Bra forskningsrapport från Polen

PHI-grävaren OT har mejlat över en sprillans ny forskningsrapport från Polen.Studien är utförd av 8 polska forskare med för oss phi,are kända Beata Cieślak i spetsen.Den är publicerad i en specialutgåva där enbart Beatas och 2 andra forskningsrapporter platsar.

Special Issue "Microbial Biofilms and Antibiofilm Agents 2.0"

Studien handlar om antibiotikaresistens där bakterier växt sig så motståndskraftiga att vissa läkemedel tappat sin effektivitet att bota.Forskarna testade då ett nytt sätt att ta sig an detta problem. Beskrivet i abstractets inledning : The constantly growing resistance of bacteria to antibiotics and other antibacterial substances has led us to an era in which alternative antimicrobial therapies are urgently required. 

One promising approach is to target bacterial pathogens using metal complexes.

Man laborerade med olika metallföreningar som skydd eller bärare? av de läkande substanserna.

Ett för mig nytt sätt att lösa problematiken.Eftersom huvudämnet i studien är cytoxicitet,dvs läkemedlets förmåga att angripa och förgöra ex cancerceller har forskarna haft stor användning av PHI`s HoloMonitorsystem.

Man anger redan i abstractet hur viktigt dess användande var. "highlighting".

Men till studien då :

Tuning Anti-Biofilm Activity of Manganese(II) Complexes: Linking Biological Effectiveness of Heteroaromatic Complexes of Alcohol, Aldehyde, Ketone, and Carboxylic Acid with Structural Effects and Redox Activity

Published: 3 May 2021

 

Abstract

The constantly growing resistance of bacteria to antibiotics and other antibacterial substances has led us to an era in which alternative antimicrobial therapies are urgently required. One promising approach is to target bacterial pathogens using metal complexes. Therefore, we investigated the possibility of utilizing series of manganese(II) complexes with heteroaromatic ligands: Alcohol, aldehyde, ketone, and carboxylic acid as inhibitors for biofilm formation of Pseudomonas aeruginosa. To complete the series mentioned above, Mn-dipyCO-NO₃ with dipyridin-2-ylmethanone (dipyCO) was isolated, and then structurally (single-crystal X-ray analysis) and physicochemically characterized (FT-IR, TG, CV, magnetic susceptibility). The antibacterial activity of the compounds against representative Gram-negative and Gram-positive bacteria was also evaluated. It is worth highlighting that the results of the cytotoxicity assays performed (MTT, DHI HoloMonitorM4) indicate high cell viability of the human fibroblast (VH10) in the presence of the Mn(II) complexes. Additionally, the inhibition effect of catalase activity by the complexes was studied. This paper focused on such aspects as studying different types of intermolecular interactions in the crystals of the Mn(II) complexes as well as their possible effect on anti-biofilm activity, the structure–activity relationship of the Mn(II) complexes, and regularity between the electrochemical properties of the Mn(II) complexes and anti-biofilm activity.

Introduction

The problem of global antibiotic resistance among bacteria is growing to an unimaginable extent in the 21st century. Microbial resistance is a consequence of not only the massive use of antibiotics, including their often unjustified or excessive use, but also of the ability of pathogens to adapt to different conditions and develop self-defense mechanisms such as living in biofilms. 

Biofilms include bacterial microcolonies adhering to the surface and surrounded by a viscous extracellular matrix. When attached, bacteria reproduce and anchor in fairly complex structures that appear to enable communication and transfer of nutrients, waste, and signaling compounds (quorum sensing). Bacterial biofilms are extremely difficult to eliminate with conventional antibiotics and therefore pose a huge burden to healthcare. Pseudomonas aeruginosa is a particularly dangerous microorganism—an opportunistic pathogen that shows great ease in biofilm formation. 

This bacterium has become extremely dangerous in the hospital environment over the past few decades. P. aeruginosa is responsible for causing various infections, often of a complicated course, in people with weakened immunity, e.g., cystic fibrosis, oncological, or transplant patients. Hence, there is an urgent need to look for new strategies to develop new compounds to combat multi-drug-resistant and infection biofilms. Researchers are using various approaches to identify new antibiotics or new compounds with antimicrobial activity. Various inhibitors of different pathways, antimicrobial peptides, as well as organic and inorganic synthetic compounds with antibacterial activity, have been identified and studied. After the success of transition metal coordination compounds as antibacterial and anticancer agents, the use of this group of compounds brings enormous potential in medicine, biotechnology, and pharmacy applications.

Herein, we presented the biological efficacy of a series of manganese(II) complexes in the inhibition of bacterial biofilm formation. In this regard, our studies focused on the following aspects: (i) Evaluation of antibacterial and anti-biofilm activities; (ii) cytotoxicity assays for a series of Mn(II) complexes using the MTT test and digital holographic imaging (DHI) platform HoloMonitorM4 to evaluate the effects of the complexes examined on cell viability in the primary human fibroblast (VH10) as a model; (iii) inhibition effect on CAT activity by a series of Mn(II) complexes; (iv) structure–activity relationship of a series of Mn(II) complexes supported by heteroaromatic alcohol, aldehyde, ketone, and acid; (v) regularity between electrochemical properties of Mn(II) complexes and anti-biofilm activity.

2.6. Cytotoxicity Activity

The cytotoxicity test of the manganese complexes against primary human fibroblasts (VH10) was performed with the MTT test. The studies have shown that the manganese complexes are not cytotoxic to primary human fibroblasts (IC₅₀ higher or equal 1000 µM) .

Digital holographic imaging (DHI) is an innovative and useful screening tool for metabolic activity investigation of human fibroblasts in the presence of metal complexes. DHI uses phase-shift imaging combined with computer algorithms to construct holographic images and enable the simultaneous study of multiple cellular parameters such as cell count, confluence, optical thickness, cell optical volume, and cell diameter. The DHI platform HoloMonitorM4 allowed us to evaluate the effects of the Mn(II) complexes examined on cell viability of the human fibroblast as a model. 

In our experiment, the parameters such as a cell count and confluence were taken into account.

The analysis of the data indicates that the VH10 cell count did not change over time generally (20 h exposition) for all of the tested complexes. Nevertheless, a temporary drop in the cell count over time may have been caused by the migration of cells outside the field of view (statistically a balance is maintained). A similar effect was observed with regard to the confluence parameter over time. This parameter does not change statistically. The DHI analysis suggests that the compounds do not exhibit cytotoxicity against the human fibroblasts at the tested concentrations. The results obtained correlate well with the study gained by the MTT method.

 

 

3. Materials and Methods

3.4. Cytotoxicity Activity (DHI Assay)

The manganese(II) complexes’ cytotoxicity was assessed by digital holographic imaging (DHI), a noninvasive, live cell imaging technique. The fibroblasts (VH10, primary human foreskin fibroblasts, CVCL_RW72) were treated with the Mn complexes at concentrations ranging from 33.7 to 500 µM, incubated, and detected by the Holomonitor M4 (Phase Holographic Imaging AB, Lund, Sweden), a phase-contrast microscope with a digital holographic function. The cells were seeded 24 h before the treatment with about 5% confluence in 1.8 mL/well of medium. The VH10 cells were cultured in Dulbecco’s modified eagle medium (DMEM, Sigma–Aldrich, Saint Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) and penicillin–streptomycin (Sigma–Aldrich, Saint Louis, MO, USA) at a concentration of 10 mL/L. For all experiments, the VH10 cells were seeded in 24-well culture plates (Lumox^® 24-multiwell plate (cat. 94.6000.014), Sarstedt, Numbrecht, Germany) in combination with HoloLids (Phase Holographic Imaging AB, Lund, Sweden), and placed in incubator. During the experiments, the Holomonitor was inside a cell culture incubator in a water-saturated atmosphere at 37 °C in 5% CO₂. The cells were set to be imaged with a 10-min interval for 20 h. Three independent experiments per cell line were performed. 

 

4. Conclusions

The cytotoxicity studies, which are of paramount relevance to possible applicability, indicated the non-toxic effect of the Mn(II) complexes on primary human fibroblasts. For this purpose, the MTT method and an innovative DHI platform HoloMonitorM4 were used. It is worth noting the electrochemical studies, which show the varied oxidation efficiency of the manganese(II) complexes and can adequately reflect their biological activity. Interestingly, the regularity between the electrochemical and anti-biofilm studies is observed. It suggests that the metal center can interact with biological targets based on redox reactions. Taking into account the properties determined, it can be assumed that the Mn(II) complexes may contribute to oxidative stress by disturbance of antioxidant protection for bacterial biofilm. Indeed, the studies of the effect of the complexes on CAT activity demonstrate that the Mn-imCHO-Cl, Mn-pyCOOH-H₂O, [Mn-pyCOOH-H₂O]_n, and Mn-dipyCO-NO₃ complexes attenuate the catalase activity, with the exception of the Mn-pyOH-NO₃, [Mn-pyOH-SO₄]_n, and Mn-imCHO-NO₃ complexes, and the presence of which seems to promote the catalase activity. 

The analysis of the bioassay results suggests two probable actions of the evaluated compounds:

- The first one regards the induction of oxidative stress in bacterial cells by the inhibition effect on the CAT enzyme;

- The other one is connected with the participation of the complexes in the disturbance of adhesion of bacterial cells (supramolecular interactions).

To investigate these aspects in detail, further specific tests are essential. To summarize, the Mn(II) complexes might be a suitable candidates for the development of a new anti-biofilm agents. 

The complexes Mn-pyOH-NO₃, [Mn-pyOH-SO₄]_n, and Mn-imCHO-NO₃ seem to be most promising. In order to evaluate the practical potential of these compounds for biotechnological application, further studies regarding the growth inhibition mechanism should be conducted.

Ovanstående klipp är urval där HoloMonitor omnämns.För att läsa hela studien finns denna pdf.

Avslutningsvis en snygg illustration utförd av ett Fluomikroskop.

Epifluorescence microscopy images of P. aeruginosa PAO1 biofilm treated with 0.5 mM of the manganese complexes. Biofilm was stained with nucleic acid stains using the FilmTracer™ LIVE/DEAD Biofilm Viability kit (live cells are represented by the color green; dead cells are represented by the color red). The epifluorescence microscopy images were captured at 1000× magnification.

Min kommentar

Än en gång visar forskare hur användbart och nödvändigt HoloMonitor är för deras forskning.

Beata m kollegor är vana HoloMonitoranvändare och har listat ut instrumentets alla möjligheter,det bidrog förmodligen till de resultat man nu fått publicerade.Metallföreningar tillsammans med läkemedelssubstans kan vara en lösning på den alltmer växande antibiotikaresistensen.

                        Dobra robota Beata z kolegami.

 

                                                       Mvh the99 

 

Som service till alla ev HoloMonitornyfikna forskare : PHIAB Webshop