Exposure to sublethal concentrations of chlorine enhances the cytotoxicity of Acanthamoeba castellanii.

Free-living amoebae belonging to the genus Acanthamoeba are the causative agents of infections in humans and animals. Many studies are being conducted to find effective compounds against amoebae, but their sublethal concentration effects on surviving amoebae seem to have been overlooked. Chlorine is a common disinfection agent commonly added to public water facilities and supplies. In this study, the cytopathic and phagocytic properties of Acanthamoeba castellanii trophozoites following exposure to sublethal concentrations of chlorine were examined. Two hours of exposure to 5 ppm hypochlorite calcium was considered the sublethal concentration for A. castellanii trophozoites. To compare the pathogenic potential of treated and untreated Acanthamoeba trophozoites, cytotoxicity, adhesion assays in RAW 264.7 macrophages, osmo, and thermotolerance tests were carried out. Bacterial uptake was assessed in treated cells to evaluate their phagocytic characteristics. Oxidative stress biomarkers and antioxidant activities were compared in treated and untreated trophozoites. Finally, the mRNA expression of the mannose-binding protein (MBP), cysteine protease 3 (CP3), and serine endopeptidase (SEP) genes was determined in cells. In all the experiments, untreated trophozoites were considered the control. In comparison to untreated trophozoites, in chlorine-treated trophozoites, cytopathic effects were more extensive and resulted in the detachment of macrophage monolayers. Treated trophozoites could not grow at high temperatures (43 °C). Besides, they showed osmotolerance to 0.5 M D-mannitol but not to 1 M. Results demonstrated a higher bacterial uptake rate by chlorine-treated trophozoites than untreated cells. The treated and untreated cells had significantly different glutathione and glutathione/glutathione disulfide ratios. Antioxidant enzyme activities, total antioxidant capacity, and malondialdehyde levels were increased significantly in chlorine-treated cells. Quantifying mRNA expression in chlorine-treated trophozoites revealed that virulence genes were upregulated. Chlorine can form resistance and virulent amoebae if it is not used at a proper concentration and exposure time. Identification of stress responses, their mechanisms in Acanthamoeba, and their relation to amoeba virulence would give us a better perception of their pathophysiology.

Assessment of genotypes, endosymbionts and clinical characteristics of Acanthamoeba recovered from ocular infection.

INTRODUCTION: Acanthamoeba is an emerging pathogen, infamous for its resilience against antiprotozoal compounds, disinfectants and harsh environments. It is known to cause keratitis, a sight-threatening, painful and difficult to treat corneal infection which is often reported among contact lens wearers and patients with ocular trauma. Acanthamoeba comprises over 24 species and currently 23 genotypes (T1-T23) have been identified. AIMS: This retrospective study was designed to examine the Acanthamoeba species and genotypes recovered from patients with Acanthamoeba keratitis (AK), determine the presence of endosymbionts in ocular isolates of Acanthamoeba and review the clinical presentations. METHODOLOGY: Thirteen culture-confirmed AK patients treated in a tertiary eye care facility in Hyderabad, India from February to October 2020 were included in this study. The clinical manifestations, medications and visual outcomes of all patients were obtained from medical records. The Acanthamoeba isolates were identified by sequencing the ribosomal nuclear subunit (rns) gene. Acanthamoeba isolates were assessed for the presence of bacterial or fungal endosymbionts using molecular assays, PCR and fluorescence in situ hybridization (FISH). RESULTS: The mean age of the patients was 33 years (SD ± 17.4; 95% CI 22.5 to 43.5 years). Six (46.2%) cases had AK associated risk factors; four patients had ocular trauma and two were contact lens wearers. A. culbertsoni (6/13, 46.2%) was the most common species, followed by A. polyphaga and A. triangularis. Most of the isolates (12/13) belonged to genotype T4 and one was a T12; three sub-clusters T4A, T4B, and T4F were identified within the T4 genotype. There was no significant association between Acanthamoeba types and clinical outcomes. Eight (61.5%) isolates harboured intracellular bacteria and one contained Malassezia restricta. The presence of intracellular microbes was associated with a higher proportion of stromal infiltrates (88.9%, 8/9), epithelial defect (55.6%, 5/9) and hypopyon (55.6%, 5/9) compared to 50% (2/4), 25% (1/4) and 25% (1/4) AK cases without intracellular microbes, respectively. CONCLUSIONS: Genotype T4 was the predominant isolate in southern India. This is the second report of T12 genotype identified from AK patient in India, which is rarely reported worldwide. The majority of the Acanthamoeba clinical isolates in this study harboured intracellular microbes, which may impact clinical characteristics of AK.

Aerosol reduction efficacy of different intra-oral suction devices during ultrasonic scaling and high-speed handpiece use.

BACKGROUND: The COVID-19 pandemic led to significant changes in the provision of dental services, aimed at reducing the spread of respiratory pathogens through restrictions on aerosol generating procedures (AGPs). Evaluating the risk that AGPs pose in terms of SARS-CoV-2 transmission is complex, and measuring dental aerosols is challenging. To date, few studies focus on intra-oral suction. This study sought to assess the effectiveness of commonly used intra-oral suction devices on aerosol mitigation. METHODS: Ultrasonic scaling and high-speed handpiece procedures were undertaken to generate aerosol particles. Multiple particle sensors were positioned near the oral cavity. Sensor data were extracted using single board computers with custom in-house Bash code. Different high-volume and low-volume suction devices, both static and dynamic, were evaluated for their efficacy in preventing particle escape during procedures. RESULTS: In all AGPs the use of any suction device tested resulted in a significant reduction in particle counts compared with no suction. Low-volume and static suction devices showed spikes in particle count demonstrating moments where particles were able to escape from the oral cavity. High-volume dynamic suction devices, however, consistently reduced the particle count to background levels, appearing to eliminate particle escape. CONCLUSIONS: Dynamic high-volume suction devices that follow the path of the aerosol generating device effectively eliminate aerosol particles escaping from the oral cavity, in contrast to static devices which allow periodic escape of aerosol particles. Measuring the risk of SARS-CoV-2 transmission in a dental setting is multi-factorial; however, these data suggest that the appropriate choice of suction equipment may further reduce the risk from AGPs.

The legacy of industrial pollution in estuarine sediments: spatial and temporal variability implications for ecosystem stress.

The direct impacts of anthropogenic pollution are widely known public and environmental health concerns, and details on the indirect impact of these are starting to emerge, for example affecting the environmental microbiome. Anthropogenic activities throughout history with associated pollution burdens are notable contributors. Focusing on the historically heavily industrialised River Clyde, Scotland, we investigate spatial and temporal contributions to stressful/hostile environments using a geochemical framework, e.g. pH, EC, total organic carbon and potentially toxic elements: As, Co, Cr, Cu, Ni, Pb and Zn and enrichment indicators. With regular breaches of the sediment quality standards in the estuarine system we focused on PTE correlations instead. Multivariate statistical analysis (principle component analysis) identifies two dominant components, PC1: As, Cr, Cu, Pb and Zn, as well as PC2: Ni, Co and total organic carbon. Our assessment confirms hot spots in the Clyde Estuary indicative of localised inputs. In addition, there are sites with high variability indicative of excessive mixing. We demonstrate that industrialised areas are dynamic environmental sites dependant on historical anthropogenic activity with short-scale variation. This work supports the development of 'contamination' mapping to enable an assessment of the impact of historical anthropogenic pollution, identifying specific 'stressors' that can impact the microbiome, neglecting in estuarine recovery dynamics and potentially supporting the emergence of antimicrobial resistance in the environment.

Alopecia areata: A multifactorial autoimmune condition.

Alopecia areata is an autoimmune disease that results in non-scarring hair loss, and it is clinically characterised by small patches of baldness on the scalp and/or around the body. It can later progress to total loss of scalp hair (Alopecia totalis) and/or total loss of all body hair (Alopecia universalis). The rapid rate of hair loss and disfiguration caused by the condition causes anxiety on patients and increases the risks of developing psychological and psychiatric complications. Hair loss in alopecia areata is caused by lymphocytic infiltrations around the hair follicles and IFN-γ. IgG antibodies against the hair follicle cells are also found in alopecia areata sufferers. In addition, the disease coexists with other autoimmune disorders and can come secondary to infections or inflammation. However, despite the growing knowledge about alopecia areata, the aetiology and pathophysiology of disease are not well defined. In this review we discuss various genetic and environmental factors that cause autoimmunity and describe the immune mechanisms that lead to hair loss in alopecia areata patients.

Variability in nitrate-reducing oral bacteria and nitric oxide metabolites in biological fluids following dietary nitrate administration: An assessment of the critical difference.

There is conflicting evidence on whether dietary nitrate supplementation can improve exercise performance. This may arise from the complex nature of nitric oxide (NO) metabolism which causes substantial inter-individual variability, within-person biological variation (CVB), and analytical imprecision (CVA) in experimental endpoints. However, no study has quantified the CVA and CVB of NO metabolites or the factors that influence their production. These data are important to calculate the critical difference (CD), defined as the smallest difference between sequential measurements required to signify a true change. The main aim of the study was to evaluate the CVB, CVA, and CD for markers of NO availability (nitrate and nitrite) in plasma and saliva before and after the ingestion of nitrate-rich beetroot juice (BR). We also assessed the CVB of nitrate-reducing bacteria from the dorsal surface of the tongue. It was hypothesised that there would be substantial CVB in markers of NO availability and the abundance of nitrate-reducing bacteria. Ten healthy male participants (age 25 ±â€¯5 years) completed three identical trials at least 6 days apart. Blood and saliva were collected before and after (2, 2.5 and 3 h) ingestion of 140 ml of BR (∼12.4 mmol nitrate) and analysed for [nitrate] and [nitrite]. The tongue was scraped and the abundance of nitrate-reducing bacterial species were analysed using 16S rRNA next generation sequencing. There was substantial CVB for baseline concentrations of plasma (nitrate 11.9%, nitrite 9.0%) and salivary (nitrate 15.3%, nitrite 32.5%) NO markers. Following BR ingestion, the CVB for nitrate (plasma 3.8%, saliva 12.0%) and salivary nitrite (24.5%) were lower than baseline, but higher for plasma nitrite (18.6%). The CD thresholds that need to be exceeded to ensure a meaningful change from baseline are 25, 19, 37, and 87% for plasma nitrate, plasma nitrite, salivary nitrate, and salivary nitrite, respectively. The CVB for selected nitrate-reducing bacteria detected were: Prevotella melaninogenica (37%), Veillonella dispar (35%), Haemophilus parainfluenzae (79%), Neisseria subflava (70%), Veillonella parvula (43%), Rothia mucilaginosa (60%), and Rothia dentocariosa (132%). There is profound CVB in the abundance of nitrate-reducing bacteria on the tongue and the concentration of NO markers in human saliva and plasma. Where these parameters are of interest following experimental intervention, the CD values presented in this study will allow researchers to interpret the meaningfulness of the magnitude of the change from baseline.

Acanthamoeba proteases contribute to macrophage activation through PAR1 , but not PAR2.

AIM: Acanthamoeba infections are characterized by an intense localized innate immune response associated with an influx of macrophages. Acanthamoeba protease production is known to affect virulence. Herein, the ability of Acanthamoeba trophozoite proteases, of either the laboratory Neff strain or a recently isolated clinical strain, to stimulate IL-12 and IL-6 and to activate protease-activated receptors, PAR1 and PAR2 expressed on murine macrophages, was investigated. METHOD AND RESULTS: Using selected protease inhibitors, leupeptin and E64, we showed that Acanthamoeba proteases can stimulate IL-12 and IL-6 by murine macrophages. Subsequently, using specific antagonists to inhibit PAR1 , and bone marrow-derived macrophages from PAR2 gene-deficient mice, we demonstrate that PAR1 , but not PAR2 contributes to macrophage IL-12 production in response to Acanthamoeba. In contrast, Acanthamoeba-induced IL-6 production is PAR1 and PAR2 independent. CONCLUSION: This study shows for the first time the involvement of PARs, expressed on macrophages, in the response to Acanthamoeba trophozoites and might provide useful insight into Acanthamoeba infections and their future treatments.

Control of cell behaviour through nanovibrational stimulation: nanokicking.

Mechanical signals are ubiquitous in our everyday life and the process of converting these mechanical signals into a biological signalling response is known as mechanotransduction. Our understanding of mechanotransduction, and its contribution to vital cellular responses, is a rapidly expanding field of research involving complex processes that are still not clearly understood. The use of mechanical vibration as a stimulus of mechanotransduction, including variation of frequency and amplitude, allows an alternative method to control specific cell behaviour without chemical stimulation (e.g. growth factors). Chemical-independent control of cell behaviour could be highly advantageous for fields including drug discovery and clinical tissue engineering. In this review, a novel technique is described based on nanoscale sinusoidal vibration. Using finite-element analysis in conjunction with laser interferometry, techniques that are used within the field of gravitational wave detection, optimization of apparatus design and calibration of vibration application have been performed. We further discuss the application of nanovibrational stimulation, or 'nanokicking', to eukaryotic and prokaryotic cells including the differentiation of mesenchymal stem cells towards an osteoblast cell lineage. Mechanotransductive mechanisms are discussed including mediation through the Rho-A kinase signalling pathway. Optimization of this technique was first performed in two-dimensional culture using a simple vibration platform with an optimal frequency and amplitude of 1 kHz and 22 nm. A novel bioreactor was developed to scale up cell production, with recent research demonstrating that mesenchymal stem cell differentiation can be efficiently triggered in soft gel constructs. This important step provides first evidence that clinically relevant (three-dimensional) volumes of osteoblasts can be produced for the purpose of bone grafting, without complex scaffolds and/or chemical induction. Initial findings have shown that nanovibrational stimulation can also reduce biofilm formation in a number of clinically relevant bacteria. This demonstrates additional utility of the bioreactor to investigate mechanotransduction in other fields of research.This article is part of a discussion meeting issue 'The promises of gravitational-wave astronomy'.

Acanthamoeba Activates Macrophages Predominantly through Toll-Like Receptor 4- and MyD88-Dependent Mechanisms To Induce Interleukin-12 (IL-12) and IL-6.

Acanthamoeba castellanii is a ubiquitous free-living amoeba with a worldwide distribution that can occasionally infect humans, causing particularly severe infections in immunocompromised individuals. Dissecting the immunology of Acanthamoeba infections has been considered problematic due to the very low incidence of disease, despite the high exposure rates. While macrophages are acknowledged as playing a significant role in Acanthamoeba infections, little is known about how this facultative parasite influences macrophage activity. Therefore, in this study we investigated the effects of Acanthamoeba on the activation of resting macrophages. Consequently, murine bone marrow-derived macrophages were cocultured with trophozoites of either the laboratory Neff strain or a clinical isolate of A. castellaniiIn vitro real-time imaging demonstrated that trophozoites of both strains often established evanescent contact with macrophages. Both Acanthamoeba strains induced a proinflammatory macrophage phenotype characterized by the significant production of interleukin-12 (IL-12) and IL-6. However, macrophages cocultured with the clinical isolate of Acanthamoeba produced significantly less IL-12 and IL-6 than the Neff strain. The utilization of macrophages derived from MyD88-, TRIF-, Toll-like receptor 2 (TLR2)-, TLR4-, and TLR2/4-deficient mice indicated that Acanthamoeba-induced proinflammatory cytokine production was through MyD88-dependent, TRIF-independent, TLR4-induced events. This study shows for the first time the involvement of TLRs expressed on macrophages in the recognition of and response to Acanthamoeba trophozoites.

Acanthamoeba castellanii Genotype T4 Stimulates the Production of Interleukin-10 as Well as Proinflammatory Cytokines in THP-1 Cells, Human Peripheral Blood Mononuclear Cells, and Human Monocyte-Derived Macrophages.

Free-living amoebae of the genus Acanthamoeba can cause severe and chronic infections in humans, mainly localized in immune privileged sites, such as the brain and the eye. Monocytes/macrophages are thought to be involved in Acanthamoeba infections, but little is known about how these facultative parasites influence their functions. The aim of this work was to investigate the effects of Acanthamoeba on human monocytes/macrophages during the early phase of infection. Here, THP-1 cells, primary human monocytes isolated from peripheral blood, and human monocyte-derived macrophages were either coincubated with trophozoites of a clinical isolate of Acanthamoeba (genotype T4) or stimulated with amoeba-derived cell-free conditioned medium. Production of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-6 [IL-6], and IL-12), anti-inflammatory cytokine (IL-10), and chemokine (IL-8) was evaluated at specific hours poststimulation (ranging from 1.5 h to 23 h). We showed that both Acanthamoeba trophozoites and soluble amoebic products induce an early anti-inflammatory monocyte-macrophage phenotype, characterized by significant production of IL-10; furthermore, challenge with either trophozoites or their soluble metabolites stimulate both proinflammatory cytokines and chemokine production, suggesting that this protozoan infection results from the early induction of coexisting, opposed immune responses. Results reported in this paper confirm that the production of proinflammatory cytokines and chemokines by monocytes and macrophages can play a role in the development of the inflammatory response during Acanthamoeba infections. Furthermore, we demonstrate for the first time that Acanthamoeba stimulates IL-10 production in human innate immune cells, which might both promote the immune evasion of Acanthamoeba and limit the induced inflammatory response.

The role of chemokines and their receptors during protist parasite infections.

Protists are a diverse collection of eukaryotic organisms that account for a significant global infection burden. Often, the immune responses mounted against these parasites cause excessive inflammation and therefore pathology in the host. Elucidating the mechanisms of both protective and harmful immune responses is complex, and often relies of the use of animal models. In any immune response, leucocyte trafficking to the site of infection, or inflammation, is paramount, and this involves the production of chemokines, small chemotactic cytokines of approximately 8-10 kDa in size, which bind to specific chemokine receptors to induce leucocyte movement. Herein, the scientific literature investigating the role of chemokines in the propagation of immune responses against key protist infections will be reviewed, focussing on Plasmodium species, Toxoplasma gondii, Leishmania species and Cryptosporidium species. Interestingly, many studies find that chemokines can in fact, promote parasite survival in the host, by drawing in leucocytes for spread and further replication. Recent developments in drug targeting against chemokine receptors highlights the need for further understanding of the role played by these proteins and their receptors in many different diseases.

Biochemical and immunological characterization of Toxoplasma gondii macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) is a proinflammatory molecule in mammals that, unusually for a cytokine, exhibits tautomerase and oxidoreductase enzymatic activities. Homologues of this well conserved protein are found within diverse phyla including a number of parasitic organisms. Herein, we produced recombinant histidine-tagged Toxoplasma gondii MIF (TgMIF), a 12-kDa protein that lacks oxidoreductase activity but exhibits tautomerase activity with a specific activity of 19.3 µmol/min/mg that cannot be inhibited by the human MIF inhibitor ISO-1. The crystal structure of the TgMIF homotrimer has been determined to 1.82 Å, and although it has close structural homology with mammalian MIFs, it has critical differences in the tautomerase active site that account for the different inhibitor sensitivity. We also demonstrate that TgMIF can elicit IL-8 production from human peripheral blood mononuclear cells while also activating ERK MAPK pathways in murine bone marrow-derived macrophages. TgMIF may therefore play an immunomodulatory role during T. gondii infection in mammals.

The effects of nitrate on the oral microbiome: a systematic review investigating prebiotic potential.

Background: Nitrate (NO3-) has been suggested as a prebiotic for oral health. Evidence indicates dietary nitrate and nitrate supplements can increase the proportion of bacterial genera associated with positive oral health whilst reducing bacteria implicated in oral disease(s). In contrast, chlorhexidine-containing mouthwashes, which are commonly used to treat oral infections, promote dysbiosis of the natural microflora and may induce antimicrobial resistance. Methods: A systematic review of the literature was undertaken, surrounding the effects of nitrate on the oral microbiota. Results: Overall, n = 12 in vivo and in vitro studies found acute and chronic nitrate exposure increased (representatives of) health-associated Neisseria and Rothia (67% and 58% of studies, respectively) whilst reducing periodontal disease-associated Prevotella (33%). Additionally, caries-associated Veillonella and Streptococcus decreased (25% for both genera). Nitrate also altered oral microbiome metabolism, causing an increase in pH levels (n = 5), which is beneficial to limit caries development. Secondary findings highlighted the benefits of nitrate for systemic health (n = 5). Conclusions: More clinical trials are required to confirm the impact of nitrate on oral communities. However, these findings support the hypothesis that nitrate could be used as an oral health prebiotic. Future studies should investigate whether chlorhexidine-containing mouthwashes could be replaced or complemented by a nitrate-rich diet or nitrate supplementation.

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.

Periodontal treatment causes a longitudinal increase in nitrite-producing bacteria.

BACKGROUND: The oral microbiome-dependent nitrate (NO3 -)-nitrite (NO2 -)-nitric oxide (NO) pathway may help regulate blood pressure. NO2 --producing bacteria in subgingival plaque are reduced in relative abundance in patients with untreated periodontitis compared with periodontally healthy patients. In periodontitis patients, the NO2 --producing bacteria increase several months after periodontal treatment. The early effects of periodontal treatment on NO2 --producing bacteria and the NO3 --NO2 --NO pathway remain unknown. The aim of this study was to determine how periodontal treatment affects the oral NO2 --producing microbiome and salivary NO3 - and NO2 - levels over time. METHODS: The subgingival microbiota of 38 periodontitis patients was analysed before (baseline [BL]) and 1, 7 and 90 days after periodontal treatment. Changes in NO2 --producing bacteria and periodontitis-associated bacteria were determined by 16s rRNA Illumina sequencing. Saliva samples were collected at all-time points to determine NO3 - and NO2 - levels using gas-phase chemiluminescence. RESULTS: A significant increase was observed in the relative abundance of NO2 --producing species between BL and all subsequent timepoints (all p < 0.001). Periodontitis-associated species decreased at all timepoints, relative to BL (all p < 0.02). NO2 --producing species negatively correlated with periodontitis-associated species at all timepoints, with this relationship strongest 90 days post-treatment (ρ = -0.792, p < 0.001). Despite these findings, no significant changes were found in salivary NO3 - and NO2 - over time (all p > 0.05). CONCLUSIONS: Periodontal treatment induced an immediate increase in the relative abundance of health-associated NO2 --producing bacteria. This increase persisted throughout periodontal healing. Future studies should test the effect of periodontal treatment combined with NO3 - intake on periodontal and cardiovascular health.

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.

The role of naturally acquired intracellular Pseudomonas aeruginosa in the development of Acanthamoeba keratitis in an animal model.

BACKGROUND: Acanthamoeba is an environmental host for various microorganisms. Acanthamoeba is also becoming an increasingly important pathogen as a cause of keratitis. In Acanthamoeba keratitis (AK), coinfections involving pathogenic bacteria have been reported, potentially attributed to the carriage of microbes by Acanthamoeba. This study assessed the presence of intracellular bacteria in Acanthamoeba species recovered from domestic tap water and corneas of two different AK patients and examined the impact of naturally occurring intracellular bacteria within Acanthamoeba on the severity of corneal infections in rats. METHODOLOGY/PRINCIPAL FINDINGS: Household water and corneal swabs were collected from AK patients. Acanthamoeba strains and genotypes were confirmed by sequencing. Acanthamoeba isolates were assessed for the presence of intracellular bacteria using sequencing, fluorescence in situ hybridization (FISH), and electron microscopy. The viability of the bacteria in Acanthamoeba was assessed by labelling with alkyne-functionalized D-alanine (alkDala). Primary human macrophages were used to compare the intracellular survival and replication of the endosymbiotic Pseudomonas aeruginosa and a wild type strain. Eyes of rats were challenged intrastromally with Acanthamoeba containing or devoid of P. aeruginosa and evaluated for the clinical response. Domestic water and corneal swabs were positive for Acanthamoeba. Both strains belonged to genotype T4F. One of the Acanthamoeba isolates harboured P. aeruginosa which was seen throughout the Acanthamoeba's cytoplasm. It was metabolically active and could be seen undergoing binary fission. This motile strain was able to replicate in macrophage to a greater degree than strain PAO1 (p<0.05). Inoculation of Acanthamoeba containing the intracellular P. aeruginosa in rats eyes resulted in a severe keratitis with increased neutrophil response. Acanthamoeba alone induced milder keratitis. CONCLUSIONS/SIGNIFICANCE: Our findings indicate the presence of live intracellular bacteria in Acanthamoeba can increase the severity of acute keratitis in vivo. As P. aeruginosa is a common cause of keratitis, this may indicate the potential for these intracellular bacteria in Acanthamoeba to lead to severe polymicrobial keratitis.

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.

Sublethal Exposure to Plasma-Activated Water Influences the Morphological Characteristics, Phagocytic Ability, and Virulence of Acanthamoeba castellanii.

PURPOSE: This study aimed to examine the ultrastructure, cytotoxicity, phagocytosis, and antioxidant responses of Acanthamoeba castellanii trophozoites exposed to sublethal plasma-activated water. METHODS: Trophozoites were exposed to a sublethal treatment of PAW and compared to untreated viable trophozoites via adhesion assays on macrophage monolayers, osmo- and thermotolerance tests. Bacterial uptake was assessed in treated cells to evaluate their phagocytic characteristics. Oxidative stress biomarkers and antioxidant activities were compared in treated and untreated trophozoites. Finally, the expression of the mannose-binding protein (MBP), cysteine protease 3 (CP3), and serine endopeptidase (SEP) genes was determined in cells. RESULTS: In PAW-treated trophozoites, cytopathic effects were more extensive and resulted in the detachment of macrophage monolayers. Treated trophozoites could not grow at high temperatures (43 °C). Moreover, they showed osmotolerance to 0.5 M D-mannitol but not to 1 M. Results demonstrated a higher bacterial uptake rate by PAW-treated trophozoites than untreated cells. Activities of superoxide dismutase and catalase and catalase were significantly greater in the treated trophozoites, and the glutathione and glutathione/glutathione disulfide were significantly lower in the PAW-treated cells. Exposure to PAW also significantly increased the malondialdehyde level and total antioxidant capacity. Treatment with PAW led to significantly higher expression of virulent genes like MBP, CP3, and SEP. CONCLUSION: PAW is a double-edged sword against A. castellanii. PAW is an effective antiamoebic agent in proper usage, whereas its sublethal exposure may reduce its effectiveness and increase amoebas' pathogenicity. An agent's adequate concentration and exposure time are essential to achieve optimum results.

Towards direct detection of tetracycline residues in milk with a gold nanostructured electrode.

Tetracycline antibiotics are used extensively in veterinary medicine, but the majority of the administrated dose is eliminated unmodified from the animal through various excretion routes including urine, faeces and milk. In dairy animals, limits on residues secreted in milk are strictly controlled by legislation. Tetracyclines (TCs) have metal chelation properties and form strong complexes with iron ions under acidic conditions. In this study, we exploit this property as a strategy for low cost, rapid electrochemical detection of TC residues. TC-Fe(III) complexes in a ratio of 2:1 were created in acidic conditions (pH 2.0) and electrochemically measured on plasma-treated gold electrodes modified with electrodeposited gold nanostructures. DPV measurements showed a reduction peak for the TC-Fe(III) complex that was observed at 50 mV (vs. Ag/AgCl QRE). The limit of detection in buffer media was calculated to be 345 nM and was responsive to increasing TC concentrations up to 2 mM, added to 1 mM FeCl3. Whole milk samples were processed to remove proteins and then spiked with tetracycline and Fe(III) to explore the specificity and sensitivity in a complex matrix with minimal sample preparation, under these conditions the LoD was 931 nM. These results demonstrate a route towards an easy-to-use sensor system for identification of TC in milk samples taking advantage of the metal chelating properties of this antibiotic class.