Non-toxic plant metabolites regulate Staphylococcus viability and biofilm formation: a natural therapeutic strategy useful in the treatment and prevention of skin infections

In the present study, the efficacy of generally recognised as safe (GRAS) antimicrobial plant metabolites in regulating the growth of Staphylococcus aureus and S. epidermidis was investigated. Thymol, carvacrol and eugenol showed the strongest antibacterial action against these microorganisms, at a subinhibitory concentration (SIC) of ≤ 50 μg ml^?1. Genistein, hydroquinone and resveratrol showed antimicrobial effects but with a wide concentration range (SIC = 50–1,000 μg ml^?1), while catechin, gallic acid, protocatechuic acid, p-hydroxybenzoic acid and cranberry extract were the most biologically compatible molecules (SIC ≥ 1000 μg ml^?1). Genistein, protocatechuic acid, cranberry extract, p-hydroxybenzoic acid and resveratrol also showed anti-biofilm activity against S. aureus, but not against S. epidermidis in which, surprisingly, these metabolites stimulated biofilm formation (between 35% and 1,200%). Binary combinations of cranberry extract and resveratrol with genistein, protocatechuic or p-hydroxibenzoic acid enhanced the stimulatory effect on S. epidermidis biofilm formation and maintained or even increased S. aureus anti-biofilm activity.     

Extracellular protease in <Emphasis Type="Italic">Actinomycetes</Emphasis> culture supernatants inhibits and detaches <Emphasis Type="Italic">Staphylococcus</Emphasis><Emphasis Type="Italic">aureus</Emphasis> biofilm formation

Bacterial biofilms are associated with chronic infections due to their resistance to antimicrobial agents. Staphylococcus aureus is a versatile human pathogen and can form biofilms on human tissues and diverse medical devices. To identify novel biofilm inhibitors of S. aureus, the supernatants from a library of 458 Actinomycetes strains were screened. The culture supernatants (1% v/v) of more than 10 Actinomycetes strains inhibited S. aureus biofilm formation by more than 80% without affecting the growth. The culture supernatants of these biofilm-reducing Actinomycetes strains contained a protease (equivalent to 0.1 μg proteinase K ml⁻¹), which both inhibited S. aureus biofilm formation and detached pre-existing S. aureus biofilms. This study suggests that protease treatment could be a feasible tool to reduce and eradicate S. aureus biofilms.     

Intra-clade metabolomic profiling of MAR4 Streptomyces from the Macaronesia Atlantic region reveals a source of anti-biofilm metabolites

The search for new and effective strategies to reduce bacterial biofilm formation is of utmost importance as bacterial resistance to antibiotics continues to emerge. The use of anti-biofilm agents that can disrupt recalcitrant bacterial communities can be an advantageous alternative to antimicrobials, as their use does not lead to the development of resistance mechanisms. Six MAR4 Streptomyces strains isolated from the Madeira Archipelago, at the unexplored Macaronesia Atlantic ecoregion, were used to study the chemical diversity of produced hybrid isoprenoids. These marine actinomycetes were investigated by analysing their crude extracts using LC–MS/MS and their metabolomic profiles were compared using multivariate statistical analysis (principal component analysis), showing a separation trend closely related to their phylogeny. Molecular networking unveiled the presence of a class of metabolites not previously described from MAR4 strains and new chemical derivatives belonging to the napyradiomycin and marinone classes. Furthermore, these MAR4 strains produce metabolites that inhibit biofilm formation of Staphylococcus aureus and Marinobacter hydrocarbonoclasticus. The anti-biofilm activity of napyradiomycin SF2415B3 ( 1 ) against S. aureus was confirmed.     

In vitro and in vivo antibiofilm activity of a coral associated actinomycete against drug resistant Staphylococcus aureus biofilms

Staphylococcus aureus is now amongst the most important pathogenic bacteria responsible for bloodstream nosocomial infections and for biofilm formation on indwelling medical devices. Its increasing resistance to common antibiotics, partly attributed to its ability to form biofilms, is a challenge for the development of new antimicrobial agents. Accordingly, the goal of this study was to evaluate the effect of a coral associated actinomycete (CAA) - 3 on S. aureus biofilms both in vitro and in vivo. Methanolic extracts of CAA-3 showed a reduction in in vitro biofilm formation by S. aureus ATCC 11632, methicillin resistant S. aureus ATCC 33591 and clinical isolates of S. aureus at the biofilm inhibitory concentration (BIC) of 0.1 mg ml^?1. Furthermore, confocal laser scanning microscope (CLSM) studies provide evidence of CAA-3 inhibiting intestinal colonisation of S. aureus in the nematode Caenorhabditis elegans. To conclude, this study for the first time, reports CAA as a promising source of anti-biofilm compounds, for developing novel drugs against highly resistant staphylococcal biofilms.     

Thermostable xylanase inhibits and disassembles Pseudomonas aeruginosa biofilms

Pseudomonas aeruginosa biofilms are problematic and play a critical role in the persistence of chronic infections because of their ability to tolerate antimicrobial agents. In this study, various cell-wall degrading enzymes were investigated for their ability to inhibit biofilm formation of two P. aeruginosa strains, PAO1 and PA14. Xylanase markedly inhibited and detached P. aeruginosa biofilms without affecting planktonic growth. Xylanase treatment broke down extracellular polymeric substances and decreased the viscosity of P. aeruginosa strains. However, xylanase treatment did not change the production of pyochelin, pyocyanin, pyoverdine, the Pseudomonas quinolone signal, or rhamnolipid. In addition, the anti-biofilm activity of xylanase was thermally stable for > 100 days at 45°C. Also, xylanase showed anti-biofilm activity against one methicillin-resistance Staphylococcus aureus and two Escherichia coli strains.     

Inhibitory effect of α-mangostin on Acinetobacter baumannii biofilms – an in vitro study

The present study was designed to investigate the anti-biofilm potential of alpha-mangostin (α-MG) against Acinetobacter baumannii (AB). The biofilm inhibitory concentration (BIC) of α-MG against AB was found to be 2 μg ml^?1. α-MG (0.5, 1 and 2 μg ml^?1) exhibited non-bactericidal concentration-dependent anti-biofilm activities against AB. However, α-MG failed to disintegrate the mature biofilms of AB even at a 10-fold increased concentration from its BIC. Results from qRT-PCR and in vitro bioassays further demonstrated that α-MG downregulated the expression of bfmR, pgaA, pgaC, csuA/B, ompA, bap, katE, and sodB genes, which correspondingly affects biofilm formation and its associated virulence traits. The present study suggests that α-MG exerts its anti-biofilm property by interrupting initial biofilm formation and the cell-to-cell signaling mechanism of AB. Additional studies are required to understand the mode of action responsible for the anti-biofilm property.     

Staphylococcus aureus biofilm susceptibility to small and potent β2,2-amino acid derivatives

Small antimicrobial β^2,2-amino acid derivatives (Mw < 500 Da) are reported to display high antibacterial activity against suspended Gram-positive strains combined with low hemolytic activity. In the present study, the anti-biofilm activity of six β^2,2-amino acid derivatives (A1–A6) against Staphylococcus aureus (ATCC 25923) was investigated. The derivatives displayed IC₅₀ values between 5.4 and 42.8 μM for inhibition of biofilm formation, and concentrations between 22.4 and 38.4 μM had substantial effects on preformed biofilms. The lead derivative A2 showed high killing capacity (log R), and it caused distinct ultrastructural changes in the biofilms as shown by electron and atomic force microscopy. The anti-biofilm properties of A2 was preserved under high salinity conditions. Extended screening showed also high activity of A2 against Escherichia coli (XL1 Blue) biofilms. These advantageous features together with high activity against preformed biofilms make β^2,2-amino acid derivatives a promising class of compounds for further development of anti-biofilm agents.     

In vitro activity of the antimicrobial peptide AP7121 against the human methicillin-resistant biofilm producers Staphylococcus aureus and Staphylococcus epidermidis

Abstract

In vitro activity against methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis biofilm producers from blood cultures of patients with prosthetic hip infections was evaluated. The Minimum Inhibitory Concentration (MIC) for AP7121 was determined and the bactericidal activity of AP7121 (MICx1, MICx4) against planktonic cells was studied at 4, 8 and 24?h. The biofilms formed were incubated with AP7121 (MICx1, MICx4) for 1 and 24?h. The anti-adhesion effect of an AP7121-treated inert surface over the highest MIC isolate was studied with scanning electron microscopy (SEM). The bactericidal activity of AP7121 against all the planktonic staphylococcal cells was observed at 4?h at both peptide concentrations. Dose-dependent anti-biofilm activity was detected. AP7121 (MICx4) showed bactericidal activity at 24?h in all isolates. SEM confirmed prevention of biofilm formation. This research showed the in vitro anti-biofilm activity of AP7121 against MRSA and S. epidermidis and the prevention of biofilm formation by them on an abiotic surface.     

The HIV aspartyl protease inhibitor ritonavir impairs planktonic growth,biofilm formation and proteolytic activity in Trichosporon spp.

This study evaluated the effect of the protease inhibitor ritonavir (RIT) on Trichosporon asahii and Trichosporon inkin. Susceptibility to RIT was assessed by the broth microdilution assay and the effect of RIT on protease activity was evaluated using azoalbumin as substrate. RIT was tested for its anti-biofilm properties and RIT-treated biofilms were assessed regarding protease activity, ultrastructure and matrix composition. In addition, antifungal susceptibility, surface hydrophobicity and biofilm formation were evaluated after pre-incubation of planktonic cells with RIT for 15 days. RIT (200 μg ml^?1) inhibited Trichosporon growth. RIT (100 μg ml^?1) also reduced protease activity of planktonic and biofilm cells, decreased cell adhesion and biofilm formation, and altered the structure of the biofilm and the protein composition of the biofilm matrix. Pre-incubation with RIT (100 μg ml^?1) increased the susceptibility to amphotericin B, and reduced surface hydrophobicity and cell adhesion. These results highlight the importance of proteases as promising therapeutic targets and reinforce the antifungal potential of protease inhibitors.     

A combination of ellagic acid and tetracycline inhibits biofilm formation and the associated virulence of Propionibacterium acnes in vitro and in vivo

Propionibacterium acnes is an opportunistic pathogen which has become notorious owing to its ability to form a recalcitrant biofilm and to develop drug resistance. The current study aimed to develop anti-biofilm treatments against clinical isolates of P. acnes under in vitro and in vivo conditions. A combination of ellagic acid and tetracycline (ETC; 250 μg ml^?1 + 0.312 μg ml^?1) was determined to effectively inhibit biofilm formation by P. acnes (80–91%) without affecting its growth, therefore potentially limiting the possibility of the bacterium attaining resistance. In addition, ETC reduced the production of extracellular polymeric substances (EPS) (20–26%), thereby making P. acnes more susceptible to the human immune system and antibiotics. The anti-biofilm potential of ETC was further substantiated under in vivo conditions using Caenorhabditis elegans. This study reports a novel anti-biofilm combination that could be developed as an ideal therapeutic agent with broad cosmeceutical and pharmaceutical applicability in the era of antibiotic resistance.     

Holo and apo-transferrins interfere with adherence to abiotic surfaces and with adhesion/invasion to HeLa cells in Staphylococcus spp.

Staphylococcus aureus and Staphylococcus epidermidis are the major cause of infections associated with implanted medical devices. Colonization on abiotic and biotic surfaces is often sustained by biofilm forming strains. Human natural defenses can interfere with this virulence factor. We investigated the effect of human apo-transferrin (apo-Tf, the iron-free form of transferrin, Tf) and holo-transferrin (holo-Tf, the iron-saturated form) on biofilm formation by CA-MRSA S. aureus USA300 type (ST8-IV) and S. epidermidis (a clinical isolate and ATCC 35984 strain). Furthermore S. aureus adhesion and invasion assays were performed in a eukaryotic cell line. A strong reduction in biofilm formation with both Tfs was obtained albeit at very different concentrations. In particular, the reduction in biofilm formation was higher with apo-Tf rather than obtained with holo-Tf. Furthermore, while S. aureus adhesion to eukaryotic cells was not appreciably affected, their invasion was highly inhibited in the presence of holo-Tf, and partially inhibited by the apo form. Our results suggest that Tfs could be used as antibacterial adjuvant therapy in infection sustained by staphylococci to strongly reduce their virulence related to adhesion and cellular invasion.     

Antibiofilm action of a toluidine blue O-silver nanoparticle conjugate on Streptococcus mutans: a mechanism of type I photodynamic therapy

The objective of this study was to evaluate the anti-biofilm efficacy of photodynamic therapy by conjugating a photosensitizer (TBO) with silver nanoparticles (AgNP). Streptococcus mutans was exposed to laser light (630 nm) for 70 s (9.1 J cm^?2) in the presence of a toluidine blue O–silver nanoparticle conjugate (TBO–AgNP). The results showed a reduction in the viability of bacterial cells by 4 log₁₀. The crystal violet assay, confocal laser scanning microscopy and scanning electron microscopy revealed that the TBO–AgNP conjugates inhibited biofilm formation, increased the uptake of propidium iodide and leakage of the cellular constituents, respectively. Fluorescence spectroscopic studies confirmed the generation of OH^? as a major reactive oxygen species, indicating type I phototoxicity. Both the conjugates down-regulated the expression of biofilm related genes compared to TBO alone. Hence TBO–AgNP conjugates were found to be more phototoxic against S. mutans biofilm than TBO alone.     

Anti-biofilm activity of biogenic selenium nanoparticles and selenium dioxide against clinical isolates of Staphylococcus aureus,Pseudomonas aeruginosa,and Proteus mirabilis

The aim of the present study was to investigate the anti-biofilm activity of biologically synthesized selenium nanoparticles (Se NPs) against the biofilm produced by clinically isolated bacterial strains compared to that of selenium dioxide. Thirty strains of Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis were isolated from various specimens of the patients hospitalized in different hospitals (Kerman, Iran). Quantification of the biofilm using microtiter plate assay method introduced 30% of S. aureus, 13% of P. aeruginosa and 17% of P. mirabilis isolates as severely adherent strains. Transmission electron micrograph (TEM) of the purified Se NPs (produced by Bacillus sp. MSh-1) showed individual and spherical nano-structure in the size range of 80–220 nm. Obtained results of the biofilm formation revealed that selenium nanoparticles inhibited the biofilm of S. aureus, P. aeruginosa, and P. mirabilis by 42%, 34.3%, and 53.4%, respectively, compared to that of the non-treated samples. Effect of temperature and pH on the biofilm formation in the presence of Se NPs and SeO₂ was also evaluated.     

Inhibitory effects of antibiofilm compound 1 against Staphylococcus aureus biofilms

A novel benzimidazole molecule that was identified in a small‐molecule screen and is known as antibiofilm compound 1 (ABC‐1) has been found to prevent bacterial biofilm formation by multiple bacterial pathogens, including Staphylococcus aureus, without affecting bacterial growth. Here, the biofilm inhibiting ability of 156 μM ABC‐1 was tested in various biofilm‐forming strains of S. aureus. It was demonstrated that ABC‐1 inhibits biofilm formation by these strains at micromolar concentrations regardless of the strains' dependence on Polysaccharide Intercellular Adhesin (PIA), cell wall‐associated protein dependent or cell wall‐ associated extracellular DNA (eDNA). Of note, ABC‐1 treatment primarily inhibited Protein A (SpA) expression in all strains tested. spa gene disruption showed decreased biofilm formation; however, the mutants still produced more biofilm than ABC‐1 treated strains, implying that ABC‐1 affects not only SpA but also other factors. Indeed, ABC‐1 also attenuated the accumulation of PIA and eDNA on cell surface. Our results suggest that ABC‐1 has pleotropic effects on several biofilm components and thus inhibits biofilm formation by S. aureus.     

Inhibitory Activity by Barley Coffee Components Towards Streptococcus Mutans Biofilm

It was shown that barley coffee (BC) interferes with Streptococcus mutans adsorption to hydroxyapatite. After BC component fractionation by dialysis and gel filtration chromatography (GFC), it was found that the low molecular mass (<1,000 Da) fraction (LMM fraction) containing polyphenols, zinc and fluoride ions and, above all, a high molecular mass (HMM > 1,000 kDa) melanoidin fraction display strong anti-adhesive properties towards S. mutans. In this study, we have further examined the potential of BC, BC LMM fraction and BC HMM melanoidin fraction as caries controlling agents by evaluating their anti-biofilm activity.The effects of BC and BC fractions on biofilm formation by S. mutans ATCC 25175 and its detachment from pre-developed biofilms were evaluated by microtiter plate assay. It was found that BC and its fractions, at concentrations ranging from 60 to 15 mg ml⁻¹ that are devoid of antimicrobial activity, inhibited S. mutans biofilm formation. An increase of S. mutans ATCC 25175 detachment from 24 h developed biofilm was observed at the highest tested concentrations. Interestingly, BC and BC fractions also showed anti-biofilm activity towards a variety of S. mutans clinical strains isolated from saliva, plaque and caries lesions of adult donors. In general, the HMM melanoidin fraction was more active than the LMM fraction. These findings, classifying BC LMM fraction and BC HMM melanoidin fractions as natural anti-biofilm agents, represent the basis for studying their possible use as anti-caries agents.     

One pot synthesis and anti-biofilm potential of copper nanoparticles (CuNPs) against clinical strains of Pseudomonas aeruginosa

Pseudomonas aeruginosa, an opportunistic pathogen frequently associated with nosocomial infections, is emerging as a serious threat due to its resistance to broad spectrum antimicrobials. The biofilm mode of growth confers resistance to antibiotics and novel anti-biofilm agents are urgently needed. Nanoparticle based treatments and therapies have been of recent interest because of their versatile applications. This study investigates the anti-biofilm activity of copper nanoparticles (CuNPs) synthesized by the one pot method against P. aeruginosa. Standard physical techniques including UV–visible and Fourier transform infrared spectroscopy, X-ray diffraction and transmission electron microscopy were used to characterize the synthesized CuNPs. CuNP treatments at 100 ng ml^?1 resulted in a 94, 89 and 92% reduction in biofilm, cell surface hydrophobicity and exopolysaccharides respectively, without bactericidal activity. Evidence of biofilm inhibition was also seen with light and confocal microscope analysis. This study highlights the anti-biofilm potential of CuNPs, which could be utilized as coating agents on surgical devices and medical implants to manage biofilm associated infections.     

Synergistic antibacterial and anti-biofilm activity of nisin like bacteriocin with curcumin and cinnamaldehyde against ESBL and MBL producing clinical strains

Abstract

Bacteriocins are small peptides that can inhibit the growth of a diverse range of microbes. There is a need to identify bacteriocins that are effective against biofilms of resistant clinical strains. The present study focussed on the efficacy of purified nisin like bacteriocin-GAM217 against extended spectrum β-lactamase (ESBL) and metallo-beta-lactamase (MBL) producing clinical strains. Bacteriocin-GAM217 when combined with curcumin and cinnamaldehyde, synergistically enhanced antibacterial activity against planktonic and biofilm cultures of Staphylococcus epidermidis and Escherichia coli. Bacteriocin-GAM217 and phytochemical combinations inhibited biofilm formation by >80%, and disrupted the biofilm for selected ESBL and MBL producing clinical strains. The anti-adhesion assay showed that these combinatorial compounds significantly lowered the attachment of bacteria to Vero cells and that they elicited membrane permeability and rapid killing as viewed by confocal microscopy. This study demonstrates that bacteriocin-GAM217 in combination with phytochemicals can be a potential anti-biofilm agent and thus has potential for biomedical applications.     

Inhibitory effects of the essential oils α-longipinene and linalool on biofilm formation and hyphal growth of Candida albicans

Candida albicans is one of the most common fungal pathogens, and causes systemic and invasive infections in humans. C. albicans biofilms are composed of yeast and hyphal and pseudohyphal elements, and the transition of yeast to the hyphal stage could be a virulence factor. In this study, diverse essential oils were initially investigated for anti-biofilm activity against C. albicans strains, and cascarilla bark oil and helichrysum oil and their components α-longipinene (a major constituent of both) and linalool were found to markedly inhibit biofilm formation without affecting planktonic cell growth. Moreover, α-longipinene and linalool were found to synergistically reduce biofilm formation. Notably, treatments with cascarilla bark oil, helichrysum oil, α-longipinene, or linalool clearly inhibited hyphal formation, and this appeared to be largely responsible for their anti-biofilm effect. Furthermore, the two essential oils, α-longipinene and linalool, reduced C. albicans virulence in Caenorhabditis elegans.