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. 2017 Apr 21:8:625.
doi: 10.3389/fmicb.2017.00625. eCollection 2017.

Protective Effect of Carvacrol against Gut Dysbiosis and Clostridium difficile Associated Disease in a Mouse Model

Shankumar Mooyottu  1 , Genevieve Flock  1 , Abhinav Upadhyay  1 , Indu Upadhyaya  1 , Kendra Maas  2 , Kumar Venkitanarayanan  1
Affiliations
Free PMC article

Protective Effect of Carvacrol against Gut Dysbiosis and Clostridium difficile Associated Disease in a Mouse Model

Shankumar Mooyottu et al. Front Microbiol. .
Free PMC article

Abstract

This study investigated the effect of carvacrol (CR), a phytophenolic compound on antibiotic-associated gut dysbiosis and C. difficile infection in a mouse model. Five to six-week-old C57BL/6 mice were randomly divided into seven treatment groups (challenge and control) of eight mice each. Mice were fed with irradiated feed supplemented with CR (0, 0.05, and 0.1%); the challenge groups were made susceptible to C. difficile by orally administering an antibiotic cocktail in water and an intra-peritoneal injection of clindamycin. Both challenge and control groups were infected with 105CFU/ml of hypervirulent C. difficile (ATCC 1870) spores or PBS, and observed for clinical signs for 10 days. Respective control groups for CR, antibiotics, and their combination were included for investigating their effect on mouse enteric microflora. Mouse body weight and clinical and diarrhea scores were recorded daily post infection. Fecal samples were collected for microbiome analysis using rRNA sequencing in MiSeq platform. Carvacrol supplementation significantly reduced the incidence of diarrhea and improved the clinical and diarrhea scores in mice (p < 0.05). Microbiome analysis revealed a significant increase in Proteobacteria and reduction in the abundance of protective bacterial flora in antibiotic-treated and C. difficile-infected mice compared to controls (p < 0.05). However, CR supplementation positively altered the microbiome composition, as revealed by an increased abundance of beneficial bacteria, including Firmicutes, and significantly reduced the proportion of detrimental flora such as Proteobacteria, without significantly affecting the gut microbiome diversity compared to control. Results suggest that CR could potentially be used to control gut dysbiosis and reduce C. difficile infection.

Keywords: Clostridium difficile; carvacrol; gut dysbiosis; microbiome; mouse model.

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Figures

Figure 1
Figure 1
Effect of CR supplementation on the incidence of C. difficile associated diarrhea in mice. The incidence of diarrhea in different treatement groups was recorded after C. difficile challenge. Groups: (1) Negative Control: Mice treated with no CR, no antibiotics and no C. difficile (2) CR control: Mice fed with 0.1% CR in feed, (3) Ant Control: Mice administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, (4) Ant + CR Control: Mice fed with CR (0.1%) supplemented feed and administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, (5) Ant + CD: administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, and infected by C. difficile (6) (Ant + CD + 0.05% CR): Mice fed with CR (0.05%), administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, and infected with C. difficile) (Ant + CD + 0.1% CR): Mice fed with CR (0.1%), administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, and infected with C. difficile. (* treatments significantly differed from infected control group (Ant + CD) p < 0.05; m indicates the number of mortalities recorded; cumulative incidence of diarrhea per total number of animals used in the experiment is shown in parenthesis).
Figure 2
Figure 2
Effect of CR supplementation on the severity of C. difficile associated disease in mice. The severeity of C. difficile associated disease in different treatment groups was determined based on a clinical score sheet. Groups: (1) Negative Control: Mice treated with no CR, no antibiotics and no C. difficile (2) CR control: Mice fed with 0.1% CR in feed, (3) Ant Control: Mice administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, (4) Ant + CR Control: Mice fed with CR (0.1%) supplemented feed and administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, (5) Ant + CD: administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, and infected by C. difficile 6) (Ant + CD + 0.05% CR): Mice fed with CR (0.05%), administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, and infected with C. difficile) (Ant + CD + 0.1% CR): Mice fed with CR (0.1%), administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, and infected with C. difficile. (*The clinincal scores of positive control group (Ant + CD were significantly greater than that of Ant + CD + CR 0.1% and control groups, p < 0.05).
Figure 3
Figure 3
Effect of CR supplementation on relative weight loss in C. difficile infected and non-infected mice. The body weights of the animals were recorded daily and the relative percentage weight with respect to the initial weight prior to the infection was calculated. Groups: (1) Negative Control: Mice treated with no CR, no antibiotics and no C. difficile (2) CR control: Mice fed with 0.1% CR in feed, (3) Ant Control: Mice administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, (4) Ant + CR Control: Mice fed with CR (0.1%) supplemented feed and administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, (5) Ant + CD: administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, and infected by C. difficile (6) (Ant + CD + 0.05% CR): Mice fed with CR (0.05%), administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, and infected with C. difficile) (Ant + CD + 0.1% CR): Mice fed with CR (0.1%), administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, and infected with C. difficile. (*The relative weight loss of positive control group (Ant + CR) was significantly greater than Ant + CD + CR 0.05%, Ant + CD + CR 0.1% and control groups, p < 0.05).
Figure 4
Figure 4
Effect of CR supplementation on the abundance of major gut microbiota (phyla level) in the antibiotic treated and C. difficile challenged mice. The relative abundance of OTUs at different taxonomic levels was determined by gut microbiome analysis. Groups: (1) Negative Control: Mice treated with no CR, no antibiotics and no C. difficile (2) CR control: Mice fed with 0.1% CR in feed, (3) Ant Control: Mice administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, (4) Ant + CR Control: Mice fed with CR (0.1%) supplemented feed and administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, (5) Ant + CD: administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, and infected by C. difficile (6) (Ant + CD + 0.05% CR): Mice fed with CR (0.05%), administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, and infected with C. difficile) (Ant + CD + 0.1% CR): Mice fed with CR (0.1%), administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, and infected with C. difficile.
Figure 5
Figure 5
Effect of CR supplementation on the abundance of Enterobacteriaceae, Lactobacillaceae and Lachnospiraceae in the antibiotic treated and C. difficile challenged mice. The relative abundance of OTUs at family level (Family. Enterobacteriaceae, Lactobacillaceae and Lachnospraceae) was by determined using gut microbiome analysis. Groups: (1) Negative Control: Mice treated with no CR, no antibiotics and no C. difficile (2) CR control: Mice fed with 0.1% CR in feed, (3) Ant Control: Mice administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, (4) Ant + CR Control: Mice fed with CR (0.1%) supplemented feed and administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, (5) Ant + CD: administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, and infected by C. difficile (6) (Ant + CD + 0.05% CR): Mice fed with CR (0.05%), administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, and infected with C. difficile (7) (Ant + CD + 0.1% CR): Mice fed with CR (0.1%), administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, and infected with C. difficile. (Treatments significantly differed from respective control groups, p < 0.05).
Figure 6
Figure 6
Effect of CR supplementation on the diversity of gut microbiota of antibiotic treated and C. difficile challenged mice. Alpha diversity of the gut microbiome was calculated by using inverse Simpson to measure the richness and evenness of the OTUs. Groups: (1) Negative Control: Mice treated with no CR, no antibiotics and no C. difficile (2) CR control: Mice fed with 0.1% CR in feed, (3) Ant Control: Mice administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, (4) Ant + CR Control: Mice fed with CR (0.1%) supplemented feed and administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, (5) Ant + CD: administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, and infected by C. difficile (6) (Ant + CD + 0.05% CR): Mice fed with CR (0.05%), administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, and infected with C. difficile (7) (Ant + CD + 0.1% CR): Mice fed with CR (0.1%), administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, and infected with C. difficile.
Figure 7
Figure 7
Effect of CR supplementation on the diversity of gut microbiota of antibiotic treated and C. difficile challenged mice. Relationships between treatment groups based on the abundance of species present in each sample were plotted. Groups: (1) Negative Control: Mice treated with no CR, no antibiotics and no C. difficile (2) CR Control: Mice fed with 0.1% CR in feed, (3) Ant Control: Mice administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, (4) Ant + CR Control: Mice fed with CR (0.1%) supplemented feed and administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, (5) Ant + CD: administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, and infected by C. difficile (6) (Ant + CD + 0.05% CR): Mice fed with CR (0.05%), administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, and infected with C. difficile (7) (Ant + CD + 0.1% CR): Mice fed with CR (0.1%), administered with antibiotic cocktail in water and an intra-peritoneal injection of clindamycin, and infected with C. difficile. Circle indicates close clustering of CR Control samples and Nagative Control samples.
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