Combination of Sanguisorbigenin and Conventional Antibiotic Therapy for Methicillin-Resistant Staphylococcus aureus: Inhibition of Biofilm Formation and Alteration of Cell Membrane Permeability

Methicillin-resistant Staphylococcus aureus (MRSA) infection is challenging to eradicate because of antibiotic resistance and biofilm formation. Novel antimicrobial agents and alternative therapies are urgently needed. This study aimed to evaluate the synergy of sanguisorbigenin (SGB) isolated from Sanguisorba officinalis L. with six conventional antibiotics to achieve broad-spectrum antibacterial action and prevent the development of resistance. A checkerboard dilution test and time-to-kill curve assay were used to determine the synergistic effect of SGB combined with antibiotics against MRSA. SGB showed significant synergy with antibiotics and reduced the minimum inhibitory concentration of antibiotics by 2–16-fold. Biofilm inhibition assay, quantitative RT-PCR, crystal violet absorption, and transmission electron microscopy were performed to evaluate the synergy mechanism. The results indicated that SGB could inhibit biofilm formation and alter cell membrane permeability in MRSA. In addition, SGB was found to exhibit quite low cytotoxicity and hemolysis. The discovery of the superiority of SGB suggests that SGB may be an antibiotic adjuvant for use in combination therapy and as a plant-derived antibacterial agent targeting biofilms.     

The Effect of Rotating Magnetic Field on Susceptibility Profile of Methicillin-Resistant Staphylococcus aureus Strains Exposed to Activity of Different Groups of Antibiotics

Methicillin-resistant strains of Staphylococcus aureus (MRSA) have become a global issue for healthcare systems due to their resistance to most β-lactam antibiotics, frequently accompanied by resistance to other classes of antibiotics. In this work, we analyzed the impact of combined use of rotating magnetic field (RMF) with various classes of antibiotics (β-lactams, glycopeptides, macrolides, lincosamides, aminoglycosides, tetracyclines, and fluoroquinolones) against nine S. aureus strains (eight methicillin-resistant and one methicillin-sensitive). The results indicated that the application of RMF combined with antibiotics interfering with cell walls (particularly with the β-lactam antibiotics) translate into favorable changes in staphylococcal growth inhibition zones or in minimal inhibitory concentration values compared to the control settings, which were unexposed to RMF. As an example, the MIC value of cefoxitin was reduced in all MRSA strains by up to 42 times. Apart from the β-lactams, the reduced MIC values were also found for erythromycin, clindamycin, and tetracycline (three strains), ciprofloxacin (one strain), gentamicin (six strains), and teicoplanin (seven strains). The results obtained with the use of in vitro biofilm model confirm that the disturbances caused by RMF in the bacterial cell walls increase the effectiveness of the antibiotics towards MRSA. Because the clinical demand for new therapeutic options effective against MRSA is undisputable, the outcomes and conclusions drawn from the present study may be considered an important road into the application of magnetic fields to fight infections caused by methicillin-resistant staphylococci.     

Staphylococcus aureus and MRSA Growth and Biofilm Formation after Treatment with Antibiotics and SeNPs

Methicillin-resistant Staphylococcus aureus (MRSA) is a dangerous pathogen resistant to β-lactam antibiotics. Due to its resistance, it is difficult to manage the infections caused by this strain. We examined this issue in terms of observation of the growth properties and ability to form biofilms in sensitive S. aureus and MRSA after the application of antibiotics (ATBs)—ampicillin, oxacillin and penicillin—and complexes of selenium nanoparticles (SeNPs) with these ATBs. The results suggest the strong inhibition effect of SeNPs in complexes with conventional ATBs. Using the impedance method, a higher disruption of biofilms was observed after the application of ATB complexes with SeNPs compared to the group exposed to ATBs without SeNPs. The biofilm formation was intensely inhibited (up to 99% ± 7% for S. aureus and up to 94% ± 4% for MRSA) after application of SeNPs in comparison with bacteria without antibacterial compounds whereas ATBs without SeNPs inhibited S. aureus up to 79% ± 5% and MRSA up to 16% ± 2% only. The obtained results provide a basis for the use of SeNPs as a tool for the treatment of bacterial infections, which can be complicated because of increasing resistance of bacteria to conventional ATB drugs.     

Molecular Insights into an Antibiotic Enhancer Action of New Morpholine-Containing 5-Arylideneimidazolones in the Fight against MDR Bacteria

In the search for an effective strategy to overcome antimicrobial resistance, a series of new morpholine-containing 5-arylideneimidazolones differing within either the amine moiety or at position five of imidazolones was explored as potential antibiotic adjuvants against Gram-positive and Gram-negative bacteria. Compounds (7–23) were tested for oxacillin adjuvant properties in the Methicillin-susceptible S. aureus (MSSA) strain ATCC 25923 and Methicillin-resistant S. aureus MRSA 19449. Compounds 14–16 were tested additionally in combination with various antibiotics. Molecular modelling was performed to assess potential mechanism of action. Microdilution and real-time efflux (RTE) assays were carried out in strains of K. aerogenes to determine the potential of compounds 7–23 to block the multidrug efflux pump AcrAB-TolC. Drug-like properties were determined experimentally. Two compounds (10, 15) containing non-condensed aromatic rings, significantly reduced oxacillin MICs in MRSA 19449, while 15 additionally enhanced the effectiveness of ampicillin. Results of molecular modelling confirmed the interaction with the allosteric site of PBP2a as a probable MDR-reversing mechanism. In RTE, the compounds inhibited AcrAB-TolC even to 90% (19). The 4-phenylbenzylidene derivative (15) demonstrated significant MDR-reversal “dual action” for β-lactam antibiotics in MRSA and inhibited AcrAB-TolC in K. aerogenes. 15 displayed also satisfied solubility and safety towards CYP3A4 in vitro.     

Antibacterial Activity of New Oxazolidin-2-One Analogues in Methicillin-Resistant Staphylococcus aureus Strains

Staphylococcus aureus is one of the most common causes of nosocomial infections. The purpose of this study was the synthesis and in vitro evaluation of antimicrobial activity of 10 new 3-oxazolidin-2-one analogues on 12 methicillin resistant S. aureus (MRSA) clinical isolates. S. aureus confirmation was achieved via catalase and coagulase test. Molecular characterization of MRSA was performed by amplification of the mecA gene. Antimicrobial susceptibility was evaluated via the Kirby-Bauer disc diffusion susceptibility test protocol, using commonly applied antibiotics and the oxazolidinone analogues. Only (R)-5-((S)-1-dibenzylaminoethyl)-1,3-oxazolidin-2-one (7a) exhibited antibacterial activity at 6.6 μg. These results, allow us to infer that molecules such as 7a can be potentially used to treat infections caused by MRSA strains.     

Structural Studies on 4,5‐Disubstituted 2‐Aminoimidazole‐Based Biofilm Modulators that Suppress Bacterial Resistance to β‐Lactams

A library of 4,5‐disubstituted 2‐aminoimidazole triazole amide (2‐AITA) conjugates has been successfully assembled. Upon biological screening, this class of small molecules was discovered as enhanced biofilm regulators through non‐microbicidal mechanisms against methicillin‐resistant Staphylococcus aureus (MRSA) and multidrug‐resistant Acinetobacter baumannii (MDRAB), with active concentrations in the low micromolar range. The library was also subjected to synergism and resensitization studies with β‐lactam antibiotics against MRSA. Lead compounds were identified that suppress the antibiotic resistance of MRSA by working synergistically with oxacillin, a β‐lactam antibiotic resistant to penicillinase. A further structure–activity relationship (SAR) study on the parent 2‐AITA compound delivered a 2‐aminoimidazole diamide (2‐AIDA) conjugate with significantly increased synergistic activity with oxacillin against MRSA, decreasing the MIC value of the β‐lactam antibiotic by 64‐fold. Increased anti‐biofilm activity did not necessarily lead to increased suppression of antibiotic resistance, which indicates that biofilm inhibition and resensitization are most likely occurring via distinct mechanisms.     

Identification and Determination of 1,3-Thiazinane-4-carboxylic Acid in Human Urine—Chromatographic Studies

It is well established that homocysteine (Hcy) and its thiolactone (HTL) are reactive towards aldehydes in an aqueous environment, forming substituted thiazinane carboxylic acids. This report provides evidence that Hcy/HTL and formaldehyde (FA) adduct, namely 1,3-thiazinane-4-carboxylic acid (TCA) is formed in vivo in humans. In order to provide definitive proof, a gas chromatography–mass spectrometry (GC–MS) based method was elaborated to identify and quantify TCA in human urine. The GC–MS assay involves chemical derivatization with isobutyl chloroformate (IBCF) in the presence of pyridine as a catalyst, followed by an ethyl acetate extraction of the obtained isobutyl derivative of TCA (TCA-IBCF). The validity of the method has been demonstrated based upon United States Food and Drug Administration recommendations. The assay linearity was observed within a 1–50 µmol L⁻¹ range for TCA in urine, while the lowest concentration on the calibration curve was recognized as the limit of quantification (LOQ). Importantly, the method was successfully applied to urine samples delivered by apparently healthy volunteers (n = 15). The GC–MS assay may provide a new analytical tool for routine clinical analysis of the role of TCA in living systems in the near future.     

Rotating Magnetic Field Increases β-Lactam Antibiotic Susceptibility of Methicillin-Resistant Staphylococcus aureus Strains

Methicillin-resistant strains of Staphylococcus aureus (MRSA) have developed resistance to most β-lactam antibiotics and have become a global health issue. In this work, we analyzed the impact of a rotating magnetic field (RMF) of well-defined and strictly controlled characteristics coupled with β-lactam antibiotics against a total of 28 methicillin-resistant and sensitive S. aureus strains. The results indicate that the application of RMF combined with β-lactam antibiotics correlated with favorable changes in growth inhibition zones or in minimal inhibitory concentrations of the antibiotics compared to controls unexposed to RMF. Fluorescence microscopy indicated a drop in the relative number of cells with intact cell walls after exposure to RMF. These findings were additionally supported by the use of SEM and TEM microscopy, which revealed morphological alterations of RMF-exposed cells manifested by change of shape, drop in cell wall density and cytoplasm condensation. The obtained results indicate that the originally limited impact of β-lactam antibiotics in MRSA is boosted by the disturbances caused by RMF in the bacterial cell walls. Taking into account the high clinical need for new therapeutic options, effective against MRSA, the data presented in this study have high developmental potential and could serve as a basis for new treatment options for MRSA infections.     

Biofilm Matrix and Its Regulation in Pseudomonas aeruginosa

Biofilms are communities of microorganisms embedded in extracellular polymeric substances (EPS) matrix. Bacteria in biofilms demonstrate distinct features from their free-living planktonic counterparts, such as different physiology and high resistance to immune system and antibiotics that render biofilm a source of chronic and persistent infections. A deeper understanding of biofilms will ultimately provide insights into the development of alternative treatment for biofilm infections. The opportunistic pathogen Pseudomonas aeruginosa, a model bacterium for biofilm research, is notorious for its ability to cause chronic infections by its high level of drug resistance involving the formation of biofilms. In this review, we summarize recent advances in biofilm formation, focusing on the biofilm matrix and its regulation in P. aeruginosa, aiming to provide resources for the understanding and control of bacterial biofilms.     

2-(2-Methyl-2-nitrovinyl)furan but Not Furvina Interfere with Staphylococcus aureus Agr Quorum-Sensing System and Potentiate the Action of Fusidic Acid against Biofilms

Quorum sensing (QS) plays an essential role in the production of virulence factors, in biofilm formation and antimicrobial resistance. Consequently, inhibiting QS is being considered a promising target for antipathogenic/anti-virulence therapies. This study aims to screen 2-nitrovinylfuran derivatives structurally related to Furvina (a broad-spectrum antibiotic already used for therapeutic purposes) for their effects on QS and in biofilm prevention/control. Furvina and four 2-nitrovinylfuran derivatives (compounds 1–4) were tested to assess the ability to interfere with QS of Staphylococcus aureus using bioreporter strains (S. aureus ALC1742 and ALC1743). The activity of Furvina and the most promising quorum-sensing inhibitor (QSI) was evaluated in biofilm prevention and in biofilm control (combined with fusidic acid). The biofilms were further characterized in terms of biofilm mass, viability and membrane integrity. Compound 2 caused the most significant QS inhibition with reductions between 60% and 80%. Molecular docking simulations indicate that this compound interacts preferentially with the protein hydrophobic cleft in the LytTR domain of AgrA pocket. Metabolic inactivations of 40% for S. aureus ALC1742 and 20% for S. aureus ALC1743 were reached. A 24 h-old biofilm formed in the presence of the QSI increased the metabolic inactivation by fusidic acid to 80%, for both strains. The overall results highlight the effects of compound 2 as well as the potential of combining QSI with in-use antibiotics for the management of skin and soft tissues infections.     

Spike Formation Is a Turning Point Determining Wheat Root Microbiome Abundance,Structures and Functions

Root selection of their associated microbiome composition and activities is determined by the plant’s developmental stage and distance from the root. Total gene abundance, structure and functions of root-associated and rhizospheric microbiomes were studied throughout wheat growth season under field conditions. On the root surface, abundance of the well-known wheat colonizers Proteobacteria and Actinobacteria decreased and increased, respectively, during spike formation, whereas abundance of Bacteroidetes was independent of spike formation. Metagenomic analysis combined with functional co-occurrence networks revealed a significant impact of plant developmental stage on its microbiome during the transition from vegetative growth to spike formation. For example, gene functions related to biofilm and sensorial movement, antibiotic production and resistance and carbons and amino acids and their transporters. Genes associated with these functions were also in higher abundance in root vs. the rhizosphere microbiome. We propose that abundance of transporter-encoding genes related to carbon and amino acid, may mirror the availability and utilization of root exudates. Genes related to antibiotic resistance mechanisms were abundant during vegetative growth, while after spike formation, genes related to the biosynthesis of various antibiotics were enriched. This observation suggests that during root colonization and biofilm formation, bacteria cope with competitor’s antibiotics, whereas in the mature biofilm stage, they invest in inhibiting new colonizers. Additionally, there is higher abundance of genes related to denitrification in rhizosphere compared to root-associated microbiome during wheat growth, possibly due to competition with the plant over nitrogen in the root vicinity. We demonstrated functional and phylogenetic division in wheat root zone microbiome in both time and space: pre- and post-spike formation, and root-associated vs. rhizospheric niches. These findings shed light on the dynamics of plant–microbe and microbe–microbe interactions in the developing root zone.     

The Neosartorya fischeri Antifungal Protein 2 (NFAP2): A New Potential Weapon against Multidrug-Resistant Candida auris Biofilms

Candida auris is a potential multidrug-resistant pathogen able to persist on indwelling devices as a biofilm, which serve as a source of catheter-associated infections. Neosartorya fischeri antifungal protein 2 (NFAP2) is a cysteine-rich, cationic protein with potent anti-Candida activity. We studied the in vitro activity of NFAP2 alone and in combination with fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin against C. auris biofilms. The nature of interactions was assessed utilizing the fractional inhibitory concentration index (FICI), a Bliss independence model, and LIVE/DEAD viability assay. NFAP2 exerted synergy with all tested antifungals with FICIs ranging between 0.312–0.5, 0.155–0.5, 0.037–0.375, 0.064–0.375, and 0.064–0.375 for fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin, respectively. These results were confirmed using a Bliss model, where NFAP2 produced 17.54 μM²%, 2.16 μM²%, 33.31 μM²%, 10.72 μM²%, and 111.19 μM²% cumulative synergy log volume in combination with fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin, respectively. In addition, biofilms exposed to echinocandins (32 mg/L) showed significant cell death in the presence of NFAP2 (128 mg/L). Our study shows that NFAP2 displays strong potential as a novel antifungal compound in alternative therapies to combat C. auris biofilms.