Antimicrobial activity of LFchimera synthetic peptide against plant pathogenic bacteria

The present study was designed to evaluate potential antibacterial activities of synthetic LFchimera against five plant pathogenic bacteria such as Ralstonia solanacearum, Erwinia amylovora, Xanthomonas campestris, Pseudomonas syringae and Pectobacterium carotovorum. The agar disc-diffusion method with different concentrations (0.2, 0.4, 0.6 and 0.8 μM) of peptide was used to study the antibacterial activity of LFchimera against bacteria. The Minimum Inhibitory Concentration (MIC) of the LFchimera peptide were tested using serial dilution method at concentration ranging from 0 to 10 μM. The Results from agar disc-diffusion method revealed that LFchimera was effective against all bacterial strain in a dose-dependent manner. LFchimera showed highest activity in 0.8 μM which was significant compared to the standard antibiotic. LFchimera pepetide showed low MIC values (4 μM) against all tested bacteria. LFchimera peptide was found to show antibacterial activity against important phytopathogenic bacteria and can improve the potential of an antimicrobial peptide in plant disease management.     

伊犁黑蜂蜂胶对口腔主要致龋细菌及生物膜生长的实验研究

目的研究伊犁黑蜂蜂胶对口腔常见致龋细菌及其生物膜生长的影响,观察其防龋效果。方法 (1)通过液体稀释法测定伊犁黑蜂蜂胶对口腔常见致龋菌的最小抑菌浓度(MIC)及最小杀菌浓度(MBC);(2)生物膜形成抑制试验测定伊犁黑蜂蜂胶对口腔常见致龋菌的最小生物膜形成抑制浓度(MBIC50);(3)通过结晶紫染色法测定伊犁黑蜂蜂胶对口腔常见致龋菌的最小生物膜清除浓度(MBEC);结果(1)伊犁黑蜂蜂胶对变形链球菌、远缘链球菌、嗜酸乳杆菌、血链球菌、粘性放线菌和内氏放线菌的最低抑菌浓度分别是0.78、0.39、1.56、0.39、0.20和0.20 mg/mL,最低杀菌浓度分别为1.56、0.78、3.125、0.78、0.39和0.39mg/mL;(2)伊犁黑蜂蜂胶对变形链球菌、远缘链球菌、血链球菌、粘性放线菌和内氏放线菌的MBIC50分别是0.39、0.39、0.39、0.05和0.10mg/mL;(3)伊犁黑蜂蜂胶对变形链球菌、远缘链球菌、血链球菌、粘性放线菌和内氏放线菌的MBEC分别是6.25、1.56、3.1256、0.78和0.78mg/mL。结论伊犁黑蜂蜂胶对口腔主要致龋细菌的生长具有抑制作用,能够抑制主要致龋细菌单菌生物膜的形成,并具有一定清除作用,是具有一定防龋效果的天然药物。     

Colonisation of the oral cavity by periodontopathic bacteria in complete denture wearers

doi: 10.1111/j.1741‐2358.2011.00506.x Colonisation of the oral cavity by periodontopathic bacteria in complete denture wearers Objective: The purpose of this study was to investigate colonisation by periodontopathic bacteria and the sites of colonisation in elderly upper and lower complete denture wearers. We also investigated the relationship between level of oral hygiene and colonisation by periodontopathic bacteria. Materials and methods: Forty edentulous and 37 dentate volunteers participated in this study. Samples were collected from whole saliva, and levels of Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia and Fusobacterium nucleatum were determined by PCR Invader technology. Detection of these species on oral mucosal and denture surfaces was performed by PCR. Fisher’s exact test was used for the statistical analysis. Cluster analysis was employed to investigate trends in the periodontopathic bacteria flora in each sampling area. Results: Detection rates of periodontopathic bacteria in whole saliva were lower under edentulous conditions than under dentulous conditions, except for A. actinomycetemcomitans and F. nucleatum (p < 0.01). Detection rate of F. nucleatum was the highest in all areas. A positive correlation was observed between DNA quantification of P. gingivalis and number of Candida species in saliva. Cluster analysis of the test species identified two clusters. Tongue‐coating status was associated with the detection rate of all periodontopathic bacteria investigated, and denture plaque status was associated with the detection rate of T. denticola and F. nucleatum. Conclusion: Results indicate the presence of periodontopathic bacteria under edentulous conditions and that the status of oral hygiene of the mucosal or denture surfaces affects colonisation by T. denticola and F. nucleatum.     

The serine protease Esperase HPF inhibits the formation of multispecies biofilm

The antifouling (AF) potential of the serine protease Esperase HPF (subtilisin) was evaluated for the ability to prevent the formation of a four-species bacterial biofilm. The effects of enzyme activity, time and application of the enzyme were tested on the density and the oxidative metabolism of biofilm developed in microtiter wells. Esperase HPF did not inhibit the oxidative metabolism of the bacterial biofilm or planktonic growth, but the enzyme inhibited biofilm formation by its proteolytic activity as inactivated enzyme had no effect. The effective enzyme concentration was determined over a period of 72 h, as by then all the tested concentrations inhibited biofilm formation maximally. The effective concentrations of the enzymes in solution were the same regardless of time of application (ie before or after biofilm formation), but immobilisation of the enzymes caused a lower effective concentration. Esperase HPF is an attractive alternative to the biocidal compounds used in AF coatings today.     

Beta-hexosaminidase activity of the oral pathogen Tannerella forsythia influences biofilm formation on glycoprotein substrates

Tannerella forsythia is an important pathogen in periodontal disease. Previously, we showed that its sialidase activity is key to utilization of sialic acid from a range of human glycoproteins for biofilm growth and initial adhesion. Removal of terminal sialic acid residues often exposes β-linked glucosamine or galactosamine, which may also be important adhesive molecules. In turn, these residues are often removed by a group of enzymes known as β-hexosaminidases. We show here that T. forsythia has the ability to cleave glucosamine and galactosamine from model substrates and that this activity can be inhibited by the hexosaminidase inhibitor PugNAc (O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino N-phenyl carbamate). We now demonstrate for the first time that β-hexosaminidase activity plays a role in biofilm growth on glycoprotein-coated surfaces because biofilm growth and initial cell adhesion are inhibited by PugNAc. In contrast, adhesion to siallo-glycoprotein-coated surfaces is unaltered by PugNAc in the absence of sialidase activity (using a sialidase-deficient mutant) or surprisingly on the clinically relevant substrates saliva or serum. These data indicate that β-hexosaminidase activity has a significant role in biofilm formation in combination with sialidase activity in the biofilm lifestyle of T. forsythia.     

Dose-response effect of chlorhexidine on a multispecies oral biofilm formed on pure titanium and on a titanium-zirconium alloy

Abstract

This study aimed to test the dose-response effect of chlorhexidine on multispecies biofilms formed on commercially pure titanium (cpTi) and titanium-zirconium (TiZr) alloy. Biofilms were formed on cpTi and TiZr discs and treated two times per day with five different chlorhexidine concentrations (0.12, 0.20, 0.50, 1, 2%). The biofilms were collected for microbiological, biochemical and microscopic analyses. The significance of differences among groups was evaluated by linear regression, ANOVA, Bonferroni and Tukey tests. The mean number of colony-forming units decreased as the chlorhexidine concentration increased for both cpTi and TiZr (p?<?0.05). The maximum effect was observed with the 0.5% concentration. Confocal microscopy images suggested an increase in the number of dead bacterial cells with increased chlorhexidine concentration. The biofilm pH increased after chlorhexidine exposure (p?<?0.05). Chlorhexidine showed an antimicrobial dose-response effect in controlling biofilm on cpTi and TiZr. 0.5% chlorhexidine can be used to achieve the maximum antimicrobial effect on both materials.     

Adhesion and biofilm formation on polystyrene by drinking water-isolated bacteria

This study was performed in order to characterize the relationship between adhesion and biofilm formation abilities of drinking water-isolated bacteria (Acinetobacter calcoaceticus, Burkholderia cepacia, Methylobacterium sp., Mycobacterium mucogenicum, Sphingomonas capsulata and Staphylococcus sp.). Adhesion was assessed by two distinct methods: thermodynamic prediction of adhesion potential by quantifying hydrophobicity and the free energy of adhesion; and by microtiter plate assays. Biofilms were developed in microtiter plates for 24, 48 and 72 h. Polystyrene (PS) was used as adhesion substratum. The tested bacteria had negative surface charge and were hydrophilic. PS had negative surface charge and was hydrophobic. The free energy of adhesion between the bacteria and PS was > 0 mJ/m² (thermodynamic unfavorable adhesion). The thermodynamic approach was inappropriate for modelling adhesion of the tested drinking water bacteria, underestimating adhesion to PS. Only three (B. cepacia, Sph. capsulata and Staphylococcus sp.) of the six bacteria were non-adherent to PS. A. calcoaceticus, Methylobacterium sp. and M. mucogenicum were weakly adherent. This adhesion ability was correlated with the biofilm formation ability when comparing with the results of 24 h aged biofilms. Methylobacterium sp. and M. mucogenicum formed large biofilm amounts, regardless the biofilm age. Given time, all the bacteria formed biofilms; even those non-adherents produced large amounts of matured (72 h aged) biofilms. The overall results indicate that initial adhesion did not predict the ability of the tested drinking water-isolated bacteria to form a mature biofilm, suggesting that other events such as phenotypic and genetic switching during biofilm development and the production of extracellular polymeric substances (EPS), may play a significant role on biofilm formation and differentiation. This understanding of the relationship between adhesion and biofilm formation is important for the development of control strategies efficient in the early stages of biofilm development.     

New insights on molecular regulation of biofilm formation in plant-associated bacteria

Biofilms are complex bacterial assemblages with a defined three-dimensional architecture, attached to solid surfaces, and surrounded by a self-produced matrix generally composed of exopolysaccharides, proteins, lipids and extracellular DNA. Biofilm formation has evolved as an adaptive strategy of bacteria to cope with harsh environmental conditions as well as to establish antagonistic or beneficial interactions with their host. Plant-associated bacteria attach and form biofilms on different tissues including leaves, stems,vasculature, seeds and roots. In this review, we examine the formation of biofilms from the plant-associated bacterial perspective and detail the recently-described mechanisms of genetic regulation used by these organisms to orchestrate biofilm formation on plant surfaces. In addition, we describe plant host signals that bacterial pathogens recognize to activate the transition from a planktonic lifestyle to multicellular behavior.     

In vitro activity of allicin against Staphylococcus epidermidis and influence of subinhibitory concentrations on biofilm formation

AIMS: The aim of this study is to determine the in vitro activity of allicin against Staphylococcus epidermidis and to evaluate the influence of allicin on biofilm formation. METHODS AND RESULTS: In vitro activity of allicin (diallyl thiosulphinate) against 38 strains of S. epidermidis was investigated. The activity of allicin was similar against S. epidermidis methicillin susceptible and methicillin resistant strains [minimum inhibitory concentration (MIC)90=8 mg l(-1)]. In general, subinhibitory concentrations (sub-MIC) of allicin diminished biofilm formation in the five strains analysed. CONCLUSION: The results confirm the antibacterial effect of allicin. Sub-MICs of allicin also diminished the biofilm formations by S. epidermidis. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study shows that allicin is active in vitro against S. epidermidis and that sub-MICs of allicin may play a role in the prevention of adherence of this bacteria to medical devices.     

Relationship between thigmotropism and Candida biofilm formation in vitro

The biofilm formation of the oral fungal pathogen Candida on denture acrylic strips coated with saliva or serum was examined in relation to the ability to induce hyphae by thigmotropic reaction, using C. albicans (4 isolates), C. glabrata (3 isolates) and C. tropicalis (3 isolates). Both the degree of biofilm formation and the amount of hyphae exhibiting thigmotropism varied depending upon both the species and strains of Candida. Although there was no significant correlation between the amount of hyphae induced by thigmotropic reaction of fungal isolates and biofilm formation on uncoated control specimens (r = 0.577; p < 0.05), the ability of hyphae induced by thigmotropic reaction significantly correlated with the amount of both saliva- and serum-admixed biofilms (r = 0.734; p < 0.05 and r = 0.793; p < 0.01, respectively). Taken together our in vitro data suggested that the hyphal induction by thigmotropic reaction is of importance in candidal biofilm formation on saliva- or serum-coated acrylic surfaces. This revised version was published online in August 2006 with corrections to the Cover Date.     

Quorum-quenching potential of recombinant PvdQ-engineered bacteria for biofilm formation

International Microbiology - Quorum sensing (QS) is a core mechanism for bacteria to regulate biofilm formation, and therefore, QS inhibition or quorum quenching (QQ) is used as an effective and...     

Microcolony and biofilm formation as a survival strategy for bacteria

Bacterial communities such as biofilms are widely recognized as being important for survival and persistence of bacteria in harsh environments. Mechanistic models of biofilm growth indicate that the way in which the surface is seeded can effect the morphology of simulated biofilms. Experimental studies indicate that genes which are important for chemotaxis also influence biofilm formation, perhaps by influencing aggregation on a surface. Understanding aggregation and microcolony formation could therefore help clarify factors influencing biofilm formation and illuminate how groups influence the fitness of bacteria. In this paper I develop an individual based model to examine how different behaviors involved in microcolony formation on a surface determine patterns of group sizes and link patterns to bacterial fitness.     

The inhibitory effects of mushroom extracts on sucrose-dependent oral biofilm formation

Mushrooms contain large quantities of α-glucans. Shiitake (Lentinula edodes), Japan’s most popular edible mushroom, has been reported to contain about 6% (weight/dried weight) of α-(1,3)-glucan. This glucan is one of the major components of oral biofilm formed by the cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus. We found that extracts from shiitake and other edible mushrooms could reduce preformed biofilms of S. mutans and S. sobrinus in the presence of dextranase. We also investigated the α-glucanase activities of shiitake mushroom extracts and their effects on biofilm formation. The extracts possessed α-glucanase activity and degraded water-insoluble glucans from mutans streptococci. The extracts strongly inhibited the sucrose-dependent formation of biofilms by S. mutans and S. sobrinus in the presence of dextranase. Our results suggest that some components of mushrooms, including α-glucanases, might inhibit the sucrose-induced formation of oral biofilms.     

Salivary detection of periodontopathic bacteria in periodontally healthy children

BACKGROUND: Salivary occurrence of periodontopathic bacteria is of interest especially in children as a risk indicator for the transmission, development and control of periodontal disease. We assessed the prevalence of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, Prevotella nigrescens and Treponema denticola as microbial complexes in the saliva of children with mixed dentition and healthy gingiva. MATERIALS AND METHODS: Paraffin-stimulated saliva samples were collected from 41 children (22 boys and 19 girls), aged 6-13 years old. Gingival health was determined during the initial screening exam. The test bacteria were identified using a 16S rRNA-based PCR analysis. RESULTS: P. nigrescens was the most frequent species (80%), followed by T. denticola (32%), A. actinomycetemcomitans (24%) and P. gingivalis (12%). P. intermedia and T. forsythia were not detected. P. nigrescens was also common species in combinations. Paired and triple bacterial combinations were found in 24% and 20% of all children, respectively. There was no positive association between bacterial combinations in colonization and subject's gender (P>0.05, Fisher exact test). CONCLUSION: The salivary presence of P. nigrescens, T. denticola, A. actinomycetemcomitans and P. gingivalis but not P. intermedia and T. forsythia can occur in childhood without clinical signs of gingival disease. Thus, the possible risk of bacterial transmissions through saliva and, the need to screen for periodontal pathogens should be considered before mixed dentition.     

Detection of collagenase activity in oral bacteria

Collagenolytic activity of 12 species of oral bacteria was assessed using two methods of detection. Except for two species, all bacterial strains tested were capable of degrading at least one general protein substrate. Results of collagenolytic activity in a growth assay indicate that Bacteroides gingivalis is the only bacterium capable of degrading collagen when the substrate is sterilized using ethylene oxide. However, if the substrate is sterilized by autoclaving, in the presence or absence of the growth medium, other bacterial species could be shown to be collagenolytic. Collagenolytic activity was also demonstrated when whole or broken cells were used in a [14C]collagen assay. Results from this assay and from inhibition studies indicate that collagenolytic activity can either be the result of the combined activities of both a specific collagenase and nonspecific proteases (B. gingivalis) or nonspecific proteases only (other strains in this study), although in the latter case, the time taken to hydrolyze collagen can be 10 times longer than with a specific collagenase.     

Mixed-species biofilm formation by lactic acid bacteria and rice wine yeasts

We found that species combinations such as Lactobacillus casei subsp. rhamnosus IFO3831 and Saccharomyces cerevisiae Kyokai-10 can form a mixed-species biofilm in coculture. Moreover, the Kyokai-10 yeast strain can form a biofilm in monoculture in the presence of conditioned medium (CM) from L. casei IFO3831. The active substance(s) in bacterial CM is heat sensitive and has a molecular mass of between 3 and 5 kDa. In biofilms from cocultures or CM monocultures, yeast cells had a distinct morphology, with many hill-like protrusions on the cell surface.     

Pili contribute to biofilm formation in vitro in Mycobacterium tuberculosis

Organized bacterial communities, or biofilms, provide an important reservoir for persistent cells that are inaccessible or tolerant to antibiotics. Curli pili are cell-surface structures produced by certain bacteria and have been implicated in biofilm formation in these species. In order to determine whether these structures, which were suggested to be encoded by the Rv3312A (mtp) gene, have a similar role in Mycobacterium tuberculosis, we generated a Δmtp mutant and a mtp-complemented strain of a clinical isolate of M. tuberculosis and analyzed these strains for their ability to produce pili in comparison to the wild-type strain. Phenotypic analysis by transmission electron microscopy proved the essentiality of mtp for piliation in M. tuberculosis. We then compared biofilm formation of the derived strains in detergent-free Sauton’s media. Biofilm mass was quantified spectrophotometrically using crystal violet. Furthermore, we examined mtp gene expression by quantitative real-time PCR in wild-type cells grown under biofilm versus planktonic growth conditions. We found a 68.4 % reduction in biofilm mass in the mutant compared to the wild-type strain (P = 0.002). Complementation of the mutant resulted in a restoration of the wild-type biofilm phenotype (P = 0.022). We, however, found no significant difference between mtp expression in cells of the biofilm to those growing planktonically. Our findings highlight a crucial, but non-specific, role of pili in the biofilm lifestyle of M. tuberculosis and indicate that they may represent an important target for the development of therapeutics to attenuate biofilm formation, thereby potentially reducing persistence.     

Effect of temperature on biofilm formation by Antarctic marine bacteria in a microfluidic device

Polar biofilms have become an increasingly popular biological issue because new materials and phenotypes have been discovered in microorganisms in the polar region. Various environmental factors affect the functionality and adaptation of microorganisms. Because the polar region represents an extremely cold environment, polar microorganisms have a functionality different from that of normal microorganisms. Thus, determining the effective temperature for the development of polar biofilms is crucial. Here, we present a simple, novel one-pot assay for analysis of the effect of temperature on formation of Antarctic bacterial biofilm using a microfluidic system where continuous temperature gradients are generated. We find that a specific range of temperature is required for the growth of biofilms. Thus, this microfluidic approach provides precise information regarding the effective temperature for polar biofilm development with a new high-throughput screening format.     

Community structure and activity dynamics of nitrifying bacteria in a phosphate-removing biofilm

The microbial community structure and activity dynamics of a phosphate-removing biofilm from a sequencing batch biofilm reactor were investigated with special focus on the nitrifying community. O(2), NO(2)(-), and NO(3)(-) profiles in the biofilm were measured with microsensors at various times during the nonaerated-aerated reactor cycle. In the aeration period, nitrification was oxygen limited and restricted to the first 200 microm at the biofilm surface. Additionally, a delayed onset of nitrification after the start of the aeration was observed. Nitrate accumulating in the biofilm in this period was denitrified during the nonaeration period of the next reactor cycle. Fluorescence in situ hybridization (FISH) revealed three distinct ammonia-oxidizing populations, related to the Nitrosomonas europaea, Nitrosomonas oligotropha, and Nitrosomonas communis lineages. This was confirmed by analysis of the genes coding for 16S rRNA and for ammonia monooxygenase (amoA). Based upon these results, a new 16S rRNA-targeted oligonucleotide probe specific for the Nitrosomonas oligotropha lineage was designed. FISH analysis revealed that the first 100 microm at the biofilm surface was dominated by members of the N. europaea and the N. oligotropha lineages, with a minor fraction related to N. communis. In deeper biofilm layers, exclusively members of the N. oligotropha lineage were found. This separation in space and a potential separation of activities in time are suggested as mechanisms that allow coexistence of the different ammonia-oxidizing populations. Nitrite-oxidizing bacteria belonged exclusively to the genus Nitrospira and could be assigned to a 16S rRNA sequence cluster also found in other sequencing batch systems.