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. 2022 Oct 18;10(10):2056.
doi: 10.3390/microorganisms10102056.

Screening of Bacteria Inhibiting Clostridium perfringens and Assessment of Their Beneficial Effects In Vitro and In Vivo with Whole Genome Sequencing Analysis

Zipeng Jiang  1   2   3   4   5 , Weifa Su  1   2   3   4   5 , Mingzhi Yang  1   2   3   4   5 , Wentao Li  1   2   3   4   5 , Tao Gong  1   2   3   4   5 , Yu Zhang  1   2   3   4   5 , Chaoyue Wen  1   2   3   4   5 , Xinxia Wang  1   2   3   4   5 , Yizhen Wang  1   2   3   4   5 , Mingliang Jin  1   2   3   4   5 , Zeqing Lu  1   2   3   4   5
Affiliations

Screening of Bacteria Inhibiting Clostridium perfringens and Assessment of Their Beneficial Effects In Vitro and In Vivo with Whole Genome Sequencing Analysis

Zipeng Jiang et al. Microorganisms. .

Abstract

Various countries and organizations call for banning the use of antibiotic growth promoters (AGPs) as prophylaxis and for growth promotion in the livestock industry. Hence, seeking a substitute for antibiotics is strongly required by the livestock industry to maintain the productivity level and profits. Probiotics could represent one viable solution because of their beneficial effects on host health and maintaining the intestinal microbiota balance. In the present study, we aimed to isolate bacterial strains with probiotics properties from JinHua pig (a Chinese native pig breed) gastrointestinal tract that have antagonistic activity against to common disease-causing bacteria on farms. The four most potent strains were isolated (PP31, BA11, BA40, BV5) by the agar well diffusion method and further characterized by acid, bile salt, trypsin tolerance, whole genome sequencing (WGS), and suppressing Clostridium perfringens adhesion to IPEC-J2 cells. According to these results, BA40 had the highest number and variety of probiotic secondary metabolic secretion genes and capacity to exclude the attachment of Clostridium perfringens to IPEC-J2 cells as same as PB6. The animal experiment in vivo illustrated that BA40 and PB6 could reduce the phenomenon induced by Clostridium perfringens challenge of body weight loss, colon length decrease, pro-inflammatory cytokine increase, and Clostridium perfringens and Escherichia coli increase. The present study provides evidence that BA40 could represent a novel probiotic candidate as PB6, which exhibited some probiotic features and mitigated the burden of Clostridium perfringens associated gut disease.

Keywords: JinHua pig; gut microbiota; immunity; mice model; probiotics; whole genome sequencing.

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Conflict of interest statement

The authors declare that there is no conflict of interest.

Figures

Figure 1
Figure 1
Experimental design.
Figure 2
Figure 2
Agar well diffusion assay illustrating the growth inhibition of pathogenic bacteria by cell-free supernatants extracted from the isolated strains. (A) Escherichia coli plate. (B) Salmonella enterica plate. (C) Staphylococcus aureus plate. (D) Clostridium perfringens plate. (E) Inhibition zone diameter of Escherichia coli (F) Inhibition zone diameter of Salmonella enterica (G) Inhibition zone diameter of Staphylococcus aureus (H) Inhibition zone diameter of Clostridium perfringens. a, b, c Means values with dissimilar letters were significantly different (p < 0.05). All values contained three repetitions.
Figure 3
Figure 3
Probiotic properties of isolated strains. (A) The growth curves. (B) The ability of acid tolerance. (C) The ability of bile salt tolerance. (D) The ability of trypsin tolerance. a, b, c, d Means values with dissimilar letters were significantly different (p < 0.05). All values contained three repetitions.
Figure 4
Figure 4
The inhibitory results of isolate strains on the adhesion of Clostridium perfringens. a, b, c Means values with dissimilar letters were significantly different (p < 0.05). All values contained three repetitions.
Figure 5
Figure 5
Annotated chord diagrams based on database matching for classification. (A) Correspondence between annotated information on bacterial genomes and metabolic pathways obtained by KEGG database. (B) The COG database was compared to classify the predicted proteins into gene families and to give the corresponding functional annotation information for the family.
Figure 6
Figure 6
The protective effect of probiotic candidates against Clostridium perfringens infection in mice. (A) Bodyweight (BW). (B) At the end of experiment, mice weight. (C) The spleen index. (D) The liver index. (E) The colon images (F) The colon length. a, b, c, d Means values with dissimilar letters were significantly different (p < 0.05). All values contained six repetitions. Partial data of this figure were published in previous study. Adapted with permission from ref. [29]. Copyright 2021 Jiang, Li, Su, Wen, Gong, Zhang, Wang, Jin and Lu.
Figure 7
Figure 7
The population of intestinal microbiota of mice. (A) Clostridium perfringens in the ileum. (B) Clostridium perfringens in the cecum. (C) Escherichia coli in the ileum. (D) Escherichia coli in the cecum. (E) Lactobacillus in the ileum. (F) Lactobacillus in the cecum. Results are presented as mean ± SD (The data were presented as log10 gene copies/g of intestinal digesta). a, b, c, d Means values with dissimilar letters were significantly different (p < 0.05).
Figure 8
Figure 8
The effect of isolated probiotics treatment on inflammatory cytokines, immunoglobulin, DAO and DLA in mice. (A) IL-1β concentrations. (B) IL-6 concentrations. (C) TNF-α concentrations. (D) IgA concentrations. (E) IgG concentrations. (F) sIgA concentrations. (G) iNOS concentrations. (H) NO concentrations. (I) DAO concentrations. (J) DLA concentrations. Results are presented as mean ± SD. a, b, c, d Means values with dissimilar letters were significantly different (p < 0.05). All values contained six repetitions.
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