Reduced Biofilm Biomass Research Articles

Synergistic antibacterial effects of reuterin and catechin against Streptococcus mutans

Reuterin is a broad-spectrum antimicrobial agent widely used to control pathogenic bacteria. Antimicrobial properties of catechin have been well described. However, no information is available regarding their combination effects. The aim of the present study is to evaluate the antibacterial effects of reuterin and catechin alone or in combination against Streptococcus mutans (S. mutans). We found a clear synergistic effect of reuterin in combination with catechin on inhibiting growth of S. mutans. Combined treatment with reuterin and catechin resulted in reduced biofilm biomass and water-insoluble glucan content, and visibly damaged the biofilm structure. Moreover, the expression of biofilm-associated genes was significantly down-regulated after exposure to the combination. Our results demonstrate that the combination of reuterin and catechin exhibits synergistic antimicrobial activity against S. mutans by repressing growth, biofilm formation, water-insoluble glucan production, and virulence genes expression. These findings support this novel combination can be used as a natural antibacterial agent for the prevention of oral diseases.

Tedizolid-Rifampicin Combination Prevents Rifampicin-Resistance on in vitro Model of Staphylococcus aureus Mature Biofilm.

Staphylococcus aureus infections associated with implanted medical devices are difficult to treat and require long-lasting antibiotic therapies, especially when device removal is not possible or easy such as in the case of joint prostheses. Biofilm formation is a major cause of treatment failure and infection recurrence. This study aimed to test, for the first time, the in vitro combination of tedizolid plus rifampicin on methicillin-sensitive (MSSA ATCC 6538) and methicillin-resistant (MRSA ATCC 43300) S. aureus mature biofilm. Here, we demonstrated that the combination of tedizolid with rifampicin significantly disaggregated pre-formed biofilm of both strains, reduced their metabolic activity and exerted bactericidal activity at clinically meaningful concentrations. Notably, tedizolid was able to completely prevent the emergence of resistance to rifampicin. Moreover these effects were similar to those obtained with daptomycin plus rifampicin, a well-known and widely used combination. Preliminary results on some MRSA clinical isolates confirmed the efficacy of this combination in reducing biofilm biomass and preventing rifampicin resistance onset. Further in vivo studies are needed to confirm the validity of this promising therapeutic option that can be useful against biofilm-associated S. aureus infections.

Effect of cell-free spent media prepared from Aggregatibacter actinomycetemcomitans on the growth of Candida albicans and Streptococcus mutans in co-species biofilms.

This study explored the influence of cell-free spent media prepared from Aggregatibacter actinomycetemcomitans LuxS mutant (Aa-LuxS), its wild type strain (Aa-WT), and the laboratory strain (Aa-Y4), on the interaction between Candida albicans and Streptococcus mutans while growing in co-species biofilm for 48h. By analyzing the results of crystal violet staining, [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] (MTT) assays, and quantitative real-time polymerase chain reaction (qPCR), we found that the presence of Aa-LuxS in treated biofilms did not affect biofilm development, while added Aa-WT or Aa-Y4 resulted in a significant decrease in both biofilm mass and the number of cells. The inhibitory effect of Aa-WT or Aa-Y4 was not dependent on the protein concentration in the spent media tested (1 and 10%). Gene transcription analyses indicated that Aa-WT/Aa-Y4 exhibits comparable inhibitory effects on the expression of hyphal-associated genes (ALS3 and HWP1), but not on the expression of YWP1, which encodes a yeast form of C.albicans. In contrast, except for gtfD, the expression of S.mutans gtfB/C genes encoding glucosyltransferase was not affected in Aa-WT and Aa-Y4 treated biofilms compared to the levels found in Aa-LuxS treated biofilms. Our results indicate that AI-2-containing spent media derived from Aa can reduce biofilm biomass without significantly inhibiting the survival rate of S.mutans.

Antibiofilm activity of shikonin against Listeria monocytogenes and inhibition of key virulence factors

Shikonin is a bioactive compound found in the roots of Lithospermum erythrorhizon and has demonstrated antibacterial activity. In the current study, we aimed to examine the antibiofilm activity of shikonin against Listeria monocytogenes, and to investigate its effects on key L. monocytogenes virulence factors associated with human infection. Present results showed that the minimum inhibitory concentration of shikonin against L. monocytogenes ranged from 25 to 100 μg/mL. Crystal violet staining confirmed that sub-inhibitory concentrations (SICs) of shikonin significantly reduced biofilm biomass in as little as 24 h. Decreased biofilm adhesion, altered biofilm morphology, and disruption of biofilm architecture following treatment with SICs of shikonin were also confirmed by light microscopy and field-emission scanning electron microscopy. Further, at SICs, shikonin effectively reduced the ability of L. monocytogenes to adhere to and invade HT-29 cells and prevented the secretion of listeriolysin O. Moreover, the transcription of biofilm-associated genes and key virulence genes that contribute to infection was also suppressed by shikonin at SICs. Therefore, present results suggest that shikonin could be used as an alternative antibacterial agent to combat L. monocytogenes biofilm formation and infection.

N-Nonyloxypentyl-l-Deoxynojirimycin Inhibits Growth, Biofilm Formation and Virulence Factors Expression of Staphylococcus aureus.

Staphylococcus aureus is one of the major causes of hospital- and community-associated bacterial infections throughout the world, which are difficult to treat due to the rising number of drug-resistant strains. New molecules displaying potent activity against this bacterium are urgently needed. In this study, d- and l-deoxynojirimycin (DNJ) and a small library of their N-alkyl derivatives were screened against S. aureus ATCC 29213, with the aim to identify novel candidates with inhibitory potential. Among them, N-nonyloxypentyl-l-DNJ (l-NPDNJ) proved to be the most active compound against S. aureus ATCC 29213 and its clinical isolates, with the minimum inhibitory concentration (MIC) value of 128 μg/mL. l-NPDNJ also displayed an additive effect with gentamicin and oxacillin against the gentamicin- and methicillin-resistant S. aureus isolate 00717. Sub-MIC values of l-NPDNJ affected S. aureus biofilm development in a dose-dependent manner, inducing a strong reduction in biofilm biomass. Moreover, real-time reverse transcriptase PCR analysis revealed that l-NPDNJ effectively inhibited at sub-MIC values the transcription of the spa, hla, hlb and sea virulence genes, as well as the agrA and saeR response regulator genes.

Environmental Phage-Based Cocktail and Antibiotic Combination Effects on Acinetobacter baumannii Biofilm in a Human Urine Model.

The emergence of multidrug-resistant (MDR) bacterial infections poses a catastrophic threat to medicine. The development of phage-based therapy combined with antibiotics might be an advantageous weapon in the arms race between human and MDR bacteria. A cocktail composed of the MDR Acinetobacter baumannii infecting bacteriophages with high lytic activity was used in combination with antibiotics to destroy a bacterial biofilm in human urine. A. baumannii exhibited varying susceptibility to the host range of bacteriophages used in this study, ranging from 56% to 84%. This study demonstrated that bacteriophages could reduce biofilm biomass in a human urine model, and some of the antibiotics commonly used in the treatment of urinary tract infection (UTI) act synergistically with phage cocktails. Additionally, the combined treatment showed a significantly greater reduction of biofilm biomass and clearance of persister cells.

Dose dependent enhanced antibacterial effects and reduced biofilm activity against Bacillus subtilis in presence of ZnO nanoparticles.

Bacterial biofilms are self-produced matrix of sticky extracellular polymeric substances. They result in fouling in the food industry, water treatment plants, and possess significant environmental and industrial impacts. Nanoparticles have shown immense potential and have been effective in combating bacterial biofilm, which is the common cause of drug resistance development, biofouling in water treatment plants and the food industry. Hence, in order to explore the same, Zinc oxide nanoparticles have been synthesized by chemical synthesis method and their action against Bacillus subtilis biofilm formation was evaluated in this study by crystal violet and ROS assay. The dose-dependent reduction in biofilm biomass and density was observed as a result of nanoparticle exposure. There was considerable reduction in biofilm formation after treatment with ZnO nanoparticles. Change in surface morphology of the Bacillus subtilis cells was observed which could be due to oxidative stress induced by ZnO nanoparticles. The oxidative stress was estimated by measurement of catalase activity that also showed dose-dependent decrease.

Effects of river ice break‐up on organic‐matter dynamics and feeding ecology of aquatic insects

AbstractDisturbance shapes the structure and function of aquatic communities and ecosystems, but the dynamics of ice are a less studied dimension of the disturbance‐regime of rivers. We investigated effects of a river‐ice regime on organic‐matter dynamics and feeding ecology of aquatic insects. Samples of biofilm and aquatic insects for gut content analysis were collected monthly from Big Creek, a sixth ‐ order tributary of the Middle Fork Salmon River in central Idaho, USA, during winter 2010–2011. Our results indicate that river ice affects both quantity and quality of organic matter available to, and used by, consumers. Specifically, scour from December and February ice break‐up events reduced biofilm biomass by one‐half and one‐third, respectively, whereas quality (chlorophyll‐a: ash‐free dry mass) increased. Diets of scrapers, Rhithrogena (Heptageniidae) and Bibiocephala (Blephariceridae), collector‐gatherers, Baetis (Baetidae), and collector‐filterers, Simulium (Simulidae) appeared to follow patterns of organic matter. Following ice break‐up events, diets of these taxa had increased proportions of diatom frustules, which are high‐quality food resources due to their relatively high nutrient content. Other taxa, such as collector‐gatherers, non‐Tanypodinae (Chironomidae), and the collector‐filterer, Arctopsyche grandis (Hydropsychidae), consistently consumed high proportions of diatom frustules and insect material, respectively, suggesting they were able to feed more selectively throughout winter. Our study indicates that ice regimes in temperate rivers can affect organic‐matter dynamics and feeding ecology of aquatic insects, a possibility that deserves additional investigation, particularly in light of potential changes to the ice regimes of rivers with changing climate.

Remarkable capacity of brosimine b to disrupt methicillin-resistant Staphylococcus aureus (MRSA) preformed biofilms

Methicillin-resistant Staphylococcus aureus (MRSA) is a major public health concern representing about 60% of S. aureus isolated from hospitalized patients in countries such as USA and Brazil in the last years. Additionally, the ability to adhere to surfaces and the development of biofilms are important properties of pathogenic bacteria involved in medical device-associated infections, and staphylococci are recognized as the major etiologic agents in these situations. The aim of this study is to evaluate three Brosimum acutifolium flavonoids, 4′-hydroxy-7,8(2″,2″-dimethylpyran)flavan (1), brosimine b (2) and 4-hydroxy-lonchocarpin (3), regarding their antibiofilm, antibacterial and antioxidant activities. Flavonoids 1 and 2 were able to reduce S. aureus viability within preformed biofilms in 73% at 50 μM while 2 also reduced biofilm biomass in 48% at 100 μM. Flavonoid 3 was not able to reduce biofilm biomass at assessed concentrations. When tested against methicillin-resistant S. aureus (MRSA) strains, 2 (100 μM) reduced 70%–98% of viable bacteria within 24h-old biofilms. The minimum inhibitory concentration against the methicillin-sensitive Staphylococcus aureus ATCC 25904 was 50 μM for the three compounds. In preliminary assays to evaluate cytotoxicity, 1 was highly hemolytic at concentrations above 50 μM while 2 and 3 did not cause significant hemolysis at 100 μM. The antioxidant activity was observed only in the ethanolic extract and 2. In vivo toxicity evaluations using Galleria mellonella larvae as alternative host model resulted in 83.3% survival for treatment with 1, 76.7% for 2, and 100% for 3 at 500 mg/kg. This study highlights the potential of these flavonoids, especially 2, as antibiofilm agent to control preformed S. aureus biofilms.

Chemical composition and anti-biofilm activity of essential oil from Citrus medica L. var. sarcodactylis Swingle against Listeria monocytogenes

Listeria monocytogenes is an important foodborne pathogen that could cause listeriosis and poses potential threats to human health. Citrus medica L. var. sarcodactylis Swingle, also known as Fingered Citron, is a citrus species widely cultivated in South China. The aim of this work was to investigate the anti-biofilm efficacy of essential oil from Fingered Citron (FCEO) against L. monocytogenes. The crystal violet assay showed that FCEO exhibited strong anti-biofilm activity on both inhibiting biofilm formation and eradicating preformed biofilm. XTT assay indicated that FCEO significantly increased the metabolic active cells in the biofilm. Confocal scanning laser microscopy images and COMSTAT assay revealed that FCEO destroyed the intact architecture of biofilm, reduced biofilm biomass, thickness and substratum coverage. The morphological changes of cells in the biofilm were observed by scanning electron microscopy, which showed that FCEO could cause the wrinkle, collapse, lysis and cytoplasmic content leakage of cells. Our results also indicated that FCEO displayed its anti-biofilm activity through both biofilm dispersal and cell killing. Our findings provide evidence that FCEO exhibits strong anti-biofilm activity and might be used as a potential agent to control L. monocytogenes biofilm.

Effects of S. mutans gene-modification and antibacterial monomer dimethylaminohexadecyl methacrylate on biofilm growth and acid production

ObjectivesAntibacterial quaternary ammonium monomers (QAMs) are used in resins. The rnc gene in Streptococcus mutans (S. mutans) plays a key role in resisting antibiotics. The objectives of this study were to investigate for the first time: (1) the effects of rnc deletion on S. mutans biofilms and acid production; (2) the combined effects of rnc deletion with dimethylaminohexadecyl methacrylate (DMAHDM) on biofilm-inhibition efficacy. MethodsParent S. mutans strain UA159 (ATCC 700610) and the rnc-deleted S. mutans were used. Bacterial growth, minimum inhibitory concentration (MIC), and minimal bactericidal concentration (MBC) were measured to analyze the bacterial susceptibility of the parent and rnc-deleted S. mutans against DMAHDM, with the gold-standard chlorhexidine (CHX) as control. Biofilm biomass, polysaccharide and lactic acid production were measured. ResultsThe drug-susceptibility of the rnc-deleted S. mutans to DMAHDM or CHX was 2-fold higher than parent S. mutans. The drug-susceptibility did not increase after 10 passages (p < 0.05). Deleting the rnc gene increased the biofilm susceptibility to DMAHDM or CHX by 2-fold. The rnc-deletion in S. mutans reduced biofilm biomass, polysaccharide and lactic acid production, even at no drugs. DMAHDM was nearly 40 % more potent than the gold-standard CHX. The combination of rnc deletion+DMAHDM treatment achieved the greatest reduction in biofilm biomass, polysaccharide synthesis, and lactic acid production. SignificanceGene modification by deleting the rnc in S. mutans reduced the biofilm growth and acid production, and the rnc deletion+DMAHDM method showed the greatest biofilm-inhibition efficacy, for the first time. The dual strategy of antibacterial monomer+bacterial gene modification shows great potential to control biofilms and inhibit caries.

Chemical Composition and Antimicrobial Effectiveness of Ocimum gratissimum L. Essential Oil Against Multidrug-Resistant Isolates of Staphylococcus aureus and Escherichia coli.

The study investigated the antimicrobial activity of the essential oil extract of Ocimum gratissimum L. (EOOG) against multiresistant microorganisms in planktonic and biofilm form. Hydrodistillation was used to obtain the EOOG, and the analysis of chemical composition was done by gas chromatography coupled with mass spectrometry (GC/MS) and flame ionization detection (GC/FID). EOOG biological activity was verified against isolates of Staphylococcus aureus and Escherichia coli, using four strains for each species. The antibacterial action of EOOG was determined by disk diffusion, microdilution (MIC/MBC), growth curve under sub-MIC exposure, and the combinatorial activity with ciprofloxacin (CIP) and oxacillin (OXA) were determined by checkerboard assay. The EOOG antibiofilm action was performed against the established biofilm and analyzed by crystal violet, colony-forming unit count, and SEM analyses. EOOG yielded 1.66% w/w, with eugenol as the major component (74.83%). The MIC was 1000 µg/mL for the most tested strains. The growth curve showed a lag phase delay for both species, mainly S. aureus, and reduced the growth level of E. coli by half. The combination of EOOG with OXA and CIP led to an additive action for S. aureus. A significant reduction in biofilm biomass and cell viability was verified for S. aureus and E. coli. In conclusion, EOOG has relevant potential as a natural alternative to treat infections caused by multiresistant strains.

Marine bacterial DNase curtails virulence and disrupts biofilms of Candida albicans and non-albicans Candida species

Candida is one of the most prevalent fungal pathogens in clinical settings which form antibiotic-resistant biofilms on biomedical devices. Hence, there is a need for non-antimicrobial alternatives to combat these infections. The present study investigates the anti-biofilm effect of marine bacterial DNase by targeting the eDNA present in the biofilms of Candida spp. A strain of Vibrio alginolyticus (AMSII) which showed enhanced DNase activity was isolated from marine sediment. Treatment of young and mature Candida biofilms with purified marine bacterial DNase (MBD) caused a 60-80% reduction in biofilm biomass, similar to treatment with DNase I from Bovine pancreas. Scanning electron microscopy showed that MBD significantly reduced the formation of biofilms on urinary catheters and more importantly prevented the virulent yeast to hyphae dimorphic switch in C. albicans. The present study identified a potential non-antibiotic alternative therapy to eradicate Candida biofilms and can be used to develop enzyme fabricated antifouling indwelling medical devices.

Antimicrobial and antibiofilm potential of bacteriocin loaded nano-vesicles functionalized with rhamnolipids against foodborne pathogens

Synthetic lipid membranes are considered as promising food-grade nano vesicles (NVs), but their functionalization is required to empower them as active-nano vesicle systems. In this study, rhamnosomes (RS) were developed by engineering the membrane of nano-liposomes with antimicrobial surfactant i.e. rhamnolipids (RL). Intrinsically active rhamnosome nano-vesicles (RSNVs) were loaded with bacteriocin (nisin Z) to acquire broad-spectrum antimicrobial activity. Addition of rhamnolipids into the lipid bilayer exhibited an increase in the encapsulation efficiency of nisin from 47 ± 4% to 88 ± 7%. Scanning electron microscope (SEM) and Zeta-sizer revealed that the surface functionalization with RL increased the size of vesicles from 145 ± 2 nm to 209 ± 4 nm. Whereas, the zeta potential of nisin-loaded RSNVs was −20 ± 0.25 mV as compared to −3.47 ± 0.13 mV for loaded nano-liposomes, indicating higher physical stability of RSNVs. RSNVs not only demonstrated higher antimicrobial activity than liposomes but also enhanced the activity of nisin against Gram-positive and Gram-negative resistant foodborne pathogens including L. monocytogenes, S. aureus, E. coli and P. aeruginosa. Almost 80% reduction in biofilm biomass was observed when treated with nisin-loaded RSNVs due to its improved binding with bacterial surface. Thus, surface-active nanoliposomes (intrinsically active) with higher stability can provide an innovative strategy to control the biofilm-forming food associated pathogens.

Inhibitory Effect of Thymoquinone on Listeria monocytogenes ATCC 19115 Biofilm Formation and Virulence Attributes Critical for Human Infection.

This study aimed to determine the antimicrobial activity of thymoquinone (TQ) against Listeria monocytogenes, and to examine its inhibitory effects on biofilm formation, motility, hemolysin production, and attachment-invasion of host cells. The minimum inhibitory concentrations (MICs) of TQ against eight different L. monocytogenes strains ranged from 6.25—12.50 μg/mL. Crystal violet staining showed that TQ clearly reduced biofilm biomass at sub-MICs in a dose-dependent manner. Scanning electron microscopy suggested that TQ inhibited biofilm formation on glass slides and induced an apparent collapse of biofilm architecture. At sub-MICs, TQ effectively inhibited the motility of L. monocytogenes ATCC 19115, and significantly impacted adhesion to and invasion of human colon adenocarcinoma cells as well as the secretion of listeriolysin O. Supporting these findings, real-time quantitative polymerase chain reaction analysis revealed that TQ down-regulated the transcription of genes associated with motility, biofilm formation, hemolysin secretion, and attachment-invasion in host cells. Overall, these findings confirm that TQ has the potential to be used to combat L. monocytogenes infection.

Laboratory-to-field extrapolation: Increase in carbamazepine toxicity in a higher tier, multiple-stress experiment

The toxicity and environmental risk of chemicals, such as the antiepileptic drug carbamazepine (CBZ), is commonly assessed using standardized laboratory tests and laboratory-to-field extrapolation. To investigate the toxicity of CBZ to aquatic key organisms in a more complex and environmentally relevant scenario, we conducted a 32-day multiple-stress experiment in artificial indoor streams. We exposed the non-biting midge Chironomus riparius, the blackworm Lumbriculus variegatus, and the New Zealand mud snail Potamopyrgus antipodarum to 80 and 400 μg CBZ/L in six artificial indoor streams. In addition to hydraulic stress, species’ interaction, and low organic content in the sediment, organisms were co-exposed to the herbicide terbutryn (TBY) as a second chemical stressor at a concentration of 6 μg/L. The exposure to CBZ under multiple stress conditions resulted in a 10- to more than 25-fold higher toxicity in C. riparius and P. antipodarum when compared to a previous, standardized laboratory experiment. The co-exposure to TBY enhanced the adverse effects of CBZ on snails (reduced production of embryos). This effect was additive as the single exposure to TBY also reduced the reproduction of snails, most likely through the reduction of biofilm biomass. The emergence of C. riparius declined at a CBZ concentration of 400 μg/L (without the co-exposure to TBY) and at 80 μg/L in combination with TBY. The difference in sensitivity between laboratory and indoor stream experiments is indicative of a potential underestimation of risk when toxicity data are extrapolated to field conditions. The present results suggest the inclusion of non-chemical and chemical stressors in environmental hazard and risk assessments.

Antibacterial and anti-biofilm activities of walnut pellicle extract (Juglans regia L.) against coagulase-negative staphylococci

Juglans regia L. (common walnut) is a deciduous tree belonging to Juglandaceae family. Since ancient time, walnut was widely used in traditional medicine for its antioxidant, antidiabetic, antimicrobial, anti-inflammatory, anti-atherogenic and liver-protective effects. In this work, the antibacterial and anti-biofilm activities of walnuts pellicle extract against coagulase-negative staphylococci were evaluated. Qualitative chemical analysis was performed by the thin layer chromatography. UPLC-Ms/Ms was used to identify the chemical composition of J. regia extract. The total flavonoid and phenolic contents were determined by the Aluminium chloride and Folin-Ciocalteu methods, respectively. The extract showed antibacterial activity with MIC ranging from 3.60 to 461.75 µg/ml and MBC ranging from 461.75 to >461.75 µg/ml. Furthermore, it significantly reduced biofilm biomass and cell viability in a dose-dependent manner. Biological activities of J. regia extract may be due to its high flavonoid and phenolic contents. The obtained results are promising and they deserve further scientific investigations.

Antimicrobial and biofilm anti-adhesion activities of silver nanoparticles and farnesol against endodontic microorganisms for possible application in root canal treatment

ObjectiveThis study aimed to evaluate the antimicrobial and biofilm anti-adhesion activities of poly(vinyl alcohol)-coated silver nanoparticles (AgNPs-PVA) and farnesol against Enterococcus faecalis, Candida albicans or Pseudomonas aeruginosa. DesignMinimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) of the solutions, as well as the effect on the biofilm biomass were evaluated. The biofilm anti-adhesion activity was evaluated using bovine root dentine treated with the solutions after 3 min of contact and analyzed by scanning electron microscopy (SEM) and by colony-forming units per milliliter (CFU mL−1) counting. Data were analyzed using ANOVA and Tukey's, the paired Student’s t-test or Kruskal-Wallis and Dunn’s tests (α = 0.05). ResultsThe MIC and MMC values (MIC/MMC) of the AgNPs-PVA and farnesol against E. faecalis were 42.5/50 μM and 0.85/1.0%, respectively. For C. albicans, the values were 27.5/37.5 μM and 1.75/2.5%; and for P. aeruginosa, 32.5/32.5 μM and 2.5/2.75%, respectively. Both solutions showed reduced biofilm biomass (p < 0.05). SEM analysis showed that dentine blocks treated with both solutions had lower biofilm formation than the control (saline), except for C. albicans. In the CFU mL−1 counting, biofilm cells were viable in the all groups in comparison with control (p > 0.05). ConclusionsAgNPs-PVA and farnesol showed antimicrobial and biofilm anti-adhesion activities, as well as potential for use as coadjuvant in endodontic treatment, and may be an option as auxiliary procedure for root canal disinfection or to inhibit biofilm formation.

Inhibitory effect of bacteriocins from enterococci on developing and preformed biofilms of Listeria monocytogenes, Listeria ivanovii and Listeria innocua.

The biofilm-forming ability of Listeria spp. is a concern to the food industry and health sectors. The aim of this study was to verify the inhibitory activity of bacteriocins produced by enterococci (Enterococcus faecium 20, 22 and 24 and Enterococcus faecalis 27) on developing biofilm and preformed biofilm of Listeria species. Bacteriocins were partially purified from cell free supernatant (CFS). L. monocytogenes 2032, L. innocua 2050 and L. ivanovii 2056 were selected to analyse the inhibitory effect of bacteriocins on biofilm biomass (crystal violet staining) and biofilm viability (XTT-reduction). The biomass of the developing and preformed biofilms of Listeria species were reduced (p < 0.05) in the presence of all bacteriocins tested. Overall, the reduction in biofilm biomass of developing biofilms was up to 87.4% for bacteriocin produced by E. faecium 22 (CFS22) against L. ivanovii and up to 87.1% for CFS22 against L. monocytogenes. These findings are in accordance with those observed in confocal microscopy analysis. Most of the CFS-containing bacteriocin (CFS22, CFS24, CFS27) were effective at decreasing the viability of biofilm cells from all Listeria species. The highest reduction in viability was observed for L. monocytogenes preformed biofilm cells (up to 98.7%), evidenced by fluorescence microscopy of propidium iodide-labelled cells. Scanning electron microscopy showed that cells of biofilm-treated bacteriocins displayed degenerative changes that may be indicative of cellular leakages. This study suggests that bacteriocins produced by enterococci have prospective applications to prevent biofilm formation and/or to reduce cell viability of formed biofilms of distinct Listeria species.

Enzyme responsive copolymer micelles enhance the anti-biofilm efficacy of the antiseptic chlorhexidine

Staphylococcal biofilms cause many infectious diseases and are highly tolerant to the effects of antimicrobials; this is partly due to the biofilm matrix, which acts as a physical barrier retarding the penetration and reducing susceptibility to antimicrobials, thereby decreasing successful treatment outcomes. In this study, both single and mixed micellar systems based on poly vinyl caprolactam (PCL)-polyethylene glycol (PEG) copolymers were optimised for delivery of chlorhexidine (CHX) to S. aureus, MRSA and S. epidermidis biofilms and evaluated for their toxicity using Caenorhabditis elegans. The respective polyethylene glycol (PEG) and poly vinyl caprolactam (PCL) structural components promoted stealth properties and enzymatic responsive release of CHX inside biofilms, leading to significantly enhanced penetration (56%) compared with free CHX and improving the efficacy against Staphylococcus aureus biofilms grown on an artificial dermis (2.4 log reduction of CFU). Mixing Soluplus-based micelles with Solutol further enhanced the CHX penetration (71%) and promoted maximum reduction in biofilm biomass (>60%). Nematodes-based toxicity assay showed micelles with no lethal effects as indicated by their high survival rate (100%) after 72 h exposure. This study thus demonstrated that bio-responsive carriers can be designed to deliver a poorly water-soluble antimicrobial agent and advance the control of biofilm associated infections.