Novel Strategy for Biofilm Inhibition by Using Small Molecules Targeting Molecular Chaperone DnaK

ABSTRACTBiofilms are complex communities of microorganisms that attach to surfaces and are embedded in a self-produced extracellular matrix. Since these cells acquire increased tolerance against antimicrobial agents and host immune systems, biofilm-associated infectious diseases tend to become chronic. We show here that the molecular chaperone DnaK is important for biofilm formation and that chemical inhibition of DnaK cellular functions is effective in preventing biofilm development. Genetic, microbial, and microscopic analyses revealed that deletion of thednaKgene markedly reduced the production of the extracellular functional amyloid curli, which contributes to the robustness ofEscherichia colibiofilms. We tested the ability of DnaK inhibitors myricetin (Myr), telmisartan, pancuronium bromide, and zafirlukast to prevent biofilm formation ofE. coli. Only Myr, a flavonol widely distributed in plants, inhibited biofilm formation in a concentration-dependent manner (50% inhibitory concentration [IC50] = 46.2 μM); however, it did not affect growth. Transmission electron microscopy demonstrated that Myr inhibited the production of curli. Phenotypic analyses of thermosensitivity, cell division, intracellular level of RNA polymerase sigma factor RpoH, and vulnerability to vancomycin revealed that Myr altered the phenotype ofE. coliwild-type cells to make them resemble those of the isogenicdnaKdeletion mutant, indicating that Myr inhibits cellular functions of DnaK. These findings provide insights into the significance of DnaK in curli-dependent biofilm formation and indicate that DnaK is an ideal target for antibiofilm drugs.

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Peptide Mix from Olivancillaria hiatula Interferes with Cell-to-Cell Communication in Pseudomonas aeruginosa

BioMed Research International ◽

10.1155/2019/5313918 ◽

2019 ◽

Vol 2019 ◽

pp. 1-12

Author(s):

Edward Ntim Gasu ◽

Hubert Senanu Ahor ◽

Lawrence Sheringham Borquaye

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Biofilm Formation ◽

Antimicrobial Agents ◽

Cell Communication ◽

Microtiter Plate ◽

Antibiofilm Activity ◽

Dependent Manner ◽

Biofilm Inhibition ◽

Swarming Motility

Bacteria in biofilms are encased in an extracellular polymeric matrix that limits exposure of microbial cells to lethal doses of antimicrobial agents, leading to resistance. In Pseudomonas aeruginosa, biofilm formation is regulated by cell-to-cell communication, called quorum sensing. Quorum sensing facilitates a variety of bacterial physiological functions such as swarming motility and protease, pyoverdine, and pyocyanin productions. Peptide mix from the marine mollusc, Olivancillaria hiatula, has been studied for its antibiofilm activity against Pseudomonas aeruginosa. Microscopy and microtiter plate-based assays were used to evaluate biofilm inhibitory activities. Effect of the peptide mix on quorum sensing-mediated processes was also evaluated. Peptide mix proved to be a good antibiofilm agent, requiring less than 39 μg/mL to inhibit 50% biofilm formation. Micrographs obtained confirmed biofilm inhibition at 1/2 MIC whereas 2.5 mg/mL was required to degrade preformed biofilm. There was a marked attenuation in quorum sensing-mediated phenotypes as well. At 1/2 MIC of peptide, the expression of pyocyanin, pyoverdine, and protease was inhibited by 60%, 72%, and 54%, respectively. Additionally, swarming motility was repressed by peptide in a dose-dependent manner. These results suggest that the peptide mix from Olivancillaria hiatula probably inhibits biofilm formation by interfering with cell-to-cell communication in Pseudomonas aeruginosa.

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Antibodies to PhnD Inhibit Staphylococcal Biofilms

Infection and Immunity ◽

10.1128/iai.02168-14 ◽

2014 ◽

Vol 82 (9) ◽

pp. 3764-3774 ◽

Cited By ~ 15

Author(s):

Hubert Lam ◽

Augustus Kesselly ◽

Svetlana Stegalkina ◽

Harry Kleanthous ◽

Jeremy A. Yethon

Keyword(s):

Biofilm Formation ◽

Bloodstream Infections ◽

Well Being ◽

Human Neutrophils ◽

Target Antigen ◽

Lead Target ◽

Biofilm Inhibition ◽

Content Type ◽

Specific Antibodies ◽

Biofilm Development

ABSTRACTBiofilm formation on central lines or peripheral catheters is a serious threat to patient well-being. Contaminated vascular devices can act as a nidus for bloodstream infection and systemic pathogen dissemination. Staphylococcal biofilms are the most common cause of central-line-associated bloodstream infections, and antibiotic resistance makes them difficult to treat. As an alternative to antibiotic intervention, we sought to identify anti-staphylococcal biofilm targets for the development of a vaccine or antibody prophylactic. A screening strategy was devised using a microfluidic system to test antibody-mediated biofilm inhibition under biologically relevant conditions of shear flow. Affinity-purified polyclonal antibodies to target antigen PhnD inhibited bothStaphylococcus epidermidisandS. aureusbiofilms. PhnD-specific antibodies blocked biofilm development at the initial attachment and aggregation stages, and deletion ofphnDinhibited normal biofilm formation. We further adapted our microfluidic biofilm system to monitor the interaction of human neutrophils with staphylococcal biofilms and demonstrated that PhnD-specific antibodies also serve as opsonins to enhance neutrophil binding, motility, and biofilm engulfment. These data support the identification of PhnD as a lead target for biofilm intervention strategies performed either by vaccination or through passive administration of antibodies.

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Tannic Acid Inhibits Staphylococcus aureus Surface Colonization in an IsaA-Dependent Manner

Infection and Immunity ◽

10.1128/iai.00877-12 ◽

2012 ◽

Vol 81 (2) ◽

pp. 496-504 ◽

Cited By ~ 59

Author(s):

David E. Payne ◽

Nicholas R. Martin ◽

Katherine R. Parzych ◽

Alex H. Rickard ◽

Adam Underwood ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Tannic Acid ◽

Biofilm Formation ◽

Antibiofilm Activity ◽

Host Immune System ◽

Dependent Manner ◽

Content Type ◽

Surface Colonization ◽

Biofilm Development ◽

Insight Into

ABSTRACTStaphylococcus aureusis a human commensal and pathogen that is capable of forming biofilms on a variety of host tissues and implanted medical devices. Biofilm-associated infections resist antimicrobial chemotherapy and attack from the host immune system, making these infections particularly difficult to treat. In order to gain insight into environmental conditions that influenceS. aureusbiofilm development, we screened a library of small molecules for the ability to inhibitS. aureusbiofilm formation. This led to the finding that the polyphenolic compound tannic acid inhibitsS. aureusbiofilm formation in multiple biofilm models without inhibiting bacterial growth. We present evidence that tannic acid inhibitsS. aureusbiofilm formation via a mechanism dependent upon the putative transglycosylase IsaA. Tannic acid did not inhibit biofilm formation of anisaAmutant. Overexpression of wild-type IsaA inhibited biofilm formation, whereas overexpression of a catalytically dead IsaA had no effect. Tannin-containing drinks like tea have been found to reduce methicillin-resistantS. aureusnasal colonization. We found that black tea inhibitedS. aureusbiofilm development and that anisaAmutant resisted this inhibition. Antibiofilm activity was eliminated from tea when milk was added to precipitate the tannic acid. Finally, we developed a rodent model forS. aureusthroat colonization and found that tea consumption reducedS. aureusthroat colonization via anisaA-dependent mechanism. These findings provide insight into a molecular mechanism by which commonly consumed polyphenolic compounds, such as tannins, influenceS. aureussurface colonization.

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Apple Flavonoid Phloretin Inhibits Escherichia coli O157:H7 Biofilm Formation and Ameliorates Colon Inflammation in Rats

Infection and Immunity ◽

10.1128/iai.05580-11 ◽

2011 ◽

Vol 79 (12) ◽

pp. 4819-4827 ◽

Cited By ~ 94

Author(s):

Jin-Hyung Lee ◽

Sushil Chandra Regmi ◽

Jung-Ae Kim ◽

Moo Hwan Cho ◽

Hyungdon Yun ◽

...

Keyword(s):

Escherichia Coli ◽

Epithelial Cells ◽

Biofilm Formation ◽

Pathogenic Bacteria ◽

Antimicrobial Agents ◽

Trinitrobenzene Sulfonic Acid ◽

Content Type ◽

E Coli ◽

Colon Inflammation ◽

K 12

ABSTRACTPathogenic biofilms have been associated with persistent infections due to their high resistance to antimicrobial agents, while commensal biofilms often fortify the host's immune system. Hence, controlling biofilm formation of both pathogenic bacteria and commensal bacteria is important in bacterium-related diseases. We investigated the effect of plant flavonoids on biofilm formation of enterohemorrhagicEscherichia coliO157:H7. The antioxidant phloretin, which is abundant in apples, markedly reducedE. coliO157:H7 biofilm formation without affecting the growth of planktonic cells, while phloretin did not harm commensalE. coliK-12 biofilms. Also, phloretin reducedE. coliO157:H7 attachment to human colon epithelial cells. Global transcriptome analyses revealed that phloretin repressed toxin genes (hlyEandstx2), autoinducer-2 importer genes (lsrACDBF), curli genes (csgAandcsgB), and dozens of prophage genes inE. coliO157:H7 biofilm cells. Electron microscopy confirmed that phloretin reduced fimbria production inE. coliO157:H7. Also, phloretin suppressed the tumor necrosis factor alpha-induced inflammatory responsein vitrousing human colonic epithelial cells. Moreover, in the rat model of colitis induced by trinitrobenzene sulfonic acid (TNBS), phloretin significantly ameliorated colon inflammation and body weight loss. Taken together, our results suggest that the antioxidant phloretin also acts as an inhibitor ofE. coliO157:H7 biofilm formation as well as an anti-inflammatory agent in inflammatory bowel diseases without harming beneficial commensalE. colibiofilms.

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Regulation of Cell Division, Biofilm Formation, and Virulence by FlhC in Escherichia coli O157:H7 Grown on Meat

Applied and Environmental Microbiology ◽

10.1128/aem.00069-11 ◽

2011 ◽

Vol 77 (11) ◽

pp. 3653-3662 ◽

Cited By ~ 10

Author(s):

Preeti Sule ◽

Shelley M. Horne ◽

Catherine M. Logue ◽

Birgit M. Prüß

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Real Time ◽

Real Time Pcr ◽

Biofilm Formation ◽

Parent Strain ◽

Cellular Functions ◽

Content Type ◽

E Coli ◽

K 12

ABSTRACTTo understand the continuous problems thatEscherichia coliO157:H7 causes as food pathogen, this study assessed global gene regulation in bacteria growing on meat. Since FlhD/FlhC ofE. coliK-12 laboratory strains was previously established as a major control point in transducing signals from the environment to several cellular processes, this study compared the expression pattern of anE. coliO157:H7 parent strain to that of its isogenicflhCmutant. This was done with bacteria that had been grown on meat. Microarray experiments revealed 287 putative targets of FlhC. Real-time PCR was performed as an alternative estimate of transcription and confirmed microarray data for 13 out of 15 genes tested (87%). The confirmed genes are representative of cellular functions, such as central metabolism, cell division, biofilm formation, and pathogenicity. An additional 13 genes from the same cellular functions that had not been hypothesized as being regulated by FlhC by the microarray experiment were tested with real-time PCR and also exhibited higher expression levels in theflhCmutant than in the parent strain. Physiological experiments were performed and confirmed that FlhC reduced the cell division rate, the amount of biofilm biomass, and pathogenicity in a chicken embryo lethality model. Altogether, this study provides valuable insight into the complex regulatory network of the pathogen that enables its survival under various environmental conditions. This information may be used to develop strategies that could be used to reduce the number of cells or pathogenicity ofE. coliO157:H7 on meat by interfering with the signal transduction pathways.

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Small-Molecule Modulators of Listeria monocytogenes Biofilm Development

Applied and Environmental Microbiology ◽

10.1128/aem.07227-11 ◽

2011 ◽

Vol 78 (5) ◽

pp. 1454-1465 ◽

Cited By ~ 32

Author(s):

Uyen T. Nguyen ◽

Iwona B. Wenderska ◽

Matthew A. Chong ◽

Kalinka Koteva ◽

Gerard D. Wright ◽

...

Keyword(s):

Fatty Acids ◽

Listeria Monocytogenes ◽

Protein Kinase ◽

Biofilm Formation ◽

De Novo ◽

Saturated Fatty Acids ◽

Antibiofilm Activity ◽

Dependent Manner ◽

Content Type ◽

Biofilm Development

ABSTRACTListeria monocytogenesis an important food-borne pathogen whose ability to form disinfectant-tolerant biofilms on a variety of surfaces presents a food safety challenge for manufacturers of ready-to-eat products. We developed here a high-throughput biofilm assay forL. monocytogenesand, as a proof of principle, used it to screen an 80-compound protein kinase inhibitor library to identify molecules that perturb biofilm development. The screen yielded molecules toxic to multiple strains ofListeriaat micromolar concentrations, as well as molecules that decreased (≤50% of vehicle control) or increased (≥200%) biofilm formation in a dose-dependent manner without affecting planktonic cell density. Toxic molecules—including the protein kinase C antagonist sphingosine—had antibiofilm activity at sub-MIC concentrations. Structure-activity studies of the biofilm inhibitory compound palmitoyl-d,l-carnitine showed that whileListeriabiofilm formation was inhibited with a 50% inhibitory concentration of 5.85 ± 0.24 μM,d,l-carnitine had no effect, whereas palmitic acid had stimulatory effects. Saturated fatty acids between C9:0and C14:0wereListeriabiofilm inhibitors, whereas fatty acids of C16:0or longer were stimulators, showing chain length specificity.De novo-synthesized short-chain acyl carnitines were less effective biofilm inhibitors than the palmitoyl forms. These molecules, whose activities against bacteria have not been previously established, are both useful probes ofL. monocytogenesbiology and promising leads for the further development of antibiofilm strategies.

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TosR-Mediated Regulation of Adhesins and Biofilm Formation in UropathogenicEscherichia coli

mSphere ◽

10.1128/msphere.00222-18 ◽

2018 ◽

Vol 3 (3) ◽

Cited By ~ 7

Author(s):

Courtney L. Luterbach ◽

Valerie S. Forsyth ◽

Michael D. Engstrom ◽

Harry L. T. Mobley

Keyword(s):

Urinary Tract ◽

Cross Talk ◽

Biofilm Formation ◽

Dependent Manner ◽

Rna Seq ◽

Concentration Dependent Manner ◽

Content Type ◽

E Coli ◽

Expression Of Genes ◽

Invasive Infections

ABSTRACTUropathogenicEscherichia colistrains utilize a variety of adherence factors that assist in colonization of the host urinary tract. TosA (typeonesecretionA) is a nonfimbrial adhesin that is predominately expressed during murine urinary tract infection (UTI), binds to kidney epithelial cells, and promotes survival during invasive infections. ThetosRCBDAEFoperon encodes the secretory machinery necessary for TosA localization to theE. colicell surface, as well as the transcriptional regulator TosR. TosR binds upstream of thetosoperon and in a concentration-dependent manner either induces or repressestosAexpression. TosR is a member of the PapB family of fimbrial regulators that can participate in cross talk between fimbrial operons. TosR also binds upstream of thepapoperon and suppresses PapA production. However, the scope of TosR-mediated cross talk is understudied and may be underestimated. To quantify the global effects of TosR-mediated regulation on theE. coliCFT073 genome, we induced expression oftosR, collected mRNA, and performed high-throughput RNA sequencing (RNA-Seq). These findings show that production of TosR affected the expression of genes involved with adhesins, including P, F1C, and Auf fimbriae, nitrate-nitrite transport, microcin secretion, and biofilm formation.IMPORTANCEUropathogenicE. colistrains cause the majority of UTIs, which are the second most common bacterial infection in humans. During a UTI, bacteria adhere to cells within the urinary tract, using a number of different fimbrial and nonfimbrial adhesins. Biofilms can also develop on the surfaces of catheters, resulting in complications such as blockage. In this work, we further characterized the regulator TosR, which links both adhesin production and biofilm formation and likely plays a crucial function during UTI and disseminated infection.

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Antibiofilm activity of aminopropanol derivatives against Pseudomonas aeruginosa

Farmatsevtychnyi zhurnal ◽

10.32352/0367-3057.1.17.12 ◽

2018 ◽

pp. 93-100

Author(s):

D. M. Dudikova ◽

Z. S. Suvorova ◽

V. V. Nedashkivska ◽

A. O. Sharova ◽

M. L. Dronova ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Biofilm Formation ◽

Antimicrobial Agents ◽

Bacterial Biofilm ◽

Antibiofilm Activity ◽

Dependent Manner ◽

Inhibition Activity ◽

Chronic Infections ◽

High Concentration ◽

Biofilm Inhibition

Bacterial biofilm, particularly formed by Pseudomonas aeruginosa, are a cause of severe chronic infectious diseases. Bacteria within a biofilm are phenotypically more resistant to antibiotics and the macroorganism immune system, making it an important virulence factor for many microbes. The aminopropanol derivatives with adamantyl (KVM-97) and N-alkylaryl radicals (KVM-194, KVM-204, KVM-261, and KVM-262) were used as study object. The aim of this study was to investigate the antibiofilm activity of compounds on biofilm formation and on mature biofilm of P. aeruginosa. The effects of the aminopropanol derivatives on the biofilm mass were evaluated by using crystal violet assay. Ciprofloxacin, meropenem, ceftazidime, gentamicin were used as reference substances. Reported results demonstrate that all compounds displayed antibiofilm activity at the tested concentrations. Remarkable reduction in biofilm formation of P. aeruginosa was found after treatment with KVM-97, KVM-261 and KVM-262 in high concentration (5× MIC), biofilm inhibition activity were 84.3%, 90.5% and 83.3% respectively. After a treatment with KVM-204 at 250 μg/ml (5× MIC) 76.6% of the preformed 24-hr biofilms were destroyed. Furthermore, compounds KVM-97, KVM-194, and KVM-261 in both concentrations showed potent antibiofilm activity against the P. aeruginosa, inhibition activity values being between 56.7 and 65.7%. All tested compounds in dose-dependent manner exhibited pronounced inhibition activity against mature 5-days P. аeruginosa biofilm. It was also observed that tested compounds show high antibiofilm activity in comparison to reference antimicrobials. The aminopropanol derivatives may provide templates for a new group of antimicrobial agents and potential future therapeutics for treating chronic infections.

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Eucalyptol inhibits biofilm formation of Streptococcus pyogenes and its mediated virulence factors

Journal of Medical Microbiology ◽

10.1099/jmm.0.001253 ◽

2020 ◽

Vol 69 (11) ◽

pp. 1308-1318

Author(s):

Karuppiah Vijayakumar ◽

Vajravelu Manigandan ◽

Danaraj Jeyapragash ◽

Veeraiyan Bharathidasan ◽

Balaiyan Anandharaj ◽

...

Keyword(s):

Gene Expression ◽

Streptococcus Pyogenes ◽

Biofilm Formation ◽

Type Species ◽

Inhibitory Concentration ◽

Antibiofilm Activity ◽

Dependent Manner ◽

Biofilm Inhibition ◽

Content Type ◽

Link Type

Introduction. Streptococcus pyogenes is a diverse virulent synthesis pathogen responsible for invasive systemic infections. Establishment of antibiotic resistance in the pathogen has produced a need for new antibiofilm agents to control the biofilm formation and reduce biofilm-associated resistance development. Aim. The present study investigates the in vitro antibiofilm activity of eucalyptol against S. pyogenes . Methodology. The antibiofilm potential of eucalyptol was assessed using a microdilution method and their biofilm inhibition efficacy was visualized by microscopic analysis. The biochemical assays were performed to assess the influence of eucalyptol on virulence productions. Real-time PCR analysis was performed to evaluate the expression profile of the virulence genes. Results. Eucalyptol showed significant antibiofilm potential in a dose-dependent manner without affecting bacterial growth. Eucalyptol at 300 µg ml−1 (biofilm inhibitory concentration) significantly inhibited the initial stage of biofilm formation in S. pyogenes . However, eucalyptol failed to diminish the mature biofilms of S. pyogenes at biofilm inhibitory concentration and it effectively reduced the biofilm formation on stainless steel, titanium, and silicone surfaces. The biochemical assay results revealed that eucalyptol greatly affects the cell-surface hydrophobicity, auto-aggregation, extracellular protease, haemolysis and hyaluronic acid synthesis. Further, the gene-expression analysis results showed significant downregulation of virulence gene expression upon eucalyptol treatment. Conclusion. The present study suggests that eucalyptol applies its antibiofilm assets by intruding the initial biofilm formation of S. pyogenes . Supplementary studies are needed to understand the mode of action involved in biofilm inhibition.

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Oxantel Disrupts Polymicrobial Biofilm Development of Periodontal Pathogens

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01375-13 ◽

2013 ◽

Vol 58 (1) ◽

pp. 378-385 ◽

Cited By ~ 8

Author(s):

Stuart Dashper ◽

Neil O'Brien-Simpson ◽

Sze Wei Liu ◽

Rita Paolini ◽

Helen Mitchell ◽

...

Keyword(s):

Bone Loss ◽

Biofilm Formation ◽

Pathogenic Bacteria ◽

Large Scale ◽

Alveolar Bone ◽

Similar Degree ◽

Dependent Manner ◽

Gene Products ◽

Content Type ◽

Biofilm Development

ABSTRACTBacterial pathogens commonly associated with chronic periodontitis are the spirocheteTreponema denticolaand the Gram-negative, proteolytic speciesPorphyromonas gingivalisandTannerella forsythia. These species rely on complex anaerobic respiration of amino acids, and the anthelmintic drug oxantel has been shown to inhibit fumarate reductase (Frd) activity in some pathogenic bacteria and inhibitP. gingivalishomotypic biofilm formation. Here, we demonstrate that oxantel inhibitedP. gingivalisFrd activity with a 50% inhibitory concentration (IC50) of 2.2 μM and planktonic growth ofT. forsythiawith a MIC of 295 μM, but it had no effect on the growth ofT. denticola. Oxantel treatment caused the downregulation of sixP. gingivalisgene products and the upregulation of 22 gene products. All of these genes are part of a regulon controlled by heme availability. There was no large-scale change in the expression of genes encoding metabolic enzymes, indicating thatP. gingivalismay be unable to overcome Frd inhibition. Oxantel disrupted the development of polymicrobial biofilms composed ofP. gingivalis,T. forsythia, andT. denticolain a concentration-dependent manner. In these biofilms, all three species were inhibited to a similar degree, demonstrating the synergistic nature of biofilm formation by these species and the dependence ofT. denticolaon the other two species. In a murine alveolar bone loss model of periodontitis oxantel addition to the drinking water ofP. gingivalis-infected mice reduced bone loss to the same level as the uninfected control.

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