Genetic and Physiological Characterization of the Antibacterial Activity of Bacillus subtilis subsp. inaquosorum Strain T1 Effective Against pirABVp-Bearing Vibrio parahaemolyticus

ABSTRACTAcute hepatopancreatic necrosis disease (AHPND) is caused by PirAB toxin-producing Vibrio parahaemolyticus and has devastated the global shrimp aquaculture industry. One approach for preventing growth of AHPND-producing Vibrio spp. is through the application of beneficial bacteria capable of inhibiting these pathogens. In this study we focus on the inhibitory activity of Bacillus subtilis subsp. inaquosorum strain T1, which hinders V. parahaemolyticus growth in co-culture experiments in a density-dependent manner; inhibition was also obtained using cell-free supernatants from T1 stationary phase cultures. Using a mariner-based transposon mutagenesis, 17 mutants were identified having complete or partial loss of inhibitory activity. Of those having total activity loss, 13 had insertions within a 42.6 kb DNA region comprising 15 genes whose deduced products were homologous to non-ribosomal polypeptide synthetases (NRPSs), polyketide synthases (PKSs) and related activities, which were mapped as one transcriptional unit. Mutants with partial activity contained insertions in spo0A and oppA, indicating stationary phase control. Expression of lacZ transcriptional fusions to NRPS and PKS genes was negligible during growth and at their highest during early stationary phase. Inactivation of sigH resulted in loss of inhibitor activity, indicating a role for σH in transcription. Disruption of abrB resulted in NRPS and PKS gene overexpression during growth as well as enhanced growth inhibition. This is the first study examining expression and control of an NRPS-PKS region unique to the inaquosorum subspecies of B. subtilis and an understanding of factors involved in T1 inhibitor production will enable its development for use as a potential tool against AHPND Vibrio pathogens in shrimp aquaculture.IMPORTANCEThe shrimp aquaculture industry has been impacted by the rise of acute hepatopancreatic necrosis disease (AHPND), resulting in significant financial losses annually. Caused by strains of the bacterial pathogen, Vibrio parahaemolyticus, treatment of AHPND involves the use of antibiotics, which leads to a rise in antibiotic resistant strains. An alternative approach is through the application of beneficial microorganisms having inhibitory activities against AHPND-generating pathogens. In this study, we examine the genetic basis for the ability of Bacillus subtilis strain T1 to inhibit growth of an AHPND Vibrio strain and show that activity is associated with genes having the potential for synthesizing antibacterial compounds. We found that expression of these genes is under stationary phase control and showed that inactivation of a global transition state regulator results in enhancement of inhibitory activity against the AHPND Vibrio. Our approach for understanding the factors involved in production B. subtilis strain T1 inhibitory activity may allow for development of this strain for use as a potential tool for the prevention of AHPND outbreaks.

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Antibacterial Activity of Bacillus inaquosorum Strain T1 against pirABVp-Bearing Vibrio parahaemolyticus: Genetic and Physiological Characterization

Applied and Environmental Microbiology ◽

10.1128/aem.01950-20 ◽

2020 ◽

Vol 86 (21) ◽

Author(s):

Sarah E. Avery ◽

Susannah P. Ruzbarsky ◽

Amanda M. Hise ◽

Harold J. Schreier

Keyword(s):

Stationary Phase ◽

Gene Cluster ◽

Inhibitory Activity ◽

Vibrio Parahaemolyticus ◽

Phase Control ◽

Transcriptional Unit ◽

Shrimp Aquaculture ◽

Content Type ◽

Aquaculture Industry ◽

Acute Hepatopancreatic Necrosis Disease

ABSTRACT Acute hepatopancreatic necrosis disease (AHPND) is caused by PirAB toxin-producing Vibrio parahaemolyticus and has devastated the global shrimp aquaculture industry. One approach for preventing the growth of AHPND-producing Vibrio spp. is through the application of beneficial bacteria capable of inhibiting these pathogens. In this study, we focused on the inhibitory activity of Bacillus inaquosorum strain T1, which hinders V. parahaemolyticus growth in coculture experiments in a density-dependent manner; inhibition was also observed using cell-free supernatants from T1 stationary-phase cultures. Using mariner-based transposon mutagenesis, 17 mutants having a complete or partial loss of inhibitory activity were identified. Of those displaying a total loss of activity, 13 had insertions within a 42.6-kb DNA region comprising 15 genes whose deduced products were homologous to nonribosomal polypeptide synthetases (NRPSs), polyketide synthases (PKSs), and related activities, which were mapped as one transcriptional unit. Mutants with partial activity contained insertions in spo0A and oppA, indicating stationary-phase control. The levels of expression of NRPS and PKS lacZ transcriptional fusions were negligible during growth and were the highest during early stationary phase. Inactivation of sigH resulted in a loss of inhibitor activity, indicating a role for σH in transcription. Disruption of abrB resulted in NRPS and PKS gene overexpression during growth as well as enhanced growth inhibition. Our characterization of the expression and control of an NRPS-PKS gene cluster in B. inaquosorum T1 provides an understanding of the factors involved in inhibitor production, enabling this strain’s development for use as a tool against AHPND-causing Vibrio pathogens in shrimp aquaculture. IMPORTANCE The shrimp aquaculture industry has been significantly impacted by acute hepatopancreatic necrosis disease (AHPND), resulting in significant financial losses annually. AHPND is caused by strains of the bacterial pathogen Vibrio parahaemolyticus, and treatment of AHPND involves the use of antibiotics, which leads to a rise in the number of antibiotic-resistant strains. Alternative treatments include the application of beneficial microorganisms having inhibitory activities against pathogens causing AHPND. In this study, we examined the ability of Bacillus inaquosorum strain T1 to inhibit the growth of an AHPND-causing Vibrio strain, and we show that this activity involves a gene cluster associated with antibacterial compound production. We found that gene expression is under stationary-phase control and that enhanced activity occurs upon inactivation of a global transition state regulator. Our approach for understanding the factors involved in producing B. inaquosorum strain T1 inhibitory activity will allow for the development of this strain as a tool for AHPND prevention and treatment.

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Modeling inhibitory activity of a novel antimicrobial peptide AMPNT-6 from Bacillus subtilis against Vibrio parahaemolyticus in shrimp under various environmental conditions

Food Control ◽

10.1016/j.foodcont.2013.03.010 ◽

2013 ◽

Vol 33 (1) ◽

pp. 249-253 ◽

Cited By ~ 11

Author(s):

Yuehua Pu ◽

Lijun Sun ◽

Yaling Wang ◽

Deng Qi ◽

Danmin Chen ◽

...

Keyword(s):

Bacillus Subtilis ◽

Antimicrobial Peptide ◽

Environmental Conditions ◽

Inhibitory Activity ◽

Vibrio Parahaemolyticus

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Inhibitory activity of a novel antibacterial peptide AMPNT-6 from Bacillus subtilis against Vibrio parahaemolyticus in shrimp

Food Control ◽

10.1016/j.foodcont.2012.07.025 ◽

2013 ◽

Vol 30 (1) ◽

pp. 58-61 ◽

Cited By ~ 30

Author(s):

Defeng Xu ◽

Yaling Wang ◽

Lijun Sun ◽

Huanming Liu ◽

Jianrong Li

Keyword(s):

Bacillus Subtilis ◽

Inhibitory Activity ◽

Vibrio Parahaemolyticus ◽

Antibacterial Peptide

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Antibacterial and antiproteinase activities of a double whey acidic protein domain-containing protein from Penaeus vannamei Boone, 1931 (Decapoda, Penaeidae)

Crustaceana ◽

10.1163/15685403-00003962 ◽

2020 ◽

Vol 93 (1) ◽

pp. 51-69

Author(s):

Taksaorn Sakunwattana ◽

Phattarunda Jaree ◽

Vichien Rimphanitchayakit ◽

Anchalee Tassanakajon ◽

Sirinit Tharntada

Keyword(s):

Bacillus Subtilis ◽

Heat Shock ◽

Vibrio Parahaemolyticus ◽

Whey Acidic Protein ◽

Acidic Protein ◽

Protein Domain ◽

Penaeus Vannamei ◽

Growth Inhibitory ◽

Growth Inhibitory Activity ◽

Acute Hepatopancreatic Necrosis Disease

Abstract Double whey acidic protein domain-containing proteins (DWDs) or secretory leukocyte protease inhibitor (SLPI)-like proteins, have been identified in many species of Crustacea. The PvDWD1 from Penaeus vannamei was the first SLPI-like protein identified in a crustacean. Herein, we report that the expression of PvDWD1 was up-regulated at 6 h after acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio parahaemolyticus (VPAHPND) challenge. Similarly, after a different stress other than bacterial infection, the chronic non-lethal heat shock (NLHS) induction, the expression of the PvDWD1 gene was increased at 6 and 24 h after chronic NLHS treatment. The recombinant PvDWD1 (rPvDWD1) protein was over-expressed and tested for its deleterious activity against the bacteria. The rPvDWD1 protein exhibited growth inhibitory activity against Bacillus megaterium and VPAHPND. Moreover, the rPvDWD1 protein possessed an antiproteinase activity against the secreted proteinases from Bacillus subtilis and VPAHPND. The PvDWD1 perhaps functions in shrimp immunity through antimicrobial and antiproteinase properties.

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The Study of Bacillus Subtils Antimicrobial Activity on Some of the Pathological Isolates

International Journal of Drug Delivery Technology ◽

10.25258/ijddt.9.2.12 ◽

2019 ◽

Vol 9 (02) ◽

Author(s):

Hussein A Kadhum ◽

Thualfakar H Hasan2

Keyword(s):

Staphylococcus Aureus ◽

Pseudomonas Aeruginosa ◽

Antimicrobial Activity ◽

Bacillus Subtilis ◽

Bacterial Growth ◽

Broad Spectrum ◽

Inhibitory Activity ◽

Bacterial Pathogens ◽

Inhibitory Ability ◽

Selection Of

The study involved the selection of two isolates from Bacillus subtilis to investigate their inhibitory activity against some bacterial pathogens. B sub-bacteria were found to have a broad spectrum against test bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa. They were about 23-30 mm and less against Klebsiella sp. The sensitivity of some antibodies was tested on the test samples. The results showed that the inhibitory ability of bacterial growth in the test samples using B. subtilis extract was more effective than the antibiotics used.

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PHÂN LẬP VÀ TUYỂN CHỌN CÁC CHỦNG BACILLUS ỨC CHẾ VI KHUẨN VIBRIO PARAHAEMOLYTICUS GÂY BỆNH TÔM CHẾT SỚM Ở TỈNH THỪA THIÊN HUẾ

HUE UNIVERSITY JOURNAL OF SCIENCE AGRICULTURE AND RURAL DEVELOPMENT ◽

10.26459/jard.v100i1.2981 ◽

2015 ◽

Vol 100 (1) ◽

Author(s):

Nguyễn Thị Bích Đào ◽

Trần Quang Khánh Vân ◽

Nguyễn Văn Khanh ◽

Nguyễn Quang Linh

Keyword(s):

Bacillus Subtilis ◽

Vibrio Parahaemolyticus ◽

Bacillus Amyloliquefaciens ◽

Bacillus Spp ◽

Bacillus Subtilis B1

Khi tình hình bệnh hội chứng tôm chết sớm (EMS) đã gây thiệt hại vô cùng to lớn đối với Nuôi trồng thủy sản thì các giải pháp được đề nghị và áp dụng nhằm hạn chế dịch bệnh. Trong đó, việc tìm hiểu và đưa vi khuẩn có lợi để cạnh tranh và ức chế loài vi khuẩn gây bệnh rất được quan tâm, được cho là giải pháp có nhiều triển vọng phù hợp với điều kiện môi trường, đảm bảo sức khỏe cho con người, cũng như hạn chế được dịch bệnh. Đặc biệt, đưa vi khuẩn Bacillus spp. qua đường tiêu hóa của tôm ngay từ khi mới thả đã hạn chế được mật độ vi khuẩn Vibrio. Nghiên cứu này đã phân lập được các chủng Bacillus subtilis B1, Bacillus subtilis B2, Bacillus amyloliquefaciens B4và thử khả năng đối kháng với vi khuẩn Vibrio parahaemolyticus V1 ở các nồng độ 103, 104, 105, 106 CFU theo dõi ở các thời điểm 6h, 12h, 24h, 48h và 72h. Kết quả cho thấy cả ba chủng vi khuẩn Bacillus trên phân lập được đều có khả năng ức chế tốt vi khuẩn Vibrio parahaemolyticus V1, trong đó vi khuẩn Bacillus amyloliquefaciens B4 làtốt nhất với đường kính vòng kháng khuẩn 52,67 ± 4,31mm ở thời điểm 48h; hai chủng Bacillus subtilis B1, Bacillus subtilis B2 lầnlượt là 49,67 ± 3,15 mm, 44,07 ± 5,19 mm, với mức sai số có ý nghĩa thống kê p < 0,05.

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Effects of Feed Mixed with Lactic Acid Bacteria and Carbon, Nitrogen, Phosphorus Supplied to the Water on the Growth and Survival Rate of White Leg Shrimp (Penaeus vannamei) Infected with Acute Hepatopancreatic Necrosis Disease Caused by Vibrio parahaemolyticus

Biology ◽

10.3390/biology10040280 ◽

2021 ◽

Vol 10 (4) ◽

pp. 280

Author(s):

Linh Nguyen Thi Truc ◽

Tuu Nguyen Thanh ◽

To Tran Thi Hong ◽

Day Pham Van ◽

Minh Vo Thi Tuyet ◽

...

Keyword(s):

Lactic Acid ◽

Survival Rate ◽

Lactic Acid Bacteria ◽

Vibrio Parahaemolyticus ◽

Control Group ◽

Penaeus Vannamei ◽

Growth And Survival ◽

Acute Hepatopancreatic Necrosis Disease ◽

Nitrogen Phosphorus ◽

Shrimp Health

This study aimed to evaluate the growth, survival rate, and resistance to acute hepatopancreatic necrosis disease (AHPND) of white leg shrimp (Penaeus vannamei) by using Lactobacillus plantarum, Lactobacillus fermentum, and Pediococcus pentosaceus mixed with feed, and at the same time supplying CNP in a ratio of 15:1:0.1 to the water. As a result, the treatments that shrimp were fed with feed containing lactic acid bacteria (LAB), especially L. plantarum, have increased shrimp growth, total hemocyte cells, granulocyte cells, and hyaline cells significantly (p < 0.05) in comparison to the control group. The supply of CNP to the water has promoted the intensity of V. parahaemolyticus effects on shrimp health and significantly decreased total hemocyte cells, granulocyte cells, and hyaline cells by 30–50% in the period after three days of the challenge, except in L. plantarum treatment, which had only a 20% decrease compared to other treatments. In CNP supplying treatments, the AHPND infected rate and mortality of shrimp were higher than those in other treatments. In summary, the supply of CNP had significantly reduced the shrimp’s immune response and promoted the susceptibility of shrimp to AHPND in both cases of use with and without LAB-containing diets.

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Synergistic Antimicrobial Effect of a Seaweed-Probiotic Blend Against Acute Hepatopancreatic Necrosis Disease (AHPND)-Causing Vibrio parahaemolyticus

Probiotics and Antimicrobial Proteins ◽

10.1007/s12602-019-09616-8 ◽

2019 ◽

Vol 12 (3) ◽

pp. 906-917 ◽

Cited By ~ 1

Author(s):

Shen-Yin Lim ◽

Keat Wei Loo ◽

Wey-Lim Wong

Keyword(s):

Vibrio Parahaemolyticus ◽

Antimicrobial Effect ◽

Acute Hepatopancreatic Necrosis Disease

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Draft Genome Sequence of the Shrimp Pathogen Vibrio parahaemolyticus ST17.P5-S1, Isolated in Peninsular Malaysia

Microbiology Resource Announcements ◽

10.1128/mra.01053-18 ◽

2018 ◽

Vol 7 (11) ◽

Cited By ~ 2

Author(s):

Sridevi Devadas ◽

Subha Bhassu ◽

Tze Chiew Christie Soo ◽

Fatimah M. Yusoff ◽

Mohamed Shariff

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Vibrio Parahaemolyticus ◽

East Coast ◽

Draft Genome ◽

Antibiotic Resistance Genes ◽

Peninsular Malaysia ◽

Penaeus Vannamei ◽

Content Type ◽

Acute Hepatopancreatic Necrosis Disease

We sequenced the genome of Vibrio parahaemolyticus strain ST17.P5-S1, isolated from Penaeus vannamei cultured in the east coast of Peninsular Malaysia. The strain contains several antibiotic resistance genes and a plasmid encoding the Photorhabdus insect-related (Pir) toxin-like genes, pirAvp and pirBvp, associated with acute hepatopancreatic necrosis disease (AHPND).

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