Spore Preparation Protocol for Enrichment of Clostridia from Murine Intestine

Velazquez, Eric M.; Rivera-Chávez, Fabian; Bäumler, Andreas J.

doi:10.21769/bioprotoc.2296

Cited by 3 publications

(5 citation statements)

References 4 publications

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“…To further understand the microbial trigger in a symbiotic community leading to Rdh7 regulation, we isolated spore- forming bacteria (Figure 3E) by treating mouse feces with chloroform as it has been previously described (Velazquez et al, 2017). GF mice were inoculated with the fecal spore preparation and after 2 weeks, 16S fecal microbiome analysis confirmed that these mice were colonized specifically with spore-forming bacteria belonging to class Clostridia (Figure 3F).…”

Section: Resultsmentioning

confidence: 99%

“…To enrich for spore-forming bacteria, we followed Velazquez et.al`s protocol (Velazquez et al, 2017), where cecum content of female mice were extracted and treated with 3% chloroform in order to kill all vegetative bacteria, except for spores. Once the aqueous layer containing the spores was removed, germ-free mice were gavaged with 200µl of the same.…”

Section: Star Methodsmentioning

confidence: 99%

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Commensals Suppress Intestinal Epithelial Cell Retinoic Acid Synthesis to Regulate Interleukin-22 Activity and Prevent Microbial Dysbiosis

et al. 2018

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Summary Retinoic acid (RA), a vitamin A metabolite, regulates transcriptional programs that drive protective or pathogenic immune responses in the intestine, in a manner dependent on RA concentration. Vitamin A is obtained from diet and is metabolized by intestinal epithelial cells (IECs), which operate in intimate association with microbes and immune cells. Here we found that commensal bacteria belonging to class Clostridia modulate RA concentration in the gut by suppressing the expression of retinol dehydrogenase 7 (Rdh7) in IECs. Rdh7 expression and associated RA amounts were lower in the intestinal tissue of conventional mice, as compared to germ-free mice. Deletion of Rdh7 in IECs diminished RA signaling in immune cells, reduced the IL-22-dependent antimicrobial response, and enhanced resistance to colonization by Salmonella Typhimurium. Our findings define a regulatory circuit wherein bacterial regulation of IEC-intrinsic RA synthesis protects microbial communities in the gut from excessive immune activity, achieving a balance that prevents colonization by enteric pathogens.

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Section: Resultsmentioning

confidence: 99%

Section: Star Methodsmentioning

confidence: 99%

Commensals Suppress Intestinal Epithelial Cell Retinoic Acid Synthesis to Regulate Interleukin-22 Activity and Prevent Microbial Dysbiosis

et al. 2018

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“…The prepared cecal contents were next incubated by shaking at 200 × g at 37 °C for 30 min. After allowing the chloroform to settle to the bottom of the tube at room temperature (approximately 20 min), the top aqueous layer was removed and the SFB was resuspended with 200 μL PBS in a sterile 1.5 mL tube 35,49 . For transplantation, mice were pretreated with vancomycin for 14 days and then treated with 200 μL SFB daily for consecutive 7 days.…”

Section: Bacterial Spore Preparation and Transplantationmentioning

confidence: 99%

Gut microbiota-mediated secondary bile acid alleviates Staphylococcus aureus-induced mastitis through the TGR5-cAMP-PKA-NF-κB/NLRP3 pathways in mice

Zhao

Hao

et al. 2023

npj Biofilms Microbiomes

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Although emerging evidence shows that gut microbiota-mediated metabolic changes regulate intestinal pathogen invasions, little is known about whether and how gut microbiota-mediated metabolites affect pathogen infection in the distal organs. In this study, untargeted metabolomics was performed to identify the metabolic changes in a subacute ruminal acidosis (SARA)-associated mastitis model, a mastitis model with increased susceptibility to Staphylococcus aureus (S. aureus). The results showed that cows with SARA had reduced cholic acid (CA) and deoxycholic acid (DCA) levels compared to healthy cows. Treatment of mice with DCA, but not CA, alleviated S. aureus-induced mastitis by improving inflammation and the blood-milk barrier integrity in mice. DCA inhibited the activation of NF-κB and NLRP3 signatures caused by S. aureus in the mouse mammary epithelial cells, which was involved in the activation of TGR5. DCA-mediated TGR5 activation inhibited the NF-κB and NLRP3 pathways and mastitis caused by S. aureus via activating cAMP and PKA. Moreover, gut-dysbiotic mice had impaired TGR5 activation and aggravated S. aureus-induced mastitis, while restoring TGR5 activation by spore-forming bacteria reversed these changes. Furthermore, supplementation of mice with secondary bile acids producer Clostridium scindens also activated TGR5 and alleviated S. aureus-induced mastitis in mice. These results suggest that impaired secondary bile acid production by gut dysbiosis facilitates the development of S. aureus-induced mastitis and highlight a potential strategy for the intervention of distal infection by regulating gut microbial metabolism.

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“…A recent study shows that acquisition of Clostridia species by neonatal mice confers niche protection against S. Typhimurium infection (Kim et al, 2017). Since Clostridia belong to the phylum Firmicutes, a group of spore-forming bacteria, we killed vegetative bacterial cells in the adult chicken microbiota transplant using chloroform treatment (Velazquez et al, 2017). The chloroform-treated microbiota transplant was then used to inoculate germ-free mice and microbiota profiling was performed 7 days later on cecal contents to determine its composition.…”

Section: Spore-forming Bacteria and Enterobacteriaceae Confer Niche Pmentioning

confidence: 99%

“…The spore component was produced by chloroform treatment as described previously (Velazquez et al, 2017). Briefly, the cecal content was incubated for 30 min with 3% chloroform.…”

Section: Isolation and Fractionation Of Chicken Microbiotamentioning

confidence: 99%

Commensal Enterobacteriaceae Protect against Salmonella Colonization through Oxygen Competition

Litvak

Mon

Nguyen

et al. 2019

Cell Host & Microbe

205

118

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Article Commensal Enterobacteriaceae Protect against Salmonella Colonization through Oxygen Competition Graphical Abstract Highlights d S. Enteritidis virulence factors increase epithelial oxygenation in neonatal chick guts d S. Enteritidis expands in the neonatal chick gut through aerobic respiration d Spore-forming bacteria and commensal Enterobacteriaceae confer niche protection d E. coli requires aerobic respiration to block S. Enteritidis colonization SUMMARYNeonates are highly susceptible to infection with enteric pathogens, but the underlying mechanisms are not resolved. We show that neonatal chick colonization with Salmonella enterica serovar Enteritidis requires a virulence-factor-dependent increase in epithelial oxygenation, which drives pathogen expansion by aerobic respiration. Co-infection experiments with an Escherichia coli strain carrying an oxygen-sensitive reporter suggest that S. Enteritidis competes with commensal Enterobacteriaceae for oxygen. A combination of Enterobacteriaceae and spore-forming bacteria, but not colonization with either community alone, confers colonization resistance against S. Enteritidis in neonatal chicks, phenocopying germ-free mice associated with adult chicken microbiota. Combining spore-forming bacteria with a probiotic E. coli isolate protects germ-free mice from pathogen colonization, but the protection is lost when the ability to respire oxygen under micro-aerophilic conditions is genetically ablated in E. coli. These results suggest that commensal Enterobacteriaceae contribute to colonization resistance by competing with S. Enteritidis for oxygen, a resource critical for pathogen expansion.

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Spore Preparation Protocol for Enrichment of Clostridia from Murine Intestine

Cited by 3 publications

References 4 publications

Commensals Suppress Intestinal Epithelial Cell Retinoic Acid Synthesis to Regulate Interleukin-22 Activity and Prevent Microbial Dysbiosis

Commensals Suppress Intestinal Epithelial Cell Retinoic Acid Synthesis to Regulate Interleukin-22 Activity and Prevent Microbial Dysbiosis

Gut microbiota-mediated secondary bile acid alleviates Staphylococcus aureus-induced mastitis through the TGR5-cAMP-PKA-NF-κB/NLRP3 pathways in mice

Commensal Enterobacteriaceae Protect against Salmonella Colonization through Oxygen Competition

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