Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Jun 12:10:1248.
doi: 10.3389/fmicb.2019.01248. eCollection 2019.

A Newly Isolated Bacillus subtilis Strain Named WS-1 Inhibited Diarrhea and Death Caused by Pathogenic Escherichia coli in Newborn Piglets

Yunping Du  1 , Zhichao Xu  1 , Guolian Yu  2 , Wei Liu  2 , Qingfeng Zhou  2 , Dehong Yang  2 , Jie Li  3 , Li Chen  1 , Yun Zhang  1 , Chunyi Xue  1 , Yongchang Cao  1
Affiliations

A Newly Isolated Bacillus subtilis Strain Named WS-1 Inhibited Diarrhea and Death Caused by Pathogenic Escherichia coli in Newborn Piglets

Yunping Du et al. Front Microbiol. .

Abstract

Bacillus subtilis is recognized as a safe and reliable human and animal probiotic and is associated with bioactivities such as production of vitamin and immune stimulation. Additionally, it has great potential to be used as an alternative to antimicrobial drugs, which is significant in the context of antibiotic abuse in food animal production. In this study, we isolated one strain of B. subtilis, named WS-1, from apparently healthy pigs growing with sick cohorts on one Escherichia coli endemic commercial pig farm in Guangdong, China. WS-1 can strongly inhibit the growth of pathogenic E. coli in vitro. The B. subtilis strain WS-1 showed typical Bacillus characteristics by endospore staining, biochemical test, enzyme activity analysis, and 16S rRNA sequence analysis. Genomic analysis showed that the B. subtilis strain WS-1 shares 100% genomic synteny with B. subtilis with a size of 4,088,167 bp. Importantly, inoculation of newborn piglets with 1.5 × 1010 CFU of B. subtilis strain WS-1 by oral feeding was able to clearly inhibit diarrhea (p < 0.05) and death (p < 0.05) caused by pathogenic E. coli in piglets. Furthermore, histopathological results showed that the WS-1 strain could protect small intestine from lesions caused by E. coli infection. Collectively, these findings suggest that the probiotic B. subtilis strain WS-1 acts as a potential biocontrol agent protecting pigs from pathogenic E. coli infection. Importance: In this work, one B. subtilis strain (WS-1) was successfully isolated from apparently healthy pigs growing with sick cohorts on one E. coli endemic commercial pig farm in Guangdong, China. The B. subtilis strain WS-1 was identified to inhibit the growth of pathogenic E. coli both in vitro and in vivo, indicating its potential application in protecting newborn piglets from diarrhea caused by E. coli infections. The isolation and characterization will help better understand this bacterium, and the strain WS-1 can be further explored as an alternative to antimicrobial drugs to protect human and animal health.

Keywords: Bacillus subtilis; Escherichia coli; biocontrol agent; genomic analysis; newborn piglets.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Ten unknown bacterial strains from healthy pig feces significantly inhibited the growth of E. coli in vitro. (A,B) Inhibiting effect on E. coli of total 35 unknown bacterial strains isolated from the healthy pig feces. E. coli strain 4–1 was maintained on bouillon agar medium (108 CFU/ml); the Oxford cup with 80 μl single bacterial culture fluid was placed in the middle. After 24 h, the inhibitory ring was observed and measured with ruler. (C) The No. 1 unknown bacterial strain significantly inhibited the growth of E. coli. E. coli strain 4–1 was maintained on bouillon agar medium (108 CFU/ml); the Oxford cups with 80 μl of No. 1 unknown bacterial culture fluid containing different amounts of 1.8 × 108, 3.6 × 108, and 7.2 × 108 CFU/ml were placed in the middle. The inhibitory rings were observed and measured with a ruler. Data are representative of three independent experiments. Data are represented as mean ± SD, n = 3. ****p < 0.0001.
Figure 2
Figure 2
The endospore staining of No. 1 unknown bacterial strain. Endospore staining to analyze the characteristic of No. 1 unknown bacterial strain. The arrows show the forming spores of this unknown bacterium.
Figure 3
Figure 3
Phylogenetic tree constructed on the basis of the 16S rRNA gene from B. subtilis strain WS-1 and 13 B. subtilis strains from different countries. The dendrogram was constructed using the neighbor joining method in the MEGA software package, version 5. Reference sequences obtained from GenBank are indicated by strain name. The scale bar represents 0.01 nucleotide substitutions per site.
Figure 4
Figure 4
Genome visualization and genomic synteny of the B. subtilis strain WS-1. (A) Genome visualization of B. subtilis strain WS-1. The genome of B. subtilis strain WS-1 was sequenced and analyzed using the PacBio platform. (B) Genomic synteny of B. subtilis strain WS-1 was analyzed based on the alignment results with B. subtilis.
Figure 5
Figure 5
Measurement of bacterial growth. The growth curves of B. subtilis strain WS-1. Bacteria were grown in bouillon fluid medium at 37°C for 48 h with agitation, and the Colony Forming Unit (CFU) was determined at 0, 6, 12, 18, 24, 30, 36, 42, and 48 h. The data are representative of three independent experiments. Data are represented as mean ± SD, n = 3.
Figure 6
Figure 6
B. subtilis strain WS-1 inhibited diarrhea and death caused by E. coli in newborn piglets. Newborn piglets were first orally fed with TSB medium or TSB medium containing B. subtilis strain WS-1. At 3 days, all piglets were orally challenged with E. coli strain 4–1. The diarrhea rate (A) and survival rate (B) of newborn piglets post-challenge with E. coli between the control group and the B. subtilis strain WS-1 treatment group were recorded daily from the first day to the sixth day after challenge. The data are representative of two independent experiments. Data are represented as mean ± SD, n = 6 or n = 7.
Figure 7
Figure 7
Intestinal changes in newborn piglets post-challenge with E. coli of the control group and the B. subtilis strain WS-1 treatment group. (A–C) Hematoxylin and eosin (H&E)-stained intestinal tissue section (blunt intestinal villus was indicated by arrows) of a control piglet at 3 d.p.c. (D–F) H&E-stained intestinal tissue section of a B. subtilis strain WS-1 treatment piglet at 3 d.p.c. The data are representative of two independent experiments. Data are represented as mean ± SD, n = 1 or n = 2.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Amorim C., Silverio S. C., Goncalves R. F. S., Pinheiro A. C., Silva S., Coelho E., et al. . (2019). Downscale fermentation for xylooligosaccharides production by recombinant Bacillus subtilis 3610. Carbohydr. Polym. 205, 176–183. 10.1016/j.carbpol.2018.09.088, PMID: - DOI - PubMed
    1. Bartolini M., Cogliati S., Vileta D., Bauman C., Ramirez W., Grau R. (2019). The stress-responsive alternative sigma factor SigB plays a positive role in the antifungal proficiency of Bacillus subtilis. Appl. Environ. Microbiol. 85. 10.1128/aem.00178-19 - DOI - PMC - PubMed
    1. Besemer J., Lomsadze A., Borodovsky M. (2001). GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. Nucleic Acids Res. 29, 2607–2618. 10.1093/nar/29.12.2607, PMID: - DOI - PMC - PubMed
    1. Bian X., Evivie S. E., Muhammad Z., Luo G. W., Liang H. Z., Wang N. N., et al. . (2016). In vitro assessment of the antimicrobial potentials of Lactobacillus helveticus strains isolated from traditional cheese in Sinkiang China against food-borne pathogens. Food Funct. 7, 789–797. 10.1039/C5FO01041A, PMID: - DOI - PubMed
    1. Borsodi A. K., Pollak B., Keki Z., Rusznyak A., Kovacs A. L., Sproer C., et al. . (2011). Bacillus alkalisediminis sp. nov., an alkaliphilic and moderately halophilic bacterium isolated from sediment of extremely shallow soda ponds. Int. J. Syst. Evol. Microbiol. 61, 1880–1886. 10.1099/ijs.0.019489-0, PMID: - DOI - PubMed

LinkOut - more resources