Identification, Characterization, and Pathogenic Investigation of Aeromonas veronii, Aeromonas hydrophila, Plesiomonas shigelloides, and Citrobacter freundii from a Case of Natural Mixed Infection in Common Carp (Cyprinus carpio)

1. Introduction

Common carp (Cyprinus carpio) is a world-widely cultured fish, which 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 creates ideal conditions for explosive epidemics of diseases [2]. It has been reported that C. carpio breeding industry was threatened by a variety of bacterial organisms, such as Aeromonas hydrophila, Pseudomonas fluorescens, Pseudomonas alcaligenes, and Shewanella putrefaciens [3,4,5,6]. The current key issues that need to be solved urgently are identifying the most commonly disease-causing bacteria in common carp and finding out effective medicine for treatment.

Aeromonas veronii is Gram-negative bacterium that widely distributed in water environment and can cause ulcerative diseases in many kinds of fish [7,8,9,10,11,12,13,14]. The main clinical symptoms of A. veronii were hemorrhagic septicemia, fin rot, exophthalmia, and abdominal distention [14]. A. hydrophila is another number of Aeromonas species that commonly infect aquatic animals. A. hydrophila caused hemorrhagic septicemia and lead to massive mortalities in fishes in previous reports [15,16,17,18,19]. Plesiomonas shigelloides and Citrobacter freundii were both classified into the Enterobacteriaceae family. P. shigelloides was reported to be pathogenic and the diseased fish showed protruding eyeballs, swollen anus, and ascites in the abdominal cavity, swelling and hemorrhage [20,21,22,23,24]. C. freundii infection was associated with enteritis, necrosis, body reddening, hemorrhage, and septicemia [25,26,27,28,29,30]. The four kinds of bacteria are all opportunistic pathogen and can be commonly found in normal aquaculture water and healthy fish. They are more likely to cause diseases when fish under stresses, such as temperature change, hypoxia, and parasite infection [31,32].

Here, we report a case of bacterial disease naturally occurred in common carp that rearing in the aquaculture base of Henan Normal University in China. The infected fish showed obvious hemorrhage on the body surface, extended abdomen, and flatulence in the intestine. Bacteria were isolated from the ascites and were identified as A. veronii, A. hydrophila, P. shigelloides, and C. freundii by 16S rRNA sequencing and biochemical tests. The drug resistance and pathogenicity of four types of bacteria were also analyzed to provide reference for the prevention and treatment measures of similar cases.

2. Materials and Methods

2.1. Fish

The diseased common carp (18.3±2.6 g) was from the aquaculture base of Henan Normal University in Xinxiang city, Henan province, China. The fish were kept in a concrete pond with water depth of 1.5 m. Moribund fish were transported immediately to the laboratory for diagnosis and pathogen isolation. Healthy common carp (16±2.1 g) with no history of disease were provided by a local fish farm in Xinxiang city. Healthy fish were acclimated in aquaria for two weeks prior to infection assay and fed with commercial fed once daily. The water was replaced daily, and water temperature was maintained at 28 ± 1℃. Experiments involving live fish were conducted in accordance with the US National Research Council’s “Guide for the Care and Use of Laboratory Animals”.

2.2. Isolation, Characterization, and Identification of Bacteria

The ascites of moribund fish were collected and streaked on Luria-Bertani (LB) agar plates with inoculating loop. The plates were incubated at 28℃ for 24 h. The dominant colonies from plates were re-streaked on the LB agar plates three times to obtain pure culture. Total genomic DNA of the isolate was extracted by bacteria DNA extraction kits (Sangon biotech, China). Primers 16S rRNA-F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 16S rRNA-R (5′-GGTTACCTTGTTACGACTT-3′) were used for PCR amplification of 16S rRNA. The PCR amplification conditions of 16S rRNA were initial denaturation at 94℃ for 5 min, followed by 32 cycles of denaturation at 94℃ for 30 s, annealing at 57℃ for 30 s, extension at 72℃ for 1.5 min and a final extension at 72℃ for 10 min. The genes obtained by PCR amplification were cloned to pMD-19 T vector (TakaRa, Japan) and sequenced. The biochemical characteristics were examined using commercial reagent (Hangzhou Microorganism Reagent Co., Ltd., China), and following incubated at 28℃ for 24 h or 48 h. All biochemical tests were repeated for three times, which were performed including oxidase, growth condition at 1% of NaCl, serine dihydrolase, lysine decarboxylase, Voges–Proskauer, indole production, sucrose, lactose, citrate, cellobiose, mannitol, salicin, etc.

2.3. Antibiotic Susceptibility Testing

The antibiotic susceptibility of aeromonad was determined by the Kirby-Bauer disk diffusion method[33]. Bacterial strains were streaked on Muller Hilton agar plates, and the various antibiotic disks (Difco Laboratories, USA) were applied on the streaked cultures. Disks of gentamycin (10 μg), kanamycin (30 μg), neomycin (30 μg), ceftizoxime (30 μg), florfenicol (30 μg), ampicillin (10 μg), trimethoprim-sulfamethoxazole (23.75/1.25 μg), tetracycline (30 μg), streptomycin (10 μg), norfloxacin (10 μg), and enrofloxacin (10 μg) were used. After 18 h of incubation at 28℃, 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

The infection assay was carried out in healthy common carp. The bacterial concentration determined by plating 10 -fold serial dilutions onto LB agar plates. The median lethal dosage (LD₅₀) was calculated based on the total cumulative mortality (%) as described by Reed and Muench (1938) [34]. For LD₅₀ determination, 240 carp individuals were randomly divided into six groups, including five infected groups and one control group. Infected groups (40 fish per group) were intraperitoneally injected with A. veronii, A. hydrophila, P. shigelloides, C. freundii and a mixture of four kinds of bacteria (equal numbers of each kind) at the concentration of 1.0 × 10⁴, 1.0 × 10⁵, 1.0 × 10⁶ and 1.0 × 10⁷ colony-forming unit (CFU)/fish (10 fish injected for each concentration), respectively. The control group was injected with the same dose of sterile physiological saline. The experiment was repeated once. The mortalities and clinical signs of all the groups were recorded every day for 15 d post-infection. Morbid fish were subjected to routine bacteriological examination for re-isolation and re-identification of the organism.

3. Results

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

The clinical signs of diseased fish showed hemorrhage on gill cover and lower jaw, extended abdomen, swollen anus, ascites, and intestinal flatulence (Figure 1). The bacteria isolated from the diseased common carp were Gram negative. Colonies were buff, translucent, circular, convex and intact edge on LB. Ten colonies were selected and 16S rRNA were obtained and sequenced, the results showed that five strains of the isolates were A. veronii, two strains were P. shigelloides, two strains were C. freundii, and one strain was A. hydrophila.

3.2. Biochemical Characteristics

The isolates of A. veronii and A. hydrophila had positive biochemical reactions for indole, Voges–Proskauer, arginine dihydrolase, lysine, glucose, sucrose, and mannose, but negative for inositol. A. hydrophila could utilize arabinose, but could not A. veronii. Both A. veronii and A. hydrophila were able to grow in peptone water under 8% NaCI. C. freundii made utilization of citrate, glucose, raffinose, sorbitol, xylose, and urea, while P. shigelloides was incapable of metabolizing those molecules. P. shigelloides produced indole, lysine decarboxylase, and ornithine decarboxylase. Both C. freundii and P. shigelloides possessed motility (Table 1).

3.3. Experimental Infections

To confirm the pathogenicity of the four types of bacteria, the challenge assay was carried out in healthy common carp. Fish were intraperitoneally injected with different dose of each bacteria or a mixture of four bacteria. The experimentally infected fish showed identical symptoms as observed in the naturally infected common carp, which showed hemorrhage on body surface, abdominal distention, ascites, and flatulence in the intestine (Figure 2). 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 respectively. The LD₅₀ of mixture group was 5.41×10⁴. There were no clinical symptoms or death in the control group (Table 2). Furthermore, four kinds of bacteria were re-isolated from the experimental 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 four bacteria were valued by the size of the inhibition zones around each disc, 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 3).

4. Discussion

A. veronii, A. hydrophila, C. freundii, and P. shigelloides were all opportunistic pathogen widely distributed in water environment and fish population, causing fish diseases under stressful conditions, such as overcrowding, low oxygen concentration, and high water temperature [35]. The disease occurred in this case maybe because of the high water temperature and overfeeding, as the fish stop dying after stop feeding. C. freundii and P. shigelloides had been reported to cause severe hemorrhagic septicemia in internal organs of Chinese sturgeons that cultured in a research Institute [36], and co-infection by Aeromonas bestiarum, Aeromonas sobria, and P. shigelloides was observed in Mexican golden trout, leading to red and inflamed lesions in the abdomen and mouth, and hemorrhage in fins and gills [31]. However, it’s the first time that co-infection of A. veronii, A. hydrophila, C. freundii, and P. shigelloides in common carp was reported. Experimental infections showed that fish injected with four strains of bacteria all developed the similar clinical symptoms, which were hemorrhage, abdominal distention, ascites, and flatulence in the intestine (Figure 2), the same with the sign in naturally infected fish (Figure 1). So it was difficult to identify which kind of bacteria was the main cause of the disease from symptoms. But since half of the ten isolated strains were A. veronii, and the LD₅₀ of mixture group was close to that of A. veronii group, it was most likely that A. veronii were the main pathogen.

Biochemical Characteristics of four bacteria were in accordance with the description of A. veronii, A. hydrophila, C. freundii, and P. shigelloides in Bergey’s Manual [37]. A. veronii, A. hydrophila, and C. freundii all can utilize glucose to produce gas (Table 1), that explained why the intestine of diseased fish was filled with gas (Figure 2). The flatulence was more obvious in the mixed infection group, indicating more gas was produced in mixed infection than that of single bacterial infection. The LD₅₀ determination showed that C. freundii was the most virulent bacteria, and the virulence of A. hydrophila was the weakest. However, the mixed infection didn’t enhance the bacterial virulence, and the virulence of mixed bacteria was equivalent to that of A. veronii (Table 2).

Antimicrobial susceptibility testing indicated that the isolates of P. shigelloides, A. veronii, and A. hydrophila were susceptible to streptomycin, enrofloxacin, florfenicol, gentamicin, kanamycin, neomycin, norfloxacin, co-trimoxazole, cefazoxime, and resistant to ampicillin (Table 3). Simillarly, P. shigelloides isolated from Chinese sturgeon was sensitive to gentamicin and neomycin, and resistant to norfloxacin and ampicillin [36]. In our case, 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 3). In previous study, C. freundii isolated from largemouth bass was sensitive to gentamicin, norfloxacin, tetracycline, and co-trimoxazole, but resistant to kanamycin. C. freundii from angelfish was sensitive to tetracycline, resistant to enrofloxacin, florfenicol, and ampicillin. It seems that C. freundii strains from different fish showed different drug-resistant characterizations. The susceptibility test is essential for the drug selection in treatment.

5. Conclusions

In this study, we isolated A. veronii, A. hydrophila, C. freundii, and P. shigelloides from diseased common carp. The biological characteristics of four types of 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.