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INTRODUCTION
Amebiasis is an infection that may have or not clinical manifestations; 90% of amebic infections are asymptomatic and self-limiting (estimated, 50 million cases per year). Only 10% of infected persons develop an acute intestinal or extraintestinal disease1. According to the World Health Organization (WHO), Entamoeba histolytica is the third leading parasitic cause of death (100,000 deaths/year)2, and after malaria, amebiasis is the second most lethal disease caused by a protozoan parasite. This infection has a worldwide distribution, with a higher incidence in developing countries and high prevalence in countries with poor sanitary and socioeconomic conditions3. The risk groups include male homosexuals, travelers and recent immigrants, and institutionalized populations4. Food and water contamination combined with a poor hygiene are the main sources of transmission. Water-associated outbreaks of E. histolytica disease as well as sexually transmitted infections have been reported5. Metronidazole is the first-line drug for the treatment of invasive amebiasis, although it has undesirable side effects (nausea, headache, and among other adverse manifestations) and is potentially carcinogenic6. Unfortunately, E. histolytica, as many other parasites, has developed mechanisms to evade the drug pressure7 and became resistant8,9. Due to the medical importance of E. histolytica, it is necessary to search for alternative compounds that inhibit this parasite. Investigations have been conducted with probiotic bacteria to find therapeutic alternatives for amebiasis. Prebiotics, probiotics, and synbiotics are appealing as preventive and therapeutic agents for human medical disorders. Their efficacy depends on disease etiology and the probiotic strain used10. Probiotics are living microorganisms that confer health benefits to the host when administered in adequate amounts; however, dead bacteria and their components can also exhibit probiotic properties11. Recently, increasing evidence supports the notion that certain probiotic-derived components, such as bacteriocins, lipoteichoic acids, surface layer protein, and secreted protein, have a similar protective role on intestinal barrier function as that of live probiotics. These bioactive components have been named postbiotics in the most recent publications12. Postbiotics are soluble factors (products or metabolic byproducts) secreted by live bacteria or released after bacterial lysis, and they provide physiological benefits to the host13,14. However, most investigations in this field are focused on infections of bacterial origin and not on those of parasitic origin.
Typical characteristics of E. histolytica are observed by specific stains and the optical, fluorescence, transmission electron, or scanning microscopes have been used to examine this material15. However, these cells can be currently analyzed using new microscopic tools such as atomic force microscopy (AFM), which allows to observe structures at a nanometric resolution and analyze physical-mechanical properties to study biological material16. In the present study, the in vitro effect of lyophilized postbiotics on E. histolytica trophozoites was determined, and cell membranes were analyzed by AFM to evaluate morphometric changes.
METHODS
E. histolytica culture medium
The TYI-S-33 medium for E. histolytica (HM1-IMSS strain) culture and maintenance was prepared according to Diamond et al.17
Probiotic bacteria culture and obtention of postbiotics
Lactobacillus plantarum (229 strain) and Lactobacillus casei (CCRC 10697 strain) were cultivated in Man-Rogosa-Sharpe broth (Difco, Becton Dickinson Co., Sparks, MD, USA) and incubated (LabNet®) for 24-48 h at 37°C. After incubation, the extracellular factor supernatant was obtained by centrifugation (IEC CL30 Centrifuge Thermo SCIENTIFIC®) at 2500 rpm for 20 min. The recovered material was filtered using Millipore container (0.22 µm, GP Millipore Express ®PLUS Membrane, Massachusetts, USA). Then, 140.26 g of ammonium sulfate were added to 80% saturation, stirring during 24 h at 4°C in an ice bath. The mixture was centrifuged at 2547 rpm for 1 h. The obtained protein precipitate was resuspended with 0.2 M phosphate buffer at pH 7.2 and was dialyzed into membranes (spectra/Por membrane tubing). A protein qualitative determination was made to dialyzed material by Biuret test. The dialyzed material was placed in glass containers (LABCONCO®) for lyophilization (LABCONCO Corporation, Kansas City, Missouri 63132, USA) at 0.133 mBar pressure and −40°C collector temperature for 12 h.
Bioassay
The in vitro effect of lyophilized postbiotics on E. histolytica trophozoites was evaluated. E. histolytica trophozoites (2 × 104 cell/mL) were axenically grown in 13 × 150 mm borosilicate tubes containing 5 mL of TYI-S-33 medium and 0.55 mL of adult bovine serum-antibiotic mixture (400 IU penicillin and 4 mg streptomycin). Different concentrations (0.1, 0.3, and 0.5 mg/mL) of lyophilized postbiotics from each strain were added; 5 mm coverslips were placed inside the tubes and incubated for 72 h at 37°C. Nisin, at the same concentrations as the postbiotics, and metronidazole (0.1 mg/mL) were also incubated in separate assays with trophozoites. Each coverslip was removed for AFM analysis. Tubes were chilled in an ice-water bath for 20 min at 4°C. The total number of cells per mL was determined using a hemocytometer (Neubauer chamber, American®).
Preparation of trophozoite samples and AFM analysis
The trophozoites adhered to the coverslips surface were fixed with 2.5% glutaraldehyde tempered at 37°C during 30 min. Coverslips were rinsed with deionized water to remove glutaraldehyde excess and placed with double-sided tape on the special sample holder to observe by AFM. An AFM Ntegra Prima NT-MDT sitting on an active damping table was used to observe the samples employing a silicon nitride (Si3N4) rectangular cantilever (RTESPA-300, tip radius 8 nm) with a length of 125 µm and nominal spring constant of 40 N/m. AFM images (height, amplitude, and phase) were obtained under normal conditions while operating the instrument in semi-contact mode (tapping). On the other hand, roughness analysis was performed comparing the obtained images using a cantilever as mentioned above with a nominal resonant frequency of 300 kHz. The analysis was carried out using the software package NOVA version 1.1.0.1921 and WSxM v5.0 Develop 8.0.
RESULTS
Bioassay
Table 1 shows the in vitro effect of postbiotics on E. histolytica trophozoites. The lyophilized postbiotics showed variable percentages of inhibition for each strain evaluated independently. The percentage inhibition of postbiotics synergy at a concentration of 0.5 mg/mL was significantly higher than the control and similar to metronidazole and was the same as Nisin. Significant difference between treatments was found.
Table 1 Effect of Lactobacillus postbiotics on cell growth of E. histolytica trophozoites after 72 h incubation
| Treatment | Concentration (mg/mL) | Growth (1 × 104 cell/mL) | Growth inhibition (%)a at 72 h |
|---|---|---|---|
| Lactobacillus casei postbiotics | 0.1 | 3.68 | 63.65 |
| 0.3 | 8.1 | 20 | |
| 0.5 | 4.71 | 53.48 | |
| Lactobacillus plantarum postbiotics | 0.1 | 9.01 | 11 |
| 0.3 | 5.1 | 49.63 | |
| 0.5 | 8.05 | 20.49 | |
| Postbiotics synergy | 0.1 | 6.37 | 37 |
| 0.3 | 4 | 60.49 | |
| 0.5 | 1.05 | 89.63t* | |
| Nisin | 0.1 | 2 | 80.25 |
| 0.3 | 1.7 | 83.21 | |
| 0.5 | 1.05 | 89.63t* | |
| Metronidazole | 0.1 | 0 | 100 |
| Control | 10.13 | |
aMean values determined in three independent events done in triplicate.
*The data are highly significant according to the ANOVA and Tukey test. ANOVA: Analysis of variance.
AFM analysis
We analyzed the in situ cell morphology of E. histolytica trophozoites using tapping mode (Fig. 1). The typical pleomorphic structure and a measure range of 10-60 µm in control trophozoites were observed, as reported in literature4 (Column A). Furthermore, phase and magnitude images showed homogeneity in cell membrane composition, with a magnitude of 40 nA. The three-dimensional (3D) micrograph of control trophozoite showed a pleomorphic structure, maximum height of 5 µm and a membrane topography with indentations and pores (Column A), while the trophozoite with treatment in the 3D micrograph presented a rounded and smaller structure and a uniform membrane with less pores (Column B).
Figure 1 E. histolytica images by atomic force microscopy. (A) Control trophozoite: the cell membranes porosity and pleomorphic structure can be appreciated. (B) Trophozoites size decreased with postbiotics synergy treatment at 0.5 mg/mL concentration.
Table 2 shows the AFM analysis where there was an increase in roughness of 28.32% and a decrease in size of 56% after postbiotic synergy treatment at 0.5 mg/mL concentration compared to the control.
Table 2 Morphometric analysis of Entamoeba histolytica trophozoites using software package NOVA version 1.1.0.1921 and WSxM v5.0 Develop 8.0
| E. histolytica treatment | Morphometric properties | ||
|---|---|---|---|
| Morphology | Size (μm) | Roughness (nm) | |
| Control trophozoite | Pleomorphic structure | 74.14 | 359.5462 |
| With postbiotics synergy (0.5 mg/mL) | Round and small structure | 32.6 | 461.3557 |
aMean values determined in three independent events done in triplicate.
*The data are highly significant according to the ANOVA and Tukey test. ANOVA: Analysis of variance.
DISCUSSION
To provide a safer, non-toxic, and effective alternative to antiprotozoal drugs, recent studies have been aimed to evaluate the effect of probiotics against intestinal diseases. The WHO suggested using probiotic metabolites with microbial interference capacity to prevent pathogen intestinal colonization, producing antagonistic substances14. Many investigations have determined the inhibitory activity or microbial interference of probiotic metabolites against bacteria of medical importance. However, in few studies, this activity has been evaluated on medically important protozoa18. A recent study19 reported in vitro coculture of E. invadens in the presence of L. casei and Enterococcus faecium. The percentage of survival reduced gradually up to 80%, similar to what we found in this work. Another study using Lactobacillus fermentum and Lactobacillus delbrueckii on in vitro growth of E. histolytica reported the effective inhibiting parasite proliferation20. Some probiotic-derived components (postbiotics) such as bacteriocins have antimicrobial action on microorganisms of medical importance without toxicity to eukaryotic cells. A recent study reported the in vitro and in vivo activity of L. salivarius bacteriocins and supernatant on E. histolytica21. In the present work, we report the inhibitory activity of postbiotics on in vitro growth of E. histolytica trophozoites. Results showed that postbiotics synergy produced a higher percentage of inhibition than postbiotics separately, suggesting that Lactobacillus postbiotics could inhibit proliferation of E. histolytica trophozoites. These results support considering the use of postbiotics as a therapeutic alternative against amebiasis, additionally decreasing metronidazole consumption, due to current undesirable side effects in patients. As perspectives, we suggest to continue in vivo studies of the effect of Lactobacillus postbiotics on E. histolytica to demonstrate whether the in vitro inhibitory activity observed in the laboratory can be reproduced in an animal model and may have a future application on disease control in patients with amebiasis. These metabolites could be considered a natural alternative, safe, and without toxicity in the future as a substitute compound for the first-choice drugs for treatment, such as metronidazole, avoiding adverse clinical effects. On the other hand, there are few investigations on E. histolytica analysis by AFM. In the first report, our research group studied E. histolytica trophozoite, precyst, and cyst in situ characterization and made clear some differences (roughness, composition, elasticity, and size) between each stage16,22. In this study, the surviving cells were analyzed by AFM, and we observed a decrease in cell size and a modified typical cell morphology using postbiotics synergy treatment. This agrees with bacteriocin properties causing cellular alterations in sensitive cells according to each bacteriocin type. This is the first report demonstrating inhibition of E. histolytica trophozoites using a synergy of Lactobacillus postbiotics, analyzing parasite membrane morphometric alterations by AFM.
In summary, postbiotic synergy derived from L. casei and L. plantarum exerts significant antiamoebic activity on E. histolytica HM1-IMSS in vitro growth under axenic conditions and induce morphometric alterations in the cell membrane of trophozoites.
