Acanthamoeba as a protective reservoir for Pseudomonas aeruginosa 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.

Zooming in on the intracellular microbiome composition of bacterivorous Acanthamoeba 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 < 0.007), Enterobacteriales showed higher abundance in Indian corneal isolates (P < 0.017), and Bacteroidota was abundant in Australian water isolates (P < 0.019). The bacterial beta diversity of Acanthamoeba isolates from keratitis patients in India and Australia significantly differed (P < 0.05), while alpha diversity did not vary based on the country of origin or source of isolation (P > 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.

Diagnostic Considerations for Non-Acanthamoeba 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.

Alkyl-carbon chain length of two distinct compounds and derivatives are key determinants of their anti-Acanthamoeba activities.

The opportunistic pathogen, Acanthamoeba castellanii is the causative agent for the sight threatening infection Acanthamoeba keratitis (AK). It is commonly associated with contact lens wearers, and prevalence is increasing at an alarming rate due to an inadequate preventive strategy to protect the lens from this protist. This problem is compounded by the lack of an effective acanthamoebocide, particularly with cysticidal activity in the contact lens solutions. We have used cytotoxicity assays and a variety of biophysical approaches to show that two molecules with tails made of alkyl carbon, alkylphosphocholines (APCs) and quaternary ammonium compounds (QACs) had significant chain-length dependent efficacy against A. castellanii trophozoites, the latter producing death via permeabilization, and DNA complexing. QACs were more effective than APCs and had activity against cysts. Conversely, the QAC with 12 alkyl carbon chain, was non toxic, its presence increased A. castellanii trophozoites biomass and delayed encystation by 96 h. Interestingly, it was unable to induce excystation and increased trophozoite sensitivity to APC16. These results present a mono- and multi-inhibitor management strategy effective against trophozoites and cysts that may be useful for formulating into contact lense cleaning solutions and reducing AK incidence.

Paradigms of Protist/Bacteria Symbioses Affecting Human Health: Acanthamoeba species and Trichomonas vaginalis.

Ever since the publication of the seminal paper by Lynn Margulis in 1967 proposing the theory of the endosymbiotic origin of organelles, the study of the symbiotic relationships between unicellular eukaryotes and prokaryotes has received ever-growing attention by microbiologists and evolutionists alike. While the evolutionary significance of the endosymbiotic associations within protists has emerged and is intensively studied, the impact of these relationships on human health has been seldom taken into account. Microbial endosymbioses involving human eukaryotic pathogens are not common, and the sexually transmitted obligate parasite Trichomonas vaginalis and the free-living opportunistic pathogen Acanthamoeba represent two unique cases in this regard, to date. The reasons of this peculiarity for T. vaginalis and Acanthamoeba may be due to their lifestyles, characterized by bacteria-rich environments. However, this characteristic does not fully explain the reason why no bacterial endosymbiont has yet been detected in unicellular eukaryotic human pathogens other than in T. vaginalis and Acanthamoeba, albeit sparse and poorly investigated examples of morphological identification of bacteria-like microorganisms associated with Giardia and Entamoeba were reported in the past. In this review article we will present the body of experimental evidences revealing the profound effects of these examples of protist/bacteria symbiosis on the pathogenesis of the microbial species involved, and ultimately their impact on human health.