2025 Volume 60 Issue 2 Pages 33-40
One hundred sixty-four Edwardsiella specimens isolated from cultured fishes, red seabream Pagrus major, Japanese flounder Paralichthys olivaceus, thread-sail filefish Stephanolepis cirrhifer, greater amberjack Seriola dumerili and Japanese eel Anguilla japonica were analyzed by PCR for identification, and 124 of these isolates were examined using biochemical tests for characterization. Hundred and four strains from red seabream, three from Japanese flounder, and six from other fish species were identified as E. anguillarum, whereas one strain from seabream and 50 strains from flounder were identified as E. piscicida. Among the E. anguillarum strains, all strains from Japanese eel were motile and ornithine decarboxylase (ODC)-positive, whereas all strains from other fish species were non-motile and ODC-negative. The findings of this study reveal the existence of two distinct types of E. anguillarum differing in motility and ODC production. Susceptibility testing using nine antimicrobials (tetracycline, erythromycin, lincomycin, ampicillin, sulfamonomethoxine, florfenicol, thiamphenicol, oxolinic acid, and fosfomycin) showed that all strains were sensitive to fosfomycin, which had a relatively narrow range of minimum inhibitory concentrations compared to other drugs.
Edwardsiellosis is a serious systemic bacterial disease caused by Edwardsiella spp., affecting various fresh and saltwater fish species worldwide (Xu and Zhang, 2014). Members of the Edwardsiella genus are gram-negative, rod-shaped, and facultative anaerobic bacteria. Recently, based on genomic information and phylogenetic analysis, the genus Edwardsiella was reclassified into five species, including three fish pathogens (E. piscicida, E. anguillarum, and E. ictaluri) and two non-fish pathogens (E. tarda and E. hoshinae) (Abayneh et al., 2013; Shao et al., 2015; Leung et al., 2019).
Previously, Edwardsiella strains isolated from marine fish were classified as E. tarda (Reichley et al., 2017; Buján et al., 2018). Following further characterization of the bacterial species based on multilocus sequence analysis, a portion of the bacterial group isolated from fish originally identified as E. tarda was reclassified as E. piscicida (Abayneh et al., 2013). Based on the sequence of the DNA gyrase subunit B (gyrB) gene, a similar species was found within the E. piscicida group, and a specific polymerase chain reaction (PCR) was developed to distinguish Edwardsiella family members (Griffin et al., 2014). A similar species was proposed as E. anguillarum, and strains isolated from red seabream Pagrus major were identified as E. anguillarum (Shao et al., 2015). The majority of Edwardsiella strains obtained from Japanese aquacultured fish including red seabream, Japanese flounder, and Japanese eel have been classified as E. piscicida or E. anguillarum. The fish-pathogenic Edwardsiella species E. piscicida and E. anguillarum are motile (Shao et al., 2015); however, non-motile strains have been observed, highlighting the importance of characterizing Japanese strains.
Currently, only fosfomycin (FOM) is approved for edwardsiellosis in marine fish belonging to the family Perciformes, including seabream, in Japan, whereas no drugs have been approved for flatfish (Heterosomata). The approved antimicrobials for treating edwardsiellosis in eels are oxolinic acid (OA), florfenicol (FF), sulfamonomethoxine-ormetoprim combination drug (ektecin), and oxytetracycline hydrochloride. Because there is no effective vaccine for controlling edwardsiellosis, and antimicrobials have long been used to treat the disease, multidrug-resistant strains have been identified (Aoki and Kitao, 1981; Matsuoka and Wada, 1996; Kanai, 2002; Fukuda, 2003; Morii et al., 2007). This pathogen causes significant problems in aquaculture. It is important to investigate and monitor the occurrence and biological characteristics of this bacterium. However, information on the antimicrobial susceptibility of Edwardsiella strains from marine fish in Japan is limited.
Ehime Prefecture has the largest fish aquaculture facility in Japan, accounting for approximately 25% of the national fish production; combined marine and inland culture generated approximately 250,000 tons in 2022 (Ministry of Agriculture, Forestry and Fisheries, 2022). Red seabream production is particularly high, accounting for more than half of the total domestic production. Edwardsiellosis is the most serious disease affecting red seabream farming; diseased fish lose commercial value due to their disfigured appearance. This disease has also been observed in other prefectural marine aquaculture and eel farming systems.
In this study, we identified Edwardsiella strains obtained from cultured fish using PCR and investigated their biochemical properties, focusing on strains from red seabream that are severely affected by edwardsiellosis on fish farms in Ehime Prefecture, Japan. Furthermore, the antimicrobial susceptibility associated with bacterial species and hosts was examined.
Edwardsiella strains (a total of 164; red seabream P. major, n = 105; Japanese flounder Paralichthys olivaceus, n = 53; thread-sail filefish Stephanolepis cirrhifer, n = 2; greater amberjack Seriola dumerili, n = 1; and Japanese eel Anguilla japonica, n = 3) were obtained from the ulcers, kidneys, and spleens of diseased fish from 57 farms in the Ehime area from 2005 to 2018 (Table 1).
| Source of isolation | No. of strains in year | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Common name | Scientific name | 2005 | 2007 | 2012 | 2013 | 2014 | 2015 | 2016 | 2017 | 2018 | Total |
| Red seabream | Pagrus major | 10 | 10 | 12 | 18 | 21 | 18 | 16 | 105 | ||
| Japanese flounder | Paralichthys olivaceus | 1 | 10 | 7 | 11 | 7 | 10 | 7 | 53 | ||
| Thread-sail filefish | Stephanolepis cirrhifer | 1 | 1 | 2 | |||||||
| Greater amberjack | Seriola dumerili | 1 | 1 | ||||||||
| Japanese eel | Anguilla japonica | 2 | 1 | 3 | |||||||
| Total | 1 | 2 | 12 | 22 | 19 | 29 | 28 | 28 | 23 | 164 | |
The strains were cultured on trypticase soy agar (TSA) at 28°C for 24 h, and total DNA was extracted from pure subcultured colonies of the strains using InstaGene Matrix (Bio-Rad Laboratories) following the manufacturer’s protocol. PCR was performed using primers specific for E. anguillarum and E. piscicida, as previously described (Griffin et al., 2014). PCR was performed using the T-100 thermal cycler (Bio-Rad Laboratories) with Takara Ex Taq (Takara Bio) under the following conditions: initial cycle at 95°C for 5 min, 35 cycles of 95°C for 15 s, 58°C for 15 s, 72°C for 15 s, and a final extension step at 72°C for 5 min. The PCR products were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining.
Biochemical characterizationOf the strains used for species identification by PCR, 124 (105 from red seabream, 13 from Japanese flounder, two from thread-sail filefish, one from greater amberjack, and three from the Japanese eel) were used in the characterization test.
Each strain was streaked onto TSA and incubated at 28°C for 24 h. Then, single colonies were stabbed into the sulfide-indole-motility semisolid medium (Nissui), and motility was determined after incubation at 28°C for 24 h. Other single colonies on TSA were suspended in 5 mL of 0.85% sterile saline, turbidity was adjusted to the equivalent of 0.5 McFarland standard (MacFaddin, 1980), and then subjected to the API20E (bioMerieux) test according to the manufacturer’s protocol. The results were obtained after 24 h of incubation at 28°C.
Antimicrobial susceptibility testingThe 124 strains used in the biochemical characterization tests were used for antimicrobial susceptibility testing. The minimum inhibitory concentrations (MICs) of nine antimicrobial agents (tetracycline [TC], ampicillin [ABPC], erythromycin [EM], lincomycin [LCM], OA, thiamphenicol [TP], FF, FOM, and sulfamonomethoxine [SMMX]) were determined using the broth microdilution method on frozen plates with cation-adjusted Mueller–Hinton broth (CAMHB) (Eiken Chemical Co., Ltd.) according to the manufacturer’s instructions. For the FOM susceptibility tests, CAMHB was supplemented with 25 mg/L glucose-6-phosphate according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2023). Pure cultured colonies of the bacterial strains were suspended in 3 mL of 0.85% sterile saline at a density of 1.0 McFarland standards. Bacterial growth inhibition was confirmed after 48 h of growth at 28°C. The MIC was defined as the lowest concentration that did not produce any visible growth. The breakpoints for the antimicrobial agents were estimated only for E. anguillarum and were defined as the midpoints between the peaks when MICs were bimodally distributed (MacGowan and Wise, 2001). The strains that showed MIC values above breakpoint were considered as antimicrobial resistant. The MIC value with more than 50% of the strains in the test population inhibited were defined as MIC50, which corresponds to the median MIC value. MIC90 represents the MIC value at which more than 90% of the strains in the test population were inhibited. The MIC50 was the value at position n × 0.5, the MIC90 was calculated accordingly, using n × 0.9. The antimicrobial susceptibility of E. piscicida was tentatively evaluated based on the breakpoints of E. anguillarum.
PCR-based species identification confirmed that 104 of the 105 strains isolated from red seabream belonged to E. anguillarum, whereas the remaining strain belonged to E. piscicida. Three of the 53 strains from Japanese flounder were identified as E. anguillarum, whereas the remaining 50 strains were identified as E. piscicida. All six strains isolated from thread-sail filefish, greater amberjack, and Japanese eel were identified as E. anguillarum.
Biochemical characterizationThe results of the motility and biochemical property tests for the strains are shown in Table 2. All three E. anguillarum strains obtained from Japanese eels were motile, whereas the remaining E. anguillarum strains obtained from other fish species (red seabream, Japanese flounder, thread-sail filefish, and greater amberjack) were non-motile. The E. piscicida strains obtained from red seabream and Japanese flounder were motile.
| Test items | Type of strains | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Present study | Shao et al., (2015) | ||||||||
| I (n = 110) | II (n = 3) | III (n = 11) | IV | V | VI | ||||
| Motility | – | (0) | + | (3) | + | (11) | + | + | + |
| β-galactosidase | – | (0) | – | (0) | – | (0) | – | – | – |
| Arginine dihydrolase | – | (4) | – | (0) | – | (0) | – | – | – |
| Lysine decarboxylase | + | (110) | + | (3) | + | (11) | + | + | + |
| Ornithine decarboxylase | – | (0) | + | (3) | + | (11) | + | + | + |
| Citrate utilization | + | (75) | + | (2) | + | (9) | + | + | – |
| H2S production | + | (110) | + | (3) | + | (11) | + | + | + |
| Urease | – | (4) | – | (0) | – | (0) | – | – | – |
| Tryptophan deaminase | – | (0) | – | (0) | – | (0) | – | – | – |
| Indole production | + | (110) | + | (3) | + | (11) | + | + | + |
| Acetoin production | – | (0) | – | (0) | – | (0) | + | – | – |
| Gelatinase | – | (0) | – | (0) | – | (0) | – | – | – |
| D-Glucose | + | (110) | + | (3) | + | (11) | + | + | + |
| D-Mannitol | + | (110) | + | (3) | – | (0) | + | – | – |
| Inositol | – | (0) | – | (0) | – | (0) | – | – | – |
| D-Sorbitol | – | (0) | – | (0) | – | (0) | – | – | – |
| L-Rhamnose | – | (0) | – | (0) | – | (0) | – | – | – |
| Saccharose | – | (3) | – | (0) | – | (0) | – | – | – |
| D-Melibiose | – | (0) | – | (0) | – | (0) | – | – | – |
| D-Amygdalin | – | (0) | – | (0) | – | (0) | – | – | – |
| L-Arabinose | + | (106) | + | (3) | – | (0) | + | – | – |
The number of positive strains for each item is shown in parentheses.
Details of each type are shown below.
I : E. anguillarum from seabream, flounder, filefish and amberjack
II : E. anguillarum from Japanese eel
III : E. piscicida from seabream and flounder
IV : E. anguillarum ET080813T
V : E. piscicida ET883T
VI : E. tarda ATCC 15947T
In the characterization test using API20E, all 124 strains were negative for β-galactosidase, tryptophan deaminase, acetoin production, gelatinase, inositol, D-sorbitol, L-rhamnose, D-melibiose, and D-amygdalin, whereas lysine decarboxylase, H2S production, indole production, and D-glucose were positive. Four E. anguillarum strains isolated from seabream tested positive for both arginine dihydrolase and urease. In addition, three of these strains utilized saccharose. Most strains were positive for citrate utilization, although several negative strains were identified, regardless of the bacterial species.
All E. anguillarum strains obtained from Japanese eels and E. piscicida were positive for ornithine decarboxylase (ODC), whereas E. anguillarum strains obtained from other fish species were negative for ODC. All E. anguillarum strains were positive for D-mannitol and L-arabinose, whereas E. piscicida was negative for these compounds.
Antimicrobial susceptibility testThe distribution of the MIC values (MICs) of the antimicrobial agents against all Edwardsiella strains used in this study is shown in Fig. 1. The MICs for E. anguillarum are listed in Table 3. The MICs of all strains showed a bimodal distribution for the four antimicrobials (TC, ABPC, OA, and TP) (Fig. 1). The MICs of E. anguillarum strains showed a similar bimodal distribution for these antimicrobials (Table 3).
| antimicrobials | MIC (μg mL−1) | MIC50 (μg mL−1) | MIC90 (μg mL−1) | Break point (BP) | CLSI BP* | No. resistant (%) | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ≤0.12 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | 64 | 128 | 256 | >256 | ||||||
| TC | 14 | 59 | 1 | 1 | 1 | 5 | 2 | 17 | 13 | 0.25 | 64 | 2 | 8 | 39 (34.5%) | ||||
| ABPC | 2 | 13 | 73 | 5 | 4 | 1 | 7 | 5 | 3 | 2 | 128 | 16 | 16 | 16 (14.2%) | ||||
| TP | 13 (≤0.25) | 92 | 3 | 1 | 4 | 0.5 | 0.5 | 2 | 16 | 5 (4.4%) | ||||||||
| FF | 82 (≤0.25) | 29 | 1 | 1 | ≤0.25 | 0.5 | 16 | |||||||||||
| OA | 111 | 1 | 1 | ≤0.12 | ≤0.12 | 0.25 | >16 <32 | 2 (1.8%) | ||||||||||
| FOM | 25 | 73 | 15 | 2 | 4 | 128 | ||||||||||||
| EM | 2 | 72 | 14 | 25 (>128) | 32 | >128 | 1-4 | |||||||||||
| LCM | 1 | 7 | 52 | 53 | 256 | >256 | 1-2 | |||||||||||
| SMMX | 16 (≤8) | 26 | 17 | 6 | 6 | 42 (>128) | 32 | >128 | >256 <512 | |||||||||
TC; tetracycline, ABPC; ampicillin, TP; thiamphenicol, FF; florfenicol, OA; oxolinic acid, FOM; fosfomycin, EM; erythromycin, LCM; lincomycin, SMMX; Sulfamonomethoxine
Lower brackets indicate MIC values outside the measurement range.
The MICs of TC showed a clear bimodal distribution, with approximately 37% (46/124) of the strains being resistant to TC (Fig. 1). TC resistance was observed in E. anguillarum obtained from red seabream and eel and in E. piscicida obtained from seabream and flounder. The MICs for ABPC showed bimodal behavior, with approximately 13% (16/124) of the strains being resistant (Fig. 1). Although a relatively large number of antimicrobial-resistant bacteria (ARB) have been identified, regardless of their source, ABPC resistance was found only in E. anguillarum strains from seabream.
The MIC values for TP showed moderate bimodal distribution, with approximately 13% (16/124) of the strains being resistant, including all the E. anguillarum from eels and E. piscicida (Fig. 1). Although no clear bimodal distribution was observed for the MICs of FF, the four E. piscicida strains isolated from flounder showed a tendency toward resistance, with MIC values of four (Fig. 1). Most strains were susceptible to OA, although 10 strains were determined to be resistant, including eight strains of E. piscicida. All strains showed susceptibility to FOM, with MIC values ranging from one to four. (Fig. 1). The MICs for EM and LCM were 16 and 64 μg/mL or higher, respectively, and 21% (26/124) of the strains showed MIC values over 128 μg/mL in EM. No clear bimodality was found for SMMX, although a single peak was observed at an MIC value of 16 μg/mL, and approximately 40% (50/124) of the strains showed MICs greater than 128 μg/mL (Fig. 1). The MIC distribution of strains exceeding these measurement ranges were unknown, hence no breakpoints were set.
The frequencies of resistance of E. anguillarum 113 strains to TC, ABPC, TP, and OA were 34.5%, 14.2%, 4.4%, and 1.8%, respectively. In addition, the MIC50 and MIC90 values for TC and ABPC were widely different, and they were separated by 8-step dilutions (Table 3).
Most of the red seabream and Japanese flounder sampled in this study were cultured in the same bay, and serious edwardsiellosis was found in both fish species during periods of high water temperatures. More than 99% (104/105) and 94% (50/53) of the strains isolated from seabream and flounder were E. anguillarum and E. piscicida, respectively. Although the susceptibility of fish to Edwardsiella spp. has been reported to vary according to the bacterial species, only a few strains have been investigated for seabream isolates (Buján et al., 2018; Sugiura et al., 2022). The large-scale strain identification in this study provides further evidence that susceptibility varies among fish species. In exceptional cases, when fish farms with edwardsiellosis are close to each other, red seabream and Japanese flounder are occasionally infected with E. piscicida and E. anguillarum, respectively.
Shao et al., (2015) defined E. anguillarum as a motile bacterium using the hanging drop method (Jain et al., 2020). However, the FPC503 strain tested in their study was confirmed to be non-motile in several other studies (Matsuyama et al., 2005; Nakamura et al., 2013). The other non-motile Edwardsiella strains, later identified as E. anguillarum, have also been isolated from crimson seabream Evynnis japonica, red seabream, and yellowtail Seriola quinqueradiata in Japan (Kusuda et al., 1977; Yasunaga et al., 1982), and white grouper Epinephelus aeneus in Israel (Reichley et al., 2017). In the present study, the strains isolated from eels were motile, whereas those from red seabream, Japanese flounder, thread-sail filefish, and greater amberjack were non-motile. For a biochemical analysis using API20E, Shao et al. (2015) reported that E. anguillarum was positive for ODC and acetoin production. In contrast, in the present study, the eel-derived strains were positive for ODC, whereas the other strains were negative, and none of the E. anguillarum strains produced acetoin. These results indicate that E. anguillarum differs in its motility and biochemical properties depending on the strain, with all red seabream-derived strains being non-motile and ODC-negative. For convenience, we classified E. anguillarum from seabream, flounder, filefish and amberjack as Type I, from Japanese eel as Type II, and E. piscicida from seabream and flounder as Type III (Table 2).
In antimicrobial susceptibility testing, many ARBs were identified regardless of the source of isolation, although ABPC resistance was found only in E. anguillarum isolated from red seabream; E. piscicida strains tended to be highly resistant to TP and OA. According to a survey of the MIC values of E. tarda, which was presumed to be E. anguillarum, isolated from cultured red seabream in Ehime Prefecture from 2009 to 2012, 9.0% (6/67) and 4.5% (3/67) of the strains were resistant to ABPC and TP, respectively, and no resistance to FOM was observed (Yamashita et al., 2014). In the present study, 14.2% (16/113) and 4.4% (5/113) of the strains were resistant to ABPC and TP, respectively, and a slight increase in the resistance to ABPC was observed. The large difference between the MIC50 and MIC90 values also suggests that there are many ARBs for these antimicrobials. No strains resistant to FOM were found, indicating that susceptibility to antimicrobials was maintained. Due to outbreaks of pasteurellosis, vibriosis, and edwardsiellosis in seabream farming (Yasunaga and Yamamoto, 1977; Yasunaga et al., 1983), antibiotics such as TC, ABPC, TP, and OA are occasionally administered, which may influence the emergence of resistant E. anguillarum strains. Bacteria with multidrug resistant plasmids may be resistant to unused antibiotics (Aoki and Kitao, 1981). Edwardsiella species is naturally resistant to macrolides and lincosamides (Stock and Wiedemann, 2001); the MICs of EM and LCM in this study were higher than 16 and 32 μg/mL, respectively.
Edwardsiella spp. cause infections in various animals, including fish and humans, and numerous antimicrobial prophylactic strategies have been developed to prevent this disease. Edwardsiella spp. are susceptible to several antimicrobials (Stock and Wiedemann, 2001). On the other hand, the appearance of ARB associated with the use of antimicrobials in aquaculture has emerged as a serious problem (Santos and Ramos, 2018). Comparing the results of the present study in seabream farming with those of previous reports (Yamashita et al., 2014), it is estimated that the appearance of ARB has not changed significantly. The emergence of ARB has been known to be influenced by the selection of antibiotics and the administration method (Santos and Ramos, 2018). Currently approved antibiotics in Japan are limited to FOM, and new treatment strategies and preventive measures are needed to address ARBs. Epidemiological studies are needed to maintain effective pharmacological treatment and provide guidelines for drug administration based on current trends.
We express our gratitude to the private farms for providing the fish samples. This research was partially supported by a grant from the Agriculture, Forestry, Fisheries, and Food Industry Science and Technology Research Promotion Project (grant number 27016 B). We thank Editage (www.editage.jp) for the English language editing.
