Journal: Bioactive Materials

Article Title: Felodipine enhances aminoglycosides efficacy against implant infections caused by methicillin-resistant Staphylococcus aureus , persisters and biofilms

doi: 10.1016/j.bioactmat.2021.11.019

Figure Lengend Snippet: Changes in the transcriptome of MRSA treated with felodipine or DMSO (control). (A) Heatmap of correlation between samples. The different colors of the squares represent the different correlation coefficients of the two samples. (B) Volcano map of differentially expressed genes (DEGs). Each dot represents a specific gene. Red indicates significant upregulation, blue indicates significant downregulation, and gray indicates non-significant differential expression. (C) Clustering heatmap of some important DEGs. The shade of color indicates the amount of gene expression. Each group contains data for three independent samples. (D) Go enrichment analysis of DEGs. Each bubble represents a GO Term. The size of the bubble is proportional to the number of genes enriched to the GO Term. The different colors represent the three major classifications of GO. BP = biological process; CC = cellular component; MF = molecular function. (E) Validation of gene expression by RT-PCR. Felodipine inhibited the gene expression associated with energy metabolism, biofilm formation, aminoglycosides resistance, and bacterial virulence. And felodipine increased the gene expression associated protein degradation. Data analysis was performed using the comparative CT method, with 16S rRNA serving as the comparator. The results are presented as fold-changes relative to the control, which was set to a value of 1. Data are expressed as the mean ± SD. n = 3; **p < 0.01; ***p < 0.001.

Article Snippet: The LIVE/DEAD™ BacLight™ Bacterial Viability Kit and Membrane Potential Kit were purchased from Thermo Fisher Scientific (USA), while the Microbial Viability Assay Kit, Biofilm Formation Assay Kit, and Biofilm TestPiece Assay Kit were purchased from Dojindo (Japan).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Bioactive Materials

Article Title: Felodipine enhances aminoglycosides efficacy against implant infections caused by methicillin-resistant Staphylococcus aureus , persisters and biofilms

doi: 10.1016/j.bioactmat.2021.11.019

Figure Lengend Snippet: Felodipine combined with gentamicin against biofilm. (A) Minimum biofilm inhibitory concentration (MBIC) testing of felodipine against MRSA and MRSE. Biofilms was stained with crystal violet. (B) Biofilm mass was quantified by measuring sample absorbance at 595 nm using a spectrophotometer. Data are expressed as the mean ± SD; n = 3; ***p < 0.001. (C) After treated with felodipine or gentamicin, the biofilm formation on the surface of medical implant (Ti6Al4V disks) was stained with a bacterial viability kit and detected by CLSM. (D) SEM was conducted to observe the effect of felodipine or gentamicin in preventing biofilm formation. (E) Crystal violet staining was applied to examine the antibacterial efficacy of felodipine against established biofilms on the surface of implants. (F) Biofilms were quantified by measuring the absorbance of the samples at 595 nm using spectrophotometer. Data are expressed as the mean ± SD; n = 3; ***p < 0.001. (G) After treatment with different concentration of felodipine or gentamicin, the number of bacteria within the established biofilm was enumerated by the spreading plate method. Data are expressed as the mean ± SD; n = 3; ***p < 0.001. (H) After treatment with different concentration of felodipine or gentamicin, the number of MRSA persisters was counted at indicated time. (I) After treatment with felodipine or gentamicin, the number of MRSE persisters was counted at indicated time.

Article Snippet: The LIVE/DEAD™ BacLight™ Bacterial Viability Kit and Membrane Potential Kit were purchased from Thermo Fisher Scientific (USA), while the Microbial Viability Assay Kit, Biofilm Formation Assay Kit, and Biofilm TestPiece Assay Kit were purchased from Dojindo (Japan).

Techniques: Concentration Assay, Staining, Spectrophotometry

Journal: Bioactive Materials

Article Title: Felodipine enhances aminoglycosides efficacy against implant infections caused by methicillin-resistant Staphylococcus aureus , persisters and biofilms

doi: 10.1016/j.bioactmat.2021.11.019

Figure Lengend Snippet: Felodipine induces proteolysis of MRSA and decreases energy metabolism. (A) PCA analysis of samples from each group. The relationship between samples is presented in different dimensions. Each point represents one replicate of a treatment group, with different colors for different groups. The samples within the felodipine treatment group had good similarity. (B) The distribution of differentially expressed proteins (DEPs) are presented in volcano plot. Blue dots indicate down-regulated proteins, red dots indicate up-regulated proteins. (C) Cluster heat map of some important DEPs. The protein level of aacA-aphD was reduced after treatment with felodipine. (D) KEGG pathway enrichment analysis of DEPs. The ordinate is the top 20 pathways that are significantly enriched. Pathways associated with TCA cycle, purine metabolism, arginine and proline metabolism were significantly influenced. (E) Parallel reaction monitoring was conducted to quantify the expression levels of some DEPs associated with energy metabolism, biofilm formation, aminoglycosides resistance, bacterial virulence. (F) Metabolon-based energy metabolism detection of MRSA after treatment with felodipine for 8h. Box plot shows the distribution of metabolites for each sample. Each group contains 6 biological replicates of the sample. **p < 0.01. ***p < 0.001 . (G) Structural model of the ClpP complexed with Felodipine. In the close-up view, felodipine binds to the H pockets [Protein Data Bank (PDB) ID: 5 W18]. The amino acid residues involved includes Tyr61, Tyr63, and His83.

Article Snippet: The LIVE/DEAD™ BacLight™ Bacterial Viability Kit and Membrane Potential Kit were purchased from Thermo Fisher Scientific (USA), while the Microbial Viability Assay Kit, Biofilm Formation Assay Kit, and Biofilm TestPiece Assay Kit were purchased from Dojindo (Japan).

Techniques: Expressing

Journal: Bioactive Materials

Article Title: Felodipine enhances aminoglycosides efficacy against implant infections caused by methicillin-resistant Staphylococcus aureus , persisters and biofilms

doi: 10.1016/j.bioactmat.2021.11.019

Figure Lengend Snippet: Felodipine in combination with gentamicin alleviates murine periprosthetic joint infection. (A) Schematic diagram of the treatment of implant infections in mice. (B) After treatment with felodipine (40 mg/kg, s.c.), gentamicin (80 mg/kg, s.c.), or the combination of felodipine (40 mg/kg, s.c.) and gentamicin (80 mg/kg, s.c.), the infected joint was examined using X-ray and Micro-CT to evaluate periosteal reaction, osteolysis and the position of Ti6Al4V rod. (C) Radiographic scores of infected joints. The evidence for scoring includes five aspects. Each aspect was scored on a five-point scale (0–4), where 4 represented the most severe. (D) Bone mineral density and (E) bone volume fraction (BV/TV) were measured using Micro-CT. Data are expressed as the means ± SD; n = 10; ns, not significant; **p < 0.01, ***p < 0.001. (F) Bacterial morphology and biofilm viability on the surface of Ti6Al4V rod were detected by SEM and CLSM. (G) Distribution of CFU density per implant was determined by spread plate method. Data are expressed as the means ± SD; n = 10; *p < 0.05; ***p < 0.001. (H) Distribution of CFU density per soft tissue was determined by spread plate method. Data are expressed as the means ± SD; n = 10; ns, not significant; **p < 0.01, ***p < 0.001.

Article Snippet: The LIVE/DEAD™ BacLight™ Bacterial Viability Kit and Membrane Potential Kit were purchased from Thermo Fisher Scientific (USA), while the Microbial Viability Assay Kit, Biofilm Formation Assay Kit, and Biofilm TestPiece Assay Kit were purchased from Dojindo (Japan).

Techniques: Infection, Micro-CT

Journal: Bioactive Materials

Article Title: Felodipine enhances aminoglycosides efficacy against implant infections caused by methicillin-resistant Staphylococcus aureus , persisters and biofilms

doi: 10.1016/j.bioactmat.2021.11.019

Figure Lengend Snippet: Scheme illustration of drug repurposing screens and synergistic drug-combination. Felodipine in combination with gentamicin alleviate chronic implant infections caused by biofilms and persisters. The proposed mechanism showed that felodipine activate ClpP protease and reduce the protein level of aminoglycosides modifying enzyme (aacA-aphD).

Article Snippet: The LIVE/DEAD™ BacLight™ Bacterial Viability Kit and Membrane Potential Kit were purchased from Thermo Fisher Scientific (USA), while the Microbial Viability Assay Kit, Biofilm Formation Assay Kit, and Biofilm TestPiece Assay Kit were purchased from Dojindo (Japan).

Techniques:

Journal: Bioactive Materials

Article Title: Felodipine enhances aminoglycosides efficacy against implant infections caused by methicillin-resistant Staphylococcus aureus , persisters and biofilms

doi: 10.1016/j.bioactmat.2021.11.019

Figure Lengend Snippet: Changes in the transcriptome of MRSA treated with felodipine or DMSO (control). (A) Heatmap of correlation between samples. The different colors of the squares represent the different correlation coefficients of the two samples. (B) Volcano map of differentially expressed genes (DEGs). Each dot represents a specific gene. Red indicates significant upregulation, blue indicates significant downregulation, and gray indicates non-significant differential expression. (C) Clustering heatmap of some important DEGs. The shade of color indicates the amount of gene expression. Each group contains data for three independent samples. (D) Go enrichment analysis of DEGs. Each bubble represents a GO Term. The size of the bubble is proportional to the number of genes enriched to the GO Term. The different colors represent the three major classifications of GO. BP = biological process; CC = cellular component; MF = molecular function. (E) Validation of gene expression by RT-PCR. Felodipine inhibited the gene expression associated with energy metabolism, biofilm formation, aminoglycosides resistance, and bacterial virulence. And felodipine increased the gene expression associated protein degradation. Data analysis was performed using the comparative CT method, with 16S rRNA serving as the comparator. The results are presented as fold-changes relative to the control, which was set to a value of 1. Data are expressed as the mean ± SD. n = 3; **p < 0.01; ***p < 0.001.

Article Snippet: The LIVE/DEAD™ BacLight™ Bacterial Viability Kit and Membrane Potential Kit were purchased from Thermo Fisher Scientific (USA), while the Microbial Viability Assay Kit, Biofilm Formation Assay Kit, and Biofilm TestPiece Assay Kit were purchased from Dojindo (Japan).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Bioactive Materials

Article Title: Felodipine enhances aminoglycosides efficacy against implant infections caused by methicillin-resistant Staphylococcus aureus , persisters and biofilms

doi: 10.1016/j.bioactmat.2021.11.019

Figure Lengend Snippet: Felodipine combined with gentamicin against biofilm. (A) Minimum biofilm inhibitory concentration (MBIC) testing of felodipine against MRSA and MRSE. Biofilms was stained with crystal violet. (B) Biofilm mass was quantified by measuring sample absorbance at 595 nm using a spectrophotometer. Data are expressed as the mean ± SD; n = 3; ***p < 0.001. (C) After treated with felodipine or gentamicin, the biofilm formation on the surface of medical implant (Ti6Al4V disks) was stained with a bacterial viability kit and detected by CLSM. (D) SEM was conducted to observe the effect of felodipine or gentamicin in preventing biofilm formation. (E) Crystal violet staining was applied to examine the antibacterial efficacy of felodipine against established biofilms on the surface of implants. (F) Biofilms were quantified by measuring the absorbance of the samples at 595 nm using spectrophotometer. Data are expressed as the mean ± SD; n = 3; ***p < 0.001. (G) After treatment with different concentration of felodipine or gentamicin, the number of bacteria within the established biofilm was enumerated by the spreading plate method. Data are expressed as the mean ± SD; n = 3; ***p < 0.001. (H) After treatment with different concentration of felodipine or gentamicin, the number of MRSA persisters was counted at indicated time. (I) After treatment with felodipine or gentamicin, the number of MRSE persisters was counted at indicated time.

Article Snippet: The LIVE/DEAD™ BacLight™ Bacterial Viability Kit and Membrane Potential Kit were purchased from Thermo Fisher Scientific (USA), while the Microbial Viability Assay Kit, Biofilm Formation Assay Kit, and Biofilm TestPiece Assay Kit were purchased from Dojindo (Japan).

Techniques: Concentration Assay, Staining, Spectrophotometry

Journal: Bioactive Materials

Article Title: Felodipine enhances aminoglycosides efficacy against implant infections caused by methicillin-resistant Staphylococcus aureus , persisters and biofilms

doi: 10.1016/j.bioactmat.2021.11.019

Figure Lengend Snippet: Felodipine induces proteolysis of MRSA and decreases energy metabolism. (A) PCA analysis of samples from each group. The relationship between samples is presented in different dimensions. Each point represents one replicate of a treatment group, with different colors for different groups. The samples within the felodipine treatment group had good similarity. (B) The distribution of differentially expressed proteins (DEPs) are presented in volcano plot. Blue dots indicate down-regulated proteins, red dots indicate up-regulated proteins. (C) Cluster heat map of some important DEPs. The protein level of aacA-aphD was reduced after treatment with felodipine. (D) KEGG pathway enrichment analysis of DEPs. The ordinate is the top 20 pathways that are significantly enriched. Pathways associated with TCA cycle, purine metabolism, arginine and proline metabolism were significantly influenced. (E) Parallel reaction monitoring was conducted to quantify the expression levels of some DEPs associated with energy metabolism, biofilm formation, aminoglycosides resistance, bacterial virulence. (F) Metabolon-based energy metabolism detection of MRSA after treatment with felodipine for 8h. Box plot shows the distribution of metabolites for each sample. Each group contains 6 biological replicates of the sample. **p < 0.01. ***p < 0.001 . (G) Structural model of the ClpP complexed with Felodipine. In the close-up view, felodipine binds to the H pockets [Protein Data Bank (PDB) ID: 5 W18]. The amino acid residues involved includes Tyr61, Tyr63, and His83.

Article Snippet: The LIVE/DEAD™ BacLight™ Bacterial Viability Kit and Membrane Potential Kit were purchased from Thermo Fisher Scientific (USA), while the Microbial Viability Assay Kit, Biofilm Formation Assay Kit, and Biofilm TestPiece Assay Kit were purchased from Dojindo (Japan).

Techniques: Expressing

Journal: Bioactive Materials

Article Title: Felodipine enhances aminoglycosides efficacy against implant infections caused by methicillin-resistant Staphylococcus aureus , persisters and biofilms

doi: 10.1016/j.bioactmat.2021.11.019

Figure Lengend Snippet: Felodipine in combination with gentamicin alleviates murine periprosthetic joint infection. (A) Schematic diagram of the treatment of implant infections in mice. (B) After treatment with felodipine (40 mg/kg, s.c.), gentamicin (80 mg/kg, s.c.), or the combination of felodipine (40 mg/kg, s.c.) and gentamicin (80 mg/kg, s.c.), the infected joint was examined using X-ray and Micro-CT to evaluate periosteal reaction, osteolysis and the position of Ti6Al4V rod. (C) Radiographic scores of infected joints. The evidence for scoring includes five aspects. Each aspect was scored on a five-point scale (0–4), where 4 represented the most severe. (D) Bone mineral density and (E) bone volume fraction (BV/TV) were measured using Micro-CT. Data are expressed as the means ± SD; n = 10; ns, not significant; **p < 0.01, ***p < 0.001. (F) Bacterial morphology and biofilm viability on the surface of Ti6Al4V rod were detected by SEM and CLSM. (G) Distribution of CFU density per implant was determined by spread plate method. Data are expressed as the means ± SD; n = 10; *p < 0.05; ***p < 0.001. (H) Distribution of CFU density per soft tissue was determined by spread plate method. Data are expressed as the means ± SD; n = 10; ns, not significant; **p < 0.01, ***p < 0.001.

Article Snippet: The LIVE/DEAD™ BacLight™ Bacterial Viability Kit and Membrane Potential Kit were purchased from Thermo Fisher Scientific (USA), while the Microbial Viability Assay Kit, Biofilm Formation Assay Kit, and Biofilm TestPiece Assay Kit were purchased from Dojindo (Japan).

Techniques: Infection, Micro-CT

Journal: Biomedical Optics Express

Article Title: Single-objective high-resolution confocal light sheet fluorescence microscopy for standard biological sample geometries

doi: 10.1364/BOE.420788

Figure Lengend Snippet: Digital Confocal Axial Plane Optical Microscopy for high-resolution imaging of living samples. (A) DC-APOM employs the same objective lens for light sheet illumination and detection, to image a vertical xz-plane with high-NA oil-immersion objectives. The vertical image is rotated by 90° into a horizontal image that can be projected onto a camera sensor using a remote objective lens (Supplement 1, Fig. S5). Blue lines at the sample and on the camera sensor indicate the beam that is scanned across the sample to form a light sheet. A red box indicates the rows of pixels on the camera sensor that are currently read out relative to the position of the scanned beam. While the beam is moved across the sample, the virtual confocal slit follows. (B) Optical system of DC-APOM. The beam path is described briefly in the Methods, and in detail in Appendix A. The following designations are used: SMF - single mode fiber; CL - collimator; L1, L2, L3, L4, L5, L6 - lenses; GX - X-axis galvo; SL - scan lens; TL1, TL2, TL3, TL4 - tube lenses 1; O1, O2 - objectives; DM - dichroic mirror; EF - emission filter; M1, M2, M3, M4, M5, M6 - mirrors; PBS - polarizing beam splitter; QWP – quarter-wave plate. The blue and green arrows indicate the direction of light propagation for the excitation and emission, respectively. (C) Samples that are difficult to image at cellular resolution, such as densely-packed bacterial biofilms can be fully resolved and reconstructed in 3D using DC-APOM imaging. In the V. cholerae biofilm shown here, each cell is coloured according to its distance to the center of mass of the whole biofilm colony. (D) Widefield xy-image and APOM xz-image of the same nanohole array target used for alignment of the system. The holes in the chromium layer of the target have a diameter of 400 nm and a spacing of 7 µm. (E) Schematic drawings of examples for sample geometries that can be used for DC-APOM imaging. The only constraint on the sample geometry is the necessity for a coverslip.

Article Snippet: Three-dimensional images of biofilms ( were reconstructed from image stacks using Matlab (Mathworks) and BiofilmQ [ 54 ] as described previously [ 52 ] .

Techniques: Microscopy, Imaging

Journal: Biomedical Optics Express

Article Title: Single-objective high-resolution confocal light sheet fluorescence microscopy for standard biological sample geometries

doi: 10.1364/BOE.420788

Figure Lengend Snippet: Optimization of illumination and detection conditions for DC-APOM. (A) Comparison of the performance for three different objectives with high numerical aperture, using spinning disk confocal and DC-APOM in xz-images of fixed V. cholerae biofilms. For DC-APOM, a pair of two identical objectives was used in each configuration. The insets show the point spread function measured directly on the coverslip using 100 nm fluorescent beads embedded either in medium matching the refractive index of the immersion oil (marked here as oil) or in water. (B) Comparison of the effect of different illumination beam profiles on image contrast (left axis, red) and on signal-to-background ratio (right axis, blue). The confocal slit width is varied for light sheets based on Gaussian beams of a given width: FWHMxy = 0.6 µm (circles), FWHMxy = 1.3 µm (diamonds), FWHMxy = 2.2 µm (squares). Similarly, the confocal slit width is varied for Bessel beams of given width: FWHMxy = 0.4 µm (pyramids), FWHMxy = 0.6 µm (circles), FWHMxy = 1.3 µm (squares). Measurements connected by a line are acquired for the same biofilm. Image contrast is quantified using the variance of Laplacian (LAPV) score. The LAPV and SBR values for an infinitely large confocal slit width correspond to measurements for static light sheets and all LAPV scores are normalized to this point.

Article Snippet: Three-dimensional images of biofilms ( were reconstructed from image stacks using Matlab (Mathworks) and BiofilmQ [ 54 ] as described previously [ 52 ] .

Techniques:

Journal: Biomedical Optics Express

Article Title: Single-objective high-resolution confocal light sheet fluorescence microscopy for standard biological sample geometries

doi: 10.1364/BOE.420788

Figure Lengend Snippet: Photobleaching performance. Photobleaching is quantified using fluorescence intensity values I normalized to the first imaging cycle I0, for DC-APOM based on Bessel or Gaussian beams, and for spinning disk confocal. To compare the photobleaching, 3D image volumes of the same resolution were acquired using all imaging modalities, with the same signal-level in the images. Lines denote the mean of n = 10 biofilm volumes and the shaded area indicates the standard deviation.

Article Snippet: Three-dimensional images of biofilms ( were reconstructed from image stacks using Matlab (Mathworks) and BiofilmQ [ 54 ] as described previously [ 52 ] .

Techniques: Fluorescence, Imaging, Standard Deviation

Journal: Biomedical Optics Express

Article Title: Single-objective high-resolution confocal light sheet fluorescence microscopy for standard biological sample geometries

doi: 10.1364/BOE.420788

Figure Lengend Snippet: Imaging of dynamic three-dimensional processes at high resolution with DC-APOM. (A) Time-lapse of the dispersal process of a V. cholerae biofilm colony (cells express sfGFP), induced by removing glucose from the growth medium. Different cells in the outer periphery of the biofilm depart from the biofilm colony over the course of several minutes. The bottom of the biofilm remains firmly attached to the glass coverslip. (B) Time-lapse at high time resolution of a V. cholerae biofilm (cells express sfGFP) exposed to an osmotic shock, caused by replacing the growth medium with distilled water, showing a massive disruption of the biofilm structure over the course of several seconds. (C) Image overlay showing the interaction of murine macrophages stained with CellTracker (magenta) and E. coli cells expressing sfGFP (green). Individual E. coli cells that have been phagocytosed by the macrophages are visible inside phagosomes. The magnified inset shows the two fluorescence channels separately. The dotted gray lines in the images denote the position of the glass coverslip.

Article Snippet: Three-dimensional images of biofilms ( were reconstructed from image stacks using Matlab (Mathworks) and BiofilmQ [ 54 ] as described previously [ 52 ] .

Techniques: Imaging, Staining, Expressing, Fluorescence