Diagnostic considerations for non-<i>Acanthamoeba</i> amoebic keratitis and clinical outcomes

Cases of amoebic keratitis involving species other than Acanthamoeba are hypothesised to be underdiagnosed and poorly understood. Amoebic keratitis is debilitating and associated with chronic visual impairment. Understanding associated symptoms of non-Acanthamoeba amoebic keratitis could facilitate new diagnostic procedures and enable prompt treatment, ultimately leading to improved patient outcomes. Thus, a review of the literature was undertaken surrounding non-Acanthamoeba amoebic keratitis. Cases were geographically widespread and mostly confined to contact lens wearers ≤ 30 years old exposed to contaminated water sources and/or demonstrating poor lens hygiene. Vermamoeba vermiformis (previously Hartmanella vermiformis) was the most common causative agent, and a moderate number of mixed keratitis cases were also reported. A crucial disease indicator was early onset stromal deterioration/ulcerations, reported in 10 of the studies, usually only occurring in advanced Acanthamoeba keratitis. Mixed infections were the most difficult to treat, often requiring keratoplasty after unsuccessful combination treatment regimens. New diagnostic measures for non-Acanthamoeba amoebic keratitis should consider early onset stromal disease as a key disease indicator. Deep corneal scrapes are also necessary for accurate amoebic identification. Moreover, a combination approach to diagnosis is advised and should involve culture, microscopy and PCR techniques. In vitro drug sensitivity tests should also be conducted to help develop patient-specific treatment regimes

Microbial interactions that contribute to gill disease in aquaculture

The rapid growth in the human population has led to an increased requirement for readily available food sources. The aquaculture industry is a fundamental source for maintaining food supplies; however, it is subjected to mounting pressures to meet supply demands. Thus, limiting factors that negatively impact the cultivation of farmed aquatic organisms is essential. Gill disease is an increasing area of concern, resulting in substantial losses in farmed fish. Several microbial pathogens are known to cause gill disease and, in many instances, multiple pathogens or factors can be involved in the disease, resulting in complex gill disease (CGD). The role of mixed infections in gill disease is largely unknown, as such this review aims to examine data on previous infections and highlight the variety of microbes that might be involved in gill disease. The influence of climate change in the context of CGD is also discussed given the strong links between physicochemical extremes and numerous microbial gill pathogens. Understanding these factors will allow for improved diagnostic and therapeutic strategies to be implemented

Zooming in on the intracellular microbiome composition of bacterivorous <i>Acanthamoeba</i> isolates

Acanthamoeba, a free-living amoeba in water and soil, is an emerging pathogen causing severe eye infection known as Acanthamoeba keratitis. In its natural environment, Acanthamoeba performs a dual function as an environmental heterotrophic predator and host for a range of microorganisms that resist digestion. Our objective was to characterize the intracellular microorganisms of phylogenetically distinct Acanthamoeba spp. isolated in Australia and India through directly sequencing 16S rRNA amplicons from the amoebae. The presence of intracellular bacteria was further confirmed by in situ hybridization and electron microscopy. Among the 51 isolates assessed, 41% harboured intracellular bacteria which were clustered into four major phyla: Pseudomonadota (previously known as Proteobacteria), Bacteroidota (previously known as Bacteroidetes), Actinomycetota (previously known as Actinobacteria), and Bacillota (previously known as Firmicutes). The linear discriminate analysis effect size analysis identified distinct microbial abundance patterns among the sample types; Pseudomonas species was abundant in Australian corneal isolates (P &lt; 0.007), Enterobacteriales showed higher abundance in Indian corneal isolates (P &lt; 0.017), and Bacteroidota was abundant in Australian water isolates (P &lt; 0.019). The bacterial beta diversity of Acanthamoeba isolates from keratitis patients in India and Australia significantly differed (P &lt; 0.05), while alpha diversity did not vary based on the country of origin or source of isolation (P &gt; 0.05). More diverse intracellular bacteria were identified in water isolates as compared with clinical isolates. Confocal and electron microscopy confirmed the bacterial cells undergoing binary fission within the amoebal host, indicating the presence of viable bacteria. This study sheds light on the possibility of a sympatric lifestyle within Acanthamoeba, thereby emphasizing its crucial role as a bunker and carrier of potential human pathogens

Sodium metabisulfite inhibits <i>Acanthamoeba</i> trophozoite growth through thiamine depletion

Acanthamoeba keratitis (AK) is a severe infection of the cornea. Prevention and treatment are difficult due to the inefficacy of currently available compounds. The impact of many commonly used compounds for routine examinations of Acanthamoeba is unexplored but might offer insight useful in combatting AK. In this study, we demonstrate that sodium metabisulfite, a common preservation constituent of eye care solutions, was found to be active against Acanthamoeba trophozoites at concentrations lower than that commonly found in eye drops (IC50 0.03 mg/mL). We demonstrate that sodium metabisulfite depletes thiamine from growth medium and that Acanthamoeba is a thiamine auxotroph, requiring thiamine salvage for growth. The inhibitory effects of sodium metabisulfite can be overcome by thiamine supplementation. These results are consistent with the lack of key enzymes for thiamine biosynthesis in the genome of Acanthamoeba, an area which might prove exploitable using new or existing compounds. Indeed, this study highlights sodium metabisulfite as a useful inhibitor of Acanthamoeba castellanii trophozoites in vitro and that it acts, at least in part, by limiting available thiamine

Acanthamoeba as a protective reservoir for <i>Pseudomonas aeruginosa</i> in a clinical environment

Pseudomonas aeruginosa is a growing concern in healthcare associated infections and poses significant risk to those with serious underlying health conditions. The antimicrobial resistance traits of the pathogen and ability to form biofilms make effective mitigation and disinfection strategies difficult. Added to this challenge is the role that free-living amoebae such as Acanthamoeba play in the detection, disinfection, and transmission of P. aeruginosa. P. aeruginosa can survive intracellularly within amoebae, which has the potential to limit detectability and permit transmission into high-risk areas. Herein, we have screened for the presence of Acanthamoeba species and P. aeruginosa within a functioning general hospital in Scotland using a culture and molecular approach, noting their presence at several sites over a 4-month period, particularly within floor drains connecting patient rooms. In addition, microbiome analysis revealed amoebae harbour a unique microbial community comprised primarily of Pseudomonas species that were not readily detected using microbiome sequencing techniques on environmental swabs. Having demonstrated that both organisms were consistently present in hospital settings, we investigated the relationship between Acanthamoeba and P. aeruginosa in the laboratory, showing that i) Acanthamoeba growth rate is increased in the presence of Pseudomonas biofilms and viable Pseudomonas persist within the amoebae and ii) hydrogen peroxide-based disinfectants are significantly less effective against an isolate of P. aeruginosa in the presence of Acanthamoeba than when the bacteria are incubated alone. These findings suggest that amoebae, and other protists, can influence the detection and persistence of P. aeruginosa in high-risk areas and should be considered when implementing mitigation strategies

Sodium Metabisulfite Inhibits Acanthamoeba Trophozoite Growth through Thiamine Depletion

Acanthamoeba keratitis (AK) is a severe infection of the cornea. Prevention and treatment are difficult due to the inefficacy of currently available compounds. The impact of many commonly used compounds for routine examinations of Acanthamoeba is unexplored but might offer insight useful in combatting AK. In this study, we demonstrate that sodium metabisulfite, a common preservation constituent of eye care solutions, was found to be active against Acanthamoeba trophozoites at concentrations lower than that commonly found in eye drops (IC50 0.03 mg/mL). We demonstrate that sodium metabisulfite depletes thiamine from growth medium and that Acanthamoeba is a thiamine auxotroph, requiring thiamine salvage for growth. The inhibitory effects of sodium metabisulfite can be overcome by thiamine supplementation. These results are consistent with the lack of key enzymes for thiamine biosynthesis in the genome of Acanthamoeba, an area which might prove exploitable using new or existing compounds. Indeed, this study highlights sodium metabisulfite as a useful inhibitor of Acanthamoeba castellanii trophozoites in vitro and that it acts, at least in part, by limiting available thiamine

Sodium metabisulfite inhibits acanthamoeba trophozoite growth through thiamine depletion

Acanthamoeba keratitis (AK) is a severe infection of the cornea. Prevention and treatment are difficult due to the inefficacy of currently available compounds. The impact of many commonly used compounds for routine examinations of Acanthamoeba is unexplored but might offer insight useful in combatting AK. In this study, we demonstrate that sodium metabisulfite, a common preservation constituent of eye care solutions, was found to be active against Acanthamoeba trophozoites at concentrations lower than that commonly found in eye drops (IC50 0.03 mg/mL). We demonstrate that sodium metabisulfite depletes thiamine from growth medium and that Acanthamoeba is a thiamine auxotroph, requiring thiamine salvage for growth. The inhibitory effects of sodium metabisulfite can be overcome by thiamine supplementation. These results are consistent with the lack of key enzymes for thiamine biosynthesis in the genome of Acanthamoeba, an area which might prove exploitable using new or existing compounds. Indeed, this study highlights sodium metabisulfite as a useful inhibitor of Acanthamoeba castellanii trophozoites in vitro and that it acts, at least in part, by limiting available thiamine