Characterization of Entamoeba fatty acid elongases; validation as targets and provision of promising leads for new drugs against amebiasis.

Entamoeba histolytica is a protozoan parasite belonging to the phylum Amoebozoa that causes amebiasis, a global public health problem. E. histolytica alternates its form between a proliferative trophozoite and a dormant cyst. Trophozoite proliferation is closely associated with amebiasis symptoms and pathogenesis whereas cysts transmit the disease. Drugs are available for clinical use; however, they have issues of adverse effects and dual targeting of disease symptoms and transmission remains to be improved. Development of new drugs is therefore urgently needed. An untargeted lipidomics analysis recently revealed structural uniqueness of the Entamoeba lipidome at different stages of the parasite's life cycle involving very long (26-30 carbons) and/or medium (8-12 carbons) acyl chains linked to glycerophospholipids and sphingolipids. Here, we investigated the physiology of this unique acyl chain diversity in Entamoeba, a non-photosynthetic protist. We characterized E. histolytica fatty acid elongases (EhFAEs), which are typically components of the fatty acid elongation cycle of photosynthetic protists and plants. An approach combining genetics and lipidomics revealed that EhFAEs are involved in the production of medium and very long acyl chains in E. histolytica. This approach also showed that the K3 group herbicides, flufenacet, cafenstrole, and fenoxasulfone, inhibited the production of very long acyl chains, thereby impairing Entamoeba trophozoite proliferation and cyst formation. Importantly, none of these three compounds showed toxicity to a human cell line; therefore, EhFAEs are reasonable targets for developing new anti-amebiasis drugs and these compounds are promising leads for such drugs. Interestingly, in the Amoebazoan lineage, gain and loss of the genes encoding two different types of fatty acid elongase have occurred during evolution, which may be relevant to parasite adaptation. Acyl chain diversity in lipids is therefore a unique and indispensable feature for parasitic adaptation of Entamoeba.

HIF-1α modulates sex-specific Th17/Treg responses during hepatic amoebiasis.

BACKGROUND & AIMS: An invasive form of intestinal Entamoeba (E.) histolytica infection, which causes amoebic liver abscess, is more common in men than in women. Immunopathological mechanisms are responsible for the more severe outcome in males. Here, we used a mouse model of hepatic amoebiasis to investigate the contribution of hepatic hypoxia-inducible factor (HIF)-1α to T helper 17 (Th17)/regulatory T cell (Treg) responses in the context of the sex-specific outcome of liver damage. METHODS: C57BL/6J mice were infected intrahepatically with E. histolytica trophozoites. HIF-1α expression was determined by qPCR, flow cytometry and immunohistochemistry. Tregs and Th17 cells were analysed by immunohistochemistry and flow cytometry. Finally, male and female hepatocyte-specific Hif1α knockout mice were generated, and the effect of HIF-1α on abscess development, the cytokine milieu, and Th17/Treg differentiation was examined. RESULTS: E. histolytica infection increased hepatic HIF-1α levels, along with the elevated frequencies of hepatic Th17 and Treg cells. While the Th17 cell population was larger in male mice, Tregs characterised by increased expression of Foxp3 in female mice. Male mice displayed increased IL-6 expression, contributing to immunopathology; this increase in IL-6 expression declined upon deletion of hepatic HIF-1α. In both sexes, hepatic deletion of HIF-1α reduced the Th17 cell frequency; however, the percentage of Tregs was reduced in female mice only. CONCLUSIONS: Hepatic HIF-1α modulates the sex-specific outcome of murine E. histolytica infection. Our results suggest that in male mice, Th17 cells can be modulated by hepatic HIF-1α via IL-6, indicating marked involvement in the immunopathology underlying abscess development. Strong expression of Foxp3 by hepatic Tregs from female mice suggests a potent immunosuppressive function, leading to initiation of liver regeneration. LAY SUMMARY: Infection with the parasite Entamoeba histolytica activates immunopathological mechanisms in male mice, which lead to liver abscesses that are larger than those in female mice. In the absence of the protein HIF-1α in hepatocytes, abscess formation is reduced; moreover, the sex difference in abscess size is abolished. These results suggest that HIF-1α modulates the immune response involved in the induction of immunopathology, resulting in differential disease susceptibility in males and females.

Morphological and Motility Features of the Stable Bleb-Driven Monopodial Form of Entamoeba and Its Importance in Encystation.

Entamoeba histolytica and its reptilian counterpart and encystation model Entamoeba invadens formed a polarized monopodial morphology when treated with pentoxifylline. This morphology was propelled by retrograde flow of the cell surface resulting from a cyclic sol-gel conversion of cytoplasm and a stable bleb at the leading edge. Pentoxifylline treatment switched the unpolarized, adherent trophozoites to the nonadherent, stable bleb-driven form and altered the motility pattern from slow and random to fast, directionally persistent, and highly chemotactic. Interestingly, exogenously added adenosine produced multiple protrusions and random motility, an opposite phenotype to that of pentoxifylline. Thus, pentoxifylline, an adenosine antagonist, may be inducing the monopodial morphology by preventing lateral protrusions and restricting the leading edge to one site. The polarized form of E. invadens was aggregation competent, and time-lapse microscopy of encystation revealed its appearance during early hours, mediating the cell aggregation by directional cell migration. The addition of purine nucleotides to in vitro encystation culture prevented the formation of polarized morphology and inhibited the cell aggregation and, thus, the encystation, which further showed the importance of the polarized form in the Entamoeba life cycle. Cell polarity and motility are essential in the pathogenesis of Entamoeba parasites, and the stable bleb-driven polarized morphology of Entamoeba may also be important in invasive amoebiasis.

Entamoeba histolytica and Entamoeba dispar: Morphological and Behavioral Differences Induced by Fibronectin through GTPases Activation and Actin-Binding Proteins.

Early steps of tissue invasion by Entamoeba histolytica are mediated by adhesion and migration through matrix components such as fibronectin with the participation of the actin cytoskeleton. Striking differences in their produced structures, movement, and migration were found. These observations suggest differential changes in their ability to organize the actin cytoskeleton and, therefore, to modify its morphology after adhesion to fibronectin. To understand these observations, we explore deeper the cytoskeleton pathway of E. histolytica compared to Entamoeba dispar, analyzing the activation and involvement of actin cytoskeleton regulatory proteins such as small GTPases (Rho, Rac1 and Cdc42), myosin IB, paxillin, alpha-actinin, and ARP2/3 during interaction with fibronectin. Results showed a higher activation of Rac1 in E. histolytica compared to E. dispar, while Cdc42 and RhoA were equally activated in both amebae; besides, variations in the amount of myosin IB, paxillin, and ARP2/3 were detected among these species, coinciding and reflected in formation of lamellipodia in E. histolytica and filopodia in E. dispar. These could partially explain the higher invasive capacity of E. histolytica compared to E. dispar, due to its pleomorphic ability, high motility, migration, activation, and abundance of proteins involved in the cytoskeleton arrangement.

De novo synthesis of sphingolipids plays an important role during in vitro encystment of Entamoeba invadens.

Entamoeba invadens is a protozoan, which causes multiple damages in reptiles and is considered a prototype for the study of the Entamoeba encystment in vitro. Here we report for the first time the role of the de novo synthesis pathway of sphingolipids during the encystment of E. invadens. In silico analysis showed that this parasite has six putative genes coding for ceramide synthases (CerS), all of them coding for proteins containing the Lag1p motif, a region conserved in the ceramide synthases of multiple organisms, suggesting that they might be bona fide CerS. The six genes of E. invadens are differentially expressed at different time intervals in both stages trophozoite and cyst, based on the results obtained through qRT-PCR assays, the genes involved in the synthesis of sphingolipids with long-chain fatty acids CerS 2,3,4 (EIN_046610, EIN_097030, EIN_130350) have maximum points of relative expression in both stages of the E. invadens life cycle, which strongly suggest that the signaling exerted from the synthesis pathway of sphingolipids is essential for the encystment of E. invadens, since the generation of the more abundant sphingomyelin (SM) subspecies with long-chain fatty acids are fundamental for the parasite to reach its conversion from trophozoite to cyst. When myriocin was used as an inhibitor of serine palmitoyl CoA transferase (SPT), first enzyme in the de novo biosynthesis of sphingolipids, the trophozoites of E. invadens were unable to reach the encystment. Since the effect of myriocin was reversed with exogenous d-erythrosphingosine (DHS), it was demonstrated that the inhibition was specific and it was confirmed that the synthesis of sphingolipids play an essential role during the encystment process of E. invadens.

Unique Features of Entamoeba Sulfur Metabolism; Compartmentalization, Physiological Roles of Terminal Products, Evolution and Pharmaceutical Exploitation.

Sulfur metabolism is essential for all living organisms. Recently, unique features of the Entamoeba metabolic pathway for sulfated biomolecules have been described. Entamoeba is a genus in the phylum Amoebozoa and includes the causative agent for amoebiasis, a global public health problem. This review gives an overview of the general features of the synthesis and degradation of sulfated biomolecules, and then highlights the characteristics that are unique to Entamoeba. Future biological and pharmaceutical perspectives are also discussed.

[CHARACTERISTICS OF PARASITIC PROTOZOA METABOLISM AND POSSIBILITIES OF ANTIPROTOZOA DRUG DEVELOPMENT (REVIEW)].

One of the most poorly studied areas of protozoology is metabolic processes of parasitic protozoa. Study of the biochemistry of parasites required for the development of effective chemotherapy of protozoal diseases. Some amitochondrial parasites of humans, such as Giardia intestinalis, Entamoeba histolytica, Trichomonas sp., living in an environment with low oxygen content, have specialized cellular organelles-hydrogenosomes (like mitochondria provide cells with simple energy). The study of the functioning of these organelles allows us to consider them as targets for the development of аntiprotozoal drugs. The target for chemotherapy in the treatment of trypanosomiasis can be processes related to the characteristics of the glycolytic pathway or a decrease in the level of energy substrate, such as glucose. This leads to a rapid decrease in ATP levels in the cell of the parasite, an overall loss of mobility and disappearance of trypanosomes from the bloodstream of the infected host. Also, glucose transporters located in the membrane of the parasite can be targets for drugs.

Ethanol and isopropanol in concentrations present in hand sanitizers sharply reduce excystation of Giardia and Entamoeba and eliminate oral infectivity of Giardia cysts in gerbils.

Enteric protozoan parasites, which are spread by the fecal-oral route, are important causes of diarrhea (Giardia duodenalis) and amebic dysentery (Entamoeba histolytica). Cyst walls of Giardia and Entamoeba have a single layer composed of fibrils of ß-1,3-linked GalNAc and ß-1,4-linked GlcNAc (chitin), respectively. The goal here was to determine whether hand sanitizers that contain ethanol or isopropanol as the active microbicide might reduce transmission of these parasites. We found that treatment with these alcohols with or without drying in a rotary evaporator (to model rapid evaporation of sanitizers on hands) kills 85 to 100% of cysts of G. duodenalis and 90 to 100% of cysts of Entamoeba invadens (a nonpathogenic model for E. histolytica), as shown by nuclear labeling with propidium iodide and failure to excyst in vitro. Alcohols with or without drying collapsed the cyst walls of Giardia but did not collapse the cyst walls of Entamoeba. To validate the in vitro results, we showed that treatment with alcohols eliminated oral infection of gerbils by 1,000 G. duodenalis cysts, while a commercial hand sanitizer (Purell) killed E. invadens cysts that were directly applied to the hands. These results suggest that expanded use of alcohol-based hand sanitizers might reduce the transmission of Giardia and Entamoeba.

Entamoeba invadens: Identification of a SERCA protein and effect of SERCA inhibitors on encystation.

Calcium has an important role on signaling of different cellular processes, including growth and differentiation. Signaling by calcium also has an essential function in pathogenesis and differentiation of the protozoan parasites Entamoeba histolytica and Entamoeba invadens. However, the proteins of these parasites that regulate the cytoplasmic concentration of this ion are poorly studied. In eukaryotic cells, the calcium-ATPase of the SERCA type plays an important role in calcium homeostasis by catalyzing the active efflux of calcium from cytoplasm to endoplasmic reticulum. Here, we reported the identification of SERCA of E. invadens (EiSERCA). This protein contains a putative sequence for endoplasmic reticulum retention and all domains involved in calcium transport identified in mammalian SERCA. By immunofluorescence assays, an antibody against SERCA of E. histolytica detected EiSERCA in a vesicular network in the cytoplasm of E. invadens trophozoites, co-localizing with calreticulin. Interestingly, EiSERCA was redistributed close to plasma membrane during encystation, suggesting that this pump could participate in regulate the calcium concentration during this process. In addition, thapsigargin and cyclopiazonic acid, both specific inhibitors of SERCA, affected the number and structure of cysts, supporting the hypothesis that calcium flux mediated by SERCA has an important role in the life cycle of Entamoeba.

Synthesis, characterization, antiamoebic activity and cytotoxicity of new pyrazolo[3, 4-d]pyrimidine-6-one derivatives.

A new series of pyrazolo[3,4-d]pyrimidine-6-one derivatives (2a-2j) were prepared by using the Biginelli multicomponent cyclocondensation of 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one (1a), different aromatic aldehydes, and urea with a catalytic amount of HCl at reflux temperature. These compounds were characterized by IR, (1)H NMR, (13)C NMR, and Mass spectral data. In vitro antiamoebic activity was performed against HM1:IMSS strain of Entamoeba histolytica. The results showed that the compounds 2b, 2i, and 2j with IC(50) values of 0.37 µM, 0.04 µM, and 0.06 µM, respectively, exhibited better antiamoebic activity than the standard drug metronidazole (IC(50) = 1.33 µM). The toxicological studies of these compounds on human breast cancer MCF-7 cell line showed that the compounds 2b, 2i, and 2j exhibited >80% viability at the concentration range of 1.56-50 µM.

Surface properties related to concanavalin A-induced agglutination. A comparative study of several Entamoeba strains.

Pathogenic strains of Entamoeba histolytica are more easily agglutinated with concanavalin A (Con A) than strains isolated from human asymptomatic carriers. All three pathogenic strains studied here were found to agglutinate with low concentrations of Con A in contrast to various nonpathogenic axenic strains of amebas, characterized by their ability to grow at room temperature. Our present observations suggest that the extreme susceptibility of pathogenic strains of E. histolytica to agglutinate with Con A is related to their higher capacity for lectin binding and to their lack of detectable repulsive charges at the cell surface. The amount of fluorescein-tagged Con A bound to the surface was much higher in pathogenic strains. Only nonpathogenic strains showed a detectable negative surface charge as studied both by means of cell microelectrophoresis and by labeling cells with cationized ferritin at 0 degrees C. The mobility of surface Con A receptors estimated as the percentage of caps was comparable in all strains. Results of one strain cultured in axenic and monoxenic conditions suggested that bacteria can modify the behaviour of E. histolytica trophozoites by altering surface properties of the amebas.

First synthesis and antiprotozoal activities of divinyl sulfone-modified carbohydrates.

Divinyl sulfone-modified carbohydrates have been synthesized for the first time by reacting easily available carbohydrate epoxides with thioethanol in a regiospecific fashion. One of the modified divinyl sulfones initiated significant cell death in Entamoeba species and the influence of the anomeric configurations on the biological activities of these sugar-derived divinyl sulfones has been highlighted. The most active compound in this series was found to be devoid of any toxicity.

Hypericum erectum alcoholic extract inhibits Toxoplasma growth and Entamoeba encystation: an exploratory study on the anti-protozoan potential.

Hypericum erectum is an important ethnobotanical medicine in East Asian tradition. To explore the anti-parasitic potential of H. erectum, inhibitory effects on the growth of intracellular parasite Toxoplasma and on the encystation of intestinal parasite Entamoeba were examined. The constituents in H. erectum alcoholic extracts and fractions separated by solvent-partitioning were analysed by high resolution LC-MS. Toxoplasma gondii growth inhibition assay was performed using GFP-labelled T. gondii strain PTG-GFP by measuring the fluorescence intensity. Anti-Toxoplasma drug pyrimethamine was used as a positive control. T. gondii-induced immune reaction was assessed by quantitative PCR and fluorescence microscopy, using co-culture of PTG-GFP and monocyte-macrophage cell line Raw264. The inhibitory effect on the encystation of Entamoeba invadens was measured by flow-cytometry, where paromomycin was used as a positive control. H. erectum methanol (MeOH) extract (50 µg/mL) and ethyl acetate (EtOAc) fraction (50 µg/mL) inhibited the growth of T. gondii, while 50%MeOH extract and hydrophilic fractions were ineffective. Co-culture with T. gondii reduced the viability of macrophages, however macrophages were protected in the presence of H. erectum MeOH extract or EtOAc fraction (above 10 µg/mL). The MeOH extract and EtOAc fraction also effectively suppressed the encystation of E. invadens at 1 mg/mL. Hypericine, a major constituent in MeOH extract and EtOAc fraction, inhibited T. gondii growth and E. invadens encystation. Our results demonstrated that H. erectum effectively inhibited Toxoplasma growth and Entamoeba encystation. These activities are partly mediated by hypericin. In addition, it was suggested the extract and fraction may protect innate immune cells from Toxoplasma-induced damages, thereby enhancing parasite clearance. Further investigation is warranted to address the in vivo effectiveness of H. erectum as an anti-protozoal medicine.

Giardia, Entamoeba, and Trichomonas enzymes activate metronidazole (nitroreductases) and inactivate metronidazole (nitroimidazole reductases).

Infections with Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis, which cause diarrhea, dysentery, and vaginitis, respectively, are each treated with metronidazole. Here we show that Giardia, Entamoeba, and Trichomonas have oxygen-insensitive nitroreductase (ntr) genes which are homologous to those genes that have nonsense mutations in metronidazole-resistant Helicobacter pylori isolates. Entamoeba and Trichomonas also have nim genes which are homologous to those genes expressed in metronidazole-resistant Bacteroides fragilis isolates. Recombinant Giardia, Entamoeba, and Trichomonas nitroreductases used NADH rather than the NADPH used by Helicobacter, and two recombinant Entamoeba nitroreductases increased the metronidazole sensitivity of transformed Escherichia coli strains. Conversely, the recombinant nitroimidazole reductases (NIMs) of Entamoeba and Trichmonas conferred very strong metronidazole resistance to transformed bacteria. The Ehntr1 gene of the genome project HM-1:IMSS strain of Entamoeba histolytica had a nonsense mutation, and the same nonsense mutation was present in 3 of 22 clinical isolates of Entamoeba. While ntr and nim mRNAs were variably expressed by cultured Entamoeba and Trichomonas isolates, there was no relationship to metronidazole sensitivity. We conclude that microaerophilic protists have bacterium-like enzymes capable of activating metronidazole (nitroreductases) and inactivating metronidazole (NIMs). While Entamoeba and Trichomonas displayed some of the changes (nonsense mutations and gene overexpression) associated with metronidazole resistance in bacteria, these changes did not confer metronidazole resistance to the microaerophilic protists examined here.

Entamoeba invadens: sphingolipids metabolic regulation is the main component of a PKC signaling pathway in controlling cell growth and proliferation.

The sphingolipids biosynthesis pathway generates bioactive molecules crucial to the regulation of physiological processes. We have recently reported that DAG (diacylglycerol) generated during sphingomyelin synthesis, plays an important role in PKC (protein kinase C) activation, necessary for the transit through the cell cycle (G1 to S transition) and cell proliferation (Cerbon and Lopez-Sanchez, 2003. Diacylglycerol generated during sphingomyelin synthesis is involved in protein kinase C activation and cell proliferation in Madin-Darby canine kidney cells. Biochem. J. 373, 917-924). Since pathogenic Entamoeba invadens synthesize the sphingolipids inositol-phosphate ceramide (IPC) and ethanolamine-phosphate ceramide (EPC) as well as sphingomyelin (SM), we decided to investigate when during growth initiation, the synthesis of sphingolipids takes place, DAG is generated and PKC is activated. We found that during the first 6h of incubation there was a significant increase in the synthesis of all three sphingolipids, accompanied by a progressive increment (up to 4-fold) in the level of DAG, and particulate PKC activity was increased 4-8 times. The enhanced DAG levels coincided with decrements in the levels of sphingoid bases, conditions adequate for the activation of PKC. Moreover, we found that inhibition of sphingolipid synthesis with myriocin, specific inhibitor of the synthesis of sphinganine, reduce DAG generation, PKC activation and cell proliferation. All these inhibitory processes were restored by metabolic complementation with exogenous D-erythrosphingosine, indicating that the DAG generated during sphingolipid synthesis was necessary for PKC activation and cell proliferation. Also, we show that PI (phosphatidylinositol), PE (phosphatidylethanolamine) and PC (phosphatidylcholine) are the precursors of their respective sphingolipids (IPC, EPC and SM), and therefore sources of DAG to activate PKC.

Involvement of serine proteases in the excystation and metacystic development of Entamoeba invadens.

Although the functions of cysteine proteases involved in the pathogenicity and differentiation of Entamoeba histolytica have been demonstrated, little is known about the functions of serine proteases. We examined the involvement of serine proteases in amoebic excystation and metacystic development using inhibitors specific for serine proteases. Entamoeba invadens IP-1 strain was used as the model of excystation and metacystic development of E. histolytica. Four serine protease inhibitors, phenylmethanesulfonyl fluoride (PMSF), 4-(2-aminoethyl) bezensulfonylfluoride hydrochloride, 3, 4-dichloroisocoumarin, and N-tosyl-phe-chloromethylketone, decreased the number of metacystic amoebae in a dose-dependent manner, without showing cytotoxicity to cysts. PMSF inhibited not only the increase but also the development of metacystic amoebae as determined by the change of nucleus number from four- to one-nucleate amoebae. The protease activity in cyst lysates was also inhibited by PMSF and the band of protease on gelatin sodium dodecyl sulfate polyacrylamide gel electrophoresis was weaker than controls when treated with PMSF. Three serine protease families, S28 (three types), S9 (two), and S26 (one) were retrieved from the database of E. invadens. Phylogenetic analysis revealed that amebic enzymes from the serine protease families formed different clades from those from other organisms. The expression levels of these serine proteases in cysts 5 h after the induction of excystation as assessed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) were higher than those observed prior to induction assayed by real-time RT-PCR; the increase in one type of S9 (named S9-3) expression was the highest. The expression of S9 enzymes also increased from cysts to trophozoites higher than the other family serine proteases. Thus, the results show that Entamoeba uses their serine proteases in the excystation and metacystic development, which leads to successful infection.

[Diabetic foot infections. Prevalence and antibiotic sensitivity of the causative microorganisms]. / Infecciones del pie diabético. Prevalencia de los distintos microorganismos y sensibilidad a los antimicrobianos.

BACKGROUND: Foot infections are a common reason for hospitalization and a cause of complications in patients with diabetes. The aim of this study was to determine the prevalence of microorganisms found on culture in complicated diabetic foot infections in hospitalized patients, and the sensitivity of the causative microorganisms to antimicrobial agents. METHODS: Between December 2001 and December 2005 in our department, 84 samples in 62 diabetic patients with moderate/severe infection were collected for microbiological study. RESULTS: At least one microorganism was isolated in 88% of samples. The most frequently isolated germ group was gram-positive bacteria (55% of the samples), with Staphylococcus aureus (33%) in the first position, followed by Pseudomonas aeruginosa (12%), Enterococcus spp. (9%), and Escherichia coli (8%). Culture for anaerobic microorganisms was only performed in half the samples; 25% were positive, and Peptostreptococcus spp. predominated. Among the multiresistant microorganisms, methicillin-resistant staphylococci aureus (MRSA) were the most common, accounting for 38% of the isolated strains of S. aureus, ie, 12% of all samples. As to the gram-negative microorganisms, nearly 30% of E. coli strains were resistant to amoxicillin/clavulanic acid and ciprofloxacin. CONCLUSION: Most of the cultures in our study were monomicrobial, with S. aureus being the most prevalent microorganism, followed by enterobacteria and P. aeruginosa. The main resistant microorganism in diabetic foot infections requiring hospitalization was methicillin-resistant golden staphylococcus, which was found in 12% of the series.

The in vitro activity of selected mouthrinses on the reference strains of Trichomonas tenax and Entamoeba gingivalis

Protozoa, such as Trichomonas tenax, Entamoeba gingivalis and Leishmania braziliensis, may be present in the mouth but their role in the pathophysiology of oral diseases is not clear yet. The use of various types of mouthrinses plays an important role in maintaining proper oral hygiene and in removing some of the microbial components from the oral cavity. The purpose of this study was to investigate the effects of selected mouthrinses on the reference strains of Trichomonas tenax and Entamoeba gingivalis which can be a part of the oral cavity microbiota. Two standard strains Trichomonas tenax (ATCC 30207) and Entamoeba gingivalis (ATCC 30927) were used and metronidazole as a drug used in the treatment of infections caused by protozoa as well as fourteen agents used as mouthwashes were tested, with two pure compounds acting as mouthrinse ingredients, i.e. 20% benzocaine and 0.2% chlorhexidine, as well as 12 commercially-available formulas: Azulan, Colgate Plax Complete Care Sensitive, Corsodyl 0.2%, Curasept ADS 205, Dentosept, Dentosept A, Eludril Classic, Listerine Total Care, Octenidol, Oral-B Pro-Expert Clinic Line, Sylveco and Tinctura salviae. The protozoonicidal activity of the preparations was evaluated on the basis of the ratio of dead to living ratios after incubation in an incubator (37°C) for 1, 10 and 30 min. Protozoa were counted in the Bürker chamber in each case up to 100 cells in an optical microscope (over 400×). The criterion for the death of protozoa was the lack of movement and changes in the shape and characteristics of cell disintegration. The curves of activity were obtained after experiments conducted for 5­7 different solutions of each preparation. On the basis of the curves, the solution killing 50% of the population (CL50) was calculated. All mouthrinses tested in this work in their undiluted form acted lethally on both protozoa. Benzocaine, used as a local anesthetic, has etiotropic properties which can be useful for supporting antiprotozoal treatment. Chlorhexidine confirmed its high efficiency in the eradication of potentially pathogenic protozoa. The use of mouthrinses is an important complement for other procedures intended to maintain correct oral hygiene.

Growth-promoting effects of the hydrogen-sulfide compounds produced by Desulfovibrio desulfuricans subsp. desulfuricans co-cultured with Escherichia coli (DH5α) on the growth of Entamoeba and Endolimax species isolates from swine.

Certain Desulfovibrio sp. (anaerobic sulfate-reducing bacteria) are indigenous to swine cecum and colon, which are also common habitats for parasitic amoebae such as Entamoeba polecki and Entamoeba suis. In this study, we evaluated the growth-promoting effects of D. desulfuricans co-cultured with Escherichia coli (DH5α) and its products [e.g., hydrogen sulfide (H2S) and certain iron-sulfide (FeS) compounds] using Robinson's medium, on the 4 amoeba isolates from swine-Entamoeba polecki subtype (ST)-1, E. polecki ST-3, Entamoeba suis, and Endolimax sp., and, consequently, a continuous culture system for these amoebae was established. However, this novel culture system was required to regulate the excess H2S dissolved in the medium by increasing air space as amoeba isolates thrive only in large air spaces (30-40%). The effects of air space and H2S and FeS compounds on the growth of E. polecki ST-1 (TDP-5) were determined. E. polecki ST-1 (TDP-5) thrived well in culture bottles with an air space of 30-40% (aerobic) (H2S: ~250-400 µmoles/L), but did not grow at all in an air space < 5% (microaerobic) ( H2S:~800 µmoles/L) and in anaerobic vessels (H2S: 20-30 µmoles/L). In both H2S-depleted and FeS compound-depleted conditions, the amoebae sp. could not thrive either. It was hypothesized that an appropriate concentration of H2S and FeS compounds might function as important physiologically active components of electron carriers such as FeS and ferredoxin.

Drug Resistance in Protozoan Parasites: An Incessant Wrestle for Survival.

Nowadays, drug resistance in parasites is considered to be one of the foremost concerns in health and disease management. It is interconnected worldwide and undermines the health of millions of people, threatening to grow worse. Unfortunately, it does not receive serious attention from every corner of society. Consequently, drug resistance in parasites is gradually complicating and challenging the treatment of parasitic diseases. In this context, we have dedicated ourselves to review the incidence of drug resistance in the protozoan parasites Plasmodium, Leishmania, Trypanosoma, Entamoeba and Toxoplasma gondii. Moreover, understanding the role of ATP-binding cassette (ABC) transporters in drug resistance is essential in the control of parasitic diseases. Therefore, we also focused on the involvement of ABC transporters in drug resistance, which will be a superior approach to find ways for better regulation of diseases caused by parasitic infections.