Prevalence and characterization of ciprofloxacin-resistant Salmonella enterica spp. isolated from food animals during 2010–2023 in South Korea
Md. Sekendar Ali, Hee-Seung Kang, Bo-Youn Moon, Ye-Eun Heo, Min Young Kim, Ji-Hyun Choi, Yu-Jeong Hwang, Ji-In Kim, Yeon-Hee Lee, Jae-Myung Kim, Suk-Kyung Lim
TL;DR
This study analyzed ciprofloxacin-resistant Salmonella in food animals in South Korea from 2010 to 2023, identifying resistance patterns and genetic markers.
Contribution
The study provides a detailed characterization of ciprofloxacin resistance in Salmonella from food animals in South Korea over a 13-year period.
Findings
3.1% of Salmonella isolates showed resistance to ciprofloxacin.
Common mutations in QRDRs included S83F, D87N, and D87G in gyrA.
ST198 S. Kentucky was the most prevalent sequence type among resistant isolates.
Abstract
We isolated 6,561 Salmonella strains from food animals, cattle (n = 217), pigs (n = 1526), chickens (n = 3942), and ducks (n = 876). Isolates were evaluated for antimicrobial sensitivity, mutations in quinolone resistance determination regions (QRDRs), and plasmid-mediated quinolone resistance (PMQR) genes. Clonal relationship and genetic diversity were assessed by multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Overall, 3.1% of isolates exhibited resistance to ciprofloxacin. Commonly identified mutations in QRDRs were S83F, D87N, and D87G in gyrA; T57S and S80I in parC; and L416F in parE. Furthermore, mutations differed by serotypes. In S. Albany, S83F mutation in gyrA and T57S in parC were prevalent, while in S. Kentucky, S83F and D87N in gyrA, T57S and S80I in parC; and in S. Indiana, S83F and D87G in gyrA, T57S and S80R in parC, and L416F in parE were…
Genes, proteins, chemicals, diseases, species, mutations and cell lines named across the full text — each resolved to its canonical identifier and authoritative record.
| Minimum inhibitory concentration (MIC) of ciprofloxacin (µg/mL) | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| ≤0.12 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | 16 | >16 | ≥1 | ||
| Cattle | 2010–2012 ( | 80.0 (60) | 12.0 (9) | 4.0 (3) | 4.0 (3) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 4.0 (3) |
| 2013–2015 ( | 61.7 (37) | 15.0 (9) | 16.7 (10) | 6.7 (4) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 6.7 (4) | |
| 2016–2018 ( | 48.8 (21) | 9.3 (4) | 41.9 (18) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | |
| 2019–2021 ( | 32.0 (8) | 44.0 (11) | 24.0 (6) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | |
| 2022–2023 ( | 71.4 (10) | 14.3 (2) | 14.3 (2) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | |
| Subtotal ( | 62.7 (136) | 16.1 (35) | 18.0 (39) | 3.2 (7) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 3.2 (7) | |
| Pigs | 2010–2012 ( | 73.8 (225) | 11.2 (34) | 14.1 (43) | 1.0 (3) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1.0 (3) |
| 2013–2015 ( | 78.1 (281) | 6.9 (25) | 14.4 (52) | 0.6 (2) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0.6 (2) | |
| 2016–2018 ( | 70.5 (208) | 11.9 (35) | 16.3 (48) | 0.7 (2) | 0.7 (2) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1.3 (4) | |
| 2019–2021 ( | 69.5 (282) | 11.8 (48) | 14.0 (57) | 2.5 (10) | 1.7 (7) | 0.2 (1) | 0.2 (1) | 0 (0) | 0 (0) | 4.6 (19) | |
| 2022–2023 ( | 67.5 (108) | 12.5 (20) | 17.5 (28) | 1.9 (3) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1.9 (3) | |
| Subtotal ( | 72.3 (1104) | 10.6 (162) | 14.9 (228) | 1.3 (20) | 0.6 (9) | 0.1 (1) | 0.1 (1) | 0 (0) | 0 (0) | 2.0 (31) | |
| Chickens | 2010–2012 ( | 57.1 (317) | 22.3 (124) | 13.3 (74) | 5.6 (31) | 1.6 (9) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 7.2 (40) |
| 2013–2015 ( | 37.0 (234) | 45.2 (286) | 13.4 (85) | 1.6 (10) | 2.2(14) | 0.5 (3) | 0.2 (1) | 0 (0) | 0 (0) | 4.4 (28) | |
| 2016–2018 ( | 36.1 (250) | 54.5 (377) | 8.2 (57) | 0.3 (2) | 0.7 (5) | 0 (0) | 0.1 (1) | 0 (0) | 0 (0) | 1.2 (8) | |
| 2019–2021 ( | 41.2 (445) | 33.8 (365) | 21.2 (229) | 0.6 (6) | 0.6 (7) | 0.2 (2) | 0.8 (9) | 0.6 (7) | 0.7 (8) | 3.6 (39) | |
| 2022–2023 ( | 27.6 (271) | 56.4 (554) | 14.5 (143) | 1.0 (10) | 0.1 (1) | 0 (0) | 0.3 (3) | 0.1 (1) | 0 (0) | 1.5 (15) | |
| Subtotal ( | 38.5 (1517) | 43.2 (1704) | 14.9 (588) | 1.5 (59) | 0.9 (36) | 0.1 (5) | 0.4 (14) | 0.2 (8) | 0.2 (8) | 3.2 (130) | |
| Ducks | 2016–2018 ( | 78.7 (74) | 5.3 (5) | 14.9 (14) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1.1 (1) | 1.1 (1) |
| 2019–2021 ( | 59.2 (302) | 21.0 (107) | 15.1 (77) | 0.6 (3) | 1.6 (8) | 1.0 (5) | 1.0 (5) | 0.2 (1) | 0.4 (2) | 4.7 (24) | |
| 2022–2023 ( | 51.5 (140) | 33.5 (91) | 9.9 (27) | 0.4 (1) | 3.0 (8) | 0 (0) | 0.4 (1) | 1.1 (3) | 0 (0) | 4.8 (13) | |
| Subtotal ( | 58.9 (516) | 23.2 (203) | 13.5 (118) | 0.5 (4) | 1.8 (16) | 0.6 (5) | 0.7 (6) | 0.5 (4) | 0.3 (3) | 4.3 (38) | |
| Subtotal | 2010–2012 ( | 64.4 (602) | 17.9 (167) | 12.8 (120) | 4.0 (37) | 1.0 (9) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 4.9 (46) |
| 2013–2015 ( | 52.4 (552) | 30.4 (320) | 14.0 (147) | 1.5 (16) | 1.3 (14) | 0.3 (3) | 0.1 (1) | 0 (0) | 0 (0) | 3.2 (34) | |
| 2016–2018 ( | 49.2 (553) | 37.3 (419) | 12.2 (137) | 0.4 (4) | 0.6 (7) | 0 (0) | 0.1 (1) | 0 (0) | 0.1 (1) | 1.2 (13) | |
| 2019–2021 ( | 51.3 (1037) | 26.4 (531) | 18.3 (369) | 0.9 (19) | 1.1 (22) | 0.5 (10) | 0.7 (15) | 0.4 (8) | 0.5 (11) | 4.1 (82) | |
| 2022–2023 ( | 37.0 (529) | 46.7 (667) | 14.0 (200) | 1.0 (14) | 0.6 (9) | 0.1 (1) | 0.3 (4) | 0.3 (4) | 0 (0) | 2.2 (31) | |
| Total ( | 49.9 (3273) | 32.0 (2104) | 14.8 (973) | 1.4 (90) | 0.9 (61) | 0.2 (11) | 0.3 (21) | 0.2 (12) | 0.2 (11) | 3.1 (206) | |
| Serotypes (No.) | Minimum inhibitory concentration of ciprofloxacin (µg/mL) | |||||
|---|---|---|---|---|---|---|
| 1 | 2 | 4 | 8 | 16 | >16 | |
| 31 (34.4) | 14 | – | – | – | – | |
| 29 | 12 | 4 | – | – | – | |
| – | – | – | 11 | 11 | 1 | |
| 4 | 5 | 4 | 5 | 1 | – | |
| 2 | 1 | 1 | – | – | 7 | |
| 5 | 4 | 1 | – | – | – | |
| – | 7 | – | 1 | – | – | |
| 5 | 1 | – | – | – | – | |
| 4 | – | – | – | – | – | |
| 3 | 2 | – | 1 | – | – | |
| 2 | 1 | – | 2 | – | – | |
| – | 4 | – | – | – | – | |
| – | 4 | – | – | – | – | |
| 2 | – | – | – | – | – | |
| – | 2 | – | – | – | – | |
| 1 | 1 | – | – | – | – | |
| – | 1 | – | – | – | – | |
| 1 | – | – | – | – | – | |
| – | 1 | – | – | – | – | |
| – | – | – | 1 | – | – | |
| Unidentified ( | 1 | 1 | 1 | – | – | 3 |
| Total ( | 90 (43.7) | 61 (30.0) | 11 (5.3) | 21 (10.2) | 12 (5.8) | 11 (5.3) |
| CIP MIC (µg/mL) (No. of isolates) | Serotypes (No.) |
|
|
|
| PMQR gene (No.) |
|---|---|---|---|---|---|---|
| 4 ( | S83F | WT | T57S, P112L | WT | ||
| S83F | WT | T57S | WT | |||
| D87N | WT | T57S | WT | |||
| D87G | WT | T57S | WT | |||
| S83Y | WT | WT | WT | |||
| D87Y | WT | WT | WT | |||
| Unidentified ( | S83F | WT | T57S | WT | ||
| 8 ( | S83F | WT | T57S | WT | ||
| S83F | WT | T57S | WT | |||
| S83F | WT | T57S | WT | |||
| S83F | WT | WT | WT | |||
| S83F, D87N | WT | T57S, S80I | WT | – | ||
| S83F, D87Y | WT | T57S, S80I | WT | – | ||
| S83F, D87Y | WT | S80I | WT | – | ||
| S83F, D87N | WT | T57S, S80I | WT | – | ||
| 16 ( | S83F | WT | T57S | WT | ||
| S83F, D87N | WT | T57S, S80I | WT | – | ||
| S83F, D87Y | WT | T57S, S80I | WT | – | ||
| >16 ( | S83F, D87G | WT | T57S, S80R | L416F | ||
| S83F, D87G | WT | T57S, S80R | L416F | – | ||
| S83F, D87N | WT | T57S, S80I | WT |
| Serotypes | Isolates | Year | Province | Farm ID | Source | MIC (µg/mL) | Non-fluoroquinolone resistance patterns | MLST | PFGE pattern | |
|---|---|---|---|---|---|---|---|---|---|---|
| Ciprofloxacin | Cefotaxime | |||||||||
| 13-S03-57 | 2013 | Incheon | AQ | Chicken feces | 8 | ≤0.5 | AMP, GEN, STR, FIS, TET | 198 | KX7KA8 | |
| 19-S03-03 | 2019 | Gyeonggi | D | Chicken carcass | 16 | ≤0.5 | AMP, FIS, TET | 198 | KX1KA1 | |
| 19-S03-04 | 2019 | Gyeonggi | E | Chicken carcass | 8 | ≤0.5 | AMP, FIS, TET | 198 | KX1KA1 | |
| 19-S03-05 | 2019 | Gyeonggi | AK | Chicken carcass | 16 | ≤0.5 | AMP, FIS, TET | 198 | KX1KA1 | |
| 19-S03-06 | 2019 | Gyeonggi | AK | Chicken carcass | 8 | ≤0.5 | AMP, FIS, TET | 198 | KX1KA1 | |
| 19-S03-12 | 2019 | Gyeongbuk | K | Chicken carcass | 16 | ≤0.5 | AMP, FIS, TET | 198 | KX1KA1 | |
| 20-A02-18 | 2020 | Chungbuk | T | Pig feces | 8 | ≤0.5 | AMP, FIS, TET | 198 | KX4KA4 | |
| 20-A03-19 | 2020 | Chungbuk | AI | Chicken feces | 8 | ≤0.5 | AMP, FIS, TET | 198 | KX4KA4 | |
| 20-S03-14 | 2020 | Gyeonggi | O | Chicken carcass | 16 | ≤0.5 | AMP, FIS, TET | 198 | KX1KA2 | |
| 20-S03-15 | 2020 | Gyeonggi | Y | Chicken carcass | 16 | ≤0.5 | AMP, FIS, TET | 198 | KX1KA2 | |
| 20-S03-20 | 2020 | Chungbuk | AJ | Chicken carcass | 16 | ≤0.5 | AMP, FIS, TET | 198 | KX4KA4 | |
| 20-S03-21 | 2020 | Chungbuk | AG | Chicken carcass | 8 | ≤0.5 | AMP, FIS, TET | 198 | KX4KA1 | |
| 20-S03-22 | 2020 | Chungbuk | S | Chicken carcass | 8 | ≤0.5 | – | 198 | KX6KA6 | |
| 21-A03-35 | 2021 | Gyeonggi | A | Chicken feces | 16 | ≤0.5 | – | 198 | KX2KA1 | |
| 21-A03-36 | 2021 | Gyeonggi | A | Chicken feces | 8 | ≤0.5 | – | 198 | KX2KA1 | |
| 21-A03-37 | 2021 | Gyeonggi | A | Chicken feces | 8 | ≤0.5 | AMP, FIS, TET | 198 | KX1KA1 | |
| 21-A03-38 | 2021 | Gyeonggi | A | Chicken feces | 8 | ≤0.5 | AMP, FIS, TET | 198 | KX1KA1 | |
| 21-A04-47 | 2021 | Gyeongnam | V | Duck feces | >16 | >8* | AMP, FEP, CTX, CAZ, CHL, FIS, TET, SXT | 198 | KX4KA5 | |
| 22-E03-52 | 2022 | Jeonbuk | AD | Diseased chicken | 8 | >8* | AMP, FEP, CTX, CAZ, TET | 198 | KX5KA7 | |
| 22-E04-49 | 2022 | Chungbuk | M | Diseased duck | 16 | ≤0.5 | AMP, FIS, TET | 198 | KX1KA3 | |
| 22-E04-50 | 2022 | Chungbuk | M | Diseased duck | 16 | ≤0.5 | AMP, FIS, TET | 198 | KX1KA3 | |
| 22-E04-51 | 2022 | Chungbuk | M | Diseased duck | 16 | ≤0.5 | AMP, FIS, TET | 198 | KX1KA3 | |
| 23-A03-54 | 2023 | Chungnam | F | Chicken feces | 16 | >8* | AMP, FEP, CTX, CAZ, TET | 198 | – | |
| 18-S04-01 | 2018 | Chungbuk | AB | Duck carcass | >16 | ≤0.5 | AMP, CHL, STR, FIS, TET | 17 | IX1IA4 | |
| 19-A03-08 | 2019 | Gangwon | AC | Chicken feces | >16 | ≤0.5 | AMP, CHL, GEN, STR, FIS, TET | 17 | IX1IA1 | |
| 19-A03-09 | 2019 | Gangwon | AC | Chicken feces | >16 | ≤0.5 | AMP, CHL, GEN, STR, FIS, TET | 17 | IX1IA1 | |
| 19-E03-10 | 2019 | Chungbuk | H | Diseased chicken | >16 | ≤0.5 | AMP, CHL, GEN, STR, FIS, TET | 17 | IX1IA3 | |
| 20-A03-29 | 2020 | Chungnam | Z | Chicken feces | >16 | ≤0.5 | AMP, CHL, GEN, STR, FIS, TET | 17 | IX2IA1 | |
| 20-A03-30 | 2020 | Chungnam | U | Chicken feces | >16 | ≤0.5 | AMP, CHL, GEN, STR, FIS, TET | 17 | IX3IA2 | |
| 20-A04-33 | 2020 | Gyeongnam | X | Duck feces | 4 | ≤0.5 | FIS, SXT | 17 | IX5IA7 | |
| 20-S03-16 | 2020 | Gyeonggi | AA | Chicken carcass | >16 | ≤0.5 | AMP, CHL, GEN, STR, FIS, TET | 17 | IX1IA1 | |
| 20-S03-17 | 2020 | Gyeonggi | AA | Chicken carcass | >16 | ≤0.5 | AMP, CHL, GEN, STR, FIS, TET | 17 | IX1IA1 | |
| 20-S04-23 | 2020 | Chungbuk | Q | Duck carcass | >16 | ≤0.5 | – | 17 | IX6IA5 | |
| 21-A03-40 | 2021 | Chungbuk | P | Chicken feces | >16 | ≤0.5 | AMP, CHL, GEN, STR, FIS, TET | 17 | IX4IA6 | |
| 19-A04-11 | 2019 | Jeonnam | C | Duck feces | 8 | ≤0.5 | AMP, CHL, FIS, TET, SXT | 292 | AX4AA1 | |
| 20-A03-31 | 2020 | Jeonnam | L | Chicken feces | 4 | ≤0.5 | CHL, FIS, SXT | 292 | AX5AA4 | |
| 20-A04-32 | 2020 | Jeonnam | AE | Duck feces | 8 | ≤0.5 | AMP, CHL, FIS, TET, SXT | 292 | AX3AA3 | |
| 20-E04-24 | 2020 | Chungbuk | I | Diseased duck | 8 | ≤0.5 | AMP, CHL, FIS, TET, SXT | 292 | AX1AA1 | |
| 20-E04-25 | 2020 | Chungbuk | I | Diseased duck | 8 | ≤0.5 | AMP, CHL, FIS, TET, SXT | 292 | AX1AA1 | |
| 20-E04-26 | 2020 | Chungbuk | AF | Diseased duck | 4 | ≤0.5 | AMP, CHL, FIS, TET, SXT | 292 | NDAA5 | |
| 20-E04-27 | 2020 | Chungbuk | AF | Diseased duck | 4 | ≤0.5 | AMP, CHL, FIS, TET, SXT | 292 | AX1AA5 | |
| 20-E04-28 | 2020 | Chungbuk | I | Diseased duck | 16 | ≤0.5 | AMP, CHL, FIS, TET, SXT | 292 | AX1AA1 | |
| 21-S04-42 | 2021 | Jeonbuk | J | Duck carcass | 4 | ≤0.5 | AMP, CHL, FIS, SXT | 292 | AX2AA2 | |
| 21-E04-43 | 2021 | Jeonnam | B | Diseased duck | 8 | ≤0.5 | AMP, CHL, FIS, TET, SXT | 292 | AX1AA1 | |
| 14-A03-58 | 2014 | Gyeongbuk | AN | Chicken feces | 4 | ≤0.5 | AMP, CHL, GEN, STR, FIS, TET, SXT | 50 | – | |
| 15-A03-60 | 2015 | Jeonbuk | AP | Chicken feces | 4 | ≤0.5 | AMP, CHL, STR, FIS, TET, SXT | 50 | – | |
| 15-S03-59 | 2015 | Chungbuk | AO | Chicken carcass | 4 | ≤0.5 | AMP, CHL, GEN, STR, FIS, TET, SXT | 50 | – | |
| 21-E02-34 | 2021 | Gyeongbuk | AL | Diseased pig | 4 | 1 | AMP, CHL, STR, FIS, TET, SXT | 8316 | – | |
| 23-A03-55 | 2023 | Jeonbuk | N | Chicken feces | 8 | ≤0.5 | AMP, CHL, TET | 13 | – | |
| 23-S03-56 | 2023 | Jeonbuk | N | Chicken carcass | 8 | ≤0.5 | AMP, CHL, TET | 13 | – | |
| 22-S04-53 | 2022 | Gyeongnam | W | Duck carcass | 8 | ≤0.5 | STR | 33 | – | |
| 18-A03-02 | 2018 | Gyeongbuk | AH | Chicken feces | 8 | ≤0.5 | AMP, CHL, STR, FIS, TET, SXT | ND | – | |
| 21-A03-44 | 2021 | Gyeongbuk | R | Chicken feces | 4 | ≤0.5 | STR, FIS, TET, SXT | 4 | MX1MA1 | |
| 21-A03-39 | 2021 | Gyeonggi | A | Chicken feces | 8 | ≤0.5 | AMP, FIS, TET | 198 | – | |
| Unknown | 21-A04-41 | 2021 | Jeonbuk | G | Duck feces | 4 | ≤0.5 | AMP, CHL, FIS, SXT | 292 | – |
- —Animal and Plant Quarantine Agency, Ministry of Agriculture, Food, and Rural Affairs, Republic of Korea
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Taxonomy
TopicsSalmonella and Campylobacter epidemiology · Antibiotic Resistance in Bacteria · Vibrio bacteria research studies
Introduction
Salmonella is one of the most common pathogens causing foodborne illness in humans globally. According to the global burden of disease study, approximately 535,000 cases of non-typhoidal human salmonellosis were detected in 2017 worldwide (Stanaway et al. 2019). In Korea, there were an average of 857 reported cases per year from 2009 to 2020 of individuals infected with Salmonella (Koh et al. 2022). Although non-typhoidal salmonellosis usually recovers spontaneously, suitable antimicrobial therapy is essential for severe cases, particularly those with compromised immune systems, pediatrics, and geriatrics (Sánchez-Vargas et al. 2011). Nevertheless, numerous countries, including developing and developed ones, have reported antimicrobial-resistant Salmonella infections, resulting in mortality and morbidity (Majowicz et al. 2010; Kariuki et al. 2015; Parisi et al. 2018). The use of antimicrobials in both humans and food animals leads to the spread of antimicrobial resistance; consequently, the effectiveness of the most commonly used antimicrobials, such as penicillin, tetracycline, and chloramphenicol, has become limited (Kuang et al. 2018). As a result, fluoroquinolones, including ciprofloxacin, have become a preferred treatment for invasive salmonellosis due to their effectiveness in treating a wide range of infections (Cuypers et al. 2018). However, the widespread use of quinolone and fluoroquinolone inevitably triggers the development of antimicrobial resistance with novel resistance mechanisms (Li et al. 2018).
The prevalence of ciprofloxacin-resistant Salmonella isolated from food animals has been described worldwide (Hur et al. 2012). Moreover, numerous studies on the occurrence of ciprofloxacin resistance in Salmonella isolates from humans have been reported in China (Kuang et al. 2018), Poland (Wołkowicz et al. 2021), and Korea (Lee et al. 2021). In addition, it was shown that ciprofloxacin resistance can be transmitted to humans through direct contact or consumption of Salmonella-contaminated meat of food-producing animals (Bulut 2014).
Recently, the prevalence of fluoroquinolone-resistant Salmonella has been augmented in food animals in Korea (Kim et al. 2019; Moon et al. 2021; Zhang et al. 2021). Understanding the phenotypic and genotypic characteristics of Salmonella in food animals is essential for ascertaining the potential risk to humans. Therefore, the purpose of this study was to determine the prevalence at the national level and molecular characteristics of ciprofloxacin-resistant Salmonella spp. isolated from food animals during 2010–2023 in South Korea.
Materials and methods
Bacterial isolation and identification
2.1.
Salmonella isolation, identification, and serotyping were carried out according to the previously delineated method (Ali et al. 2023). The Salmonella strains were obtained from feces and carcasses of food-producing animals (cattle, pigs, chickens, and ducks) from 16 laboratories/centers participating in the Korean Veterinary Antimicrobial Resistance Monitoring System (KVARS) between 2010 and 2023. The isolation process involves the sample pre-enrichment in buffered peptone water followed by incubation in a modified semisolid Rappaport Vassiliadis medium (MSRV: Becton Dickinson, CA, USA). The diffused regions at MSRV were cultured using CHROMagar (Merck, Darmstadt, Germany), and the colonies were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (Biomerieux, Marcy L’Etoile, France). Salmonella serotype was determined using polymerase chain reaction (PCR) (Ranieri et al. 2013; Mechesso et al. 2022) and finally confirmed by the classic White-Kauffman-Le Minor scheme (Grimont and Weill 2007). One isolate from each sample was used for subsequent analysis. However, we do not have information on the history of antimicrobial usage in food-producing animals considered for this investigation.
Antimicrobial susceptibility testing
2.2.
The antimicrobial susceptibility of the isolates was assessed by the broth microdilution method (Ali et al. 2024) using the Sensititre^TM^ panel KRNV6F (Thermo Fischer Scientific, Waltham, MA, USA) based on the manufacturer’s protocol. The obtained minimum inhibitory concentrations (MICs) were interpreted according to the guidelines provided by the Clinical and Laboratory Standards Institute (CLSI) (2023). The MIC ≥4 µg/mL of ciprofloxacin was set as a threshold value to indicate a high level of ciprofloxacin resistance (Raveendran et al. 2008). The high-level ciprofloxacin-resistant isolates were further characterized using molecular techniques.
Mechanisms of ciprofloxacin resistance
2.3.
Genes encoding for DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) in quinolone resistance determining regions (QRDRs) were performed using multiplex PCR. The PCR products were subjected to sequencing using an automated ABI Prism 3700 analyzer (Applied Biosystems, Foster, CA, USA). The primers and PCR conditions are summarized in the Supplementary Table S1. We used the Basic Local Alignment Search Tool (BLAST) to identify gene mutations in QRDRs. We compared the sequences with those available in the GenBank nucleotide database at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST). The detection of plasmid-mediated quinolone resistance (PMQR) genes (qnrB1, qnrS, and aac(6′)-Ib-cr) was performed by PCR using the previously described conditions and primers (Table S1).
Mechanism of third-generation cephalosporin resistance
2.4.
The double-disc synergy was performed to detect the presence of extended-spectrum β-lactamase (ESBL) genes among the ciprofloxacin-resistant Salmonella using cefotaxime-cefotaxime/clavulanic acid discs, following the guidelines provided by the Clinical and Laboratory Standards Institute (CLSI) (2023). The PCR was conducted using previously described primers to identify the presence of ESBL (blaCTX-M) genes in cefotaxime-resistant Salmonella isolates (Na et al. 2020). First, we detected the CTX-M gene using a universal primer, followed by the detection of the CTX-M-9 group gene using blaCTX-M group-specific primers. Then, we performed the PCR and sequencing using primers covering the whole CTX-M-9 group gene. The sequencing of PCR products was performed using an ABI3730XL DNA sequencing analyzer (Solgnet Daejeon, South Korea) to identify the blaCTX-M by searching the homologous sequence in the GenBank database utilizing the Basic Local Alignment Search Tool (BLAST) on the National Center for Biotechnology Information website (http://www.ncbi.nlm.gov/BLAST). The primers and PCR conditions are detailed in Table S1.
Multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE)
2.5.
The clonal relationship of Salmonella isolates was determined using MLST, following the previous method (Kidgell et al. 2002). A total of seven housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA, and* thrA*) were amplified and sequenced in this classical approach. The allelic profiles and sequence types (STs) for Salmonella spp. were identified using the online MLST database (http://pubmlst.org/organisms/Salmonella-spp). The genetic diversity of Salmonella isolates was evaluated using PFGE of genomic DNA digested with the enzymes XbaI and AvrII (TaKaRa Bio, Inc., Shiga, Japan) (Ali et al. 2023). The PFGE band profiles were assessed by Bionumerics software (version 5.1), and the level of similarity was evaluated using the unweighted pair-group technique with an algorithm-based arithmetic average and Dice similarity index.
Results
Prevalence of Salmonella
3.1.
A total of 6561 Salmonella strains have been isolated from feces and carcasses of food-producing animals (Table 1). Overall, ciprofloxacin resistance was found in 3.1% of the isolates. The resistance rate was varied by different year groups. The level of resistance was high during 2010–2012 but was lower in the period of 2016–2018. However, the proportion of isolates that showed MIC ≤0.12 µg/mL decreased from 64.4% to 37% in 2010–2012 and 2022–2023. Furthermore, isolates that showed high levels of MIC were increased.
Although no resistant isolate was observed before 2013, a total of 1.7% and 0.6% of isolates showed MIC ≥8 µg/mL in 2019–2021 and 2022–2023, respectively (Table 1). The rate of resistance also differed among different animal species. Resistance was lower in pig isolates compared to the isolates obtained from other animals. Even though the resistance rate was 3.2% in cattle, all isolates showed MIC 1 µg/mL. Generally, the resistance rate was higher in chicken and duck isolates. Furthermore, the number of isolates showing an elevated level of MIC 8–≥16 µg/mL was higher in chickens (0.8%) and ducks (1.5%) compared to cattle (0%) and pigs (0.1%).
Distribution of ciprofloxacin-resistant Salmonella by serotypes
3.2.
Ciprofloxacin-resistant Salmonella was observed in 20 serotypes (Table 2). The most frequently observed serotypes were S. Gallinarium (21.8%) and S. Typhimurium (21.8%), followed by S. Kentucky (11.2%), S. Albany (9.2%), S. Indiana (5.3%), and S. Montevideo (4.9%). Moreover, the level of MIC was different by serotype. Most serotypes showed MIC at 1–2 µg/mL, especially 100% of S. Gallinarium and 91.1% of S. Typhimurium isolates. Of note, six serotypes had high MIC (8 µg/mL), and very high MIC (≥16 µg/mL) was observed in S. Kentucky, S. Indiana, and S. Albany.
QRDR mutation and detection of qnr genes
3.3.
All ciprofloxacin-resistant isolates showed mutations in gyrA and parC (Table 3). However, no mutation was found in gyrB. The most commonly observed mutations in gyrA were S83F (89.1%, 49/55), followed by D87N (40%, 22/55), D87G (20%, 11/55), D87Y (7.3%, 4/55) and S83Y (5.5%, 3/55). The predominant mutations identified in parC were T57S (89.1%, 49/55) and S80I (43.6%, 24/55). We found a stepwise pattern among the QRDR mutations. The number of mutation sites increases in high-MIC isolates. The MICs of ≥16 µg/mL isolates possessed mutations at 4–5 sites. In addition, mutation patterns were different by serotype. In S. Albany, the S83F mutation in gyrA and T57S in parC were common, while in S. Kentucky, S83F and D87N in gyrA, T57S and S80I in parC, and in S. Indiana, S83F and D87G in gyrA, T57S and S80R in parC, and L416F in parE were frequently detected.
Overall, all isolates with MIC ≥4 µg/mL carried the qnr gene. Moreover, a total of three PMQR genes (qnrB1, qnrS, and aac(6′)-Ib-cr) were detected (Table 3). Among them, qnrS (32.7%, 18/55) was predominantly found, followed by aac(6′)-Ib-cr (12.7%, 7/55) and qnrB1 (7.3%, 4/55). In the serotype level, qnrS was mostly identified in S. Albany (8 isolates), aac(6′)-Ib-cr in S. Indiana (6 isolates), and qnrB1 in S. Albany (2 isolates).
Molecular characterization
3.4.
The high-level (MIC ≥4 µg/mL) ciprofloxacin-resistant isolates were distributed nationwide and obtained from 39 farms located in all provinces except one (Table 4). Overall, only one isolate was identified in each farm except one (A), which detected five isolates: four S. Kentucky and one S. Seremban. Since high-level ciprofloxacin-resistant S. Kentucky isolates were first detected in chickens in 2013, ciprofloxacin resistance was observed in various other serotypes, including S. Kentucky and S. Indiana, which were mainly observed in chickens, while S. Albany was mostly identified in ducks. The ciprofloxacin-resistant isolates also exhibited resistance to non-fluoroquinolone. Most of the isolates demonstrated resistance to three more antimicrobials, commonly ampicillin, sulfisoxazole, and tetracycline. Of note, three isolates were resistant to third-generation cephalosporin. These isolates carried blaCTX-M-65 in S. Kentucky from one duck and two chickens.
The MLST analysis revealed specific ST by serotypes. All S. Kentucky was assigned to ST198, S. Albany to ST292, and S. Indiana to ST17 (Table 4). At the animal species level, S. Kentucky ST198 and S. Indiana ST17 were prevalent among chickens, while S. Albany ST292 was predominantly found in ducks. However, PFGE with two restriction enzymes (XbaI and AvrII) digestion differentiated the same STs (Figure S1). A total of 26 unique pulsotypes were detected, comprising eleven types in S. Kentucky, eight in S. Indiana, and seven in S. Albany among the 55 ciprofloxacin-resistant Salmonella isolates. Overall, PFGE patterns were heterogeneous except for S. Kentucky. One PFGE type, KX1KA1, was observed in 30.4% of S. Kentucky isolates. Furthermore, identical ST and PFGE types were found in the same and different farms. The KX1KA1 pattern in S. Kentucky was mainly observed in farms AK (n = 2), A (n = 2), D (n = 1), E (n = 1), and K (n = 1); in S. Albany, AX1AA1 was frequently detected in farms I (n = 3) and B (n = 1); and in S. Indiana, IX1IA1 was detected in farms AC (n = 2) and AA (n = 2).
Discussion
This extensive, long-term investigation identified the high-level ciprofloxacin resistance in various serotypes, especially S. Kentucky, S. Indiana, and S. Albany, mainly from chicken sources. The major resistance mechanism is the mutation at QRDR. In addition, specific serotypes and clones S. Kentucky ST198, S. Indiana ST17, and S. Albany 292 were widely detected. Of note, three ciprofloxacin-resistant S. Kentucky carried the blaCTX-M-65.
Overall, ciprofloxacin resistance was detected in 3.1% of the isolates. The resistance rate of the isolates differed by the various collection year groups, animal species, and level of MICs. Isolates from food animals had varying levels of MIC for ciprofloxacin, consistent with findings from previous studies (Jiang et al. 2014; Bai et al. 2015). Noticeably, susceptibility was decreased compared to 10 years ago. Furthermore, high-level ciprofloxacin-resistant isolates (≥4 µg/mL) have been detected in food animals since 2013. The high incidence of ciprofloxacin resistance in Salmonella strains isolated from food animals was also found in China (14.8%) (Chen et al. 2021), Ukraine (6.3%) (Kozytska et al. 2023), and South Africa (8%) (Mthembu et al. 2019). In Korea, Sin et al. (2020) reported the occurrence of ciprofloxacin-resistant Salmonella (3.5%) in food-producing animals is consistent with our findings.
The level of ciprofloxacin resistance was different by serotypes in this study. The resistance level for MIC 1–2 µg/mL was mainly detected in serotypes S. Gallinarium (29.8%) and S. Typhimurium (27.2%). In note, high-level resistance (MIC 4–>16 µg/mL) was detected in S. Kentucky (41.8%), S. Albany (18.2%), and S. Indiana (14.5%). This finding concurs with previous studies showing that resistance levels can vary depending on the serotypes (Zhang et al. 2017; Abd El-Aziz et al. 2021; Awosile et al. 2023). The high prevalence of ciprofloxacin resistance in S. Gallinaiurm (26.9%) from chickens has been reported in South Korea, consistent with our findings (Seo et al. 2019). Furthermore, ciprofloxacin resistance was detected in 72.4% of S. Gallinaium in India (Bangera et al. 2019) and 48% of S. Typhimurium in Malaysia (Syed Abu Thahir et al. 2023) isolated from chickens, which is greater than our current findings. Moreover, it was shown that the majority of the S. Kentucky isolates recovered from poultry (71.4%) in 28 European Union (EU) member states demonstrated high-level ciprofloxacin resistance (EFSA and ECDC 2020). In addition, Zengfeng et al. (2022) found that 89.9% of the S. Indiana isolates from food-producing animals in China exhibited resistance to ciprofloxacin. Our previous studies also reported the occurrence of high-level ciprofloxacin resistance in S. Kentucky (Moon et al. 2021) and S. Indiana (Moon et al. 2021) isolated from chickens.
Moreover, this study reported ciprofloxacin resistance in other serotypes, including S. Albany, S. Infantis, S. Agona, and S. Seremban. The prevalence of ciprofloxacin-resistant S. Albany has recently emerged significantly in food animals (Kaichao et al. 2020). In addition, the emergence of high-level ciprofloxacin resistance in the serotypes S. Infantis and S. Agona has been increased in humans and food-producing animals, including chickens, pigs, and cattle (Kuang et al. 2018; Lai et al. 2023). Furthermore, although the ciprofloxacin-resistant S. Seremban is less frequent, it has also been reported in humans and food animals (Hendriksen 2010).
Salmonella isolates in this investigation were obtained from various sources, with commonly observed ciprofloxacin resistance in chickens. The high prevalence of ciprofloxacin-resistant Salmonella isolated from poultry is consistent with findings from previous investigations (Kim et al. 2019; Moon et al. 2021). The reason might be due to the increased consumption of fluoroquinolones in poultry, particularly with about 70% of the total used 35–40 tons of enrofloxacin annually in Korean livestock (Animal and Plant Quarantine Agency (APQA) 2022).
The resistance of fluoroquinolones in Salmonella was caused by mutations in the subunits constituting topoisomerase II (gyrA and gyrB) and IV (parC and parE) in the QRDRs (Abd El-Aziz et al. 2021). In this study, all resistant isolates had mutations in gyrA, and most of the resistant isolates possessed mutations in parC. Moreover, the mutations in gyrA were most commonly detected at S83F, D87N, and D87G, whereas the most frequent mutations in parC occurred at T57S and S80I. Salmonella showing resistance to ciprofloxacin typically possesses a minimum of two mutations in gyrA, in addition to mutations in other QRDRs (Abd El-Aziz et al. 2021). These additional mutations in the QRDR genes triggered the obtaining of a significant level of resistance to fluoroquinolones. Cao et al. (2017) demonstrated that the sites, types, and number of amino acid substitutions in QRDR genes varied among serovars, suggesting that resistance can differ regarding types and numbers of mutations in QRDRs. The L416F change was found in parE in our investigation, while L416F has previously been reported in Salmonella strains isolated from China (Yang et al. 2023).
In this study, all isolates with a MIC of ≥4 µg/mL possessed the qnr genes. In addition, different PMQR genes, qnrS, qnrB1, and aac(6′)-Ib-cr, have been detected. Moreover, the presence of the qnr genes differed by serotype. Most of the serotypes carried one or more qnr or aac(6′)-Ib-cr genes. Nonetheless, no qnr genes were detected in S. Kentucky. Consistent with this investigation, qnrS and qnrB1 genes were detected in nontyphoidal Salmonella strains isolated from humans and food animals, including chickens, pigs, and cattle in China (Chen et al. 2024), Ethiopia (Eguale et al. 2017), and Romania (Colobatiu et al. 2015). The qnr genes were not associated with high-level resistance or with serotypes. However, their presence can complement other mechanisms to enhance the accumulation of the mutational target sites, leading to the selection of high-level resistance (Shaheen et al. 2021). It was shown that the occurrence of the aac(6′)-Ib-cr gene can complement the high-level quinolone resistance (Hooper and Jacoby 2015). Microorganisms that possess this gene may become more challenging to treat and pose a critical risk to human and animal health (Wong et al. 2014). A previous investigation reported that the acquisition of plasmids encoding aac(6′)-Ib-cr by Salmonella resulted in elevated ciprofloxacin MICs, and this resistance determinant was found to be responsible for a significant rise in ciprofloxacin resistance observed in human cases of salmonellosis (Lee et al. 2021). Moreover, this mobile genetic element, found in livestock globally, can be plasmid-borne or situated in a chromosome, indicating a high likelihood of spreading (Biao et al. 2022).
We found that ciprofloxacin-resistant Salmonella also showed resistance to other commonly used antimicrobials in livestock. Interestingly, most of them, except S. Kentucky, demonstrated resistance to ampicillin, sulfisoxazole, tetracycline, and chloramphenicol. This is consistent with the previous findings, which showed that ciprofloxacin-resistant S. Indiana strains obtained from chickens demonstrated multidrug resistance, including ampicillin, chloramphenicol, tetracycline, and trimethoprim-sulfamethoxazole (Bai et al. 2016). In addition, it was revealed that ciprofloxacin-resistant S. Indiana and S. Typhimurium isolated from food animals simultaneously exhibited resistance to these antimicrobials (Chen et al. 2024; Han et al. 2024). Moreover, concurring with the previous investigations, our research demonstrated that Salmonella strains recovered from humans and food-producing animals exhibited an identical resistance pattern with ampicillin, chloramphenicol, gentamicin, tetracycline, and trimethoprim/sulfamethoxazole (Wołkowicz et al. 2021; Borah et al. 2022).
It was observed that three ciprofloxacin-resistant isolates carrying blaCTX-M-65 in S. Kentucky from two chickens and one duck were detected in this study. The prevalence of blaCTX-M-65-carrying Salmonella in food animals, particularly chickens, has been documented worldwide, including in China (Lin et al. 2015), Australia (Sia et al. 2021), the EU (EFSA and ECDC 2020), and Korea (Kang et al. 2024). Moreover, Salmonella strains obtained from food animals and humans exhibit similar genetic characteristics of cephalosporin resistance (Chen et al. 2019). The development and transmission of the blaCTX-M-65 gene may exacerbate the cephalosporin resistance in human salmonellosis. Furthermore, the infection caused by ciprofloxacin and third-generation cephalosporin co-resistant Salmonella in individuals further aggravates the situation (Jiang et al. 2024).
The MLST analysis revealed the occurrence of several STs representing specific serotypes. Among them, S. Kentucky ST198 and S. Indiana ST17 were predominantly detected in chickens, while S. Albany ST292 was commonly identified in ducks. Previous studies reported the frequent prevalence of ciprofloxacin-resistant S. Kentucky ST198 clones in chickens in Korea (Moon et al. 2021), China (Xiong et al. 2020), and Spain (Samper-Cativiela et al. 2022). Furthermore, multidrug-resistant S. Albany ST292 has been detected in humans and food animals, posing human health hazards (Monte et al. 2019). Recently, ciprofloxacin-resistant S. Indiana ST17 has also frequently been identified in food-producing animals, including chickens and ducks (Wang et al. 2020; Du et al. 2022), which could be spread to humans and other animals, posing potential health problems.
The PFGE analysis detected a total of 26 unique pulsotypes, including novel types, among the ciprofloxacin-resistant Salmonella isolates. Several types were found in different farms located in various provinces throughout the country, possibly because of the extensive spread of these clones. Previous studies have shown that multidrug-resistant Salmonella clones are widely spread in both humans and food animals (Borah et al. 2022; Manzari et al. 2022). Our analysis revealed that several new clones emerged and have the potential to be transmitted to humans or other animals.
Conclusion
The present study provides important insights regarding the prevalence and mechanisms of ciprofloxacin-resistant Salmonella isolates obtained from food animals. Ciprofloxacin-resistant isolates commonly contained S83F and D87N mutations in gyrA; T57S and S80I in parC; and L416F in parE as well as carrying PMQR genes (qnrB1, qnrS, and aac(6′)-Ib-cr). Moreover, particular serotypes and clones such as S. Kentucky ST198, S. Albany ST292, and S. Indiana ST17 were predominantly detected that could potentially contribute to the dissemination of fluoroquinolone-resistant Salmonella in livestock. In addition, this resistance can be transmitted to humans through direct contact or the food chain. Thus, the findings of this study suggest the importance of regular monitoring and strict regulation of antimicrobial use in food animals. Moreover, further research on vertical and horizontal antimicrobial resistance gene transmission is required to determine the potential health hazards for humans and other animals.
Supplementary Material
Supplemental Material
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