Biofilm Cell Viability Research Articles

Inactivation Efficacy of 405 nm LED Against Cronobacter sakazakii Biofilm.

The objectives of this study were to evaluate the inactivation efficacy of a 405-nm light-emitting diode (LED) against Cronobacter sakazakii biofilm formed on stainless steel and to determine the sensitivity change of illuminated biofilm to food industrial disinfectants. The results showed that LED illumination significantly reduced the population of viable biofilm cells, showing reduction of 2.0 log (25°C), 2.5 log (10°C), and 2.0 log (4°C) between the non-illuminated and LED-illuminated groups at 4 h. Images of confocal laser scanning microscopy and scanning electron microscopy revealed the architectural damage to the biofilm caused by LED illumination, which involved destruction of the stereoscopic conformation of the biofilm. Moreover, the loss of biofilm components (mainly polysaccharide and protein) was revealed by attenuated total reflection Fourier-transformed infrared spectroscopy, and the downregulation of genes involved in C. sakazakii biofilm formation was confirmed by real time quantitative PCR analysis, with greatest difference observed in fliD. In addition, the sensitivity of illuminated-biofilm cells to disinfectant treatment was found to significantly increased, showing the greatest sensitivity change with 1.5 log reduction between non-LED and LED treatment biofilms in the CHX-treated group. These results indicated that 405 nm LED illumination was effective at inactivating C. sakazakii biofilm adhering to stainless steel. Therefore, the present study suggests the potential of 405 nm LED technology in controlling C. sakazakii biofilms in food processing and storage, minimizing the risk of contamination.

Potential synergistic activity of quercetin with antibiotics against multidrug-resistant clinical strains of Pseudomonas aeruginosa.

Development of drug resistance in opportunistic pathogens is one of the major healthcare challenges associated with infection management. Combination therapy has many advantages due to the simultaneous action of two drugs on two separate cellular targets. However, selection of the drugs should offer safety and synergistic interaction against most of the strains. Here, the efficacy of antibiotics in combination with quercetin, a natural flavonoid capable of targeting quorum sensing was tested against biofilm-forming Pseudomonas aeruginosa strains previously isolated from catheter associated urinary tract infection. Based on the antibiotic susceptibility pattern, synergistic effect of quercetin with selected antibiotics (levofloxacin, ceftriaxone, gentamycin, tobramycin and amikacin) was tested at the fractional concentrations of MIC by the checkerboard method and the fractional inhibitory concentration index (FICi) was calculated to estimate the synergistic effect. Effect of the synergistic combinations were further tested using time-kill assay, and against biofilm formation and biofilm cell viability. Cytotoxicity assays were performed using Human Embryonic Kidney 293T cells (HEK-293T) using the effective drug combinations with respective controls. The biofilm formation and biofilm cell viability were drastically affected with quercetin and selected antibiotics combinations with ≥80% inhibition. In vitro infection studies showed that all the strains could exert significant cell killing (68 to 85%) and the drug combinations decreased the infection rate significantly by reducing the cell killing effect of P. aeruginosa (p<0.05). The synergistic effect of quercetin is attributed to its quorum sensing inhibitory properties. These findings indicate that quercetin along with existing antibiotics can potentiate the treatment against P. aeruginosa infection and may reduce the selection pressure due to antibiotic overuse.

The Response of Pseudomonas aeruginosa PAO1 to UV-activated Titanium Dioxide/Silica Nanotubes.

Pseudomonas aeruginosa is a bacterium of high clinical and biotechnological importance thanks to its high adaptability to environmental conditions. The increasing incidence of antibiotic-resistant strains has created a need for alternative methods to increase the chance of recovery in infected patients. Various nanomaterials have the potential to be used for this purpose. Therefore, we aimed to study the physiological response of P. aeruginosa PAO1 to titanium dioxide/silica nanotubes. The results suggest that UV light-irradiated nanomaterial triggers strong agglomeration in the studied bacteria that was confirmed by microscopy, spectrophotometry, and flow cytometry. The effect was diminished when the nanomaterial was applied without initial irradiation, with UV light indicating that the creation of reactive oxygen species could play a role in this phenomenon. The nanocomposite also affected biofilm formation ability. Even though the biomass of biofilms was comparable, the viability of cells in biofilms was upregulated in 48-hour biofilms. Furthermore, from six selected genes, the mexA coding efflux pump was upregulated, which could be associated with an interaction with TiO2. The results show that titanium dioxide/silica nanotubes may alter the physiological and metabolic functions of P. aeruginosa PAO1.

Combination of Miconazole and Domiphen Bromide Is Fungicidal against Biofilms of Resistant Candida spp.

The occurrence and recurrence of mucosal biofilm-related Candida infections, such as oral and vulvovaginal candidiasis, are serious clinical issues. Vaginal infections caused by Candida spp., for example, affect 70 to 75% of women at least once during their lives. Miconazole (MCZ) is the preferred topical treatment against these fungal infections, yet it has only moderate antibiofilm activity. Through screening of a drug-repurposing library, we identified the quaternary ammonium compound domiphen bromide (DB) as an MCZ potentiator against Candida biofilms. DB displayed synergistic anti-Candida albicans biofilm activity with MCZ, reducing the number of viable biofilm cells 1,000-fold. In addition, the MCZ-DB combination also resulted in significant killing of biofilm cells of azole-resistant C. albicans, C. glabrata, and C. auris isolates. In vivo, the MCZ-DB combination had significantly improved activity in a vulvovaginal candidiasis rat model compared to that of single-compound treatments. Data from an artificial evolution experiment indicated that the development of resistance against the combination did not occur, highlighting the potential of MCZ-DB combination therapy to treat Candida biofilm-related infections.

Biofilm Formation and Methylene Blue-mediated Photodynamic Inactivation of Vibrio Parahaemolyticus in the Sea Food Industry

This study was conducted to better understand the mechanism of Vibrio Parahaemolyticus biofilm formation and to assess the inactivation effects of methylene blue-mediated photodynamic inactivation (PDI) technology as a preventative measure. Optical microscopy, following crystal violet staining, was used to observe the kinetics of V. parahaemolyticus biofilm formation. The crystal violet-based assay was performed in microtiter plates, and it was employed to determine which factors were most influential in the formation of the biofilms. Colony counting and confocal laser scanning microscopy (CLSM) were used to test the inactivation effect of methylene blue-mediated photodynamic technology on the biofilms. V. parahaemolyticus has the ability to form biofilms, as evidenced by their immediate adherence to glass surfaces and rapid maturity, within 24 h. High (7%) or low (0.5%) salinity was not conducive to the formation of biofilms, and rotational speed greater than 130 rpm also inhibited the process. A 4.05 log reduction in the concentration of viable biofilm cells was obtained with 100 μg/mL methylene blue and 20 min irradiation (24.996 J/cm2), but planktonic cells were more susceptible to the methylene blue-mediated photodynamic reaction (5.46 log reduction). The results presented here show that the methylene blue-mediated PDI technology is an effective means to inactivate V. parahaemolyticus by disrupting its membrane integrity and to inhibit the pathogen’s formation of protective biofilms. This technology is a valid tool that can be used to enhance food safety in the sea food industry.

In situ synthesis and long-term stabilization of nanosilver in poly(vinyl acetate-co-butyl acrylate-co-neodecanoate) matrix for antibacterial applications

Although the synthesis and the study of properties of silver nanoparticles (AgNP) have been the subject of much research, it is also of great interest the development of new hybrid nanomaterials based on AgNP and the corresponding stabilization by means of new substrates, especially polymeric ones like vinylacrylic and derivatives, extensively used in industrial applications. These types of substrates in addition to stabilizing these nanoparticles take advantage of the synergy of properties between both types of materials. The purpose of this research is the in situ synthesis of AgNP in poly (vinyl acetate-co-butyl acrylate-co-neodecanoate) (VAVTD) terpolymer water emulsion as stabilization matrix and the corresponding characterization. UV-VIS, X-ray photoelectron spectroscopy (XPS), transmission (TEM) and scanning electron microscopy (SEM) were used to characterize these AgNP polymer hybrids. Additionally, preliminary work was carried out to assess the biofilm formation of a multiple antibiotic resistance (MAR) strain of Pseudomonas aeruginosa on these AgNP/VAVTD hybrids in order to explore their use in antibacterial applications. The results show that VAVTD terpolymer water emulsion, in low concentrations, is a very good substrate to stabilize AgNP particles as evidenced by the SEM and TEM studies, and the relatively small displacement in the corresponding UV spectra of AgNP particles, even after 45 d of ageing. It was also found a significant reduction of P. aeruginosa cell viability in biofilms formed at 1 and 7 d (p < 0.05) on VAVDT/AgNP hybrid materials, suggesting potential antibacterial properties. Our results also provide important insights to the current knowledge in control strategies for MAR bacterial infections, such as those caused by P. aeruginosa, while avoiding the spreading of resistant strains in healthcare facilities and multiple environments related to human everyday live, especially for surfaces that require antibacterial protection.

Tailoring Nanoparticle-Biofilm Interactions to Increase the Efficacy of Antimicrobial Agents Against Staphylococcus aureus.

BackgroundConsidering the timeline required for the development of novel antimicrobial drugs, increased attention should be given to repurposing old drugs and improving antimicrobial efficacy, particularly for chronic infections associated with biofilms. Methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) are common causes of biofilm-associated infections but produce different biofilm matrices. MSSA biofilm cells are typically embedded in an extracellular polysaccharide matrix, whereas MRSA biofilms comprise predominantly of surface proteins and extracellular DNA (eDNA). Nanoparticles (NPs) have the potential to enhance the delivery of antimicrobial agents into biofilms. However, the mechanisms which influence the interactions between NPs and the biofilm matrix are not yet fully understood.MethodsTo investigate the influence of NPs surface chemistry on vancomycin (VAN) encapsulation and NP entrapment in MRSA and MSSA biofilms, mesoporous silica nanoparticles (MSNs) with different surface functionalization (bare-B, amine-D, carboxyl-C, aromatic-A) were synthesised using an adapted Stöber method. The antibacterial efficacy of VAN-loaded MSNs was assessed against MRSA and MSSA biofilms.ResultsThe two negatively charged MSNs (MSN-B and MSN-C) showed a higher VAN loading in comparison to the positively charged MSNs (MSN-D and MSN-A). Cellular binding with MSN suspensions (0.25 mg mL−1) correlated with the reduced viability of both MSSA and MRSA biofilm cells. This allowed the administration of low MSNs concentrations while maintaining a high local concentration of the antibiotic surrounding the bacterial cells.ConclusionOur data suggest that by tailoring the surface functionalization of MSNs, enhanced bacterial cell targeting can be achieved, leading to a novel treatment strategy for biofilm infections.

In vitro Mixed Biofilm of Streptococcus suis and Actinobacillus pleuropneumoniae Impacts Antibiotic Susceptibility and Modulates Virulence Factor Gene Expression

Streptococcus suis (S. suis) and Actinobacillus pleuropneumoniae (A. pleuropneumoniae) are primary swine pathogens that have been frequently co-isolated from pigs suffering from severe respiratory disease. The purpose of this study was to investigate the biological impacts of the interactions between S. suis and A. pleuropneumoniae. A single- and dual-species culture model was established in vitro via S. suis HA9801 (serotype 2) and A. pleuropneumoniae CVCC265 (serotype 1). The single or mixed biofilms were imaged by confocal laser scanning microscopy. The biomass and viable cells in biofilms were quantified by crystal violet staining and determination of colony-forming units. The antibiotic susceptibility was determined by a microdilution broth method. The differences in gene transcription in pure- or mixed-species biofilms of S. suis and A. pleuropneumoniae was evaluated by quantitative PCR. S. suis and A. pleuropneumoniae formed two-species biofilms when co-cultured in vitro. When co-cultured with S. suis, biofilm formation by A. pleuropneumoniae was significantly increased with the absence of NAD that is necessary for the growth of A. pleuropneumoniae. Moreover, compared with monocultures, the antibiotic resistance of S. suis and A. pleuropneumoniae was both enhanced in the co-culture model. When grown in dual-species biofilms, for A. pleuropneumoniae, genes associated with virulence factors, including exotoxins and adhesins, were significantly upregulated. For S. suis, virulence factor-related genes cps2, gdh, mrp, and sly were highly induced. These results suggest that the interspecies interactions between S. suis and A. pleuropneumoniae may be cooperative under specific conditions and may play an important role in the disease progression and persistent infection.

Tea extracts differentially inhibit Streptococcus mutans and Streptococcus sobrinus biofilm colonization depending on the steeping temperature

This study aimed to evaluate the effects of tea extracts on oral biofilm colonization depending on steeping temperature. S. mutans and S. sobrinus were cultured and treated with green or black tea extracts prepared under different steeping conditions. Biofilm formation, glucosyltransferase (GTF) levels, bacterial growth, and acidogenicity were evaluated. Biofilms were also assessed by gas chromatography-mass spectrometry and confocal laser scanning microscopy. All extracts with hot steeping showed higher inhibitory effects on biofilm formation and cell viability and lower GTF levels compared with those with cold steeping (p < 0.05). Hot steeping significantly reduced bacterial growth (p < 0.05) and maintained the pH. Catechins were only identified from hot steeping extracts. Within the limits of this study, extracts with cold steeping showed lower inhibitory effects on oral biofilms. The different effects between steeping extracts may be attributed to the difference in catechins released from tea extracts under the different steep conditions.

Cultivation of Productive Biofilms in Flow Reactors and Their Characterization by CLSM.

Biofilms, a natural form of immobilized whole cells, are currently being investigated as a robust biocatalyst for the production of chemicals. Fluidic conditions and reactor geometry severely influence biofilm growth, development, and reaction performance. However, there is a missing link between the in situ characterization of biofilms on microscale setups and macroscale reactors because of the difference in reactor geometry and fluidic conditions. In this protocol, we describe the assembly and operation of flow cell and flow reactor setups with identical system geometry and segmented flow conditions to link biofilm characterization to reactor performance. The flow cell setup enables the in situ characterization of biofilm growth, structural development, and cell viability by utilizing confocal laser scanning microscopy (CLSM). Whereas, the laboratory scale flow reactor allows the determination of overall biofilm dry mass, catalytic activity, and final product titer during biocatalysis. Finally, CLSM image acquisition and the following data analysis are briefly described.

Bacteriocin of Pediococcus acidilactici HW01 Inhibits Biofilm Formation and Virulence Factor Production by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a potential source of food contamination that leads to food spoilage and infections as a result of the generation of biofilm and virulence factors. In the present study, we demonstrate that bacteriocin produced by Pediococcus acidilactici HW01 (HW01 bacteriocin) effectively inhibited the biofilm formation of Ps. aeruginosa (66.41, 45.77, and 21.73% of biofilm formation at 0.5, 1, and 2mg/mL of HW01 bacteriocin, respectively) as well as the production of virulence factors. By means of a microtiter plate method and scanning electron microscopy, HW01 bacteriocin inhibited biofilm formation by Ps. aeruginosa in a dose-dependent manner. Although the viability of biofilm cells of Ps. aeruginosa was reduced in the presence of HW01 bacteriocin, the viability of planktonic cells of Ps. aeruginosa was not affected by HW01 bacteriocin (2.0 × 109CFU/mL vs. 2.4 × 109CFU/mL in the absence and the presence of HW01 bacteriocin, respectively). Additionally, HW01 bacteriocin decreased the twitching motility of Ps. aeruginosa as well as the production of virulence factors, such as pyocyanin, protease, and rhamnolipid. Furthermore, HW01 bacteriocin significantly inhibited Ps. aeruginosa biofilm formation on the surface of stainless steel (57% reduction at 24h and 83% reduction at 72h). These results indicate that HW01 bacteriocin is an effective antagonist of Ps. aeruginosa as a result of its ability to inhibit biofilm formation and the production of virulence factors.

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.

Reserpine attenuates biofilm formation and virulence of Staphylococcus aureus

This study investigated the effects of reserpine, the main bioactive compound of Rauwolfia serpentina, on biofilm formation and biofilm-associated virulence factors production in a Gram-positive pathogen, Staphylococcus aureus. Crystal violet assay, MTT assay, Congo red binding, CLSM studies were performed to assess the antibiofilm activity. Molecular docking was performed to explain the possible mode of action, catheter model was used to evaluate its application potential and the combinatorial study was performed in search of an improved therapeutic formulation. Reserpine affected biofilm formation, EPS production, biofilm cell viability and virulence factor production. It could eradicate 72.7% biofilm at ½ × MIC dose and could also stop the metabolic activity of 50.6% bacterial cells in a biofilm. Staphylococcus aureus biofilm- and virulence-regulatory proteins like AgrA, AtlE, Bap, IcaA, SarA and SasG were found to interact with reserpine which might lead to the attenuation of its pathogenicity. Reserpine along with other commercial antibiotics could generate a hightened antibiofilm response, and also eradicated a good percentage of bacterial biofilm from a urinary catheter model. These findings suggested reserpine as a good alternative entity to generate new improved therapeutic formulations.

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.

Streptococcus mutans Biofilm Formation and Cell Viability on Polymer-infiltrated Ceramic and Yttria-stabilized Polycrystalline Zirconium Dioxide Ceramic.

The aim of this study was to investigate the biofilm formation and cell viability of a polymer-infiltrated ceramic (PIC) and an yttria-stabilized polycrystalline zirconium dioxide ceramic (Y-TZP). The null hypothesis was that there would be no difference in biofilm formation and cell viability between the materials. Streptococcus mutans biofilm was analyzed with scanning electron microscopy (SEM), confocal laser scanning microscopy, and colony counting (colony-forming units/mL). The cell viability (fibroblasts) of both materials was measured with 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium) (MTT) test. Roughness measurements were also performed. The PIC displayed higher roughness but showed similar colony-forming units and biovolume values to those of Y-TZP. SEM showed a higher amount of adhered fibroblasts on the PIC surface on the first day and similar amounts on both materials after seven days. Moreover, the materials were biocompatible with human fibroblasts. PIC and Y-TZP are biocompatible and present the same characteristics for biofilm formation; therefore, they are indicated for indirect restorations and implant abutments.

Pseudomonas aeruginosa Increases the Sensitivity of Biofilm-Grown Staphylococcus aureus to Membrane-Targeting Antiseptics and Antibiotics.

Pseudomonas aeruginosa and Staphylococcus aureus often cause chronic, recalcitrant infections in large part due to their ability to form biofilms. The biofilm mode of growth enables these organisms to withstand antibacterial insults that would effectively eliminate their planktonic counterparts. We found that P. aeruginosa supernatant increased the sensitivity of S. aureus biofilms to multiple antimicrobial compounds, including fluoroquinolones and membrane-targeting antibacterial agents, including the antiseptic chloroxylenol. Treatment of S. aureus with the antiseptic chloroxylenol alone did not decrease biofilm cell viability; however, the combination of chloroxylenol and P. aeruginosa supernatant led to a 4-log reduction in S. aureus biofilm viability compared to exposure to chloroxylenol alone. We found that the P. aeruginosa-produced small molecule 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO) is responsible for the observed heightened sensitivity of S. aureus to chloroxylenol. Similarly, HQNO increased the susceptibility of S. aureus biofilms to other compounds, including both traditional and nontraditional antibiotics, which permeabilize bacterial membranes. Genetic and phenotypic studies support a model whereby HQNO causes an increase in S. aureus membrane fluidity, thereby improving the efficacy of membrane-targeting antiseptics and antibiotics. Importantly, our data show that P. aeruginosa exoproducts can enhance the ability of various antimicrobial agents to kill biofilm populations of S. aureus that are typically difficult to eradicate. Finally, our discovery that altering membrane fluidity shifts antimicrobial sensitivity profiles of bacterial biofilms may guide new approaches to target persistent infections, such as those commonly found in respiratory tract infections and in chronic wounds.IMPORTANCE The thick mucus in the airways of cystic fibrosis (CF) patients predisposes them to frequent, polymicrobial respiratory infections. Pseudomonas aeruginosa and Staphylococcus aureus are frequently coisolated from the airways of individuals with CF, as well as from diabetic foot ulcers and other wounds. Both organisms form biofilms, which are notoriously difficult to eradicate and promote chronic infection. In this study, we have shown that P. aeruginosa-secreted factors can increase the efficacy of compounds that alone have little or no bactericidal activity against S. aureus biofilms. In particular, we discovered that P. aeruginosa exoproducts can potentiate the antistaphylococcal activity of phenol-based antiseptics and other membrane-active drugs. Our findings illustrate that polymicrobial interactions can dramatically increase antibacterial efficacy in vitro and suggest that altering membrane physiology promotes the ability of certain drugs to kill bacterial biofilms-knowledge that may provide a path for the discovery of new biofilm-targeting antimicrobial strategies.

Biofilm development and cell viability: An undervalued mechanism in the persistence of the fish pathogen Tenacibaculum maritimum

Tenacibaculum maritimum is a bacterial pathogen that causes tenacibaculosis outbreaks in major commercial marine fish worldwide. However, scarce knowledge exists regarding T. maritimum life strategies that could act as potential mechanisms for environmental persistence and transmission, such as surface-attached biofilms. This study used high-throughput analyses to quantitatively and qualitatively characterize biofilm formation and activity in T. maritimum strains ACC50.1, PC424.1, PC503.1, and CECT 4276. Additionally, cell-surface properties of the T. maritimum strains, such as hydrophobicity, were determined before inducing biofilm formation. We also briefly examined if pathogenicity against fish varied among T. maritimum strains with differing cell-surface properties. All strains were moderately hydrophobic and rapidly formed biofilms on polystyrene surfaces in a three-step process, with live and dead cells in multilayered-like bacterial aggregates. Virulence in turbot (Scophthalmus maximus) was assessed through a prolonged-immersion challenge. Turbot exhibited the classical signs of tenacibaculosis and mortalities for the T. maritimum strains PC424.1, CECT 4276, and ACC50.1, but no symptoms or mortalities were recorded against PC503.1 or non-challenged controls. Interestingly, while all T. maritimum strains tended to develop profuse biofilms over time, all biofilms showed a decreasing tendency in cell viability. (i.e., a range of fluorescent ratios of live-to-dead sessile cells of ca. 17–51 at 24 h versus 12–22 at 96 h). The fast kinetics of T. maritimum biofilm formation suggests that aquaculture settings can harbor these cellular accretions as transient reservoirs for virulent strains, possibly contributing to tenacibaculosis prevalence to a degree thus far unknown.

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.

Effect of Helicobacter pylori biofilm formation on susceptibility to amoxicillin, metronidazole and clarithromycin

The human gastric pathogen Helicobacter pylori forms biofilms in vitro and in vivo. We previously demonstrated that H. pylori biofilm formation in vitro decreased its susceptibility to clarithromycin (CAM). The aim of this study was to evaluate the effects of biofilm formation on amoxicillin (AMPC) and metronidazole (MNZ) susceptibility. In addition, we assessed the influence of biofilms of CAM resistant H. pylori on CAM susceptibility. It was shown that high levels of efflux pump gene transcripts were detected in biofilm cells of all H. pylori strains used in this study. H. pylori biofilm biomass was significantly decreased compared to initial biomass after treatment with the minimum inhibitory concentration (MIC) of AMPC. Similarly, the biofilm biomass of H. pylori decreased after treatment with MIC of MNZ, although the difference was not statistically significant. However, minimum bactericidal concentrations (MBCs) of AMPC or MNZ to biofilm cells were higher than those of planktonic cells. The biofilm biomasses of all of the CAM resistant strains were significantly decreased compared to initial biomass after treatment with 2x MIC of CAM. However, the viability of the CAM treated biofilm cells with 2x MIC of CAM was not significantly reduced compared to initial cell numbers with the exception of one strain. The viability of biofilm cells of all strains was higher than that of planktonic cells after treatment with various concentrations of CAM. These results indicate that biofilm cells were more resistant to these antibiotics than planktonic cells and that the assessment of the ability to form biofilms in H. pylori is important for eradication of this microorganism.

Anti-staphylococcal activity of Syagrus coronata essential oil: Biofilm eradication and in vivo action on Galleria mellonela infection model

In this study, essential oil extracted from Syagrus coronata seeds (SCEO) was evaluated for antibacterial and antibiofilm activities against Staphylococcus aureus; in addition, Galleria mellonella model was used as an in vivo infection model. SCEO was mainly composed by fatty acids (89.79%) and sesquiterpenes (8.5%). The major components were octanoic acid, dodecanoic acid, decanoic acid and γ-eudesmol. SCEO showed bactericidal activity (minimal bactericidal concentration from 312 to 1250 μg/mL) against all tested S. aureus clinical isolates, which showed distinct biofilm-forming and multiple drug resistance phenotypes. SCEO weakly reduced biomass but remarkably decreased cell viability in pre-formed biofilms of S. aureus isolate UFPEDA-02 (ATCC-6538). Electron microscopy analysis showed that SCEO treatments decreased the number of bacterial cells (causing structural alterations) and lead to loss of the roughness in the multiple layers of the three-dimensional biofilm structure. In addition, overproduction of exopolymeric matrix was observed. SCEO at 31.2 mg/kg improved the survival of G. mellonela larvae inoculated with UFPEDA-02 isolate and reduced the bacterial load in hemolymph and melanization. In conclusion, SCEO is an antibacterial agent against S. aureus strains with different resistance phenotypes and able to disturb biofilm architecture. Our results show SCEO as a potential candidate to drug development.