Journal: Pharmaceuticals

Article Title: Amoebicidal Effect of COVID Box Molecules against Acanthamoeba : A Study of Cell Death

doi: 10.3390/ph17060808

Figure Lengend Snippet: Evaluation of the effect of IC 90 of Terconazole on actin distribution of the trophozoites of Acanthamoeba culbertsoni ; cells show an undeveloped network with a dramatical decrease in cell size ( D – F ). The phalloidin-TRITC dye stains the polymerized actin cytoskeleton showing the normal organization of the networks with an orange fluorescence in the negative control cells ( A – C ). Mounting DAPI solution for DNA staining shows a blue fluorescence ( A , D ); Phalloidin-TRITC channel ( B , D ); overlay channel ( C , F ). All images (63×) were obtained using a Leica DMI 4000 B inverted confocal light microscope.

Article Snippet: Further strains were used to fine-tune the most active amoebicidal agents: A. polyphaga (genotype T4, type strain: ATCC 30461), A. griffini (genotype T3, clinical strain), A. castellanii L-10 (genotype T4, clinical strain), and A. culbertsoni (genotype T10, type strain: ATCC 30171).

Techniques: Fluorescence, Negative Control, Staining, Light Microscopy

Journal: Pharmaceuticals

Article Title: Amoebicidal Effect of COVID Box Molecules against Acanthamoeba : A Study of Cell Death

doi: 10.3390/ph17060808

Figure Lengend Snippet: Trophozoites of Acanthamoeba culbertsoni incubated with IC 90 of Terconazole for 24 h. Compared to the negative control ( A – C ), treated cells demonstrated an undeveloped tubulin network, with lower emitted red fluorescence ( D – F ). Mounting DAPI solution for DNA staining showed a blue fluorescence. Dapi channel ( A , D ); Alexa 594 channel ( B , E ); overlay channel ( C , F ) Images (63×) were obtained using a Leica DMI 4000 B inverted confocal light microscope.

Article Snippet: Further strains were used to fine-tune the most active amoebicidal agents: A. polyphaga (genotype T4, type strain: ATCC 30461), A. griffini (genotype T3, clinical strain), A. castellanii L-10 (genotype T4, clinical strain), and A. culbertsoni (genotype T10, type strain: ATCC 30171).

Techniques: Incubation, Negative Control, Fluorescence, Staining, Light Microscopy

Journal: Pharmaceuticals

Article Title: Amoebicidal Effect of COVID Box Molecules against Acanthamoeba : A Study of Cell Death

doi: 10.3390/ph17060808

Figure Lengend Snippet: Evaluation of the mitochondrial membrane potential using JC-1 dye in Acanthamoeba culbertsoni trophozoites incubated with IC 90 of Terconazole ( D – F ) for 24 h. Control cells ( A – C ). Images (63×) are based on Leica SPE confocal microscopy. Data showed in the graph are presented as means ± SD, ** p < 0.01; the results demonstrate significant differences when comparing cells treated with Terconazole to negative control cells ( G ). Differences between the mean values of ratio fluorescence intensity red/green were assessed using one-way analysis of variance (ANOVA). The mean fluorescence intensity of stained cells for each assay was determined using EVOS M5000. All experiments were conducted in triplicate.

Article Snippet: Further strains were used to fine-tune the most active amoebicidal agents: A. polyphaga (genotype T4, type strain: ATCC 30461), A. griffini (genotype T3, clinical strain), A. castellanii L-10 (genotype T4, clinical strain), and A. culbertsoni (genotype T10, type strain: ATCC 30171).

Techniques: Membrane, Incubation, Control, Confocal Microscopy, Negative Control, Fluorescence, Staining

Journal: Pharmaceuticals

Article Title: Amoebicidal Effect of COVID Box Molecules against Acanthamoeba : A Study of Cell Death

doi: 10.3390/ph17060808

Figure Lengend Snippet: Evaluation of reactive oxygen species (ROS) production using a CellROX ® Deep Red fluorescent probe in Acanthamoeba culbertsoni trophozoites, incubated with IC 90 of Terconazole ( B ) for 24 h. Control cells ( A ). The images (63×) were obtained using Leica SPE confocal microscopy. Data shown in the graph are presented as means ± SD, *** p < 0.001, obtained with the software of the EVOS™ FL Cell Imaging System M5000; the results demonstrate significant differences when comparing cells treated with nitroxoline to negative control ( C ). Differences between the values were assessed using one-way analysis of variance (ANOVA).

Article Snippet: Further strains were used to fine-tune the most active amoebicidal agents: A. polyphaga (genotype T4, type strain: ATCC 30461), A. griffini (genotype T3, clinical strain), A. castellanii L-10 (genotype T4, clinical strain), and A. culbertsoni (genotype T10, type strain: ATCC 30171).

Techniques: Incubation, Control, Confocal Microscopy, Software, Imaging, Negative Control

Journal: Infection and Immunity

Article Title: Zinc-limited Mycobacterium tuberculosis stimulate distinct responses in macrophages compared with standard zinc-replete bacteria

doi: 10.1128/iai.00578-24

Figure Lengend Snippet: Macrophages more readily phagocytose Zn 2+ -limited Mtb than Zn 2+ -replete Mtb. (A) Intracellular CFUs of Aux-ZRM and Aux-ZLM in RAW 264.7 cells at 4 and 24 hpi. (B) The percentage and (C) relative bacterial burden of RAW 264.7 macrophages infected with DsRed-expressing Aux-ZRM and Aux-ZLM at 4 and 24 hpi, as determined by flow cytometry. (D) Intracellular CFUs of Rv-ZRM and Rv-ZLM in murine BMDMs at 4 hpi. (E) Intracellular CFUs of Aux-ZRM and Aux-ZLM in THP-1 macrophages at 4 hpi. (F) The percentage and (G) relative bacterial burden of THP-1 macrophages infected with GFP-expressing Aux-ZRM and Aux-ZLM at 2 hpi, as determined by flow cytometry. Bacterial burden is relative to the burden of macrophages infected with Aux-ZRM. The error bars are the standard deviations of six replicates. Representative data from at least two independent experiments are shown. **: P -value < 0.01. ***: P -value < 0.001. ****: P -value < 0.0001. CFUs: colony forming units; Aux-ZRM and Aux-ZLM: Mtb mc 2 6206 grown in Zn 2+ -replete or limited media, respectively; Rv-ZRM and Rv-ZLM: Mtb H37Rv grown in Zn 2+ -replete or limited media, respectively; BMDM: bone marrow-derived macrophages. MΦ: macrophage. a.u.: arbitrary units.

Article Snippet: The fluorescent strain of Mtb-Aux expressing DsRed (pMSP12::DsRed, Addgene plasmid # 30171) and GFP (Live-Dead reporter; a gift from Dr. Christopher Sassetti) were used for the flow cytometry assays and confocal microscopy when indicated ( , ).

Techniques: Infection, Expressing, Flow Cytometry, Derivative Assay

Journal: Pathogens

Article Title: Epidemiology of and Genetic Factors Associated with Acanthamoeba Keratitis

doi: 10.3390/pathogens13020142

Figure Lengend Snippet: The traditional classification of Acanthamoeba .

Article Snippet: , A. culbertsoni , T10 , ATCC 30171 , [ ] .

Techniques:

Journal: ISME Communications

Article Title: Zooming in on the intracellular microbiome composition of bacterivorous Acanthamoeba isolates

doi: 10.1093/ismeco/ycae016

Figure Lengend Snippet: List of Acanthamoeba isolates positive for 16S rRNA used for profiling of intracellular bacterial microbiome composition.

Article Snippet: A total of 51 isolates were included with 33 isolates from Australia (19 corneal, 9 water, and 5 nasal mucosa isolates), 13 from India (all corneal isolates), and five were ATCC strains (two isolates obtained from human corneal samples in the UK, ATCC 30873 and 30868), one isolate derived from swimming pool water in France (ATCC 30841), another isolated from cell culture in India (ATCC 30171), and one strain cultured from freshwater in the USA (ATCC 30871).

Techniques: Cell Culture

Journal: ISME Communications

Article Title: Zooming in on the intracellular microbiome composition of bacterivorous Acanthamoeba isolates

doi: 10.1093/ismeco/ycae016

Figure Lengend Snippet: List of Acanthamoeba isolates positive for 16S rRNA used for profiling of intracellular bacterial microbiome composition.

Article Snippet: Additionally, Acanthamoeba culbertsoni (ATCC 30171) harboured intracellular bacteria ( ).

Techniques: Cell Culture

Journal: ISME Communications

Article Title: Zooming in on the intracellular microbiome composition of bacterivorous Acanthamoeba isolates

doi: 10.1093/ismeco/ycae016

Figure Lengend Snippet: Venn-diagram showing unique and shared ASVs (relative abundance >0) among different Acanthamoeba groups as per source of isolation and origin of country ( A ). The top 20 most abundant ASVs clustered into six different bacterial families cross all Acanthamoeba isolates as per source of isolation and origin of country ( B ).

Article Snippet: Additionally, Acanthamoeba culbertsoni (ATCC 30171) harboured intracellular bacteria ( ).

Techniques: Isolation

Journal: ISME Communications

Article Title: Zooming in on the intracellular microbiome composition of bacterivorous Acanthamoeba isolates

doi: 10.1093/ismeco/ycae016

Figure Lengend Snippet: Beta diversity of bacterial microbiome composition in Acanthamoeba corneal isolates was compared between the countries of origin, India ( n = 8) and Australia ( n = 7), and also within Australian isolates based on their sources: corneal ( n = 7) and water isolates ( n = 5). Two-dimensional PCoA plots comparing Bray–Curtis dissimilarity index ( A ) and weighted UniFrac distance metric ( B ) show significant differences ( P < 0.05) between Indian and Australian corneal isolates of Acanthamoeba spp., but no significant differences ( P > 0.05) between Australian water and corneal isolates ( C , D ). The axes represent the first two principal coordinates of the PCoA plot, with each point on the plot representing the bacterial microbiome of an individual Acanthamoeba strain. The ASVs data were transformed to relative abundance before plotting to account for differences in sequencing depth and some of the sample points are overlapped on the plots due to the very similar bacterial microbiome composition.

Article Snippet: Additionally, Acanthamoeba culbertsoni (ATCC 30171) harboured intracellular bacteria ( ).

Techniques: Transformation Assay, Sequencing

Journal: ISME Communications

Article Title: Zooming in on the intracellular microbiome composition of bacterivorous Acanthamoeba isolates

doi: 10.1093/ismeco/ycae016

Figure Lengend Snippet: Alpha diversity of bacterial microbiome composition of Acanthamoeba strains by group; ( A) Shannon index, and ( B) number of observed ASVs. A global Kruskal–Wallis test was used to perform statistical analysis among the three groups, whereas a Wilcoxon rank sum test was performed between the two groups. Acanthamoeba isolates; Australia water ( n = 5), Australia cornea ( n = 7), and India cornea ( n = 8). The boxplots show the smallest and largest values (the 25th and 75th quartiles), the median, and outliers.

Article Snippet: Additionally, Acanthamoeba culbertsoni (ATCC 30171) harboured intracellular bacteria ( ).

Techniques:

Journal: ISME Communications

Article Title: Zooming in on the intracellular microbiome composition of bacterivorous Acanthamoeba isolates

doi: 10.1093/ismeco/ycae016

Figure Lengend Snippet: Intracellular bacterial microbiome composition of Acanthamoeba isolates by groups; Indian corneal isolates, Australian corneal, and water isolates. Stacked bar plots visually represent the average relative abundance (%) of 16S V1–3 rRNA gene sequences assigned to bacterial phyla ( A ), families ( B ), and genera ( C ). For visualization, taxa with <1% relative abundance have been grouped together. In cases where the genus level classification was not possible, a higher taxonomic level is mentioned and “ Candidatus ” was mentioned for Candidatus Jidaibacter acanthamoeba. ( D ) Heatmap representing the top 20 most abundant ASVs (log10). ASVs (genus level) are shown in y-axis and x-axis represents individual samples included for intracellular microbiome profiling of Acanthamoeba isolates targeting 16S rRNA, V1–3 (refer for details of Acanthamoeba isolates). White cells correspond no ASVs detected. For visualization, “ Candidatus ” was labelled for Candidatus Jidaibacter acanthamoeba in B, C, and D.

Article Snippet: Additionally, Acanthamoeba culbertsoni (ATCC 30171) harboured intracellular bacteria ( ).

Techniques:

Journal: ISME Communications

Article Title: Zooming in on the intracellular microbiome composition of bacterivorous Acanthamoeba isolates

doi: 10.1093/ismeco/ycae016

Figure Lengend Snippet: Representative FISH micrographs showing the presence of intracellular bacteria in Acanthamoeba trophozoites investigated in this study. Probes EUK516 conjugated with Cy5, targeting Acanthamoeba , and EUB conjugated with Cy3, targeting most of bacterial strains were used for all Acanthamoeba strains positive for bacterial 16S rRNA. DAPI was used in mounting medium when visualized by a fluorescence microscope. Probe pB-914 labelled with 6-FAM was used for isolates containing high abundance of bacteria belong to Enterobacteriaceae family. ( A ) Rod shaped bacteria were observed throughout the cytoplasm of Acanthamoeba trophozoites (Indian corneal isolate) and a few cocci bacteria were also observed (yellow arrows). The white arrow represents bacterium cell undergoing binary fission. ( B ) Bacteria showing binary fission (white arrows) in vacuole like structure of Acanthamoeba recovered from water sample (R3). ( C and D ) Corneal isolates of Acanthamoeba spp. (Ac-112 and L-579/20, respectively) with intracellular bacteria. ( E ) Intracellular bacteria labelled with probes EUB and pB-914 simultaneously in Acanthamoeba sp. isolated from an AK patient. ( F ) Clinical (Ac-102) isolate of Acanthamoeba trophozoite depicting rod shaped intracellular bacteria. Indicators: white arrow, bacterial cell undergoing binary fission; yellow arrow: cocci shaped bacteria. Scale bar in each panel represents 10 μm.

Article Snippet: Additionally, Acanthamoeba culbertsoni (ATCC 30171) harboured intracellular bacteria ( ).

Techniques: Bacteria, Fluorescence, Microscopy, Isolation

Journal: ISME Communications

Article Title: Zooming in on the intracellular microbiome composition of bacterivorous Acanthamoeba isolates

doi: 10.1093/ismeco/ycae016

Figure Lengend Snippet: Representative images of TEM showing Acanthamoeba isolates containing intracellular bacteria. ( A ) Overview of an Acanthamoeba trophozoite (Indian corneal isolate) harbouring intracellular bacteria. ( A.i-ii ) Higher magnification showing rod (white arrow) and cocci (blue arrow) shaped bacteria inside early phagocytic ( i ) or PV ( ii ), and bacterial cells were also observed in trophozoite cytoplasm (ii). A bacterium undergoing binary fission (asterisk) and digested bacteria (arrowhead) appear disintegrated surrounded by multiple layers (yellow arrow) (ii). ( B ) Engulfed bacteria appeared disintegrated and digested inside PV surrounded by multiple layers (Australian water isolate). ( C ) Rod and spherical shaped bacterial cells close to host NM appears enclosed by double-membranous vacuole and disintegrated (arrowhead). And a bacterial cell is undergoing binary fission (Australian corneal isolate). ( D ) Engulfed bacteria appeared disintegrated and digested inside PV close to host NM. Both digested and undigested bacteria in the same PV consisting multiple layers of membrane. ( E ) Digested and undigested cocci bacteria in the same PV. Symbols = EPV: early phagocytic vacuole; PV: phagocytic vacuole; M: mitochondria; N: nucleus; NM: nuclear membrane; NP: nuclear pore; DV: digestive vacuole; CV: contractile vacuole; white arrow: rod bacteria; blue arrow: spherical bacteria; arrowhead: digested bacteria; yellow arrow: surrounded by multiple layers; asterisk ( * ): binary fission; alpha (α): electron translucent space. The lengths of bars in the bottom right corner of each image represent 500 nm except A (1 μm), and D (1 μm).

Article Snippet: Additionally, Acanthamoeba culbertsoni (ATCC 30171) harboured intracellular bacteria ( ).

Techniques: Bacteria, Membrane