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The accelerating spread of antibiotic resistance has significantly weakened the clinical efficacy of existing antibiotics, posing a severe threat to public health. There is an urgent need to develop novel antimicrobial alternatives that can bypass the mechanisms of antibiotic resistance and effectively kill multidrug-resistant (MDR) pathogens. Antimicrobial peptides (AMPs) are one of the most promising candidates to treat MDR pathogenic infections since they display broad-spectrum antimicrobial activities and are less prone to achieve drug resistance. In this study, we investigated the antibacterial capability and mechanisms of two machine learning-driven linear peptide compounds termed YI12 and FK13. We reveal that YI12 and FK13 exhibit broad-spectrum antibacterial properties against clinically significant bacterial pathogens, inducing no or minimal hemolysis in mammalian red blood cells. We further ascertain that YI12 and FK13 are resilient to heat and acid-base conditions, and exhibit susceptibility to hydrolytic enzymes and divalent cations under physiological conditions. Initial mechanistic investigations reveal that YI12 and FK13 compromise bacterial membrane integrity, leading to membrane potential dissipation and excessive reactive oxygen species (ROS) generation. Collectively, our findings highlight the prospective utility of these two cationic amphiphilic peptides as broad-spectrum antibacterial agents. Full article

(This article belongs to the Special Issue New Approaches to Combating Multidrug-Resistant Pathogens)

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Characterization of YI12 and FK13 peptides. (A) Chemical structures of two AMPs. (B) Helical wheel projections of YI12 and FK13. Hydrophilic residues—circle shape, hydrophobic residues—diamond shape, potentially positively charged residues—pentagonal shape, potentially negatively charged residues—triangle shape (none). Hydrophobicity is color-coded: the most hydrophobic residues are green, and the amount of green decreases with hydrophobicity. Hydrophilic residues are coded in red, and the amount of red decreases with hydrophilicity. The residues that may be charged are light blue. Full article ">Figure 2
The growth patterns and time-killing curves of the two MDR bacteria exposed to YI12 and FK13, respectively. (A–D) Growth patterns of MRSA T144 and E. coli B2 under YI12 and FK13. (E,F) Time-dependent killing curves of MRSA T144 and E. coli B2 by FK13. Bacteria isolates were grown to stationary phase and mixed with 0-, 1-, 5-, and 10-fold MIC of FK13. Data are representative of three independent experiments and shown as mean ± SEM. PBS, phosphate-buffered saline. Full article ">Figure 3
CD spectrum of YI12 and FK13 under different solutions. Use PBS (10 mM, pH = 7.4), LPS (50 µM), SDS (50 mM), and 50% TFEA. Values are averages of three scans of samples with a peptide concentration of 100 μg/mL. Full article ">Figure 4
Hemolytic activity evaluation of YI12 and FK13 toward mammalian RBCs. Sterilized PBS served as a negative control, and ddH2O served as a positive control. Full article ">Figure 5
YI12 and FK13 disrupt bacterial membrane permeability. (A,D) Outer membrane permeability of E. coli B2 after exposure to YI12 and FK13, respectively, determined using a fluorescent probe N-phenyl-1-naphthylamine (NPN, excitation wavelength 350 nm, emission wavelength 420 nm); (B,C,E,F) YI12 and FK13 disrupt bacterial membrane permeability. Detections were performed with the fluorescent probe propidium iodide (PI, excitation 535 nm, emission 615 nm). Statistical analysis was conducted through nonparametric one-way analysis (ns, not significant; ** p < 0.01, *** p < 0.001, **** p < 0.0001). Full article ">Figure 6
Changes in membrane potential of E. coli B2 after exposure to YI12 (A) and FK13 (C), and MRSA T144 after exposure to YI12 (B) and FK13 (D). MRSA T144 and E. coli B2 membrane potential was determined by monitoring the fluorescence intensity of 3,3′-dipropylthiodicarbocyanine iodine (DiSC3(5)). Excitation wavelength 622 nm, emission wavelength 670 nm. Full article ">Figure 7
YI12 and FK13 trigger the generation of ROS in E. coli B2 and MRSA T144. Generation of ROS in E. coli B2 (A,D) and MRSA T144 (B,E), determined by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, excitation wavelength 488 nm, emission wavelength 525 nm). The addition of the ROS scavenger NAC significantly inhibited the effects of YI12 and FK13 on MRSA T144 and E. coli B2 (C,F). Statistical analysis was conducted through nonparametric one-way analysis (ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Full article ">

21 pages, 5445 KiB

Open AccessArticle

Characterization of Two Novel Endolysins from Bacteriophage PEF1 and Evaluation of Their Combined Effects on the Control of Enterococcus faecalis Planktonic and Biofilm Cells

by Chen Wang, Junxin Zhao, Yunzhi Lin, Su Zar Chi Lwin, Mohamed El-Telbany, Yoshimitsu Masuda, Ken-ichi Honjoh and Takahisa Miyamoto

Antibiotics 2024, 13(9), 884; https://doi.org/10.3390/antibiotics13090884 (registering DOI) - 13 Sep 2024

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Abstract

Endolysin, a bacteriophage-derived lytic enzyme, has emerged as a promising alternative antimicrobial agent against rising multidrug-resistant bacterial infections. Two novel endolysins LysPEF1-1 and LysPEF1-2 derived from Enterococcus phage PEF1 were cloned and overexpressed in Escherichia coli to test their antimicrobial efficacy against multidrug-resistant E. faecalis strains and their biofilms. LysPEF1-1 comprises an enzymatically active domain and a cell-wall-binding domain originating from the NLPC-P60 and SH3 superfamilies, while LysPEF1-2 contains a putative peptidoglycan recognition domain that belongs to the PGRP superfamily. LysPEF1-1 was active against 89.86% (62/69) of Enterococcus spp. tested, displaying a wider antibacterial spectrum than phage PEF1. Moreover, two endolysins demonstrated lytic activity against additional gram-positive and gram-negative species pretreated with chloroform. LysPEF1-1 showed higher activity against multidrug-resistant E. faecalis strain E5 than LysPEF1-2. The combination of two endolysins effectively reduced planktonic cells of E5 in broth and was more efficient at inhibiting biofilm formation and removing biofilm cells of E. faecalis JCM 7783^T than used individually. Especially at 4 °C, they reduced viable biofilm cells by 4.5 log after 2 h of treatment on glass slide surfaces. The results suggest that two novel endolysins could be alternative antimicrobial agents for controlling E. faecalis infections. Full article

(This article belongs to the Special Issue Phage Applications from Diagnostics to Treatment of Bacterial Infections in a One Health World)

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19 pages, 1650 KiB

Open AccessArticle

Evaluating Nanoparticulate Vaccine Formulations for Effective Antigen Presentation and T-Cell Proliferation Using an In Vitro Overlay Assay

by Dedeepya Pasupuleti, Priyal Bagwe, Amarae Ferguson, Mohammad N. Uddin, Martin J. D’Souza and Susu. M. Zughaier

Vaccines 2024, 12(9), 1049; https://doi.org/10.3390/vaccines12091049 (registering DOI) - 13 Sep 2024

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Abstract

Inducing T lymphocyte (T-cell) activation and proliferation with specificity against a pathogen is crucial in vaccine formulation. Assessing vaccine candidates’ ability to induce T-cell proliferation helps optimize formulation for its safety, immunogenicity, and efficacy. Our in-house vaccine candidates use microparticles (MPs) and nanoparticles (NPs) to enhance antigen stability and target delivery to antigen-presenting cells (APCs), providing improved immunogenicity. Typically, vaccine formulations are screened for safety and immunostimulatory effects using in vitro methods, but extensive animal testing is often required to assess immunogenic responses. We identified the need for a rapid, intermediate screening process to select promising candidates before advancing to expensive and time-consuming in vivo evaluations. In this study, an in vitro overlay assay system was demonstrated as an effective high-throughput preclinical testing method to evaluate the immunogenic properties of early-stage vaccine formulations. The overlay assay’s effectiveness in testing particulate vaccine candidates for immunogenic responses has been evaluated by optimizing the carboxyfluorescein succinimidyl ester (CFSE) T-cell proliferation assay. DCs were overlaid with T-cells, allowing vaccine-stimulated DCs to present antigens to CFSE-stained T-cells. T-cell proliferation was quantified using flow cytometry on days 0, 1, 2, 4, and 6 upon successful antigen presentation. The assay was tested with nanoparticulate vaccine formulations targeting Neisseria gonorrhoeae (CDC F62, FA19, FA1090), measles, H1N1 flu prototype, canine coronavirus, and Zika, with adjuvants including Alhydrogel^® (Alum) and AddaVax™. The assay revealed robust T-cell proliferation in the vaccine treatment groups, with variations between bacterial and viral vaccine candidates. A dose-dependent study indicated immune stimulation varied with antigen dose. These findings highlight the assay’s potential to differentiate and quantify effective antigen presentation, providing valuable insights for developing and optimizing vaccine formulations. Full article

(This article belongs to the Special Issue Advances in the Use of Nanoparticles for Vaccine Platform Development)

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Live cell imaging of DAPI-stained naïve T-cells interacting with activated dendritic cells. (A): Overview of the culture showing DAPI-stained T-cells (blue) interacting with activated dendritic cells that are stimulated with ICG-coated BSA MPs (green) across the field. Scale bar: 100 µm. (B): Close-up view highlighting a DAPI-stained T-cell engaging with a dendritic cell, indicated by the black arrow. Scale bar: 100 µm. (C): Magnified image displaying multiple T-cells in the process of interacting with dendritic cells. Black arrows indicate T-cells undergoing division. Scale bar: 100 µm. (D): Detailed image of T-cells post-division, as indicated by black arrows, continuing their interaction with dendritic cells. Scale bar: 100 µm. Full article ">Figure 2
Representative flow cytometry data of T-lymphocyte profiling. (A). Gating strategy for separating T lymphocytes from the forward scattering vs. side scattering plot. T-cells were gated, capturing 16.5% of the total population of the scatter plot. (B). Singlets are shown on the forward scattering a vs. forward scattering height plot from the T lymphocyte gating. (C). Histograms gated 1, 2, 3, and 4 according to daughter T-cell proliferation over time intervals: 0–1, 1–2, 2–4, and 4–6 days, respectively. Gates were established in accordance with the proliferation pattern of bacterial and viral-based vaccine candidates. Gates were left unchanged for the corresponding blank MP/NP groups. Full article ">Figure 3
Quantitative comparisons of CFSE (FITC-A filter) expressions due to T-cells proliferated as days passed. CFSE is expressed by the proliferating T-cells in response to antigen presentation by the DCs upon stimulation by various treatment groups. (A). Comparison of all blank groups involved in the experiment, including blank CFSE-stained T-cells only, blank BSA MPs, and blank PLGA NPs. (B). comparison of all viral antigen-based vaccine candidates. (C). comparison of all bacterial antigen-based vaccine candidates. All treatments are at 200 µg per well dose. Data are expressed as mean ± SEM, ordinary one-way ANOVA test, post-hoc Tukey’s multiple comparison test. ns, non-significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Full article ">Figure 4
Dose-dependent study results quantifying T-cell proliferation against various concentrations of vaccine candidates. (A). T-cell proliferation analysis measured on days 1, 2, 4, and 6 when treated with vaccine candidate against N. gonorrhoeae strain FA1090 at concentrations of 200 µg, 160 µg, 120 µg, 80 µg, and 40 µg vaccine MPs per well. (B). T-cell proliferation analysis was measured on days 1, 2, 4, and 6 when treated with a vaccine candidate against the measles virus at concentrations of 200 µg, 160 µg, 120 µg, 80 µg, and 40 µg vaccine NPs per well. (C,D). T-cell proliferation trends quantified in response to H1N1 virus particle vaccine candidate and N. gonorrhoeae strain CDC F62 bacterial particle vaccine candidates on day 6. Both were tested at concentrations of 200 µg, 160 µg, 120 µg, 80 µg, and 40 µg per well on day 6. Data are expressed as mean ± SEM, one-way ANOVA, post hoc Tukey’s multiple comparisons test; ns, non-significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Full article ">

17 pages, 4616 KiB

Open AccessArticle

Fluopsin C Promotes Biofilm Removal of XDR Acinetobacter baumannii and Presents an Additive Effect with Polymyxin B on Planktonic Cells

by Leandro Afonso, Kathlen Giovana Grzegorczyk, Julio Martins Salomão, Kawany Roque Basso, Leonardo Cruz Alves, Maria Clara Davis Silva, Andreas Lazaros Chryssafidis, Bárbara Gionco-Cano, Sueli Fumie Yamada-Ogatta and Galdino Andrade

Antibiotics 2024, 13(9), 875; https://doi.org/10.3390/antibiotics13090875 - 12 Sep 2024

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Abstract

Acinetobacter baumannii emerged as one of the most important pathogens for the development of new antimicrobials due to the worldwide detection of isolates resistant to all commercial antibiotics, especially in nosocomial infections. Biofilm formation enhances A. baumannii survival by impairing antimicrobial action, being an important target for new antimicrobials. Fluopsin C (FlpC) is an organocupric secondary metabolite with broad-spectrum antimicrobial activity. This study aimed to evaluate the antibiofilm activity of FlpC in established biofilms of extensively drug-resistant A. baumannii (XDRAb) and the effects of its combination with polymyxin B (PolB) on planktonic cells. XDRAb susceptibility profiles were determined by Vitek 2 Compact, disk diffusion, and broth microdilution. FlpC and PolB interaction was assessed using the microdilution checkerboard method and time–kill kinetics. Biofilms of XDRAb characterization and removal by FlpC exposure were assessed by biomass staining with crystal violet. Confocal Laser Scanning Microscopy was used to determine the temporal removal of the biofilms using DAPI, and cell viability using live/dead staining. The minimum inhibitory concentration (MIC) of FlpC on XDRAb was 3.5 µg mL⁻¹. Combining FlpC + PolB culminated in an additive effect, increasing bacterial susceptibility to both antibiotics. FlpC-treated 24 h biofilms reached a major biomass removal of 92.40 ± 3.38% (isolate 230) using 7.0 µg mL⁻¹ FlpC. Biomass removal occurred significantly over time through the dispersion of the extracellular matrix and decreasing cell number and viability. This is the first report of FlpC’s activity on XDRAb and the compound showed a promissory response on planktonic and sessile cells, making it a candidate for the development of a new antimicrobial product. Full article

(This article belongs to the Special Issue Antimicrobial and Antibiofilm Activity by Natural Compounds)

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(A) Dark-green, thin, prismatic crystals of Fluopsin C purified from Pseudomonas aeruginosa LV strain supernatant by two sequential flash chromatography processes. (B) Purified Fluopsin C chromatogram at 262 nm using a C18 (5 µm × 4.6 mm × 250 mm) column washed with a gradient system of acidified ultrapure water (1% v/v acetic acid) to acetonitrile. High-purity (90.45%) Fluopsin C molecule was detected at 14.3 min. Full article ">Figure 2
Screening for Fluopsin C susceptibility of A. baumannii clinical isolates and the reference strain ATCC 19606 by the disk diffusion method. The disks on the left were impregnated with 10 µg of Fluopsin C in DMSO, and the disks on the right were impregnated with 10 µL of DMSO. (A) ATCC 19606; (B) CI 223; (C) CI 224; (D) CI 226; (E) CI 227; (F) CI 230; (G) CI 232. Full article ">Figure 3
Time–kill kinetics of XDR A. baumannii CI 226 (A) and 230 (B) treated with MIC values to Fluopsin C (FlpC, 3.5 µg mL−1), polymyxin B (PolB, 226 = 8 µg mL−1 and 230 = 4 µg mL−1) and Fluopsin C/polymyxin B combination (FlpC + PolB, 1.75/1 µg mL−1). Full article ">Figure 4
Effect of different concentrations of Fluopsin C on 24 h established biofilms of XDR A. baumannii clinical isolates 226, 227, and 230 (moderate biofilm producers). Different letters indicate significant differences between biofilm removal, considering p < 0.05 as significant in the Tukey multiple comparison test. Full article ">Figure 5
Temporal effect of exposure of A. baumannii ATCC 19606 and XDR CI 230 24 h biofilms to Fluopsin C at MIC for planktonic cells (3.5 µg mL−1). Fluorescence microscopy tridimensional reconstruction of ATCC 19606 24 h biofilms treated for 12 h (A,B), 18 h (C,D), and for 24 h (E,F), and CI 230 24 h biofilms treated for 12 h (G,H), 18 h (I,J), and 24 h (K,L). Controls consisted of the addition of TSB-G without Fluopsin C (A,C,E for ATCC 19606 and G,I,K for CI 230). Treatments were based on the addition of TSB-G plus 3.5 µg mL−1 and incubation at 37 °C during different times (B,D,F for ATCC 19606 and H,J,L for CI 230). (M) Comparison of fluorescence intensity (gray-scale pixels) of ATCC 19606 and CI 230 24 h biofilms treated over 12, 18, or 24 h with 3.5 µg mL−1 FlpC. Asterisks represent significative differences between control and treatment by Sidak’s multiple-comparisons test where p ≤ 0.05 (*); p ≤ 0.001 (***). Full article ">Figure 6
Live/dead staining micrography of 24 h biofilms of XDR A. baumannii CI 230 treated with Fluopsin C. (A) Untreated biofilm controls; (B) treatment with 3.5 µg mL−1 Fluopsin C for 12 h. Full article ">

20 pages, 1336 KiB

Open AccessArticle

Diversification of Pseudomonas aeruginosa Biofilm Populations under Repeated Phage Exposures Decreases the Efficacy of the Treatment

by Mark Grevsen Martinet, Mara Lohde, Doaa Higazy, Christian Brandt, Mathias W. Pletz, Mathias Middelboe, Oliwia Makarewicz and Oana Ciofu

Microorganisms 2024, 12(9), 1880; https://doi.org/10.3390/microorganisms12091880 - 12 Sep 2024

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Abstract

Phage therapy has been proposed as a therapeutic alternative to antibiotics for the treatment of chronic, biofilm-related P. aeruginosa infections. To gain a deeper insight into the complex biofilm–phage interactions, we investigated in the present study the effect of three successive exposures to lytic phages of biofilms formed by the reference strains PAO1 and PA14 as well as of two sequential clinical P. aeruginosa isolates from the sputum of a patient with cystic fibrosis (CF). The Calgary device was employed as a biofilm model and the efficacy of phage treatment was evaluated by measurements of the biomass stained with crystal violet (CV) and of the cell density of the biofilm bacterial population (CFU/mL) after each of the three phage exposures. The genetic alterations of P. aeruginosa isolates from biofilms exposed to phages were investigated by whole-genome sequencing. We show here that the anti-biofilm efficacy of the phage treatment decreased rapidly with repeated applications of lytic phages on P. aeruginosa strains with different genetic backgrounds. Although we observed the maintenance of a small subpopulation of sensitive cells after repeated phage treatments, a fast recruitment of mechanisms involved in the persistence of biofilms to the phage attack occurred, mainly by mutations causing alterations of the phage receptors. However, mutations causing phage-tolerant phenotypes such as alginate-hyperproducing mutants were also observed. In conclusion, a decreased anti-biofilm effect occurred after repeated exposure to lytic phages of P. aeruginosa biofilms due to the recruitment of different resistance and tolerance mechanisms. Full article

(This article belongs to the Special Issue Biotechnological Applications of Bacteriophages and Enteric Viruses)

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Reduction in the biofilms of P. aeruginosa PAO1 and PA14 depends on NP3 and NP1 phage concentrations. (A) The viable cells were determined as CFU/mL in four independent experiments, and the reduction was calculated as a percentage of the viable bacteria after treatment in relation to the untreated controls; means and standard error of means (SEM) are shown. (B) The reduction in biomass was determined via crystal violet staining of the phage-treated biofilms and untreated biofilms (indicated here as the horizontal lines). The measurements were performed 8 times, and means and standard deviations (SD) are shown. Full article ">Figure 2
Effect of NP3 phage on PAO1 and PA14 biofilms after repeated treatments compared to the corresponding control biofilms. (A) PAO1 biomass is measured as CV absorbance. (B) Viable bacteria fraction of the resolved PAO1 biofilms as CFU/mL. (C) PA14 biomass measured as CV absorbance. (D) Viable bacteria fraction of the resolved PA14 biofilms as CFU/mL. In (A,C) mean and standard deviation (SD) and in (B,D) box blots with the 5–95%c percentile (whiskers), the 25–75% quadrille (box) with the median (line within the box) and mean (+ within the box) and outliers (grey dots) of four biological and four technical replicates are presented. Significance was assumed for p-values below or equal to 0.05, indicated as follows: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. Full article ">Figure 3
Effect of phages M32 on PAO1 and NP1 on PA14 biofilms after repeated treatments compared to the corresponding control biofilms. (A) PAO1 biomass is measured as CV absorbance. (B) Viable bacteria fraction of the resolved PAO1 biofilms as CFU/mL. (C) PA14 biomass measured as CV absorbance. (D) Viable bacteria fraction of the resolved PA14 biofilms as CFU/mL. In (A,C) mean and standard deviation (SD) and in (B,D) box blots with the 5–95%c percentile (whiskers), the 25–75% quadrille (box) with the median (line within the box) and mean (+ within the box) and outliers (grey dots) of four biological and four technical replicates are presented. Significance was assumed for p-values below or equal to 0.05, indicated as follows: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. Full article ">Figure 4
Effect of NP3 phage on biofilms of clinical isolates CF341_06 and CF341_08 after repeated treatments compared to the corresponding control biofilms. (A) CF341_06 biomass measured as CV absorbance. (B) Viable bacteria fraction of the resolved CF341_08 biofilms as CFU/mL. (C) CF341_08 biomass measured as CV absorbance. (D) Viable bacteria fraction of the resolved CF341_06 biofilms as CFU/mL. In (A,C) mean and standard deviation (SD) and in (B,D) box blots with the 5–95%c percentile (whiskers), the 25–75% quadrille (box) with the median (line within the box) and mean (+ within the box) and outliers (grey dots) of four biological and four technical replicates are presented. Significance was assumed for p-values below or equal to 0.05, indicated as follows: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. Full article ">Figure 5
Altered genes were identified in the clones of the different strains treated with the respective phages as indicated in each diagram in (A–E). The colors in (A–E) correspond to different pathways (see legend (F)). The number of clones with altered genes in the controls (untreated) and treated clones exhibiting phage-resistant and -sensitive or-tolerant phenotypes. The phage-resistant clones are marked in grey. WP_0031… is the coding side WP_003138482.1 corresponding to glycosyltransferase family 4 protein. Full article ">

13 pages, 2161 KiB

Open AccessArticle

The Accurate and Exclusive Quantification of Somatic Cells in Raw Milk with an OPD-Cu²⁺ System-Based Colorimetric Method

by Menghui Xie, Meng Wang, Siyuan Liu, Yingying Liu, Ziquan Wang, Guoping Zhou and Zhiwei Sui

Foods 2024, 13(18), 2890; https://doi.org/10.3390/foods13182890 - 12 Sep 2024

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Abstract

The somatic cell count (SCC) refers to the number of somatic cells present in each milliliter of raw milk and serves as a crucial indicator of dairy cow udder health and raw milk quality. Traditional SCC detection methods are often time-consuming, expensive, and susceptible to bacterial interference, rendering them unsuitable for the rapid and unbiased assessment of raw milk quality. Consequently, there is an urgent need for a low-cost, accurate, and user-friendly SCC quantification method. Here, a method based on an OPD-Cu²⁺ system for SCC quantification was developed. It was found that OPD oxidation signals exhibited a linear correlation with SCC. Following optimization, the detection system was established with a Cu²⁺ concentration of 25 μM, an OPD concentration of 2 mM, and an incubation time of 15 min. Furthermore, the method demonstrated significant resistance to bacterial interference, though it produced weaker signals in response to bacteria. The somatic cell recovery rate in milk after pretreatment was 88.9%, and SCC was quantified accurately within 45 min, with a linear range of 10⁴–10⁶ cells/mL. In summary, the method developed is cost-effective, straightforward, and facilitates precise somatic cell quantification, offering significant practical value and a new approach for SCC detection in raw milk. Full article

(This article belongs to the Topic Omics Technologies and Other Quality and Safety Methodologies in Nutrition and Health)

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Schematic diagram of somatic cell detection by the OPD-Cu2+ system-based colorimetric method. The somatic cells effectively consumed Cu2+, inhibiting the Cu2+-triggered oxidation of OPD to OPDox. Consequently, the OPDox signals significantly decreased. Full article ">Figure 2
Flow chart of the OPD-Cu2+ system-based colorimetric method for SCC quantification. Full article ">Figure 3
The somatic cells directly decreased the OPD-Cu2+ solution absorbance value. The absorbance values at 417 nm of OPD (a), OPD-Cu2+ (b), OPD–somatic cells (c), and OPD–Cu2+–somatic cells (d) were recorded. Insets: corresponding photos. Full article ">Figure 4
Optimization of the OPD-Cu2+ system detection conditions. (A) Optimization of Cu2+ concentration for somatic cell detection with the OPD-Cu2+ system. The optimal Cu2+ concentration was established by comparing the detection results of the somatic cell and bacterial samples. (B) Optimization of OPD concentration for somatic cell detection with the OPD-Cu2+ system. OPD was added to determine the concentration that produced the strongest signal. The optimized OPD concentration was subsequently validated with various concentrations of cell samples. Full article ">Figure 5
Standard curve for measuring SCC via the OPD-Cu2+ system. ΔA—the change in the absorbance signal between the blank control and the cell samples. A0—the absorbance signal of the blank control. Full article ">Figure 6
Quantification capability of the OPD-Cu2+ system-based colorimetric method in milk samples. (A) Relationship between the colorimetric detection and microscopic counting methods for the quantification of somatic cells in Tris-HCl. (B) Relationship between colorimetric detection and microscopic counting methods for the quantification of somatic cells in artificially contaminated milk samples. Full article ">

12 pages, 4259 KiB

Open AccessArticle

Streptococcus suis Induces Macrophage M1 Polarization and Pyroptosis

by Siqi Li, Tianfeng Chen, Kexin Gao, Yong-Bo Yang, Baojie Qi, Chunsheng Wang, Tongqing An, Xuehui Cai and Shujie Wang

Microorganisms 2024, 12(9), 1879; https://doi.org/10.3390/microorganisms12091879 - 12 Sep 2024

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Abstract

Streptococcus suis is an important bacterial pathogen that affects the global pig industry. The immunosuppressive nature of S. suis infection is recognized, and our previous research has confirmed thymus atrophy with a large number of necrotic cells. In this current work, we aimed to uncover the role of pyroptosis in cellular necrosis in thymic cells of S. suis-infected mice. Confocal microscopy revealed that S. suis activated the M1 phenotype and primed pyroptosis in the macrophages of atrophied thymus. Live cell imaging further confirmed that S. suis could induce porcine alveolar macrophage (PAM) pyroptosis in vitro, displaying cell swelling and forming large bubbles on the plasma membrane. Meanwhile, the levels of p-p38, p-extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) were increased, which indicated the mitogen-activated protein kinase (MAPK) and AKT pathways were also involved in the inflammation of S. suis-infected PAMs. Furthermore, RT-PCR revealed significant mRNA expression of pro-inflammatory mediators, including interleukin (IL)-1β, IL-6, IL-18, tumor necrosis factor (TNF)-α and chemokine CXCL8. The data indicated that the inflammation induced by S. suis was in parallel with pro-inflammatory activities of M1 macrophages, pyroptosis and MAPK and AKT pathways. Pyroptosis contributes to necrotic cells and thymocyte reduction in the atrophied thymus of mice. Full article

(This article belongs to the Special Issue The Pathogenic Epidemiology of Important Swine Diseases)

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Thymic macrophage polarization in S. suis infection. Using confocal laser scanning microscopy, appropriate FITC-conjugated antibodies were used to label cells in sections from thymus of infected mice; M1 macrophage marker IL-1β (green); M2 macrophage marker CD206 (green). Macrophages were stained with F4/80 antibody (red) and cell nuclei (blue) were stained with DAPI; BF: Bright-field. Scale bars, 5 μm. Full article ">Figure 2
S. suis induces pyroptosis in macrophages of the thymus of infected mice. Confocal laser scanning immunostaining was performed to visualize thymic macrophages (F4/80 antibody; purple) and pyroptosis (GSDMD-N; red) in atrophied and control thymus of mice, and nuclei were stained with DAPI (blue). BF: Bright-field. Scale bars, 5 μm. Full article ">Figure 3
S. suis induces pro-inflammatory mediators in thymus of infected mice. (A) Total tissue RNA was extracted at 12 hpi and 1, 2 and 4 dpi, and the levels of IL-6, (B) CXCL8, (C) IL-1β, (D) IL-18, (E) and TNF-α mRNA expression were analyzed using real-time RT-PCR. Experiments were repeated three times. Differences were analyzed using unpaired Student’s t-test; data are mean ± SD; *: p < 0.05; **: p < 0.01; ***: p < 0.001. Full article ">Figure 4
S. suis-induced LDH release and damage to primary PAMs. Primary PAMs were infected with S. suis 700794 at an MOI = 1 for 1, 2, 3, 8, 12 or 24 h, with a negative control included as 0 h. (A) LDH release induced by S. suis in primary PAMs was quantified by a standard LDH-release assay. (B) Electron micrographs of infected primary PAMs showing S. suis-induced cellular damage at 8 hpi, and yellow circle showed vacuoles of PAMs. Experiments were repeated three times, and results are expressed as means ± S.D., as determined by one-way ANOVA; *: p < 0.05; scale bars, 2 μm. Full article ">Figure 5
Live-cell imaging of pyroptosis after in vitro infection with S. suis. PAMs were cultured in glass-bottom cell culture dishes and treated with S. suis (MOI = 1). (A) Microscopy images of iPAMs treated with S. suis incubated in medium containing Hoechst 33342 (cell nuclei; blue) and propidium iodide (PI; red), the yellow arrows indicate pyrolyzed cells. (B) Microscopy images of primary PAMs treated with S. suis, and rectangle indicates pyrolyzed cells. Numbers at upper left indicate time post-infection (h:min:s). Full article ">Figure 6
The MAPK and AKT pathways were activated by S. suis in porcine macrophages. Cells were infected with S. suis at an MOI = 1 for 15, 30, 60 and 120 min. The cell lysates were collected, and levels of proteins related to inflammatory functional indexes were determined by Western blot (A). Representative Western blots show the expression level of (B) p-p38, (C) p38, (D) p-ERK1/2, (E) ERK1/2 and (F) AKT. The mean ± SD of experiments are shown; *: p < 0.05; **: p < 0.01. Full article ">

12 pages, 1131 KiB

Open AccessArticle

Characteristics, Management, and Outcomes of Acute Life-Threatening Asthma in Adult Intensive Care

by Adam J. R. Watson, Thomas Roe, Oliver Arscott, Charlotte Thomas, James Ward, Ryan Beecham, David Browning, Kordo Saeed and Ahilanandan Dushianthan

Clin. Pract. 2024, 14(5), 1886-1897; https://doi.org/10.3390/clinpract14050149 - 12 Sep 2024

Viewed by 241

Abstract

Background: There is limited evidence regarding the management of acute life-threatening asthma in intensive care units (ICUs), and few guidelines have details on this. We aimed to describe the characteristics, management, and outcomes of adults with life-threatening asthma requiring ICU admission. Methods: In this single-centre retrospective observational study, we included consecutive adults with acute asthma requiring ICU admission between 1 January 2016 and 31 December 2023. Our primary outcome was requirement for invasive mechanical ventilation (IMV). Results: We included 100 patients (median age 42.5 years, 67% female). The median pH, PaCO₂, and white cell count (WCC) on ICU admission were 7.37, 39 mmHg, and 13.6 × 109/L. There were 30 patients (30%) who required IMV, and the best predictors of IMV requirement were pH (AUC 0.772) and PaCO₂ (AUC 0.809). In univariate analysis, IMV requirement was associated with both increasing WCC (OR 1.14) and proven bacterial infection (OR 8.50). A variety of respiratory support strategies were utilised, with 38 patients (38%) receiving only non-invasive respiratory support. Conclusions: Our data highlight key characteristics which may be risk factors for acute asthma requiring ICU admission and suggest that pH, PaCO₂, and WCC are prognostic markers for disease severity. Our overall outcomes were good, with an IMV requirement of 30% and a 28-day mortality of 1%. Full article

(This article belongs to the Special Issue 2024 Feature Papers in Clinics and Practice)

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Figure 1

Figure 1
Flow diagram of eligible, included, and excluded patients. Full article ">Figure 2
Proportion of patients admitted to ICU over time according to maximum level of respiratory support. Full article ">Figure 3
AUROC analysis for pH, PaCO2, and WCC on ICU admission as predictors of IMV requirement. Red solid line represents random predictor. Full article ">

18 pages, 18776 KiB

Open AccessArticle

Optimization of Helicobacter pylori Biofilm Formation in In Vitro Conditions Mimicking Stomach

by Paweł Krzyżek, Paweł Migdał, Barbara Krzyżanowska and Anna Duda-Madej

Int. J. Mol. Sci. 2024, 25(18), 9839; https://doi.org/10.3390/ijms25189839 - 11 Sep 2024

Viewed by 269

Abstract

Helicobacter pylori is one of the most common bacterial pathogens worldwide and the main etiological agent of numerous gastric diseases. The frequency of multidrug resistance of H. pylori is growing and the leading factor related to this phenomenon is its ability to form biofilm. Therefore, the establishment of a proper model to study this structure is of critical need. In response to this, the aim of this original article is to validate conditions of the optimal biofilm development of H. pylori in monoculture and co-culture with a gastric cell line in media simulating human fluids. Using a set of culture-based and microscopic techniques, we proved that simulated transcellular fluid and simulated gastric fluid, when applied in appropriate concentrations, stimulate autoaggregation and biofilm formation of H. pylori. Additionally, using a co-culture system on semi-permeable membranes in media imitating the stomach environment, we were able to obtain a monolayer of a gastric cell line with H. pylori biofilm on its surface. We believe that the current model for H. pylori biofilm formation in monoculture and co-culture with gastric cells in media containing host-mimicking fluids will constitute a platform for the intensification of research on H. pylori biofilms in in vitro conditions that simulate the human body. Full article

(This article belongs to the Special Issue Pathogenicity and Antibiotic Resistance of Helicobacter pylori)

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Figure 1

Figure 1
Assessment of the impact of a one-week culture on the physiological parameters of H. pylori 2CML in BHI + 5% FCS and the concentration gradient of STF (0–10%, with 1% intervals). An increasing concentration of STF is marked by the intensified gray of bars. (A) Autoaggregation measured microscopically by estimating the degree of the observation field coverage, n = 3. (B) Biofilm formation determined by a crystal violet staining method and spectrophotometric measurements, n = 9. (C) Viability of bacterial cells determined by fluorescent staining with a LIVE/DEAD kit and a ratio of green/red fluorescence, n = 3. (D) Density of planktonic cells assessed by spectrophotometry, n = 9. (E) A set of images showing the growth of H. pylori 2CML under the most optimal conditions of the current model—BHI + 5% FCS + 5% STF (marked with red bars). Scale bars, 20 µm. * indicates statistical difference (p < 0.05) with the control—0%. Full article ">Figure 2
Assessment of the impact of a one-week culture on the physiological parameters of H. pylori 2CML in STF and the concentration gradient of FCS (0–10%, with 1% intervals). An increasing concentration of FCS is marked by the intensified gray of bars. (A) Autoaggregation measured microscopically by estimating the degree of the observation field coverage, n = 3. (B) Biofilm formation determined by a crystal violet staining method and spectrophotometric measurements, n = 9. (C) Viability of bacterial cells determined by fluorescent staining with a LIVE/DEAD kit and the ratio of green/red fluorescence, n = 3. (D) Density of planktonic cells assessed by spectrophotometry, n = 9. (E) A set of images showing growth of H. pylori 2CML under the most optimal conditions of the current model—STF + 8% FCS (marked with red bars). Scale bars, 20 µm. * indicates statistical difference (p < 0.05) with the control—0%. Because of the low values for all the physiological parameters during 1- and 2-day cultures, these data were not recorded. Full article ">Figure 3
Assessment of the impact of a one-week culture on the physiological parameters of H. pylori 2CML in BHI + 5% FCS and the concentration gradient of SGF (0–2%, with 0.2% intervals). An increasing concentration of SGF is marked by the intensified gray of bars. (A) Autoaggregation measured microscopically by estimating the degree of the observation field coverage, n = 3. (B) Biofilm formation determined by a crystal violet staining method and spectrophotometric measurements, n = 9. (C) Viability of bacterial cells determined by fluorescent staining with a LIVE/DEAD kit and a ratio of green/red fluorescence, n = 3. (D) Density of planktonic cells assessed by spectrophotometry, n = 9. (E) A set of images showing the growth of H. pylori 2CML under the most optimal conditions of the current model—BHI + 5% FCS + 1% SGF (marked with red bars). Scale bars, 20 µm. * indicates statistical difference (p < 0.05) with the control—0%. Full article ">Figure 4
Assessment of the impact of a one-week culture on the physiological parameters of H. pylori 2CML in STF and the concentration gradient of SGF (0–2%, with 0.2% intervals). An increasing concentration of SGF is marked by the intensified gray of bars. (A) Autoaggregation measured microscopically by estimating the degree of the observation field coverage, n = 3. (B) Biofilm formation determined by a crystal violet staining method and spectrophotometric measurements, n = 9. (C) Viability of bacterial cells determined by fluorescent staining with a LIVE/DEAD kit and a ratio of green/red fluorescence, n = 3. (D) Density of planktonic cells assessed by spectrophotometry, n = 9. (E) A set of images showing the growth of H. pylori 2CML under the most optimal conditions of the current model—STF + 1% SGF (marked with red bars). Scale bars, 20 µm. * indicates statistical difference (p < 0.05) with the control—0%. Because of the low values for all the physiological parameters during 1- and 2-day cultures, these data were not recorded. Full article ">Figure 5
Representative images showing a co-culture between H. pylori 2CML and KATO III cells growing on semi-permeable membranes in host-mimicking fluids. (A) A set of images obtained by fluorescence microscopy with different components of the co-culture model being stained (FM 1–43 to visualize bacterial biomass (biofilm), DAPI to show cell nuclei of eukaryotic cells, and WGA-conjugated Texas Red to indicate the presence of mucus glycoproteins (mucins)). Scale bars, 20 µm. (B) A set of images obtained by scanning electron microscopy. On the top, a control constituting non-infected KATO III cells; on the bottom, a co-infection of KATO III with H. pylori 2CML, where red arrows indicate areas of biofilm development. Scale bars, 20 µm. Full article ">Figure 6
Representative images showing a co-culture between H. pylori 2CML and KATO III cells growing in microfluidic conditions in host-mimicking fluids. (A) Time-dependent development of H. pylori biofilm attached to KATO III cells. (B) An image showing a one-day co-culture with a marking of specific components of this model, where red circles indicate KATO III cells and yellow dashed lines highlight areas of the development of H. pylori biofilm, being in close contact with these cells. Scale bars, 20 µm. To see stacked time-lapse sequences from the above experiments, please see <a href="#app1-ijms-25-09839" class="html-app">Videos S1 and S2</a>. Full article ">

17 pages, 3341 KiB

Open AccessArticle

In Vitro Evaluation of Antipseudomonal Activity and Safety Profile of Peptidomimetic Furin Inhibitors

by Sara Maluck, Rivka Bobrovsky, Miklós Poór, Roman W. Lange, Torsten Steinmetzer, Ákos Jerzsele, András Adorján, Dávid Bajusz, Anita Rácz and Erzsébet Pászti-Gere

Biomedicines 2024, 12(9), 2075; https://doi.org/10.3390/biomedicines12092075 - 11 Sep 2024

Viewed by 398

Abstract

Inhibitors of the serine protease furin have been widely studied as antimicrobial agents due to their ability to block the cleavage and activation of certain viral surface proteins and bacterial toxins. In this study, the antipseudomonal effects and safety profiles of the furin inhibitors MI-1851 and MI-2415 were assessed. Fluorescence quenching studies suggested no relevant binding of the compounds to human serum albumin and α₁-acid glycoprotein. Both inhibitors demonstrated significant antipseudomonal activity in Madin–Darby canine kidney cells, especially compound MI-1851 at very low concentrations (0.5 µM). Using non-tumorigenic porcine IPEC-J2 cells, neither of the two furin inhibitors induced cytotoxicity (CCK-8 assay) or altered significantly the intracellular (Amplex Red assay) or extracellular (DCFH-DA assay) redox status even at a concentration of 100 µM. The same assays with MI-2415 conducted on primary human hepatocytes also resulted in no changes in cell viability and oxidative stress at up to 100 µM. Microsomal and hepatocyte-based CYP3A4 activity assays showed that both inhibitors exhibited a concentration-dependent inhibition of the isoenzyme at high concentrations. In conclusion, this study indicates a good safety profile of the furin inhibitors MI-1851 and MI-2415, suggesting their applicability as antimicrobials for further in vivo investigations, despite some inhibitory effects on CYP3A4. Full article

(This article belongs to the Special Issue Drug Discovery for Infectious Diseases—Second Edition)

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Graphical abstract

14 pages, 2917 KiB

Open AccessArticle

Enrichment of Cis-Acting Regulatory Elements in Differentially Methylated Regions Following Lipopolysaccharide Treatment of Bovine Endometrial Epithelial Cells

by Naveed Jhamat, Yongzhi Guo, Jilong Han, Patrice Humblot, Erik Bongcam-Rudloff, Göran Andersson and Adnan Niazi

Int. J. Mol. Sci. 2024, 25(18), 9832; https://doi.org/10.3390/ijms25189832 - 11 Sep 2024

Viewed by 346

Abstract

Endometritis is an inflammatory disease that negatively influences fertility and is common in milk-producing cows. An in vitro model for bovine endometrial inflammation was used to identify enrichment of cis-acting regulatory elements in differentially methylated regions (DMRs) in the genome of in vitro-cultured primary bovine endometrial epithelial cells (bEECs) before and after treatment with lipopolysaccharide (LPS) from E. coli, a key player in the development of endometritis. The enriched regulatory elements contain binding sites for transcription factors with established roles in inflammation and hypoxia including NFKB and Hif-1α. We further showed co-localization of certain enriched cis-acting regulatory motifs including ARNT, Hif-1α, and NRF1. Our results show an intriguing interplay between increased mRNA levels in LPS-treated bEECs of the mRNAs encoding the key transcription factors such as AHR, EGR2, and STAT1, whose binding sites were enriched in the DMRs. Our results demonstrate an extraordinary cis-regulatory complexity in these DMRs having binding sites for both inflammatory and hypoxia-dependent transcription factors. Obtained data using this in vitro model for bacterial-induced endometrial inflammation have provided valuable information regarding key transcription factors relevant for clinical endometritis in both cattle and humans. Full article

(This article belongs to the Section Biochemistry)

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Motif enrichment in significant differentially methylated regions (DMRs). Enriched motifs found using MEME software v5.3.0 in hyper (left) and hypo (right) DMR sequences. Only the most significant motifs for known transcription factors with p-value < 0.01 are shown. Full article ">Figure 2
Enrichment of transcription factor binding sites in DMRs and their effect on gene expression. (A) Heatmap showing binding motifs of transcription factors enriched in hyper and hypo differentially methylated regions (DMRs). (B) Sequence logos depicting the consensus sequences of the binding-site regions in DMRs that contain CpG sites. (C) Heatmap showing motifs enriched in the promoter regions of significant differentially expressed genes in RNAseq data. (D) Hyper and hypo DMRs sharing significantly enriched transcription factor motifs. (E) Co-localization of flanking motifs in the DMRs with motifs enriched in both hyper and hypo as shown in heatmap (A). Full article ">Figure 3
Effects of methylation changes on the activity of transcription factors. (A) MA-plot showing expression and fold change differences of genes between control and LPS samples. (B) Genome-wide methylation levels of binding sites for transcription factors with |log2 Fold change| > 0.5 as depicted in the MA-plot. Median values are represented by horizontal lines in each box. Asterisk represents significant difference between the distribution of methylation levels in control and LPS groups (Wilcoxon signed-rank test, p < 0.001). (C) Relative FPKM expression of transcription factors in control and LPS samples. Relative expression of genes shown was calculated using SUZ12 endogenous gene from the bEECs (p < 0.05). Analysis with TBP and ACTB presented similar expression differences between control and LPS groups. Full article ">

13 pages, 6936 KiB

Open AccessArticle

In Vitro Antimicrobial Synergistic Activity and the Mechanism of the Combination of Naringenin and Amikacin Against Antibiotic-Resistant Escherichia coli

by Lankun Yi, Mingze Cao, Xu Chen, Yubin Bai, Weiwei Wang, Xiaojuan Wei, Yuxiang Shi, Yongying Zhang, Tenghe Ma, Zhen Zhu and Jiyu Zhang

Microorganisms 2024, 12(9), 1871; https://doi.org/10.3390/microorganisms12091871 - 11 Sep 2024

Viewed by 306

Abstract

Bacterial drug resistance is becoming an increasingly serious problem, and the development of antibacterial synergists is urgently needed. Combining existing antibiotics with promising nonantibiotic agents is one strategy that has been shown to be effective at overcoming the widespread emergence of antibiotic-resistant pathogens. In this study, we investigated the antibacterial activities and mechanism of naringenin (NG) combined with amikacin (AMK) against multidrug-resistant Escherichia coli (E. coli). We first measured the fractional inhibitory concentration (FIC) of NG combined with antibiotics via the checkerboard method. The results indicated that the combination of NG and AMK had a synergistic effect on E. coli ATCC 25922 and E. coli C7F3. In addition, this synergistic effect was verified by time-kill assays. Moreover, scanning electron microscopy (SEM) was used to observe cell morphology. The results showed that the cell wall of E. coli was destroyed. Furthermore, we assessed the leakage of alkaline phosphatase (AKP), K⁺, and protein. The extracellular AKP activity increased after the combinational group of 1/2MIC NG and 1/2MIC AMK, suggesting an impairment in cell wall permeability. An increase in the leakage of intracellular K⁺ and protein indicated an increase in cell inner membrane permeability. These results revealed that NG and AMK inhibited E. coli by damaging cell walls and membranes. In addition, PI uptake rapidly increased after treatment with NG and AMK. Confocal laser scanning microscopy (CLSM) revealed that NG caused cell wall and cell membrane damage in E. coli. In summary, our results provide a new strategy for responding to the development of E. coli drug resistance. Full article

(This article belongs to the Special Issue Potential Antimicrobial Synergistic Interactions of Natural Products)

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Figure 1
Time-kill curves of 1/2MIC NG and 1/2MIC AMK alone and in combination against E. coli ATCC 25922 (A) and E. coli C7F3 (B). Each value is presented as the mean ± SD (n = 3). Full article ">Figure 2
Effects of 1/2MIC NG and 1/2MIC AMK alone or in combination on the morphology of E. coli ATCC 25922 (A–D) and E. coli C7F3 (E–H) cells. (A,E): control; (B,F): 1/2MIC AMK; (C,G): 1/2MIC NG; and (D,H): 1/2MIC NG + 1/2MIC AMK. Magnification = 20,000×. Ten visual fields were observed for each replicate and representative images were selected. Full article ">Figure 3
Effects of 1/2MIC NG and 1/2MIC AMK alone or in combination on AKP leakage of E. coli ATCC 25922 (A) and E. coli C7F3 (B). Each value is presented as the mean ± SD (n = 3). ns p-value > 0.05, * p-value < 0.05, and **** p-value < 0.0001. Full article ">Figure 4
Effects of 1/2MIC NG and 1/2MIC AMK alone or in combination on protein leakage of E. coli ATCC 25922 (A) and E. coli C7F3 (B). Each value is presented as the mean ± SD (n = 3). * p-value < 0.05, ** p-value < 0.01, and **** p-value < 0.0001. Full article ">Figure 5
Effects of 1/2MIC NG and 1/2MIC AMK alone or in combination on K+ leakage of E. coli ATCC 25922 (A) and E. coli C7F3 (B). Each value is presented as the mean ± SD (n = 3). ns p-value > 0.05, ** p-value < 0.01, and **** p-value < 0.0001. Full article ">Figure 6
CLSM images of E. coli ATCC 25922 and E. coli C7F3 treated with 1/2MIC NG and 1/2MIC AMK alone or in combination. (A,E): control; (B,F): 1/2MIC AMK; (C,G): 1/2MIC NG; and (D,H): 1/2MIC NG + 1/2MIC AMK. Scale Bar: 50 µm. Full article ">Figure 7
A model of NG and AMK combination therapy on E. coli cells. When AMK is used alone, the cell wall and cell membrane of drug-resistant bacteria are intact, making it difficult for AMK to enter the bacteria to exert its antibacterial effect. However, when NG is used in combination with AMK, NG will destroy the integrity of the E. coli cell wall and cell membrane, allowing AMK to smoothly enter the interior of the bacteria in large quantities, inhibit ribosomes from synthesizing proteins, and quickly kill drug-resistant E. coli. Full article ">

19 pages, 4491 KiB

Open AccessArticle

Myrtus communis L. Essential Oil Exhibits Antiviral Activity against Coronaviruses

by Dar-Yin Li, Matthew G. Donadu, Taylor Shue, Georgios Dangas, Antonis Athanasiadis, Shuiyun Lan, Xin Wen, Basem Battah, Stefania Zanetti, Vittorio Mazzarello, Stefan G. Sarafianos, Marco Ferrari and Eleftherios Michailidis

Pharmaceuticals 2024, 17(9), 1189; https://doi.org/10.3390/ph17091189 - 10 Sep 2024

Viewed by 390

Abstract

Human coronaviruses are a continuous threat to the human population and have limited antiviral treatments, and the recent COVID-19 pandemic sparked interest in finding new antiviral strategies, such as natural products, to combat emerging coronaviruses. Rapid efforts in the scientific community to identify effective antiviral agents for coronaviruses remain a focus to minimize mortalities and global setbacks. In this study, an essential oil derived from Myrtus communis L. (MEO) is effective against HCoV-229E and HCoV-OC43 virus infections in comparison to two FDA-approved drugs, Remdesivir and Nirmatrelvir. Gas-chromatography and mass spectrometry were used to identify the chemical composition of MEO. Slight antioxidant activity was observed in MEO, indicating a role in oxidative stress. A dose–response curve measuring the EC₅₀ indicates a high potency against HCoV-229E and HCoV-OC43 virus infections on Huh7.5 cells with low cytotoxicity using a PrestoBlue cell viability assay. Our findings demonstrate that MEO exhibits potent antiviral activity against HCoV-229E and HCoV-OC43 on Huh7.5 cells within a low-cytotoxicity range, but not on SARS-CoV-2. Artificial bacterial chromosome plasmids that expressed SARS-CoV-2 used for replicon—to determine viral replication and viral assembly/egress on HEK293T/17 cells—and virus-like particles on Huh7.5-AT cells—to determine viral entry and assembly/egress—showed no antiviral activity with MEO in comparison to Remdesivir. This study reveals the potential effectiveness of MEO as an alternative natural remedy to treat human coronaviruses and a potential antiviral agent for future coronavirus infections. Full article

(This article belongs to the Special Issue The 20th Anniversary of Pharmaceuticals—Advances in Natural Products)

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Figure 1

Figure 1
MEO inhibits HCoV-229E infection. (A) Experimental design of MEO experiment for dose–response curve. Huh7.5 cells were seeded in two 96-well plates with Remdesivir and Nirmatrelvir as antiviral controls. The experiment was performed in triplicate, and the starting concentration for MEO was 1:1000 (0.8469 mg/mL) and received a 1:2 serial dilution. The starting concentrations for Remdesivir and Nirmatrelvir were 250 nM and 25 µM, respectively, and were diluted with a 1:2 serial dilution. After HCoV-229E was added to the plates an hour after drug treatment, cells were fixed with 4% PFA one day post-infection. Immunofluorescent staining (IF) was performed to visualize infected cells using Cytation 7. (B) Dose–response curve showing that MEO has antiviral activity against HCoV-229E with EC50 = 0.1204 mg/mL MEO concentration starting at 1:1000 (0.8469 mg/mL) with a 1:2 serial dilution and infected with 1:10 HCoV-229E virus. (C) Cytotoxicity assay measuring cell viability in MEO-treated Huh7.5 cells. A 1:10 (84.69 mg/mL) starting concentration for MEO was used with a 1:2 serial dilution. Huh7.5 cells treated with MEO were normalized to the untreated Huh7.5 cells. Full article ">Figure 2
Immunofluorescent staining for HCoV-229E Spike protein. Huh7.5 cells without MEO treatment showed a mean fluorescence intensity of 22 with HCoV-229E. At a 1:2000 (0.42345 mg/mL) Myrtus concentration, the HCoV-229E viral infection has a mean fluorescence intensity of 7. Nuclei were stained with Hoechst, and HCoV-229E was stained with HCoV-229E spike protein and Alexa Fluor 488, labeled goat anti-mouse secondary antibody, and was imaged at a 1000 µm scale. Full article ">Figure 3
MEO inhibits HCoV-OC43 infection. (A) Experimental layout of MEO with HCoV-OC43 infection to determine EC50. Two collagen-coated 96-wells plates were seeded with Huh7.5 cells to evaluate the dose–response curve of Remdesivir, Nirmatrelvir, and MEO. Each drug was conducted in triplicates with the starting concentration for MEO to be 1:1000 (0.8469 mg/mL) and serially diluted 1:2. The starting concentrations for Remdesivir and Nirmatrelvir were 7 µM and 25 µM, respectively, and were diluted with a 1:2 serial dilution. An hour after drug treatment, HCoV-OC43 was added, and cells were fixed with 4% PFA at 3 days post-infection. Immunofluorescent staining (IF) was performed to visualize infected cells using the Cytation 7. (B) A dose–response curve showed that MEO has antiviral activity against HCoV-OC43 with EC50 = 1.405 mg/mL MEO concentration starting at a concentration of 1:1000 (0.8469 mg/mL) with a 1:2 serial dilution and infected with 1:20 HCoV-OC43E virus. Full article ">Figure 4
Immunofluorescent staining for HCoV-OC43 spike protein. Huh7.5 cells without MEO treatment showed a mean fluorescence intensity of 12 with HCoV-OC43. At a 1:1000 (0.8469 mg/mL) MEO concentration, HCoV-OC43 viral infection has a mean fluorescence intensity of 4. Nuclei were stained with Hoechst and HCoV-OC43 was stained with anti-coronavirus antibody, OC-43 strain, clone 541-8F, and Alexa Fluor 488, labeled goat anti-mouse secondary antibody, and was imaged at a 1000 µm scale. Full article ">Figure 5
MEO does not inhibit SARS-CoV-2 viral replication based on SARS-CoV-2 replicon. (A) Normalized SARS-CoV-2 replicon transfection to determine if MEO inhibits viral replication. Using GFP reporter in the SARS-CoV-2 replicon to calculate the percent of transfected cells, there was no difference in the number of transfected cells compared to normalized and untreated wells. NLuc activity from the SARS-CoV-2 replicon plasmid was used to quantify the amount of viral replication under MEO-treated conditions. Titration of MEO with SARS-CoV-2 replicon transfected cells showed no difference in viral replication via NLuc activity. Cell viability is not affected by SARS-CoV-2 replicon, nor by MEO cytotoxicity at the tested MEO concentrations. The relative luminescence graph validates that viral replicon is not inhibited by MEO. (B) Remdesivir substantially inhibited SARS-CoV-2 viral replication and normalized transfection level similar to the relative luminescence units, as untreated wells received about an eight-fold increase compared to 7.5 µM. At 15 µM of Remdesivir, cell viability dropped below 50%, exhibiting Remdesivir cytotoxicity. Full article ">Figure 6
No inhibition against SARS-CoV-2 VLPs for MEO. (A) Immunofluorescence images showed no difference in VLP inhibition for untreated and MEO-treated wells at 1:200 (4.2345 mg/mL) concentration. Remdesivir inhibited SARS-CoV-2 VLP in a dose-dependent manner, which acted as positive control. To determine the percentage of cells infected with SARS-CoV-2 VLPs, the cells were counterstained with Hoechst and imaged for GFP reporter signal from the replicon plasmids and Hoechst dye using Cytation 7. All images are in 1000 µm scale. (B) The VLP transduction assay with MEO treatment showed similar infection levels as untreated wells. At 1:100 (8.469 mg/mL) concentration of MEO, cells were not viable, and viral replication remained consistent. (C) Remdesivir showed potent inhibition against SARS-CoV-2 VLP as an inhibitor of SARS-CoV-2 viral replication. Full article ">Figure 7
MEO treatment has little impact SARS-CoV-2 Omicron BA.1 VLP formation/release. SARS-CoV-2 BA.1 VLPs were produced in HEK293T/17 cells that were pre-treated with either a 1:1000 (0.8469 mg/mL) or 1:500 (1.6938 mg/mL) concentration of MEO in DMEM (10% FBS + 1% NEAA) to assess the effect of MEO on the formation of infectious VLPs. VLPs were harvested via centrifugation and concentrated 20× using 100,000 MW Amicon filter units. VLPs were titrated on Huh7.5-AT cells at a starting dilution of 1:5 and continued with a 1:2 dilution. At one day post-transduction, the cells were counterstained with 1:5000 Hoechst dye and imaged using Cytation 7 for the number of GFP+ and total cells. Then, the cell culture supernatant was measured for NLuc activity. (A) Hoechst staining shows that there was a slight decrease in cell viability for VLPs formed in the presence of MEO treatment in a dose-dependent manner (p < 0.0001). (B) Analysis of the GFP+ cells representing cells successfully transduced with VLP and undergoing replication of the replicon plasmid showed minor differences between MEO-treated and untreated VLPs, but only at the highest dilutions of VLP delivery (p = 0.01). (C) NLuc activity shows no difference between MEO VLPs and untreated VLPs. Overall, MEO treatment during VLP production has no effect on nascent VLP particles. Full article ">

15 pages, 1706 KiB

Open AccessArticle

The Effect of the Lysine Acetylation Modification of ClpP on the Virulence of Vibrio alginolyticus

by Shi Wang, Yingying Jiang, Weijie Zhang, Yingzhu Wei, Xing Xiao, Zhiqing Wei, Xiaoxin Wen, Yuhang Dong, Jichang Jian, Na Wang and Huanying Pang

Molecules 2024, 29(17), 4278; https://doi.org/10.3390/molecules29174278 - 9 Sep 2024

Viewed by 273

Abstract

Acetylation modification has become one of the most popular topics in protein post-translational modification (PTM) research and plays an important role in bacterial virulence. A previous study indicated that the virulence-associated caseinolytic protease proteolytic subunit (ClpP) is acetylated at the K165 site in Vibrio alginolyticus strain HY9901, but its regulation regarding the virulence of V. alginolyticus is still unknown. We further confirmed that ClpP undergoes lysine acetylation (Kace) modification by immunoprecipitation and Western blot analysis and constructed the complementation strain (C-clpP) and site-directed mutagenesis strains including K165Q and K165R. The K165R strain significantly increased biofilm formation at 36 h of incubation, and K165Q significantly decreased biofilm formation at 24 h of incubation. However, the acetylation modification of ClpP did not affect the extracellular protease (ECPase) activity. In addition, we found that the virulence of K165Q was significantly reduced in zebrafish by in vivo injection. To further study the effect of lysine acetylation on the pathogenicity of V. alginolyticus, GS cells were infected with four strains, namely HY9901, C-clpP, K165Q and K165R. This indicated that the effect of the K165Q strain on cytotoxicity was significantly reduced compared with the wild-type strain, while K165R showed similar levels to the wild-type strain. In summary, the results of this study indicate that the Kace of ClpP is involved in the regulation of the virulence of V. alginolyticus. Full article

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Figure 1

Figure 1
(A) Multiple sequence alignment of ClpP from different Vibrio species. Vibrio alginolyticus ClpP is highlighted in red. The degree of consistency of ClpP with other sequences is indicated by the values in the red box. (B) The 3D visualized protein model of ClpP at the K165 site in V. alginolyticus HY9901. The K165 active residue is indicated in red. (C) The 3D visualized protein model of the acetylation of ClpP at the K165 site. The predicted acetylated residue is represented in red. (D) The 3D visualized protein model of the deacetylation of ClpP at the K165 site. The predicted deacetylated residue is highlighted in red. Full article ">Figure 2
(A) SDS-PAGE of ClpP and Western blot of anti-ClpP. The uninduced whole bacterial protein of ClpP is shown in lane 1, the IPTG-induced whole bacterial protein of ClpP is shown in lane 2, the uninduced supernatant protein after the crushing of ClpP is shown in lane 3, the IPTG-induced supernatant protein after the crushing of ClpP is shown in lane 4, the purified supernatant protein of ClpP is shown in lanes 5 and 6 and the Western blot result of the anti-ClpP antibody is shown in lane 7. (B) The lysine acetylation modification of the ClpP natural protein and recombinant protein was identified by immunoprecipitation and Western blotting. The ClpP natural protein was enriched by IP with a specific antibody (anti-ClpP), followed by Western blot with the ClpP protein-specific antibody (in lane 1) and Western blot with anti-acetylation mouse mAb (in lane 2). Full article ">Figure 3
(A) Effect of lysine acetylation on the growth of V. alginolyticus. (B) Effects of C-clpP, K165Q and K165R on the biofilm formation of V. alginolyticus at different time points. The average SD was obtained from three independent experiments. ** p < 0.01, significantly different compared with the WT strain. Full article ">Figure 4
(A) The histogram of the effect of the lysine acetylation of the ClpP protein on the ECPase activity of V. alginolyticus. All values are mean ± SD, n = 3. (B) Effect of lysine acetylation on LDH activity in GS cells. (C) Effect of lysine acetylation on NO release in GS cells. (D) Effect of lysine acetylation on GSH content in GS cells. The average SD was obtained from three independent experiments. ** p < 0.01, significantly different compared with the WT strain. Full article ">

16 pages, 5087 KiB

Open AccessArticle

Persistence and Culturability of Escherichia coli under Induced Toxin Expression

by Yousr Dhaouadi, Mohamad Javad Hashemi and Dacheng Ren

Antibiotics 2024, 13(9), 863; https://doi.org/10.3390/antibiotics13090863 - 9 Sep 2024

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Abstract

Background/Objectives: Bacteria are well known to enter dormancy under stress conditions. However, the mechanisms of different dormancy-related phenotypes are still under debate and many questions remain unanswered. This study aims to better understand the effects of toxin gene expression on the dormancy of Escherichia coli. Methods: The effects of toxin gene expression on growth, persistence, and culturability were characterized. Specifically, we detailed dose- and time-dependent dormancy of E. coli and its susceptibility to ofloxacin via arabinose-induced hipA toxin gene expression under the P_BAD promoter. A new plot was developed to better describe the dynamic changes in culturability and persistence. The expression level of hipA was determined using qPCR and cellular activities were monitored using fluorescence imaging and flow cytometry. Results: High-level persister formation and strong tolerance to ofloxacin were observed after high-level hipA induction. The new plot reveals more information than the changes in persistence alone, e.g., reduced culturability of E. coli and thus deeper dormancy under high-level hipA induction. Consistently, controlled hipA induction led to decreased cellular activities at promoter P_rrnBP1 and an increase in the non-culturable subpopulation. Conclusions: Overall, this study provides new insights into dormancy induced by toxin gene expression and a more comprehensive view of persistence and culturability. The findings may help develop better control agents against dormant bacterial cells. Full article

(This article belongs to the Section Genetic and Biochemical Studies of Antibiotic Activity and Resistance)

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