Development and characterization of essential oil component-based polymer films: a potential approach to reduce bacterial biofilm

The development of new polymeric materials aimed to control the bacterial biofilm appears to be an important practical approach. The goal of the present study was to prepare and characterize poly(ethylene-co-vinyl acetate) copolymer (EVA) films containing citronellol, eugenol, and linalool and evaluate their efficiency on growth and biofilm formation of Listeria monocytogenes, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa in monospecies and dual species. The results showed that the addition of oil components influenced the elastic modulus (15 % decrease), the tensile stress (30 % decrease), the elongation at break (10 % increase), and the contact angle values (10–20° decrease) while leaving the homogeneity of the surface unaltered. Among the polymeric films, EVA + citronellol and EVA + eugenol at 7 wt% had the best inhibitory effect. After 24–48 h of incubation, EVA + citronellol was more effective against the growth (30–60 % reduction) than EVA + eugenol (15–30 % inhibition). However, this inhibition decreased after 240 h of incubation. On the contrary, the biofilm evaluation revealed a strong inhibition trend also after prolonged incubation time: the amount of biomass per square centimeter formed on copolymer with oil components was significantly less (40–70 % decrease) than that on pure copolymer control for L. monocytogenes, S. aureus, and E. coli. When polymeric materials were simultaneously inoculated with combinations of S. aureus and E. coli, the biomass accumulated was higher for EVA + citronellol and lower for EVA + eugenol than that in monoculture biofilm. The findings were similar to the results obtained by 2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide assay that measures the metabolic activity of viable cells.     

In vitro anti‐biofilm activity of Boswellia spp. oleogum resin essential oils

Aims: To evaluate the anti‐biofilm activity of the commercially available essential oils from two Boswellia species. Methods and Results: The susceptibility of staphylococcal and Candida albicans biofilms was determined by methyltiazotetrazolium (MTT) staining. At concentrations ranging from 217·3 μg ml^?1 (25% v/v) to 6·8 μg ml^?1 (0·75% v/v), the essential oil of Boswellia papyrifera showed considerable activity against both Staphylococcus epidermidis DSM 3269 and Staphylococcus aureus ATCC 29213 biofilms. The anti‐microbial efficacy of this oil against S. epidermidis RP62A biofilms was also tested using live/dead staining in combination with fluorescence microscopy, and we observed that the essential oil of B. papyrifera showed an evident anti‐biofilm effect and a prevention of adhesion at sub‐MIC concentrations. Boswellia rivae essential oil was very active against preformed C. albicans ATCC 10231 biofilms and inhibited the formation of C. albicans biofilms at a sub‐MIC concentration. Conclusions: Essential oils of Boswellia spp. could effectively inhibit the growth of biofilms of medical relevance. Significance and Impact of the Study: Boswellia spp. essential oils represent an interesting source of anti‐microbial agents in the development of new strategies to prevent and treat biofilms.     

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.     

Design,synthesis and antistaphylococcal activity of marine pyrrole alkaloid derivatives

Abstract

A novel set of 16 hybrids of bromopyrrole alkaloids with aroyl hydrazone were designed, synthesized and evaluated for antibacterial and antibiofilm activities against methicillin-resistant Staphylococcus aureus (MRSA; ATCC 43866), methicillin-susceptible Staphylococcus aureus (MSSA; ATCC 35556) and Staphylococcus epidermidis (SE, S. epidermidis ATCC 35984). Of the 16 tested hybrids, 14 exhibited equal or superior antibiofilm activity against MSSA and MRSA relative to standard vancomycin. Compound 4m showed highest potency with antibiofilm activity of 0.39?µg/mL and 0.78?µg/mL against MSSA and MRSA, respectively. Thus, this compound could act as a potential lead for further development of new antistaphylococcal drugs.     

Impact of the <Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis> LytSR two-component regulatory system on murein hydrolase activity,pyruvate utilization and global transcriptional profile

Background  

Staphylococcus epidermidis has emerged as one of the most important nosocomial pathogens, mainly because of its ability to colonize implanted biomaterials by forming a biofilm. Extensive studies are focused on the molecular mechanisms involved in biofilm formation. The LytSR two-component regulatory system regulates autolysis and biofilm formation in Staphylococcus aureus. However, the role of LytSR played in S. epidermidis remained unknown.     

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.     

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.     

Enterocin B3A-B3B produced by LAB collected from infant faeces: potential utilization in the food industry for <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> biofilm management

Enterococcus faecalis B3A-B3B produces the bacteriocin B3A-B3B with activity against Listeria monocytogenes, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium perfringens, but apparently not against fungi or Gram-negative bacteria, except for Salmonella Newport. B3A-B3B enterocin has two different nucleotides but similar amino acid composition to the class IIb MR10A-MR10B enterocin. B3A-B3B consists of two peptides of predicted molecular mass of 5176.31 Da (B3A) and 5182.21 Da (B3B). Importantly, B3A-B3B impeded biofilm formation of the foodborne pathogen L. monocytogenes 162 grown on stainless steel. The antimicrobial treatment of stainless steel with nisin (1 or 16 mg ml^?1) decreased the cell numbers by about 2 log CFU ml^?1, thereby impeding the biofilm formation by L. monocytogenes 162 or its nisin-resistant derivative strain L. monocytogenes 162R. Furthermore, the combination of nisin and B3A-B3B enterocin reduced the MIC required to inhibit this pathogen grown in planktonic or biofilm cultures.     

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.     

Characterization,Release Profile and Antimicrobial Properties of Bioactive Polyvinyl Alcohol-Alyssum homolocarpum Seed Gum- Nisin Composite Film

A common goal of active packaging is to improve the shelf life, safety, or quality of packaged foods. The integrity of an active package must be remained in order to prevent the growth of microorganisms on the surface of food. Therefore, active polyvinyl alcohol-Alyssum homolocarpum seed gum (PVA-AHSG) composite films with different nisin concentrations (3000, 5000 and 10,000 IU) were prepared and their physico-chemical and antimicrobial properties were determined. Addition of nisin to the composite films increased their water vapor permeability (WVP), elongation at break (EB) and opacity, whereas their total color difference (ΔE), glass transition temperature (Tg), melting temperature (Tm), tensile strength (TS) and young modulus (YM) were decreased. Increasing the nisin concentration remarkably increased the chain mobility, interactions between polymers and water molecules and also the free volume of polymer matrix. The antimicrobial potential of film against L. monocytogene, S. aureus and E. coli as well as the release of nisin into phosphate buffer solution (pH 7.2) were investigated. Films containing nisin had inhibition effect against gram positive pathogens among which L. monocytogenes was the most sensitive bacterium. In liquid media, all films containing nisin prevented the growth of L. monocytogenes and S. aureus, but it was only the film with 10,000 IU nisin content which was able to control 100% of the microbial population during incubation time. Nisin release and diffusion coefficient (D) increased as its concentration increased in the film matrix due to the interaction of nisin with film polymer chains. Therefore, the resultant film had appropriate controlled release property and suitable microbial inhibitory against gram positive bacteria.

Composite bioactive films based on polyvinyl alcohol-Alyssum homolocarpum seed gum blend and Nisin: Physiochemical characterization and antimicrobial properties. Monjazeb et al. (2017).

     

苦参碱对表皮葡萄球菌生物被膜作用初探

通过中药有效成分苦参碱对表皮葡萄球菌生物被膜抑制作用的研究,为表皮葡萄球菌生物被膜引起的相关感染提供新的治疗途径。利用XTT减低法评价苦参碱对表皮葡萄球菌初始粘附及生物被膜内细菌代谢的影响,镜下观察该药对表皮葡萄球菌生物被膜的形态学影响。结果表明:苦参碱对表皮葡萄球菌生物被膜菌的SMIC50和SMIC80分别为62.5 mg/L和500 mg/L;1 000 mg/L浓度的苦参碱对表皮葡萄球菌早期粘附有抑制作用;250 mg/L浓度的苦参碱对表皮葡萄球菌生物被膜的形态有显著影响。因此可见,苦参碱对表皮葡萄球菌生物被膜的形成与粘附均有抑制作用。     

Chemical Composition Determination and Evaluation of the Antimicrobial Activity of Essential Oil from Croton blanchetianus (Euphorbiaceae) against Clinically Relevant Bacteria

In this study, the chemical composition of the essential oil (EO) extracted from Croton blanchetianus Baill leaves was identified, and antimicrobial and antibiofilm activities against Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli strains were determined. Moreover, the effects of EO in combination with ampicillin and tetracycline were investigated. Thirty-four components, mainly mono-and sesquiterpenes that represented 94.05 % of the chemical composition, were identified in the EO. The EO showed bacteriostatic and bactericidal activities against all strains tested. Furthermore, the EO showed a synergistic effect with ampicillin and tetracycline. EO significantly inhibited biofilm formation and reduced the number of viable cells in biofilms. The EO may be a promising natural product for preventing bacterial biofilm infections.     

Differential Roles of Poly-N-Acetylglucosamine Surface Polysaccharide and Extracellular DNA in Staphylococcus aureus and Staphylococcus epidermidis Biofilms

Staphylococcus aureus and Staphylococcus epidermidis are major human pathogens of increasing importance due to the dissemination of antibiotic-resistant strains. Evidence suggests that the ability to form matrix-encased biofilms contributes to the pathogenesis of S. aureus and S. epidermidis. In this study, we investigated the functions of two staphylococcal biofilm matrix polymers: poly-N-acetylglucosamine surface polysaccharide (PNAG) and extracellular DNA (ecDNA). We measured the ability of a PNAG-degrading enzyme (dispersin B) and DNase I to inhibit biofilm formation, detach preformed biofilms, and sensitize biofilms to killing by the cationic detergent cetylpyridinium chloride (CPC) in a 96-well microtiter plate assay. When added to growth medium, both dispersin B and DNase I inhibited biofilm formation by both S. aureus and S. epidermidis. Dispersin B detached preformed S. epidermidis biofilms but not S. aureus biofilms, whereas DNase I detached S. aureus biofilms but not S. epidermidis biofilms. Similarly, dispersin B sensitized S. epidermidis biofilms to CPC killing, whereas DNase I sensitized S. aureus biofilms to CPC killing. We concluded that PNAG and ecDNA play fundamentally different structural roles in S. aureus and S. epidermidis biofilms.     

Anti‐biofilm activity of ultrashort cinnamic acid peptide derivatives against medical device‐related pathogens

The threat of antimicrobial resistance has placed increasing emphasis on the development of innovative approaches to eradicate multidrug‐resistant pathogens. Biofilm‐forming microorganisms, for example, Staphylococcus epidermidis and Staphylococcus aureus, are responsible for increased incidence of biomaterial infection, extended hospital stays and patient morbidity and mortality. This paper highlights the potential of ultrashort tetra‐peptide conjugated to hydrophobic cinnamic acid derivatives. These peptidomimetic molecules demonstrate selective and highly potent activity against resistant biofilm forms of Gram‐positive medical device‐related pathogens. 3‐(4‐Hydroxyphenyl)propionic)‐Orn‐Orn‐Trp‐Trp‐NH₂ displays particular promise with minimum biofilm eradication concentration (MBEC) values of 125 µg/ml against methicillin sensitive (ATCC 29213) and resistant (ATCC 43300) S. aureus and activity shown against biofilm forms of Escherichia coli (MBEC: 1000 µg/ml). Kill kinetics confirms complete eradication of established 24‐h biofilms at MBEC with 6‐h exposure. Reduced cell cytotoxicity, relative to Gram‐positive pathogens, was proven via tissue culture (HaCaT) and haemolysis assays (equine erythrocytes). Existing in nature as part of the immune response, antimicrobial peptides display great promise for exploitation by the pharmaceutical industry in order to increase the library of available therapeutic molecules. Ultrashort variants are particularly promising for translation as clinical therapeutics as they are more cost‐effective, easier to synthesise and can be tailored to specific functional requirements based on the primary sequence allowing factors such as spectrum of activity to be varied. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Discovery of quinoline small molecules with potent dispersal activity against methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis biofilms using a scaffold hopping strategy

Staphylococcus aureus and Staphylococcus epidermidis are recognized as the most frequent cause of biofilm-associated nosocomial and indwelling medical device infections. Biofilm-associated infections are known to be highly resistant to our current arsenal of clinically used antibiotics and antibacterial agents. To exacerbate this problem, no therapeutic option exists that targets biofilm-dependent machinery critical to Staphylococcal biofilm formation and maintenance. Here, we describe the discovery of a series of quinoline small molecules that demonstrate potent biofilm dispersal activity against methicillin-resistant S. aureus and S. epidermidis using a scaffold hopping strategy. This interesting class of quinolines also has select synthetic analogues that demonstrate potent antibacterial activity and biofilm inhibition against S. aureus and S. epidermidis.     

Nisin-activated hydrophobic and hydrophilic surfaces: assessment of peptide adsorption and antibacterial activity against some food pathogens

An effective antimicrobial packaging or food contact surface should be able to kill or inhibit micro-organisms that cause food-borne illnesses. Setting up such systems, by nisin adsorption on hydrophilic and hydrophobic surfaces, is still a matter of debate. For this purpose, nisin was adsorbed on two types of low-density polyethylene: the hydrophobic native film and the hydrophilic acrylic acid-treated surface. The antibacterial activity was compared for those two films and it was highly dependent on the nature of the surface and the nisin-adsorbed amount. The hydrophilic surfaces presented higher antibacterial activity and higher amount of nisin than the hydrophobic surfaces. The effectiveness of the activated surfaces was assessed against Listeria innocua and the food pathogens Listeria monocytogenes, Bacillus cereus, and Staphylococcus aureus. S. aureus was more sensitive than the three other test bacteria toward both nisin-functionalized films. Simulation tests to mimic refrigerated temperature showed that the films were effective at 20 and 4 °C with no significant difference between the two temperatures after 30 min of exposure to culture media.     

Sophorolipid biosurfactants: Possible uses as antibacterial and antibiofilm agent

Biosurfactants are amphipathic, surface-active molecules of microbial origin which accumulate at interfaces reducing interfacial tension and leading to the formation of aggregated micellular structures in solution. Some biosurfactants have been reported to have antimicrobial properties, the ability to prevent adhesion and to disrupt biofilm formation. We investigated antimicrobial properties and biofilm disruption using sophorolipids at different concentrations. Growth of Gram negative Cupriavidus necator ATCC 17699 and Gram positive Bacillus subtilis BBK006 were inhibited by sophorolipids at concentrations of 5% v/v with a bactericidal effect. Sophorolipids (5% v/v) were also able to disrupt biofilms formed by single and mixed cultures of B. subtilis BBK006 and Staphylococcus aureus ATCC 9144 under static and flow conditions, as was observed by scanning electron microscopy. The results indicated that sophorolipids may be promising compounds for use in biomedical application as adjuvants to other antimicrobial against some pathogens through inhibition of growth and/or biofilm disruption.     

Human cathelicidin peptide LL37 inhibits both attachment capability and biofilm formation of Staphylococcus epidermidis

Aims: The aim of this work was to investigate the possible effect of human cathelicidin antimicrobial peptide LL37 on biofilm formation of Staphylococcus epidermidis, a major causative agent of indwelling device‐related infections. Methods and Results: We performed initial attachment assay and biofilm formation solid surface assay in microtitre plates, as well as growth experiment in liquid medium using laboratory strain Staph. epidermidis ATCC35984. We found that already a low concentration of the peptide LL37 (1 mg l^?1) significantly decreased both the attachment of bacteria to the surface and also the biofilm mass. No growth inhibition was observed even at 16 mg l^?1 concentration of LL37, indicating a direct effect of the peptide on biofilm production. Conclusions: As biofilm protects bacteria during infections in humans and allows their survival in a hostile environment, inhibition of biofilm formation by LL37 may have a key role to prevent bacterial colonization on indwelling devices. Significance and Impact of the Study: Our findings suggest that this host defence factor can be a potential candidate in prevention and treatment strategies of Staph. epidermidis infections in humans.     

A novel application of Fourier-transformed infrared spectroscopy: classification of slime from staphylococci

Abstract

It has been proposed that the virulence of nosocomial Staphylococcus infections associated with indwelling medical devices is related to the ability of the bacterium to colonise these materials by forming a biofilm composed of multilayered cell clusters embedded in a slime matrix. However, the pathogenic role of exopolysaccharide biofilms is not fully understood. A new method was sought for differentiating the structure of slime from two closely related bacterial strains, Staphylococcus aureus and Staphylococcus epidermidis. Using PCR it was confirmed that these strains were positive for the icaA and icaD genes and the complete ica operon (2.7 kb). Monosaccharide analysis by thin-layer chromatography revealed an identical profile for both strains, with xylose and glucose present among the four visible bands. Using Fourier-transformed infrared spectroscopy and hierarchical cluster analysis, three of four S. aureus samples (75%), and four of five S. epidermidis samples were grouped according to species. A novel FTIR approach in classifying slime produced by S. aureus and S. epidermidis is reported.     

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.