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Communication

Mixed Infection in Common Carp (Cyprinus carpio) Caused by Aeromonas veronii, Aeromonas hydrophila, Plesiomonas shigelloides, and Citrobacter freundii

Engineering Lab of Henan Province for Aquatic Animal Disease Control, College of Fisheries, Henan Normal University, Xinxiang 453007, China
*
Author to whom correspondence should be addressed.
Animals 2025, 15(6), 805; https://doi.org/10.3390/ani15060805
Submission received: 29 January 2025 / Revised: 10 March 2025 / Accepted: 10 March 2025 / Published: 12 March 2025
(This article belongs to the Section Aquatic Animals)

Simple Summary

Aeromonas veronii, Aeromonas hydrophila, Plesiomonas shigelloides, and Citrobacter freundii are opportunistic pathogens and cause fish diseases in aquaculture. The four kinds of bacteria were isolated from a group of diseased common carp. Artificial infection indicated that the four isolates were all highly pathogenic to fish. Similar cases of mixed bacterial infection in farmed common carp have rarely been reported previously, and our study on bacterial drug resistance provides a reference for treatment of bacterial fish diseases caused by these pathogens.

Abstract

Aeromonas veronii, Aeromonas hydrophila, Plesiomonas shigelloides, and Citrobacter freundii are opportunistic pathogens that are widely distributed in aquatic environments and fish populations, causing fish diseases under stressful conditions. In this study, bacteria were isolated from a group of diseased common carp, Cyprinus carpio, with symptoms of hemorrhaging along the body surface, abdominal distention, and flatulence in the intestine. According to the results of biochemical characteristics and 16S rRNA sequencing analyses, the isolates were identified as A. veronii, A. hydrophila, P. shigelloides, and C. freundii. The virulence genes from these four representative bacterial strains were amplified. The experimentally co-infected fish showed similar symptoms to those observed in the naturally infected common carp. The LD50 of C. freundii, P. shigelloides, A. veronii, A. hydrophila, and the mixture group were 1.95 × 104, 4.74 × 104, 5.12 × 104, 1.53 × 105, and 5.41 × 104 CFU, respectively. The antibiotic resistance results showed that P. shigelloides, A. veronii, and A. hydrophila were most sensitive to ceftizoxime, florfenicol, enrofloxacin, and norfloxacin. C. freundii was most sensitive to ceftizoxime and gentamicin.

1. Introduction

The common carp (Cyprinus carpio) is a fish that is cultured worldwide and is also one of the most important economic fish species in China [1]. The total production of farmed C. carpio was 2.87 million tons in 2023, according to the China Fishery Statistical Yearbook. However, improper management and high-density culture create the ideal conditions for explosive epidemics of various diseases. It has been reported that the C. carpio breeding industry is threatened by a variety of bacterial organisms [2,3,4,5]. Aeromonas veronii is a Gram-negative bacterium that is widely distributed in aquatic environments and can cause ulcerative diseases in many fishes [6,7,8,9]. The main clinical symptoms of A. veronii infection are hemorrhagic septicemia, fin rot, exophthalmia, and abdominal distention [9]. A. hydrophila is another Aeromonas species that is highly similar to A. veronii in pathogenicity [10]. Plesiomonas shigelloides and Citrobacter freundii both belong to the Enterobacteriaceae family. P. shigelloides has been reported to cause protruding eyeballs, swollen anus, ascites in the abdominal cavity, swelling, and hemorrhaging in diseased fish [11,12]. C. freundii infection is associated with enteritis, necrosis, body reddening, hemorrhaging, and septicemia [13,14,15,16]. The four species of bacteria are all opportunistic pathogens and can be commonly found in normal aquaculture water and healthy fish. They are more likely to cause diseases when fish are exposed to various stressors, such as temperature change, hypoxia, and parasite infection [17,18].
Common carp disease has historically been attributed to many bacteria. However, the mixed infection of A. veronii, A. hydrophila, P. shigelloides, and C. freundii in common carp has not been reported before. Research into the pathogenicity and drug resistance of these four bacteria would help to provide a reference for the prevention and treatment of similar cases.

2. Materials and Methods

2.1. Fish

The diseased common carp (n = 46, 18.3 ± 2.6 g) were obtained from the aquaculture base of Henan Normal University in Xinxiang city, Henan province, China. The fish were kept in a concrete pond (30 m2) with a water depth of 1.5 m and at a water temperature of approximately 32 °C. Moribund fish were transported immediately to the laboratory for diagnosis and pathogen isolation. For the experimental infection, healthy common carp (16 ± 2.1 g) with no history of disease were obtained from a local fish farm in Xinxiang city. The healthy fish were acclimated in aquaria (200 L each) for two weeks prior to the infection assay and fed with commercial feed at 1% of their body weight once daily. Half of the total volume of water was replaced daily, and the water temperature was maintained at 28 ± 1 °C. The experiments involving live fish were conducted in accordance with the American National Research Council’s “Guide for the Care and Use of Laboratory Animals”.

2.2. Isolation and Identification of Bacteria

Ten bacterial strains were isolated from the ascites of moribund fish and identified through the 16S rRNA genes, following the method used in previous reports [7]. The biochemical characteristics were examined using a commercial reagent (Hangzhou Microorganism Reagent Co., Ltd., Hangzhou, China). The 10 strains identified were intraperitoneally injected into healthy fish (10 fish each strain) at 1.0 × 105 colony-forming units (CFU) per fish. The highest mortality strain in each bacterial species, namely A. hydrophila XX236, A. veronii XX237, C. freundii XX238, and P. shigelloides XX239, was chosen as a representative strain of each species and used for the following studies (Table S1). The primers used to detect the virulence genes were designed according to the previous reports [7,12,14,19]. The amplified virulence genes were verified by sequencing.

2.3. Antibiotic Susceptibility Testing

The antibiotic susceptibility of aeromonad was determined by the Kirby–Bauer disk diffusion method [20]. The bacterial strains were streaked on Mueller–Hinton agar plates, and the various antibiotic disks (Difco Laboratories, Detroit, MI, USA) were applied on the streaked cultures. After 18 h of incubation at 28 °C, the sizes of the zone of bacterial growth inhibition were measured. The isolates were classified as sensitive (S), moderately sensitive (M), or resistant (R) according to the National Committee for Clinical Laboratory Standards.

2.4. Experimental Infections

For median lethal dosage (LD50) determination, 210 carp individuals were randomly divided into six groups, including five infected groups (40 fish per group) and one control group (10 fish). The infected groups were intraperitoneally injected with A. veronii, A. hydrophila, P. shigelloides, C. freundii, and a mixture (equal colony numbers of each bacterial species) at the concentrations of 1.0 × 104, 1.0 × 105, 1.0 × 106, and 1.0 × 107 CFU/fish (10 fish each), respectively. All the bacterial strains were grown in LB medium for 24 h at 28 °C before use. The control group was injected with the same dose of sterile physiological saline. The mortality and clinical signs of all groups were recorded every day for 15 d post-infection. The LD50 was calculated based on the total cumulative mortality (%) as described by Reed and Muench (1938) [7]. Moribund fish underwent routine bacteriological examination to re-isolate and re-identify the present organisms.

3. Results

3.1. Clinical Signs and Isolation of Bacteria from Diseased Common Carp

All of the diseased fish showed hemorrhaging along the gill cover and lower jaw, extended abdomen, swollen anus (Figure 1A), ascites, and intestinal flatulence (Figure 1B,C). All the colonies were smooth-edged, circular, and translucent buff or cream in color (Figure 1D). Ten colonies were selected, and the 16S rRNA were obtained and sequenced. The biochemical characteristics of the ten bacterial strains were also tested, and the results indicated the presence of the representative strains (Table S1). A. veronii (five strains), P. shigelloides (two strains), C. freundii (two strains), and A. hydrophila (one strain) were detected.

3.2. Phylogenetic Tree and Virulence Related Genes

Based on the 16s rRNA sequences of A. hydrophila XX236, A. veronii XX237, C. freundii XX238, and P. shigelloides XX239, a phylogenetic tree was constructed to elucidate the genetic relationships among the four isolates and the other representative species. The isolates of XX236, XX237, XX238, XX239 were grouped with A. hydrophila, A. veronii, C. freundii, and P. shigelloides, respectively (Figure S1).
For the virulence gene detection, act, aerA, ahyB, fla, and hlyA were detected in A. hydrophila. Act, aerA, ahyB, alt, fla, gcaT, and hlyA were present in A. veronii. P. shigelloides had actP, ahpA, astA, astB, astD, astE, flaA, and phlA genes, while C. freundii was positive for the cfa, ompX, ureD, ureE, ureF, and viaB genes (Figure 2).

3.3. Experimental Infections

To confirm the pathogenicity of the representative strains, a challenge assay was carried out in the healthy common carp. The fish were intraperitoneally injected with different doses of each bacterial strain or a mixture. The fish injected with Aeromonas (Figure 3(A1,B1)) had protruding eyes, ulceration on the abdomen, and redness of the anus. The P. shigelloides-infected fish showed an extended and ulcerated abdomen, as well as a fin hemorrhage (Figure 3(C1)). Fin hemorrhage was also observed in the C. freundii-infected fish (Figure 3(D1)). The mixed infection group had all the above symptoms (Figure 3(E1)). All the artificially infected fish showed ascites and flatulence in the intestine (Figure 3(A2–E2)). The LD50 of C. freundii, P. shigelloides, A. veronii, and A. hydrophila were 1.95 × 104, 4.74 × 104, 5.12 × 104, and 1.53 × 105 CFU, respectively. The LD50 of the mixture group was 5.41 × 104 CFU (Table 1). Furthermore, the same bacterial species were re-isolated from the experimentally infected fish, as confirmed by colonial morphology observation, physiological and biochemical characteristics analyses, and 16S rRNA sequencing analyses.

3.4. Determination of Antimicrobial Resistance

The antibiotic resistance patterns of the four bacteria, measured by the size of the inhibition zones around each disk, showed that P. shigelloides, A. veronii, and A. hydrophila were sensitive to streptomycin, enrofloxacin, florfenicol, gentamicin, kanamycin, neomycin, norfloxacin, co-trimoxazole, ceftizoxime, and resistant to ampicillin. C. freundii was sensitive to streptomycin, gentamicin, and ceftizoxime, moderately susceptible to kanamycin, neomycin, norfloxacin, and ampicillin, and resistant to enrofloxacin, florfenicol, tetracycline, and co-trimoxazole (Table 2).

4. Discussion

The co-infection cases of two (C. freundii and P. shigelloides) or three (A. bestiarum, A. sobria, and P. shigelloides) bacteria in fish have been reported [17,21]; however, we report, for the first time, on a mixed infection with four bacterial species (A. veronii, A. hydrophila, C. freundii, and P. shigelloides) in common carp. The phylogenetic tree analysis further confirmed the identification of those strains besides the biological characterization. The experimental infections showed that fish injected with the different bacterial strains developed varying clinical symptoms (Figure 3(A1–D2)), but the artificially co-infected fish showed similar symptoms with the naturally infected fish, which were hemorrhaging, abdominal distention, ascites, and flatulence (Figure 1 and Figure 3(E1,E2)), indicating that the clinical signs in observed in the naturally infected fish may be a result of co-infection. However, it was difficult to identify which bacteria was the main cause of the disease by the symptoms alone. But since half of the ten isolated strains were A. veronii, and the LD50 of mixture group (5.41 × 104 CFU) was close to that of the A. veronii group (5.12 × 104 CFU), it is most likely that A. veronii was the main pathogen. The clinical symptoms of ulceration, redness of the anus, intestinal hyperemia, and flatulence commonly caused by A. veronii were also the main symptoms in the co-infected fish.
Virulence genes are important contributors to the pathogenicity of bacteria [7,12,22]. The high pathogenicity of A. hydrophila, A. veronii, C. freundii, and P. shigelloides may be associated with the virulence-related gene families that we detected (act, aer, ahp, ahy, alt, ast, fla, gca, hly, phl, cfa, omp, and ure). The LD50 determination showed that C. freundii was the most virulent bacteria, and A. hydrophila was the weakest. The virulence of the mixed bacteria group was close to the average amount (Table 1).
The results of the antimicrobial susceptibility testing indicated that P. shigelloides, A. veronii, and A. hydrophila were not significantly different from those previously reported [22]. However, the results for C. freundii differed significantly from the other fish species [14,18]. The susceptibility test is essential for the selection of drugs for treatment.

5. Conclusions

In this study, we isolated A. veronii, A. hydrophila, C. freundii, and P. shigelloides from diseased common carp. The virulence genes in four bacteria were determined. Artificial infection in fish showed four strains of bacteria were all highly pathogenic and could cause the disease with hemorrhage and abdominal distention syndrome, indicating the naturally infected fish maybe died of mixed infection. The drug sensitivity of the bacteria was also tested. The findings in this study will provide reference for the diagnosis, treatment, and prevention of the disease caused by mixed bacterial infection.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ani15060805/s1, Table S1. Mortality rate of isolated strains. Table S2. Biochemical characteristics of isolated strains. Figure S1. Neighbor-joining tree based on 16S rRNA sequence Numbers at nodes indicate bootstrap percentages derived from 1000 replications made by MEGA7 [23], numbers at the end of bacteria species names denote the Genbank accession number. Red triangles indicate the strains of present study. Reference [23] are cited in the supplementary materials.

Author Contributions

C.P.: Formal Analysis, Funding Acquisition, Methodology, Software, Supervision, Validation, Writing; J.Z. (Jinghang Zhang): Conceptualization, Data Curation, Investigation, Project Administration, Software, Visualization; D.Q.: Conceptualization, Data Curation, Investigation, Software, Visualization; H.W.: Data Curation, Investigation; X.Z.: Formal Analysis, Validation; X.J.: Formal Analysis, Validation; L.Z.: Formal Analysis, Validation; J.Z. (Jie Zhang): Formal Analysis, Validation; L.L.: Formal Analysis, Validation; X.K.: Conceptualization, Resources, Supervision. All authors have read and agreed to the published version of the manuscript.

Funding

This work was sponsored by the Key scientific research projects of colleges and universities in Henan Province (22A240002).

Institutional Review Board Statement

This study was approved by the Laboratory Animal Ethics Committee of the College of Fisheries of Henan Normal University (Approved ID: 2023081101, Data: 11 August 2023).

Informed Consent Statement

Not applicable.

Data Availability Statement

Data will be made available by the authors upon request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Symptoms of naturally infected fish (left) and colonies on LB plate (right). (A) The external symptoms of diseased fish. (B,C) Symptoms of internal organs. (D) Colonies on LB plate. Black arrow shows hemorrhaging along the body surface, and red arrow shows the flatulence in intestine.
Figure 1. Symptoms of naturally infected fish (left) and colonies on LB plate (right). (A) The external symptoms of diseased fish. (B,C) Symptoms of internal organs. (D) Colonies on LB plate. Black arrow shows hemorrhaging along the body surface, and red arrow shows the flatulence in intestine.
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Figure 2. Detection of virulence genes in A. hydrophila (A), A. veronii (B), P. shigelloides (C), and C. freundii (D). M: Maker.
Figure 2. Detection of virulence genes in A. hydrophila (A), A. veronii (B), P. shigelloides (C), and C. freundii (D). M: Maker.
Animals 15 00805 g002
Figure 3. Symptoms on the epithelial surface and internal organs of experimentally infected fish. (A1,A2), A. veronii infection, (B1,B2), A. hydrophila infection, (C1,C2), P. shigelloides infection, (D1,D2), C. freundii infection, (E1,E2), mixed infection.
Figure 3. Symptoms on the epithelial surface and internal organs of experimentally infected fish. (A1,A2), A. veronii infection, (B1,B2), A. hydrophila infection, (C1,C2), P. shigelloides infection, (D1,D2), C. freundii infection, (E1,E2), mixed infection.
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Table 1. Cumulative mortality of experimentally infected fish by bacterial isolates.
Table 1. Cumulative mortality of experimentally infected fish by bacterial isolates.
GroupConcentration (CFU)FishDead Fish Number on Day After ChallengeAccumulative MortalityLD50 Value (CFU)
123456789101112131415
Control0100000000000000000%
P. shigelloides1.0 × 10710000041131000000100%4.74 × 104
1.0 × 1061000002103020010090%
1.0 × 1051000000003120100070%
1.0 × 1041000000001000100020%
C. freundii1.0 × 10710000042310000000100%1.95 × 104
1.0 × 10610000011231110000100%
1.0 × 1051000000101511000090%
1.0 × 1041000000000300000030%
A. veronii1.0 × 1071024300000000000090%5.12 × 104
1.0 × 1061010004201000100090%
1.0 × 1051010000102100200070%
1.0 × 1041000000100100000020%
A. hydrophila1.0 × 10710100031200102000100%1.53 × 105
1.0 × 1061000001011401000080%
1.0 × 1051000000000101200040%
1.0 × 1041000000000000100010%
Mixed infection1.0 × 10710000031122100000100%5.41 × 104
1.0 × 1061000001101212010090%
1.0 × 1051000001001001101050%
1.0 × 1041000000000012000030%
Table 2. Susceptibility of bacterial isolates to antibiotics.
Table 2. Susceptibility of bacterial isolates to antibiotics.
AntibioticDrug Concentration (μg/disc)Inhibition Zone Diameter (mm)
A. veroniiA. hydrophilaC. freundiiP. shigelloides
Streptomycin 1019 S15 S15 S15 S
Enrofloxacin 1040 S23 S10 R30 S
Florfenicol 3030 S32 S0 R28 S
Gentamicin 1021 S19 S20 S19 S
Kanamycin 3020 S20 S15 I18 S
Neomycin 3017 S18 S17 I18 S
Tetracycline 3011 R25 S0 R21 S
Norfloxacin 1034 S23 S14 I27 S
Co-trimoxazole 23.75/1.2519 S20 S0 R13 I
Ceftizoxime 3044 S40 S32 S37 S
Ampicillin 1010 R10 R14 I0 R
Note: “S” is sensitive, “I” is intermediate, and “R” is resistant.
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MDPI and ACS Style

Zhang, J.; Qiao, D.; Wang, H.; Zhao, X.; Jiang, X.; Zhu, L.; Zhang, J.; Li, L.; Kong, X.; Pei, C. Mixed Infection in Common Carp (Cyprinus carpio) Caused by Aeromonas veronii, Aeromonas hydrophila, Plesiomonas shigelloides, and Citrobacter freundii. Animals 2025, 15, 805. https://doi.org/10.3390/ani15060805

AMA Style

Zhang J, Qiao D, Wang H, Zhao X, Jiang X, Zhu L, Zhang J, Li L, Kong X, Pei C. Mixed Infection in Common Carp (Cyprinus carpio) Caused by Aeromonas veronii, Aeromonas hydrophila, Plesiomonas shigelloides, and Citrobacter freundii. Animals. 2025; 15(6):805. https://doi.org/10.3390/ani15060805

Chicago/Turabian Style

Zhang, Jinghang, Dan Qiao, Haoyu Wang, Xianliang Zhao, Xinyu Jiang, Lei Zhu, Jie Zhang, Li Li, Xianghui Kong, and Chao Pei. 2025. "Mixed Infection in Common Carp (Cyprinus carpio) Caused by Aeromonas veronii, Aeromonas hydrophila, Plesiomonas shigelloides, and Citrobacter freundii" Animals 15, no. 6: 805. https://doi.org/10.3390/ani15060805

APA Style

Zhang, J., Qiao, D., Wang, H., Zhao, X., Jiang, X., Zhu, L., Zhang, J., Li, L., Kong, X., & Pei, C. (2025). Mixed Infection in Common Carp (Cyprinus carpio) Caused by Aeromonas veronii, Aeromonas hydrophila, Plesiomonas shigelloides, and Citrobacter freundii. Animals, 15(6), 805. https://doi.org/10.3390/ani15060805

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