Synergistic Antimicrobial Activity of Supplemented Medical-Grade Honey against Pseudomonas aeruginosa Biofilm Formation and Eradication.

Biofilms hinder wound healing. Medical-grade honey (MGH) is a promising therapy because of its broad-spectrum antimicrobial activity and the lack of risk for resistance. This study investigated the inhibitory and eradicative activity against multidrug-resistant Pseudomonas aeruginosa biofilms by different established MGH-based wound care formulations. Six different natural wound care products (Medihoney, Revamil, Mebo, Melladerm, L-Mesitran Ointment, and L-Mesitran Soft) were tested in vitro. Most of them contain MGH only, whereas some were supplemented. L-Mesitran Soft demonstrated the most potent antimicrobial activity (6.08-log inhibition and 3.18-log eradication). Other formulations ranged between 0.89-log and 4.80-log inhibition and 0.65-log and 1.66-log eradication. Therefore, the contribution of different ingredients of L-Mesitran Soft was investigated in more detail. The activity of the same batch of raw MGH (1.38-log inhibition and 2.35-log eradication), vitamins C and E (0.95-log inhibition and 0.94-log eradication), and all ingredients except MGH (1.69-log inhibition and 0.75-log eradication) clearly support a synergistic activity of components within the L-Mesitran Soft formulation. Several presented clinical cases illustrate its clinical antimicrobial efficacy against Pseudomonas aeruginosa biofilms. In conclusion, MGH is a potent treatment for Pseudomonas biofilms. L-Mesitran Soft has the strongest antimicrobial activity, which is likely due to the synergistic activity mediated by its supplements.

Influence of the lung microbiome on antibiotic susceptibility of cystic fibrosis pathogens.

The lungs of patients with cystic fibrosis (CF) are colonised by a microbial community comprised of pathogenic species, such as Pseudomonas aeruginosa and Staphylococcus aureus, and microorganisms that are typically not associated with worse clinical outcomes (considered as commensals). Antibiotics directed at CF pathogens are often not effective and a discrepancy is observed between activity of these agents in vitro and in the patient. This review describes how interspecies interactions within the lung microbiome might influence the outcome of antibiotic treatment targeted at common CF pathogens. Protective mechanisms by members of the microbiome such as antibiotic degradation (indirect pathogenicity), alterations of the cell wall, production of matrix components decreasing antibiotic penetration, and changes in metabolism are discussed. Interspecies interactions that increase bacterial susceptibility are also addressed. Furthermore, we discuss how experimental conditions, such as culture media, oxygen levels, incorporation of host-pathogen interactions, and microbial community composition may influence the outcome of microbial interaction studies related to antibiotic activity. Hereby, the importance to create in vitro conditions reflective of the CF lung microenvironment is highlighted. Understanding the role of the CF lung microbiome in antibiotic efficacy may help find novel therapeutic and diagnostic approaches to better tackle chronic lung infections in this patient population.

Antimicrobial Tolerance and Metabolic Adaptations in Microbial Biofilms.

Active bacterial metabolism is a prerequisite for optimal activity of many classes of antibiotics. Hence, bacteria have developed strategies to reduce or modulate metabolic pathways to become tolerant. This review describes the tight relationship between metabolism and tolerance in bacterial biofilms, and how physicochemical properties of the microenvironment at the host-pathogen interface (such as oxygen and nutritional content) are key to this relationship. Understanding how metabolic adaptations lead to tolerance brings us to novel approaches to tackle antibiotic-tolerant biofilms. We describe the use of hyperbaric oxygen therapy, metabolism-stimulating metabolites, and alternative strategies to redirect bacterial metabolism towards an antibiotic-susceptible phenotype.

Pectin-bioactive glass self-gelling, injectable composites with high antibacterial activity.

The present work focuses on the development of novel injectable, self-gelling composite hydrogels based on two types of low esterified amidated pectins from citrus peels and apple pomace. Sol-gel-derived, calcium-rich bioactive glass (BG) fillers in a particle form are applied as delivery vehicles for the release of Ca2+ ions to induce internal gelation of pectins. Composites were prepared by a relatively simple mixing technique, using 20% w/v BG particles of two different sizes (2.5 and <45 µm). Smaller particles accelerated pectin gelation slightly faster than bigger ones, which appears to result from the higher rate of Ca2+ ion release. µCT showed inhomogeneous distribution of the BG particles within the hydrogels. All composite hydrogels exhibited strong antibacterial activity against methicilin-resistant Staphylococcus aureus. The mineralization process of pectin-BG composite hydrogels occurred upon incubation in simulated body fluid for 28 days. In vitro studies demonstrated cytocompatibility of composite hydrogels with MC3T3-E1 osteoblastic cells.

Biogenic selenium nanoparticles synthesized by Stenotrophomonas maltophilia SeITE02 loose antibacterial and antibiofilm efficacy as a result of the progressive alteration of their organic coating layer.

Increasing emergence of drug-resistant microorganisms poses a great concern to clinicians; thus, new active products are urgently required to treat a number of infectious disease cases. Different metallic and metalloid nanoparticles have so far been reported as possessing antimicrobial properties and proposed as a possible alternative therapy against resistant pathogenic microorganisms. In this study, selenium nanoparticles (SeNPs) synthesized by the environmental bacterial isolate Stenotrophomonas maltophilia SeITE02 were shown to exert a clear antimicrobial and antibiofilm activity against different pathogenic bacteria, either reference strains or clinical isolates. Antimicrobial and antibiofilm capacity seems to be strictly linked to the organic cap surrounding biogenic nanoparticles, although the actual role played by this coating layer in the biocidal action remains still undefined. Nevertheless, evidence has been gained that the progressive loss in protein and carbohydrate content of the organic cap determines a decrease in nanoparticle stability. This leads to an alteration of size and electrical properties of SeNPs along with a gradual attenuation of their antibacterial efficacy. Denaturation of the coating layer was proved even to have a negative effect on the antibiofilm activity of these nanoparticles. The pronounced antimicrobial efficacy of biogenic SeNPs compared to the denatured ones can - in first instance - be associated with their smaller dimensions. This study showed that the native organic coating layer of biogenic SeNPs functions in avoiding aggregation and maintaining electrostatic stability of the nanoparticles, thus allowing them to maintain efficient antimicrobial and antibiofilm capabilities.

Whey Protein Complexes with Green Tea Polyphenols: Antimicrobial, Osteoblast-Stimulatory, and Antioxidant Activities.

Polyphenols are known for their antimicrobial activity, whilst both polyphenols and the globular protein ß-lactoglobulin (bLG) are suggested to have antioxidant properties and promote cell proliferation. These are potentially useful properties for a tissue-engineered construct, though it is unknown if they are retained when both compounds are used in combination. In this study, a range of different microbes and an osteoblast-like cell line (human fetal osteoblast, hFOB) were used to assess the combined effect of: (1) green tea extract (GTE), rich in the polyphenol epigallocatechin gallate (EGCG), and (2) whey protein isolate (WPI), rich in bLG. It was shown that approximately 20-48% of the EGCG in GTE reacted with WPI. GTE inhibited the growth of Gram-positive bacteria, an effect which was potentiated by the addition of WPI. GTE alone also significantly inhibited the growth of hFOB cells after 1, 4, and 7 days of culture. Alternatively, WPI significantly promoted hFOB cell growth in the absence of GTE and attenuated the effect of GTE at low concentrations (64 µg/mL) after 4 and 7 days. Low concentrations of WPI (50 µg/mL) also promoted the expression of the early osteogenic marker alkaline phosphatase (ALP) by hFOB cells, whereas GTE inhibited ALP activity. Therefore, the antioxidant effects of GTE can be boosted by WPI, but GTE is not suitable to be used as part of a tissue-engineered construct due to its cytotoxic effects which negate any positive effect WPI has on cell proliferation.

Optimization and characterization of a murine lung infection model for the evaluation of novel therapeutics against Burkholderia cenocepacia.

Several B. cenocepacia mouse models are available to study the pulmonary infection by this Burkholderia cepacia complex (BCC) species. However, a characterized B. cenocepacia mouse model to evaluate the efficacy of potential new antibacterial therapies is not yet described. Therefore, we optimized and validated the course of infection (i.e. bacterial proliferation in lung, liver and spleen) and the efficacy of a reference antibiotic, tobramycin (TOB), in a mouse lung infection model. Furthermore, the local immune response and histological changes in lung tissue were studied during infection and treatment. A reproducible lung infection was observed when immunosuppressed BALB/c mice were infected with B. cenocepacia LMG 16656. Approximately 50 to 60% of mice infected with this BCC species demonstrated a dissemination to liver and spleen. TOB treatment resulted in a two log reduction in lung burden, prevented dissemination of B. cenocepacia to liver and spleen and significantly reduced levels of proinflammatory cytokines. As this mouse model is characterized by a reproducible course of infection and efficacy of TOB, it can be used as a tool for the in vivo evaluation of new antibacterial therapies.

Taking the Silver Bullet Colloidal Silver Particles for the Topical Treatment of Biofilm-Related Infections.

Biofilms are aggregates of bacteria residing in a self-assembled matrix, which protects these sessile cells against external stress, including antibiotic therapies. In light of emerging multidrug-resistant bacteria, alternative strategies to antibiotics are emerging. The present study evaluated the activity of colloidal silver nanoparticles (AgNPs) of different shapes against biofilms formed by Staphylococcus aureus (SA), methicillin-resistant SA (MRSA), and Pseudomonas aeruginosa (PA). Colloidal quasi-spherical, cubic, and star-shaped AgNPs were synthesized, and their cytotoxicity on macrophages (THP-1) and bronchial epithelial cells (Nuli-1) was analyzed by the lactate dehydrogenase assay. The antibiofilm activity was assessed in vitro by the resazurin assay and in an in vivo infection model in Caenorhabditis elegans. Cubic and star-shaped AgNPs induced cytotoxicity, while quasi-spherical AgNPs were not toxic. Quasi-spherical AgNPs showed substantial antibiofilm activity in vitro with 96% (±2%), 97% (±1%), and 98% (±1%) biofilm killing of SA, MRSA, and PA, respectively, while significantly reducing mortality of infected nematodes. The in vivo antibiofilm activity was linked to the accumulation of AgNPs in the intestinal tract of C. elegans as observed by 3D X-ray tomography. Quasi-spherical AgNPs were physically stable in suspension for over 6 months with no observed loss in antibiofilm activity. While toxicity and stability limited the utilization of cubic and star-shaped AgNPs, quasi-spherical AgNPs could be rapidly synthesized, were stable and nontoxic, and showed substantial in vitro and in vivo activity against clinically relevant biofilms. Quasi-spherical AgNPs hold potential as pharmacotherapy, for example, as topical treatment for biofilm-related infections.

Evaluation of combination therapy for Burkholderia cenocepacia lung infection in different in vitro and in vivo models.

Burkholderia cenocepacia is an opportunistic pathogen responsible for life-threatening infections in cystic fibrosis patients. B. cenocepacia is extremely resistant towards antibiotics and therapy is complicated by its ability to form biofilms. We investigated the efficacy of an alternative antimicrobial strategy for B. cenocepacia lung infections using in vitro and in vivo models. A screening of the NIH Clinical Collection 1&2 was performed against B. cenocepacia biofilms formed in 96-well microtiter plates in the presence of tobramycin to identify repurposing candidates with potentiator activity. The efficacy of selected hits was evaluated in a three-dimensional (3D) organotypic human lung epithelial cell culture model. The in vivo effect was evaluated in the invertebrate Galleria mellonella and in a murine B. cenocepacia lung infection model. The screening resulted in 60 hits that potentiated the activity of tobramycin against B. cenocepacia biofilms, including four imidazoles of which econazole and miconazole were selected for further investigation. However, a potentiator effect was not observed in the 3D organotypic human lung epithelial cell culture model. Combination treatment was also not able to increase survival of infected G. mellonella. Also in mice, there was no added value for the combination treatment. Although potentiators of tobramycin with activity against biofilms of B. cenocepacia were identified in a repurposing screen, the in vitro activity could not be confirmed nor in a more sophisticated in vitro model, neither in vivo. This stresses the importance of validating hits resulting from in vitro studies in physiologically relevant model systems.

Screening a repurposing library for potentiators of antibiotics against Staphylococcus aureus biofilms.

Staphylococcus aureus biofilms are involved in a wide range of infections that are extremely difficult to treat with conventional antibiotic therapy. We aimed to identify potentiators of antibiotics against mature biofilms of S. aureus Mu50, a methicillin-resistant and vancomycin-intermediate-resistant strain. Over 700 off-patent drugs from a repurposing library were screened in combination with vancomycin in a microtitre plate (MTP)-based biofilm model system. This led to the identification of 25 hit compounds, including four phenothiazines among which thioridazine was the most potent. Their activity was evaluated in combination with other antibiotics both against planktonic and biofilm-grown S. aureus cells. The most promising combinations were subsequently tested in an in vitro chronic wound biofilm infection model. Although no synergistic activity was observed against planktonic cells, thioridazine potentiated the activity of tobramycin, linezolid and flucloxacillin against S. aureus biofilm cells. However, this effect was only observed in a general biofilm model and not in a chronic wound model of biofilm infection. Several drug compounds were identified that potentiated the activity of vancomycin against biofilms formed in a MTP-based biofilm model. A selected hit compound lost its potentiating activity in a model that mimics specific aspects of wound biofilms. This study provides a platform for discovering and evaluating potentiators against bacterial biofilms and highlights the necessity of using relevant in vitro biofilm model systems.

Novel hamamelitannin analogues for the treatment of biofilm related MRSA infections-A scaffold hopping approach.

Antimicrobial research is increasingly being focused on the problem of resistance and biofilm formation. Hamamelitannin (HAM) was recently identified as an antimicrobial potentiator for conventional antibiotics towards Staphylococcus aureus. This paper describes the synthesis and biological evaluation of novel hamamelitannin analogues with alternative central scaffolds. Via a ligand-based approach, several interesting compounds with improved synthetic accessibility were identified as potentiators for vancomycin in the treatment of MRSA infections.

Design, synthesis and biological evaluation of novel hamamelitannin analogues as potentiators for vancomycin in the treatment of biofilm related Staphylococcus aureus infections.

Staphylococcus aureus is a frequent cause of biofilm-related infections. Bacterial cells within a biofilm are protected from attack by the immune system and conventional antibiotics often fail to penetrate the biofilm matrix. The discovery of hamamelitannin as a potentiator for antibiotics, recently led to the design of a more drug-like lead. In the present study, we want to gain further insight into the structure-activity relationship (S.A.R.) of the 5-position of the molecule, by preparing a library of 21 hamamelitannin analogues.

Biofilm-grown Burkholderia cepacia complex cells survive antibiotic treatment by avoiding production of reactive oxygen species.

The presence of persister cells has been proposed as a factor in biofilm resilience. In the present study we investigated whether persister cells are present in Burkholderia cepacia complex (Bcc) biofilms, what the molecular basis of antimicrobial tolerance in Bcc persisters is, and how persisters can be eradicated from Bcc biofilms. After treatment of Bcc biofilms with high concentrations of various antibiotics often a small subpopulation survived. To investigate the molecular mechanism of tolerance in this subpopulation, Burkholderia cenocepacia biofilms were treated with 1024 µg/ml of tobramycin. Using ROS-specific staining and flow cytometry, we showed that tobramycin increased ROS production in treated sessile cells. However, approximately 0.1% of all sessile cells survived the treatment. A transcriptome analysis showed that several genes from the tricarboxylic acid cycle and genes involved in the electron transport chain were downregulated. In contrast, genes from the glyoxylate shunt were upregulated. These data indicate that protection against ROS is important for the survival of persisters. To confirm this, we determined the number of persisters in biofilms formed by catalase mutants. The persister fraction in ΔkatA and ΔkatB biofilms was significantly reduced, confirming the role of ROS detoxification in persister survival. Pretreatment of B. cenocepacia biofilms with itaconate, an inhibitor of isocitrate lyase (ICL), the first enzyme in the glyoxylate shunt, reduced the persister fraction approx. 10-fold when the biofilms were subsequently treated with tobramycin. In conclusion, most Bcc biofilms contain a significant fraction of persisters that survive treatment with high doses of tobramycin. The surviving persister cells downregulate the TCA cycle to avoid production of ROS and at the same time activate an alternative pathway, the glyoxylate shunt. This pathway may present a novel target for combination therapy.

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.

Role of PvdQ in Pseudomonas aeruginosa virulence under iron-limiting conditions.

PvdQ, an acylase from Pseudomonas aeruginosa PAO1, has been shown to have at least two functions. It can act as a quorum quencher due to its ability to degrade long-chain N-acylhomoserine lactones (AHLs), e.g. 3-oxo-C12-HSL, leading to a decrease in virulence factors. In addition, PvdQ is involved in iron homeostasis by playing a role in the biosynthesis of pyoverdine, the major siderophore of P. aeruginosa. In accordance with earlier studies on RNA level, we could show at the protein level that PvdQ is only expressed when iron is present at very low concentrations. We therefore set out to investigate the two functions of PvdQ under iron-limiting conditions. Gene deletion of pvdQ does not affect growth of P. aeruginosa but abrogates pyoverdine production, and results in an accumulation of 3-oxo-C12-HSL. Phenotypic analyses of our DeltapvdQ mutant at low iron concentrations revealed that this mutant is impaired in swarming motility and biofilm formation. Additionally, a plant and a Caenorhabditis elegans infection model demonstrated that the deletion of pvdQ resulted in reduced virulence. None of the phenotypes in the present study could be linked to the presence or absence of AHLs. These results clearly indicate that under iron-limiting conditions PvdQ plays a major role in swarming motility, in biofilm development and in infection that is more likely to be linked to the pyoverdine pathway rather than the LasI/LasR/3-oxo-C12-HSL quorum-sensing circuit.

Development and evaluation of different normalization strategies for gene expression studies in Candida albicans biofilms by real-time PCR.

BACKGROUND: Candida albicans biofilms are commonly found on indwelling medical devices. However, the molecular basis of biofilm formation and development is not completely understood. Expression analysis of genes potentially involved in these processes, such as the ALS (Agglutinine Like Sequence) gene family can be performed using quantitative PCR (qPCR). In the present study, we investigated the expression stability of eight housekeeping genes potentially useful as reference genes to study gene expression in Candida albicans (C. albicans) biofilms, using the geNorm Visual Basic Application (VBA) for Microsoft Excel. To validate our normalization strategies we determined differences in ALS1 and ALS3 expression levels between C. albicans biofilm cells and their planktonic counterparts. RESULTS: The eight genes tested in this study are ranked according to their expression stability (from most stable to least stable) as follows: ACT1 (beta-actin)/PMA1 (adenosine triphosphatase), RIP (ubiquinol cytochrome-c reductase complex component), RPP2B (cytosolic ribosomal acidic protein P2B), LSC2 (succinyl-CoA synthetase beta-subunit fragment), IMH3 (inosine-5'-monophosphate dehydrogenase fragment), CPA1 (carbamoyl-phosphate synthethase small subunit) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Our data indicate that five genes are necessary for accurate and reliable normalization of gene expression data in C. albicans biofilms. Using different normalization strategies, we found a significant upregulation of the ALS1 gene and downregulation of the ALS3 gene in C. albicans biofilms grown on silicone disks in a continous flow system, the CDC reactor (Centre for Disease Control), for 24 hours. CONCLUSION: In conclusion, we recommend the use of the geometric mean of the relative expression values from the five housekeeping genes (ACT1, PMA1, RIP, RPP2B and LSC2) for normalization, when analysing differences in gene expression levels between C. albicans biofilm cells and planktonic cells. Validation of the normalization strategies described above showed that the ALS1 gene is overexpressed and the ALS3 gene is underexpressed in C. albicans biofilms grown on silicone in the CDC reactor for 24 hours.