Table I
The composition of the AST-GP67 card, drug names, and MIC ranges tested.
| Antibiotics | Abbreviations | Measurement range (μg/ml) |
|---|---|---|
| 1 Linezolid | LZD | 0.5–8 |
| 2 Ampicillin | AMP | 2–32 |
| 3 Penicillin | PEN | 0.12–64 |
| 4 Erythromycin | ERY | 0.25–8 |
| 5 Clindamycin | CLI | 0.25–8 |
| 6 Tigecycline | TGC | 0.12–2 |
| 7 Tetracycline | TET | 1–16 |
| 8 Gentamicin – high concentration | GEH | S/R |
| 9 Streptomycin – high concentration | STH | S/R |
| 10 Ciprofloxacin | CIP | 0.5–8 |
| 11 Levofloxacin | LVX | 0.12–8 |
| 12 Moxifloxacin | MFX | 0.25–8 |
| 13 Vancomycin | VAN | 0.5 |
| 14 Nitrofurantoin | NIT | 16–512 |
Fig. 1.
Electrophoresis of polymerase chain reaction products of the optrA gene in the selected strains: M is a DNA Marker, C is a blank control; B10, A6, B2, A15, L13, L4 are strain numbers. Marker bands from top to bottom are in the order of 2,000 bp, 1,000 bp, 750 bp, 500 bp, 250 bp, 100 bp.
Table II
Characteristics of 30 optrA-positive linezolid-resistant Enterococcus faecalis.
| Isolate | Ward | Type of specimen | Isolation time | ST | LZD MIC (ug/ml) | Resistance genes | Virulence genes |
|---|---|---|---|---|---|---|---|
| 1 A1 | urology | blood | 2018/10/19 | 16 | 8 | aac(6’)-aph(2”), str, erm(A), erm(B), lsa(A), fexA, cat, tet(M), dfrG | elrA, srtA, ace, agg, ccf, cob, cad, camE, ebpA, ebpC, tpx, efaAfs, hylA, cylA, cylL, cylM |
| 2 A2 | urology | urine | 2018/11/02 | 585 | 8 | aac(6’)-aph(2”), erm(A), erm(B), fexA, tet(M), dfrG | elrA, srtA, ace, agg, ccf, cob, cad, camE, ebpA, ebpC, tpx, efaAfs, cylA, cylL, cylM, fsrB, gelE |
| 3 A3 | urology | urine | 2018/11/18 | 330 | 16 | aac(6’)-aph(2”), aph(3’)-III, Str, erm(A), lnu(G), fexA, cat, tet(M), dfrG | elrA, srtA, ace, ccf, cob, cad, camE, ebpA, ebpC, tpx, efaAfs, fsrB, gelE, hylA, hylB |
| 4 A5 | urology | urine | 2019/01/12 | 974 | 8 | erm(A), erm(B), fexA, tet(M), dfrG | elrA, srtA, ace, agg, ccf, cob, cad, camE, ebpA, ebpC, tpx, efaAfs, fsrB, gelE, hylA, hylB, cylA, cylL, cylM |
| 5 A6 | ICU | drainage | 2019/01/17 | 123 | 8 | aph(3’)-III, erm(B), cat, fexA, tet(L), dfrG | elrA, srtA, ace, ccf, cob, cad, camE, ebpA, ebpC, tpx, efaAfs, fsrB, gelE, hylA, hylB |
| 6 A7 | urology | urine | 2019/03/13 | 16 | 8 | ant(6’)-la, erm(A), erm(B), lsa(A), fexA, cat, tet(M), dfrG | elrA, srtA, ace, agg, ccf, cob, cad, camE, cylA, cylL, cylM, ebpA, ebpC, tpx, efaAfs, hylA |
| 7 A8 | ICU | urine | 2019/03/17 | 16 | 8 | aac(6’)-aph(2”), ant(6’)-la, erm(A), erm(B), lsa(E), fexA, cat, tet(M) | elrA, srtA, ace, agg, ccf, cob, cad, camE, cylA, cylL, cylM, ebpA, ebpC, tpx, efaAfs, hylA |
| 8 A9 | urology | urine | 2019/04/09 | 16 | 8 | aac(6’)-aph(2”), erm(B), lsa(A), fexA, tet(M), dfrG | elrA, srtA, ace, ccf, cob, cad, camE, cylA, cylL, cylM, ebpA, ebpC, tpx, efaAfs, hylA, fsrB, gelE |
| 9 A10 | urology | urine | 2019/04/22 | 69 | 8 | aac(6’)-aph(2”), aph(3’)-III, erm(A), erm(B), lsa(A), lsa(E), fexA, cat(PC233), tet(M), dfrG | elrA, srtA, ace, agg, ccf, cob, cad, camE, cylA, cylL, cylM, ebpA, ebpC, tpx, efaAfs, hylB, fsrB, gelE |
| 10 A13 | urology | urine | 2019/07/01 | 16 | 8 | erm(A), lsa(A), fexA, tet(M) | elrA, srtA, ace, agg, ccf, cob, cad, camE, cylA, cylL, cylM, ebpA, ebpC, tpx, efaAfs, hylA |
| 11 A15 | urology | urine | 2019/07/23 | 16 | 8 | aac(6’)-aph(2”), aph(3’)-III, erm(A), erm(B), lsa(A), fexA, tet(M), dfrG | elrA, srtA, ace, agg, ccf, cob, cad, camE, ebpA, ebpC, tpx, efaAfs, hylA, cylA, cylL, cylM |
| 12 A20 | general surgery | drainage | 2019/12/20 | 902 | 8 | aac(6’)-aph(2”), ant(6’)-la, str, erm(A), lsa(A), cat, tet(M), dfrG | elrA, srtA, ace, ccf, cob, cad, camE, ebpA, ebpC, tpx, efaAfs, hylA, hylB |
| 13 A22 | hepatobiliary surgery | drainage | 2020/07/07 | 535 | 16 | erm(B), fexA, cat, tet(M) | elrA, srtA, ace, ccf, cob, cad, camE, ebpA, ebpC, tpx, gelE, efaAfs, hylB, hylA, fsrB |
| 14 A23 | urology | urine | 2020/07/29 | 1287 | 8 | aac(6’)-aph(2”), ant(6’)-la, erm(A), erm(B), lsa(A), fexA, tet(M), dfrG | srtA, ace, agg, ccf, cob, cad, camE, ebpA, ebpC, tpx, gelE, efaAfs, hylB, fsrB |
| 15 A26 | ICU | drainage | 2020/06/05 | 619 | 16 | aac(6’)-aph(2”), ant(6’)-la, aph(3’)-III, erm(B), lnu(G), fexA, tet(M), dfrG | elrA, srtA, ace, ccf, cob, cad, camE, ebpA, ebpB, tpx, gelE, efaAfs, hylB, hylA, fsrB |
| 16 L3 | burn | wound secretion | 2020/11/06 | 480 | 8 | aph(3’)-III, ant(6’)-la, erm(A), erm(B), fexA, tet(M), dfrG | elrA, srtA, ace, ccf, cob, cad, camE, ebpA, ebpB, fsrB, tpx, efaAfs, gelE, hylA, hylB |
| 17 L4 | hepatobiliary surgery | pus | 2020/10/21 | 902 | 8 | ant(6’)-la, erm(A), lsa(E), cat, fexA, tet(M), dfrG | hylB, ace, efaAfs, elrA, srtA, ccf, cob, cad, camE, ebpA, ebpC, tpx |
| 18 L9 | nephrology | urine | 2020/12/01 | 585 | 8 | aac(6’)-aph(2”), aph(3’)-III, str, lsa(E), fexA, cat, tet(M), dfrG | elrA, srtA, ace, agg, ccf, cob, cad, camE, cylB, cylL, cylM, ebpA, ebpC, fsrB, tpx, efaAfs, gelE |
| 19 L13 | urology | urine | 2020/09/27 | 480 | 8 | aac(6’)-aph(2”), ant(6’)-la, aph(3’)-III, lsa(E), erm(A), erm(B), fexA, cat, tet(M), dfrG | hylA, ace, efaAfs, elrA, srtA, ccf, cob, cad, camE, ebpA, ebpB, tpx, agg |
| 20 B2 | urology | urine | 2021/08/12 | 16 | 8 | aac(6’)-aph(2”), ant(6’)-la, aph(3’)-III, lsa(A), lsa(E), lnu(B), erm(A), erm(B), fexA, tet(L), tet(M), dfrG | elrA, srtA, ace, agg, ccf, cob, cad, camE, ebpA, ebpC, tpx, efaAfs, hylA, cylA, cylL, cylM |
| 21 B3 | dermatovenereology | wound secretion | 2021/08/30 | 207 | 8 | aac(6’)-aph(2”), aph(3’)-III, erm(A), erm(B), lsa(A), lsa(E), fexA, tet(M), dfrG | elrA, srtA, ace, agg, ccf, cob, cad, camE, ebpA, ebpC, tpx, efaAfs, hylB, fsrB, gelE |
| 22 B7 | urology | urine | 2021/10/09 | 632 | 8 | ant(6’)-la, erm(A), erm(B), fexA, tet(M), dfrG | elrA, srtA, ace, agg, ccf, cob, cad, camE, ebpA, ebpC, tpx, efaAfs, hylB, fsrB, gelE |
| 23 B9 | urology | urine | 2021/10/14 | 816 | 16 | cat(pC233), fexA, tet(M) | elrA, srtA, ace, agg, ccf, cob, cad, camE, ebpA, ebpB, tpx, efaAfs, hylB, hylA, fsrB, gelE, cylA, cylL, cylM |
| 24 B10 | ICU | bile | 2021/10/23 | 968 | 8 | aac(6’)-aph(2”), aph(3’)-III, ant(6’)-la, erm(A), lsa(A), lsa(E), cat, fexA, tet(M), dfrG | elrA, srtA, ace, ccf, cob, cad, camE, ebpA, ebpC, tpx, efaAfs, hylA |
| 25 B11 | burn | wound secretion | 2021/10/23 | 300 | 8 | ant(6’)-la, erm(A), erm(B), lsa(A), fexA, tet(M) | srtA, ace, agg, ccf, cob, cad, camE, ebpA, ebpC, tpx, efaAfs, hylA, hylB, fsrB, gelE |
| 26 B12 | urology | urine | 2021/11/06 | 179 | 8 | aac(6’)-aph(2”), aph(3’)-III, ant(6’)-la, erm(A), erm(B), lsa(A), lsa(E), cat, fexA, tet(M) | elrA, srtA, ace, agg, ccf, cob, cad, camE, cylA, cylL, cylM, ebpA, ebpC, tpx, efaAfs, hylA |
| 27 B13 | hematology | wound secretion | 2021/11/07 | 16 | 8 | aac(6’)-aph(2”), aph(3’)-III, str, erm(A), erm(B), lsa(A), lsa(E), fexA, cat, tet(M), dfrG | elrA, srtA, ace, ccf, cob, cad, camE, cylA, cylL, cylM, ebpA, ebpC, tpx, efaAfs, hylA |
| 28 B15 | urology | drainage | 2021/12/05 | 660 | 8 | ant(6’)-la, aph(3’)-III, erm(A), erm(B), fexA, tet(M), dfrG | elrA, srtA, ace, ccf, cob, cad, camE, ebpA, ebpC, tpx, efaAfs, hylB, fsrB, gelE |
| 29 B18 | TCM | pus | 2021/08/13 | 631 | 8 | aph(3’)-III, ant(6’)-la, erm(A), erm(B), cat, fexA, tet(M) | elrA, srtA, ace, ccf, cob, cad, camE, cylA, cylL, cylM, ebpA, ebpB, ebpC, tpx, efaAfs, hylB, fsrB, gelE |
| 30 B19 | gastroenterology | urine | 2021/12/03 | 585 | 8 | ant(6’)-la, str, erm(A), erm(B), cat, fexA, tet(M), dfrG | elrA, srtA, ace, agg, ccf, cob, cad, camE, cylA, cylL, cylM, ebpA, ebpC, tpx, efaAfs, fsrB, gelE |
Fig. 2.
Phylogenetic tree of 30 optrA gene-positive LREfs. The columns to the right side of the phylogenetic tree sequentially refer to strain names, sequence types, source departments, specimen types, and linezolid minimal inhibitory concentrations from left to right.
Table III
Resistance phenotypes, genotypes and STs of recipient bacteria, donor bacteria and transconjugants.
| Strain | MIC (μg/ml) | ST | Conjugation frequency | Resistance genes | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| RA | FFC | LZD | CHL | ERY | CLI | TET | GEH | STH | ||||
| Enterococcus faecalis JH2–2 | 128 | 8 | 2 | S | ≤ 0.25 | ≤ 6 | ≤ 0.12 | < 500 (S) | ≤ 1,000 (S) | 8 | – | – |
| Enterococcus faecalis B2 | 2 | 128 | 8 | 6 (R) | ≥ 8 (R) | ≥ 8 (R) | ≥ 16 (R) | < 500 (S) | ≥ 1,000 (R) | 16 | – | aac(6’)-aph(2”), ant(6’)-la, aph(3’)-III, lsa(A), lsa(E), lnu(B), erm(A), erm(B), optrA, fexA, tet(L), tet(M), dfrG |
| Enterococcus faecalis TC-B2 | 128 | 128 | 8 | 6 (R) | ≥ 8 (R) | ≥ 8 (R) | ≥ 16 (R) | ≥ 500 (R) | ≥ 1,000 (R) | 8 | 1.2 × 10−4 | aac(6’)-aph(2”), ant(6’)-la, aph(3’)-III, lsa(A), lsa(E), lnu(B), erm(A), erm(B), optrA, fexA, dfrG |
| Enterococcus faecalis B3 | 2 | 128 | 8 | 6 (R) | ≥ 8 (R) | ≥ 8 (R) | ≥ 17 (R) | ≥ 500 (R) | < 1,000 (S) | 207 | – | aac(6’)-aph(2”), ant(6’)-la, aph(3’)-III, erm(A), erm(B), lnu(B), lsa(A), lsa(E), optrA, fexA, tet(L), tet(M), dfrG |
| Enterococcus faecalis TC-B3 | 128 | 128 | 8 | 6 (R) | ≥ 8 (R) | ≥ 8 (R) | ≥ 16 (S) | ≥ 500 (S) | < 1,000 (S) | 8 | 2.0 × 10−4 | aac(6’)-aph(2”), ant(6’)-la, aph(3’)-III, lsa(A), lsa(E), lnu(B), erm(A), erm(B), optrA, fexA, dfrG |
| Enterococcus faecalis B10 | 0.5 | 128 | 8 | 7 (R) | ≥ 8 (R) | ≥ 8 (R) | ≥ 16 (R) | ≥ 500 (R) | ≥ 1,000 (R) | 968 | – | aac(6’)-aph(2”), aph(3’)-III, ant(6’)-la, erm(A), erm(B), lsa(A), lsa(E), lnu(B), cat, optrA, fexA, tet(L), tet(M), dfrG |
| Enterococcus faecalis TC-B10 | 128 | 128 | 8 | 6 (R) | ≥ 8 (R) | ≥ 8 (R) | ≥ 16 (R) | ≥ 500 (R) | < 1,000 (S) | 8 | 1.2 × 10−5 | aac(6’)-aph(2”), aph(3’)-III, ant(6’)-la, erm(A), erm(B), lsa(A), lsa(E), lnu(B), optrA, fexA, dfrG |
Fig. 3.
Evolutionary tree constructed using SNP comparison files of 30 optrA gene-positive LREfs. A9, A8, A13, A15, A7, A1, B2, B12, B13 on same branch.
Fig. 4.
Genetic background of the optrA gene located on plasmids PB2-1 (above) and PTC-B2 (below). Genes and their orientations are indicated and labeled with arrows; colored arrows indicate genes and gray arrows indicate hypothetical proteins of unknown function, with ferrodoxin representing the gene encoding ferredoxin proteins.
Table IV
Genomic characterization of Enterococcus faecalis B2 and Enterococcus faecalis TC-B2.
| Genomic Features | Enterococcus faecalis B2 | Enterococcus faecalis TC-B2 |
|---|---|---|
| Genome size (bp) | 3,171,404 | 2,989,374 |
| Number of plasmids | 2 | 1 |
| Size range of plasmid(s) (bp) | 62,351 (PB2-1) | 105,222 |
| GC content of plasmid(s) (%) | 34.89 (PB2-1) | 34.32 |
| Coding genes of plasmid(s) | 70 (PB2-1) | 115 |
| Location of the optrA gene | 46108–48021 | 97914–99827 |