Interaction of a novel Bacillus velezensis (BvL03) against Aeromonas hydrophila in vitro and in vivo in grass carp
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This study evaluated the inhibition and interaction of Bacillus velezensis BvL03 as a probiotic agent against Aeromonas hydrophila. Strain BvL03 isolated from sediment samples of fish ponds had excellent antimicrobial activity against several fish pathogenic bacteria, especially Aeromonas, including A. hydrophila, A. veronii, A. caviae, and A. sobria. The successful amplification of lipopeptide antimicrobial chemical biosynthetic genes, including iturin family (ituA, ituB, and ituD), bacillomycin family (bacA, bacD, and bacAB), surfactin family (srfAB, srfC, and srfAA), and subtilosin family (albF and sunT) from the genome of BvL03 strain, confirmed its predominant antimicrobial activity. The challenge test suggested that BvL03 significantly decreased fish mortality when challenged with A. hydrophila, which had a cumulative mortality of 12.5% in the treatment group. Toxicity and hemolytic activity of A. hydrophila after co-cultured with BvL03 were relieved as confirmed by the cell experiments, when the initial inoculated concentration of BvL03 was 109 cfu/mL or higher. Moreover, the BvL03 strain labeled with GFP protein (BvL03-GFP) and AhX040 strain labeled with mCherry protein (AhX040-mCherry) were injected into grass carps. The fluorescence levels were monitored by using In Vivo Imaging System (IVIS), in which the green color was steadily increasing, whereas the red color was gradually weakening. Whole genome sequencing revealed that strain BvL03 possesses 15 gene clusters related to antibacterial compounds, including 5 NRPS gene clusters and 3 PKS gene clusters. These results suggested that B. velezensis BvL03 has the potential to be developed as a probiotic candidate against A. hydrophila infection in aquaculture.
KeywordsBacillus velezensis Aeromonas hydrophila Antibacterial activity Grass carps Fluorescence labeling Interaction
L.N.C. and L.F.P. designed the experiments. L.N.C., L.F.P., Y.H.Y., H.C.H., Y.P.L., Y.N.P., D.J.L., and S.B.H. contributed in performing the experiments. L.N.C., L.G., X.Z.D., Z.Q.Y., Y.J.S., and Y.B.H. analyzed the data. L.N.C., L.G., and W.T.H. wrote a draft of the manuscript. G.F.Y. and L.Q.X. supervised the research.
This study was funded by the National Natural Science Foundation of China (31770106), the National Basic Research Program of China (“973” program; 2012CB722301), the Major Research Projects in Hunan Province (2017NK1030), and “Hunan Province Biological Development Engineering and New Product Development Collaborative Innovation Center project” (20134486).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Research involving human participants and/or animals
Ethical approval. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the Animal Care Committee of Hunan Normal University at which the studies were conducted.
- Adorian TJ, Jamali H, Farsani HG, Darvishi P, Hasanpour S, Bagheri T, Roozbehfar R (2019) Effects of probiotic bacteria Bacillus on growth performance, digestive enzyme activity, and hematological parameters of Asian sea bass, Lates calcarifer (Bloch). Probiotics Antimicro 11(1):248–255. https://doi.org/10.1007/s12602-018-9393-z CrossRefGoogle Scholar
- Chandrarathna H, Nikapitiya C, Dananjaya S, Wijerathne C, Wimalasena S, Kwun H, Heo G, Lee J, De Zoysa M (2018) Outcome of co-infection with opportunistic and multidrug resistant Aeromonas hydrophila and A. veronii in zebrafish: identification, characterization, pathogenicity and immune responses. Fish Shellfish Immun 80:573–581. https://doi.org/10.1016/j.fsi.2018.06.049 CrossRefGoogle Scholar
- Ditu LM, Chifiriuc MC, Bezirtzoglou E, Voltsi C, Bleotu C, Pelinescu D, Mihaescu G, Lazar V (2011) Modulation of virulence and antibiotic susceptibility of enteropathogenic Escherichia coli strains by Enterococcus faecium probiotic strain culture fractions. Anaerobe 17(6):448–451. https://doi.org/10.1016/j.anaerobe.2011.05.019 CrossRefPubMedGoogle Scholar
- Elsabagh M, Mohamed R, Moustafa EM, Hamza A, Farrag F, Decamp O, Dawood MAO, Eltholth M (2018) Assessing the impact of Bacillus strains mixture probiotic on water quality, growth performance, blood profile and intestinal morphology of Nile tilapia, Oreochromis niloticus. Aquac Nutr 24(6):1613–1622. https://doi.org/10.1111/anu.12797 CrossRefGoogle Scholar
- Gong L, He H, Li D, Cao L, Khan TA, Li Y, Pan L, Yan L, Ding X, Sun Y, Zhang Y, Yi G, Hu S, Xia L (2019) A new isolate of Pediococcus pentosaceus (SL001) with antibacterial activity against fish pathogens and potency in facilitating the immunity and growth performance of grass carps. Front Microbiol 10:1384. https://doi.org/10.3389/fmicb.2019.01384 CrossRefPubMedPubMedCentralGoogle Scholar
- Harun-Or-Rashid M, Kim HJ, Yeom SI, Yu HA, Manir MM, Moon SS, Kang YJ, Chung YR (2018) Bacillus velezensis YC7010 enhances plant defenses against brown planthopper through transcriptomic and metabolic changes in rice. Front Plant Sci 9:1904. https://doi.org/10.3389/fpls.2018.01904 CrossRefPubMedPubMedCentralGoogle Scholar
- Hasan MT, Jang WJ, Lee BJ, Kim KW, Hur SW, Lim SG, Bai SC, Kong IS (2019) Heat-killed Bacillus sp. SJ-10 probiotic acts as a growth and humoral innate immunity response enhancer in olive flounder (Paralichthys olivaceus). Fish Shellfish Immun 88:424–431. https://doi.org/10.1016/j.fsi.2019.17018 CrossRefGoogle Scholar
- Huang MB, Baker CN, Banerjee S, Tenover FC (1992) Accuracy of the E test for determining antimicrobial susceptibilities of Staphylococci, Enterococci, Campylobacter jejuni, and Gram-negative bacteria resistant to antimicrobial agents. J Clin Microbiol 30(12):3243–3248PubMedPubMedCentralGoogle Scholar
- Huys G, Bartie K, Cnockaert M, Hoang Oanh DT, Phuong NT, Somsiri T, Chinabut S, Yusoff FM, Shariff M, Giacomini M, Teale A, Swings J (2007) Biodiversity of chloramphenicol-resistant mesophilic heterotrophs from Southeast Asian aquaculture environments. Res Microbiol 158(3):228–235. https://doi.org/10.1016/j.resmic.2006.12.011 CrossRefPubMedGoogle Scholar
- Madani NSH, Adorian TJ, Farsani HG, Hoseinifar SH (2018) The effects of dietary probiotic Bacilli (Bacillus subtilis and Bacillus licheniformis) on growth performance, feed efficiency, body composition and immune parameters of whiteleg shrimp (Litopenaeus vannamei) postlarvae. Aquac Res 49(5):1926–1933. https://doi.org/10.1111/are.13648 CrossRefGoogle Scholar
- Rosa IA, Rodrigues P, Bianchini AE, Silveira BP, Ferrari FT, Bandeira Junior G, Vargas APC, Baldisserotto B, Heinzmann BM (2019) Extracts of hesperozygis ringens (Benth.) epling: in vitro and in vivo antibacterial activity against fish pathogenic bacteria. J Appl Microbiol 126(5):1353–1361. https://doi.org/10.1111/jam.14219 CrossRefPubMedGoogle Scholar
- Shan S, Wang W, Song C, Wang M, Sun B, Li Y, Fu Y, Gu X, Ruan W, Rasmann S (2019) The symbiotic bacteria Alcaligenes faecalis of the entomopathogenic nematodes Oscheius spp. exhibit potential biocontrol of plant- and entomopathogenic fungi. Microb Biotechnol 12(3):459–471. https://doi.org/10.1111/1751-7915.13365 CrossRefPubMedPubMedCentralGoogle Scholar
- Su X, Zhu G, Huang Z, Wang X, Guo Y, Li B, Du Y, Yang W, Gao J (2019) Fine mapping and molecular marker development of the Sm gene conferring resistance to gray leaf spot (Stemphylium spp.) in tomato. Theor Appl Genet 132(4):871–882. https://doi.org/10.1007/s00122-018-3242-z CrossRefPubMedGoogle Scholar
- Thurlow CM, Williams MA, Carrias A, Ran C, Newman M, Tweedie J, Allison E, Jescovitch LN, Wilson AE, Terhune JS, Liles MR (2019) Bacillus velezensis AP193 exerts probiotic effects in channel catfish (Ictalurus punctatus) and reduces aquaculture pond eutrophication. Aquaculture 503:347–356. https://doi.org/10.1016/j.aquaculture.2018.11.051 CrossRefGoogle Scholar
- Van TT, Chin J, Chapman T, Tran LT, Coloe PJ (2008) Safety of raw meat and shellfish in Vietnam: an analysis of Escherichia coli isolations for antibiotic resistance and virulence genes. Int J Food Microbiol 124(3):217–223. https://doi.org/10.1016/j.ijfoodmicro.2008.03.029 CrossRefPubMedGoogle Scholar
- Vignesh V, Sathiyanarayanan G, Parthiban K, Kumar KS, Thirumurugan R (2018) Functional assessment of subtilosin A against Aeromonas spp. causing gastroenteritis and hemorrhagic septicaemia. Indian J Biotechnol 17(1):27–32Google Scholar
- Wang C, Liu Y, Sun GX, Li X, Liu ZP (2019) Growth, immune response, antioxidant capability, and disease resistance of juvenile Atlantic salmon (Salmo salar L.) fed Bacillus velezensis V4 and Rhodotorula mucilaginosa compound. Aquaculture 500:65–74. https://doi.org/10.1016/j.aquaculture.2018.09.052 CrossRefGoogle Scholar
- Yi C, Liu C, Chuang K, Chang Y, Hu S (2019) A potential probiotic Chromobacterium aquaticum with bacteriocin-like activity enhances the expression of indicator genes associated with nutrient metabolism, growth performance and innate immunity against pathogen infections in zebrafish (Danio rerio). Fish Shellfish Immun 93:124–134. https://doi.org/10.1016/j.fsi.2019.07.042 CrossRefGoogle Scholar
- Yi Y, Zhang Z, Zhao F, Liu H, Yu L, Zha J, Wang G (2018) Probiotic potential of Bacillus velezensis JW: antimicrobial activity against fish pathogenic bacteria and immune enhancement effects on Carassius auratus. Fish Shellfish Immun 78:322–330. https://doi.org/10.1016/j.fsi.2018.04.055 CrossRefGoogle Scholar
- Zalila-Kolsi I, Mahmoud AB, Ali H, Sellami S, Nasfi Z, Tounsi S, Jamoussi K (2016) Antagonist effects of Bacillus spp. strains against Fusarium graminearum for protection of durum wheat (Triticum turgidum L. subsp. durum). Microbiol Res 192:148–158. https://doi.org/10.1016/j.micres.2016.06.012 CrossRefPubMedGoogle Scholar
- Zhou S, Song D, Zhou X, Mao X, Zhou X, Wang S, Wei J, Huang Y, Wang W, Xiao SM, Qin Q (2019) Characterization of Bacillus subtilis from gastrointestinal tract of hybrid Hulong grouper (Epinephelus fuscoguttatus) and its effects as probiotic additives. Fish Shellfish Immun 84:1115–1124. https://doi.org/10.1016/j.fsi.2018.10.058 CrossRefGoogle Scholar