Antibacterial effect of bestatin during periodontitis

Periodontitis is a polymicrobial disease inciting inflammatory destruction of the tooth-supporting tissues, i.e., periodontium. The initiation of this infectious disease is ascribed to the formation of subgingival biofilms. These biofilms cause stimulation of myriad of chronic inflammatory reactions by the affected tissue. The Gram-negative anaerobe Porphyromonas gingivalis is commonly found as part of the microbiota of subgingival biofilms, and is involved in the occurrence of the disease. P. gingivalis possesses numerous virulence factors supporting its survival, regulating its communication with other species in the biofilm, degrading host tissues. Fusobacterium nucleatum is pivotal for formation of biofilm and promotes growth and invasion properties of P. gingivalis. Bestatin is an aminopeptide inhibitor, produced by actinomycetes. It possesses antibacterial properties against P. gingivalis and F. nucleatum. The following review focuses on action of bestatin on the mentioned bacteria.     

Fluorescence change of <Emphasis Type="Italic">Fusobacterium nucleatum</Emphasis> due to <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis>

The aim of this study was to measure changes in the fluorescence of Fusobacterium nucleatum interacting with Porphyromonas gingivalis for excitation with blue light at 405-nm. P. gingivalis was mono- and co-cultivated in close proximity with F. nucleatum. The fluorescence of the bacterial colonies was photographed using a QLF-D (Quantitative Light-induced Fluorescence-Digital) Biluminator camera system with a 405 nm light source and a specific filter. The red, green and blue intensities of fluorescence images were analyzed using the image analysis software. A fluorescence spectrometer was used to detect porphyrin synthesized by each bacterium. F. nucleatum, which emitted green fluorescence in single cultures, showed intense red fluorescence when it was grown in close proximity with P. gingivalis. F. nucleatum co-cultivated with P. gingivalis showed the same pattern of fluorescence peaks as for protoporphyrin IX in the red part of the spectrum. We conclude that the green fluorescence of F. nucleatum can change to red fluorescence in the presence of adjacent co-cultured with P. gingivalis, indicating that the fluorescence character of each bacterium might depend on the presence of other bacteria.     

Decrease of insoluble glucan formation in <Emphasis Type="Italic">Streptococcus mutans</Emphasis> by co-cultivation with <Emphasis Type="Italic">Enterococcus faecium</Emphasis> T7 and glucanase addition

Objectives

To develop preventive canine oral health bio-materials consisting of probiotics and glucanase to reduce insoluble glucan and volatile sulfur compound formation.

Results

Co-cultivation of Enterococcus faecium T7 with Streptococcus mutans at inoculation ratio of 3:1 (v/v) resulted in 25% reduction in the growth of Streptococcus mutans. Amounts of soluble and insoluble glucans produced by S. mutans were decreased to 70 and 55%, respectively. Insoluble glucan was decreased from 0.6 µg/ml in S. mutans culture to 0.03 µg/ml in S. mutans co-cultivated with E. faecium T7 in the presence of Lipomyces starkeyi glucanase. Volatile sulfur compound, a main component of halitosis produced by Fusobacteria nucleatum, was decreased by co-cultivating F. nucleatum with E. faecium.

Conclusion

E. faecium and glucanase can be combined as potentially active ingredients of oral care products for pets by reducing plaque-forming bacteria growth and their by-products that cause cavity and periodontal disease.

     

Subtilosin Prevents Biofilm Formation by Inhibiting Bacterial Quorum Sensing

Subtilosin, the cyclic lantibiotic protein produced by Bacillus subtilis KATMIRA1933, targets the surface receptor and electrostatically binds to the bacterial cell membrane. In this study, subtilosin was purified using ammonium sulfate ((NH₄)₂SO₄) precipitation and purified via column chromatography. Subtilosin’s antibacterial minimum and sub-minimum inhibitory concentrations (MIC and sub-MIC) and anti-biofilm activity (biofilm prevention) were established. Subtilosin was evaluated as a quorum sensing (QS) inhibitor in Gram-positive bacteria using Fe(III) reduction assay. In Gram-negative bacteria, subtilosin was evaluated as a QS inhibitor utilizing Chromobacterium voilaceum as a microbial reporter. The results showed that Gardnerella vaginalis was more sensitive to subtilosin with MIC of 6.25 μg/mL when compared to Listeria monocytogenes (125 μg/mL). The lowest concentration of subtilosin, at which more than 90% of G. vaginalis biofilm was inhibited without effecting the growth of planktonic cells, was 0.78 μg/mL. About 80% of L. monocytogenes and more than 60% of Escherichia coli biofilm was inhibited when 15.1 μg/mL of subtilosin was applied. Subtilosin with 7.8–125 μg/mL showed a significant reduction in violacein production without any inhibitory effect on the growth of C. violaceum. Subtilosin at 3 and 4 μg/mL reduced the level of Autoinducer-2 (AI-2) production in G. vaginalis. However, subtilosin did not influence AI-2 production by L. monocytogenes at sub-MICs of 0.95–15.1 μg/mL. To our knowledge, this is the first report exploring the relationship between biofilm prevention and quorum sensing inhibition in G. vaginalis using subtilosin as a quorum sensing inhibitor.     

Investigation of microorganisms involved in kefir biofilm formation

Kefir is a natural fermentation agent composed of various microorganisms. To address the mechanism of kefir grain formation, we investigated the microbial role in forming kefir biofilms. The results showed that a biofilm could be formed in kefir-fermented milk and the biofilm forming ability reached the maximum at 13 days. The strains Kluyveromyces marxianus, Lactococcus lactis, Leuconostoc mesenteroides, Lactobacillus kefiri, Lactobacillus sunkii and Acetobacter orientalis were isolated from kefir biofilms by the streak-plate method. These microorganisms were analysed with respect to biofilm forming properties, including their surface characterisation (hydrophobicity and zeta potentials) and the microbial aggregation. The results indicated that Klu. marxianus possessed the strongest biofilm forming properties with the strongest hydrophobicity, lowest zeta potential and greatest auto-aggregation ability. When Klu. marxianus and Ac. orientalis were co-cultured with kefir LAB strains respectively, it was found that mixing Klu. marxianus with Lb. sunkii produced the highest co-aggregation ability. These results elucidated the mechanism of kefir biofilm formation and the microorganisms involved.     

Dual-species biofilm of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> on stainless steel surface

Listeria monocytogenes is a Gram-positive bacterium commonly associated with foodborne diseases. Due its ability to survive under adverse environmental conditions and to form biofilm, this bacterium is a major concern for the food industry, since it can compromise sanitation procedures and increase the risk of post-processing contamination. Little is known about the interaction between L. monocytogenes and Gram-negative bacteria on biofilm formation. Thus, in order to evaluate this interaction, Escherichia coli and L. monocytogenes were tested for their ability to form biofilms together or in monoculture. We also aimed to evaluate the ability of L. monocytogenes 1/2a and its isogenic mutant strain (ΔprfA ΔsigB) to form biofilm in the presence of E. coli. We assessed the importance of the virulence regulators, PrfA and σ^B, in this process since they are involved in many aspects of L. monocytogenes pathogenicity. Biofilm formation was assessed using stainless steel AISI 304 #4 slides immersed into brain heart infusion broth, reconstituted powder milk and E. coli preconditioned medium at 25 °C. Our results indicated that a higher amount of biofilm was formed by the wild type strain of L. monocytogenes than by its isogenic mutant, indicating that prfA and sigB are important for biofilm development, especially maturation under our experimental conditions. The presence of E. coli or its metabolites in preconditioned medium did not influence biofilm formation by L. monocytogenes. Our results confirm the possibility of concomitant biofilm formation by L. monocytogenes and E. coli, two bacteria of major significance in the food industry.     

Resveratrol,pterostilbene, and baicalein: plant-derived anti-biofilm agents

Microbial adhesion to surfaces and the subsequent biofilm formation may result in contamination in food industry and in healthcare-associated infections and may significantly affect postoperative care. Some plants produce substances with antioxidant and antimicrobial properties that are able to inhibit the growth of food-borne pathogens. The aim of our study was to evaluate antimicrobial and anti-biofilm effect of baicalein, resveratrol, and pterostilbene on Candida albicans, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Escherichia coli. We determined the minimum inhibitory concentrations (MIC), the minimum adhesion inhibitory concentration (MAIC), and the minimum biofilm eradication concentration (MBEC) by crystal violet and XTT determination. Resveratrol and pterostilbene have been shown to inhibit the formation of biofilms as well as to disrupt preformed biofilms. Our results suggest that resveratrol and pterostilbene appear potentially very useful to control and inhibit biofilm contaminations by Candida albicans, Staphylococcus epidermidis, and Escherichia coli in the food industry.     

Type I fimbriae mediate in vitro adherence of porcine F18ac+ enterotoxigenic <Emphasis Type="Italic">Escherichia coli</Emphasis> (ETEC)

Type I fimbriae commonly expressed by Escherichia coli mediate initial attachment of bacteria to host epithelial cells. However, the role of type I fimbriae in the adherence of porcine enterotoxigenic E. coli (ETEC) to host receptors is unclear. In this study, we examined the role of type I fimbriae in the adherence and biofilm formation of F18ac+ ETEC by constructing mutant strains with deletion of type I fimbrial major subunit (fimA) or minor subunit (fimH). The data indicated that the isogenic ΔfimA and ΔfimH mutants showed significantly lower adherence to porcine epithelial IPEC-1 and IPEC-J2 cells as compared to the F18ac+ ETEC parent strain. In addition, the adherence of F18ac+ ETEC to both cell lines was blocked by the presence of 0.5% D-mannose in the cell culture medium. In addition, both mutant strains impaired their ability to form biofilm in vitro. Interestingly, the deletion of fimA or fimH genes resulted in remarkable up-regulation of the expression of adhesin involved in diffuse adherence (AIDA-I). These results indicated that type I fimbriae may be required for efficient adherence of F18ac+ ETEC to pig epithelial cells and, perhaps, biofilm formation.     

Assessment of genetic diversity among four orchids based on ddRAD sequencing data for conservation purposes

Genetic diversity was assessed in the four orchid species using NGS based ddRAD sequencing data. The assembled nucleotide sequences (fastq) were deposited in the SRA archive of NCBI Database with accession number (SRP063543 for Dendrobium, SRP065790 for Geodorum, SRP072201 for Cymbidium and SRP072378 for Rhynchostylis). Total base pair read was 1.1 Mbp in case of Dendrobium sp., 553.3 Kbp for Geodorum sp., 1.6 Gbp for Cymbidium, and 1.4 Gbp for Rhynchostylis. Average GC% was 43.9 in Geodorum, 43.7% in Dendrobium, 41.2% in Cymbidium and 42.3% in Rhynchostylis. Four partial gene sequences were used in DnaSP5 program for nucleotide diversity and phylogenetic relationship determination (Ycf2 gene of Dendrobium, matK gene of Geodorum, psbD gene of Cymbidium and Ycf2 gene of Ryhnchostylis). Nucleotide diversity (per site) Pi (π) was 0.10560 in Dendrobium, 0.03586 in Geodorum, 0.01364 in Cymbidium and 0.011344 in Rhynchostylis. Neutrality test statistics showed the negative value in all the four orchid species (Tajima’s D value ?2.17959 in Dendrobium, ?2.01655 in Geodorum, ?2.12362 in Rhynchostylis and ?1.54222 in Cymbidium) indicating the purifying selection. Result for these gene sequences (matK and Ycf2 and psbD) indicate that they were not evolved neutrally, but signifying that selection might have played a role in evolution of these genes in these four groups of orchids. Phylogenetic relationship was analyzed by reconstructing dendrogram based on the matK, psbD and Ycf2 gene sequences using maximum likelihood method in MEGA6 program.     

Enterocin B3A-B3B produced by LAB collected from infant faeces: potential utilization in the food industry for <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> biofilm management

Enterococcus faecalis B3A-B3B produces the bacteriocin B3A-B3B with activity against Listeria monocytogenes, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium perfringens, but apparently not against fungi or Gram-negative bacteria, except for Salmonella Newport. B3A-B3B enterocin has two different nucleotides but similar amino acid composition to the class IIb MR10A-MR10B enterocin. B3A-B3B consists of two peptides of predicted molecular mass of 5176.31 Da (B3A) and 5182.21 Da (B3B). Importantly, B3A-B3B impeded biofilm formation of the foodborne pathogen L. monocytogenes 162 grown on stainless steel. The antimicrobial treatment of stainless steel with nisin (1 or 16 mg ml^?1) decreased the cell numbers by about 2 log CFU ml^?1, thereby impeding the biofilm formation by L. monocytogenes 162 or its nisin-resistant derivative strain L. monocytogenes 162R. Furthermore, the combination of nisin and B3A-B3B enterocin reduced the MIC required to inhibit this pathogen grown in planktonic or biofilm cultures.     

Molecular analyses of <Emphasis Type="Italic">Ficus erecta</Emphasis> and its allies within the subsection <Emphasis Type="Italic">Frutescentiae</Emphasis> (Moraceae)

Ficus (Moraceae) is a keystone group in tropical and subtropical forests with remarkable diversity of species and taxonomical challenges as a consequence of fig–pollinator coevolution. Ficus subsect. Frutescentiae includes about 30 species that are predominantly shrubs or small trees with Terminalia branching. Many of these species are difficult to delimit morphologically, and the group includes a tangle of uncertain taxa and incorrectly applied names. We conducted a phylogenetic analysis with internal and external transcribed spacer data (ITS and ETS) and data from 18 polymorphic microsatellite loci to evaluate the species status of the most perplexing members of this subsection. The results confirm the monophyly of subsect. Frutescentiae, with F. pedunculosa as sister to the rest. The F. erecta complex comprises approximately 17 taxa: F. erecta, F. abelii, F. boninsimae, F. nishimurae, F. iidaiana, F. gasparriniana var. laceratifolia, F. gasparriniana var. viridescens, F. pyriformis, F. stenophylla, F. fusuiensis, F. fengkaiensis, F. sinociliata, F. tannoensis, F. vaccinioides, F. formosana, F. pandurata, and F. periptera. The last five of these were supported as good species, while the others were not well supported by the present evidence. Evidence also supported the status of the non-F. erecta complex species including. F. pedunculosa, F. ischnopoda, F. heteromorpha, and F. variolosa. Ficus filicauda and F. neriifolia are possibly conspecific. The species status of F. potingensis should be restored and it should be treated as a member of section Eriosycea. Identification of the remaining taxa (F. gasparriniana var. esquirolii, F. ruyuanensis, F. daimingshanensis, F. chapaensis, F. changii, F. trivia, and F. tuphapensis) and their relationships to the F. erecta complex were not clarified. As a whole, only ten species in this subsection are confirmed, one is excluded, one is synonymous, and the others are either unresolved or short of samples. There appears to be a consistent genetic background among these unresolved groups, which suggests that repeated hybridization (as a result of pollinator host shifts) has filled up the interspecific gaps during the fig–pollinator coevolution process.     

Vanillin Resistance Induced by BssS Overexpression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Overexpression of the BssS gene, a biofilm formation regulator, in planktonic Escherichia coli cells has been shown to confer the vanillin-resistant phenotype Van^r to the bacteria. The MG1655P_L-tac-bssS strain started growing in liquid aerated LB medium with 2 g/L vanillin after a lag phase of 17 ± 2 h, whereas the original MG1655 strain did not grow under these conditions. The role of aldehyde reductase YqhD, a vanillin- degrading enzyme, in Van^r phenotype formation has been assessed. However, the Van^r trait in the MG1655P_L-tac-bssS strain primarily depended on autoinducer-2 (AI-2), which formed in E. coli cells with an intact luxS gene. We supposed that BssS acts together with autoinducer-2 (which presumably accumulated during the prolonged lag phase) to induce vanillin resistance determined by changes in the expression of a range of genes.     

Effect of Homocysteine on Biofilm Formation by Mycobacteria

Mycobacteria show peculiar aggregated outgrowth like biofilm on the surface of solid or liquid media. Biofilms harbor antibiotic resistant bacteria in a self-produced extracellular matrix that signifies the bacterial fate to sedentary existence. Despite years of research, very little is known about the mechanisms that contribute to biofilm formation. LuxS has been previously known to play a role in biofilm formation in Autoinducer-2 dependent manner. We here show the effect of LuxS product-homocysteine, on the biofilm forming ability of non-tuberculous mycobacteria, Mycobacterium smegmatis and Mycobacterium bovis BCG showing AI-2 independent phenotypic effect of LuxS. Exogenous supplementation of homocysteine in the culture media leads to aberrant cording, pellicle outgrowth, and biofilm formation. Thus, our study contributes to the better understanding of the mechanism of mycobacterial biofilm formation and sheds light on the role of LuxS product homocysteine. In addition, we highlight the contribution of activated methyl cycle in bacterial quorum sensing.     

Inhibition of biofilm in <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> Q-426 by diketopiperazines

Biofilm formation can make significant effects on bacteria habits and biological functions. In this study, diketopiperazines (DKPs) produced by strain of Bacillus amyloliquefaciens Q-426 was found to inhibit biofilm formed in the gas–liquid interface. Four kinds of DKPs were extracted from B. amyloliquefaciens Q-426, and we found that 0.04 mg ml^?1 DKPs could obviously inhibit the biofilm formation of the strain. DKPs produced by B. amyloliquefaciens Q-426 made a reduction on extracellular polymeric substance (EPS) components, polysaccharides, proteins, DNAs, etc. Real-time PCR was performed to determine that whether DKPs could make an obvious effect on the expression level for genes related to biofilm formation in the strain. The relative expression level of genes tasA, epsH, epsG and remB which related to proteins, extracellular matrix, and polysaccharides, were downregulated with 0.04 mg ml^?1 DKPs, while the expression level of nuclease gene nuc was significantly upregulated. The quantitative results of the mRNA expression level for these genes concerted with the quantitative results on EPS levels. All of the experimental results ultimately indicated that DKPs could inhibit the biofilm formation of the strain B. amyloliquefaciens Q-426.     

Isolation and characterization of marine biofilm forming bacteria from a ship’s hull

Background

Diverse aquatic microorganisms are capable of colonizing living and non-living surfaces leading to the formation of biofilms. Commonly visualized as a slimy layer, these biofilms are filled with hundreds of other microorganisms compared to free living planktonic cells. Microbial surface colonization and surface-associated metabolic activities also exert several macroscale deleterious effects, including biofouling, biocorrosion and the persistence and transmission of harmful or pathogenic microorganisms and virulence determinants. The present study deals with the isolation and screening of marine bacteria for biofilm formation. The screened isolates were characterized and identified as Pychrobacter celer, Pychrobacter alimentarius and Kocuria rhizophila by 16S rRNA sequencing.

Methods

Biofilm forming bacteria were isolated by spread plate technique and subjected to screening by microtiter plate assay. The potent biofilm formers were identified by molecular characterization using 16S rRNA gene sequencing.

Results

Twelve bacterial isolates were obtained by pour plate technique and subjected to biofilm assay. Among the 12 isolates three isolates which showed maximum biofilm formation were subjected to molecular characterizationby 16S rRNA gene sequencing method. The isolates were identified as Pychrobacter celer, Pychrobacter alimentarius and Kocuria rhizophila. The EPS produced by the three biofilm forming bacteria was extracted and the protein and carbohydrate content determined.

Conclusion

Among the isolates screened, isolate 8 (Kocuria rhizophila) produced maximum protein and carbohydrate which was also in accordance with the results of microtiter plate assay.

     

Chromosome number variability in central european members of the<Emphasis Type="Italic">Festuca ovina</Emphasis> and<Emphasis Type="Italic">F. pallens</Emphasis> groups (sect.<Emphasis Type="Italic">Festuca</Emphasis>)

Chromosome numbers for 98 plants ofF. pallens, 19 ofF. psammophila, F. belensis andF. vaginata, and 44 ofF. ovina (originating from Austria, the Czech Republic, Germany, Slovakia and Latvia) are given. In addition to theF. ovina andF. pallens groups, chromosome counts for the following taxa are also reported:F. alpestris (2n=14) reported for the first time in this work,F. amethystina subsp.amethystina (2n=28),F. brevipila (2n=42),F. cinerea (2n=28),F. rupicola subsp.rupicola (2n=42) andF. versicolor subsp.versicolor (2n=14).InF. pallens, two ploidy levels (2n=2x=14+0-1B, 2n=4x=28+0-1B) as well as two natural triploid plants (2n=21+0-1B), were found. In addition to the fourF. pallens types that have been distinguished in Austria, one new tetraploid type (F. pallens “scabrifolia”) from the Czech Republic and Germany is reported and its taxonomy is discussed. The distributions of the Oberösterreich-Niederösterreich and Pannonisches-HügellandF. pallens types outside of Austria are documented.Only the diploid chromosome number (2n=14) was found inF. psammophila andF. vaginata. Chromosome numbers forF. psammophila subsp.muellerstollii andF. belensis (both 2n=14) were determined here for the first time. Two ploidy levels, 2n=14+0-5B corresponding toF. ovina subsp.ovina and 2n=28 corresponding toF. ovina subsp.guestphalica andF. cf.duernsteinensis were confirmed inF. ovina. Differences in chromosome structure (simple and multiple secondary constrictions) betweenF. pallens as opposed toF. psammophila andF. vaginata are discussed. A complete survey of published chromosome counts for Central European species from theF. ovina andF. pallens groups is included.     

Unexpected presence of <Emphasis Type="Italic">Fagus orientalis</Emphasis> complex in Italy as inferred from 45,000-year-old DNA pollen samples from Venice lagoon

Background

Phylogeographic analyses on the Western Euroasiatic Fagus taxa (F. orientalis, F. sylvatica, F. taurica and F. moesiaca) is available, however, the subdivision of Fagus spp. is unresolved and there is no consensus on the phylogeny and on the identification (both with morphological than molecular markers) of Fagus Eurasiatic taxa.For the first time molecular analyses of ancient pollen, dated at least 45,000 years ago, were used in combination with the phylogeny analysis on current species, to identify the Fagus spp. present during the Last Interglacial period in Italy.In this work we aim at testing if the trn L-trn F chloroplast DNA (cpDNA) region, that has been previously proved efficient in discriminating different Quercus taxa, can be employed in distinguishing the Fagus species and in identifying the ancient pollen.

Results

86 populations from 4 Western Euroasistic taxa were sampled, and sequenced for the trn L-trn F region to verify the efficiency of this cpDNA region in identifying the Fagus spp.. Furthermore, Fagus crenata (2 populations), Fagus grandifolia (2 populations), Fagus japonica, Fagus hayatae, Quercus species and Castanea species were analysed to better resolve the phylogenetic inference.Our results show that this cpDNA region harbour some informative sites that allow to infer relationships among the species within the Fagaceae family. In particular, few specific and fixed mutations were able to discriminate and identify all the different Fagus species.Considering a short fragment of 176 base pairs within the trn L intron, 2 transversions were found able in distinguishing the F. orientalis complex taxa (F. orientalis, F. taurica and F. moesiaca) from the remaining Fagus spp. (F. sylvatica, F. japonica, F. hayataea, F. crenata and F. grandifolia). This permits to analyse this fragment also in ancient samples, where DNA is usually highly degraded.The sequences data indicate that the DNA recovered from ancient pollen belongs to the F. orientalis complex since it displays the informative sites characteristic of this complex.

Conclusion

The ancient DNA sequences demonstrate for the first time that, in contrast to current knowledge based on palynological and macrofossil data, the F. orientalis complex was already present during the Tyrrhenian period in what is now the Venice lagoon (Italy).This is a new and important insight considering that nowadays West Europe is not the natural area of Fagus orientalis complex, and up to now nobody has hypothesized the presence during the Last Interglacial period of F. orientalis complex in Italy.