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Environmental Science & Technology Oct 2023Opportunistic pathogens (OPs) are of concern in drinking water distribution systems because they persist despite disinfectant residuals. While many OPs garner protection...
Opportunistic pathogens (OPs) are of concern in drinking water distribution systems because they persist despite disinfectant residuals. While many OPs garner protection from disinfectants via a biofilm lifestyle, () also gains disinfection resistance by being harbored within free-living amoebae (FLA). It has been long established, but poorly understood, that grown within FLA show increased infectivity toward subsequent FLA or human cells (i.e., macrophage), via a process we previously coined "protozoan-priming". The objectives of this study are (i) to identify in a key genetic determinant of how protozoan-priming increases its infectivity, (ii) to determine the chemical stimulus within FLA to which responds during protozoan-priming, and (iii) to determine if more infectious forms of also exhibit enhanced disinfectant resistance. Using as a FLA host, the priming effect was isolated to 's locus, which is activated upon sensing elevated magnesium concentrations. Supplementing growth medium with 8 mM magnesium is sufficient to produce grown with an infectivity equivalent to that of grown via the protozoan-primed route. Both forms with increased infectivity (FLA-grown and Mg-supplemented) exhibit greater monochloramine resistance than grown in standard media, indicating that passage through FLA not only increases 's infectivity but also enhances its monochloramine resistance. Therefore, laboratory-based testing of disinfection strategies should employ conditions that simulate or replicate intracellular growth to accurately assess disinfectant resistance.
Topics: Humans; Legionella pneumophila; Magnesium; Water Microbiology; Disinfectants; Amoeba
PubMed: 37756220
DOI: 10.1021/acs.est.3c04013 -
FEMS Microbiology Letters Jan 2023Legionella pneumophila is an opportunistic pathogen responsible for Legionnaires' disease or Legionellosis. This bacterium is found in the environment interacting with...
Legionella pneumophila is an opportunistic pathogen responsible for Legionnaires' disease or Legionellosis. This bacterium is found in the environment interacting with free-living amoebae such as Acanthamoeba castellanii. Until now, proteomic analyses have been done in amoebae infected with L. pneumophila but focused on the Legionella-containing vacuole. In this study, we propose a global proteomic analysis of the A. castellanii proteome following infection with L. pneumophila wild-type (WT) or with an isogenic ΔdotA mutant strain, which is unable to replicate intracellularly. We found that infection with L. pneumophila WT leads to reduced levels of A. castellanii proteins associated with lipid homeostasis/metabolism, GTPase regulation, and kinase. The levels of organelle-associated proteins were also decreased during infection. Legionellapneumophila WT infection leads to increased levels of proteins associated with polyubiquitination, folding or degradation, and antioxidant activities. This study reinforces our knowledge of this too little explored but so fundamental interaction between L. pneumophila and A. castellanii, to understand how the bacterium could resist amoeba digestion.
Topics: Humans; Legionnaires' Disease; Acanthamoeba castellanii; Proteomics; Legionella pneumophila; Homeostasis
PubMed: 37653467
DOI: 10.1093/femsle/fnad086 -
Acta Tropica Nov 2023Acanthamoeba keratitis (AK) is an eye disease often occurring in contact lens wearers. AK treatment is prolonged and requires multiple drugs, which can lead to adverse...
Acanthamoeba keratitis (AK) is an eye disease often occurring in contact lens wearers. AK treatment is prolonged and requires multiple drugs, which can lead to adverse effects. Our study aimed to compare the in vitro activities and safety of Miltefosine with that of conventional antimicrobial agents used to treat AK. Acanthamoeba castellanii genotype T4 was obtained from a patient with keratitis and subjected to in vitro susceptibility testing with various antimicrobial agents, including Chlorhexidine (CHX), Pentamidine isethionate (PI)Polyhexamethylene biguanide (PHMB), and Miltefosine to assess their efficacy against Acanthamoeba trophozoites and cyst. The cytotoxicity of the agents was evaluated in Vero cells, and their selectivity indexes (SI) were calculated. Chlorhexidine exhibited the highest amoebicidal activity with the highest selectivity index against the trophozoite and cyst, ranging from 1.17 to 8.35. The selectivity index of PHMB is slightly comparable to Chlorhexidine, exhibiting significant anti-Acanthamoeba activity. On the other hand, Pentamidine isethionate and Miltefosine displayed low SI among the compounds. Pentamidine isethionate was effective at high concentrations, which was toxic. Miltefosine exhibited the lowest cytotoxicity; nevertheless, due to the lowest anti-Acanthamoeba activity presented a low selectivity against the parasite. Further studies on more clinical samples and prolonged incubation time should be done to investigate the effectiveness and toxicity of drugs in both in vitro and in vivo conditions.
Topics: Chlorocebus aethiops; Animals; Humans; Acanthamoeba; Chlorhexidine; Trophozoites; Pentamidine; Vero Cells; Anti-Infective Agents; Acanthamoeba Keratitis; Cysts
PubMed: 37643658
DOI: 10.1016/j.actatropica.2023.107009 -
Global prevalence of free-living amoebae in solid matrices - A systematic review with meta-analysis.Acta Tropica Nov 2023The ubiquitous free-living amoebae (FLA) are microorganisms of significant medical, sanitary, and ecological importance. However, their characterization within solid... (Meta-Analysis)
Meta-Analysis Review
The ubiquitous free-living amoebae (FLA) are microorganisms of significant medical, sanitary, and ecological importance. However, their characterization within solid matrices such as soil, dust, sediment, mud, sludge, and compost remain to be systematized. In this study, we conducted a systematic review with meta-analysis to explore the global distribution of FLA in solid matrices. From the analysis of 104 out of 4,414 scientific articles retrieved from different databases, it was found that the general global prevalence of FLA in solid matrices was of 55.13% (95% confidence interval (CI) 49.32-60.94). Specifically, FLA prevalence was high in soil (72.40%, 95% CI 69.08-75.73), sediment (57.91%, 95% CI 50.01-65.81), mud (52.90%, 95% CI 24.01-81.78), dust (48.60%, 95% CI 43.00-54.19), and sewage sludge (40.19%, 95% CI 30.68-49.70). In aerosols it was comparatively lower (17.21%, 95% CI 12.76-21.66). Acanthamoeba spp. (52.23%) and Hartmanella/Vermamoeba spp. (36.06%) were found to be more prevalent, whereas Naegleria spp. (34.98%) and Balamuthia spp. (27.32%) were less prevalent. The distribution of the highest global prevalence values for species of Acanthamoeba spp., considering different publication periods of the studies, is as follows: A. hatchetti (51.46%), A. rhysodes (47.49%), A. polyphaga (36.37%), A. culbertsoni (34.31%), A. castellanii (34.21%), and A. lenticulata (32.82%). For other FLA species, the distribution is: Hartmannella/Vermamoeba vermiformis (91.57%), Naegleria fowleri (42.32%), Naegleria gruberi (32.39%), and Balamuthia mandrillaris (25%). The most prevalent Acanthamoeba genotypes were T4 (33.38%) and T3 (23.94%). Overall, the global prevalence of FLA in solid matrices is as high as or greater than that reported in water by previous systematic reviews. Thus, actions aimed at reducing exposure to FLA or exploring their ecological dynamics should consider not only water but also the various solid matrices. The finding outlined here can provide valuable insights for such actions, e.g., informing on the level of exposure to FLA, or on the microbial biodiversity of specific environmental compartments.
Topics: Animals; Amoeba; Prevalence; Sewage; Acanthamoeba; Coleoptera; Dust; Soil; Water
PubMed: 37633571
DOI: 10.1016/j.actatropica.2023.107006 -
Pathogens (Basel, Switzerland) Aug 2023spp. is the etiological agent of amoebic keratitis. In this study, the effect of taurine in physiological concentrations in tears (195 μM) on trophozoites of through...
spp. is the etiological agent of amoebic keratitis. In this study, the effect of taurine in physiological concentrations in tears (195 μM) on trophozoites of through the ex vivo amoebic keratitis model was evaluated. Trophozoites were coincubated with the Syrian golden hamster cornea () for 3 and 6 h. Group 1: Control (-). Corneas coincubated with amoebic culture medium and taurine. Group 2: Control (+). Corneas coincubated with trophozoites without taurine. Group 3: Corneas coincubated with taurine 15 min before adding trophozoites. Group 4: Trophozoites coincubated 15 min with taurine before placing them on the cornea. Group 5: Corneas coincubated for 15 min with trophozoites; subsequently, taurine was added. Results are similar for both times, as evaluated by scanning electron microscopy. As expected, in the corneas of Group 1, no alterations were observed in the corneal epithelium. In the corneas of Group 2, few adhered trophozoites were observed on the corneal surface initiating migrations through cell junctions as previously described; however, in corneas of Groups 3, 4 and 5, abundant trophozoites were observed, penetrating through different corneal cell areas, emitting food cups and destabilizing corneal surface in areas far from cell junctions. Significant differences were confirmed in trophozoites adherence coincubated with taurine ( < 0.05). Taurine does not prevent the adhesion and invasion of the amoebae, nor does it favor its detachment once these have adhered to the cornea, suggesting that taurine in the physiological concentrations found in tears stimulates pathogenic mechanisms of .
PubMed: 37624009
DOI: 10.3390/pathogens12081049 -
Microbiology (Reading, England) Aug 2023The predation and engulfment of bacteria by facilitates intimate interactions between host and prey. This process plays an important and underestimated role in the...
The predation and engulfment of bacteria by facilitates intimate interactions between host and prey. This process plays an important and underestimated role in the physiology, ecology and evolution of pathogenic bacteria. species can be reservoirs for many important human pathogens including is the leading cause of bacterial foodborne enteritis worldwide, despite being a microaerophile that is incapable of withstanding atmospheric levels of oxygen long-term. The persistence and transmission of this major pathogen in the natural environment outside its avian and mammalian hosts is not fully understood. Recent evidence has provided insight into the relationship of and spp. where are a transient host for this pathogen. Mutations to the flagella components were shown to affect interactions. Here, we show that the motility function of flagella is not a prerequisite for interactions and that specific -linked glycan modifications of the major flagellin, FlaA, are important for the recognition, interaction and phagocytosis by . Substitution of the -linked glycosylated serine 415 and threonine 477 with alanine within FlaA abolished interactions with and these mutants were indistinguishable from a Δ mutant. By contrast, mutation to serine 405 did not affect 11168H and interactions. Given the abundance of flagella glycosylation among clinically important pathogens, our observations may have a wider implication for understanding host-pathogen interactions.
Topics: Humans; Animals; Acanthamoeba castellanii; Campylobacter jejuni; Flagellin; Alanine; Polysaccharides; Mammals
PubMed: 37610804
DOI: 10.1099/mic.0.001386 -
Parasite (Paris, France) 2023Free-Living Amebae (FLA) and Cryptosporidium oocysts occasionally share the same environment. From 2004 to 2016, Cryptosporidium was responsible for 60% of 905 worldwide...
Free-Living Amebae (FLA) and Cryptosporidium oocysts occasionally share the same environment. From 2004 to 2016, Cryptosporidium was responsible for 60% of 905 worldwide waterborne outbreaks caused by protozoan parasites. The aim of this study was to evaluate interactions between C. parvum oocysts and two common FLAs (Acanthamoeba castellanii and Vermamoeba vermiformis) in a water environment. Encystment and survival of FLAs were evaluated by microscopy using trypan blue vital coloration. Oocysts were numerated on microscopy. Interactions were studied over time in conditions both unfavorable and favorable to phagocytosis. Potential phagocytosis was directly evaluated by several microscopic approaches and indirectly by numeration of microorganisms and oocyst infectivity evaluation. Occasional phagocytosis of C. parvum by FLAs was documented. However, oocyst concentrations did not decrease significantly, suggesting resistance of oocysts to phagocytosis. A temporary decrease of oocyst infectivity was observed in the presence of A. castellanii. The effect of these interactions on C. parvum infectivity is particularly interesting. The biofilm condition could favor the persistence or even the proliferation of oocysts over time. This study demonstrated interactions between C. parvum and FLAs. Further knowledge of the mechanisms involved in the decrease of oocyst infectivity in the presence of A. castellanii could facilitate the development of new therapeutic approaches.
Topics: Animals; Cryptosporidium parvum; Amoeba; Cryptosporidium; Cryptosporidiosis; Disease Outbreaks; Oocysts
PubMed: 37606589
DOI: 10.1051/parasite/2023033 -
International Journal of Molecular... Aug 2023Uncoupling proteins (UCPs) are mitochondrial inner membrane transporters that mediate free-fatty-acid-induced, purine-nucleotide-inhibited proton leak into the...
Uncoupling proteins (UCPs) are mitochondrial inner membrane transporters that mediate free-fatty-acid-induced, purine-nucleotide-inhibited proton leak into the mitochondrial matrix, thereby uncoupling respiratory substrate oxidation from ATP synthesis. The aim of this study was to provide functional evidence that the putative gene of the free-living protozoan amoeba, , encodes the mitochondrial protein with uncoupling activity characteristic of UCPs and to investigate its role during oxidative stress. We report the sequencing and cloning of a complete coding sequence, its phylogenetic analysis, and the heterologous expression of AcUCP in the strain Measurements of mitochondrial respiratory activity and membrane potential indicate that the heterologous expression of AcUCP causes AcUCP-mediated uncoupling activity. In addition, in a model of oxidative stress with increased reactive oxygen species levels (superoxide dismutase 1 knockout yeasts), AcUCP expression strongly promotes cell survival and growth. The level of superoxide anion radicals is greatly reduced in the Δ strain expressing AcUCP. These results suggest that AcUCP targeted to yeast mitochondria causes uncoupling and may act as an antioxidant system. Phylogenetic analysis shows that the UCP diverges very early from other UCPs, but clearly locates within the UCP subfamily rather than among other mitochondrial anion carrier proteins.
PubMed: 37569876
DOI: 10.3390/ijms241512501 -
Translational Vision Science &... Aug 2023To develop a feline model of acute Acanthamoeba keratitis using methods that replicate natural routes of infection transmission.
PURPOSE
To develop a feline model of acute Acanthamoeba keratitis using methods that replicate natural routes of infection transmission.
METHODS
Corneal Acanthamoeba castellanii inoculation was performed by three methods: topical inoculation with Acanthamoeba solution following corneal abrasion, placement of a contaminated contact lens for 7 days, and placement of a contaminated contact lens for 7 days following corneal abrasion. Sham inoculations with parasite-free medium and sterile contact lenses were also performed. Cats were monitored by ocular examination and in vivo corneal confocal microscopy for 21 days post-inoculation. Corneal samples were collected at intervals for microbiologic assessment, histopathology, and immunohistochemistry.
RESULTS
All cats in the corneal abrasion groups developed clinical keratitis. Clinical ocular disease was inconsistently detected in cats from the contaminated contact lens only group. Initial corneal lesions were characterized by multifocal epithelial leukocyte infiltrates. Ocular lesions progressed to corneal epithelial ulceration and diffuse stromal inflammation. After 14 days, corneal ulcerations resolved, and stromal inflammation consolidated into multifocal subepithelial and stromal infiltrates. Corneal amoebae were detected by culture, in vivo confocal microscopy, histopathology, and immunohistochemistry in cats with keratitis. Neutrophilic and lymphocytic keratoconjunctivitis with lymphoplasmacytic anterior uveitis were identified by histopathology. Coinfection with aerobic bacteria was detected in some, but not all, cats with keratitis. Ocular disease was not detected in the sham inoculation groups.
CONCLUSIONS
Feline Acanthamoeba keratitis is experimentally transmissible by contaminated contact lenses and topical inoculation following corneal epithelial trauma.
TRANSLATIONAL RELEVANCE
Experimentally induced acute Acanthamoeba keratitis in cats is clinically and histopathologically similar to its human counterpart.
Topics: Cats; Animals; Humans; Acanthamoeba Keratitis; Acanthamoeba castellanii; Cornea; Corneal Injuries; Inflammation
PubMed: 37566398
DOI: 10.1167/tvst.12.8.10 -
Molecular and Biochemical Parasitology Dec 2023Acanthamoeba are known to cause a vision threatening eye infection typically due to contact lens wear, and an infection of the central nervous system. The ability of...
Acanthamoeba are known to cause a vision threatening eye infection typically due to contact lens wear, and an infection of the central nervous system. The ability of these amoebae to switch phenotypes, from an active trophozoite to a resistant cyst form is not well understood; the cyst stage is often resistant to chemotherapy, which is of concern given the rise of contact lens use and the ineffective disinfectants available, versus the cyst stage. Herein, for the first time, a range of raloxifene sulfonate/sulfamate derivatives which target nucleotide pyrophosphatase/phosphodiesterase enzymes, were assessed using amoebicidal and excystation tests versus the trophozoite and cyst stage of Acanthamoeba. Moreover, the potential for cytopathogenicity inhibition in amoebae was assessed. Each of the derivatives showed considerable anti-amoebic activity as well as the ability to suppress phenotypic switching (except for compound 1a). Selected raloxifene derivatives reduced Acanthamoeba-mediated host cell damage using lactate dehydrogenase assay. These findings suggest that pyrophosphatase/phosphodiesterase enzymes may be valuable targets against Acanthamoeba infections.
Topics: Animals; Acanthamoeba castellanii; Raloxifene Hydrochloride; Sulfonic Acids; Trophozoites; Alkanesulfonates; Phosphoric Diester Hydrolases
PubMed: 37562558
DOI: 10.1016/j.molbiopara.2023.111582