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Applied Microbiology and Biotechnology

, Volume 103, Issue 21–22, pp 8987–8999 | Cite as

Interaction of a novel Bacillus velezensis (BvL03) against Aeromonas hydrophila in vitro and in vivo in grass carp

  • Lina Cao
  • Lifei Pan
  • Liang Gong
  • Yahui Yang
  • Haocheng He
  • Yanping Li
  • Yanan Peng
  • Dongjie Li
  • Liang Yan
  • Xuezhi Ding
  • Shengbiao Hu
  • Ziquan Yu
  • Yunjun Sun
  • Weitao Huang
  • Yibo Hu
  • Ganfeng YiEmail author
  • Liqiu XiaEmail author
Applied microbial and cell physiology

Abstract

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.

Keywords

Bacillus velezensis Aeromonas hydrophila Antibacterial activity Grass carps Fluorescence labeling Interaction 

Notes

Author contributions

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.

Funding information

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.

References

  1. Abdallah DB, Frikha-Gargouri O, Tounsi S (2018) Rizhospheric competence, plant growth promotion and biocontrol efficacy of Bacillus amyloliquefaciens subsp. plantarum strain 32a. Biol Control 124:61–67.  https://doi.org/10.1016/j.biocontrol.2018.01.013 CrossRefGoogle Scholar
  2. 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
  3. Balcazar JL, de Blas I, Ruiz-Zarzuela I, Cunningham D, Vendrell D, Muzquiz JL (2006) The role of probiotics in aquaculture. Vet Microbiol 114(3–4):173–186.  https://doi.org/10.1016/j.vetmic.2006.01.009 CrossRefPubMedGoogle Scholar
  4. Cabello FC (2006) Heavy use of prophylactic antibiotics in aquaculture: a growing problem for human and animal health and for the environment. Environ Microbiol 8(7):1137–1144.  https://doi.org/10.1111/j.1462-2920.2006.01054.x CrossRefPubMedPubMedCentralGoogle Scholar
  5. 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
  6. Chung S, Kong H, Buyer JS, Lakshman DK, Lydon J, Kim SD, Roberts DP (2008) Isolation and partial characterization of Bacillus subtilis ME488 for suppression of soilborne pathogens of cucumber and pepper. Applied Microbiology and Biotechnology 80 (1):115–123CrossRefGoogle Scholar
  7. Defoirdt T, Sorgeloos P, Bossier P (2011) Alternatives to antibiotics for the control of bacterial disease in aquaculture. Curr Opin Microbiol 14(3):251–258.  https://doi.org/10.1016/j.mib.2011.03.004 CrossRefGoogle Scholar
  8. 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
  9. 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
  10. Fan B, Wang C, Song X, Ding X, Wu L, Wu H, Gao X, Borriss R (2018) Bacillus velezensis FZB42 in 2018: the Gram-positive model strain for plant growth promotion and biocontrol. Front Microbiol 9:2491.  https://doi.org/10.3389/fmicb.2018.02491 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Gond S, Bergen M, Torres M, White J (2015) Endophytic Bacillus spp. produce antifungal lipopeptides and induce host defence gene expression in maize. Microbiol Res 172:79–87.  https://doi.org/10.1016/j.micres.2014.11.004 CrossRefPubMedGoogle Scholar
  12. 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
  13. Hadieva GF, Lutfullin MT, Pudova DS, Akosah YA, Gogoleva NE, Shagimardanova EI, Mardanova AM, Sharipova MR (2019) Data on the genome analysis of the probiotic strain Bacillus subtilis GM5. Data in brief 23:103643.  https://doi.org/10.1016/j.dib.2018.12.081 CrossRefPubMedGoogle Scholar
  14. 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
  15. 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
  16. 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
  17. 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
  18. Kang X, Zhang W, Cai X, Zhu T, Xue Y, Liu C (2018) Bacillus velezensis CC09: a potential ‘Vaccine’ for controlling wheat diseases. Mol Plant Microbe 31(6):623–632.  https://doi.org/10.1094/MPMI-09-17-21-R CrossRefGoogle Scholar
  19. Lowe TM, Eddy SR (1997) tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25(5):955–964.  https://doi.org/10.1093/nar/25.5.955 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 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
  21. Miranda CD, Zemelman R (2001) Antibiotic resistant bacteria in fish from the Concepcion Bay, Chile. Mar Pollut Bull 42(11):1096–1102CrossRefGoogle Scholar
  22. Moslehi-Jenabian S, Vogensen FK, Jespersen L (2011) The quorum sensing luxS gene is induced in Lactobacillus acidophilus NCFM in response to Listeria monocytogenes. Int J Food Microbiol 149(3):269–273.  https://doi.org/10.1016/j.ijfoodmicro.2011.06.011 CrossRefPubMedGoogle Scholar
  23. Nayak SK (2010) Probiotics and immunity: a fish perspective. Fish Shellfish Immun 29(1):2–14.  https://doi.org/10.1016/j.fsi.2010.02.017 CrossRefGoogle Scholar
  24. Ohta S, Chang T, Ikegami N, Kondo M, Miyata H (1993) Antibiotic substance produced by a newly isolated marine microalga, Chlorococcum HS-101. B Environ Contam Tox 50(2):171–178CrossRefGoogle Scholar
  25. Pengcheng W, Xiaosong L, Xiaofeng L, Zhongzhi L (2017) Validation of donor-specific tolerance of intestinal transplant by a secondary heart transplantation model. Exp Clin Transplant 15(1):89–95PubMedGoogle Scholar
  26. Perez-Ramos A, Mohedano ML, Pardo MA, Lopez P (2018) Beta-glucan-producing Pediococcus parvulus 2.6: test of probiotic and immunomodulatory properties in zebrafish models. Front Microbiol 9:1684.  https://doi.org/10.3389/fmicb.2018.01684 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Petersen A, Andersen JS, Kaewmak T, Somsiri T, Dalsgaard A (2002) Impact of integrated fish farming on antimicrobial resistance in a pond environment. Appl Environ Microbiol 68(12):6036–6042CrossRefGoogle Scholar
  28. Plaza G, Chojniak J, Rudnicka K, Paraszkiewicz K, Bernat P (2015) Detection of biosurfactants in Bacillus species: genes and products identification. J Appl Microbiol 119(4):1023–1034.  https://doi.org/10.1111/jam.12893 CrossRefPubMedGoogle Scholar
  29. Reda RM, Selim KM (2015) Evaluation of Bacillus amyloliquefaciens on the growth performance, intestinal morphology, hematology and body composition of Nile tilapia, Oreochromis niloticus. Aquac Int 23(1):203–217.  https://doi.org/10.1007/s10499-014-9809-z CrossRefGoogle Scholar
  30. Rodriguez MC, Alegre MT, Mesas JM (2007) Optimization of technical conditions for the transformation of Pediococcus acidilactici P60 by electroporation. Plasmid 58(1):44–50.  https://doi.org/10.1016/j.plasmid.2006.12.005 CrossRefPubMedGoogle Scholar
  31. 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
  32. Saraceni PR, Romero A, Figueras A, Novoa B (2016) Establishment of infection models in zebrafish larvae (Danio rerio) to study the pathogenesis of Aeromonas hydrophila. Front Microbiol 7:1219.  https://doi.org/10.3389/fmicb.2016.01219 CrossRefPubMedPubMedCentralGoogle Scholar
  33. Sajitha KL, Dev SA (2016) Quantification of antifungal lipopeptide gene expression levels in Bacillus subtilis B1 during antagonism against sapstain fungus on rubberwood. Biol Control 96:78–85.  https://doi.org/10.1016/j.biocontrol.2016.02.007 CrossRefGoogle Scholar
  34. Schmidt AS, Bruun MS, Dalsgaard I, Pedersen K, Larsen JL (2000) Occurrence of antimicrobial resistance in fish-pathogenic and environmental bacteria associated with four Danish rainbow trout farms. Appl Environ Microbiol 66(11):4908–4915CrossRefGoogle Scholar
  35. 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
  36. Simon O (2010) An interdisciplinary study on the mode of action of probiotics in pigs. J Anim Feed Sci 19(2):230–243.  https://doi.org/10.22358/jafs/66284/2010 CrossRefGoogle Scholar
  37. Singh V, Somvanshi P, Rathore G, Kapoor D, Mishra BN (2010) Gene cloning, expression, and characterization of recombinant aerolysin from Aeromonas hydrophila. Appl Biochem Biotechnol 160(7):1985–1991.  https://doi.org/10.1007/s12010-009-8752-3 CrossRefPubMedGoogle Scholar
  38. Silva M, Rosado T, Teixeira D, Candeias A, Caldeira AT (2017) Green mitigation strategy for cultural heritage: bacterial potential for biocide production. Environ Sci Pollut Res 24(5):4871–4881.  https://doi.org/10.1007/s11356-016-8175-y CrossRefGoogle Scholar
  39. 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
  40. Tamura K, Nei M, Kumar S (2004) Prospects for inferring very large phylogenies by using the neighbor-joining method. P Natl Acad Sci USA 101(30):11030–11035.  https://doi.org/10.1073/pnas.0404206101 CrossRefGoogle Scholar
  41. 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
  42. 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
  43. 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
  44. Villamil L, Reyes C, Martinez-Silva MA (2014) In vivo and in vitro assessment of Lactobacillus acidophilus as probiotic for tilapia (Oreochromis niloticus, Perciformes: Cichlidae) culture improvement. Aquac Res 45(7):1116–1125.  https://doi.org/10.1111/are.12051 CrossRefGoogle Scholar
  45. 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
  46. Wu JY, Jiang XT, Jiang YX, Lu SY, Zou F, Zhou HW (2010) Effects of polymerase, template dilution and cycle number on PCR based 16S rRNA diversity analysis using the deep sequencing method. BMC Microbiol 10:255.  https://doi.org/10.1186/1471-2180-10-255 CrossRefPubMedPubMedCentralGoogle Scholar
  47. 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
  48. 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
  49. 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
  50. Zhang D, Guo X, Wang Y, Gao T, Zhu B (2017) Novel screening strategy reveals a potent Bacillus antagonist capable of mitigating wheat take- all disease caused by Gaeumannomyces graminis var. tritici. Lett Appl Microbiol 65(6):512–519.  https://doi.org/10.1111/lam.12809 CrossRefPubMedGoogle Scholar
  51. 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
  52. Zokaeifar H, Babaei N, Saad CR, Kamarudin MS, Sijam K, Balcazar JL (2014) Detection and identification of antibiotic biosynthesis genes in Bacillus subtilis strains. Biocontrol Sci Tech 24(2):233–240.  https://doi.org/10.1080/09583157.2013.852653 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Developmental Biology of Freshwater Fish, Hunan Provincial Key Laboratory of Microbial Molecular Biology, College of Life ScienceHunan Normal UniversityChangshaPeople’s Republic of China

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