Inhibitory and anti-adherent effects of Piper betle L. leaf extract against Acanthamoeba triangularis in co-infection with Staphylococcus aureus and Pseudomonas aeruginosa: A sustainable one-health approach.

Background and Aim: Keratitis is a serious ocular infection often caused by pathogenic microorganisms such as Acanthamoeba spp. Among other harmful microbes, Acanthamoeba keratitis presents a particular challenge due to its resistance to conventional antimicrobial agents. Piper betle Linn., commonly known as betel leaf, has been traditionally used for its medicinal properties. This study aimed to assess the potential of the leaf ethanol extract of P. betle Linn. in the treatment of Acanthamoeba triangularis in monoculture and co-culture with two prevalent pathogenic bacteria, Staphylococcus aureus and Pseudomonas aeruginosa, associated with keratitis. Materials and Methods: Minimum inhibitory concentrations (MICs) of A. triangularis, S. aureus, and P. aeruginosa extracts in monoculture and coinfected conditions were examined. In addition, this study explored the potential of the extract in preventing Acanthamoeba adherence in both monoculture and co-culture environments. Scanning electron microscopy (SEM) analysis confirmed the impact of the extract on Acanthamoeba cell membranes, including acanthopodia. Furthermore, a time-kill kinetic assay was used to validate the amoebicidal activity of the extract against A. triangularis and the tested bacteria. Results: MICs for trophozoites, cysts, P. aeruginosa, and S. aureus in the monoculture were 0.25, 0.25, 0.51, and 0.128 mg/mL, respectively, whereas the MICs for Acanthamoeba coinfected with bacteria were higher than those in the monoculture. This extract inhibited the growth of A. triangularis trophozoites and cysts for up to 72 h. Moreover, P. betle extract effectively prevented the adherence of Acanthamoeba to contact lenses under monoculture conditions. SEM analysis confirmed that P. betle extract affects the cell membrane of Acanthamoeba, including Acanthopodia. In addition, the time-kill kinetic assay confirmed that the extract contained amoebicidal activity against A. triangularis, including the tested bacteria. Notably, S. aureus was more susceptible than A. triangularis and P. aeruginosa to P. betle extract treatment. Unexpectedly, our study revealed that S. aureus negatively affected A. triangularis in the co-culture after 3 days of incubation, whereas P. aeruginosa facilitated the growth of A. triangularis in the presence of the extract. Conclusion: This study provides compelling evidence of the anti-adhesive and anti-Acanthamoeba properties of P. betle leaf extract against A. triangularis under monoculture and co-culture conditions. The observed impact on Acanthamoeba cell membranes, coupled with the time-kill kinetic assay results, underscores the potential of P. betle leaf extract as a promising agent for combating Acanthamoeba-related infections in humans and animals.

Piper betle extract and its application in bovine teat dipping solution inhibit and eliminate biofilms in bovine mastitis-inducing staphylococci.

Background and Aim: Staphylococci, including Staphylococcus aureus, Staphylococcus chromogenes, and Staphylococcus haemolyticus, are significant bacteria that induce bovine mastitis, primarily because they can form biofilms in bovine teat canals. This study aimed to investigate the efficacy of Piper betle extract and a bovine teat dipping solution containing P. betle extract (BSP) against these mastitis-causing staphylococci. Materials and Methods: BSP was prepared using P. betle extract as the bioactive compound. The antibacterial activity of the plant extract and BSP against the pathogens was investigated using a broth microdilution method. The activity of the extract and BSP against the pathogen biofilms was also determined. A stability test was performed to observe the pH, color, turbidity, homogeneity, precipitation, and separation of BSP stored at 4°C and 25°C for up to 4 weeks. Results: The extract exhibited potent antibacterial activity against S. aureus and S. haemolyticus, with similar values for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) ranging from 0.03 mg/mL to 0.125 mg/mL. The MIC and MBC values of the extract against S. chromogenes were 0.5-1 mg/mL and 0.5-2 mg/mL, respectively. Moreover, BSP exhibited MIC and MBC values of 12.5-50 v/v against all tested staphylococci isolates. When used at 1/2 and 1/4 × MIC, the extract and BSP significantly inhibited the formation of staphylococcal biofilms (p < 0.05) in the tested strains. The results indicated that treatment with 1/2 × MIC of the extract and BSP resulted in biofilm inhibition ranging from 30%-66% and 19%-39%, respectively. Furthermore, the extract at 16 × MIC eliminated 54%-86% of established mature isolate biofilms, whereas BSP removed 41%-61% of mature biofilm viability. Storage of BSP at 4°C did not change the factors associated with stability from the 1st to 4th week. Conclusion: These findings suggest that BSP may exhibit potential medicinal benefits in inhibiting the growth and biofilm formation of mastitis-inducing staphylococci in bovines.

Antibacterial and antivirulence factor activities of protein hydrolysates from Phatthalung Sangyod rice (Oryza sativa L.) seeds against zoonotic and foodborne pathogens.

Background and Aim: Antimicrobial resistance is an emerging public health threat. Foodborne illnesses are typically caused by bacteria, such as Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, and Staphylococcus aureus, which are frequently resistant to common antimicrobial agents. Rice is a staple grain in most parts of the world. Our previous work showed that Phatthalung Sangyod rice seed protein hydrolysates (SYPs), especially SYP4, exhibit antifungal activity against several fungal species that are pathogenic for both humans and animals and are non-cytotoxic to animal red blood cells. In this study, we aimed to determine the effects of the bioactive peptides in SYPs against several pathogenic bacteria in humans and animals. Materials and Methods: After isolating SYP1, it was treated as follows: heated (SYP2), and hydrolyzed using pepsin (SYP3), and proteinase K (SYP4). Then, we used 500 µg of protein to evaluate the antibacterial effects on four pathogenic bacteria, including E. coli, P. aeruginosa, B. cereus, and S. aureus, using agar well diffusion. Using a broth microdilution assay, we determined the minimum inhibitory and bactericidal concentration (MIC and MBC, respectively) values of active SYPs. Using the agar well diffusion and microtube incubation methods, we also assessed the inhibitory effects of SYPs on the bacterial quorum sensing (QS) activity of Chromobacterium violaceum. Sangyod rice seed protein hydrolysates were evaluated for their ability to inhibit the biofilm formation of bacterial cells by a crytal violet assay. Furthermore, using the dropping method, we tested the inhibitory effects of SYPs on the bacterial pigments pyocyanin in P. aeruginosa and staphyloxanthin in S. aureus. Results: Our results showed that the crude protein lysate (SYP1) did not exhibit antibacterial activity against any of the test bacteria. Intriguingly, after boiling (SYP2) and enzymatic hydrolysis (SYP3 and SYP4), the protein hydrolysates were transformed into bioactive peptides and displayed antibacterial properties against all of the test bacteria at a concentration of 500 µg as determined by agar well diffusion. SYP4 demonstrated the highest antibacterial activity as it completely inhibited all test strains, with inhibition zones ranging from 16.88 ± 0.25 to 21.25 ± 0.5 mm, and also yielded the highest MIC/MBC values against P. aeruginosa, B. cereus, and E. coli, at 256 and >256 µg/mL, respectively. We observed that at least 256 µg/mL of SYP4 is required to exhibit optimal antibacterial activity. At 16-128 µg/mL, it exhibited antibiofilm activity against S. aureus. Furthermore, at 256 µg/mL, SYP4 inhibited pyocyanin in P. aeruginosa and staphyloxanthin in S. aureus. Although SYP2 and SYP3 displayed weak antibacterial activity and their MIC values could not be obtained for all bacteria, they showed strong QS inhibition in C. violaceum at 256 µg protein. Moreover, SYP2 and SYP3, at a minimum concentration of 32 µg/mL, significantly reduced violacein production. SYP3 also showed biofilm reduction activity on S. aureus at least 16-512 µg/mL. Conclusion: Sangyod Phatthalung protein hydrolysates exerted excellent inhibitory effects against the growth of bacteria and their virulence factors, such as QS, biofilm formation, and/or pigment production. These factors include zoonotic and foodborne pathogens. Therefore, daily consumption of Sangyod Phatthalung rice might reduce the risk of bacterial pathogenesis and foodborne diseases. In conclusion, functional foods or alternate methods of treating bacterial illnesses may be developed in humans and animals.

Virulence factors and quorum sensing as targets of new therapeutic options by plant-derived compounds against bacterial infections caused by human and animal pathogens.

The emergence of antibiotic-resistant bacteria and hospital-acquired bacterial infection has become rampant due to antibiotic overuse. Virulence factors are secondary to bacterial growth and are important in their pathogenesis, and therefore, new antimicrobial therapies to inhibit bacterial virulence factors are becoming important strategies against antibiotic resistance. Here, we focus on anti-virulence factors that act through anti-quorum sensing and the subsequent clearance of bacteria by antimicrobial compounds, especially active herbal extracts. These quorum sensing systems are based on toxins, biofilms, and efflux pumps, and bioactive compounds isolated from medicinal plants can treat bacterial virulence pathologies. Ideally, bacterial virulence factors are secondary growth factors of bacteria. Hence, inhibition of bacterial virulence factors could reduce bacterial pathogenesis. Furthermore, anti-virulence factors from herbal compounds can be developed as novel treatments for bacterial infection. Therefore, this narrative review aims to discuss bacterial virulence factors acting through quorum sensing systems that are preserved as targets for treating bacterial infection by plant-derived compounds.

Knema retusa is antibacterial and antibiofilm against antibiotic resistant Staphylococcus aureus and S. haemolyticus isolated in bovine mastitis.

This study aimed to assess antibacterial activity of Knema retusa wood extract (KRe) against antibiotic resistant staphylococci which are causative agents of bovine mastitis. From 75 cases of intramammary infections in dairy cows, 66 staphylococcal isolates were collected, including 11 Staphylococcus aureus isolates (17%) and 55 coagulase-negative staphylococci (83%). Sixty isolates (91%) formed strong biofilms. KRe had minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) against the isolates ranging 32-256 ug/mL and 64-512 ug/mL, respectively. Two-hour KRe exposures at 4×MIC, viabilities of S. aureus and S. haemolyticus decreased by 3 log10 compared to the control. Scanning EM (SEM) showed that KRe disrupted the bacterial cells of both species. KRe at 1/16×MIC significantly inhibited biofilm formation (P < 0.05) in both S. aureus and S. haemolyticus. At 1/2×MIC, S. aureus and S. haemolyticus biofilm inhibition ranged from 75 to 99%. Cells within established biofilms were disrupted 66-83% by KRe at 32×MIC. Moreover, 1/2×MIC KRe reduced bacterial adhesion to glass surfaces observed by SEM. According to GC-MS analysis, the major compound in KRe was endo-2-hydroxy-9,9-(ethylenedioxy)-1-carbethoxy bicyclo [3.3.1] nonane (E2N). Molecular docking analysis of E2N has a high affinity for staphylococcal accessory regulator A (SarA), binding free-energy - 6.40kcal/mol. The results suggested that KRe may have medicinal benefits by inhibiting the growth, biofilm, and adhesion of antibiotic resistant staphylococci isolated from bovine mastitis.

A Comparative Study of Chemical Profiling and Bioactivities between Thai and Foreign Hemp Seed Species (Cannabis sativa L.) Plus an In-Silico Investigation.

Hemp (Cannabis sativa L.) is a plant widely used by humans for textiles, food, and medicine. Thus, this study aimed to characterize the chemical profiling of 12 hemp seed extracts from Thai (HS-TH) and foreign (HS-FS) samples using gas chromatography-mass spectrometry (GC-MS). Their antibacterial activity and α-glucosidase inhibitory activity were assayed. Linoleic acid (17.63-86.53%) was a major component presented in Thai hemp seed extracts, while α,ß-gluco-octonic acid lactone (30.39%), clionasterol (13.42-29.07%), and glyceryl-linoleate (15.12%) were detected as the main metabolites found in foreign hemp seed extracts. Furthermore, eight extracts from both Thai and foreign hemp seed exhibited antibacterial activity against Staphylococcus aureus, Staphylococcus epidermidis, Methicillin-resistant Staphylococcus aureus, and Cutibacterium acnes, with MIC values ranging from 128 to 2048 µg/mL. Interestingly, the ethanol extract of Thai hemp seed (HS-TH-2-M-E) showed superior α-glucosidase inhibition (IC50 value of 33.27 ug/mL) over foreign species. The combination between Thai hemp species (HS-TH-2-M-E) and acarbose showed a synergistic effect against α-glucosidase. Furthermore, the docking investigation revealed that fatty acids had a greater impact on α-glucosidase than fatty acid esters and cannabinoids. The computational simulation predicts a potential allosteric binding pocket of guanosine on glucosidase and is the first description of gluco-octonic acid's anti-glucosidase activity in silico. The findings concluded that Thai hemp seed could be used as a resource for supplemental drugs or dietary therapy for diabetes mellitus.

Anti-Acanthamoeba activity of a semi-synthetic mangostin derivative and its ability in removal of Acanthamoeba triangularis WU19001 on contact lens.

Garcinia mangostana L., also known as the mangosteen tree, is a native medicinal plant in Southeast Asia having a wide variety of pharmacologically active compounds, including xanthonoid mangostin. In this study, we examined the pharmacological activities of the selected semi-synthetic mangostin derivative, namely, amoebicidal activity, encystation inhibition, excystation activity, and removal capacity of adhesive Acanthamoeba from the surface of contact lens (CL). Among the three derivatives, C1 exhibited promising anti-Acanthamoeba activity against Acanthamoeba triangularis WU19001 trophozoites and cysts. SEM images displayed morphological changes in Acanthamoeba trophozoites, including the loss of acanthopodia, pore formation in the cell membrane, and membrane damage. In addition, the treated cyst was shrunken and adopted an irregular flat cyst shape. Under a fluorescence microscope, acridine orange and propidium iodide (AO/PI) staining revealed C1 induced condensation of cytoplasm and chromatin with the loss of cell volume in the treated trophozoites, while calcofluor white staining demonstrated the leakage of cell wall in treated cysts, leading to cell death. Interestingly, at the concentration ranges in which C1 showed the anti-Acanthamoeba effects (IC50 values ranging from 0.035-0.056 mg/mL), they were not toxic to Vero cells. C1 displayed the highest inhibitory effect on A. triangularis encystation at 1/16×MIC value (0.004 mg/mL). While C1 demonstrated the excystation activity at 1/128×MIC value with a high rate of 89.47%. Furthermore, C1 exhibited the removal capacity of adhesive Acanthamoeba from the surface of CL comparable with commercial multipurpose solutions (MPSs). Based on the results obtained, C1 may be a promising lead agent to develop a therapeutic for the treatment of Acanthamoeba infections and disinfectant solutions for CL.

Robusta coffee extracts inhibit quorum sensing activity in Chromobacterium violaceum and reduce biofilms against Bacillus cereus and Staphylococcus aureus.

Background and Aim: Bacillus cereus and Staphylococcus aureus cause foodborne intoxication in humans and animals. Pathogens can produce biofilms controlled by the quorum sensing system. The study aimed to investigate the antibacterial, antibiofilm, and anti-quorum sensing activities of Coffea canephora P. ex Fr. (Robusta coffee) extracts against B. cereus and S. aureus. Materials and Methods: Ethanol extracts of fruit peels and seeds of Robusta coffee were tested for antibacterial activity against B. cereus and S. aureus using a broth microdilution assay. Reduction of the biofilm formation and elimination of the viability of mature biofilm-grown cells of B. cereus and S. aureus were determined. Inhibition of quorum sensing activity in Chromobacterium violaceum by the extracts was investigated using the disk diffusion method and flask incubation assay. Results: Fresh fruit peel extract showed the strongest antibacterial activity against B. cereus and S. aureus with minimum inhibitory concentration (MIC) values of 2 and 4 mg/mL, respectively. However, the extracts did not inhibit Escherichia coli, avian pathogenic E. coli, and Pseudomonas aeruginosa at 8 mg/mL. Significant inhibition of biofilm formation at 1/2 × MIC of the fresh peel extract was detected in B. cereus (56.37%) and S. aureus (39.69 %), respectively. At 8 × MIC of the fresh peel extract, a significant elimination of the mature biofilm viability was detected in B. cereus (92.48%) and S. aureus (74.49%), respectively. The results showed that fresh and dried peel fruit extracts at 1/2 × MIC significantly reduced violacein production with the highest percentage inhibition ranging from 44.53 to 47.48% at 24 h (p ≤ 0.05). Conclusion: The results of the present study suggest the potential therapeutic benefits of Robusta coffee extracts in inhibiting the growth, biofilm, and quorum sensing of both B. cereus and S. aureus. The results put forward an alternative strategy to control the foodborne intoxications caused by both pathogens.

Sangyod rice bran extract enhances Lacticaseibacillus paracasei growth during the exponential phase and antibacterial activity of L. paracasei supernatant against zoonotic and foodborne pathogens.

Background and Aim: Prebiotics are a group of nutrients or compounds that are degraded by the gut microbiota, including Lacticaseibacillus paracasei. The probiotic plays an important role in adhesion to the gut and is able to produce antimicrobial substances to inhibit pathogens. This study aimed to investigate the effects of Sangyod rice bran extract on the growth promotion of L. paracasei. Furthermore, antibacterial activity of the extract and L. paracasei supernatants cultured in De Man, Rogosa and Sharpe (MRS) medium plus the extract against zoonotic and foodborne pathogens was investigated. Materials and Methods: Antibacterial activity of the crude extract and the oil from Sangyod rice bran against the pathogens, including Bacillus cereus, Staphylococcus aureus, Escherichia coli, Avian pathogenic E. coli, and Pseudomonas aeruginosa was investigated using broth microdilution assay. The effects of the crude extract and the oil on the growth and adhesion of L. paracasei were further determined. The antibacterial activity of L. paracasei supernatant cultured in the medium supplemented with the extract and the oil against the pathogens was determined by agar well diffusion assay, followed by the broth microdilution assay. Finally, the chemical constituents and antioxidant activity of the crude extract and the oil from Sangyod rice bran were investigated. Results: The crude extract and the oil from Sangyod rice bran enhanced L. paracasei growth during the exponential phase. Furthermore, the crude extract at 0.25 mg/mL significantly enhanced the adhesion of L. paracasei to the surface compared with the control. Both minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values of the crude extract against B. cereus and S. aureus were 0.5 and 1.0 mg/mL, respectively. All pathogens were sensitive to the supernatant of L. paracasei with similar MIC and MBC ranging from 12.5% v/v to 50% v/v. However, the MIC and MBC values of L. paracasei supernatant grown in MRS medium plus the crude extract and oil were not significantly different compared to the supernatant obtained from MRS alone. The crude extract had free radical scavenging activities with IC50 values at 0.61 mg/mL. Conclusion: The results suggested the potential benefits of the crude extract from Sangyod rice bran for inducing the growth and the adhesion of L. paracasei and inhibiting zoonotic and foodborne pathogens.

Curcumin effect on Acanthamoeba triangularis encystation under nutrient starvation.

Background: Curcumin is an active compound derived from turmeric, Curcuma longa, and is known for its benefits to human health. The amoebicidal activity of curcumin against Acanthamoeba triangularis was recently discovered. However, a physiological change of intracellular pathways related to A. triangularis encystation mechanism, including autophagy in the surviving amoeba after curcumin treatment, has never been reported. This study aims to investigate the effect of curcumin on the survival of A. triangularis under nutrient starvation and nutrient-rich condition, as well as to evaluate the A. triangularis encystation and a physiological change of Acanthamoeba autophagy at the mRNA level. Methods: In this study, A. triangularis amoebas were treated with a sublethal dose of curcumin under nutrient starvation and nutrient-rich condition and the surviving amoebas was investigated. Cysts formation and vacuolization were examined by microscopy and transcriptional expression of autophagy-related genes and other encystation-related genes were evaluated by real-time PCR. Results: A. triangularis cysts were formed under nutrient starvation. However, in the presence of the autophagy inhibitor, 3-methyladenine (3-MA), the percentage of cysts was significantly reduced. Interestingly, in the presence of curcumin, most of the parasites remained in the trophozoite stage in both the starvation and nutrient-rich condition. In vacuolization analysis, the percentage of amoebas with enlarged vacuole was increased upon starvation. However, the percentage was significantly declined in the presence of curcumin and 3-MA. Molecular analysis of A. triangularis autophagy-related (ATG) genes showed that the mRNA expression of the ATG genes, ATG3, ATG8b, ATG12, ATG16, under the starvation with curcumin was at a basal level along the treatment. The results were similar to those of the curcumin-treated amoebas under a nutrient-rich condition, except AcATG16 which increased later. On the other hand, mRNA expression of encystation-related genes, cellulose synthase and serine proteinase, remained unchanged during the first 18 h, but significantly increased at 24 h post treatment. Conclusion: Curcumin inhibits cyst formation in surviving trophozoites, which may result from its effect on mRNA expression of key Acanthamoeba ATG-related genes. However, further investigation into the mechanism of curcumin in A. triangularis trophozoites arrest and its association with autophagy or other encystation-related pathways is needed to support the future use of curcumin.

Phytochemical, anti-Acanthamoeba, and anti-adhesion properties of Garcinia mangostana flower as preventive contact lens solution.

Acanthamoeba keratitis infection extends due to the growing number of contact lens users. Indigenous plants including Garcinia mangostana play a vital role in human health and well being. Many species of this plant have been reported with myriads of potent medicinal properties. However, the aims of this study were, for the first time, to isolate compounds from the flower of G. mangostana and to test their anti-Acanthamoeba and anti-adhesion activity against Acanthamoeba triangularis. Powdered flowers of G. mangostana were extracted and chromatographed on a silica gel column. The structures of the compounds were established with the aid of 1H NMR. More so, the anti-Acanthamoeba and anti-adhesion properties were tested on a 96-well polystyrene microtiter plate and soft contact lenses. Scanning electron microscope (SEM) was used to determine the features of A. triangularis on contact lenses. Eight pure compounds were obtained, namely 9-hydroxycalabaxanthone, tovophillin A, garcinone E, garcinone B, α-mangostin, gartinin, 8-deoxygartinin and γ-mangostin. The extract and pure compounds exhibited anti-Acanthamoeba activity with MIC values in the range of 0.25-1 mg/mL. In addition, the extract and α-mangostin displayed significant activity against the adhesion of A. triangularis trophozoites both in polystyrene plate and in contact lenses at 0.5 × MIC (0.25 mg/mL). Furthermore, α-mangostin has the potential to remove A. triangularis adhesion in contact lenses similar to a commercial multipurpose solution (MPS). SEM study confirmed that crude extract and α-mangostin are effective as solutions for contact lenses, which removed A. triangularis trophozoites within 24 h. Alpha-mangostin was non-toxic to Vero cells at a concentration below 39 µM in 24 h. Crude extract of G. mangostana flower and its α-mangostin serve as candidate compounds in the treatment of Acanthamoeba infection or as lens care solution, since they can be used as a source of natural products against Acanthamoeba and virulence factor associated with the adhesion of A. triangularis.

Conserved Candidate Antigens and Nanoparticles to Develop Vaccine against Giardia intestinalis.

Giardia intestinalis (Giardia lambia, Giardia duodenalis) infections in humans may be asymptomatic or symptomatic and associated with diarrhea (without blood), abdominal cramps, bloating, flatulence, and weight loss. The protozoan Giardia is the third most common cause of diarrhea and death in children under five, preceded only by rotavirus and by Cryptosporidium parvum and C. hominis infections. Antimicrobial drugs, particularly 5-nitroimidazole (5-NIs), are used to treat giardiasis in humans. Immunologically naive or immunocompromised host are more vulnerable to Giardia infection, whereas a degree of resistance to this protozoan is present in humans living in endemic areas. This suggests that vaccination may be a potential and appropriate means to control this parasitic disease outbreak and protect the human population. This review discusses Giardia antigens related to vaccine development. Additionally, based on the latest development of nanoparticle technology, a combination of methods for future research and development is proposed for the design of the next generation of powerful immunogens and an effective vaccine against Giardia.

Targeting Acanthamoeba proteins interaction with flavonoids of Propolis extract by in vitro and in silico studies for promising therapeutic effects.

Background : Propolis is a natural resinous mixture produced by bees. It provides beneficial effects on human health in the treatment/management of many diseases. The present study was performed to demonstrate the anti- Acanthamoeba activity of ethanolic extracts of Propolis samples from Iran. The interactions of the compounds and essential proteins of Acanthamoeba were also visualized through docking simulation. Methods: The minimal inhibitory concentrations (MICs) of Propolis extract against Acanthamoeba trophozoites and cysts was determined in vitro. In addition, two-fold dilutions of each of agents were tested for encystment, excystment and adhesion inhibitions. Three major compounds of Propolis extract such as chrysin, tectochrysin and pinocembrin have been selected in molecular docking approach to predict the compounds that might be responsible for encystment, excystment and adhesion inhibitions of A. castellanii. Furthermore, to confirm the docking results, molecular dynamics (MD) simulations were also carried out for the most promising two ligand-pocket complexes from docking studies. Results : The minimal inhibitory concentrations (MICs) 62.5 and 125 µg/mL of the most active Propolis extract were assessed in trophozoites stage of Acanthamoeba castellanii ATCC30010 and ATCC50739, respectively. At concentrations lower than their MICs values (1/16 MIC), Propolis extract revealed inhibition of encystation. However, at 1/2 MIC, it showed a potential inhibition of excystation and anti-adhesion. The molecular docking and dynamic simulation revealed the potential capability of Pinocembrin to form hydrogen bonds with A. castellanii Sir2 family protein (AcSir2), an encystation protein of high relevance for this process in Acanthamoeba. Conclusions : The results provided a candidate for the development of therapeutic drugs against Acanthamoeba infection. In vivo experiments and clinical trials are necessary to support this claim.

Antibacterial, antibiofilm, and anti-adhesion activities of Piper betle leaf extract against Avian pathogenic Escherichia coli.

Piper betle leaves have traditionally been used to treat many diseases, including bacterial infections. The present study aimed to investigate the antibacterial, antibiofilm, and anti-adhesion activities of P. betle extract against avian pathogenic Escherichia coli (APEC). The ethanol extract of P. betle leaves demonstrated strong antibacterial activity against clinical isolates of APEC with MIC and MBC values ranging from 0.5 to 1.0 mg/mL as compared with 1% DMSO, a negative control. Disruption and breakdown of the bacterial cells were detected when the cells were challenged with the extract at 2 × MIC. Bacterial cells treated with the extract demonstrated longer cells without a septum, compared to the control. The extract at 1/8, 1/4, and 1/2 × MIC significantly inhibited the formation of the bacterial biofilm of all the tested isolates except the isolate CH10 (P < 0.05) without inhibiting growth. At 1/2 × MIC, 55% of the biofilm inhibition was detected in APEC CH09, a strong biofilm producer. At 32 × MIC, 88% of the inhibition of viable cells embedded in the mature biofilm was detected in APEC CH09. Reduction in the bacterial adhesion to surfaces was shown when APEC were treated with sub-MICs of the extract as observed by SEM. Hydroxychavicol was found to be the major compound presented in the leaf extract as detected by GC-MS analysis. The information suggested potential medicinal benefits of P. betle extract to inhibit the growth, biofilm, and adhesion of avian pathogenic E. coli.

Peganum harmala Extract Has Antiamoebic Activity to Acanthamoeba triangularis Trophozoites and Changes Expression of Autophagy-Related Genes.

Peganum harmala, a well-known medicinal plant, has been used for several therapeutic purposes as it contains numerous pharmacological active compounds. Our study reported an anti-parasitic activity of P. harmala seed extract against Acanthamoeba triangularis. The stress induced by the extract on the surviving trophozoites for Acanthamoeba encystation and vacuolization was examined by microscopy, and transcriptional expression of Acanthamoeba autophagy-related genes was investigated by quantitative PCR. Our results showed that the surviving trophozoites were not transformed into cysts, and the number of trophozoites with enlarged vacuoles were not significantly different from that of untreated control. Molecular analysis data demonstrated that the mRNA expression of tested AcATG genes, i.e., ATG3, ATG8b, and ATG16, was at a basal level along the treatment. However, upregulation of AcATG16 at 24 h post treatment was observed, which may indicate an autophagic activity of this protein in response to the stress. Altogether, these data revealed the anti-Acanthamoeba activity of P. harmala extract and indicated the association of autophagy mRNA expression and cyst formation under the extract stress, representing a promising plant for future drug development. However, further identification of an active compound and a study of autophagy at the protein level are needed.

Potential anti-Acanthamoeba and anti-adhesion activities of Annona muricata and Combretum trifoliatum extracts and their synergistic effects in combination with chlorhexidine against Acanthamoeba triangularis trophozoites and cysts.

Plants with medicinal properties have been used in the treatment of several infectious diseases, including Acanthamoeba infections. The medicinal properties of Cambodian plant extracts; Annona muricata and Combretum trifoliatum were investigated against Acanthamoeba triangularis. A total of 39 plant extracts were evaluated and, as a result, 22 extracts showed positive anti-Acanthamoeba activity. Of the 22 extracts, 9 and 4 extracts showed anti-Acanthamoeba activity against trophozoites and cysts of A. triangularis, respectively. The minimum inhibitory concentration of A. muricata and C. trifoliatum extracts against trophozoites and cysts was 500 and 1,000 µg/mL, respectively. The combination of A. muricata at 1/4×MIC with chlorhexidine at 1/8×MIC demonstrated a synergistic effect against trophozoites, but partial synergy against cysts. A 40% reduction in trophozoites and 60% of cysts adhered to the plastic surface treated with both extracts at 1/2×MIC were noted comparing to the control (P < 0.05). Furthermore, a reduction of 80% and 90% of trophozoites adhered to the surface was observed after pre-treatment with A. muricata and C. trifoliatum extracts, respectively. A 90% of cysts adhered to the surface was decreased with pre-treatment of A. muricata at 1/2×MIC (P < 0.05). A 75% of trophozoites and cysts from Acanthamoeba adhered to the surface were removed after treatment with both extracts at 4×MIC (P < 0.05). In the model of contact lens, 1 log cells/mL of trophozoites and cysts was significantly decreased post-treatment with both extracts compared to the control. Trophozoites showed strong loss of acanthopodia and thorn-like projection pseudopodia, while cysts demonstrated retraction and folded appearance treated with both extracts when observed by SEM, which suggests the potential benefits of the medicinal plants A. muricata and C. trifoliatum as an option treatment against Acanthamoeba infections.

Amoebicidal activity of Cassia angustifolia extract and its effect on Acanthamoeba triangularis autophagy-related gene expression at the transcriptional level.

Cassia angustifolia Vahl. plant is used for many therapeutic purposes, for example, in people with constipation, skin diseases, including helminthic and parasitic infections. In our study, we demonstrated an amoebicidal activity of C. angustifolia extract against Acanthamoeba triangularis trophozoite at a micromolar level. Scanning electron microscopy (SEM) images displayed morphological changes in the Acanthamoeba trophozoite, which included the formation of pores in cell membrane and the membrane rupture. In addition to the amoebicidal activity, effects of the extract on surviving trophozoites were observed, which included cyst formation and vacuolization by a microscope and transcriptional expression of Acanthamoeba autophagy in response to the stress by quantitative polymerase chain reaction. Our data showed that the surviving trophozoites were not transformed into cysts and the trophozoite number with enlarged vacuole was not significantly different from that of untreated control. Molecular analysis data demonstrated that the mRNA expression of AcATG genes was slightly changed. Interestingly, AcATG16 decreased significantly at 12 h post treatment, which may indicate a transcriptional regulation by the extract or a balance of intracellular signalling pathways in response to the stress, whereas AcATG3 and AcATG8b remained unchanged. Altogether, these data reveal the anti-Acanthamoeba activity of C. angustifolia extract and the autophagic response in the surviving trophozoites under the plant extract pressure, along with data on the formation of cysts. These represent a promising plant for future drug development. However, further isolation and purification of an active compound and cytotoxicity against human cells are needed, including a study on the autophagic response at the protein level.

Anti-Acanthamoeba synergistic effect of chlorhexidine and Garcinia mangostana extract or α-mangostin against Acanthamoeba triangularis trophozoite and cyst forms.

Acanthamoeba spp. can cause amoebic keratitis (AK). Chlorhexidine is effective for AK treatment as monotherapy, but with a relative failure on drug bioavailability in the deep corneal stroma. The combination of chlorhexidine and propamidine isethionate is recommended in the current AK treatment. However, the effectiveness of treatment depends on the parasite and virulence strains. This study aims to determine the potential of Garcinia mangostana pericarp extract and α-mangostin against Acanthamoeba triangularis, as well as the combination with chlorhexidine in the treatment of Acanthamoeba infection. The minimal inhibitory concentrations (MICs) of the extract and α-mangostin were assessed in trophozoites with 0.25 and 0.5 mg/mL, for cysts with 4 and 1 mg/mL, respectively. The MIC of the extract and α-mangostin inhibited the growth of A. triangularis trophozoites and cysts for up to 72 h. The extract and α-mangostin combined with chlorhexidine demonstrated good synergism, resulting in a reduction of 1/4-1/16 of the MIC. The SEM results showed that Acanthamoeba cells treated with a single drug and its combination caused damage to the cell membrane and irregular cell shapes. A good combination displayed by the extract or α-mangostin and chlorhexidine, described for the first time. Therefore, this approach is promising as an alternative method for the management of Acanthamoeba infection in the future.

Curcuma longa rhizome extract and Curcumin reduce the adhesion of Acanthamoeba triangularis trophozoites and cysts in polystyrene plastic surface and contact lens.

Curcuma longa and Curcumin have been documented to have a wide spectrum of pharmacological effects, including anti-Acanthamoeba activity. Hence, this study sought to explore the anti-adhesion activity of C. longa extract and Curcumin against Acanthamoeba triangularis trophozoites and cysts in plastic and contact lenses. Our results showed that C. longa extract and Curcumin significantly inhibited the adhesion of A. triangularis trophozoites and cysts to the plastic surface, as investigated by the crystal violet assay (P < 0.05). Also, an 80-90% decrease in adhesion of trophozoites and cysts to the plastic surface was detected following the treatment with C. longa extract and Curcumin at 1/2 × MIC, compared to the control. In the contact lens model, approximately 1 log cells/mL of the trophozoites and cysts was reduced when the cells were treated with Curcumin, when compared to the control. Pre-treatment of the plastic surface with Curcumin at 1/2-MIC reduced 60% and 90% of the adhesion of trophozoites and cysts, respectively. The reduction in 1 Log cells/mL of the adhesion of A. triangularis trophozoites was observed when lenses were pre-treated with both the extract and Curcumin. Base on the results obtained from this study, A. triangularis trophozoites treated with C. longa extract and Curcumin have lost strong acanthopodia, thorn-like projection pseudopodia observed by scanning electron microscope. This study also revealed the therapeutic potentials of C. longa extract and Curcumin, as such, have promising anti-adhesive potential that can be used in the management/prevention of A. triangularis adhesion to contact lenses.

Gordonia sediminis sp. nov., an actinomycete isolated from mangrove sediment.

The taxonomic position of an actinomycete designated AMA 120T, isolated from mangrove sediment, was clarified by phenotypic, chemotaxonomic and phylogenetic studies. The 16S rRNA gene sequence revealed that strain AMA 120T was most closely related to Gordonia rhizosphera NBRC 16068T (98.9 %), Gordonia polyisoprenivorans NBRC 16320T (98.1 %) and Gordonia bronchialis NBRC 16047T (98.1 %). A fragment of the gyrB gene of strain AMA 120T formed a distinct phyletic line with G. rhizosphera NBRC 16068T (95.4 %). Strain AMA 120T contained meso-diaminopimelic acid, arabinose and galactose as cell-wall components, and MK-9(H2) was the predominant menaquinone. The polar lipid profile for this strain consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and two unidentified phospholipids. Mycolic acid was present. The major fatty acids were C16 : 0, C18 : 1ω9c and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The DNA-DNA relatedness values between AMA 120T and close species were below 70 %. There was an obvious distinction in the average nucleotide identity distribution between strain AMA 120T and its closely related strains at around 75-92%. The DNA G+C content of strain AMA 120T was 66.6 mol%. These results, coupled with the phenotypic and chemotaxonomic data, indicated that strain AMA 120T represents a novel species of the genus Gordonia, for which the name Gordoniasediminis sp. nov. is proposed. The type strain is AMA 120T (=TBRC 7109T=NBRC 113236T).