Occurrence of virulence factors and antimicrobial susceptibility of Citrobacter freundii isolated from diseased ornamental fish in Poland
, , and Dorota Pietras-Ożga
Department of Epizootiology and Clinic of Infectious Diseases, Faculty of Veterinary Medicine, University of Life SciencesLublin, Poland
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Mar 25, 2025
About this article
Published Online: Mar 25, 2025
Page range: 17 - 26
Received: Sep 04, 2024
Accepted: Mar 12, 2025
DOI: https://doi.org/10.2478/jvetres-2025-0017
Keywords
© 2025 Anna Pastuszka et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.
Fig. 1.
Fig. 2.
Primers used for virulence and antibiotic resistance gene expression study in Citrobacter freundii isolates from ornamental fish in Poland
| Primer name | Primer sequence (5′–3′) | Target | Temperature of annealing | Amplicon size (bp) | Reference |
|---|---|---|---|---|---|
| viaB | F-TGTCGAGCAGATGGATGAGCAT |
Vi capsular polysaccharide gene (Vi antigen) | 62°C | 516 | 14 |
| LT | F-ACGGCGTTACTATCCTCTC |
Heat-labile toxin gene of |
62°C | 273 | 38 |
| STp | F-TCTTTCCCCTCTTTTAGTCAG |
Heat-stable toxin gene of |
62°C | 166 | 38 |
| hlyA | F-GTCTGCAAAGCAATCCGCTGCAAATAAA |
α-haemolysin gene | 58°C | 561 | 19 |
| hep 58–59 | F-TCATGGCTTGTTATGACTGT |
Class 1 integron-encoding gene | 55° | 200 | 44 |
| tetA | F-CGCYTATATYGCCGAYATCAC |
Tetracycline resistance genes (tetracycline efflux pumps) | 60° | 417 | 26 |
| tetB | F-GGDATTGGBCTTATYATGCC |
60% | 967 | 26 | |
| tetG | F-TATGCRTTKATGCAGGTC |
60% | 917 | 26 |
Basic local alignment search tool results of sequence analysis of 16S rRNA amplicons and genotypic profiles of Citrobacter freundii (n = 20) isolates from ornamental fish in Poland
| Isolate | Length of |
Accession number | Percentage identity | Sequence name/ID (GenBank) | Presence of virulence genes (viaB/hlyA/LT/STp primers) | Presence of Class 1 integron-encoding gene (hep58–59 primers) | Presence of tetracycline resistance genes (tetA/tetB/tetG primers) |
|---|---|---|---|---|---|---|---|
| 1,348 bp | PP264335 | 99.41% | +/–/–/– | + | –/+/+ | ||
| 1,353 bp | PP264336 | 99.11% | +/–/–/– | – | –/–/– | ||
| 1,348 bp | PP264337 | 99.63% | –/–/+/– | – | –/–/– | ||
| 1,349 bp | PP264338 | 99.26% | +/–/–/+ | + | –/–/– | ||
| 1,352 bp | PP264339 | 99.70% | –/–/–/+ | + | +/+/+ | ||
| 1,349 bp | PP264340 | 99.78% | + | +/–/+ | |||
| 1,351 bp | PP264341 | 99.85% | –/–/–/– | – | –/–/– | ||
| 1,347 bp | PP264342 | 99.41% | + | +/–/+ | |||
| 1,346 bp | PP264343 | 99.24% | – | –/–/+ | |||
| 1,354 bp | PP264344 | 99.34% | + | +/–/+ | |||
| 1,349 bp | PP264345 | 99.56% | – | +/–/+ | |||
| 1,351 bp | PP264346 | 99.41% | + | +/–/+ | |||
| 1,352 bp | PP264347 | 99.63% | –/–/–/– | – | –/–/– | ||
| 1,351 bp | PP264348 | 99.56% | – | –/–/– | |||
| 1,351 bp | PP264349 | 99.78% | –/–/–/– | – | –/–/– | ||
| 1,352 bp | PP264350 | 99.56% | –/–/–/– | – | –/–/– | ||
| 1,342 bp | PP264351 | 99.55% | – | –/–/– | |||
| 1,342 bp | PP264352 | 99.40% | + | +/–/– | |||
| 1,341 bp | PP264353 | 98.51% | + | +/–/– | |||
| 1,342 bp | PP264354 | 99.78% | –/–/–/– | – | –/–/– |
Antibiotic resistance profiles for all tested Citrobacter isolates from freshwater and ornamental fish (n = 20)
| Antibiotic | Bacterial isolates’ antibiogram profile and interpretative range for disc diffusion | |||||
|---|---|---|---|---|---|---|
| Resistant (%) | Zone diameter (mm) | Intermediate (%) | Zone diameter (mm) | Susceptible (%) | Zone diameter (mm) | |
| Ampicillin (AM10 μg) | 20(100) | ≤13 | 0 (0) | 14–16 | 0 (0) | ≥17 |
| Piperacillin-tazobactam (TZP 110 μg) | 0 (0) | ≤17 | 1 (5) | 18–20 | 19 (95) | ≥21 |
| Cefotaxime (CTX 30 μg) | 2 (10) | ≤22 | 7 (35) | 23–25 | 11 (55) | ≥26 |
| Imipenem (IPM 10 μg) | 1 (5) | ≤19 | 1 (5) | 20–22 | 18 (90) | ≥23 |
| Meropenem (MEM 10 μg) | 0 (0) | ≤19 | 1 (5) | 20–22 | 19 (95) | ≥23 |
| Gentamicin (CN 10 μg) | 0 (0) | ≤12 | 1 (5) | 13–14 | 19 (95) | ≥15 |
| Amikacin (AK 30 μg) | 2 (10) | ≤14 | 3 (15) | 15–16 | 15 (75) | ≥17 |
| Kanamycin (K 30 μg) | 5 (25) | ≤16 | 9 (45) | 14–17 | 6 (30) | ≥18 |
| Streptomycin (S 10 μg) | 9 (45) | ≤11 | 8 (40) | 12–14 | 3 (15) | ≥15 |
| Tetracycline (TE 30 μg) | 12 (60) | ≤11 | 0 (0) | 12–14 | 8 (40) | ≥15 |
| Doxycycline (DO 30 μg) | 10 (50) | ≤10 | 3 (15) | 11–13 | 7 (35) | ≥14 |
| Ciprofloxacin (CIP 5 μg) | 13 (65) | ≤21 | 6 (30) | 22–25 | 1 (5) | ≥26 |
| Nalidixic acid (NA 30 μg) | 12 (60) | ≤13 | 3 (15) | 14–18 | 5 (25) | ≥19 |
| Sulfamethoxazole-trimethoprim (SXT 25 μg) | 8 (40) | ≤10 | 0 (0) | 11–15 | 12 (60) | ≥16 |
| Chloramphenicol (C30 μg) | 9 (45) | ≤12 | 0 (0) | 13–17 | 11 (55) | ≥18 |
Origin and identification by matrix-assisted laser desorption/ionisation–time-of-flight mass spectrometry (MALDI-TOF MS) of Citrobacter freundii strains isolated from ornamental fish in Poland
| Strain signature | Host species | Habitat / year of sampling | Source of isolate | Clinical signs of disease | Presence of other pathogens in the material | MALDI-TOF MS | |
|---|---|---|---|---|---|---|---|
| Score values | Species ID (based on MALDI Biotyper 3.1 software) | ||||||
| Aquatic env. /2023 | Trunk kidney | Lethargy, debilitated condition, no appetite, haemorrhages on the fin bases and around the anus and mouth | 2.367 | ||||
| Aquatic env. / 2023 | Trunk kidney | Lethargy, gill necrosis, increased respiration, septicaemia | 2.037 | ||||
| Aquatic env. / 2023 | Skin ulcers | Haemorrhagic ulcerations in the skin, haemorrhages around the anus | - | 2.236 | |||
| Aquatic env. / 2022 | Liver | Ocular opacity, exophthalmos, abdominal distention | - | 2.151 | |||
| Aquatic env. / 2023 | Skin ulcers | Haemorrhagic ulcerations in the skin, erratic swimming, no appetite | 2.225 | ||||
| Aquatic env. / 2022 | Liver | Weakened condition, fluid accumulation in the abdominal cavity | 2.211 | ||||
| Aquatic env. / 2023 | Skin ulcers | Haemorrhagic ulcerations in the skin, haemorrhages on the fin bases and around the anus and mouth | - | 2.112 | |||
| Aquatic env. / 2022 | Trunk kidney | No appetite, swimming disorders, caudal fin with frayed edges | 2.284 | ||||
| Aquatic env. / 2023 | Liver | Lethargy, swimming disorders, caudal fin with frayed edges, haemorrhages on the fin bases | 2.216 | ||||
| Aquatic env. / 2022 | Skin ulcers | Haemorrhagic ulcerations in the skin | 2.268 | ||||
| Aquatic env. / 2023 | Trunk kidney | Emaciation, lethargy, haemorrhages around the anus and mouth | 2.340 | ||||
| Aquatic env. / 2022 | Trunk kidney | Swimming disorders, exophthalmos, fluid accumulation in the abdominal cavity | 2.402 | ||||
| Aquatic env. / 2023 | Skin ulcers | Haemorrhagic ulcerations in the skin | - | 2.059 | |||
| Aquatic env. / 2023 | Trunk kidney | Weakened condition, caudal fin with frayed edges | - | 2.229 | |||
| Aquatic env. / 2023 | Liver | Haemorrhaging in the eye and exophthalmia, abdominal distention | 2.094 | ||||
| Aquatic env. / 2022 | Skin ulcers | Skin discolouration, haemorrhagic spots and superficial ulcers | - | 2.253 | |||
| Aquatic env. / 2022 | Liver | Haemorrhages on the fin bases and around the anus and mouth | - | 2.300 | |||
| Aquatic env. / 2022 | Liver | Skin discoloration, haemorrhagic spots, fluid accumulation in the abdominal cavity | 2.254 | ||||
| Aquatic env. / 2022 | Liver | Weakened condition, no appetite, petechial haemorrhaging in the liver and intestines | 2.378 | ||||
| Aqu. Env / 2023 | Liver | Lethargy, debilitated condition, no appetite, increased respiration, hemorrhages on the fin bases | - | 2.049 | |||