College of Fisheries, Guangdong Ocean University, Zhanjiang, 524000, China; Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture, Guangdong Ocean University, Zhanjiang, 524000, China; Guangdong Key Laboratory of Control for Disease of Aquatic Animals of Guangdong Higher Education Institutes, Zhanjiang, 524088, China; Guangdong Provincial Engineering Research Centre for Aquatic Animal Health Assessment, Shenzhen, 327005, China.
College of Fisheries, Guangdong Ocean University, Zhanjiang, 524000, China; Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture, Guangdong Ocean University, Zhanjiang, 524000, China; Guangdong Key Laboratory of Control for Disease of Aquatic Animals of Guangdong Higher Education Institutes, Zhanjiang, 524088, China; Guangdong Provincial Engineering Research Centre for Aquatic Animal Health Assessment, Shenzhen, 327005, China.
Fish Shellfish Immunol. 2024 Sep;152:109795. doi: 10.1016/j.fsi.2024.109795. Epub 2024 Jul 26.
As an alternative to the criticized antibiotics, probiotics have been adopted for their eco-friendly nature and ability to enhance host growth and immunity. Nevertheless, reports suggest ineffectiveness in commercially available probiotics since most are from non-fish sources; thus, this study was envisaged to isolate and characterize new Bacillus spp. from the gut of hybrid grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂) which could serve as potential probiotics. The isolation and characterization were performed based on their morphological and biochemical properties, and 16S rRNA sequencing homology analysis. A subsequent 30-day in vivo biosafety feeding trial was conducted to ascertain isolates' non-pathogenicity, as well as their effects on fish growth, and intestinal mucosal microvilli via scanning electron microscopy (SEM) analysis. Four Bacillus spp. strains, namely, B. velezensis strain PGSAK01 (accession number OQ726606), B. stercoris strain PGSAK05 (accession number OQ726607), B. velezensis strain PGSAK17 (accession number OQ726601), and B. subtilis strain PGSAK19 (accession number OQ726605), were identified and characterized in the current study. The strains showed promising probiotic properties such higher adhesion capability, higher thermotolerance, displaying higher survivability to 0.5 % bile, lower pH tolerance, γ-haemolytic activity, and multispecies characteristics. Among the 24 antibiotics tested, while all isolates showed susceptibility to 21, the PGSAK01 strain showed resistance to furazolidone antibiotics. None of the isolates showed possession of i) virulence factor genes encoding enterotoxigenic (hblA, hblC, hblD, nheA, nheB, and entFM) and emetic (cereulide synthetase gene, ces) genes, and ii) streptomycin resistance gene (vat c), ampicillin-resistant genes (mecA and bla), and vancomycin-resistant gene (van B). Nevertheless, the PGSAK01 and PGSAK17 strains showed possession of tek K, cat, and ant(4')-Ia (adenylyltransferase) (except the PGSAK01) resistant genes. All isolates displayed better antimicrobial effects against pathogenic bacteria Streptococcus agalactiae, S. iniae, Vibrio harveyi, and V. alginolyticus. The in vivo biosafety trial involved hybrid grouper fish being grouped into five (average weight 32 ± 0.94 g), namely, the group fed the basal diet void of isolate's supplementation (control), and the remaining four groups fed the basal diet with 1 × 10 CFU/g diet of individual strain PGSAK01, PGSAK05, PGSAK17, and PGSAK19 supplementation. At the end of the study, a significantly higher WGR, K (except the PGSAK01 group), VSI; lysozyme (except PGSAK01 group), total antioxidant activity, alkaline phosphatase, superoxide dismutase enzyme activities; highly dense intestinal mucosal villi (based on the scanning electron microscopy analysis); and significantly lower malondialdehyde levels were witnessed in the isolated treated groups compared to the control, supporting the results obtained in the auto-aggregation and cell-surface hydrophobicity test. This work's results have provided thought-provoking targets; thus, studies involving extensive genome sequencing and functional annotation analysis will be explored to offer unfathomable insights into their mechanisms of action and potential health benefits, further establishing the four Bacillus strains' (PGSAK01, PGSAK05, PGSAK17, and PGSAK19) potential role in probiotic fields and functional foods.
作为被批评的抗生素的替代品，益生菌因其环保特性和增强宿主生长和免疫力的能力而被采用。然而，报告表明，市售益生菌的效果并不理想，因为大多数益生菌都不是来自鱼类；因此，本研究旨在从杂交石斑鱼（♀×♂）的肠道中分离和鉴定新的芽孢杆菌属菌株，这些菌株可能作为潜在的益生菌。分离和鉴定是基于其形态和生化特性以及 16S rRNA 测序同源性分析进行的。随后进行了为期 30 天的体内生物安全性喂养试验，以确定分离株的非致病性以及通过扫描电子显微镜（SEM）分析对鱼类生长和肠道黏膜微绒毛的影响。目前研究鉴定和表征了四种芽孢杆菌菌株，即 PGSAK01 菌株（登录号 OQ726606）、PGSAK05 菌株（登录号 OQ726607）、PGSAK17 菌株（登录号 OQ726601）和 PGSAK19 菌株（登录号 OQ726605）。这些菌株表现出有希望的益生菌特性，如更高的粘附能力、更高的耐热性、在 0.5%胆汁中更高的存活率、更低的 pH 耐受性、γ-溶血活性和多物种特性。在测试的 24 种抗生素中，虽然所有分离株对 21 种抗生素均表现出敏感性，但 PGSAK01 株对呋喃唑酮类抗生素表现出耐药性。所有分离株均未携带编码肠毒素（hblA、hblC、hblD、nheA、nheB 和 entFM）和呕吐毒素（cereulide 合成酶基因，ces）的毒力因子基因，也未携带链霉素耐药基因（vat c）、氨苄西林耐药基因（mecA 和 bla）和万古霉素耐药基因（van B）。然而，PGSAK01 和 PGSAK17 菌株携带 tek K、cat 和 ant(4')-Ia（腺苷转移酶）（除 PGSAK01 外）耐药基因。所有分离株对致病性细菌无乳链球菌、杀鲑气单胞菌、哈维氏弧菌和鳗弧菌均表现出更好的抗菌作用。体内生物安全性试验涉及将杂交石斑鱼分为五组（平均体重 32±0.94g），即不添加分离株的基础饲料组（对照组）和其余四组分别用 1×10 CFU/g 饮食的单个菌株 PGSAK01、PGSAK05、PGSAK17 和 PGSAK19 补充剂喂养。在研究结束时，与对照组相比，在添加分离株的处理组中观察到更高的 WGR、K（除 PGSAK01 组外）、VSI；溶菌酶（除 PGSAK01 组外）、总抗氧化活性、碱性磷酸酶、超氧化物歧化酶酶活性；高密度肠道黏膜绒毛（基于扫描电子显微镜分析）；以及更低的丙二醛水平，这支持了在自动聚集和细胞表面疏水性试验中获得的结果。这项工作的结果提供了发人深省的目标；因此，将探索涉及广泛基因组测序和功能注释分析的研究，以提供对其作用机制和潜在健康益处的深入了解，进一步确立四种芽孢杆菌菌株（PGSAK01、PGSAK05、PGSAK17 和 PGSAK19）在益生菌领域和功能性食品中的潜在作用。
