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 Escherichia coli | 62°C | 273 | 38 |
| STp | F-TCTTTCCCCTCTTTTAGTCAG | Heat-stable toxin gene of Escherichia coli | 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 16S rRNA sequence deposited in GenBank | 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) |
|---|---|---|---|---|---|---|---|
| C.f_01 | 1,348 bp | PP264335 | 99.41% | C. freundii ATCC 8090/NR_028894.1 | +/–/–/– | + | –/+/+ |
| C. f_02 | 1,353 bp | PP264336 | 99.11% | C. freundii ATCC 8090/NR_028894.1 | +/–/–/– | – | –/–/– |
| C. f_03 | 1,348 bp | PP264337 | 99.63% | C. freundii strain LMG 3246/NR_117752.1 | –/–/+/– | – | –/–/– |
| C. f_04 | 1,349 bp | PP264338 | 99.26% | C. freundii ATCC 8090/NR_028894.1 | +/–/–/+ | + | –/–/– |
| C. f_05 | 1,352 bp | PP264339 | 99.70% | C. freundii ATCC 8090/NR_028894.1 | –/–/–/+ | + | +/+/+ |
| C. f_06 | 1,349 bp | PP264340 | 99.78% | C. freundii ATCC 8090/NR_028894 | +/–/–/– | + | +/–/+ |
| C. f_07 | 1,351 bp | PP264341 | 99.85% | C. freundii strain LMG 3246/NR_117752.1 | –/–/–/– | – | –/–/– |
| C. f_08 | 1,347 bp | PP264342 | 99.41% | C. freundii ATCC 8090/NR_028894.1 | +/–/–/– | + | +/–/+ |
| C. f_09 | 1,346 bp | PP264343 | 99.24% | C. freundii strain NBRC 12681/NR_114345.1 | +/–/–/– | – | –/–/+ |
| C. f_10 | 1,354 bp | PP264344 | 99.34% | C. freundii ATCC 8090/NR_028894.1 | +/–/–/– | + | +/–/+ |
| C. f_11 | 1,349 bp | PP264345 | 99.56% | C. freundii ATCC 8090/NR_028894.1 | +/–/–/– | – | +/–/+ |
| C. f_12 | 1,351 bp | PP264346 | 99.41% | C. freundii ATCC 8090/NR_028894.1 | +/–/–/– | + | +/–/+ |
| C. f_13 | 1,352 bp | PP264347 | 99.63% | C. freundii ATCC 8090/NR_028894.1 | –/–/–/– | – | –/–/– |
| C. f_14 | 1,351 bp | PP264348 | 99.56% | C. freundii strain LMG 3246/NR_117752.1 | +/–/–/– | – | –/–/– |
| C. f_15 | 1,351 bp | PP264349 | 99.78% | C. freundii ATCC 8090/NR_028894.1 | –/–/–/– | – | –/–/– |
| C. f_16 | 1,352 bp | PP264350 | 99.56% | C. freundii strain LMG 3246/NR_117752.1 | –/–/–/– | – | –/–/– |
| C. f_17 | 1,342 bp | PP264351 | 99.55% | C. freundii strain LMG 3246/NR_117752.1 | +/–/–/– | – | –/–/– |
| C. f_18 | 1,342 bp | PP264352 | 99.40% | C. freundii ATCC 8090/NR_028894.1 | +/–/–/– | + | +/–/– |
| C. f_19 | 1,341 bp | PP264353 | 98.51% | C. freundii ATCC 8090/NR_028894.1 | +/–/–/– | + | +/–/– |
| C.f_20 | 1,342 bp | PP264354 | 99.78% | C. freundii strain LMG 3246/NR 117752.1 | –/–/–/– | – | –/–/– |
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) | ||||||
| Citrobacter freundii_1 | Trichopodus trichopterus | Aquatic env. /2023 | Trunk kidney | Lethargy, debilitated condition, no appetite, haemorrhages on the fin bases and around the anus and mouth | Aeromonas hydrophila | 2.367 | C. freundii 22054_1 CHB |
| Citrobacter freundii_2 | Poecilia wingei | Aquatic env. / 2023 | Trunk kidney | Lethargy, gill necrosis, increased respiration, septicaemia | Acinetobacter lwoffii | 2.037 | C. freundii 22054_1 CHB |
| Citrobacter freundii_3 | Tanichthys albonubes | Aquatic env. / 2023 | Skin ulcers | Haemorrhagic ulcerations in the skin, haemorrhages around the anus | - | 2.236 | C. freundii 22054_1 CHB |
| Citrobacter freundii_4 | Xiphophorus maculatus | Aquatic env. / 2022 | Liver | Ocular opacity, exophthalmos, abdominal distention | - | 2.151 | C. freundii 22054_1 CHB |
| Citrobacter freundii_5 | Corydoras paleatus | Aquatic env. / 2023 | Skin ulcers | Haemorrhagic ulcerations in the skin, erratic swimming, no appetite | Aeromonas veronii | 2.225 | C. freundii 13158_2 CHB |
| Citrobacter freundii_6 | Hypostomus plecostomus | Aquatic env. / 2022 | Liver | Weakened condition, fluid accumulation in the abdominal cavity | Aeromonas caviae, Vibrio albensis | 2.211 | C. freundii 13158_2 CHB |
| Citrobacter freundii_7 | Sahyadria denisonii | Aquatic env. / 2023 | Skin ulcers | Haemorrhagic ulcerations in the skin, haemorrhages on the fin bases and around the anus and mouth | - | 2.112 | C. freundii DSM 30039T HAM |
| Citrobacter freundii_8 | Chromobotia macracanthus | Aquatic env. / 2022 | Trunk kidney | No appetite, swimming disorders, caudal fin with frayed edges | Pseudomonas alcaligenes | 2.284 | C. freundii DSM 15979 DSM |
| Citrobacter freundii_9 | Xiphophorus maculatus | Aquatic env. / 2023 | Liver | Lethargy, swimming disorders, caudal fin with frayed edges, haemorrhages on the fin bases | Plesiomonas shigelloides | 2.216 | C. freundii 22054_1 CHB |
| Citrobacter freundii_10 | Betta splendens | Aquatic env. / 2022 | Skin ulcers | Haemorrhagic ulcerations in the skin | Shewanella putrefaciens | 2.268 | C. freundii 22054_1 CHB |
| Citrobacter freundii_11 | Poecilia sphenops | Aquatic env. / 2023 | Trunk kidney | Emaciation, lethargy, haemorrhages around the anus and mouth | Pseudomonas putida | 2.340 | C. freundii 22054_1 CHB |
| Citrobacter freundii_12 | Betta splendens | Aquatic env. / 2022 | Trunk kidney | Swimming disorders, exophthalmos, fluid accumulation in the abdominal cavity | Aeromonas veronii, Shewanella putrefaciens | 2.402 | C. freundii 13158_2 CHB |
| Citrobacter freundii_13 | Hemigrammus rhodostomus | Aquatic env. / 2023 | Skin ulcers | Haemorrhagic ulcerations in the skin | - | 2.059 | C. freundii 13158_2 CHB |
| Citrobacter freundii_14 | Betta splendens | Aquatic env. / 2023 | Trunk kidney | Weakened condition, caudal fin with frayed edges | - | 2.229 | C. freundii 22054_1 CHB |
| Citrobacter freundii_15 | Pseudotropheus Saulosi | Aquatic env. / 2023 | Liver | Haemorrhaging in the eye and exophthalmia, abdominal distention | Aeromonas hydrophila | 2.094 | C. freundii 13158_2 CHB |
| Citrobacter freundii_16 | Xiphophorus hellerii | Aquatic env. / 2022 | Skin ulcers | Skin discolouration, haemorrhagic spots and superficial ulcers | - | 2.253 | C. freundii 22054_1 CHB |
| Citrobacter freundii_17 | Macropodus opercularis | Aquatic env. / 2022 | Liver | Haemorrhages on the fin bases and around the anus and mouth | - | 2.300 | C. freundii 22054_1 CHB |
| Citrobacter freundii_18 | Danio rerio | Aquatic env. / 2022 | Liver | Skin discoloration, haemorrhagic spots, fluid accumulation in the abdominal cavity | Chryseobacterium indologenes | 2.254 | C. freundii 22054_1 CHB |
| Citrobacter freundii_19 | Mikrogeophagus ramirezi | Aquatic env. / 2022 | Liver | Weakened condition, no appetite, petechial haemorrhaging in the liver and intestines | Aeromonas veronii, Pseudomonas putida | 2.378 | C. freundii 22054_1 CHB |
| Citrobacter freundii_20 | Mikrogeophagus ramirezi | Aqu. Env / 2023 | Liver | Lethargy, debilitated condition, no appetite, increased respiration, hemorrhages on the fin bases | - | 2.049 | C. freundii 13158_2 CHB |