Aquaculture provides humans with over 1/3 of the high-quality protein consumed (Li et al., 2022). The bullfrog is an important aquatic economy native to eastern North America and has been widely introduced worldwide (Akmentins and Cardozo, 2010). With the progress of socio-economic development and increases in people’s standard of living, the demands for food safety and quality keep increasing. The bullfrog is increasingly consumer friendly as a good quality meat, and recently, demand for bullfrog has been increasing (Schloegel et al., 2010; Zhu et al., 2021). The aquaculture of American bullfrogs for the meat industry has expanded worldwide. China, Taiwan, Brazil, and Ecuador are well-known for their significant production, while the United States, France, Canada, Belgium, Italy, and Spain are well-known for their significant consumption (FAO, 2023). The bullfrog is an economical frog, and the development of artificial aquaculture is rapidly developing to meet the rise in market demand (Zhang et al., 2015). The bullfrog was first introduced to China as a food source from Cuba and Japan (Wu et al., 2004). Since then, the cultivation of the bullfrog has made remarkable developments in China and has been introduced to many provinces (Zhang et al., 2015). Recently, there have been frequent occurrences of bullfrog diseases, especially bacterial pandemics, such as epidemic meningitis-like disease (EMD) and red leg syndrome (RLS), which severely damage the bullfrog aquaculture industry (Pasteris et al., 2006; Trimpert et al., 2021). EMD has been frequently occurring in recent years (Zajmi et al., 2022). Disease in the bullfrog is observed as signs of torticollis, head slanting to one side, swimming in circles, and loss of appetite (Hu et al., 2017), and 60-90% of diseased animals die within several days to weeks after the onset (Hu et al., 2020). It is found that the epidemic of bacterial diseases is the main cause of major loss to economic bullfrog farming (Mauel et al., 2002; Li et al., 2018), so in order for frog aquaculture to healthily and rapidly develop, we should accelerate the study of bacterial diseases in aquaculture and find reasonable measures (Yu et al., 2021).

Elizabethkingia spp. is a pathogen that threatens the lives of humans and animals (Zajmi et al., 2022). Elizabethkingia spp. is a potential zoonotic pathogen (Vancanneyt et al., 1994); it is widely distributed in the natural environment and also exists in the hospital environment (Moore et al., 2016; Chew et al., 2018; Hem et al., 2022). It is a potentially infectious pathogen in the hospital, which can cause newborn meningitis, adult sepsis, and skin and soft tissue infection, and mortality is rather high in infected patients (Furyk et al., 2011). Elizabethkingia spp. infection has been reported worldwide, especially in patients whose immune function is compromised, causing a fatal human infection (Dziuban et al., 2018).

Besides human infection, Elizabethkingia spp. also infects birds (Vancanneyt et al., 1994), dogs (Bordelo et al., 2016), aquaculture animals such as tilapia (Jacobs and Chenia, 2011), catfish (Laith et al., 2017), and many amphibians, including tiger frogs (Xie et al., 2009), spiny frogs (Lei et al., 2019), and northern leopard frogs (Trimpert et al., 2021). According to earlier research, the Enterobacteriaceae (including Proteus vulgaris and Proteus mirabilis), Pseudomonas aeruginosa, Aeromonas, and a number of Staphylococcus epidermidis strains were considered to be the pathogens causing EMD (Cunningham et al., 1996). Another study identified E. miricola as a pathogen of EMD in black-spotted frogs (Hu et al., 2017). A recent study has shown that E. miricola was the pathogen isolated from diseased American bullfrogs (Lithobates catesbeianus) in farms in the Guangdong province (Liu et al., 2022). There are many reports on EMD, which is the most serious disease for many kinds of cultured frogs in recent years (Mauel et al., 2002; Xie et al., 2009; Lei et al., 2019), but there is no consensus on the pathogen. A widespread outbreak of disease occurred in bullfrogs on different farms in Guangxi, resulting in high mortality and severe economic losses. Nevertheless, the underlying cause of the explosion of bullfrog disease is not clear. In the present study, we investigated the pathogen of the bullfrog, characterized the pathogen, and isolated the main bacterial pathogen in meningitis. The results of the study provide a theoretical reference for further studies on bullfrog dermatology and for helping to prevent and treat EMD during bullfrog farming.

Many studies have shown that E. miricola can cause clinical meningitis in addition to pneumonia and meningitis in newborns, children, and the elderly (Dziuban et al., 2018). The immunocompromised are particularly at high risk. In addition to humans and poultry, E. miricola bacteria also infect many aquatic animals (Jacobs and Chenia, 2011; Laith et al., 2017). In recent years, E. miricola bacteria have become an emerging pathogen of frog farming, especially bullfrog farming (Liu et al., 2022). The infected bullfrog shows disease symptoms such as severe torticollis; curvature of the spine; and enlargement of the liver, spleen, and kidneys, which are also reported in several other frog species (Xie et al., 2009; Lei et al., 2019). This phenomenon also occurs in black-spotted frogs (Hu et al., 2017). In the current study, the NFEM01 strain was isolated from the brain of bullfrogs with EMD in Nanning, Guangxi. The surface of a typical NFEM01 colony was smooth and wet and the edge was neat and white, which was consistent with E. miricola isolated from the spiny frog (Lei et al., 2019). NFEM01 has beta-hemolytic activity, which indicates that NFEM01 has strong pathogenic potential.

In the API^® 20E test, the NFEM01 strain showed similar characteristics to those reported previously (Huang et al., 2019), but differences between isolates of Elizabethkingia spp. in trypsin response were observed. Some of the characteristics of Elizabethkingia spp. have been summarized. They can all produce catalase, phosphatases, galactosidases, and indole, whereas they cannot hydrolyze starch, use malonate, and ferment galactose, sorbitol, inositol, and salicylic acid. However, some features are variable in the same species and the phenotypic similarities between known species challenge the correct identification of clinical isolates (Nicholson et al., 2018). The genus Elizabethkingia is genetically heterogeneous, and the identification by phenotypic similarity is challenging for the accurate identification of clinical isolates (Bruun and Ursing, 1987).

In previous works, the 16S rRNA gene was used for the clinical reports of most cases (Frank et al., 2013; Chew et al., 2018). However, there are five copies of the 16S rRNA gene in Elizabethkingia spp., and there are some differences between them (Nicholson et al., 2018). Therefore, it is difficult to distinguish Elizabethkingia spp. from the 16S rRNA gene sequence alone, so other methods to support identification are needed (Chew et al., 2018; Lei et al., 2019). The rpoB gene sequencing is superior to other gene targets because it has a higher resolution than 16S rRNA gene sequencing, and is used for delineating new bacterial species (Adékambi et al., 2009; Turton et al., 2010). Nicholson et al. (2018) first proposed using the rpoB gene to identify Elizabethkingia species. Subsequently, Kenna et al. studied the distribution of Elizabethkingia species using rpoB gene sequencing. In their study, 43 isolates from 38 patients formed a cluster with E. miricola and E. bruuniana sp. nov. (Kenna et al., 2018). In a separate study based on 16S rRNA and rpoB gene sequencing, the authors identified six patients infected with E. bruuniana between 2005 and 2017 (Lin et al., 2019). On this basis, we further verified and confirmed the mutT gene of E. miricola by amplification and sequencing. Zhang et al. (2015) established a real-time fluorescent quantitative PCR system based on mutT gene amplification, which could specifically identify E. miricola and had no nonspecific amplification with many bacteria. In our study, a combination of 16S rRNA, rpoB, and mutT genes was used to identify E. miricola isolated from the bullfrog, eliminating a possible error caused by single 16S rRNA-based identification.

Previous antibiotic susceptibility testing revealed E. miricola was resistant to erythromycin and oxytetracycline (Colapietro et al., 2016; Han et al., 2017; Gao et al., 2021). Elizabethkingia spp. is highly resistant to various antibiotics, leading to fewer choices of therapeutic drugs. Because of this, clinically, patients with Elizabethkingia spp. infections have high mortality (Opota et al., 2017). In this study, the NFEM01 was resistant to the majority of antibiotics, including gentamicin, florfenicol, neomycin, penicillin, amoxicillin, doxycycline, and sulfamonomethoxine. Therefore, the choice of antibiotics to treat EMD is limited. The NFEM01 isolate showed high resistance to multiple antibiotics similar to previously reported isolates from the Chinese spiny frog (Lei et al., 2019). The main reason for the multi-drug resistance of Elizabethkingia spp. is that there are many natural resistance genes on its chromosome, which can produce antibiotic-inactivating enzymes and lead to corresponding antibiotic resistance, for example, Metallo-β-lactamases (MBLs) (Opota et al., 2017). The use of Chinese herbal medicines may be a potentially effective approach (Li et al., 2021).

In our study, the mortality of the infected bullfrog was 40% and 100%, respectively when 10⁵ and 10⁷ CFU·mL^-1 NFEM01 were injected. These results are similar to those of another study, which found that the mortality rate of infected black-spotted frogs was 50% and 70% when injected with E. miricola FL160902 at 10⁷ and 10⁸ CFU·mL^-1, respectively (Hu et al., 2017). Another study indicated that the mortality rates of Chinese spiny frogs after infection at 10⁶, 10⁷, and 10⁸ CFU·mL^-1 were 50%, 80%, and 100%, respectively (Lei et al., 2019). These traits suggest that the strains isolated in these studies all show strong pathogenicity to frogs. However, in previous studies, mortality after infection at 10⁸ CFU·mL^-1 was 80% and 33.3%, with relatively low lethality in the black-spotted frog (Huang et al., 2019). These studies suggest that different bacterial strains from amphibians may have different pathogenicity or that different amphibian species have differing susceptibility.

In summary, E. miricola was confirmed as the pathogenic bacterium isolated from the brain of bullfrogs with meningitis-like disease. E. miricola was first isolated from the bullfrog in Guangxi and is highly pathogenic to bullfrogs. This provides a reference for further study of the pathogenesis mechanism, propagation, and prevention of the disease.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.

The animal study was reviewed and approved by the ethics committee of Guangxi Academy of Sciences.

PL, DW, and ZX conceived and designed the research. YC, SX, WL, QY, SYH, SSH, and JS performed the experiments and analyzed the data. DW wrote the manuscript. All authors contributed to the article and approved the submitted version.