The influence of probiotic bacteria and human gut microorganisms causing opportunistic infections on Blastocystis ST3
Blastocystis subtype 3 is an intestinal protist present in humans throughout the world with a controversial pathogenic potential. It has been suggested that probiotic bacteria inhibit the multiplication of gut protozoans, while others are beneficial for their development. This study aimed to evaluate the efficacy of the lactic acid bacteria Lactobacillus rhamnosus, Lactococcus lactis and Enterococcus faecium in Blastocystis ST3 eradication and the relevance of the intestinal microorganisms Escherichia coli, Candida albicans and Candida glabrata in protozoan proliferation. Blastocystis xenic and axenic culture was co-incubated with the above-mentioned microorganisms and their cell free supernatants at different concentrations in vitro. The number of protozoan cells was counted every day.
Both experiments, with xenic and axenic cultures, showed Blastocystis inhibition by L. rhamnosus and L. lactis and their supernatants from the 2nd day of co-incubation. Furthermore, co-incubation with both E. faecium and E. coli showed a beneficial influence on Blastocystis during the first 2 days. Only after 3 days did the above-mentioned bacteria start to inhibit Blastocystis growth in both cultures. The supernatant containing the metabolites of E. coli was effective to a lesser degree. Compared to the control samples, co-incubation with both C. albicans and C. glabrata showed a faster decrease in Blastocystis proliferation, but this was not statistically significant.
This study has shown the potential of using L. rhamnosus and L. lactis, as well as E. faecium as a prophylactic treatment against Blastocystis colonization or as an additional treatment regimen in combination with standard drugs.
KeywordsBlastocystis Subtype 3 Eradication Probiotics Gut microbiota
Blastocystis is a protist present throughout the world in the intestines of both healthy and symptomatic humans and animals [1, 2]. Its pathogenic potential is still controversial. This unicellular microorganism causes gastrointestinal as well as skin disorders [3, 4]. Seventeen morphologically indistinguishable subtypes have been identified based on an analysis of a small subunit rDNA (SSU rRNA) gene sequence among Blastocystis isolated from humans and animals. It has been suggested that ST3 may be the only subtype (ST) of human origin . That is why this subtype was chosen for analysis in this study. The fecal–oral route is most likely the main mode of transmission. Children, the elderly and immunocompromised individuals appear to be highly susceptible to Blastocystis invasion , while other researchers have suggested that people between 30 and 50 years of age are most prone to being infected by Blastocystis [7, 8, 9, 10]. In the recent literature, researchers have been discussing the correlation between different Blastocystis subtypes and their pathogenic potential. The explanations for pathogenicity may include intra-subtype variations in Blastocystis protease activity, or differences in the intestinal microbiota of the individual host, which can interact to mediate host colonization and Blastocystis virulence [11, 12]. Recently, it has been found that the presence of gut microbiota seems to be essential for the pathogenic expression of enteric protozoan such as Blastocystis. Berrilli et al.  suggest the hypothesis that some intestinal bacteria inhibit multiplication of gut protozoa.
A 2014 study by Nourrisson et al.  and a 2016 study by Nagel et al.  suggest that Blastocystis may be used as an indicator of microbiota changes—a lower abundance of Faecalibacterium prausnitzii and Bifidobacterium spp. was reported to lead to the intestinal dysbiosis. On the other hand, in 2016, Audebert et al.  suggested that colonization by Blastocystis could be associated with healthy gut microbiota. Their study showed a higher bacterial diversity in Blastocystis-colonized patients compared to that identified in Blastocystis-free individuals. However, the same study showed an increasing level of the Lactobacillaceae family in patients not colonized by Blastocystis. Many researchers have reported on the inhibition of a wide range of pathogenic microorganisms like Giardia sp., Entamoeba histolytica, Eimeria sp. or Cryptosporidium parvum by probiotic bacteria [17, 18, 19, 20, 21, 22, 23]. Also there have been previous studies which have shown the effects of certain probiotic yeasts—Saccharomyces boulardii on Blastocystis development .
The most recent results of the latest studies leave the pathogenicity of Blastocystis still unclear. Researchers still do not know if Blastocystis is an agent of gut dysbiosis and is responsible for changing the microbiotic diversity, or if the metabolic dysfunctions and changes in the content of microbiota are the reason for the higher colonization by Blastocystis. There is a possibility that some species of bacteria are creating the conditions for Blastocystis colonization in the gut. It may also depend on the parasitic subtype .
The World Health Organization (WHO) defines probiotics as “live organisms which when administered in adequate amounts confer a health benefit to the host” . As an alternative bio-therapeutic for giardiosis, amoebiasis or cryptosporidiosis, there are a number of studies which have been conducted. In our study, we have aimed to explore the inhibitory effect of 3 different probiotics and 3 species causing opportunistic infections on Blastocystis proliferation for the first time.
Materials and methods
Blastocystis subtype 3 was kindly provided by C. Rune Stensvold (Statens Serum Institute, Copenhagen, Denmark) and cultured in modified Jones’ medium (pH = 7.1) [mix of 93.8 mL Na2HPO4—9.46 g/L of distilled water, 31.3 mL KH2HPO4—9.08 g/L of distilled water, 562.5 NaCl—9 g/L of distilled water, 0.1% of yeast extract (Oxoid, UK)] supplemented with 10% horse serum (Sigma-Aldrich, USA) [26, 27] at 37 °C in tightly closed polypropylene 12 mL Falcon tubes, in anaerobic conditions. Because the experiment was performed in two ways, two versions of Blastocystis culture—xenic and axenic were conducted. The xenic culture (containing bacteria from the patient gut) was subcultured every 2–3 days. The axenic culture (without bacteria) was obtained by supplementation with 100 UI/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich, USA) and incubated for 3–4 days. The cultures were then screened using standard microscopy .
Bacterial and fungal isolates and growth conditions
A lyophilized stock of the organisms was obtained from the American Type Culture Collection (ATCC). The commensal bacteria Lactobacillus rhamnosus (ATCC 7469), Lactococcus lactis (ATCC 11454), Enterococcus faecium (ATCC 6057), and the microorganisms causing opportunistic infections Escherichia coli (ATCC 25922) as well as Candida albicans (ATCC 64548) and Candida glabrata (ATCC 15126) isolates were used in the present study. All isolates were previously purchased in MicroSwab form from Merck (Warsaw, Poland). The bacterial and fungal isolates were freshly cultivated on Tryptone Soy Broth (TSB) (Merck, Poland) before the experiments and also stored in TSB with 20% glycerol at − 70 °C until needed. The bacteria were routinely cultured on TSB (pH 7.3) for 2 days at 37 °C and the fungi were cultured on Sabouraud broth (pH 5.8) for 6 days at 24.5 °C. Sabouraud broth contains mycological peptone (10 g/L), glucose (20 g/L). All of the microorganisms were incubated in tightly closed polypropylene 12 mL Falcon tubes, in anaerobic conditions.
Concentrations of microorganisms used to the experiment
OD620; colony forming units (CFU)/mL
0.54; 4.48 × 108
1.04; 8.56 × 108
1.49; 1.22 × 109
0.51; 4.02 × 108
1.06; 8.48 × 108
1.51; 1.23 × 109
0.56; 4.48 × 108
1.05; 8.72 × 108
1.52; 1.25 × 109
0.55; 4.40 × 108
1.06; 8.48 × 108
1.50; 1.22 × 109
1.75 × 104
2.85 × 105
1.85 × 106
1.55 × 104
2.7 × 105
1.8 × 106
Two isolates of fungi, Candida albicans and Candida glabrata, were harvested by centrifugation at 2300×g for 10 min and washed three times in sterile PBS. Subsequently, the fungi were concentrated into pellet form by centrifugation and suspended in Jones’ medium. The number of fungi cells was determined by counting in a Neubauer chamber (Heinz Herenz, Hamburg, Germany), and adjusted to the final concentrations (Table 1).
Preparation of cell free supernatants (CFS)
Cell free supernatant is a suspension of microorganism metabolites of chemical compounds. CFS of bacteria was obtained from TSB broth cultures after 21 h incubation at 37 °C by centrifugation at 4000×g for 10 min. Supernatant was filtered through 0.20 µm pore size filters (SARSTEDT AG & Co. KG, Germany) . The CFS of fungi was obtained from cultures on Sabouraud broth after 5 days of incubation at 24.5 °C by centrifugation at 2300×g for 10 min. The supernatant was sterilized by filtration with a 0.20 µm syringe filter. The pH of all supernatants was measured.
A stock solution of metronidazole (MTZ, Sigma-Aldrich, USA), as a reference antiprotozoan drug  was prepared by adding 50 mL of sterile distilled water to 3000 mg of the drug to give a final concentration of 60 mg/mL. This was stored in a dark bottle at 4 °C . MTZ was prepared at different concentrations directly before use in the experiment.
Experimental setup and procedure
The number of Blastocystis cells after 2 days of laboratory incubation used for the experiment was determined by counting in a Neubauer chamber. A final concentration of Blastocystis in Jones’ medium was approximately 2.8 × 105 cells/mL for the fungi experiment, 2.9 × 105 cells/mL for the bacterial influence assay and 2.5 × 106 CFU/mL for the experiment using cell free supernatants. Sterile 5 mL polypropylene tubes (Equimed, Poland) were used. One milliliter of each microorganism described above and its CFSs were inserted into 5 mL tubes (Equimed) containing 3 mL of Jones’ medium and 1 mL of Blastocystis xenic as well as axenic cultures in triplicate and the tubes were then sealed with a lid. Four milliliter of Jones medium with 1 mL of Blastocystis culture were used as control samples (pH = 6.28) in the experiment containing alive microorganisms. For the CFS assay as control samples 1 mL of Blastocystis culture, 3 mL of Jones’ medium and 1 mL of TSB (pH = 6.25) or Sabouraud broth (pH = 3.97) were used. The reference antiprotozoal drug metronidazole (MTZ) was tested against Blastocystis using three different concentrations—1 µg/mL, 5 µg/mL, and 10 µg/mL. The parasite was co-incubated for 5 days with each species of bacteria, and 7 days with each species of fungi because Candida sp. is a yeast which needs more time to multiply. Blastocystis was also co-incubated with supernatants of each microorganism for 6 days at 37 °C statically, in tightly closed polypropylene 5 mL tubes, in anaerobic conditions. Each day of incubation the number of Blastocystis cells was determined by counting in a Neubauer chamber. The viability of Blastocystis cells was assessed by using staining with 0.4% Trypan blue solution. Unstained cells were counted. The pH was measured every day of co-incubation. All experiments were repeated three times.
The influence on Blastocystis was determined by calculating the mean and SD (standard deviation) of the number of parasite cells in the 5 mL tubes with microorganisms and the mean of number of Blastocystis cells in the control samples. A cell count was taken for each bacterial and fungal concentration as well as for cell free supernatants. The results were reported as an average.
The number of viable morphological forms of Blastocystis in treatment and controls were compared using t-test (GraphPad Prism 7.04), as well as Pearson Chi square and two-way ANOVA tests were used whenever appropriate. To compare the influence of the dilutions according to the time of co-incubation, three-way ANOVA (Tukey’s test) was used. A p value of < 0.05 was considered as statistically significant.
Co-incubation of bacteria with Blastocystis ST3 xenic culture
The influence of different bacterial concentrations was statistically analyzed. The number of added E. coli cells when the optical density (OD620) was 0.5 and 1.5 (4.48 × 108 CFU/mL and 1.22 × 109 CFU/mL, respectively) had a significant influence on increased Blastocystis proliferation during the first 2 days (p = 0.0007 and p = 0.0301) as compared to the control sample. As mentioned above, the number of parasites decreased on the 5th day of co-incubation with E. coli. In this case, the concentration OD620 = 0.5 again had a significant influence (p = 0.023). The OD620 = 1.0 concentration also inhibited protozoan proliferation (p = 0.0474). Similar results were observed with regards to E. faecium. After the first 2 days, a higher number of Blastocystis cells co-incubated with the OD620 = 1.0 (8.48 × 108 CFU/mL) concentration of E. faecium was noted (p = 0.0021), and during the 5th day the OD620 = 1.5 (1.23 × 109 CFU/mL) concentration inhibited the proliferation of the protozoan cells (p = 0.0063).
Of the four tested bacteria, L. rhamnosus and L. lactis definitely inhibited Blastocystis growth from the 2nd day of co-incubation, as compared to the control samples. Inhibition was perfectly visible during all 5 days when OD620 = 1.0 concentration (8.72 × 108 CFU/mL and 8.48 × 108 CFU/mL, respectively) was added and this was statistically significant (p = 0.0076 and p = 0.0383).
Co-incubation of bacteria with Blastocystis ST3 axenic culture
The influence of different bacterial concentrations was statistically analyzed. The number of added E. coli cells when optical density (OD620) was 0.5, 1.0 and 1.5 (4.48 × 108 CFU/mL, 8.56 × 108 CFU/mL, and 1.22 × 109 CFU/mL, respectively) significantly influenced a higher Blastocystis proliferation after the first 2 days (p < 0.0001) as compared to the control sample. The number of parasites decreased on the 5th day of co-incubation with E. coli. In this case, all of the different concentrations (OD620 = 0.5, 1.0, and 1.5) again had a statistically significant influence (p < 0.0001, p = 0.0003, p < 0.0001, respectively). A similar situation with regards to E. faecium was observed. After the first 2 days, a higher number of Blastocystis cells co-incubated with the OD620 = 0.5 and OD620 = 1.0 (4.02 × 108 CFU/mL, 8.48 × 108 CFU/mL) concentration of E. faecium was noted. Moreover, during the 5th day the number of Blastocystis cells decreased but not significantly (Fig. 2).
In the experiment with L. rhamnosus and L. lactis the inhibition of Blastocystis proliferation was noted during the entire duration of the co-incubation. This was quite clearly visible during the 3rd day of co-incubation when OD620 = 0.5 and OD620 = 1.0 concentrations of L. rhamnosus (4.48 × 108 CFU/mL and 8.72 × 108 CFU/mL, respectively) were added. Statistical analysis showed significance with p-values lower than 0.05, p = 0.0337 and p = 0.0269, respectively. On the 5th day, all of the concentrations of L. rhamnosus and L. lactis significantly inhibited Blastocystis growth.
Both experiments, with xenic and axenic cultures, showed similar results with regards to Blastocystis inhibition by L. rhamnosus and L. lactis from the 2nd day of co-incubation with those bacteria. Also in both, co-incubation with E. faecium and E. coli showed a beneficial influence on Blastocystis during first 2 days. Only after 3 days did the above-mentioned bacteria start to inhibit Blastocystis growth in xenic and axenic culture. Usually the vacuolar form was observed. The cyst form occurred rare. In the cultures co-incubated with E. coli and E. faecium a high level of amoebic forms of Blastocystis was noticed.
Co-incubation of fungi with Blastocystis ST3 xenic and axenic culture
Co-incubation of cell free supernatant
pH changes during co-incubation
The pH of all of the co-cultures was measured as well as cell free supernatants and microorganisms cultures on appropriate media. The pH of CFS of E. coli was equal to 6.5, E. faecium—5.45, L. rhamnosus—5.0, L. lactis—5.26, C. albicans—4.53 and C. glabrata—4.71. The pH of E. coli culture incubated for 2 days on TSB was equal to 6.1, E. faecium—5.36, L. rhamnosus—4.94, L. lactis—5.16, and of C. albicans culture incubated for 6 days on Sabouraud broth was equal to 4.65, C. glabrata—4.68.
It can be assumed that the small pH differences did not influence on Blastocystis proliferation significantly.
Living lactic acid bacteria, L. rhamnosus and L. lactis, as well as their cell free supernatants had a similar effect to the MTZ control concentrations of 10 µg/mL and 5 µg/mL in xenic cultures of Blastocystis. Axenic culture could not be described, because of a low number of Blastocystis cells which nearly all died after the addition of different concentrations of MTZ. With regards to this, there were statistically significant differences between both E. coli and E. faecium, as well as C. albicans, C. glabrata and MTZ addition (p < 0.0001).
Natural gut microbiota plays a very important role in controlling intestinal diseases and keeping the intestines healthy. Studies have shown that intestinal microbiota could alter the Blastocystis . Consequently, suggestions have been made that the use of the antiprotozoal drug known as metronidazole could give rise to drug resistant Blastocystis subtypes . Moreover, metronidazole, which is the first-line treatment, has been shown to exhibit side effects and quite often a low effectiveness of this drug during eradication is noted . As an alternative, we have tried to explore the potential of probiotic bacteria. We studied the response of Blastocystis in vitro to two different strains of the Lactobacillaceae family—L. rhamnosus and L. lactis, as well as E. faecium. In addition, we tried to define the role of E. coli and two fungal strains from the Candidaceae family in protozoan development using concentrations similar to that in a healthy human colon.
Our study shows the strong inhibitory effect of various lactic acid bacteria (LAB) at different concentrations on the proliferation of the Blastocystis from the beginning of co-incubation in xenic and axenic culture, whereas E. faecium and E. coli exhibited anti-proliferative activity after 4 days. In the case of co-incubation with E. faecium and E. coli, a high level of amoebic forms of Blastocystis was noticed. It may mean that the bacteria mentioned above are beneficial for Blastocystis development and confirms the previous research of Rajamanikam and Govind , who suggest that the amoebic form is found during optimal conditions for Blastocystis growth and plays a role in the exacerbation of intestinal symptoms during Blastocystis colonization. In our study, fungi from the Candidaceae family had little antiprotozoal influence, as well as forcing the vacuolar forms into cysts and granular forms. Those forms are usually observed during thermal stress, as Thergarajan et al.  reported in their research.
Our experiments on axenic cultures of the parasite confirmed a strong negative influence of LAB on Blastocystis. The cell free supernatants of bacteria were used for further investigation to determine whether the bacterial metabolites showed similar results. Sarjapuram et al.  reported the inhibition of other protozoan proliferation by spent media of probiotic culture. They noticed changes of the pH of microbiological media and adjusted it to eliminate its influence on Entamoeba growth . Similarly, our study showed that not only living bacteria had a negative influence on Blastocystis, but also their metabolites. Zhang et al.  determined the optimal pH for Blastocystis growth to be 7.0 .
Our study clearly shows the inhibition of Blastocystis proliferation by LAB, which suggests that people using probiotic rich diets and having a stable gut microbiota are more resistant to protozoan colonization. Several previous studies revealed that the total bacterial population, as well as bacterial groups such as Bifidobacterium sp., Bacteroides spp., and Clostridium sp., shows a high degree of temporal stability [36, 37, 38, 39]. However, the situation is different for the Lactobacillus population. The Walter et al., Vanhoutte et al., and Scanlan et al. studies of fecal samples from most human subjects showed temporal dynamics that were characterized by fluctuations and a lack of stability [36, 38, 40]. A lack of these bacteria in the large intestine caused by a poor diet, antibiotic therapy or taking drugs for gastrointestinal disorders such as proton pomp inhibitors (PPI) may influence the susceptibility to Blastocystis invasions [41, 42]. The interesting thing is that all of the bacterial concentrations used in this study affected the parasite proliferation. This suggests that lower bacterial CFUs may also be used for Blastocystis eradication. Molan in his research  suggested, which we validated in our work, that the factor which causes that inhibition may be the bacteria themselves or their chemical compounds. Lactobacilli increase their protective or therapeutic effect through the production of antimicrobial compounds , a reduction of gut pH by stimulating the lactic acid producing microflora , competition for binding of receptor sites that pathogens occupy and competition with pathogens for available nutrients [45, 46]. Nisin-producing L. lactis strains have high antimicrobial properties . This study shows that lactic acid produced by the Lactobacillaceae family lowered the pH of the TSB medium, but not of the Jones’ medium during co-incubation with Blastocystis. That means the pH could not be a reason for the decline of Blastocystis, as has been reported about other protozoan parasites such as Entamoeba histolytica, Giardia sp. or Eimeria sp. [19, 21, 22, 48].
The other situation has been demonstrated by E. faecium and E. coli. Some strains of the first of these bacteria has been reported to be an effective probiotic species . Our study confirmed the data from the Sarjapuram  research. The authors reported that E. faecium inhibited Eimeria after 24 h of co-incubation with a total bacterial CFU of 108 cells/mL. Blastocystis ST3 seems to be more resistant to its influence. In our experiment, the more effective concentration was 1.23 × 109 CFU/mL, as well as a longer incubation time—4 or 5 days. Most likely, it lasts longer because E. faecium produces strong antimicrobial, yet no antiprotozoal compounds, and it does not compete for enteric adherence sites. More likely, this is caused by competing for nutrients. This could be due to it being a lactic acid bacteria which colonizes differently, and competes with Blastocystis differently .
In human medicine, E. faecium has been used successfully in the treatment of acute diarrheal diseases and in the prevention of antibiotic—associated diarrhea [50, 51]. Starke et al., as well as Klingspor et al. in 2015, investigated the intestinal microbiota of pigs whose components are similar to human gut microorganisms [52, 53]. They showed that the probiotic bacteria E. faecium modifies the porcine intestinal microbiota and modulates epithelial integrity, heat shock protein as well as the proinflammatory cytokine response in intestinal cells. That could lead to the eradication of intestinal pathogens, including protozoans. Our study showed the dependence of Blastocystis on a fecal bacteria presence. In axenic control cultures, there were far fewer Blastocystis cells than in xenic culture, which means the intestinal commensal bacteria have a role in parasite development. One possible explanation for Blastocystis eradication by E. faecium may be the direct growth inhibiting effect of the probiotic on other intestinal bacteria, such as E. coli, Clostridium sp. or other fecal commensal microorganisms. Bednorz et al.  showed in their data a minor influence of E. faecium on the overall population of non-pathogenic E. coli in healthy piglets. However, this same strain has a profound effect on mucosa-adherent E. coli. Russo et al. and Kaper et al. reported that E. faecium 10415 significantly reduced pathogenic organisms, such as extraintestinal E. coli (ExPEC) [55, 56]. To sum up, there may be two ways of Blastocystis eradication by E. faecium: directly by cellular compounds and nutrient competition, and indirectly by killing the beneficial intestinal bacteria.
The results of our E. coli experiment were quite interesting. The number of Blastocystis cells increased, then after the 4th day began to decrease significantly. In the Ganas et al. study from 2012, E. coli was found to strongly support the growth of the parasite—Histomonas meleagridis, which may suggest it is a beneficial bacteria for protozoan parasite development . That may be an explanation for the increase of Blastocystis proliferation, especially if E. coli is also an intestinal microorganism. The question remains as to why the number of Blastocystis cells decreased significantly after 5 days of co-incubation? One option may be the fact that the bacteria might be absorbed by Blastocystis at first while only low numbers of E. coli cells were in the incubated tubes. That supports protozoan proliferation. Another option is that E. coli produces endotoxins, such as lipopolysaccharides (LPS) which could negatively influence Blastocystis cells from inside after phagocytosis which was observed in amoebic form and destroy the parasite [57, 58, 59].
There are not many studies regarding the influence of Candida on protozoans. Mostly the researchers have focused on interactions between intestinal fungi and bacteria . Our study focused on the interaction between Blastocystis and Candida albicans and Candida glabrata to determine if people who are colonized by that fungi as the natural microbiota of the human intestine are more susceptible or resistant to Blastocystis invasions. It can therefore be assumed that both Candida should to a small degree inhibit the potential pathogenic protozoan development in the intestine just as other yeasts like Saccharomyces boulardii . Our study shows almost no inhibitory effect on Blastocystis by C. glabrata and only limited inhibition by C. albicans in 106 cells/mL concentration. Moyes et al.  reported that Candida does not produce any toxins influencing protozoa, but produces some toxins against bacteria and epithelial cells. Also, these do not change the pH of the environment. Moreover, Konno et al. and Sherrington et al. proved Candida adapt to environmental pH changes [62, 63]. A minor decrease in the number of Blastocystis cells was most likely caused by competition between the protozoan and Candida for space and nutrition .
Our study has shown the potential of using L. rhamnosus and L. lactis, as well as E. faecium as probiotics against Blastocystis colonization. The fact that these probiotic bacterial strains are able to disrupt the cell cycle of Blastocystis shows a promising future in the use of probiotics for prophylactic treatment of blastocystosis, or as an additional treatment regimen in combination with standard drugs. The obtained results did not show what is the mechanism of Blastocystis inhibition by lactic acid bacteria. This issue requires further research.
ML was responsible for idea conception, designed and performed the experiment, analysed the data and drafted and wrote this manuscript; ED was responsible for critical evaluation and manuscript review. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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