*3.1. L. acidophilus Amebicide Activity Depends on the Formation of H2O2*

*L. acidophilus* is commonly found in the gastrointestinal tract of healthy humans. It is widely used as a food preservative and as a probiotic. *L. acidophilus* antimicrobial activity is caused by the production of antimicrobial peptides, including lactacins B, organic acid production such as lactic acids and H2O2 (recently reviewed in [29]), and immune induction [30]. Whereas the antibacterial and antifungal activity [31,32] of *L. acidophilus* has been well illustrated, the antiparasitic properties of *L. acidophilus* have been less studied. Studies with mouse models of the diseases caused by *Giardia lamblia* [33], *Toxocara canis* [34], *Trichinella spiralis* [35], and *Cryptosporidium parvum* [36] reveal that a combination of probiotics and other probiotic strains is beneficial in the treatment and prevention of these parasites. In a recent study, we demonstrated that *L. acidophilus* is detrimental to *E. histolytica* but the amebicide mechanism was unknown [19]. In this study, we investigated whether H2O2 generated by *L. acidophilus* is directly responsible for the amebicide activity. We first measured the ability of *L. acidophilus* to produce H2O2 by the FOX assay. We found that overnight culture of *L. acidophilus* cultivated in MRS media with agitation produces 0.14 ± 0.3 mM H2O2. A viability assay was performed on *E. histolytica* trophozoites incubated either with *L. acidophilus* or with heat-killed *L. acidophilus*, which served as negative control. The viability of *E. histolytica* trophozoites was not affected when the parasite was incubated with *L. acidophilus* for 60 min (Figure 1). However, the viability of *E. histolytica* trophozoites was significantly decreased by 50% when the parasite was incubated with *L. acidophilus* for 120 min. In contrast, the viability of *E. histolytica* trophozoites incubated with heat-killed *L. acidophilus* for 120 min was not impaired (Figure 1). Next, we wanted to establish if the amebicidal activity of *L. acidophilus* was dependent on the formation of H2O2. We incubated *E. histolytica* and *L. acidophilus* in presence of catalase, an enzyme that catalyzes the decomposition of H2O2 to H2O and O2 [37]. We observed that the amebicidal activity of *L. acidophilus* was strongly reduced when catalase was added during the incubation of *L. acidophilus* with the parasite (Figure 1). Based on this finding, it strongly suggests that H2O2 produced by *L. acidophilus* is the primary cause of parasite death.

**Figure 1.** Viability assay of *E. histolytica* trophozoites. Note: *E. histolytica* trophozoites (WT) were incubated with live *L. acidophilus* (LA) or with heat-killed *L. acidophilus* (DN), with/without catalase (Cat) (50 μg/mL) for 60 and 120 min at 37 ◦C. The data represent two independent experiments performed in triplicate. \*\*\* *p* value < 0.001 by an unpaired Student's *t*-test.
