Article Figures & Data
Figures
- FIG 1
Bacterial strains used in this study and their pulsed-field gel electrophoresis (PFGE) patterns and drug susceptibilities. The superscript italic “a” indicates results of PFGE analysis of SmaI-digested DNA isolated from the locally spread strains. Pulse time varied from 5.3 to 34.9 s during the 20.0 h of electrophoresis. The superscript italic “b” indicates the following abbreviations and definitions: VAN, vancomycin; TEC, teicoplanin; LZD, linezolid; AMP, ampicillin; GEN, gentamicin; KAN, kanamycin; STR, streptomycin; ERY, erythromycin; CHL, chloramphenicol; TET, tetracycline; MIN, minocycline; CIP, ciprofloxacin; CRO, ceftriaxone; CMZ, cefmetazole; MEM, meropenem. To determine the MICs, E. raffinosus strains were grown for 48 h because their growth rate was low.
- FIG 2
Pulsed-field gel electrophoresis (PFGE) analysis of S1 nuclease-untreated DNA and Southern blotting of the locally spread strains. PFGE analysis of S1 nuclease-untreated DNA (left) and Southern hybridization with a vanA gene probe (right). Lanes: C, AA708, a control strain harboring the linear plasmid pELF1; MM, Lambda Ladder PFG Marker (New England BioLabs); 1, KUHS1; 2, KUHS2; 3, KUHS3; 4, KUHS4; 5, KUHS5; 6, KUHS6; 7, KUHS7; 8, KUHS8; 9, KUHS9; 10, KUHS10; 11, KUHS11; 12, KUHS12; 13, KUHS13; 14, KUHS14; 15, KUHS15; 16, KUHS16; 17, KUHS17; 18, KUHS18; 19, KUHS19; 20, KUHS20; 21, KUHS21; 22, KUHS22; 23, KUHS23; 24, KUHS24; 25, KUHS25.
- FIG 3
Schematics of the genetic structure of the vanA gene cluster (A) and pELF2 (B). The panels show the genetic structure of the vanA gene cluster carried on pELF2 (A) and a genomic comparison between pELF2 and pELF1 generated by Easyfig (B) (21). Gray lines connecting the nucleotide sequences of the two plasmids represent nucleotide identity of >67%. Arrows represent vancomycin resistance genes (orange), other drug resistance genes (red), putative replication-related genes (yellow), and putative transfer-related genes (green), respectively.
Supplemental Material
FIG S1
Time course of isolation of multispecies vancomycin-resistant enterococci (VREs) from four patients. Dates of isolation of multispecies VREs from patients A to D are shown. Characters by themselves represent Enterococcus faecium isolates, characters on gray background represent Enterococcus raffinosus isolates, and characters in boxes represent Enterococcus casseliflavus isolates. Characters in brackets represent SmaI-PFGE patterns. N.D., not detected. Download FIG S1, TIF file, 0.05 MB.
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FIG S2
Pulse-field gel electrophoresis (PFGE) analysis of SmaI-digested DNA of the locally spread strains. PFGE analysis of SmaI-digested DNA isolated from the strains was performed. Pulse times ranged from 5.3 to 34.9 s during the 20.0 h of electrophoresis. Download FIG S2, TIF file, 0.2 MB.
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FIG S3
Pulse-field gel electrophoresis (PFGE) analysis of S1 nuclease-treated DNA of the locally spread strains. PFGE analysis of S1 nuclease-treated DNA isolated from the strains was performed. Pulse times ranged from 5.3 to 66 s during the 19.5 h of electrophoresis. Lanes: MM, Lambda ladder PFG marker (New England BioLabs); 1, KUHS1; 2, KUHS2; 3, KUHS3; 4, KUHS4; 5, KUHS5; 6, KUHS6; 7, KUHS7; 8, KUHS8; 9, KUHS9; 10, KUHS10; 11, KUHS11; 12, KUHS12; 13, KUHS13; 14, KUHS14; 15, KUHS15; 16, KUHS16; 17, KUHS17; 18, KUHS18; 19, KUHS19; 20, KUHS20; 21, KUHS21; 22, KUHS22; 23, KUHS23; 24, KUHS24; 25, KUHS25; C, AA708, a control strain harboring linear plasmid pELF1. Download FIG S3, TIF file, 0.6 MB.
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FIG S4
Nucleotide sequence alignment of each end of the pELF2 and pELF1 linear plasmids. The nucleotide sequence alignments of the left (A) and right (B) ends of pELF2 and pELF1 are shown. The box indicates putative hairpin sequences of the left end (5′-TATA-3′). Black boxes indicate single nucleotide variations between pELF2 and pELF1. Download FIG S4, TIF file, 0.7 MB.
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FIG S5
Secondary structure of the right-end nucleotide sequence of pELF2. (A) The secondary structure of the 280-nucleotide sequence of the 3′ right end of pELF2 was predicted using mfold software (ΔG = −52.31 kcal/mol) (A) (22). (B) The 280-nucleotide sequence of the 3′ right end of pELF2 is shown. Arrows and Roman numerals indicate palindromic sequences. Boxes indicate predicted loop-structure sequences of each palindromic sequence. Download FIG S5, TIF file, 0.3 MB.
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TABLE S1
Whole-genome sequence statistics. The superscript italic “a” indicates reads that were checked using Nanoplot (ver. 1.25.0) (36). The superscript italic “b” indicates reads that were checked using FastQC (ver. 0.11.8) (37). Download Table S1, XLSX file, 0.01 MB.
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TABLE S2
Detailed information of WGS of KUHS13. The superscript italic “a” indicates that WGS was performed using Illumina Miniseq and Nanopore Minion and data were assembled using Canu (v1.8) and refined with Racon (v1.3.1.1) and Pilon (v1.20.1) (30–32). Download Table S2, XLSX file, 0.01 MB.
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TABLE S3
Transfer frequency of pELF2 between KUHS13 and recipients strains. The superscript italic “a” indicates the frequencies were estimated from the CFU ratio of transconjugant strains to donor strains. The values represent means of three independent experiments with standard errors. Download Table S3, XLSX file, 0.01 MB.
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TABLE S4
MICs of various antibiotic drugs. The superscript italic “a” indicates the following abbreviations and definitions: VAN, vancomycin; TEC, teicoplanin; LZD, linezolid; AMP, ampicillin; GEN, gentamicin; STR, streptomycin; KAN, kanamycin; ERY, erythromycin; CHL, chloramphenicol; TET, tetracycline; MIN, minocycline; CIP, ciprofloxacin; CRO, ceftriaxone; CMZ, cefmetazole; MEM, meropenem. The superscript italic “b” indicates the corresponding transconjugants that were obtained by filter mating with the donor KUHS13 strain. The superscript italic “c” indicates instances in which the strain was used as a recipient strain for the conjugation experiment. Download Table S4, XLSX file, 0.01 MB.
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