Bile Salt Hydrolase Activities: A Novel Target to Screen Anti- Giardia Lactobacilli?

Abstract

Giardia duodenalis is a protozoan parasite responsible for giardiasis, a disease characterized by intestinal malabsorption, diarrhea and abdominal pain in a large number of mammal species. Giardiasis is one of the most common intestinal parasitic diseases in the world and thus a high veterinary, and public health concern. It is well-established that some probiotic bacteria may confer protection against this parasite in vitro and in vivo and we recently documented the implication of bile-salt hydrolase (BSH)-like activities from strain La1 of Lactobacillus johnsonii as mediators of these effects in vitro. We showed that these activities were able to generate deconjugated bile salts that were toxic to the parasite. In the present study, a wide collection of lactobacilli strains from different ecological origins was screened to assay their anti-giardial effects. Our results revealed that the anti-parasitic effects of some of the strains tested were well-correlated with the expression of BSH-like activities. The two most active strains in vitro, La1 and Lactobacillus gasseri CNCM I-4884, were then tested for their capacity to influence G. duodenalis infection in a suckling mice model. Strikingly, only L. gasseri CNCM I-4884 strain was able to significantly antagonize parasite growth with a dramatic reduction of the trophozoites load in the small intestine. Moreover, this strain also significantly reduced the fecal excretion of Giardia cysts after 5 days of treatment, which could contribute to blocking the transmission of the parasite, in contrast of La1 where no effect was observed. This study represents a step toward the development of new prophylactic strategies to combat G. duodenalis infection in both humans and animals.

Keywords: Giardia duodenalis; Lactobacillus gasseri; Lactobacillus johnsonii; bile salt hydrolases; lactobacilli; probiotics.

FIGURE 1

Bile-salt hydrolase (BSH) and anti-Giardia in vitro activities of different lactobacilli strains. (A) Percentage of living Giardia duodenalis trophozoites when cultivated 22 h with lactobacilli supernatants. G. duodenalis trophozoites were enumerated after 22 h of co-incubation at 37°C in anaerobic conditions (values are represented with bile supplementation). Values are mean ± SEM. (B) Spearman’s rank correlation test between percentage of inhibition of lactobacilli strains and their BSH activity score. BSH score was determined depending on the size of the halo zone (+ = 1; ++ = 2; +++ = 3, Table 1) and the ability to deconjugate either tauro-conjugated bile salts or glyco-conjugated bile salts or both (addition of BSH score for each substrate specificity). A positive correlation is observed (r = 0.86; p < 0.0001). (C) Inhibition assays according to Bile Salt Hydrolase activities. Lactobacilli strains were divided into two groups: (i) BSH negative (strains with no detected BSH activity), (ii) BSH positive (strains exhibiting either TDCA, GDCA, or both BSH activities). G. duodenalis trophozoites were enumerated after 22 h of co-incubation at 37°C in anaerobic conditions (values are represented with bile supplementation). Values are in mean ± SEM. (D) Percentage of living G. duodenalis trophozoites when co-cultivated 22 h with lactobacilli strains supernatants, with or without bile supplementation (bovine bile 0.6 g/L). G. duodenalis trophozoites were enumerated after 22 h of co-incubation at 37°C in anaerobic conditions. Values are mean ± SEM. ^∗∗p ≤ 0.01; ^∗∗∗p ≤ 0.001.

FIGURE 2

Experimental design of G. duodenalis infection in OF1 suckling mice model. Lactobacilli strains were administered daily by intragastric gavage (5 × 10⁸ CFU) to 5 days old OF1 suckling mice from day 5 to day 15. Control animals received PBS/glycerol 15%. Mice were challenged with G. duodenalis WB6 trophozoites (10⁵) at day 10 by intragastric gavage. Mice were sacrificed by cervical dislocation at day 16.

FIGURE 3

In vivo activities of Lactobacillus gasseri CNCM I-4884 and La1 lactobacilli strains against G. duodenalis. (A) G. duodenalis trophozoites enumeration after administration to animals. Suckling mice received either PBS (n = 8), La1 (n = 12), L. gasseri CNCM I-4884 (n = 10) or L. curvatus CNRZ1335 (n = 9) by intragastric gavage (5 × 10⁸ CFU/mice) daily from day 5, before inoculation with G. duodenalis WB6 trophozoites (10⁵ trophozoites per animal) at day 10. Gavages were performed until day 15. Small intestines were resuspended in PBS and trophozoites were counted using a hemocytometer. Values are mean ± SEM; p < 0.05. (B) G. duodenalis cysts counting in colon and caecum. Suckling mice received either PBS/glycerol 15%, La1, L. gasseri CNCM I-4884 or L. curvatus CNRZ1335 by intragastric gavage (5 × 10⁸ CFU/mice) daily from day 5 before inoculation with G. duodenalis WB6 trophozoites (10⁵ trophozoites per animal) at day 10. Gavages were performed until day 15 (n = 8–12/group). Colons and caeca were resuspended in 5 ml of ice-chilled 2.5% potassium dichromate and cysts were counted using a hemocytometer. Values are mean ± SEM. ^∗∗p ≤ 0.01; ^∗∗∗p ≤ 0.001.