Elucidation on potential utilization of Forsythia suspensa fruits in freshwater bivalve (Hyriopsis cumingii) culture against Aeromonas veronii infection

https://doi.org/10.1016/j.indcrop.2023.116963Get rights and content

Highlights

  • Virulence of A. veronii may be linked to the ability of peptidoglycan synthesis.

  • Cephalexin was unsuitable in combination with P. mume extract against A. veronii.

  • F. suspensa extract enhanced antioxidant and immune status of the infected mussels.

  • The therapeutic rate of 0.6% F. suspensa extract was favored over rate of 1.2%.

Abstract

Plant extracts have been implicated in biological studies for their antibacterial, antioxidant, and anti-inflammatory properties. The curative efficacy of fruit extracts (Forsythia suspensa and Prunus mume) and three common antibiotics against Aeromonas veronii (alone or in extract-antibiotic combination) were clarified in vitro by microplate assays and checkerboard methods. Then the extracts with high antibacterial activity (F. suspensa) in vitro were exposed to Hyriopsis cumingii with A. veronii infection for in vivo assessments of their protective efficacy. Subsequently, oxidative stress markers, immune enzymes, and mRNA expression of immune genes in H. cumingii were investigated. Finally, principal component analysis of the physiological biomarkers and genetic markers that respond to A. veronii infection and extract exposures was conducted. The FICI values in vitro implied the following: P. mume combined with cephalexin was antagonistic, as opposed to other combinations whose effects were commutative or indifferent. In vivo results demonstrated that A. veronii caused lipid oxidative damage led to significant decreases in antioxidant defenses and induced immune responses in H. cumingii. F. suspensa significantly enhanced alanine transaminase activities and reduced malondialdehyde levels caused by A. veronii infection in gills and hepatopancreas. It also promoted antioxidant defenses, including superoxide dismutase activities and peroxidase contents, inhibiting increased alkaline phosphatase and acid phosphatase activities. Notably, 0.6% of F. suspensa extract (dry fruits: wet H. cumingii, w/w) reversed the upregulation of glutathione S-transferase and small heat shock protein mRNA levels in hepatopancreas and remarkably improved the survival rate of H. cumingii after the infection. Building on the research, F. suspensa may represent a promising substitute for offering effective protection to H. cumingii against bacterial diseases and contribute to the transformation and upgrading of the freshwater pearl culture industry.

Introduction

In recent years, plant extracts, essential oils, and other derivatives of medicinal plants have been extensively developed due to their antimicrobial and immunostimulatory properties in animals (Bandeira et al., 2017, Fratini et al., 2017, Pires et al., 2021). The various phenolic compounds presented in plant extracts are inextricably linked to antibacterial activity (Dong et al., 2021, Liang et al., 2022). For example, pomegranate extract had antibacterial activities against pathogenic bacteria and protected Yesso scallops infected by Vibrio anguillarum (Shan et al., 2021). A similar finding was consistent with Nhu et al. (2019), who confirmed that the application of plant extracts enhanced the immune response of striped catfish and reduced mortality after bacterial challenge. Moreover, these medicinal plants have drawn worldwide academic interest resulting from their low cost, convenience of preparation, and slight side effects for animals (Maphosa and Masika, 2010, Zheng et al., 2023).

Forsythia suspensa is a Chinese historical herbal plant cultivated widely in China, Korea, Japan, and numerous European countries. Its fruits have a multitude of anti-inflammatory, antioxidant, anti-bacterial, and anti-allergic traits (Chao et al., 2022, Li et al., 2022, Long et al., 2022). The study demonstrated that F. suspensa aqueous extract displayed powerful anti-tumor activity and prolonged survival time in mice in vitro and in vivo (Bao et al., 2016). Aqueous extract of F. suspensa possesses significant protective effects against neurotoxicity induced by anticancer drugs (Yi et al., 2019). Generally speaking, the major components of F. suspensa are represented by phenylethanoid glycosides, the most abundant of which is forsythoside A (Han et al., 2017).

Additionally, Prunus mume is an economic flora with particular availability in East Asia and is widely planted for its utility, nutritional, and therapeutic properties (Zhu et al., 2022). A recent study has pointed out that P. mume has the potential to prevent mucositis and diarrhea (Bailly, 2020) and the intake of its fruit extract with an amine-rich diet has been found to inhibit the production of endogenous nitrosamines, thereby lowering the risk of cancer in humans (Choi et al., 2002). In addition to supplying traditional medicine and supplements for humans, the fruits of P. mume are gradually being researched for their efficacy in improving the growth performance and health status of animals, especially rodents such as mice (Pan et al., 2016, Shin et al., 2013). However, the application of P. mume fruit extract in aquaculture species is lacking and only limited work has reported its involvement in the regulation of protein conversion rate in fish diets (Lee et al., 2015).

As the predominant material for cultivating freshwater pearls, Hyriopsis cumingii carries considerable weight globally and commercially (Saucedo et al., 2021). The system of fertilizing H. cumingii has deteriorated the habitat and allowed the growth of a huge number of underlying pathogenic bacteria. The latest report pointed to high mortality in H. cumingii as a consequence of outbreaks of Aeromonas veronii (Yang et al., 2020). In fact, A. veronii which distributes in freshwater environments broadly is an opportunistic pathogen with the ability to infect a wide range of fish species (Dien et al., 2023). Furthermore, the impact of A. veronii infection has spread to other aquatic animals, such as Pelodiscus sinensis (Dai et al., 2022) and Macrobrachium nipponense (Yi et al., 2021). One of the most widely used strategies for the treatment and prevention of A. veronii in aquaculture is the use of antibiotics, but it is extensively documented that A. veronii is resistant to a variety of antibiotics, such as ampicillin, penicillin, and norfloxacin (Li et al., 2019, Pei et al., 2021, Yang et al., 2020). Moreover, the multi-antibiotic resistance caused by the extensive use of antibiotics can spread via food chains and waterways and eventually pose a health threat to humans. Although vaccinations and probiotic supplements are being developed as new treatments for A. veronii (Jinendiran et al., 2021, Zhang et al., 2020), plant extracts stand out as an excellent alternative measure to curb antibiotic use because of their properties (Lenin et al., 2022, Zheng et al., 2023).

Therefore, we hypothesized that the fruits of F. suspensa and P. mume may also be beneficial in the prevention and control of bivalve bacterial diseases due to their excellent pharmacological properties that are conducive to human health. The first purpose of our work was to investigate the antibacterial activities of antibiotics and plant fruit extracts (F. suspensa and P. mume) against A. veronii strains GL1 and FO1. The second aim was to assess the antibacterial activity of the extracts combined with the antibiotics to examine joint effects against A. veronii. The third objective was to estimate whether F. suspensa extract exerted protective and immunological effects on GL1-infected H. cumingii. Our study is the first to employ fruit extracts in the treatment of bivalve diseases. Based on our results, we can delineate the critical role of F. suspensa fruits in the control of bivalve bacterial diseases both in vitro and in vivo.

Section snippets

Plants and antibiotics

Two dried herbal fruits, P. mume and F. suspensa, were obtained from Beibei Traditional Chinese Medicine Hospital in Chongqing, China. The extracts of the fruits were prepared by decoction (Cao et al., 2022). In brief, the fruit (200 g) was cut into small pieces and soaked in purified water (2 L) overnight. The fruits were then boiled for 30 min and the liquid was filtered for collecting dregs. The dregs were added to purified water (2 L) and the process was in triplicate. The filtrations were

Susceptibility of strains to single extract or antibiotic

The reference strain ATCC had lower MIC values of Pm and CLX than that in GL1 and FO1, while the opposite was observed in the other three substances. The MIC value of CLX in GL1 exceeded twice as high as in FO1, with a value of 53.33 mg mL−1 (Table 1). The inhibition curves of the extracts and antibiotics were plotted in Fig. 1A-E. The EC50 values against the strains were consistent with the MIC trend in Table 1. Besides, there existed no difference between GL1 and FO1 on the EC50 values except

Potential links between strain resistance and virulence

The micro-broth dilution method is considered an essential criterion for the assessment of sensitivity test results (Huang et al., 2019). The results revealed that there existed significant differences among the antibacterial properties of CLX against GL1 and FO1. CLX is a β-lactam antibiotic that works by inhibiting peptidoglycan synthesis in bacteria cell walls, finally leading to cell lysis (Lourenco et al., 2022). There were some pieces of evidence in previous reports regarding an

Conclusion

As stated above, fruit extracts such as Fs exhibited beneficial properties for aquaculture applications. In the present study, the extracts and antibiotics showed high efficacy in vitro antibacterial activity against A. veronii. However, only CLX showed a significant difference regarding inhibition rates against GL1 and FO1 represented two distinct virulence levels. In addition, Fs exposure improved the antioxidant and innate immune response of the infected H. cumingii. Even though both Fs

Ethics statement

This study has been reviewed and approved by the Institutional Ethics Committee of Southwest University (China).

CRediT authorship contribution statement

Qinglin Yang: Conceptualization, Methodology, Investigation, Software, Visualization, Writing – original draft. Xiaoqi Tang: Investigation, Formal analysis, Software, Writing – review & editing. Qingqing Zhou: Investigation, Data curation. Xicheng Zhou: Investigation. Jing Mei: Investigation. Xiaobo Yu: Resources. Yanhong Li: Resources. Weizhi Yao: Funding acquisition. Zhengli Wu: Supervision, Project administration, Funding acquisition.

Declaration of Competing Interest

All of the authors declare no conflicts of interest.

Acknowledgments

This work was supported by the Chongqing Municipal Natural Science Foundation (Grant No. CSTB2022NSCQ-MSX0305), the Special Key Project of Chongqing Technology Innovation and Application Development (Grant No. CSTB2022TIAD-ZXX0053), and the earmarked fund for CARS (Grant No. CARS-49).

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    Yang Q and Tang X contributed equally to this work.

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