Survival of foodborne pathogens in Acanthamoeba castellanii cysts on fresh and pickled cucumber and cauliflower.

Amoebicidal activity of benzothiazole on Acanthamoeba castellanii cysts and trophozoites and its cytotoxic potentials

The amoebicidal activity of three substances derived from benzothiazole on Acanthamoeba castellanii cysts and trophozoites and its cytotoxic potentials

The existence of symbiotic relationships between Acanthamoeba and a variety of bacteria is well-documented. However, the ability of Acanthamoeba interacting with host bacterial pathogens has gained particular attention. Here, to understand the interactions of Escherichia coli K1 and E. coli K5 strains with Acanthamoeba castellanii trophozoites and cysts, association assay, invasion assay, survival assay, and the measurement of bacterial numbers from cysts were performed, and nonpathogenic E. coli K12 was also applied. The association ratio of E. coli K1 with A. castellanii was 4.3 cfu per amoeba for 1 hr but E. coli K5 with A. castellanii was 1 cfu per amoeba for 1 hr. By invasion and survival assays, E. coli K5 was recovered less than E. coli K1 but still alive inside A. castellanii. E. coli K1 and K5 survived and multiplied intracellularly in A. castellanii. The survival assay was performed under a favourable condition for 22 hr and 43 hr with the encystment of A. castellanii. Under the favourable condition for the transformation of trophozoites into cysts, E. coli K5 multiplied significantly. Moreover, the pathogenic potential of E. coli K1 from A. castellanii cysts exhibited no changes as compared with E. coli K1 from A. castellanii trophozoites. E. coli K5 was multiplied in A. castellanii trophozoites and survived in A. castellanii cysts. Therefore, this study suggests that E. coli K5 can use A. castellanii as a reservoir host or a vector for the bacterial transmission.

This study examines the effects of three different drugs with metformin, acarbose and pioglitazone active ingredients used for antidiabetic purposes on Acanthamoeba cysts and trophozoites. Cultures of A. castellanii trophozoites and cysts were prepared to test the anti-amoebic activity of metformin, acarbose and pioglitazone. Cultures were then prepared for A. castellanii cyst and trophozoite forms and parasites were exposed to different concentrations (0.750 mg/mL, 0.375 mg/mL, 0.186 mg/mL and 0.093 mg/mL) of metformin, acarbose and pioglitazone. As a result of the study, the reproductive potential suppressive effects and conversion from trophozoite form to cyst form of all three substances on A. castellanii trophozoites and cysts were determined. Parasites were counted at 12, 24 and 48 hours in the cell counter after staining with trypan blue. In comparison of the effects of metformin, acarbose and pioglitazone used in the study on A. castellanii trophozoites and cysts, it was observed that all three substances were statistically effective against cysts and trophozoites at a concentration of 0.750 mg/mL. Furthermore, it was determined that all concentrations of the three active substances included in the study significantly decreased the rate of cyst formation even at the end of the 7th day. In this context, it was determined that all three substances have amebicidal effects, and they significantly inhibit the transformation of A. castellanii trophozoites to cyst form. It is thought that these active substances, which are currently used as anti-diabetic, can be used in combination with other drugs in A. castellanii infections based on our study findings.

Status of the effectiveness of contact lens disinfectants in Malaysia against keratitis-causing pathogens

Targeting cyst wall is an effective strategy in improving the efficacy of marketed contact lens disinfecting solutions against Acanthamoeba castellanii cysts.

Simple SummaryAcanthamoeba is free-living amoeba known to cause severe vision threatening eye infection. In most of the cases treating this infection is difficult due to the Acanthamoeba’s ability to switch between an active feeding stage (trophozoite) and inactive dormant stage (cyst). Arginine rich peptides are highly positively charged and can kill the microorganisms by acting on their negatively charged surface. This study assessed the anti-amoebic activity of polyhomoarginines of different lengths against Acanthamoeba castellanii trophozoites and cysts. Polyhomoarginine showed excellent anti-amoebic activity against both the stages of Acanthamoeba castellanii.Arginine-rich peptides can have broad-spectrum anti-bacterial and anti-fungal activities. Polyhomoarginine consists of highly cationic residues which can act on the negatively charged microbial cell membranes. Acanthamoeba is a free-living protozoan known to cause a rare corneal infection which is difficult to diagnose and treat. This study evaluated the activity of the polyhomoarginines against Acanthamoeba castellanii. Acanthamoeba amoebicidal, amoebistatic, encystation and excystment assays were performed using protocols described in the literature. The activity of polyhomoarginines (PHAs) of different lengths (10 to 400 residues) was measured against the trophozoites and cysts of Acanthamoeba castellanii ATCC30868 in concentrations ranging from 0.93 μM to 15 μM. Data were represented as mean ± SE and analysed using one-way ANOVA. Overall, PHAs demonstrated good anti-acanthamoeba activity against both trophozoites and cysts. PHA 30 reduced the number of viable trophozoites by 99%, inhibited the formation of cysts by 96% and the emergence of trophozoites from cysts by 67% at 3.75 μM. PHA 10 was similarly active, but at a slightly higher concentration of 15 μM, reducing the numbers of viable trophozoites by 98%, inhibiting cyst formation by 84% and preventing the emergence of trophozoites from cysts by 99%. At their greatest anti-amoeba concentrations, PHA 10 gave only 8% haemolysis at 15 μM while PHA 30 gave <40 % haemolysis at 3.75 μM. Polyhomoarginine 10 showed excellent anti-amoebic activity against both forms of Acanthamoeba castellanii and was non-toxic at its most active concentrations. This implies that polyhomoarginines can be developed into a potential therapeutic agent for Acanthamoeba keratitis. However, there is a need to carry out further pre-clinical and then in vivo experiments in the AK animal model.

Objective: This study aimed to evaluate the amoebicidal activity of o-phenylenediamine (o-PDA)-conjugated carrier systems, including maleic anhydride-vinyl acetate alternating copolymer (MAVA) and citric acid-derived carbon quantum dots (CNDs), against Acanthamoeba castellanii cysts and trophozoites. Additionally, the cytotoxicity of these compounds was assessed on the HT-22 Mouse Hippocampal Neuronal cell line. Method: Two non-toxic carrier systems were synthesized: MAVA via charge transfer complex (CTC) radical polymerization, and CNDs via rapid thermal pyrolysis of citric acid. o-PDA surface conjugation was achieved through an amide mechanism after the ring-opening reaction. The synthesized compounds were tested for their amoebicidal effects on A. castellanii cysts and trophozoites, while cytotoxicity analyses were conducted using the HT-22 neuronal cell line. Results: According to our findings, the cytotoxicity of all compounds at the tested concentrations was within an acceptable range. When examining the amoebicidal activities, it was observed that MAVA at concentrations of 0.01 g/mL and 0.005 g/mL did not have any viable trophozoites, while CNDs at a concentration of 0.01 g/mL did not have any viable cysts. Overall, all compounds showed significant amoebicidal activity. Conclusion: Our findings demonstrate a strong dose-dependent amoebicidal effect of these substances on A. castellanii cysts and trophozoites. It is believed that these substances, supported by in vivo experiments, could potentially serve as new therapeutic agents.

Acanthamoeba castellanii has ubiquitous distribution and causes primary acanthamoebic keratitis (AK). AK is a common disease in contact lens wearers and results in permanent visual impairment or blindness. In this study, we observed the cytopathic effect, in vitro cytotoxicity, and secretion pattern of cytokines in human corneal epithelial cells (HCECs) induced by A. castellanii trophozoites and/or cysts. Morphological observation revealed that panked dendritic HCECs co-cultured with amoeba cysts had changed into round shape and gradually died. Such changes were more severe in co-culture with cyst than those of co-cultivation with trophozoites. In vitro cytotoxicity assay revealed the highest cytotoxicity to HCECs in the co-culture system with amoeba cysts. A. castellanii induced the expression of IL-1α, IL-6, IL-8, and CXCL1 in HCECs. Secreted levels of IL-1α, IL-6, and IL-8 in HCECs co-cultured with both trophozoites and cysts were increased at an early incubation time (3 and 6 hr). These results suggested that cytopathic changes and pro-inflammatory cytokines release of HCECs in response to A. castellanii, especially amoebic cysts, are an important mechanism for AK development.

Several lines of evidence suggest that Acanthamoeba interact with bacteria, which may aid in pathogenic bacterial transmission to susceptible hosts, and these interactions may have influenced evolution of bacterial pathogenicity. In this study, we tested if Gram-negative Pseudomonas aeruginosa and Gram-positive Corynebacterium spp. can associate/invade and survive inside Acanthamoeba castellanii trophozoites and cysts, as well as non-phagocytic human brain microvascular endothelial cells. The results revealed that both Corynebacterium spp. and P. aeruginosa were able to associate as well as invade and/or taken up by the phagocytic A. castellanii trophozoite. In contrast, P. aeruginosa exhibited higher association as well as invasion of non-phagocytic HBMEC compared with Corynebacterium spp. Notably, P. aeruginosa remained viable during the encystment process and exhibited higher levels of recovery from mature cysts (74.54 bacteria per amoebae) compared with Corynebacterium spp. (2.69 bacteria per amoeba) (P < 0.05). As Acanthamoeba cysts can be airborne, these findings suggest that Acanthamoeba is a potential vector in the transmission of P. aeruginosa to susceptible hosts. When bacterial-ridden amoebae were exposed to favourable (nutrient-rich) conditions, A. castellanii emerged as vegetative trophozoites and remained viable, and likewise viable P. aeruginosa were also observed but rarely any Corynebacterium spp. were observed. Correspondingly, P. aeruginosa but not Corynebacterium spp. exhibited higher cytotoxicity to non-phagocytic HBMEC, producing more than 75% cell death in 24 h, compared to 20% cell death observed with Corynebacterium spp. Additionally, it was observed that the bacterial conditioned medium had no negative effect on A. castellanii growth. Further characterization of amoebal and bacterial interactions will assist in identifying the role of Acanthamoeba in the transmission and evolution of pathogenic bacteria.

The current study aimed to investigate the trophocidal and cysticidal activities of cinnamaldehyde (Cinn), a natural compound with known antimicrobial properties, against environmental and reference strains of Acanthamoeba castellanii. Additionally, we explored the potential benefits of Cinn formulated as a nanoemulsion (Cinn-NE) in enhancing its efficacy. Cinn-NE was prepared using the ultrasonic emulsification method. The amoebicidal effect of Cinn was evaluated against both trophozoites and cysts of reference and environmental strains of Acanthamoeba, along with an assessment of whether nanotechnology enhances this effect. Our results demonstrated that Cinn exhibited strong activity against both trophozoites and cysts of Acanthamoeba. Importantly, Cinn-NE showed enhanced activity compared to pure Cinn, possibly due to increased surface area and improved interaction with microbial membranes. In this study, the difference in susceptibility to Cinn and Cinn-NE between the environmental and the reference strain of A. castellanii was observed. Acanthamoeba cysts were more resistant to the effects of Cinn or Cinn-NE than trophozoites. Additionally, we found that nutrient availability in the medium influenced the susceptibility of Acanthamoeba to Cinn and Cinn-NE. A nutrient-deficient medium reduced their vulnerability to destruction, suggesting a role for nutrient availability in the response of Acanthamoeba to antimicrobial agents. In conclusion, our study highlights the potential of Cinn and Cinn-NE as effective agents for combating Acanthamoeba infections. Further research is needed to elucidate the specific mechanisms underlying the differential susceptibility to Cinn, optimize its use as a treatment or surface disinfectant, and explore Acanthamoeba's cellular and molecular responses to nutrient availability.

Amoebicidal activity of a preserved contact lens multipurpose disinfecting solution compared to a disinfectinn/neutralisation peroxide system

The Effect of Currently Available Contact Lens Disinfection Systems on Acanthamoeba castellanii and Acanthamoeba polyphaga

Physical and antimicrobial properties of anise oil loaded nanoemulsions on the survival of foodborne pathogens

PurposeTo compare LDH release assay, trypan blue and fluorescent stainings and non‐nutrient Escherichia coli (E. coli) plate assay in determining treatment efficacy of antiamoebic‐agents against Acanthamoeba castellanii trophozoites and cysts, in‐vitro.MethodsA. castellanii strain 1BU‐trophozoites/cysts were challenged with the antiamoebic‐agents 0.02% polyhexamethylen‐biguanid (PHMB), 0.1% propamidin‐isethionate (PD) and 0.0065% miltefosine (MF). Efficacies of the drugs were determined by LDH‐release and trypan blue assays. The fluorescent dyes Hoechst 33343, calcein‐AM, and ethidium‐homodimer‐1 were tested for their abilities to differentiate between viable and dead 1BU‐trophozoites/cysts following treatment, a non‐nutrient agar E. coli plate assay was applied to monitor the outgrowth of 1BU‐trophozoites/cysts challenged with antiamoebic‐agents.ResultsAll three antiamoebic‐agents induced a significant LDH‐release from trophozoites, when compared to controls (p &lt; 0.0001). There was a negligible trypan blue, Hoechst33342, calcein‐AM and ethidium‐homodimer‐1 staining in untreated 1BU‐throphozoites and cysts. Percentage of trypan blue positive trophozoites/cysts was 59.3 ± 1.5% /100% after challenge with PHMB, 100%/99.3 ± 0.5% upon PD treatment, and 95.6 ± 2.5%/77.6 ± 5.1% when using MF. Hoechst 33342 stained 100%/100%, ethidium‐homodimer‐1 100%/100%, calcein‐AM 75.33 ± 4.04%/60.67 ± 3.51% of PHMB treated trophozoites/cysts. Hoechst 33342 and ethidium‐homodimer‐1 were 100%/100%, calcein‐AM 9.67 ± 2.52%/36.67 ± 4.51% positive in PD‐treated trophozoites/cysts. MF treatment resulted in 100%/100% Hoechst 33342 and ethidium‐homodimer‐1 positive trophozoites/cysts, while none of them became positive for calcein‐AM. On non‐nutrient agar E. coli plates, in controls and MF treated 1BU trophozoites/cysts, new trophozoites appeared within 24 hr, and encystment occured after 5 weeks of cultivation. In PHMB and PD treated 1BU‐throphozoites/cysts, irregularly shaped, smaller trophozoites appeared after 72 hr, which failed to form new cysts within 5 weeks.ConclusionsNone of the enzymatic‐ and dye‐based viability assays tested here generated survival rates for trophozoites/cysts that were comparable with those yielded with the non‐nutrient agar E. coli plate assay, suggesting that the culture‐based assay is the best method to study the treatment efficacy of drugs against Acanthamoeba.