Supporting Information Molecular and Functional Characterization of a Novel Plasmid-Borne Blandm-Like Gene, Blaafm-1, in a Clini

Home , Aminocoumarin, Glycylcycline, Penam

Supporting Information

Molecular and functional characterization of a novel plasmid-borne

blaNDM-like gene, blaAFM-1, in a clinical strain of Aeromonas hydrophila

Table S1 Primers used for functional detection of the antimicrobial resistance genes in this

work

Restriction Vectors Annealing Amplicon Genes Primer sequences (5'-3') endonucleases temperature size

GCTCTAGAGAATACGACTCCAT F XbaI CCATGATTCAG blaAFM-6 pUCP24 58℃ 950 bp CGGGATCCGATCCTTCC R BamHI AGCTCGTCGCAAAC

GCTCTAGAAGCCCTTTA F XbaI CCAAACCAATAC blaOXA-1 pUCP24 62℃ 899 bp CGGGATCCCGACTTGAT R BamHI TGAAGGGTTGG

GCTCTAGACATAATTGCT F XbaI CACAGCCAAAC aac(6')-Ib10 pUCP20 52℃ 1036 bp CGGGATCCTTTAACGGC R BamHI TAACAATTAATGAGG

GCTCTAGAAGTTTATCAC F XbaI CACCGACTATTTGC aph(3')-Ia pUCP20 62℃ 1036 bp CGGGATCCTGGGTCGAT R BamHI TCCTGGGAGG

GCTCTAGACACCCTGTG F XbaI GATGAATAGC msr(E) pUCP20 62℃ 1685 bp CGGGATCCGTGCTTCAG R BamHI CAAGTGATTG

GCTCTAGAGGATCGGAG F XbaI CGAATGATATTC mph(E) pUCP20 62℃ 1266 bp CGGGATCCCAAAACAAG R BamHI GCAGTTCTCAG

The underlined sequences represent the restriction endonuclease sites and their protective bases Table S2 Antibiotic resistance genes in Aeromonas hydrophila SS332 detected using the CARD database Resistance Coverag Identity Gene ID ARO Name AMR Gene Family Drug Class Mechanism e (%) (%) CMCFKOJJ_0113 chloramphenicol catB3 phenicol antibiotic antibiotic inactivation100 100 6 acetyltransferase (CAT) CMCFKOJJ_0113 PER-3 PER beta-lactamase penam; penem; carbapenem; cephalosporin; monobactam antibiotic inactivation 100 100 9

CMCFKOJJ_0114 antibiotic target sul1 sulfonamide resistant sul sulfonamide antibiotic 98.92 100 4 replacement

macrolide CMCFKOJJ_0115 mphA phosphotransferase macrolide antibiotic antibiotic inactivation100 100 1 (MPH)

CMCFKOJJ_0219 MOX-7 MOX beta-lactamase penam; cephamycin; cephalosporin antibiotic inactivation 100 96.61 0 CMCFKOJJ_0113 aadA16 ANT(3'') aminoglycoside antibiotic antibiotic inactivation97.86 96.36 3

tetracycline antibiotic; aminocoumarin antibiotic; macrolide antibiotic; carbapenem; resistance-nodulation- CMCFKOJJ_0443 antibacterial free fatty acids; aminoglycoside antibiotic; acridine dye; diaminopyrimidine CRP cell division (RND) antibiotic efflux 100.95 89.15 6 antibiotic; penam; triclosan; glycylcycline; monobactam; fluoroquinolone antibiotic; antibiotic efflux pump phenicol antibiotic CMCFKOJJ_0351 OXA-427 OXA beta-lactamase penam; cephalosporin antibiotic inactivation 100 88.26 0 Salmonella enterica gyrA CMCFKOJJ_0102 fluoroquinolone resistant antibiotic target conferring fluoroquinolone antibiotic 102.85 75.31 4 gyrA alteration resistance to fluoroquinolones pmr CMCFKOJJ_0040 antibiotic target ugd phosphoethanolamine peptide antibiotic 100 71.65 2 alteration transferase undecaprenyl CMCFKOJJ_0328 antibiotic target bacA pyrophosphate related peptide antibiotic 100 69.6 9 alteration proteins MCR CMCFKOJJ_0201 antibiotic target MCR-7.1 phosphoethanolamine peptide antibiotic 100.19 68.7 4 alteration transferase

Escherichia coli fluoroquinolone self CMCFKOJJ_0403 parC conferring resistant parC; antibiotic target fluoroquinolone antibiotic 98.4 67.16 9 resistance to fluoroquinolone resistant alteration fluoroquinolone parC Mycobacterium tuberculosis katG CMCFKOJJ_0083 mutations antibiotic target isoniazid resistant katG isoniazid 96.35 63.81 4 conferring alteration resistance to isoniazid

ATP-binding cassette penam; pleuromutilin antibiotic; macrolide antibiotic; peptide antibiotic; acridine dye; CMCFKOJJ_0349 YojI (ABC) antibiotic efflux cephalosporin; tetracycline antibiotic; fluoroquinolone antibiotic; rifamycin antibiotic; antibiotic efflux 99.63 63.67 2 pump nitroimidazole antibiotic

ATP-binding cassette penam; pleuromutilin antibiotic; macrolide antibiotic; peptide antibiotic; acridine dye; CMCFKOJJ_0051 msbA (ABC) antibiotic efflux cephalosporin; tetracycline antibiotic; fluoroquinolone antibiotic; rifamycin antibiotic; antibiotic efflux 99.83 61.27 6 pump nitroimidazole antibiotic

trimethoprim resistant CMCFKOJJ_0240 antibiotic target dfrA3 dihydrofolate reductase diaminopyrimidine antibiotic 99.38 59.01 8 replacement dfr Staphylococcus aureus fusA with CMCFKOJJ_0318 antibiotic target mutation conferring antibiotic resistant fusA fusidic acid 101.01 58.86 6 alteration resistance to fusidic acid

major facilitator fosfomycin; benzalkonium chloride; tetracycline antibiotic; rhodamine; aminocoumarin superfamily (MFS) antibiotic; macrolide antibiotic; carbapenem; cephalosporin; antibacterial free fatty acids; CMCFKOJJ_0082 antibiotic efflux pump; bicyclomycin; nitroimidazole antibiotic; aminoglycoside antibiotic; lincosamide antibiotic; H-NS antibiotic efflux 99.27 57.35 3 resistance-nodulation- peptide antibiotic; acridine dye; diaminopyrimidine antibiotic; oxazolidinone antibiotic; cell division (RND) penam; triclosan; isoniazid; cephamycin; glycylcycline; nucleoside antibiotic; monobactam; antibiotic efflux pump fluoroquinolone antibiotic; rifamycin antibiotic; phenicol antibiotic

tetracycline antibiotic; aminocoumarin antibiotic; macrolide antibiotic; carbapenem; resistance-nodulation- CMCFKOJJ_0033 Escherichia coli cephalosporin; antibacterial free fatty acids; aminoglycoside antibiotic; acridine dye; cell division (RND) antibiotic efflux 97.98 55.53 2 acrA diaminopyrimidine antibiotic; penam; triclosan; glycylcycline; monobactam; fluoroquinolone antibiotic efflux pump antibiotic; phenicol antibiotic; rifamycin antibiotic fosfomycin; benzalkonium chloride; tetracycline antibiotic; rhodamine; macrolide antibiotic; major facilitator cephalosporin; antibacterial free fatty acids; bicyclomycin; nitroimidazole antibiotic; CMCFKOJJ_0017 rosB superfamily (MFS) lincosamide antibiotic; peptide antibiotic; acridine dye; diaminopyrimidine antibiotic; antibiotic efflux 97.16 54.66 1 antibiotic efflux pump oxazolidinone antibiotic; penam; isoniazid; glycylcycline; nucleoside antibiotic; fluoroquinolone antibiotic; phenicol antibiotic; rifamycin antibiotic

major facilitator fosfomycin; benzalkonium chloride; tetracycline antibiotic; rhodamine; aminocoumarin superfamily (MFS) antibiotic; macrolide antibiotic; carbapenem; cephalosporin; antibacterial free fatty acids; CMCFKOJJ_0024 antibiotic efflux pump; bicyclomycin; nitroimidazole antibiotic; aminoglycoside antibiotic; lincosamide antibiotic; H-NS antibiotic efflux 102.19 54.29 9 resistance-nodulation- peptide antibiotic; acridine dye; diaminopyrimidine antibiotic; oxazolidinone antibiotic; cell division (RND) penam; triclosan; isoniazid; cephamycin; glycylcycline; nucleoside antibiotic; monobactam; antibiotic efflux pump fluoroquinolone antibiotic; rifamycin antibiotic; phenicol antibiotic

ATP-binding cassette penam; rifamycin antibiotic; pleuromutilin antibiotic; macrolide antibiotic; peptide antibiotic; CMCFKOJJ_0256 macB (ABC) antibiotic efflux acridine dye; cephalosporin; tetracycline antibiotic; fluoroquinolone antibiotic; antibiotic efflux 100.47 51.16 9 pump nitroimidazole antibiotic

ATP-binding cassette penam; pleuromutilin antibiotic; macrolide antibiotic; peptide antibiotic; acridine dye; CMCFKOJJ_0082 tet(35) (ABC) antibiotic efflux cephalosporin; tetracycline antibiotic; fluoroquinolone antibiotic; rifamycin antibiotic; antibiotic efflux 94.85 50 4 pump nitroimidazole antibiotic

penam; triclosan; aminocoumarin antibiotic; macrolide antibiotic; carbapenem; acridine resistance-nodulation- CMCFKOJJ_0457 dye; antibacterial free fatty acids; diaminopyrimidine antibiotic; glycylcycline; tetracycline adeR cell division (RND) antibiotic efflux 91.9 49.78 8 antibiotic; monobactam; fluoroquinolone antibiotic; aminoglycoside antibiotic; phenicol antibiotic efflux pump antibiotic sulfonamide antibiotic; penem; tetracycline antibiotic; aminocoumarin antibiotic; resistance-nodulation- cephalosporin; macrolide antibiotic; carbapenem; antibacterial free fatty acids; CMCFKOJJ_0357 Pseudomonas cell division (RND) cephamycin; peptide antibiotic; acridine dye; diaminopyrimidine antibiotic; penam; antibiotic efflux 101.33 49.56 7 aeruginosa CpxR antibiotic efflux pump triclosan; aminoglycoside antibiotic; glycylcycline; monobactam; fluoroquinolone antibiotic; phenicol antibiotic

ATP-binding cassette penem; benzalkonium chloride; tetracycline antibiotic; rhodamine; aminocoumarin (ABC) antibiotic efflux antibiotic; cephalosporin; macrolide antibiotic; carbapenem; nitroimidazole antibiotic; pump; major facilitator aminoglycoside antibiotic; lincosamide antibiotic; glycylcycline; bicyclomycin; peptide CMCFKOJJ_0402 superfamily (MFS) TolC antibiotic; acridine dye; diaminopyrimidine antibiotic; monobactam; oxazolidinone antibiotic efflux 85.86 48.94 8 antibiotic efflux pump; antibiotic; nucleoside antibiotic; penam; triclosan; antibacterial free fatty acids; rifamycin resistance-nodulation- antibiotic; pleuromutilin antibiotic; isoniazid; fosfomycin; cephamycin; fluoroquinolone cell division (RND) antibiotic; phenicol antibiotic antibiotic efflux pump

major facilitator fosfomycin; benzalkonium chloride; tetracycline antibiotic; rhodamine; aminocoumarin superfamily (MFS) antibiotic; macrolide antibiotic; carbapenem; cephalosporin; antibacterial free fatty acids; CMCFKOJJ_0451 antibiotic efflux pump; bicyclomycin; nitroimidazole antibiotic; aminoglycoside antibiotic; lincosamide antibiotic; H-NS antibiotic efflux 100 48.17 8 resistance-nodulation- peptide antibiotic; acridine dye; diaminopyrimidine antibiotic; oxazolidinone antibiotic; cell division (RND) penam; triclosan; isoniazid; cephamycin; glycylcycline; nucleoside antibiotic; monobactam; antibiotic efflux pump fluoroquinolone antibiotic; rifamycin antibiotic; phenicol antibiotic

penam; triclosan; aminocoumarin antibiotic; macrolide antibiotic; carbapenem; acridine resistance-nodulation- CMCFKOJJ_0345 dye; antibacterial free fatty acids; diaminopyrimidine antibiotic; glycylcycline; tetracycline MexW cell division (RND) antibiotic efflux 99.41 48.12 9 antibiotic; monobactam; fluoroquinolone antibiotic; aminoglycoside antibiotic; phenicol antibiotic efflux pump antibiotic

major facilitator fosfomycin; benzalkonium chloride; tetracycline antibiotic; rhodamine; aminocoumarin superfamily (MFS) antibiotic; macrolide antibiotic; carbapenem; cephalosporin; antibacterial free fatty acids; CMCFKOJJ_0387 antibiotic efflux pump; bicyclomycin; nitroimidazole antibiotic; aminoglycoside antibiotic; lincosamide antibiotic; H-NS antibiotic efflux 91.24 48 8 resistance-nodulation- peptide antibiotic; acridine dye; diaminopyrimidine antibiotic; oxazolidinone antibiotic; cell division (RND) penam; triclosan; isoniazid; cephamycin; glycylcycline; nucleoside antibiotic; monobactam; antibiotic efflux pump fluoroquinolone antibiotic; rifamycin antibiotic; phenicol antibiotic

ATP-binding cassette penam; rifamycin antibiotic; pleuromutilin antibiotic; macrolide antibiotic; peptide antibiotic; CMCFKOJJ_0025 macB (ABC) antibiotic efflux acridine dye; cephalosporin; tetracycline antibiotic; fluoroquinolone antibiotic; antibiotic efflux 103.88 47.38 3 pump nitroimidazole antibiotic multidrug and toxic CMCFKOJJ_0133 hmrM compound extrusion acridine dye; glycylcycline; tetracycline antibiotic; fluoroquinolone antibiotic antibiotic efflux 95.26 47.06 1 (MATE) transporter CMCFKOJJ_0346 kdpE kdpDE aminoglycoside antibiotic antibiotic efflux 100 46.22 6 penam; triclosan; aminocoumarin antibiotic; macrolide antibiotic; carbapenem; acridine resistance-nodulation- CMCFKOJJ_0457 dye; antibacterial free fatty acids; diaminopyrimidine antibiotic; glycylcycline; tetracycline adeA cell division (RND) antibiotic efflux 93.18 46.07 7 antibiotic; monobactam; fluoroquinolone antibiotic; aminoglycoside antibiotic; phenicol antibiotic efflux pump antibiotic

penam; triclosan; aminocoumarin antibiotic; macrolide antibiotic; carbapenem; acridine resistance-nodulation- CMCFKOJJ_0112 dye; antibacterial free fatty acids; diaminopyrimidine antibiotic; glycylcycline; tetracycline adeG cell division (RND) antibiotic efflux 88.67 45.56 8 antibiotic; monobactam; fluoroquinolone antibiotic; aminoglycoside antibiotic; phenicol antibiotic efflux pump antibiotic

penam; triclosan; aminocoumarin antibiotic; macrolide antibiotic; carbapenem; acridine resistance-nodulation- CMCFKOJJ_0351 dye; cephalosporin; antibacterial free fatty acids; diaminopyrimidine antibiotic; smeR cell division (RND) antibiotic efflux 95.2 44.49 2 glycylcycline; cephamycin; tetracycline antibiotic; monobactam; fluoroquinolone antibiotic; antibiotic efflux pump aminoglycoside antibiotic; phenicol antibiotic

CMCFKOJJ_0375 quinolone resistance antibiotic target QnrB46 fluoroquinolone antibiotic 91.59 44.44 2 protein (qnr) protection

fosfomycin; benzalkonium chloride; tetracycline antibiotic; rhodamine; antibacterial free major facilitator fatty acids; macrolide antibiotic; cephalosporin; bicyclomycin; nitroimidazole antibiotic; CMCFKOJJ_0431 Klebsiella superfamily (MFS) lincosamide antibiotic; peptide antibiotic; acridine dye; diaminopyrimidine antibiotic; antibiotic efflux 88.99 44.33 9 pneumoniae KpnF antibiotic efflux pump oxazolidinone antibiotic; penam; aminoglycoside antibiotic; isoniazid; glycylcycline; nucleoside antibiotic; fluoroquinolone antibiotic; rifamycin antibiotic; phenicol antibiotic

CMCFKOJJ_0234 antibiotic target sul4 sulfonamide resistant sul sulfonamide antibiotic 93.38 44.03 6 replacement

fosfomycin; benzalkonium chloride; tetracycline antibiotic; rhodamine; macrolide antibiotic; major facilitator cephalosporin; antibacterial free fatty acids; bicyclomycin; nitroimidazole antibiotic; CMCFKOJJ_0111 bcr-1 superfamily (MFS) lincosamide antibiotic; peptide antibiotic; acridine dye; diaminopyrimidine antibiotic; antibiotic efflux 91.29 43.32 0 antibiotic efflux pump oxazolidinone antibiotic; penam; isoniazid; glycylcycline; nucleoside antibiotic; fluoroquinolone antibiotic; phenicol antibiotic; rifamycin antibiotic fosfomycin; benzalkonium chloride; tetracycline antibiotic; rhodamine; macrolide antibiotic; major facilitator cephalosporin; antibacterial free fatty acids; bicyclomycin; nitroimidazole antibiotic; CMCFKOJJ_0168 bcr-1 superfamily (MFS) lincosamide antibiotic; peptide antibiotic; acridine dye; diaminopyrimidine antibiotic; antibiotic efflux 91.29 43.05 7 antibiotic efflux pump oxazolidinone antibiotic; penam; isoniazid; glycylcycline; nucleoside antibiotic; fluoroquinolone antibiotic; phenicol antibiotic; rifamycin antibiotic

Streptomyces aminocoumarin self rishiriensis parY CMCFKOJJ_0347 resistant parY; antibiotic target mutant conferring aminocoumarin antibiotic 95.45 42.48 5 aminocoumarin resistant alteration resistance to parY aminocoumarin Staphylococcus aureus fusE with CMCFKOJJ_0380 antibiotic target mutation conferring antibiotic resistant fusE fusidic acid 98.31 42.29 2 alteration resistance to fusidic acid

sulfonamide antibiotic; penem; tetracycline antibiotic; aminocoumarin antibiotic; resistance-nodulation- cephalosporin; macrolide antibiotic; carbapenem; antibacterial free fatty acids; CMCFKOJJ_0364 Pseudomonas cell division (RND) cephamycin; peptide antibiotic; acridine dye; diaminopyrimidine antibiotic; penam; antibiotic efflux 100.89 40.09 9 aeruginosa CpxR antibiotic efflux pump triclosan; aminoglycoside antibiotic; glycylcycline; monobactam; fluoroquinolone antibiotic; phenicol antibiotic Table S3 Antibiotic resistance genes in pSS332-218k of Aeromonas hydrophila SS332 detected using the CARD database Coverage Identity Gene ID Cutoff ARO Name AMR Gene Family Drug Class Drug Class Resistance Mechanism (%) (%)

pleuromutilin antibiotic; macrolide antibiotic; ABC-F type ribosomal protection oxazolidinone antibiotic; tetracycline ABC-F ATP-binding cassette IBDFPOCI_00066 Perfect msrE antibiotic target protection 100 100 protein Msr€ antibiotic; streptogramin antibiotic; phenicol ribosomal protection protein antibiotic; lincosamide antibiotic

macrolide IBDFPOCI_00067 Perfect mphE macrolide antibiotic antibiotic inactivation 100 100 phosphotransferase (MPH)

Aminoglycoside N(6')- IBDFPOCI_00083 Strict AAC(6')-Ib7 aminoglycoside antibiotic antibiotic inactivation AAC(6') 75.86 98.99 acetyltransferase type 1

IBDFPOCI_00084 Strict OXA-1 Beta-lactamase OXA-1 penam; cephalosporin antibiotic inactivation OXA beta-lactamase 71.38 100

chloramphenicol IBDFPOCI_00085 Strict catB3 Chloramphenicol acetyltransferase phenicol antibiotic antibiotic inactivation 51.43 99.07 acetyltransferase (CAT)

chloramphenicol IBDFPOCI_00086 Strict catB3 Chloramphenicol acetyltransferase phenicol antibiotic antibiotic inactivation 40.48 100 acetyltransferase (CAT)

Aminoglycoside 3'- IBDFPOCI_00088 Strict APH(3')-Ia aminoglycoside antibiotic antibiotic inactivation APH(3') 100 98.52 phosphotransferase

IBDFPOCI_00093 Strict sul1 Dihydropteroate synthase sulfonamide antibiotic antibiotic target replacement sulfonamide resistant sul 37.63 99.05

IBDFPOCI_00102 Strict BRP(MBL) Bleomycin resistance protein glycopeptide antibiotic antibiotic inactivation Bleomycin resistant protein 100 97.52

penam; carbapenem; cephalosporin; IBDFPOCI_00103 Loose NDM-6 Metallo-beta-lactamase type 2 antibiotic inactivation NDM beta-lactamase 96.67 86.21 cephamycin

IBDFPOCI_00106 Strict sul1 Dihydropteroate synthase sulfonamide antibiotic antibiotic target replacement sulfonamide resistant sul 100.72 95.37

trimethoprim resistant IBDFPOCI_00108 Strict dfrA5 Dihydrofolate reductase type 1 diaminopyrimidine antibiotic antibiotic target replacement 96.82 100 dihydrofolate reductase dfr

Figure S1. Antibiotic susceptibility testing by the disc diffusion method. DH5α/pUCP24-blaAFM-1 incubated with or without 0.05 mmol/L EDTA.

Figure S2 Sequence alignment of New Delhi metallo-beta-lactamases. Residue numbers are positioned above the sequences according to AFM-1 numbers; 14 sequence fragments (F1-F14) of structurally conserved positions are boxed in black. Amino acid variants of AFM-1 in conserved sequence fragments are boxed in yellow.