Garlic Essential Oil as Promising Option for the Treatment of Acute Campylobacteriosis-Results from a Preclinical Placebo-Controlled Intervention Study

Abstract

Since human infections with Campylobacter jejuni including antibiotic-resistant strains are rising worldwide, natural compounds might constitute promising antibiotics-independent treatment options for campylobacteriosis. Since the health-beneficial properties of garlic have been known for centuries, we here surveyed the antimicrobial and immune-modulatory effects of garlic essential oil (EO) in acute experimental campylobacteriosis. Therefore, secondary abiotic IL-10^-/- mice were orally infected with C. jejuni strain 81-176 and garlic-EO treatment via the drinking water was initiated on day 2 post-infection. Mice from the garlic-EO group displayed less severe clinical signs of acute campylobacteriosis as compared to placebo counterparts that were associated with lower ileal C. jejuni burdens on day 6 post-infection. Furthermore, when compared to placebo application, garlic-EO treatment resulted in alleviated colonic epithelia cell apoptosis, in less pronounced C. jejuni induced immune cell responses in the large intestines, in dampened pro-inflammatory mediator secretion in intestinal and extra-intestinal compartments, and, finally, in less frequent translocation of viable pathogens from the intestines to distinct organs. Given its potent immune-modulatory and disease-alleviating effects as shown in our actual preclinical placebo-controlled intervention study, we conclude that garlic-EO may be considered as promising adjunct treatment option for acute campylobacteriosis in humans.

Keywords: Campylobacter jejuni; acute campylobacteriosis model; enteropathogenic infection; garlic essential oil; host-pathogen interaction; immune-modulatory effects; natural antibiotics-independent compounds; preclinical placebo-controlled intervention study; secondary abiotic IL-10-/- mice.

Figure 1

Pathogenic colonization of the gastrointestinal tract upon garlic essential oil treatment of C. jejuni infected IL-10^-/- mice. Secondary abiotic IL-10^-/- mice were orally challenged with C. jejuni strain 81-176 on days 0 and 1 followed by treatment with either garlic essential oil (white boxes) or placebo (PLC, gray boxes) via the drinking water starting on day 2 post-infection. On day 6 post-infection, C. jejuni was quantitated in defined gastrointestinal luminal samples by culture (colony forming units per gram; CFU/g). Box plots (indicating the 25th and the 75th percentiles), whiskers (indicating maximum and minimum values), medians (black bar inside box), significance levels (p values) determined by the Mann-Whitney U test and the total number of mice (in parentheses) are shown (pooled data from four independent experiments).

Figure 2

Time course of clinical conditions upon garlic essential oil treatment of C. jejuni infected IL-10^-/- mice. Secondary abiotic IL-10^-/- mice were orally challenged with C. jejuni strain 81-176 on day (d) 0 and d1 followed by treatment with either garlic essential oil (white boxes) or placebo (PLC, gray boxes) via the drinking water starting on d2 post-infection. The clinical conditions of mice were quantitated by using distinct clinical scores (see methods). Naive mice (hatched boxes) served as non-infected and untreated cohort. Box plots (indicating the 25th and the 75th percentiles), whiskers (indicating maximum and minimum values), medians (black bar inside box), significance levels (p values) determined by the Kruskal-Wallis test and Dunn’s post-correction and the number of analyzed animals (in parentheses) are shown (pooled data from four independent experiments).

Figure 3

Microscopic inflammatory sequelae upon garlic essential oil treatment of C. jejuni infected IL-10^-/- mice. Secondary abiotic IL-10^-/- mice were orally challenged with C. jejuni strain 81-176 on days 0 and 1 followed by treatment with either garlic essential oil (white boxes) or placebo (PLC, gray boxes) via the drinking water starting on day 2 post-infection. On day 6 post-infection, (A) microscopic inflammatory changes were assessed in colonic ex vivo biopsies applying histopathological scores. Furthermore, (B) the mean numbers of apoptotic colonic epithelial cells out of six high power fields (HPF, 400× magnification) were enumerated microscopically in paraffin sections that had been stained with an antibody against cleaved caspase3 (Casp3⁺). Naive mice (hatched boxes) served as non-infected and untreated cohort. Box plots (indicating the 25th and the 75th percentiles), whiskers (indicating maximum and minimum values), medians (black bar inside box), significance levels (p values) determined by the Kruskal-Wallis test and Dunn’s post-correction and the total number of mice (in parentheses) are shown (pooled data from four independent experiments).

Figure 4

Colonic immune cell responses upon garlic essential oil treatment of C. jejuni infected IL-10^-/- mice. Secondary abiotic IL-10^-/- mice were orally challenged with C. jejuni strain 81-176 on days 0 and 1 followed by treatment with either garlic essential oil (white boxes) or placebo (PLC, gray boxes) via the drinking water starting on day 2 post-infection. On day 6 post-infection, the mean numbers of (A) macrophages and monocytes (positive for F4/80) and of (B) T lymphocytes (positive for CD3) out of six high power fields (HPF, 400× magnification) were enumerated microscopically in paraffin sections following immunohistochemical staining. Naive mice (hatched boxes) served as non-infected and untreated cohort. Box plots (indicating the 25th and the 75th percentiles), whiskers (indicating maximum and minimum values), medians (black bar inside box), significance levels (p values) determined by the one-way ANOVA with Tukey post-correction and the Kruskal-Wallis test and Dunn’s post-correction, and the total number of mice (in parentheses) are shown (pooled data from four independent experiments).

Figure 5

Pro-inflammatory mediator secretion in the colon upon garlic essential oil treatment of C. jejuni infected IL-10^-/- mice. Secondary abiotic IL-10^-/- mice were orally challenged with C. jejuni strain 81–176 on days 0 and 1 followed by treatment with either garlic essential oil (white boxes) or placebo (PLC, gray boxes) via the drinking water starting on day 2 post-infection. (A) Nitric oxide and (B) IL-6 concentrations were determined in culture supernatants of colonic explants taken on day 6 post-infection. Naive mice (hatched boxes) served as non-infected and untreated cohort. Box plots (indicating the 25th and the 75th percentiles), whiskers (indicating maximum and minimum values), medians (black bar inside box), significance levels (p values) determined by the Kruskal-Wallis test and Dunn’s post-correction and the total number of mice (in parentheses) are shown (pooled data from four independent experiments).

Figure 6

Pro-inflammatory mediator secretion in the ileum upon garlic essential oil treatment of C. jejuni infected IL-10^-/- mice. Secondary abiotic IL-10^-/- mice were orally challenged with C. jejuni strain 81–176 on days 0 and 1 followed by treatment with either garlic essential oil (white boxes) or placebo (PLC, gray boxes) via the drinking water starting on day 2 post-infection. (A) Nitric oxide, (B) IL-6, (C) MCP-1, (D) TNF-α and (E) IFN-γ concentrations were determined in culture supernatants of ileal explants taken on day 6 post-infection. Naive mice (hatched boxes) served as non-infected and untreated cohort. Box plots (indicating the 25th and the 75th percentiles), whiskers (indicating maximum and minimum values), medians (black bar inside box), significance levels (p values) determined by the Kruskal-Wallis test and Dunn’s post-correction and the total number of mice (in parentheses) are shown (pooled data from four independent experiments). Outliers were excluded after identification by the Grubb’s test (α = 0.001).

Figure 7

Pro-inflammatory mediator secretion in mesenteric lymph nodes upon garlic essential oil treatment of C. jejuni infected IL-10^-/- mice. Secondary abiotic IL-10^-/- mice were orally challenged with C. jejuni strain 81-176 on days 0 and 1 followed by treatment with either garlic essential oil (white boxes) or placebo (PLC, gray boxes) via the drinking water starting on day 2 post-infection. (A) Nitric oxide and (B) IFN-γ concentrations were determined in culture supernatants of mesenteric lymph nodes (MLN) explants taken on day 6 post-infection. Naive mice (hatched boxes) served as non-infected and untreated cohort. Box plots (indicating the 25th and the 75th percentiles), whiskers (indicating maximum and minimum values), medians (black bar inside box), significance levels (p values) determined by the Kruskal-Wallis test and Dunn’s post-correction and the total number of mice (in parentheses) are shown (pooled data from four independent experiments).

Figure 8

Extra-intestinal nitric oxide secretion upon garlic essential oil treatment of C. jejuni infected IL-10^-/- mice. Secondary abiotic IL-10^-/- mice were orally challenged with C. jejuni strain 81-176 on days 0 and 1 followed by treatment with either garlic essential oil (white boxes) or placebo (PLC, gray boxes) via the drinking water starting on day 2 post-infection. Nitric oxide concentrations were determined in culture supernatants of explants taken from the (A) liver, (B) kidney, (C) lung and (D) spleen on day 6 post-infection. Naive mice (hatched boxes) served as non-infected and untreated cohort. Box plots (indicating the 25th and the 75th percentiles), whiskers (indicating maximum and minimum values), medians (black bar inside box), significance levels (p values) determined by the Kruskal-Wallis test and Dunn’s post-correction and the total number of mice (in parentheses) are shown (pooled data from four independent experiments).

Figure 9

Bacterial translocation upon garlic essential oil treatment of C. jejuni infected IL-10^-/- mice. Secondary abiotic IL-10^-/- mice were orally challenged with C. jejuni strain 81–176 on days 0 and 1 followed by treatment with either garlic essential oil (white bars) or placebo (PLC, gray bars) via the drinking water starting on day 2 post-infection. Bacterial translocation frequencies (in %) were determined in explants taken from the mesenteric lymph nodes (MLN), liver, kidney, lung, and spleen and in cardiac blood (as indicated) on day 6 post-infection by culture. Naive mice (hatched bars) served as non-infected and untreated cohort. Bars indicating the translocation frequencies (in %) and the numbers of culture-positive out of the total number of analyzed samples (in parentheses) are shown (pooled data from four independent experiments).