The anti-platelet drug ticlopidine inhibits FapC fibrillation and biofilm production: Highlighting its antibiotic activity

Multidrug resistance of bacteria and persistent infections related to biofilms, as well as the low availability of new antibacterial drugs, make it urgent to develop new antibiotics. Here, we evaluate the antibacterial and anti-biofilm properties of ticlopidine (TP), an anti-platelet aggregation drug, TP showed antibacterial activity against both gram-positive (MRSA) and gram-negative (E. coli, and P. aeruginosa) bacteria over a long treatment period. TP significantly reduced the survival of gram-negative bacteria in human blood though impact on gram-positives was more limited. TP may cause death in MRSA by inhibiting staphyloxanthin pigment synthesis, leading to oxidative stress, while scanning electron microscopy imaging indicate a loss of membrane integrity, damage, and consequent death due to lysis in gram-negative bacteria. TP showed good anti-biofilm activity against P. aeruginosa and MRSA, and a stronger biofilm degradation activity on P. aeruginosa compared to MRSA. Measuring fluorescence of the amyloid-reporter Thioflavin T (ThT) in biofilm implicated inhibition of amyloid formation as part of TP activity. This was confirmed by assays on the purified protein in P. aeruginosa, FapC, whose fibrillation kinetics was inhibited by TP. TP prolonged the lag phase of aggregation and reduced the subsequent growth rate and prolonging the lag phase to very long times provides ample opportunity to exert TP's antibacterial effect. We conclude that TP shows activity as an antibiotic against both gram-positive and gram-negative bacteria thanks to a broad range of activities, targeting bacterial metabolic processes, cellular structures and the biofilm matrix.     

Glycerol monolaurate antibacterial activity in broth and biofilm cultures

Background

Glycerol monolaurate (GML) is an antimicrobial agent that has potent activity against gram-positive bacteria. This study examines GML antibacterial activity in comparison to lauric acid, in broth cultures compared to biofilm cultures, and against a wide range of gram-positive, gram-negative, and non-gram staining bacteria.

Methodology/Principal Findings

GML is ≥200 times more effective than lauric acid in bactericidal activity, defined as a ≥3 log reduction in colony-forming units (CFU)/ml, against Staphylococcus aureus and Streptococcus pyogenes in broth cultures. Both molecules inhibit superantigen production by these organisms at concentrations that are not bactericidal. GML prevents biofilm formation by Staphylococcus aureus and Haemophilus influenzae, as representative gram-positive and gram-negative organisms, tested in 96 well microtiter plates, and simultaneously is bactericidal for both organisms in mature biofilms. GML is bactericidal for a wide range of potential bacterial pathogens, except for Pseudomonas aeruginosa and Enterobacteriaceae. In the presence of acidic pH and the cation chelator ethylene diamine tetraacetic acid, GML has greatly enhanced bactericidal activity for Pseudomonas aeruginosa and Enterobacteriaceae. Solubilization of GML in a nonaqueous delivery vehicle (related to K-Y Warming®) enhances its bactericidal activity against S. aureus. Both R and S, and 1 and 2 position lauric acid derivatives of GML exhibit bactericidal activity. Despite year-long passage of Staphylococcus aureus on sub-growth inhibitory concentrations of GML (0.5 x minimum bactericidal concentration), resistance to GML did not develop.

Conclusions/Significance

GML may be useful as a broad-spectrum human or animal topical microbicide and may be useful as an environmental surface microbicide for management of bacterial infections and contamination.     

Impact of Lactococcus lactis spp. lactis Bio Adhesion on Pathogenic Bacillus cereus Biofilm on Silicone Flowing System

Bacillus cereus is a food pathogen that can attach on most of the surfaces and form biofilms, which facilitate the persistence and resistance toward antimicrobials. The aims of this study were (i) to characterize the structural dynamics of B. cereus sessile growth in two nutritional environments (with or without a nutrient flow), and (ii) to evaluate the impact of bio adhesion of Lactococcus lactis on B. cereus biofilm. Significantly greater biofilm volume and thickness were observed under dynamic conditions than under static conditions after 48 h and B. cereus biofilm was highly organized. The variation of physico-chemical characteristics of silicone by B. cereus bio adhesion favours the adhesion of hydrophilic Lc. lactis on the surface adhered by biofilm. Lc. lactis was able to adhere to silicone surface and produce biofilm obviously exhibited a significant reduction of B. cereus adhered cells up to nine orders of magnitude after 48 h of contact with competitive activity for nutrient and oxygen. This study constitutes a step ahead in developing strategies to prevent microbial colonization of silicone with lactococcal protective biofilm.     

Optimisation of support medium for particle-based biofilm reactors

The aim of this study was to propose a method to improve the biofilm growth on different polymer materials by modifying their surface properties. The ability of two aerobic bacteria strains: Pseudomonas aeruginosa O1 and Bacillus subtilis CIP 5265 to grow on various non-coated and coated polymer materials were investigated. A layer of polymethylmethacrylate and powdered activated carbon (PMMA/PAC) was used to improve the microbial adhesion dynamics. The substratum and cell surface properties were characterized using contact angle measurements. Fluorescent microscopy and SEM were used to observe the support and the biofilm growth. It was determined that better results can be obtained increasing the difference between the surface free energies of the support and the bacteria. It was found that supports with modified surface show higher biofilm development rate and better surface colonization. The influence of the surface free energy on the detachment force and correspondingly on the biofilm formation was demonstrated.     

Production of human lysozyme in biofilm reactor and optimization of growth parameters of Kluyveromyces lactis K7

Lysozyme (1,4-β-N-acetylmuramidase) is a lytic enzyme, which degrades the bacterial cell wall. Lysozyme has been of interest in medicine, cosmetics, and food industries because of its anti-bactericidal effect. Kluyveromyces lactis K7 is a genetically modified organism that expresses human lysozyme. There is a need to improve the human lysozyme production by K. lactis K7 to make the human lysozyme more affordable. Biofilm reactor provides high biomass by including a solid support, which microorganisms grow around and within. Therefore, the aim of this study was to produce the human lysozyme in biofilm reactor and optimize the growth conditions of K. lactis K7 for the human lysozyme production in biofilm reactor with plastic composite support (PCS). The PCS, which includes polypropylene, soybean hull, soybean flour, bovine albumin, and salts, was selected based on biofilm formation on PCS (CFU/g), human lysozyme production (U/ml), and absorption of lysozyme inside the support. To find the optimum combination of growth parameters, a three-factor Box–Behnken design of response surface method was used. The results suggested that the optimum conditions for biomass and lysozyme productions were different (27 °C, pH 6, 1.33 vvm for biomass production; 25 °C, pH 4, no aeration for lysozyme production). Then, different pH and aeration shift strategies were tested to increase the biomass at the first step and then secrete the lysozyme after the shift. As a result, the lysozyme production amount (141 U/ml) at 25 °C without pH and aeration control was significantly higher than the lysozyme amount at evaluated pH and aeration shift conditions (p?<?0.05).     

Control of N-acetylglucosaminidase specific activity in myxamoebae of Dictyostelium discoideum

Myxamoebae of Dictyostelium discoideum were grown with Aerobacter aerogenes as substrate, on nutrient agar plates or in shaken culture, with mean doubling times that varied between 3 and 6 hr. Growth in axenic culture was with mean doubling times of 5–6 or 8 hr. The specific activity of N-acetylglucosaminidase was three to four times higher in myxamoebae grown axenically than in myxamoebae grown with A. aerogenes and there was no correlation between enzyme specific activity and myxamoebal growth rate. High specific activities of N-acetylglucosaminidase were also found in myxamoebae grown with gram-positive bacteria (Bacillus megaterium, Staphylococcus lactis) as substrate while low specific activities were found in myxamoebae grown with the gram-negative bacteria Acinetobacter lwoffi and Pseudomonas aeruginosa as well as with A. aerogenes. A preparation of the cell envelope of A. aerogenes was nearly as effective as the intact bacteria at depressing myxamoebal N-acetylglucosaminidase specific activity. Lipopolysaccharide extracted from the cell envelope of gram-negative bacteria, or the lipid A component of the lipopolysaccharide, also depressed N-acetylglucosaminidase specific activity when added to axenic cultures of myxamoebae.     

First ever isolation of bacterial prolipoprotein diacylglyceryl transferase in single step from Lactococcus lactis

The unique bacterial enzyme phosphatidylglycerol: prolipoprotein diacylglyceryl transferase (Lgt) is the least studied enzyme of the ubiquitous bacterial lipoprotein synthetic pathway, mostly due to the low abundance of the enzyme. So far, Lgt has been studied to a limited extent in gram-negative bacteria, mainly in Escherichia coli. We, for the first time, report the isolation of an adequate amount of Lgt from the gram-positive lactic acid bacteria, Lactococcus lactis and compare this wild-type bacterial enzyme with the E. coli enzyme. The L. lactis Lgt, when purified by cationic-exchange chromatography, showed a 20-fold increase in the specific activity compared to that of the load, and 75% of the total Lgt activity loaded was recovered. Kinetically, L. lactis Lgt was found to be similar to the E. coli enzyme with matching K_m and V_max, whereas the specific activity of the L. lactis enzyme was about 20 times less than that of the E. coli enzyme. Comparative bioinformatic analysis of L. lactis, E. coli and Staphylococcus aureus Lgt revealed that the conserved and catalytically important His-103 residue in E. coli Lgt, was altered to Tyr in L. lactis. Investigations showed that other bacteria where this alteration is visible, form a diversion within the gram-positive bacteria in evolution. Further analysis revealed Mycobacterium smegmatis to be the species which evolved with the alteration of His to Tyr.     

Individual growth detection of bacterial species in an in vitro oral polymicrobial biofilm model

Most in vitro studies on the antibacterial effects of antiseptics have used planktonic bacteria in monocultures. However, this study design does not reflect the in vivo situation in oral cavities harboring different bacterial species that live in symbiotic relationships in biofilms. The aim of this study was to establish a simple in vitro polymicrobial model consisting of only three bacterial strains of different phases of oral biofilm formation to simulate in vivo oral conditions. Therefore, we studied the biofilm formation of Actinomyces naeslundii (An), Fusobacterium nucleatum (Fn), and Enterococcus faecalis (Ef) on 96-well tissue culture plates under static anaerobic conditions using artificial saliva according to the method established by Pratten et al. that was supplemented with 1 g l^?1 sucrose. Growth was separately determined for each bacterial strain after incubation periods of up to 72 h by means of quantitative real-time polymerase chain reaction and live/dead staining. Presence of an extracellular polymeric substance (EPS) was visualized by Concanavalin A staining. Increasing incubation times of up to 72 h showed adhesion and propagation of the bacterial strains with artificial saliva formulation. An and Ef had significantly higher growth rates than Fn. Live/dead staining showed a median of 49.9 % (range 46.0–53.0 %) of living bacteria after 72 h of incubation, and 3D fluorescence microscopy showed a three-dimensional structure containing EPS. An in vitro oral polymicrobial biofilm model was established to better simulate oral conditions and had the advantage of providing the well-controlled experimental conditions of in vitro testing.     

Study of the initial phase of biofilm formation using a biofomic approach

Aims

The purpose of this work was to study the initial steps of formation of a biofilm using the BioFilm Ring Test® and the Crystal violet staining technique.

Methods and results

Eight strains of Pseudomonas aeruginosa were studied. The two methods revealed that four strains formed a rapid biofilm. The biofilm formed by these strains was detected after only 45 min with the BioFilm Ring Test® and after 6 h with the Crystal violet method. The enumeration of bacteria of the PA01 strain confirmed that, after 30 min, a significant amount of bacteria had attached on the bottom of the culture wells. After 48 h the Crystal violet method detected a biofilm with all strains. The four strains which rapidly formed a biofilm did not differ from the slow-forming strains by their mucoid character or their swarming motility or their synthesis of rhamnose. They showed higher swimming mobility.

Conclusions

Our results show that the BioFilm Ring Test® is a method specially suited for the study of the initial phase of the formation of a biofilm.

Significance and impact of study

The BioFilm Ring Test® is an easy and rapid alternative to the Crystal violet staining and the enumeration methods.     

A comparative study of the effect of probiotics on cariogenic biofilm model for preventing dental caries

Dental caries is induced by oral biofilm containing Streptococcus mutans. Probiotic bacteria were mainly studied for effect on the gastrointestinal tract and have been known to promote human health. However, the information of probiotics for oral health has been lack yet. In this study, we investigated influence of various probiotics on oral bacteria or cariogenic biofilm and evaluated candidate probiotics for dental caries among them. The antimicrobial activity of the spent culture medium of probiotics for oral streptococci was performed. Probiotics were added during the biofilm formation with salivary bacteria including S. mutans. The oral biofilms were stained with a fluorescent dye and observed using the confocal laser scanning microscope. To count bacteria in the biofilm, the bacteria were plated on MSB and BHI agar plates after disrupting the biofilm and cultivated. Glucosyltransferases (gtfs) expression of S. mutans and integration of lactobacilli into the biofilm were evaluated by real-time RT-PCR. Among probiotics, Lactobacillus species strongly inhibited growth of oral streptococci. Moreover, Lactobacillus species strongly inhibited formation of cariogenic biofilm model. The expression of gtfs was significantly reduced by Lactobacillus rhamnosus. The integration of L. rhamnosus into the biofilm model did not exhibit. However, L. acidophilus and L casei integrated into the biofilm model. These results suggest that L. rhamnosus may inhibit oral biofilm formation by decreasing glucan production of S. mutans and antibacterial activity and did not integrate into oral biofilm, which can be a candidate for caries prevention strategy.     

Role of the extracellular polymer matrix in resistance of bacterial biofilms to extreme environmental factors

Biofilms of a number of gram-positive and gram-negative bacteria (both environmental strains from the stratal waters of oil fields and collection strains) were found to exhibit higher resistance to extreme physicochemical factors (unfavorable temperature, pH, and salt concentration) than planktonic cultures. The extracellular polymers forming the structure of the biofilm matrix were shown to contribute significantly to this resistance, since suppression of matrix formation by subbacteriostatic concentrations of azithromycin (for Pseudomonas acephalitica) or mutation in the cvil gene encoding N-hexanoyl homoserine lactone synthetase (for Chromobacterium violaceum CV026) resulted in the resistance of biofilms being decreased almost to the level of planktonic cultures. The role of the biofilm matrix for bacterial survival under extreme conditions is discussed.     

A study on Candida biofilm growth characteristics and its susceptibility to aureobasidin A

Background

Biofilm is known to contribute to the antifungal resistance of Candida yeasts. Aureobasidin A (AbA), a cyclic depsipeptide targeting fungal sphingolipid biosynthesis, has been shown to be effective against several Candida species.

High-Frequency Conjugation System Facilitates Biofilm Formation and pAMβ1 Transmission by Lactococcus lactis

The importance of conjugation as a mechanism to spread biofilm determinants among microbial populations was illustrated with the gram-positive bacterium Lactococcus lactis. Conjugation triggered the enhanced expression of the clumping protein CluA, which is a main biofilm attribute in lactococci. Clumping transconjugants further transmitted the biofilm-forming elements among the lactococcal population at a much higher frequency than the parental nonclumping donor. This cell-clumping-associated high-frequency conjugation system also appeared to serve as an internal enhancer facilitating the dissemination of the broad-host-range drug resistance gene-encoding plasmid pAMβ1 within L. lactis, at frequencies more than 10,000 times higher than those for the nonclumping parental donor strain. The implications of this finding for antibiotic resistance gene dissemination are discussed.     

Heterogeneous biocatalyst for nitrile and amide transformation based on cells of nitrile-hydrolyzing bacteria and multiwalled carbon nanotubes

A heterogeneous biocatalyst for the biotransformation of nitriles and amides of carboxylic acids in the form of cells of nitrile-hydrolyzing bacteria immobilized on the carrier, was created based on multiwalled carbon nanotubes (MWCNTs). It was shown that bacterial cells form aggregates in contact with powderformed purified or unpurified MWCNTs. The amount of both gram-positive and gram-negative bacteria binding with unpurified MWCNTs was significantly higher than with purified. The nitrile hydratase and amidase activity of bacterial aggregates of purified MWCNTs was preserved to a greater extent as compared to that of unpurified MWCNTs and cells adhered to the surface of the carbonized pyrosealing material with MWCNTs. Both gram-positive Rhodococcus ruber gt1 and gram-negative Alcaligenes faecalis 2 remained viable when cultured in the presence of purified or unpurified MWCNTs. The obtained heterogeneous biocatalyst can be easily separated from the medium by filtration and can be used repeatedly.     

Susceptibility of Biofilms to Bdellovibrio bacteriovorus Attack

Biofilms are communities of microorganisms attached to a surface, and the growth of these surface attached communities is thought to provide microorganisms with protection against a range of biotic and abiotic agents. The capability of the gram-negative predatory bacterium Bdellovibrio bacteriovorus to control and reduce an existing Escherichia coli biofilm was evaluated in a static assay. A reduction in biofilm biomass was observed as early as 3 h after exposure to the predator, and an 87% reduction in crystal violet staining corresponding to a 4-log reduction in biofilm cell viability was seen after a 24-h exposure period. We observed that an initial titer of Bdellovibrio as low as 10² PFU/well or an exposure to the predator as short as 30 min is sufficient to reduce a preformed biofilm. The ability of B. bacteriovorus to reduce an existing biofilm was confirmed by scanning electron microscopy. The reduction in biofilm biomass obtained after the first 24 h of exposure to the predator remained unchanged even after longer exposure periods and reinoculation of the samples with fresh Bdellovibrio; however, no genetically stable resistant population of the host bacteria could be detected. Our data suggest that growth in a biofilm does not prevent predation by Bdellovibrio but allows a level of survival from attack greater than that observed for planktonic cells. In flow cell experiments B. bacteriovorus was able to decrease the biomass of both E. coli and Pseudomonas fluorescens biofilms as determined by phase-contrast and epifluorescence microscopy.     

Differences in Toll-like receptor expression and cytokine production after stimulation with heat-killed gram-positive and gram-negative bacteria

Innate immune surveillance in the blood is executed mostly by circulating monocytes, which recognize conserved bacterial molecules such as peptidoglycan and lipopolysaccharide. Toll-like receptors (TLR) play a central role in microbe-associated molecular pattern detection. The aim of this study was to compare the differences in TLR expression and cytokine production after stimulation of peripheral blood cells with heat-killed gram-negative and gram-positive human pathogens: Neisseria meningitidis, Escherichia coli, Staphylococcus aureus, and Streptococcus pneumoniae. We found that TLR2 expression is up-regulated on monocytes after stimulation with S. aureus, S. pneumoniae, E. coli, and N. meningitidis. Moreover, TLR2 up-regulation was positively associated with increasing concentrations of gram-positive bacteria, whereas higher concentrations of gram-negative bacteria, especially E. coli, caused a milder TLR2 expression increase when compared to low doses. Cytokines were produced in similar dose-dependent profiles regardless of the stimulatory pathogen; however, gram-negative pathogens induced higher cytokine levels when compared to gram-positive bacteria at the same density. These results indicate that gram-positive and gram-negative bacteria differ in their dose-dependent patterns of induction of TLR2 and TLR4, but not cytokine expression.     

Functional analysis of the Lactococcus lactis usp45 secretion signal in the secretion of a homologous proteinase and a heterologous α-amylase

The ups45 gene encodes the major extracellular protein from Lactococcus lactis. The deduced sequence of the 27 residue leader peptide revealed the tripartite characteristics of a signal peptide. This leader peptide directed the efficient secretion of the homologous proteinase (PrtP) in L. lactis, indicating that the putative signal peptide of PrtP can be replaced by the 27 residue Usp45 leader peptide. In addition, the 27 residue leader peptide could be used to secrete the Bacillus stearothermophilus α-amylase, encoded by the amyS gene. Fusion of the usp45 promoter region and various parts of the leader sequence to an amyS gene devoid of its signal sequence, showed that in Escherichia coli the first 19, 20, and 27 residues of the Usp45 leader are able to direct α-amylase secretion. In L. lactis the shorter signal peptides did not result in secretion of α-amylase, providing experimental evidence for the hypothesis that gram-positive bacteria require a longer signal peptide for secretion than gram-negative organisms.     

Impact of flow velocity on the dynamic behaviour of biofilm bacteria

Abstract

The impact of flow velocity (FV) on the growth dynamics of biofilms and bulk water heterotrophic plate count (HPC) bacteria in drinking water distribution systems was quantified and modeled by combining a logistic growth model with mass balance equations. The dynamic variations in the specific growth and release rates of biofilm bacteria were also quantified. The experimental results showed that the maximum biofilm biomass did not change when flow velocity was increased from 20 to 40 cm s^?1, but was significantly affected when flow velocity was further increased to 60 cm s^?1. Although the concentration of biofilm bacteria was substantially reduced by the higher shear stress, the concentration of bacteria in the bulk fluid was slightly increased. From this it is estimated that the specific growth rate and specific release rate of biofilm bacteria had doubled. The specific release (detachment) rate was dependent on the specific growth rate of the biofilm bacteria.