Eradication of Propionibacterium acnes biofilms by plant extracts and putative identification of icariin, resveratrol and salidroside as active compounds

Propionibacterium acnes is a Gram-positive bacterium that plays an important role in the pathogenesis of acne vulgaris. This organism is capable of biofilm formation and the decreased antimicrobial susceptibility of biofilm-associated cells may hamper efficient treatment. In addition, the prolonged use of systemic antibiotic therapy is likely to lead to the development and spread of antimicrobial resistance. In the present study we investigated whether P. acnes biofilms could be eradicated by plant extracts or their active compounds, and whether other mechanisms besides killing of biofilm cells could be involved. Out of 119 plant extracts investigated, we identified five with potent antibiofilm activity against P. acnes (extracts from Epimedium brevicornum, Malus pumila, Polygonum cuspidatum, Rhodiola crenulata and Dolichos lablab). We subsequently identified icariin, resveratrol and salidroside as active compounds in three of these extracts. Extracts from E. brevicornum and P. cuspidatum, as well as their active compounds (icariin and resveratrol, respectively) showed marked antibiofilm activity when used in subinhibitory concentrations, indicating that killing of microbial cells is not their only mode of action.     

Isolation and identification of quorum quenching bacteria from environmental samples

A large number of Gram-negative pathogens produce N-acylhomoserine lactones (AHLs) as signal molecules for quorum sensing (QS). This cell-cell communication system allows them to coordinate gene expression and regulate virulence. Therefore, strategies to inhibit QS are promising for the control of infectious diseases. The aim of the present study was to develop a high-throughput method for the isolation and identification of AHL-degrading bacteria from environmental samples. Samples were cultured in a microtitre plate in a minimal medium containing 1 mM N-(3-oxo-dodecanoyl)-l-homoserine lactone and 2 mM N-(3-oxo-hexanoyl)-l-homoserine lactone as the sole sources of carbon and nitrogen. Isolates growing on this minimal medium were subcultured and identified by partial 16S rRNA gene sequencing. Subsequently, the AHL-degrading capacity of each isolate was evaluated in the Pseudomonas aeruginosa QSIS2 biosensor assay, as such or after treatment with heat or proteinase K. The 16 samples tested yielded a total of 59 isolates which are, either alone or as part of a consortium, able to use AHL signal molecules as sole sources of carbon and nitrogen. Follow-up experiments have shown that in each sample there is at least one isolate with quorum quenching (QQ) activity, and that for all samples combined, 41 isolates have QQ activity. Furthermore, heat treatment did not fully inhibit QQ activity in all isolates. In some isolates, QQ activity was lost after proteinase K treatment, while others remained able to quench QS. Therefore, it is likely that some isolates produce and secrete (a) heat-stable, low molecular weight inhibitory compound(s).     

Biofilm inhibition and drug-eluting properties of novel DMAEMA-modified polyethylene and silicone rubber surfaces

Poly(2-(dimethylaminoethyl) methacrylate) (pDMAEMA) was grafted to low density polyethylene (LDPE) and silicone rubber (SR) in order to make them less susceptible to microbial biofilm formation. The direct grafting of DMAEMA using γ-rays was an efficient and fast procedure for obtaining modified materials, which could be quaternized in a second step using methyl iodide. Raman spectroscopy showed that the grafting occurred only at the surface of the LDPE, but both at the surface and in the bulk of the SR. Consequently, the grafted chains caused changes in the surface-related features of the LDPE (water contact angle and viscoelastic behavior in the dry state) and in the bulk-related properties of the SR (swelling and viscoelasticity in the swollen state). The microbiological assays revealed that the grafted DMAEMA reduced Candida albicans biofilm formation (almost no biofilm on SR), while the quaternized surfaces inhibited C. albicans and Staphylococcus aureus biofilm by more than 99% compared to pristine materials. Modified LDPE and SR were capable of holding considerable amounts of nalidixic acid, an anionic antimicrobial drug, and sustained the release for several hours. In addition, the grafted materials were cytocompatible (fibroblast cell survival?>?70%). In conclusion, these materials have the ability to inhibit microbial biofilm formation and at the same time act as drug-eluting systems, and for that reason may hold great promise for anti-biofouling applications.     

Synthesis and evaluation of thiazolidinedione and dioxazaborocane analogues as inhibitors of AI-2 quorum sensing in Vibrio harveyi

Two focused libraries based on two types of compounds, that is, thiazolidinediones and dioxazaborocanes were designed. Structural resemblances can be found between thiazolidinediones and well-known furanone type quorum sensing (QS) inhibitors such as N-acylaminofuranones, and/or acyl-homoserine lactone signaling molecules, while dioxazaborocanes structurally resemble previously reported oxazaborolidine derivatives which antagonized autoinducer 2 (AI-2) binding to its receptor. Because of this, we hypothesized that these compounds could affect AI-2 QS in Vibrio harveyi. Although all compounds blocked QS, the thiazolidinediones were the most active AI-2 QS inhibitors, with EC₅₀ values in the low micromolar range. Their mechanism of inhibition was elucidated by measuring the effect on bioluminescence in a series of V. harveyi QS mutants and by DNA-binding assays with purified LuxR protein. The active compounds neither affected bioluminescence as such nor the production of AI-2. Instead, our results indicate that the thiazolidinediones blocked AI-2 QS in V. harveyi by decreasing the DNA-binding ability of LuxR. In addition, several dioxazaborocanes were found to block AI-2 QS by targeting LuxPQ.     

Candida albicans biofilm formation on peptide functionalized polydimethylsiloxane

In order to prevent biofilm formation by Candida albicans, several cationic peptides were covalently bound to polydimethylsiloxane (PDMS). The salivary peptide histatin 5 and two synthetic variants (Dhvar 4 and Dhvar 5) were used to prepare peptide functionalized PDMS using 4-azido-2,3,5,6-tetrafluoro-benzoic acid (AFB) as an interlinkage molecule. In addition, polylysine-, polyarginine-, and polyhistidine-PDMS surfaces were prepared. Dhvar 4 functionalized PDMS yielded the highest reduction of the number of C. albicans biofilm cells in the Modified Robbins Device. Amino acid analysis demonstrated that the amount of peptide immobilized on the modified disks was in the nanomole range. Poly-d-lysine PDMS, in particular the homopeptides with low molecular weight (2500 and 9600) showed the highest activity against C. albicans biofilms, with reductions of 93% and 91%, respectively. The results indicate that the reductions are peptide dependent.     

Quality control of fifteen probiotic products containing Saccharomyces boulardii

Aims: The yeast Saccharomyces boulardii is used as a probiotic for the prevention and treatment of diarrhoea. In this study, the quality of 15 probiotic products containing S. boulardii was verified. Methods and Results: Using microsatellite typing, the identity of all Saccharomyces strains in the products was confirmed as S. boulardii. Additionally, solid‐phase cytometry (SPC) and a plate method were used to enumerate S. boulardii cells. SPC was not only able to produce results more rapidly than plating (4 h compared to 48 h) but the cell counts obtained with SPC were significantly higher than the plate counts. Finally, we found that <1% of the S. boulardii cells survived 120 min in gastric conditions and storage for 3 months at 40°C with 75% relative humidity. Conclusions: We developed a SPC method for the quantification of viable S. boulardii cells in probiotics. Additionally, we demonstrated that gastric conditions and storage have a marked effect on the viability of the yeast cells. Significance and Impact of the Study: To our knowledge, this is the first time SPC is used for the quality control of probiotics with S. boulardii. Additionally, we demonstrated the need for gastric protection and accurate storage.     

Monitoring <Emphasis Type="Italic">ALS1</Emphasis> and <Emphasis Type="Italic">ALS3</Emphasis> Gene Expression During In Vitro <Emphasis Type="Italic">Candida albicans</Emphasis> Biofilm Formation Under Continuous Flow Conditions

ALS1 and ALS3 encode cell-surface associated glycoproteins that are considered to be important for Candida albicans biofilm formation. The main goal of the present study was to monitor ALS1 and ALS3 gene expression during C. albicans biofilm formation (on silicone) under continuous flow conditions, using the Centers for Disease Control biofilm reactor (CDC reactor). For ALS1, we found few changes in gene expression until later stages of biofilm formation (72 and 96 h) when this gene appeared to be downregulated relative to the gene expression level in the start culture. We observed an induction of ALS3 gene expression in the initial stages of biofilm formation (0.5, 1, and 6 h), whereas at later stages, this gene was also downregulated relative to the gene expression level in the start culture. We also found that biofilms of an als3/als3 deletion mutant contained less filaments at several time points (1, 6, 24, and 48 h), although filamentation as such was not affected in this strain. Together, our data indicate an important role for ALS3 in the early phases of biofilm formation in the CDC reactor, probably related to adhesion of filaments, while the role of ALS1 is less clear.     

Use of the modified Robbins device to study the in vitro biofilm removal efficacy of NitrAdine, a novel disinfecting formula for the maintenance of oral medical devices

Aims:  To evaluate the use of the modified Robbins device (MRD) to test disinfection strategies against biofilms that form on oral medical devices and to test the biofilm removal efficacy of NitrAdine^TM, a disinfectant for the maintenance of oral medical devices.
Methods and Results:  Biofilms were grown on discs using the MRD and biofilms formed in this system were used to evaluate the efficacy of NitrAdine^TM and to determine the optimal disinfection conditions. Our data indicate that the use of the MRD allows for the rapid and reproducible formation of high-density biofilms. Determination of the efficacy of NitrAdine^TM revealed high activity against biofilms tested (e.g. >3 log reduction for Candida albicans and Staphylococcus aureus ) and allowed the determination of the optimal conditions for its use.
Conclusion:  The high reproducibility and flexibility of the MRD make it an excellent candidate for standardized testing of disinfectants aimed at reducing biofilms on oral medical devices. Using this system, we were able to demonstrate that NitrAdine^TM exhibits high activity against biofilms formed by the micro-organisms tested.
Significance and Impact of the Study:  Our data suggest that our procedure is appropriate for standardized testing of disinfectants aimed at reducing biofilms on oral medical devices.     

Diversity and significance of Burkholderia species occupying diverse ecological niches

Members of the genus Burkholderia are versatile organisms that occupy a surprisingly wide range of ecological niches. These bacteria are exploited for biocontrol, bioremediation and plant growth promotion purposes, but safety issues regarding human infections, especially in cystic fibrosis patients, have not been solved. This minireview gives an overview of the taxonomic and ecological diversity of the genus with particular emphasis on strains belonging to the Burkholderia cepacia complex and addresses the important question whether 'good' and 'bad' strains are actually the same.