Multisubstituted pyrimidines effectively inhibit bacterial growth and biofilm formation of Staphylococcus aureus

AbstractBiofilms are multicellular communities of microorganisms that generally attach to surfaces in a self-produced matrix. Unlike planktonic cells, biofilms can withstand conventional antibiotics, causing significant challenges in the healthcare system. Currently, new chemical entities are urgently needed to develop novel anti-biofilm agents. In this study, we designed and synthesized a set of 2,4,5,6-tetrasubstituted pyrimidines and assessed their antibacterial activity against planktonic cells and biofilms formed by Staphylococcus aureus. Compounds 9e, 10d, and 10e displayed potent activity for inhibiting the onset of biofilm formation as well as for killing pre-formed biofilms of S. aureus ATCC 25923 and Newman strains, with half-maximal inhibitory concentration (IC50) values ranging from 11.6 to 62.0 µM. These pyrimidines, at 100 µM, not only decreased the number of viable bacteria within the pre-formed biofilm by 2–3 log10 but also reduced the amount of total biomass by 30–50%. Furthermore, these compounds were effective against planktonic cells with minimum inhibitory concentration (MIC) values lower than 60 µM for both staphylococcal strains. Compound 10d inhibited the growth of S. aureus ATCC 25923 in a concentration-dependent manner and displayed a bactericidal anti-staphylococcal activity. Taken together, our study highlights the value of multisubstituted pyrimidines to develop novel anti-biofilm agents.

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An Alanine-Rich Peptide Attenuates Quorum Sensing-Regulated Virulence and Biofilm Formation in Staphylococcus aureus

Journal of AOAC International ◽

10.5740/jaoacint.18-0251 ◽

2019 ◽

Vol 102 (4) ◽

pp. 1228-1234 ◽

Cited By ~ 1

Author(s):

Raid Al Akeel ◽

Ayesha Mateen ◽

Rabbani Syed

Keyword(s):

Gene Expression ◽

Staphylococcus Aureus ◽

Quorum Sensing ◽

Biofilm Formation ◽

Bacterial Attachment ◽

The Body ◽

Dependent Manner ◽

Virulence Determinants ◽

Microarray Gene Expression ◽

Concentration Dependent Manner

Abstract Background: Alanine-rich proteins/peptides (ARP), with bioactivity of up to 20 amino acid residues, can be observed by the body easily during gastrointestinal digestion. Objective: Populus trichocarpa extract’s capability to attenuate quorum sensing-regulated virulence and biofilm formation in Staphylococcus aureus is described. Methods: PT13, an ARP obtained from P. trichocarpa, was tested for its activity against S. aureus using the broth microdilution test; a crystal-violet biofilm assay was performed under a scanning electron microscope. The production of various virulence factors was estimated with PT13 treatment. Microarray gene expression profiling of PT13-treated S. aureus was conducted and compared with an untreated control. Exopolysaccharides (EPS) was estimated to observe the PT13 inhibition activity. Results: PT13 was antimicrobial toward S. aureus at different concentrations and showed a similar growth rate in the presence and absence of PT13 at concentrations ≤8 μg/mL. Biofilm production was interrupted even at low concentrations, and biofilm-related genes were down-regulated when exposed to PT13. The genes encoding cell adhesion and bacterial attachment protein were the major genes suppressed by PT13. In addition, hemolysins, clumping activity, and EPS production of S. aureus decreased after treatment in a concentration-dependent manner. Conclusions: A long-chain PT13 with effective actions that, even at low concentration levels, not only regulated the gene expression in the producer organism but also blocked the virulence gene expression in this Gram-positive human pathogen is described. Highlights: We identified a PT13 as a potential antivirulence agent that regulated production of bacterial virulence determinants (e.g., toxins, enzymes and biofilm), downwards and it may be a promising anti-virulence agent to be further developed as an anti-infective agent.

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PYED-1 Inhibits Biofilm Formation and Disrupts the Preformed Biofilm of Staphylococcus aureus

Antibiotics ◽

10.3390/antibiotics9050240 ◽

2020 ◽

Vol 9 (5) ◽

pp. 240 ◽

Cited By ~ 1

Author(s):

Adriana Vollaro ◽

Anna Esposito ◽

Eliana Pia Esposito ◽

Raffaele Zarrilli ◽

Annalisa Guaragna ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Quorum Sensing ◽

Biofilm Formation ◽

Surface Proteins ◽

Transcriptional Analysis ◽

Capsular Polysaccharides ◽

Dependent Manner ◽

Chronic Infections ◽

Concentration Dependent Manner ◽

Expression Of Genes

Pregnadiene-11-hydroxy-16α,17α-epoxy-3,20-dione-1 (PYED-1), a heterocyclic corticosteroid derivative of deflazacort, exhibits broad-spectrum antibacterial activity against Gram-negative and Gram-positive bacteria. Here, we investigated the effect of PYED-1 on the biofilms of Staphylococcus aureus, an etiological agent of biofilm-based chronic infections such as osteomyelitis, indwelling medical device infections, periodontitis, chronic wound infections, and endocarditis. PYED-1 caused a strong reduction in biofilm formation in a concentration dependent manner. Furthermore, it was also able to completely remove the preformed biofilm. Transcriptional analysis performed on the established biofilm revealed that PYED-1 downregulates the expression of genes related to quorum sensing (agrA, RNAIII, hld, psm, and sarA), surface proteins (clfB and fnbB), secreted toxins (hla, hlb, and lukD), and capsular polysaccharides (capC). The expression of genes that encode two main global regulators, sigB and saeR, was also significantly inhibited after treatment with PYED-1. In conclusion, PYED-1 not only effectively inhibited biofilm formation, but also eradicated preformed biofilms of S. aureus, modulating the expression of genes related to quorum sensing, surface and secreted proteins, and capsular polysaccharides. These results indicated that PYED-1 may have great potential as an effective antibiofilm agent to prevent S. aureus biofilm-associated infections.

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Antimicrobial Activity of Extracts of Two Native Fruits of Chile: Arrayan (Luma apiculata) and Peumo (Cryptocarya alba)

Antibiotics ◽

10.3390/antibiotics9080444 ◽

2020 ◽

Vol 9 (8) ◽

pp. 444

Author(s):

Jitka Viktorová ◽

Rohitesh Kumar ◽

Kateřina Řehořová ◽

Lan Hoang ◽

Tomas Ruml ◽

...

Keyword(s):

Mass Spectrometry ◽

Inhibitory Concentration ◽

Dependent Manner ◽

Drug Resistant ◽

Planktonic Cells ◽

Fruit Extract ◽

Concentration Dependent Manner ◽

Autoinducer 2 ◽

Resistant Strains ◽

Drug Resistant Strains

Arrayan and peumo fruits are commonly used in the traditional medicine of Chile. In this study, the concentration of the extracts halving the bacterial viability and biofilms formation and disruption of the drug-sensitive and drug-resistant strains of Staphylococcus aureus and Pseudomonas aeruginosa was determined. The chemical composition of extracts was analyzed by high-resolution liquid chromatography coupled with mass spectrometry (U-HPLC/MS). The arrayan extract (Inhibitory concentration IC50 0.35 ± 0.01 mg/mL) was more effective than peumo extract (IC50 0.53 ± 0.02 mg/mL) in the inhibition of S. aureus planktonic cells. Similarly, the arrayan extract was more effective in inhibiting the adhesion (S. aureus IC50 0.23 ± 0.02 mg/mL, P. aeruginosa IC50 0.29 ± 0.02 mg/mL) than peumo extracts (S. aureus IC50 0.47 ± 0.03 mg/mL, P. aeruginosa IC50 0.35 ± 0.01 mg/mL). Both extracts inhibited quorum sensing in a concentration-dependent manner, and the most significant was the autoinducer-2 type communication inhibition by arrayan extract. Both extracts also disrupted preformed biofilm of P. aeruginosa (arrayan IC50 0.56 ± 0.04 mg/mL, peumo IC50 0.59 ± 0.04 mg/mL). However, neither arrayan nor peumo extracts disrupted S. aureus mature biofilm. U-HPLC/MS showed that both fruit extracts mainly possessed quercetin compounds; the peumo fruit extract also contained phenolic acids and phenylpropanoids. Our results suggested that both extracts could be used as natural antimicrobials for some skin and nosocomial infections.

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Growth Mode and Physiological State of Cells Prior to Biofilm Formation Affect Immune Evasion and Persistence of Staphylococcus aureus

Microorganisms ◽

10.3390/microorganisms8010106 ◽

2020 ◽

Vol 8 (1) ◽

pp. 106 ◽

Cited By ~ 2

Author(s):

Kirsi Savijoki ◽

Ilkka Miettinen ◽

Tuula A. Nyman ◽

Maarit Kortesoja ◽

Leena Hanski ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Immune Evasion ◽

Biofilm Formation ◽

Physiological State ◽

Detoxification Enzymes ◽

Growth Mode ◽

Cold Shock Protein ◽

Dependent Manner ◽

Ros Scavenging ◽

Planktonic Cells

The present study investigated Staphylococcus aureus ATCC25923 surfaceomes (cell surface proteins) during prolonged growth by subjecting planktonic and biofilm cultures (initiated from exponential or stationary cells) to label-free quantitative surfaceomics and phenotypic confirmations. The abundance of adhesion, autolytic, hemolytic, and lipolytic proteins decreased over time in both growth modes, while an opposite trend was detected for many tricarboxylic acid (TCA) cycle, reactive oxygen species (ROS) scavenging, Fe-S repair, and peptidolytic moonlighters. In planktonic cells, these changes were accompanied by decreasing and increasing adherence to hydrophobic surface and fibronectin, respectively. Specific RNA/DNA binding (cold-shock protein CspD and ribosomal proteins) and the immune evasion (SpA, ClfA, and IsaB) proteins were notably more abundant on fully mature biofilms initiated with stationary-phase cells (SDBF) compared to biofilms derived from exponential cells (EDBF) or equivalent planktonic cells. The fully matured SDBF cells demonstrated higher viability in THP-1 monocyte/macrophage cells compared to the EDBF cells. Peptidoglycan strengthening, specific urea-cycle, and detoxification enzymes were more abundant on planktonic than biofilm cells, indicating the activation of growth-mode specific pathways during prolonged cultivation. Thus, we show that S. aureus shapes its surfaceome in a growth mode-dependent manner to reach high levofloxacin tolerance (>200-times the minimum biofilm inhibitory concentration). This study also demonstrates that the phenotypic state of the cells prior to biofilm formation affects the immune-evasion and persistence-related traits of S. aureus.

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In VitroAnalyses of the Effects of Heparin and Parabens on Candida albicans Biofilms and Planktonic Cells

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05061-11 ◽

2011 ◽

Vol 56 (1) ◽

pp. 148-153 ◽

Cited By ~ 15

Author(s):

Marisa H. Miceli ◽

Stella M. Bernardo ◽

T. S. Neil Ku ◽

Carla Walraven ◽

Samuel A. Lee

Keyword(s):

Candida Albicans ◽

Metabolic Activity ◽

Biofilm Formation ◽

High Dose ◽

Dependent Manner ◽

Planktonic Cells ◽

Content Type ◽

Heparin Sodium ◽

The Individual

ABSTRACTInfections and thromboses are the most common complications associated with central venous catheters. Suggested strategies for prevention and management of these complications include the use of heparin-coated catheters, heparin locks, and antimicrobial lock therapy. However, the effects of heparin onCandida albicansbiofilms and planktonic cells have not been previously studied. Therefore, we sought to determine thein vitroeffect of a heparin sodium preparation (HP) on biofilms and planktonic cells ofC. albicans. Because HP contains two preservatives, methyl paraben (MP) and propyl paraben (PP), these compounds and heparin sodium without preservatives (Pure-H) were also tested individually. The metabolic activity of the mature biofilm after treatment was assessed using XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction and microscopy. Pure-H, MP, and PP caused up to 75, 85, and 60% reductions of metabolic activity of the mature preformedC. albicansbiofilms, respectively. Maximal efficacy against the mature biofilm was observed with HP (up to 90%) compared to the individual compounds (P< 0.0001). Pure-H, MP, and PP each inhibitedC. albicansbiofilm formation up to 90%. A complete inhibition of biofilm formation was observed with HP at 5,000 U/ml and higher. When tested against planktonic cells, each compound inhibited growth in a dose-dependent manner. These data indicated that HP, MP, PP, and Pure-H havein vitroantifungal activity againstC. albicansmature biofilms, formation of biofilms, and planktonic cells. Investigation of high-dose heparin-based strategies (e.g., heparin locks) in combination with traditional antifungal agents for the treatment and/or prevention ofC. albicansbiofilms is warranted.

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Antimicrobial Activity of Cyclic-Monomeric and Dimeric Derivatives of the Snail-Derived Peptide Cm-p5 against Viral and Multidrug-Resistant Bacterial Strains

Biomolecules ◽

10.3390/biom11050745 ◽

2021 ◽

Vol 11 (5) ◽

pp. 745

Author(s):

Melaine González-García ◽

Fidel Morales-Vicente ◽

Erbio Díaz Pico ◽

Hilda Garay ◽

Daniel G. Rivera ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Antimicrobial Activity ◽

Antifungal Activity ◽

Multidrug Resistant ◽

Moderate Activity ◽

Dependent Manner ◽

Bacterial Strains ◽

Acinetobacter Baumanii ◽

Concentration Dependent Manner ◽

Conventional Antibiotics

Cm-p5 is a snail-derived antimicrobial peptide, which demonstrated antifungal activity against the pathogenic strains of Candida albicans. Previously we synthetized a cyclic monomer as well as a parallel and an antiparallel dimer of Cm-p5 with improved antifungal activity. Considering the alarming increase of microbial resistance to conventional antibiotics, here we evaluated the antimicrobial activity of these derivatives against multiresistant and problematic bacteria and against important viral agents. The three peptides showed a moderate activity against Pseudomonas aeruginosa, Klebsiella pneumoniae Extended Spectrum β-Lactamase (ESBL), and Streptococcus agalactiae, with MIC values > 100 µg/mL. They exerted a considerable activity with MIC values between 25–50 µg/mL against Acinetobacter baumanii and Enterococcus faecium. In addition, the two dimers showed a moderate activity against Pseudomonas aeruginosa PA14. The three Cm-p5 derivatives inhibited a virulent extracellular strain of Mycobacterium tuberculosis, in a dose-dependent manner. Moreover, they inhibited Herpes Simplex Virus 2 (HSV-2) infection in a concentration-dependent manner, but had no effect on infection by the Zika Virus (ZIKV) or pseudoparticles of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2). At concentrations of >100 µg/mL, the three new Cm-p5 derivatives showed toxicity on different eukaryotic cells tested. Considering a certain cell toxicity but a potential interesting activity against the multiresistant strains of bacteria and HSV-2, our compounds require future structural optimization.

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Isolation, Characterization, and Comparison of Efficiencies of Bacteriophages to Reduce Planktonic and Biofilm-Associated Staphylococcus aureus

Journal of Health and Allied Sciences NU ◽

10.1055/s-0040-1715773 ◽

2020 ◽

Vol 10 (03) ◽

pp. 102-108

Author(s):

Anoopkrishna Rai ◽

Rajeshwari V. Vittal ◽

Juliet R. Mohan Raj

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Bacterial Infections ◽

Viable Cell ◽

Viable Count ◽

Dna Fingerprint ◽

Planktonic Cells ◽

Microtiter Plates ◽

Reduction Assay ◽

Need To Evaluate

Abstract Introduction In the present era, wherein occurrence of antimicrobial resistance compounded with biofilms in disease conditions has rendered present antibiotic therapy ineffective, the need for alternative strategies to treat bacterial infections has brought bacteriophages to the forefront. The antimicrobial activity of phages is often determined by a viable cell reduction assay which focuses only on planktonic forms. The physiology of an organism in biofilm differs from those that are planktonic; hence, there is a need to evaluate the activity of phages both on planktonic forms, as well as on biofilms, to select candidate therapeutic phages. Materials and Methods Bacteriophages for Staphylococcus aureus were isolated from environmental samples and characterized based on growth kinetics and DNA fingerprint patterns. Activity of isolated phages on planktonic forms was determined by viable count reduction assay. Phage ability to prevent biofilm formation and ability to disperse formed biofilms were performed in 96-well microtiter plates and biofilm estimated by crystal violet assay. Results Four bacteriophages designated, that is, P3, PD1, PE1, and PE2, were isolated and characterized. Planktonic cells of S. aureus were found to be sensitive to phages PD1, PE1, and PE2. Phages PD1 and PE2 were efficient in preventing biofilm formation and phages PD1, PE1, and P3 were efficient in dispersing formed biofilms. Conclusion The ability of some phages to disperse biofilms effectively, while unable to show the same efficiency on planktonic cells, indicates that viable count reduction assay alone may not be a sufficient tool to imply bactericidal activity of bacteriophages, especially while trying to eradicate biofilms.

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Effect of Lonicera caerulea var. emphyllocalyx Fruit on Biofilm Formed by Porphyromonas gingivalis

BioMed Research International ◽

10.1155/2019/3547858 ◽

2019 ◽

Vol 2019 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Masaaki Minami ◽

Hiroshi Takase ◽

Mineo Nakamura ◽

Toshiaki Makino

Keyword(s):

Oral Cavity ◽

Porphyromonas Gingivalis ◽

Aspiration Pneumonia ◽

Biofilm Formation ◽

Bacterial Colonization ◽

Bacterial Biofilm ◽

Dependent Manner ◽

Electron Microscopic ◽

Concentration Dependent Manner ◽

Lonicera Caerulea

Porphyromonas gingivalis is an important pathogenic anaerobic bacterium that causes aspiration pneumonia. This bacterium frequently forms biofilms in the oral cavity and in respiratory tract-associated medical devices. Bacterial colonization that occurs in association with this biofilm formation is the main reason for incurable aspiration pneumonia. The Lonicera caerulea var. emphyllocalyx (LCE) fruit has been used in folk medicine in Hokkaido, the northern part of Japan. The aim of this study was to elucidate one of the antimicrobial mechanisms of LCE methanol extract (LCEE)—the inhibitory effect of LCEE on biofilm formation by P. gingivalis. Our results show that LCEE significantly reduced biofilm formation by three different P. gingivalis isolates in a concentration- and time-dependent manner that were quantified by the adsorption of safranin red. When LCEE was added to biofilms already formed by P. gingivalis, LCEE did not degrade the biofilm. However, treatment with LCEE significantly promoted the removal of existing biofilm by vibration compared to that of control. We also confirmed biofilm formation in LCEE-treated P. gingivalis in tracheal tubes using scanning electron microscopic (SEM) analysis. Cyanidin 3-O-glucoside (C3G), one of the components of LCE, also inhibited the formation of biofilm by P. gingivalis in a concentration-dependent manner. Our results reveal that LCEE may be an effective antibacterial substance for P. gingivalis-induced aspiration pneumonia because of its role in the suppression of bacterial biofilm formation in the oral cavity.

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Protein A-Mediated Multicellular Behavior in Staphylococcus aureus

Journal of Bacteriology ◽

10.1128/jb.01222-08 ◽

2008 ◽

Vol 191 (3) ◽

pp. 832-843 ◽

Cited By ~ 170

Author(s):

Nekane Merino ◽

Alejandro Toledo-Arana ◽

Marta Vergara-Irigaray ◽

Jaione Valle ◽

Cristina Solano ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Protein A ◽

Surface Proteins ◽

Growth Media ◽

Dependent Manner ◽

Chronic Infections ◽

The Matrix ◽

Implanted Medical Devices ◽

Biofilm Development

ABSTRACT The capacity of Staphylococcus aureus to form biofilms on host tissues and implanted medical devices is one of the major virulence traits underlying persistent and chronic infections. The matrix in which S. aureus cells are encased in a biofilm often consists of the polysaccharide intercellular adhesin (PIA) or poly-N-acetyl glucosamine (PNAG). However, surface proteins capable of promoting biofilm development in the absence of PIA/PNAG exopolysaccharide have been described. Here, we used two-dimensional nano-liquid chromatography and mass spectrometry to investigate the composition of a proteinaceous biofilm matrix and identified protein A (spa) as an essential component of the biofilm; protein A induced bacterial aggregation in liquid medium and biofilm formation under standing and flow conditions. Exogenous addition of synthetic protein A or supernatants containing secreted protein A to growth media induced biofilm development, indicating that protein A can promote biofilm development without being covalently anchored to the cell wall. Protein A-mediated biofilm formation was completely inhibited in a dose-dependent manner by addition of serum, purified immunoglobulin G, or anti-protein A-specific antibodies. A murine model of subcutaneous catheter infection unveiled a significant role for protein A in the development of biofilm-associated infections, as the amount of protein A-deficient bacteria recovered from the catheter was significantly lower than that of wild-type bacteria when both strains were used to coinfect the implanted medical device. Our results suggest a novel role for protein A complementary to its known capacity to interact with multiple immunologically important eukaryotic receptors.

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Identification of ica-dependent biofilm production by Staphylococcus aureus clinical isolates and antibiofilm effects of ascorbic acid against biofilm production

Journal of Clinical Pathology ◽

10.1136/jclinpath-2019-206280 ◽

2020 ◽

Vol 73 (5) ◽

pp. 261-266

Author(s):

Sahra Kırmusaoğlu ◽

Havva Kaşıkçı

Keyword(s):

Staphylococcus Aureus ◽

Ascorbic Acid ◽

Biofilm Formation ◽

Surface Proteins ◽

Clinical Isolates ◽

High Morbidity ◽

Dependent Manner ◽

Independent Manner ◽

Biofilm Production ◽

Life Threatening

AimsStaphylococcus aureus (S. aureus) is a life-threatening pathogen with high morbidity and mortality rates which causes nosocomial and community-acquired infections. Biofilm, considered to be a common virulence factor for pathogens, plays a significant role in recurrent and untreatable infections. Biofilm formation of S. aureus is mediated by synthesis of either poly-N-acetylglucosamine in an ica-dependent manner or surface proteins in an ica-independent manner. In some cases treatment is impossible and recurrent. In this study, ica-dependent biofilm-producing S. aureus isolates were detected and the anti-biofilm effect of ascorbic acid against biofilm formation of isolates was investigated.MethodsA total of 21 methicillin-sensitive S. aureus (MSSA) clinical isolates stored in our bacterial stock were used to detect ica-dependent biofilm-producing MSSA isolates. The anti-biofilm study was undertaken with three ica-dependent biofilm-producing isolates (MSSA2–4) and ATCC 29213 (MSSA1). Biofilms and the anti-biofilm effect of ascorbic acid were detected using the microtitre plate (MtP) method. 16S-rRNA, nuc, icaA and icaD genes and expression levels of icaA and icaD of isolates were detected by RT-PCR.ResultsThe minimum inhibitory concentrations (MICs) of ascorbic acid prevented biofilm formation of MSSA1 and MSSA3. Also, 1/2 MIC of ascorbic acid prevented biofilm formation of MSSA3. It was observed that biofilm formation decreased with increased concentration. There was no significant increase in ica gene expression of MSSA1 and MSSA2. Expression of icaA and icaD of MSSA3 decreased 13% and 38%, respectively. Expression of icaA in MSSA4 decreased 12%.ConclusionThe results of our study show that ascorbic acid can be used as an anti-biofilm agent to prevent biofilm formation of S. aureus and thus biofilm-related infections.

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