Antibacterial Activity of Bifidobacterium breve Against Clostridioides difficile

ORIGINAL RESEARCH published: 07 August 2019 doi: 10.3389/fcimb.2019.00288 Antibacterial Activity of Bifidobacterium breve Against Clostridioides difficile Jingpeng Yang and Hong Yang* State Key Laboratory of Microbial Metabolism, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, China Edited by: Purna Chandra Kashyap, Mayo Clinic, United States Reviewed by: Joseph Sorg, Texas A&M University, United States Nick Wheelhouse, Edinburgh Napier University, United Kingdom *Correspondence: Hong Yang Specialty section: This article was submitted to Microbiome in Health and Disease, a section of the journal Frontiers in Cellular and Infection Microbiology Received: 13 May 2019 Accepted: 26 July 2019 Published: 07 August 2019 Citation: Yang J and Yang H (2019) Antibacterial Activity of Bifidobacterium breve Against Clostridioides difficile. Front. Cell. Infect. Microbiol. 9:288. doi: 10.3389/fcimb.2019.00288 Bifidobacterium breve (YH68) is widely used in the fields of food fermentation and biomedicine. In this study, we explored the antibacterial activity of the cell free culture supernatant (CFCS) of YH68 against Clostridioides difficile ATCC 9689 (CD) by measuring multiple indexes, including the growth, spores production, toxin A/B production, and the expression levels of the tcdA and tcdB genes of CD. In addition, we examined the changes in major cellular functional groups, structures, permeability, integrity, and the proton motive force (PMF) of the cytoplasmic membrane. The results showed that double-dilution ratio of YH68-CFCS (3 × 109 CFU/mL) was the MIC value. The cell density, spores production, and the toxin production of CD treated with YH68-CFCS were lower than that of the control (p < 0.05). In addition, the gene expression levels of tcdA and tcdB in CD treated with YH68-CFCS were significant downregulated (p < 0.05). Marked differences were observed in the cell membrane and cell wall by a FT-IR spectroscopy and SEM. Analysis of the cell membrane permeability and integrity of the CD cells revealed that YH68-CFCS induced the leakage of a large amount of intracellular K+ , inorganic phosphate, ATP, nucleic acids and proteinaceous substances. Furthermore, PMF analysis indicated that there was a significant change in 1ψ and 1pH. These findings demonstrated that the antibacterial activity of YH68-CFCS against CD involved the inhibition of growth, spore production, toxin production, and virulence genes expression; a consumption of PMF in the cytoplasmic membrane, the formation of pore in the cell membrane, together with the enhanced cell membrane permeability; and, eventually, cell completely disintegration. Keywords: Bifidobacterium breve, Clostridioides difficile, toxin production, gene expression, cytoplasmic membrane INTRODUCTION Clostridioides difficile (previously known as C. difficile) (Oren and Garrity, 2016) is gram-positive anaerobic bacterium that can produce spores, accompanied by an unique off-odor (Smits et al., 2016). C. difficile is a conditionally pathogenic bacterium, and C. difficile-induced infection (CDI) accounts for 30% of antibiotic-associated diarrhea (AAD), with manifestations varying from mild diarrhea to severe complications associated with pseudomembranous colitis, toxic megacolon and death (Gao et al., 2010; Xu et al., 2017). Metronidazole and vancomycin are often used to treat CDI, but these use of these drugs leads to serious problem such as destruction of the gut microbiota, Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 1 August 2019 | Volume 9 | Article 288 Yang and Yang Bifidobacterium breve Against Clostridioides difficile broth supplemented with 0.05% (w/v) L-cysteine (MRSC) broth for 24–48 h at 37◦ C anaerobically (AnaeroGenTM, Oxoid Ltd., Basingstoke, UK). development of multidrug-resistant strains, and recurrence of infection (rCDI) (Alcalá Hernández et al., 2017; Peng et al., 2018). Recently, several new therapeutic strategies have emerged, such as fecal microbiota transplantation (FMT) for reconstruction of the gut microbiota (Juul et al., 2018), the use of octahedron iron oxide nanocrystals (Fe3−δ O4 ) to prevent C. difficile spore germination (Lee et al., 2017), the use of Manuka honey to suppress C. difficile biofilm formation (Piotrowski et al., 2017), and the construction of genetically engineered bacteria to target virulence genes (Saeidi et al., 2011; Bender et al., 2015); nevertheless, the use of these new therapeutic methods by the public is not widespread due to a lack of safety assessment or high cost of production. Therefore, new alternative treatments that can be easily recognized and adopted for clinical therapy are urgently required. A large amount of clinical data has shown that some probiotics can affect CDI therapy (Mantegazza et al., 2017; Shen et al., 2017). Much attention has thus been paid to probiotics due to their great potential, particularly in medicinal applications. In fact, it has been well-known for a long time that some kinds of probiotics, such as lactic acid bacteria (LAB) and bifidobacteria, play essential roles in food fermentation, where these bacteria contribute to not only the development of the desired sensory properties in the final product but also inhibition of harmful microbial contamination (Smaoui et al., 2010). The use of probiotics to treat some diseases is becoming very popular, and CDI is one of these such disease that is being targeted. However, the antibacterial activity of specific probiotics against C. difficile has remained largely unknown to date, and thus, many physicians are skeptical of this probiotic therapy. Previous studies have indicated that some probiotics, such as Lactobacillus helveticus, Lactobacillus fermentum, Streptococcus thermophilus, Bifidobacterium longum, and Pediococcus pentosaceus inhibited C. difficile in vitro and in vivo. The antibacterial effect of these strains against C. difficile was mainly reflected in the inhibition of growth, suppression of sporulation, and degradation of toxin (Golic et al., 2017; Wei et al., 2018; Xu et al., 2018). However, few of investigation have focused on changes at the cellular level of C. difficile. In this study, C. difficile ATCC 9689 (CD) was treated with the cell free culture supernatant of Bifidobacterium breve (YH68-CFCS). A comprehensive investigation was carried on to explore the growth, spore formation, toxin production, and virulence gene expression level, especially the changes in the proton motive force (PMF), permeability, and integrity of the cytoplasmic membrane of CD, in order to illustrate the antibacterial activity of YH68-CFCS against CD in vitro. Inhibition Zone The cell pellets and cell-free culture supernatant (CFCS) of YH68 were collected by centrifugation at 12,000 r/min for 10 min, and the cell pellets were then resuspended in sterile saline. The antibacterial activities of the cell pellets and CFCS of YH68 against CD were t (...truncated)