalian, China) were used. Using the calcium chloride method, the recombinant plasmid was transformed into competent \u003cem\u003eE. coli\u003c/em\u003e DH5\u0026alpha; cells. Subsequently, the transformant was selected on an LB agar plate containing 100 \u0026mu;g/mL ampicillin, and the inserted DNA fragment was verified by Sanger sequencing (23).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRecombinant protein AAC(6\u003c/strong\u003e\u0026rsquo;\u003cstrong\u003e)-Io expression and purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ORF of \u003cem\u003eaac(6\u0026rsquo;)-Io\u003c/em\u003e from \u003cem\u003eB. cereus\u003c/em\u003e DW444 was amplified by PCR using the primer set \u003cem\u003eBam\u003c/em\u003eHI- enterokinase-\u003cem\u003eaac\u003c/em\u003e-F (5\u0026rsquo;- CGGGATCCGACGACGACGACAAGATGCTTTTTCAAAAAGAAGGTTTATTGATT-3\u0026rsquo;) and \u003cem\u003eHind\u003c/em\u003eIII-\u003cem\u003eaa\u003c/em\u003ec-R (5\u0026rsquo;- CCAAGCTTTTATTGTTTATATTCCATCATCCAA-3\u0026rsquo;). According to previous methods (23), the PCR products digested with restriction enzymes (\u003cem\u003eBam\u003c/em\u003eHI and \u003cem\u003eHind\u003c/em\u003eIII) (Takara Bio Inc., Dalian, China) were inserted into the pCold I expression vector, and the resulting plasmid, pCold I-enterokinase-\u003cem\u003eaac(6\u0026rsquo;)-Io\u003c/em\u003e, was transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) competent cells. The cloned fragment of the transformant was verified by Sanger sequencing (Hangzhou Tsingke Biotechnology Co., Ltd., Hangzhou, China) and was then used for recombinant protein expression. The transformant pCold I-enterokinase-\u003cem\u003eaac(6\u0026rsquo;)-Io/\u003c/em\u003eBL21 was cultivated at 37 \u0026deg;C in LB medium supplemented with 100 \u0026mu;g/mL ampicillin until the optical density (OD) at 600 nm reached 0.5. To promote the expression of His6-tagged AAC(6\u0026apos;)-Io, 1 mM isopropyl-\u0026beta;-d-thiogalactopyranoside (IPTG) was added, and the bacterial cells were continuously incubated for 18 hours at 16 \u0026deg;C. After obtaining the bacterial culture mixture, the bacterial cells were sonicated, and the soluble fraction containing His6-tagged AAC(6\u0026rsquo;)-Io was obtained by centrifugation (5000 g, 10 min, 4 \u0026deg;C). The supernatant and the precipitate from the soluble fraction were further separated by centrifugation (22,000 g, 30 min, 4 \u0026deg;C). Following the manufacturer\u0026apos;s instructions, the Histag Protein Purification Kit (Beyotime, Shanghai, China) was used to extract His6-tagged AAC(6\u0026apos;)-Io from the supernatant. Enterokinase (GenScript Inc., Nanjing, China) was utilized to digest the His6-tag for 24 hours at 16 \u0026deg;C. Sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) and Coomassie Brilliant Blue staining were used to validate the purity of AAC(6\u0026apos;)-Io. The protein content was measured spectrophotometrically with a BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, United States).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKinetic studies of AAC(6\u003c/strong\u003e\u0026rsquo;\u003cstrong\u003e)-Io\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecifically, AAC(6\u0026rsquo;)-Io catalyzes the transfer of an acetyl group of acetyl coenzyme A to position 6\u0026rsquo; of the 6-aminohexose ring of the substrate (14). The free sulfhydryl group of CoASH reacts with the 5,5\u0026apos;-dithiobis of (2-nitrobenzoic acid) (DTNB) to generate 5-thio-2-nitrobenzoic acid (TNB), whose absorbance at 412 nm can be monitored spectrophotometrically (32, 33). For a total of ten minutes, the reaction was continually observed by a Synergy Neo2 Multi-Mode Microplate Reader (Biotek, VT, USA). The kinetic assay was conducted in a total volume of 200 \u0026mu;L that contained 2 mM DTNB, 80 \u0026mu;M acetyl-CoA, 50 mM HEPES (pH 7.0), 1 mM EDTA, 25 mM 2-(N-morpholino) ethanesulfonic acid (pH 6.0), and different quantities of aminoglycosides (5\u0026ndash;150 \u0026mu;M). The assays were started by adding 4.2 \u0026mu;M of purified enzyme(34). With GraphPad Prism 9 (GraphPad Software, San Jose, CA, United States), the initial rates were plotted against substrate concentration and fitted by nonlinear regression to the Michaelis‒Menten equation to calculate \u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e and \u003cem\u003ek\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNucleotide sequence accession number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe following genomic (protein) sequences of \u003cem\u003eB. cereus\u003c/em\u003e DW444 are available in the NCBI databases:\u0026nbsp;CP130335 (chromosome), CP130336 (pDW444-290265), CP130337 (pDW444-67560), CP130338 (pDW444-77364), OR282771 (\u003cem\u003eaac(6\u0026rsquo;)-Io\u003c/em\u003e) and WKT62500 (AAC(6\u0026rsquo;)-Io).\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e\u003cstrong\u003eIdentification of the novel aminoglycoside resistance gene \u003cem\u003eaac(6\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026rsquo;\u003cstrong\u003e)-Io\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;in \u003cem\u003eB. cereus\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMore than 500 isolates, including DW444, were obtained from animal anal swabs and sewage samples collected from rural animal farms in Wenzhou, southern China, via the plate streak method (35). In addition to the \u003cem\u003ein vivo\u003c/em\u003e antibiotic susceptibility test for the isolates, the whole-genome sequences of numerous isolates were also sequenced by the Illumina NovaSeq platform. An analysis of the relationships between the bacterial resistance phenotypes and genotypes revealed that one soil bacterium, DW444, had high MICs against nearly all 35 antimicrobials of the 8 classes tested (Table 2), and even though it exhibited higher MICs (\u0026gt; 2048 \u0026mu;g/mL) for aminoglycosides such as kanamycin, amikacin, spectinomycin and ribostamycin, no gene with \u0026gt; 80% identity to the functionally characterized genes responsible for aminoglycoside resistance could be predicted from the genome sequence. This may suggest the presence of a novel resistance mechanism concerning the aminoglycoside resistance phenotype of the isolate. To investigate the potential resistance mechanism, the genome annotation results were examined, and a few hypothetical aminoglycoside resistance proteins, such as an AAC(6\u0026rsquo;)-34-like protein sharing 47.51% amino acid similarity with AAC(6\u0026rsquo;)-34 (APB03223.1), an ANT(6)-Ia-like protein sharing 42.0% amino acid similarity with ANT(6)-Ia (AHE40557.1) and an AAC(3)-VIIa-like protein with 30.0% amino acid similarity with AAC(3)-VIIa (AAA88552.1), were found (Table S1). Among the three detected genes, we selected the one with the highest similarity to known genes, that is, the \u003cem\u003eaac(6\u0026apos;)-34\u003c/em\u003e homologous gene (designated \u003cem\u003eaac(6\u0026rsquo;)-Io\u003c/em\u003e in this study), and further studied and confirmed that it has acetyltransferase activity.\u003c/p\u003e\n\u003cp\u003eTable 2 Minimum inhibitory concentrations of antimicrobials for \u003cem\u003eB. cereus\u003c/em\u003e DW444 and the recombinant and control strains (\u0026mu;g/mL).\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"607\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eClass\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003eAntibiotic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u003cem\u003eB. cereus\u0026nbsp;\u003c/em\u003eDW444\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003epMD19-T-\u003cem\u003eaac(6\u0026apos;)-Io\u003c/em\u003e/DH5\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003epMD19-T/DH5\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003eDH5\u0026alpha;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u003cem\u003eE. faecalis\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eATCC 29212\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e\u003cem\u003eS. aureus\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eATCC 29213\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eAminoglycosides\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003eAmikacin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026gt;2,048\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026lt;2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003eGentamicin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026gt;512\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003eKanamycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026gt;2,048\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e512\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026lt;2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003eNeomycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003eNetilmicin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026gt;32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026lt;0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003eParomomycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003eRibostamycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026gt;4,096\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e512\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e\u0026lt;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026lt;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026lt;2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003eSisomicin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026gt;256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e\u0026lt;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026lt;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003eSpectinomycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026gt;2,048\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003eStreptomycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e1024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003eTobramycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e512\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026beta;-Lactams\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003eAmpicillin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026gt;2,048\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e\u0026gt;2,048\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e\u0026gt;2,048\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003eAmoxicillin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e2,048\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e\u0026gt;2,048\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e\u0026gt;2,048\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026lt;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026lt;1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003eAztreonam\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026gt;16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026gt;16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026gt;16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003eCefazolin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003eCefepime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026gt;16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e0.0625\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e0.0625\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.016\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026gt;16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003eCefotaxime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026gt;16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026gt;16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003eCefoxitin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003eCeftazidime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026gt;256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026gt;256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003eCeftriaxone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026gt;128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e0.0625\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026gt;128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003eCefuroxime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026gt;1,024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003ecephalothin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e512\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026lt;4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026lt;4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003ePenicillin G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026gt;2,048\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e2,048\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e2048\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003ePiperacillin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026gt;128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e\u0026gt;128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eCarbapenems\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003eMeropenem\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026gt;32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e\u0026lt;0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e\u0026lt;0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026lt;0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003eImipenem\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026gt;64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026lt;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eQuinolones\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003eLevofloxacin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026gt;32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e\u0026lt;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e\u0026lt;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026lt;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026lt;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026lt;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003eNalidixic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e1,024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eMacrolides\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003eAzithromycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e512\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e\u0026lt;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e\u0026lt;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026lt;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026lt;1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003eErythromycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026gt;1,024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eTetracyclines\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003eTetracycline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026lt;1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003eTigecycline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026gt;32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e\u0026lt;0.0625\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e\u0026lt;0.0625\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u0026lt;0.0625\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026lt;0.0625\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026lt;0.0625\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003ePhosphonic acid derivatives\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003eFosfomycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026gt;1,024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eAmphenicols\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003eChloramphenicol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026gt;1,024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003eFlorfenicol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e512\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70px;\"\u003e\n \u003cp\u003e\u0026lt;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 65px;\"\u003e\n \u003cp\u003e\u0026lt;2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eGeneral c\u003c/strong\u003e\u003cstrong\u003eharacteristics of the \u003cem\u003eB. cereus\u003c/em\u003e DW444 genome\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the structure of the novel resistance gene \u003cem\u003eaac(6\u0026rsquo;)-Io\u003c/em\u003e, the whole genome of DW444 was sequenced. The\u003cem\u003e\u0026nbsp;\u003c/em\u003egenome consisted of a chromosome and three plasmids (Table 3). The chromosome was 5,548,786 bp in length and encoded 5,717 CDSs with a GC content of 35.24%. The three plasmids pDW444-290265, pDW444-67560, and pDW444-77364\u0026nbsp;encoded 313, 109 and 88 CDSs, respectively.\u0026nbsp;Eight genes encoding proteins sharing aa sequence similarities of \u0026ge; 80% with proteins encoded by the antimicrobial resistance genes in the CARD were found to be encoded in the chromosome, which included\u003cem\u003e\u0026nbsp;fosB\u0026nbsp;\u003c/em\u003e(fosfomycin thiol transferase), \u003cem\u003ebcl\u003c/em\u003e (class A \u003cem\u003eB. cereus\u003c/em\u003e Bc beta-lactamase), \u003cem\u003erpoB\u003c/em\u003e (rifamycin-resistant beta-subunit of RNA polymerase), \u003cem\u003ebcII\u003c/em\u003e (subclass B1 \u003cem\u003eB. cereus\u003c/em\u003e Bc beta-lactamase), \u003cem\u003ebcIII\u0026nbsp;\u003c/em\u003e(class A \u003cem\u003eB. cereus\u003c/em\u003e Bc beta-lactamase), \u003cem\u003esatA\u003c/em\u003e (streptothricin acetyltransferase), \u003cem\u003eEnterococcus faecium\u003c/em\u003e EF-Tu mutants (elfamycin resistant EF-Tu), and \u003cem\u003eisaB\u003c/em\u003e (lsa-type ABC-F protein) (Table S2). No putative antibiotic resistance gene was identified to be encoded on any of the three plasmids.\u003c/p\u003e\n\u003cp\u003eTable 3 General features of the \u003cem\u003eB. cereus\u0026nbsp;\u003c/em\u003eDW444 genome.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" align=\"\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 171px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003eChromosome\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003epDW444-67560\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003epDW444-77364\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003epDW444-290265\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 171px;\"\u003e\n \u003cp\u003eSize (bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003e5,548,786\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e67,560\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e77,364\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e290,265\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 171px;\"\u003e\n \u003cp\u003eGC\u0026nbsp;content\u0026nbsp;(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003e35.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e33.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e30.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e32.83\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 171px;\"\u003e\n \u003cp\u003ePredicted\u0026nbsp;coding\u0026nbsp;sequences (CDSs)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003e5,717\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e313\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 171px;\"\u003e\n \u003cp\u003eKnown\u0026nbsp;proteins\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003e4,249 (74.32%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e8 (9.09%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e10 (9.17%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e86 (27.48%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 171px;\"\u003e\n \u003cp\u003eHypothetical\u0026nbsp;proteins\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003e1,468 (25.68%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e80 (90.91%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e99 (90.83%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e227 (72.52%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 171px;\"\u003e\n \u003cp\u003eProtein\u0026nbsp;coding\u0026nbsp;(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003e95.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e100.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e98.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e88.92\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 171px;\"\u003e\n \u003cp\u003eAverage\u0026nbsp;ORF\u0026nbsp;length\u0026nbsp;(bp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003e810.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e655.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e599.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e675.60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 171px;\"\u003e\n \u003cp\u003eAverage\u0026nbsp;protein\u0026nbsp;length\u0026nbsp;(aa)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003e269.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e217.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e201.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e230.40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 171px;\"\u003e\n \u003cp\u003etRNAs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003e106\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 171px;\"\u003e\n \u003cp\u003erRNA\u0026nbsp;operons\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003e(16S-23S-5S) *14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eSpecies identification of the isolate revealed that the genome sequence of DW444 had the highest percentage (72.3%) of \u003cem\u003ein silico\u003c/em\u003e DNA\u0026ndash;DNA hybridization (\u003cem\u003eis\u003c/em\u003eDDH) with \u003cem\u003eB. cereus\u003c/em\u003e ATCC 14579 (GCF_006094295.1), followed by \u003cem\u003eBacillus thuringiensis\u0026nbsp;\u003c/em\u003eATCC 10792 (GCF_002119445.1, 68.3%). The ANI results demonstrated that the whole genome of DW444 shared the highest identity (99.88%) with \u003cem\u003eB. thuringiensis\u003c/em\u003e (GCF_024712805.1), and the identity with \u003cem\u003eB. cereus\u003c/em\u003e ATCC 14579 (GCF_006094295.1) was 96.84% (Table S3). According to the threshold (cutoff scores \u0026ge;70% for \u003cem\u003eis\u003c/em\u003eDDH and \u0026ge;95% for ANI) (29, 36) to specify a bacterium to a certain species, there was only one strain, \u003cem\u003eB. cereus\u003c/em\u003e ATCC 14579, in the public database, sharing both higher \u003cem\u003eis\u003c/em\u003eDDH and ANI with DW444 than the thresholds. The isolate DW444 was thus tentatively classified as the species \u003cem\u003eB. cereus\u003c/em\u003e and was therefore designated \u003cem\u003eB. cereus\u003c/em\u003e DW444.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe resistance phenotype and distribution of \u003cem\u003eaac(6\u0026rsquo;)-Io\u003c/em\u003e(-like) genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eaac(6\u0026rsquo;)-Io\u003c/em\u003e gene is 537 bp in length and encodes a predicted peptide of 179 amino acids with a predicted molecular mass of 20.72 kDa and a pI value of 5.12. The \u003cem\u003eaac(6\u0026rsquo;)-Io\u003c/em\u003e gene with its promoter region was PCR-amplified and\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ecloned and inserted into the pMD19-T vector. Compared with the control (pMD19-T/DH5\u0026alpha;), the recombinant (pMD19-T-\u003cem\u003eaac(6\u0026rsquo;)-Io\u003c/em\u003e/DH5\u0026alpha;) strains showed resistance to tobramycin, kanamycin, amikacin, netilmicin, sisomicin and ribostamycin, with increased MICs of 32-, 32-, 8-, 8-, \u0026gt;8-, and \u0026gt;256-fold, respectively, for the 6 antimicrobial agents (Table S4). Analysis of the resistance profiles of \u003cem\u003eaac(6\u0026rsquo;)-Io\u003c/em\u003e with its close relatives \u003cem\u003eaac(6\u0026rsquo;)-34\u003c/em\u003e, \u003cem\u003eaac(6\u0026rsquo;)-Ian\u003c/em\u003e, \u003cem\u003eaac(6\u0026rsquo;)-Ie\u003c/em\u003e, and \u003cem\u003eaac(6\u0026rsquo;)-Im\u003c/em\u003e revealed that among the aminoglycoside antibiotics they tested, they exhibited similar resistance characteristics, although the MICs of the aminoglycosides may vary (37, 38, 39) (Table S4).\u003c/p\u003e\n\u003cp\u003eTo date, 33 functionally characterized resistance genes sharing amino acid similarities between 47.51% (AAC(6\u0026rsquo;)-34) and 22.73% (AAC(6\u0026rsquo;)-Ib-cr3) with AAC(6\u0026rsquo;)-Io are present in the CARD, and all of them are AAC(6\u0026rsquo;)-I proteins. According to the phylogenetic analysis of these proteins, AAC(6\u0026apos;)-Io formed a new branch and was closer to AAC(6\u0026apos;)-34, AAC(6\u0026apos;)-Ian, AAC(6\u0026apos;)-Ie, and AAC(6\u0026apos;)-Im. This suggested that AAC(6\u0026rsquo;)-Io was a novel lineage of the AAC(6\u0026rsquo;)-I subfamily (Figure 1).\u003c/p\u003e\n\u003cp\u003eTo analyze the distribution of AAC(6\u0026apos;)-Io-like proteins, AAC(6\u0026apos;)-Io was used as a query to search for homologous proteins in the NCBI nr database. A total of 517 sequences with both coverage and identity \u0026gt;80% were retrieved, and all of them were from the genus \u003cem\u003eBacillus\u003c/em\u003e. The closest relative of AAC(6\u0026rsquo;)-Io was a functionally uncharacterized protein (WP_000898772.1) of GCN5-related \u003cem\u003eN\u003c/em\u003e-acetyltransferases (GNATs) from \u003cem\u003eBacillus\u003c/em\u003e spp., with an aa sequence identity of 100.0% and coverage of 100% for AAC(6\u0026rsquo;)-Io.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKinetic parameters and structural characteristics of AAC(6\u0026rsquo;)-Io and its relatives\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe enzymatic activity analysis of AAC(6\u0026rsquo;)-Io revealed that tobramycin, kanamycin, amikacin, isepamicin and netilmicin were acetylated by AAC(6\u0026rsquo;)-Io, while no acetylation was detected when using spectinomycin or\u0026nbsp;streptomycin\u0026nbsp;as a substrate (Table 4). To analyze the differences in the enzymatic activities of the functionally characterized AAC(6\u0026rsquo;) proteins with closer phylogenetic relationships (Figure 1) and greater amino acid similarity (\u0026gt; 40%) to AAC(6\u0026rsquo;)-Io, the kinetic parameters of four AAC(6\u0026rsquo;) proteins [AAC(6\u0026rsquo;)-34, AAC(6\u0026rsquo;)-Ian, AAC(6\u0026rsquo;)-Ie, and AAC(6\u0026rsquo;)-Im] were compared. The substrates of AAC(6\u0026rsquo;)-Io fall within the range of substrates of the subfamily AAC(6\u0026rsquo;)-I. Analyzing the substrate specificity of these 5 proteins (including AAC(6\u0026rsquo;)-Io of this work) revealed that among the 9 antimicrobial agents analyzed with AAC(6\u0026rsquo;)-Io, except for two (spectinomycin and streptomycin) with AAC(6\u0026rsquo;)-Io alone and both with a negative result, the other 7 antimicrobials were analyzed with the other 2-4 enzymes, and they all showed positive results. Of the other four enzymes, (AAC(6\u0026apos;)-Ian and AAC(6\u0026apos;)-Ie) were examined with all 7 substrates. AAC(6\u0026rsquo;)-Im was used to test 6 compounds, while AAC(6\u0026apos;)-34 was used to analyze only two compounds (Table S5).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Table 4 Steady-state kinetic parameters for AAC(6\u0026rsquo;)-Io.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 60px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eSubstrate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 189px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ek\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003ecat\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(s\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 160px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eK\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003em\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(\u0026mu;M)\u003cem\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 159px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ek\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003csub\u003ecat\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e/\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e (M\u003csup\u003e\u0026minus;1\u003c/sup\u003e\u003c/strong\u003e\u0026middot;\u003cstrong\u003es\u003csup\u003e\u0026minus;1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eAAC(6\u0026apos;)-Io\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003eAAC(6\u0026apos;)-34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003eAAC(6\u0026apos;)-Ie\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eAAC(6\u0026apos;)-Io\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eAAC(6\u0026apos;)-34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eAAC(6\u0026apos;)-Ie\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003eAAC(6\u0026apos;)-Io\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003eAAC(6\u0026apos;)-34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eAAC(6\u0026apos;)-Ie\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eAmikacin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e(1.17 \u0026plusmn; 0.13) \u0026times; 10\u003csup\u003e-3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e2.11 \u0026plusmn; 0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e7.6 \u0026plusmn; 0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e20.9 \u0026plusmn; 5.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e1.54 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e1.01 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eFortimycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e0.19 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e2.2 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e1.01 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eIsepamicin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e(3.28 \u0026plusmn; 0.65) \u0026times; 10\u003csup\u003e-3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e1.66 \u0026plusmn; 0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e18.7 \u0026plusmn; 9.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e0.19 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e8.87 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eKanamycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e(2.21 \u0026plusmn; 0.86) \u0026times; 10\u003csup\u003e-3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e0.31 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e1.4 \u0026plusmn; 0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e1.50 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e2.21 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eNetilmicin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e(2.67 \u0026plusmn; 0.22) \u0026times; 10\u003csup\u003e-3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e0.87 \u0026plusmn; 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e12.6 \u0026plusmn; 3.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e3.2 \u0026plusmn; 0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e2.11 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e2.27 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eSisomicin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e0.65 \u0026plusmn; 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e2.92 \u0026plusmn; 0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e4.7 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e6.5 \u0026plusmn; 2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e1.4 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e4.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eTobramycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e(3.26 \u0026plusmn; 0.37) \u0026times; 10\u003csup\u003e-3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003e0.68 \u0026plusmn; 0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003e58.6 \u0026plusmn; 9.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e2.3 \u0026plusmn; 0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003e0.56 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003e3.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eSpectinomycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eNA\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eNA\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003eNA\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eStreptomycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eNA\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 51px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eNA\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 54px;\"\u003e\n \u003cp\u003eNA\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 55px;\"\u003e\n \u003cp\u003eND\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003eND,\u0026nbsp;not detected.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u003c/sup\u003eNA, no acyl transfer activity was detected.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong the other four AAC(6\u0026rsquo;) proteins, two (AAC(6\u0026apos;)-Ian and AAC(6\u0026apos;)-Im) were free of a specific digital kinetic parameter. The enzymatic activity of AAC(6\u0026apos;)-Ian was determined by using thin-layer chromatography (38), while the activity of AAC(6\u0026apos;)-Im, due to substrate inhibition, was monitored through changes in absorbance (39). The kinetic parameters of one substrate, sisomicin, were measured for AAC(6\u0026apos;)-34 (37), which was not analyzed by AAC(6\u0026apos;)-Io in this work. AAC(6\u0026apos;)-Ie had 1.0 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e-\u003csup\u003e\u0026nbsp;\u003c/sup\u003eto\u0026nbsp;1.0 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e-fold greater catalytic efficiency (\u003cem\u003ek\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e/\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e) than did AAC(6\u0026apos;)-Io for the five substrates (tobramycin, kanamycin, amikacin, isepamicin and netilmicin), which were analyzed with both enzymes\u0026nbsp;(39, 40)\u0026nbsp;(Table 4). The lower catalytic efficiency of AAC(6\u0026rsquo;)-Io than that of AAC(6\u0026rsquo;)-Ie could be attributed to the low turnover rate (\u003cem\u003ek\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e) of AAC(6\u0026rsquo;)-Io (Table 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eTo analyze the functional essential residues or domains of the AAC(6\u0026rsquo;)-Io protein and its relatives, alignments of the deduced amino acid sequence of AAC(6\u0026rsquo;)-Io with seven other closely related AAC(6\u0026rsquo;)-I proteins were performed (Figure 2). The residues related to the function of the AAC(6)-I enzymes are conserved in these proteins, including those in this work. These residues include F\u003csup\u003e100\u003c/sup\u003e-G\u003csup\u003e102\u003c/sup\u003e and G\u003csup\u003e112\u003c/sup\u003eT\u003csup\u003e113\u003c/sup\u003e, which are Ac-CoA binding sites predicted by CDD/SPARCLE (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml.) (41), L\u003csup\u003e115\u003c/sup\u003e is the key site for the acetylation of amikacin (42), and the conserved motif (K\u003csup\u003e109\u003c/sup\u003e-Y\u003csup\u003e122\u003c/sup\u003e)\u003csup\u003e\u0026nbsp;\u003c/sup\u003efor AAC(6)-I enzymes (42).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eA novel chromosomal aminoglycoside 6'-\u003cem\u003eN\u003c/em\u003e-acetyltransferase gene, \u003cem\u003eaac(6\u0026rsquo;)-Io\u003c/em\u003e, was identified in \u003cem\u003eB. cereus\u003c/em\u003e DW444. AAC(6\u0026rsquo;)-Io shares the highest aa sequence similarity (47.51%) with the functionally characterized AAC(6\u0026rsquo;)-34 from \u003cem\u003ePaenibacillus\u003c/em\u003e sp. LC231 and confers resistance to aminoglycosides, including tobramycin, kanamycin, amikacin, isepamicin, and netilmicin. Deciphering new antibacterial resistance mechanisms in bacteria from different sources would help clinics treat infections caused by bacteria harboring the same resistance genes effectively.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cem\u003eMIC\u003c/em\u003e\u003cem\u003e:\u0026nbsp;\u003c/em\u003eMinimum inhibitory concentration\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eaa\u003c/em\u003e\u003cem\u003e:\u0026nbsp;\u003c/em\u003eAmino acid\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAMEs:\u0026nbsp;\u003c/em\u003eAminoglycoside-modifying enzymes\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAAC:\u0026nbsp;\u003c/em\u003eAminoglycoside acetyltransferase\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAPH:\u0026nbsp;\u003c/em\u003eAminoglycoside phosphotransferase\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eANT:\u0026nbsp;\u003c/em\u003eAminoglycoside nucleotidyltransferase\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMH:\u0026nbsp;\u003c/em\u003eMueller-Hinton\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCLSI:\u0026nbsp;\u003c/em\u003eClinical and Laboratory Standards Institute\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCDSs:\u0026nbsp;\u003c/em\u003eCoding sequences\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCARD:\u0026nbsp;\u003c/em\u003eComprehensive antibiotic resistance database\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eANI:\u0026nbsp;\u003c/em\u003eAverage nucleotide identity\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eORF:\u0026nbsp;\u003c/em\u003eOpen reading frame\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePCR:\u0026nbsp;\u003c/em\u003ePolymerase chain reaction\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIPTG:\u0026nbsp;\u003c/em\u003eIsopropyl-\u0026beta;-d-thiogalactopyranoside\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eOD:\u0026nbsp;\u003c/em\u003eOptical density\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNCBI:\u0026nbsp;\u003c/em\u003eNational center for biotechnology information\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSDS‒PAGE\u003c/em\u003e\u003cem\u003e:\u0026nbsp;\u003c/em\u003eSodium dodecyl sulfate‒polyacrylamide gel electrophoresis\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDTNB:\u0026nbsp;\u003c/em\u003e5,5\u0026apos;-dithiobis of (2-nitrobenzoic acid)\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTNB:\u0026nbsp;\u003c/em\u003e5-thio-2-nitrobenzoic acid\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eisDDH\u003c/em\u003e\u003cem\u003e:\u003c/em\u003e \u003cem\u003ein silico\u003c/em\u003e DNA\u0026ndash;DNA hybridization\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGNATs:\u003c/em\u003e GCN5-related N-acetyltransferases\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study used strains isolated from the environment and animals in animal farms in Wenzhou, China. The owners of the farms were informed in writing of the study and provided approval for the sampling of animals. The studies involving human participants and animals were reviewed and approved by the Animal Welfare and Ethics Committee of Wenzhou Medical University, Zhejiang Province, China (protocol number: wydw2021-0323). All authors have reviewed and approved the manuscript for publication. As our study is not a clinical trial, no clinical trial registration or approval is required.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during the current study are available in the NCBI repository, CP130335 (chromosome), CP130336 (pDW444-290265), CP130337 (pDW444-67560), CP130338 (pDW444-77364), OR282771 (\u003cem\u003eaac(6\u0026rsquo;)-Io\u003c/em\u003e), and WKT62500 (AAC(6\u0026rsquo;)-Io). The data is publicly available.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo potential conflicts of interest are reported by the authors. There are no conflicts of interest between the coauthors.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Science \u0026amp; Technology Project of Jinhua City, China (2024-4-030, 2022-2-013), and the Zhejiang Provincial Natural Science Foundation of China (LTGY24H190003, LGD22C040006), and the Science \u0026amp; Technology Project of Wenzhou City, China (N20210001).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceived and designed the experiments: QB, CC and JL; Performed the experiments: WS, LZ, CF, HL and JWL; Data analysis and interpretation: WS, CF and JWL; Drafting of the manuscript: WS, QB, CC and JL.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to acknowledge the teachers and scientists of the Science and Technology Platforms of Wenzhou Medical University who helped with the analysis of the enzyme kinetic parameters.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFavrot L, Blanchard JS, Vergnolle O. Bacterial GCN5-Related N-Acetyltransferases: From Resistance to Regulation. Biochemistry. 2016;55(7):989-1002.\u003c/li\u003e\n\u003cli\u003eSerio AW, Magalh\u0026atilde;es ML, Blanchard JS, Connolly LE. Aminoglycosides: Mechanisms of action and resistance. Antimicrobial Drug Resistance: Mechanisms of Drug Resistance, Volume 1. 2017:213-29.\u003c/li\u003e\n\u003cli\u003eHoughton JL, Green KD, Chen W, Garneau-Tsodikova S. The future of aminoglycosides: the end or renaissance? Chembiochem. 2010;11(7):880-902.\u003c/li\u003e\n\u003cli\u003eAzucena E, Mobashery S. Aminoglycoside-modifying enzymes: mechanisms of catalytic processes and inhibition. Drug Resist Updat. 2001;4(2):106-17.\u003c/li\u003e\n\u003cli\u003ePullens B, van Benthem PP. Intratympanic gentamicin for Meniere\u0026apos;s disease or syndrome. Cochrane Database Syst Rev. 2011(3):CD008234.\u003c/li\u003e\n\u003cli\u003eHainrichson M, Nudelman I, Baasov T. Designer aminoglycosides: the race to develop improved antibiotics and compounds for the treatment of human genetic diseases. Org Biomol Chem. 2008;6(2):227-39.\u003c/li\u003e\n\u003cli\u003ePokrovskaya V, Nudelman I, Kandasamy J, Baasov T. Aminoglycosides redesign strategies for improved antibiotics and compounds for treatment of human genetic diseases. Methods Enzymol. 2010;478:437-62.\u003c/li\u003e\n\u003cli\u003eHermann T. Aminoglycoside antibiotics: old drugs and new therapeutic approaches. Cell Mol Life Sci. 2007;64(14):1841-52.\u003c/li\u003e\n\u003cli\u003eGuidry CA, Davies SW, Metzger R, Swenson BR, Sawyer RG. Whence Resistance? Surg Infect (Larchmt). 2015;16(6):716-20.\u003c/li\u003e\n\u003cli\u003eWright GD. Aminoglycoside-modifying enzymes. Curr Opin Microbiol. 1999;2(5):499-503.\u003c/li\u003e\n\u003cli\u003eAhmadian L, Norouzi Bazgir Z, Ahanjan M, Valadan R, Goli HR. Role of Aminoglycoside-Modifying Enzymes (AMEs) in Resistance to Aminoglycosides among Clinical Isolates of Pseudomonas aeruginosa in the North of Iran. Biomed Res Int. 2021;2021:7077344.\u003c/li\u003e\n\u003cli\u003eRamirez MS, Tolmasky ME. Aminoglycoside modifying enzymes. Drug Resist Updat. 2010;13(6):151-71.\u003c/li\u003e\n\u003cli\u003eRamirez MS, Tolmasky ME. Amikacin: Uses, Resistance, and Prospects for Inhibition. Molecules. 2017;22(12).\u003c/li\u003e\n\u003cli\u003eVakulenko SB, Mobashery S. Versatility of aminoglycosides and prospects for their future. Clin Microbiol Rev. 2003;16(3):430-50.\u003c/li\u003e\n\u003cli\u003eTada T, Miyoshi-Akiyama T, Shimada K, Shimojima M, Kirikae T. novel 6\u0026apos;-n-aminoglycoside acetyltransferase AAC(6\u0026apos;)-Iaj from a clinical isolate of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2013;57(1):96-100.\u003c/li\u003e\n\u003cli\u003eTada T, Miyoshi-Akiyama T, Shimada K, Dahal RK, Mishra SK, Ohara H, et al. A Novel 6\u0026apos;-N-Aminoglycoside Acetyltransferase, AAC(6\u0026apos;)-Ial, from a Clinical Isolate of Serratia marcescens. Microb Drug Resist. 2016;22(2):103-8.\u003c/li\u003e\n\u003cli\u003eBottone EJ. Bacillus cereus, a volatile human pathogen. Clin Microbiol Rev. 2010;23(2):382-98.\u003c/li\u003e\n\u003cli\u003eStenfors Arnesen LP, Fagerlund A, Granum PE. From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiol Rev. 2008;32(4):579-606.\u003c/li\u003e\n\u003cli\u003ePitt TL, McClure J, Parker MD, Amezquita A, McClure PJ. Bacillus cereus in personal care products: risk to consumers. Int J Cosmet Sci. 2015;37(2):165-74.\u003c/li\u003e\n\u003cli\u003eJung J, Jin H, Seo S, Jeong M, Kim B, Ryu K, et al. Short Communication: Enterotoxin Genes and Antibiotic Susceptibility of Bacillus cereus Isolated from Garlic Chives and Agricultural Environment. Int J Environ Res Public Health. 2022;19(19).\u003c/li\u003e\n\u003cli\u003eMbhele ZN, Shobo CO, Amoako DG, Zishiri OT, Bester LA. Occurrence, Antibiotic Resistance, Virulence Factors, and Genetic Diversity of Bacillus spp. from Public Hospital Environments in South Africa. Microb Drug Resist. 2021;27(12):1692-704.\u003c/li\u003e\n\u003cli\u003eCLSI. Performance Standards for Antimicrobial Susceptibility Testing\u0026mdash;Twenty-Eighth Informational Supplement: M100-S28.2020.\u003c/li\u003e\n\u003cli\u003eShi W, Lu J, Feng C, Gao M, Li A, Liu S, et al. Functional characterization of a novel aminoglycoside phosphotransferase, APH(9)-Ic, and its variant from Stenotrophomonas maltophilia. Front Cell Infect Microbiol. 2022;12:1097561.\u003c/li\u003e\n\u003cli\u003eKoren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 2017;27(5):722-36.\u003c/li\u003e\n\u003cli\u003eWalker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One. 2014;9(11):e112963.\u003c/li\u003e\n\u003cli\u003eSeemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30(14):2068-9.\u003c/li\u003e\n\u003cli\u003eBuchfink B, Reuter K, Drost HG. Sensitive protein alignments at tree-of-life scale using DIAMOND. Nat Methods. 2021;18(4):366-8.\u003c/li\u003e\n\u003cli\u003eMcArthur AG, Waglechner N, Nizam F, Yan A, Azad MA, Baylay AJ, et al. The comprehensive antibiotic resistance database. Antimicrob Agents Chemother. 2013;57(7):3348-57.\u003c/li\u003e\n\u003cli\u003eJain C, Rodriguez RL, Phillippy AM, Konstantinidis KT, Aluru S. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat Commun. 2018;9(1):5114.\u003c/li\u003e\n\u003cli\u003ePetkau A, Stuart-Edwards M, Stothard P, Van Domselaar G. Interactive microbial genome visualization with GView. Bioinformatics. 2010;26(24):3125-6.\u003c/li\u003e\n\u003cli\u003eGilchrist CLM, Chooi YH. clinker \u0026amp; clustermap.js: automatic generation of gene cluster comparison figures. Bioinformatics. 2021;37(16):2473-5.\u003c/li\u003e\n\u003cli\u003eMagnet S, Lambert T, Courvalin P, Blanchard JS. Kinetic and mutagenic characterization of the chromosomally encoded Salmonella enterica AAC(6\u0026apos;)-Iy aminoglycoside N-acetyltransferase. Biochemistry. 2001;40(12):3700-9.\u003c/li\u003e\n\u003cli\u003eGalimand M, Fishovitz J, Lambert T, Barbe V, Zajicek J, Mobashery S, et al. AAC(3)-XI, a new aminoglycoside 3-N-acetyltransferase from Corynebacterium striatum. Antimicrob Agents Chemother. 2015;59(9):5647-53.\u003c/li\u003e\n\u003cli\u003eZhou K, Liang J, Dong X, Zhang P, Feng C, Shi W, et al. Identification and Characterization of a Novel Chromosomal Aminoglycoside 2\u0026apos;-N-Acetyltransferase, AAC(2\u0026apos;)-If, From an Isolate of a Novel Providencia Species, Providencia wenzhouensis R33. Front Microbiol. 2021;12:711037.\u003c/li\u003e\n\u003cli\u003eFigueroa-Bossi N, Balbontin R, Bossi L. Basic Bacteriological Routines. Cold Spring Harb Protoc. 2022;2022(10):Pdb prot107849.\u003c/li\u003e\n\u003cli\u003eGoris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM. DNA-DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol. 2007;57(Pt 1):81-91.\u003c/li\u003e\n\u003cli\u003ePawlowski AC, Wang W, Koteva K, Barton HA, McArthur AG, Wright GD. A diverse intrinsic antibiotic resistome from a cave bacterium. Nat Commun. 2016;7:13803.\u003c/li\u003e\n\u003cli\u003eJin W, Wachino J, Kimura K, Yamada K, Arakawa Y. New plasmid-mediated aminoglycoside 6\u0026apos;-N-acetyltransferase, AAC(6\u0026apos;)-Ian, and ESBL, TLA-3, from a Serratia marcescens clinical isolate. J Antimicrob Chemother. 2015;70(5):1331-7.\u003c/li\u003e\n\u003cli\u003eSmith CA, Bhattacharya M, Toth M, Stewart NK, Vakulenko SB. Aminoglycoside resistance profile and structural architecture of the aminoglycoside acetyltransferase AAC(6\u0026apos;)-Im. Microb Cell. 2017;4(12):402-10.\u003c/li\u003e\n\u003cli\u003eDaigle DM, Hughes DW, Wright GD. Prodigious substrate specificity of AAC(6\u0026apos;)-APH(2\u0026quot;), an aminoglycoside antibiotic resistance determinant in enterococci and staphylococci. Chem Biol. 1999;6(2):99-110.\u003c/li\u003e\n\u003cli\u003eLu S, Wang J, Chitsaz F, Derbyshire MK, Geer RC, Gonzales NR, et al. CDD/SPARCLE: the conserved domain database in 2020. Nucleic Acids Res. 2020;48(D1):D265-D8.\u003c/li\u003e\n\u003cli\u003eShaw KJ, Rather PN, Hare RS, Miller GH. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbiol Rev. 1993;57(1):138-63.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"aminoglycoside acetyltransferase, AAC(6’), antimicrobial resistance, Bacillus cereus, kinetic parameter","lastPublishedDoi":"10.21203/rs.3.rs-6041341/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6041341/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e:\u003cstrong\u003e \u003c/strong\u003eThe emergence of various resistance determinants in microbes is a growing concern for the clinical application of antimicrobial agents to treat bacterial infections. Research on the aminoglycoside resistance mechanism may help us to determine the complexity of bacterial resistance mechanisms and effective treatment of infectious diseases.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Bacteria were isolated from environmental samples via the plate streak method. The minimum inhibitory concentration (MIC) of the antibiotics was determined using the agar dilution method. Gene cloning and antibiotic susceptibility testing were conducted to confirm the function of the new resistance gene. The kinetic parameters of the enzyme were determined after the protein AAC(6’)-Io was expressed in \u003cem\u003eE. coli\u003c/em\u003e. Whole-genome sequencing and bioinformatic analysis were subsequently conducted to analyze the structure and evolution of the resistance gene-related sequences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: A novel aminoglycoside resistance gene, \u003cem\u003eaac(6')-Io,\u003c/em\u003e which was identified in the chromosome of \u003cem\u003eB. cereus\u003c/em\u003e DW444, confers resistance to tobramycin, kanamycin, amikacin, netilmicin, sisomicin and ribostamycin. Of the aminoglycoside substrates tested, AAC(6')-Io demonstrated the highest catalytic efficiency for netilmicin (\u003cem\u003ek\u003c/em\u003e\u003csub\u003ecat\u003c/sub\u003e/\u003cem\u003eK\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e, 2.11 × 10\u003csup\u003e2\u003c/sup\u003e M\u003csup\u003e−1\u003c/sup\u003e·s\u003csup\u003e−1\u003c/sup\u003e). Among the functionally characterized antimicrobial resistance proteins, AAC(6')-Io demonstrated the highest amino acid (aa) sequence similarity (47.51%) to AAC(6')-34, and it had\u003cstrong\u003e \u003c/strong\u003ethe functional essential residues or domains of the AAC(6’)-I proteins, including F\u003csup\u003e100\u003c/sup\u003e-G\u003csup\u003e102\u003c/sup\u003e and G\u003csup\u003e112\u003c/sup\u003eT\u003csup\u003e113\u003c/sup\u003e, which are Ac-CoA binding sites, and L\u003csup\u003e115\u003c/sup\u003e, which is the key site for the acetylation of amikacin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: The new aminoglycoside resistance gene \u003cem\u003eaac(6')-Io\u003c/em\u003e was described in this study, along with its molecular characteristics. Elucidating the antibiotic resistance mechanism of this pathogen will benefit the clinical application of aminoglycosides to treat infections caused by bacteria carrying its homogous genes.\u003c/p\u003e","manuscriptTitle":"A novel chromosomal aminoglycoside 6′-N-acetyltransferase, AAC(6′)-Io, confers resistance to multiple aminoglycosides identified from Bacillus cereus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-16 12:12:39","doi":"10.21203/rs.3.rs-6041341/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-03T13:07:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-27T19:44:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"180587170332839588641944006782516036976","date":"2026-02-18T18:34:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-13T16:59:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-23T15:45:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"169860207632556198604023254311948628966","date":"2025-04-16T22:33:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"308042440289220630227900651728572275442","date":"2025-04-16T18:24:51+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-14T18:58:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-04T12:19:28+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-03-31T06:05:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-28T13:01:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2025-03-28T13:00:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"17c89d2f-a02c-4d2b-98b1-e11cc6367580","owner":[],"postedDate":"April 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-03-03T13:23:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-16 12:12:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6041341","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6041341","identity":"rs-6041341","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}