Research Article: In vitro and ex vivo evaluation of the anti-Giardia duodenalis activity of the supernatant of Slab51 (SivoMixx)

Date Published: March 7, 2019

Publisher: Public Library of Science

Author(s): Stefania Perrucci, Gianluca Fichi, Enrica Ricci, Livio Galosi, Marco Lalle, Giacomo Rossi, Alessandro Giuffrè.

http://doi.org/10.1371/journal.pone.0213385

Abstract

The effects on Giardia duodenalis of Slab51 probiotic supernatants were evaluated in vitro and ex vivo. In vitro, Slab51 (101 UFC) was cultured and the obtained supernatant was filtered, adjusted at pH 7, and added (100μl/ml) as such (Slab51 FS) or after heat-treatment, to G. duodenalis cultures to evaluate its effects on G. duodenalis trophozoites growth and adherence. For comparison, negative and metronidazole (20μg/ml) treated controls were used. The morphological and ultrastructural alterations of G. duodenals trophozoites following treatment with Slab51 FS supernatant were investigated by transmission electron microscopy. Ex vivo, mice duodenal portions were cultivated in standard conditions with 5x105G. duodenalis trophozoites/ml, while to further five duodenal portions similarly cultured and infected, Slab51 FS 200μl was added. After 12 and 18h, samples were fixed in 10% buffered formalin and histologically processed to score Giardia infection and cell damage. Cell proliferation/apoptosis was scored by Ki67, TUNEL and Caspase–3 tests. All experiments were conducted in triplicate throughout the study. All data were statistically evaluated (P< 0.05). Results showed that Slab51 FS significantly reduced Giardia growth and adherence respect to negative controls, but its efficacy was overall lower than that of metronidazole. Moreover, the effects of Slab51 FS were significantly lowered by heat-treatment and this reduction was statistically higher at 90°C than at 56°C, indicating a heat-sensitive nature of active Slab51 FS compounds. At the ultrastructural level, Slab51 FS treated Giardia trophozoites were swelling, increased in size and showed alterations of their cellular membrane and vacuole patterns, loss of the nuclear envelope and nuclear architecture. In ex vivo trials, viable G. duodenalis trophozoites and enterocyte TUNEL+ and Caspase-3 expression were significantly reduced in intestinal sections added with Slab51 FS, while enterocyte Ki67 expression was significantly increased, confirming the anti-G. duodenalis activity of Slab51 FS observed in vitro. In conclusion, results from this study showed that the fresh culture supernatant of the commercial probiotic Slab51 has anti-G. duodenalis properties both in vitro and ex vivo in a mouse model.

Partial Text

Flagellated protozoans of the genus Giardia are found in the digestive tract of vertebrate hosts worldwide in which they are the cause of giardiasis [1]. Giardia duodenalis (syn. Giardia intestinalis, Giardia lamblia) is the only species found in humans and in many other wild and domestic mammals worldwide [1, 2]. Based on genetic analysis, G. duodenalis is considered a species complex, which includes at least eight distinct genetic groups or assemblages, from A to H [1]. Assemblages A and B are usually isolated from humans but can also infect other animals, being considered zoonotic [1, 3].

G. duodenalis is a common fecal-oral parasite of the small intestine and one of the most important causes of human and animal diarrheal disease worldwide. Indeed, G. duodenalis infection can be asymptomatic, or cause acute or chronic diarrhea, dehydration, intestinal malabsorption, malnutrition and steatorrhea [2, 4–7]. Chronic fatigue, post-infectious irritable bowel syndrome and intestinal dysbiosis have also been documented in humans as possible consequences of G. duodenalis infections, [28, 29], while growth retardation and cognitive malfunction have been reported in children from endemic areas [2, 30]. Probiotics may interfere with G. duodenalis infection through different mechanisms, including competition for limited adhesion sites, competition for nutrients that would otherwise be utilized by G. duodenalis, stimulation of the host immune response and by producing substances that may inhibit G. duodenalis [6, 7, 11]. Probiotic bacteria can produce compounds, which have inhibitory effects directed against pathogens, as viruses, bacteria, fungi, parasites, as well as against cancer cells [11]. Among them, the anti-G. duodenalis activity of probiotic compounds, mainly derived from Lactobacilli, has been demonstrated [11, 20, 24]. In fact, bacteriocins derived from Lactobacillus acidophilus were found able to inhibit in vitro the adhesion and the growth of G. duodenalis trophozoites [11]. Moreover, these negative effects were found associated with severe morphological changes of G. duodenalis trophozoites, a decline of the intestinal parasite density and amelioration of intestinal pathology in infected mice treated with L. acidophilus bacteriocins [11]. More recently, results from some studies suggested that the ability to deconjugate bile salts showed by some lactobacilli, as L. johnsonii strain LA1 and Lactobacillus gasseri CNCM I-4884, may represent a further mechanism contributing to the inhibition of Giardia trophozoite growth in vitro [31, 32].

 

Source:

http://doi.org/10.1371/journal.pone.0213385

 

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