Aquaculture is an industry in robust expansion, flourishing to meet the consumer demand for high-quality food protein. In 2020, it produced a global output of 87.5 million tons, with China maintaining its position as the world's largest aquaculture producer. The yield of China constitutes a significant 35% of the total global production (1). Notably, fish farming contributes to 53% total aquaculture output of China. Among these, the spotted sea bass (Lateolabrax maculatus) stands out as an economically vital species, producing nearly 200,000 tons and accounting for ~11% of the marine cultured fish products in 2021 (2). Particularly in the Doumen district of Zhuhai, Guangdong Province, China, which represents the principal brackish water region for sea bass aquaculture in the country, boasts a culture area exceeding 26 million square meters. These areas are predominantly open-air semi-enclosed ecosystem ponds. The annual output from this region alone surpasses 50% total production of China (2), significantly contributing to the nation's animal protein supply and generating substantial economic benefits for local farmers.

As agricultural intensity and stocking densities escalate, aquaculture face increasingly greater risks from infectious diseases (3, 4). Our thorough investigation indicates that the most prevalent diseases affecting spotted sea bass stem from the infection of bacteria (Aeromona, Edwardsiella, Vibrio, and Nocardia), virus (Iridovirus and Neuronecrosis virus), and parasite (Scuticociliatid ciliate, Trichodina, Dactylogyrus, and Ichthyophthirius). With the expansion of aquaculture, host species may encounter novel pathogens, posing unexpected challenges and substantial burdens to the industry (5–7). A prime example is the emergence of Acute Hepatopancreatic Necrosis Syndrome (AHPNS) in shrimp, which was first detected in China in 2009 and subsequently spread to Vietnam (2010), Malaysia (2011), Thailand (2012), and Mexico (2013) (8). This led to a significant decline in global shrimp production from 2011 to 2013 (−5.7%: 2011–2012; and −9.6%, 2012–2013) (https://wenku.baidu.com/view/179efcb8da38376bae1fae77.html?fr=sogou&_wkts_=1681790963165). Until early 2013, the causative agent of AHPNS was identified as Vibrio parahaemolyticus (5). The identification of AHPNS pathogens and in-depth study of their pathogenic mechanisms have facilitated the rapid development of detection methods and effective disease prevention and control strategies. These efforts have contributed to the slow recovery of the global shrimp industry since 2014, as evidenced by the reduction in losses or increase in production in the primarily affected Asian countries (9). Therefore, the rapid and timely identification of new pathogens in cultured animals and the analysis of their pathogenicity and drug resistance are of great significance for the prevention and control of emerging diseases.

Originally isolated from freshwater dolphins in 1976, Streptococcus iniae has emerged as one of the most serious aquatic pathogens over the past decade. It is the primary pathogen affecting a wide range of marine and freshwater fish species, including tilapia (Oreochromis spp.), rainbow trout (Oncorhynchus mykiss), red drum (Sciaenops ocellatus), and rabbit fish (Siganus spp.) (10). This bacterium was responsible for economic losses amounting to US $100 million worldwide in 1997 (10). In this study, we firstly identified S. iniae as the causative agent of spotted sea bass with characteristic symptoms such as congestive liver, splenomegaly, branchial anemia, yellowing fat, and edematous intestine (Figure 1a), resulting in nearly 10% mortality. A highly virulent strain of the bacteria was isolated from the infected spotted sea bass, and its pathological characteristics and drug resistance were analyzed. This information is instrumental for further research into its pathogenesis and for ensuring timely and effective antibiotic treatment.

S. iniae is a causative agent of streptococcicosis in various wild and farmed fish species of economic importance, ranking as one of the principal pathogens responsible for high mortality rates and significant economic losses (16). To date, S. iniae has been reported to infect over 30 different fish species, such as striped bass, tilapia, rainbow trout, red drum, red porgy, rabbitfish, olive flounder, barramundi, and Chinese sturgeon (17). However, there have been no previous reports of infection in the spotted sea bass. In our current study, we discovered for the first time that S. iniae can indeed infect spotted sea bass resulting in approximately 10% mortality. The infection happened in Zhuhai, China, a major cultivation area for this species.

Infections caused by S. iniae can manifest as acute or subacute forms, with the primary symptom being meningoencephalitis in fish. Acutely infected fish often succumb rapidly, with a mortality rate exceeding 50%, yet without displaying characteristic symptoms (18). In contrast, subacute infections result in a lower mortality rate, though daily deaths occur during the infection period. Additionally, fish infected with S. iniae display a variety of pathological symptoms (19). For instance, the infection in tilapia (Oreochromis aureus) typically involves conditions such as meningitis, epi/myocarditis, and septicemia, accompanied by splenomegaly or necrosis in the spleen and kidneys. Moreover, infected tilapia often exhibit loss of appetite, imbalance and erratic swimming behaviors, a tendency to swim near the water's surface, scale detachment, hemorrhages on various parts of the body surface, mild abdominal dropsy, mild exophthalmia (either unilateral or bilateral), along with skin erosions and ulcerations (20). Furthermore, S. iniae is also implicated in causing hemorrhagic septicemia in olive flounders (Paralichthys olivaceus) (21). In this study, we identified a subacute infection of S. iniae in spotted sea bass, presenting with typical symptoms such as hepatic congestion, splenomegaly, branchial pallor, yellowing of fat, and intestinal edema (Figure 1a), resulting in nearly 10% mortality. However, the typical symptom of exophthalmos associated with S. iniae infection was not observed. A potential pathogenic strain was isolated from the spleen, designated as strain 10S01. Further analysis of its pathological characteristics and antibiotic resistance profiles is instrumental for elucidating its pathogenesis and facilitating timely antibiotic treatment.

Bacterial identification utilizing biochemical, moleculartypic, and phenotypic analysis methods has become a standard practice, complemented by morphological analysis. Gram staining is a widely adopted differential technique in bacteriology, offering a clear contrast between bacteria and their surroundings, thereby allowing for the observation of bacterial morphology, arrangement, and other structural features (22). In this study, strain 10S01 was observed to be stained purple and arranged in chains, characteristics indicative of a typical Gram-positive Streptococcus species. The VITEK^® 2 Compact system, grounded in extensive manual verification experience, offers an automated identification solution (23). With barcode-identified operations, it ensures complete traceability and minimizes human error. Additionally, leveraging advanced colorimetric technology, VITEK^® 2 Compact simultaneously analyzes multiple reactions to enhance result accuracy. Based on Gram staining outcomes, the GP card was employed in this investigation. Due to the limitations of the system's database, strain 10S01 was identified as a Streptococcus species. It was found to metabolize eight carbon sources, including dMNE, and dTRE, and produce eight enzymes, such as LeuA. These biological traits mirror those of S. iniae strains previously isolated from cultured flounder in Jeju (24). Additionally, strain 10S01 demonstrates a tolerance to POLYB and OPTO. Notably, strain 10S01 exhibits sensitivity to BAC, contrasting with the flounder-infecting S. iniae strain 1083 which shows resistance to BAC, as reported by Lee et al. (24). Furthermore, strain 10S01 is incapable of utilizing LAC as a carbon source, whereas the majority of flounder-infecting S. iniae strains are capable of metabolizing LAC (24). The 16S rRNA gene serves as a universal marker for bacterial identification across all bacterial species (25). However, due to homology among bacteria, 16S rRNA often fails to provide species-level identification, necessitating the use of species-specific genes like gyrB for further assistance. Phylogenetic trees based on both 16S rRNA and gyrB sequences indicated that strain 10S01 clustered with S. iniae. Additionally, S. iniae has the ability to display both alpha and beta hemolysis. For instance, beta hemolysis was observed in S. iniae isolates from the Japanese flounder (Paralichthys olivaceus) as reported by Matsuoka et al. (26), and in the golden pompano (Trachinotus ovatus) from China, as documented by Cai et al. (27). In contrast, the strain 10S01 exhibits alpha hemolytic activity. Collectively, strain 10S01 has been conclusively identified as S. iniae.

Following bacterial identification, it is imperative to confirm whether the isolated strain is indeed the primary causative agent of the infection. In accordance with Koch's postulates, a deliberate infection test is essential to establish the pathogenicity of the strain in question. In this instance, the pathogenicity of strain 10S01 was assessed, revealing a relatively low lethal dose (LD50) for Spotted sea bass at 74 cfu/g-fish. This value surpasses that reported for the S. iniae strain initially isolated from red hybrid tilapia (1.5 cfu/g-fish) (28), yet it is lower than those recorded for red porgy (Pagrus pagrus, L.) (378 cfu/g-fish) (29) and Chinese sturgeon (Acipenser sinensis) (1.6 x 10⁴ CFU/g-fish) (30). Similar to natural infections, the experimentally infected fish exhibited splenomegaly and hepatic congestion. Histopathological analysis further revealed tissue lesions in the spleen, liver, brain, gills, and stomach. Notably, acute meningitis—characterized by inflammatory cell infiltration in the meningeal blood vessels and the presence of granuloma—was not observed, a feature that has been seen in red porgy (Pagrus pagrus, L.) (29) and tilapia (O. aureus) (31). Colonization assays corroborated that S. iniae can indeed invade the spleen, liver, and brain of spotted sea bass, demonstrating a predilection for splenic colonization. Consequently, it is evident that S. iniae represents the principal pathogen responsible for this outbreak, and strain 10S01 may serve as a model for investigating its pathogenic mechanisms in spotted sea bass, as well as potentially serving as a basis for developing a live attenuated vaccine.

Compared to vaccination, antibiotic therapy offers a more immediate and efficacious treatment option for emerging diseases. However, the overuse of antibiotics can lead to the emergence of resistance, posing threats to both the environment and food safety. Consequently, the use of aquatic antibiotics is subject to stringent global regulations. In China, for instance, aquaculture is permitted to utilize 11 specific antibiotics, including sulfoxamycin, flufenicol, flumequine, enrofloxacin, doxycycline hydrochloride, ciprofloxacin, neomycin sulfate, sulfamethoxine sodium, compound sulfadiazine, compound sulfadipyrimidine, and compound sulfamethoxazole (32). The U.S. Food and Drug Administration (FDA) has approved four drugs—florfenicol, oxytetracycline dihydrate, sulfadimethoxine & ormetoprim, and sulfamerazine—as Type A medicated articles for the production of medicated feed intended for food fish (source: https://www.fda.gov/animal-veterinary/product-safety-information/letter-aquaculture-professionals). In Japan, while over 20 antibacterial agents are approved for aquaculture, their use is highly specific, being designated for particular fish species and diseases (33). For example, sulfadimethoxine is allowed for use exclusively in the treatment of rainbow trout disease (34). Furthermore, analyzing antibiotic resistance is crucial for the success of chemotherapeutic treatments, thereby reducing mortality rates and lessening the impact on production. For instance, Ortega et al. (31) documented the initial infection of S. iniae in tilapia farmed in Querétaro, Mexico. The isolated pathogenic S. iniae strain was found to be sensitive to oxytetracycline, and records from the facility indicated that the infected fish were treated with this antibiotic, leading to a significant decrease in daily mortalities. (31). In our study, we discovered that the isolated strain 10S01 exhibited sensitivity to eleven tested antibiotics, a finding that aligns with the antibiotic sensitivity profiles of S. iniae strains from infected tilapia, red porgy (Pagrus pagrus L), and Chinese Sturgeon (Acipenser sinensis) (29–31, 35). Specifically, all isolates demonstrated a high degree of sensitivity to chloramphenicol, ciprofloxacin, clarithromycin, florfenicol, ofloxacin, rifampicin, and trimethoprim/sulfamethoxazole. In accordance with the antibiotics approved for use within our country, ciprofloxacin and florfenicol are recommended as the primary therapeutic agents for treating S. iniae infections in spotted sea bass.

In our investigation, we documented the initial occurrence of S. iniae infection in spotted sea bass, which resulted in lesions to the liver, spleen, gills, and intestines. Notably, this strain did not elicit the characteristic symptoms of exophthalmos and meningoencephalitis. The isolation of the highly virulent strain 10S01 paves the way for further research into its pathogenesis and the development of vaccines. Antibiotic susceptibility testing revealed that chloramphenicol, ciprofloxacin, clarithromycin, florfenicol, ofloxacin, rifampicin, and trimethoprim/sulfamethoxazole are effective against S. iniae infections in spotted sea bass. Moving forward, we advocate a three-pronged approach: (1) employing an interdisciplinary strategy to unravel the Pathogenesis of S. iniae strain 10S01, which will aid in the creation of attenuated live vaccines and genetically engineered vaccines; (2) isolating more pathogenic strains and selecting representative strains based on molecular typing to develop inactivated vaccines; (3) screening for probiotics, plants, and bacteriophages that can inhibit S. iniae infection to develop biological control strategies.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

The animal study was approved by the experimental procedures in the current study were carried out in strict compliance with the pertinent guidelines and regulations set forth by the Committee on Laboratory Animal Welfare and Ethics of South China Sea Fisheries Research Institute (nhdf2023-08). The study was conducted in accordance with the local legislation and institutional requirements.

YD: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Writing—original draft, Writing—review & editing. ZL: Data curation, Formal Analysis, Investigation, Software, Writing—review & editing. LX: Formal Analysis, Investigation, Methodology, Writing—review & editing. JJ: Investigation, Methodology, Writing—review & editing. CC: Investigation, Software, Writing—original draft. HM: Formal analysis, Investigation, Writing—original draft. JF: Conceptualization, Funding acquisition, Project administration, Supervision, Writing—review & editing.