Exopolysaccharide Synthesis Research Articles

Bacterial-derived extracellular polysaccharides reduce antimicrobial susceptibility on biotic and abiotic surfaces

ObjectiveExtracellular biofilm matrix plays a role in reducing bacterial susceptibility against antimicrobials. Since the surface where biofilm is growing modulates microbial accumulation and bacterial-derived exopolysaccharides (EPS) synthesis, this study compared the role of EPS to reduce antimicrobial susceptibility on biotic (dental surface) and abiotic (titanium (Ti) material) surfaces and the effect of remaining matrix-enriched biofilms to promote bacterial recolonization. Design48 h Streptococcus mutans UA159 strain biofilms were grown on enamel and Ti surfaces. The medium was supplemented with 1% sucrose, substrate for EPS synthesis, or with 0.5% glucose + 0.5% fructose as control. Chlorhexidine (CHX) 0.2% was used for antimicrobial treatment. Biofilms were collected and the following analyses were considered: viable bacterial counts, biofilm pH, EPS content, and biofilm structure by scanning electron microscopy and confocal laser scanning microscopy (CLSM). Substrate surfaces were analyzed by 3D laser scanning confocal microscope. ResultsEnamel surface showed a higher amount of EPS content (p < 0.05), which may be explained by the higher bacterial biomass compared to Ti material. EPS content reduced bacterial susceptibility against antimicrobial treatments for both substrates, compared to EPS control (p < 0.05). However, sucrose-treated cells presented the same magnitude of reduction for Ti or enamel. Interestingly, matrix-enriched biofilms favored bacterial recolonization for both substrates. ConclusionThe surface where the biofilm is growing modulates the amount of EPS synthesized and matrix content plays a key role in reducing antimicrobial susceptibility and promoting bacterial recolonization.

The TPR domain of PgaA is a multifunctional scaffold that binds PNAG and modulates PgaB-dependent polymer processing.

The synthesis of exopolysaccharides as biofilm matrix components by pathogens is a crucial factor for chronic infections and antibiotic resistance. Many periplasmic proteins involved in polymer processing and secretion in Gram-negative synthase dependent exopolysaccharide biosynthetic systems have been individually characterized. The operons responsible for the production of PNAG, alginate, cellulose and the Pel polysaccharide each contain a gene that encodes an outer membrane associated tetratricopeptide repeat (TPR) domain containing protein. While the TPR domain has been shown to bind other periplasmic proteins, the functional consequences of these interactions for the polymer remain poorly understood. Herein, we show that the C-terminal TPR region of PgaA interacts with the de-N-acetylase domain of PgaB, and increases its deacetylase activity. Additionally, we found that when the two proteins form a complex, the glycoside hydrolase activity of PgaB is also increased. To better understand structure-function relationships we determined the crystal structure of a stable TPR module, which has a conserved groove formed by three repeat motifs. Tryptophan quenching, mass spectrometry analysis and molecular dynamics simulation studies suggest that the crystallized TPR module can bind PNAG/dPNAG via its electronegative groove on the concave surface, and potentially guide the polymer through the periplasm towards the porin for export. Our results suggest a scaffolding role for the TPR domain that combines PNAG/dPNAG translocation with the modulation of its chemical structure by PgaB.

THE EVALUATION OF SELECTED BIOACTIVITIES OF FUNGAL MYCELIUM OF A WILD Schizophyllum commune Fr. FROM SUBMERGED CULTURES

Schizophyllum commune is a widely distributed macrofungal species assorted with bioactivities. The specific objectives were evaluation of antibacterial and antioxidant properties, synthesis of exopolysaccharides and carbohydrates contents estimation of mycelial extract of S. commune under submerged condition. Methanolic extracts of identified S. commune showed highest FRS (92.59%), chelating effect on ferrous ion (79.36%), reducing power (1.844) activities at 16, 1.5, 16 mg/ml concentrations respectively (p&lt;0.05). S. commune showed potent antibacterial activities (p&lt;0.05) against four bacterial strains except against P. aeruginosa where no activity was observed. The maximum inhibition zone was exhibited against S. aureus (20.2 mm) and minimum inhibition was found against B. subtilis (5.0 mm). This study recorded the highest dry weight (9.33 g/L) and rate of average growth (1.33 g/L/day) of S. commune in glucose (p&lt;0.05) medium but highest EPS (4.10 g/L) production was observed in xylose medium. Out of seven nitrogen sources, maximum mycelial yield (2.7 g/L), rate of average growth (0.38 g/L) and EPS (4.26 g/L) were recorded in yeast extract medium (p&lt;0.05). In glucose (0.597 g/100g) and yeast extract (0.006 g/100g) medias, the maximum amount of total carbohydrate was obtained from the EPSs of S. commune. That’s why it may be an excellent source of human medicines.

Inhibition of Biofilm Formation and Virulence Factors of Cariogenic Oral Pathogen Streptococcus mutans by Shikimic Acid.

ABSTRACTStreptococcus mutans is known as an important oral pathogen causing dental caries, a widespread oral infectious disease. S. mutans synthesize exopolysaccharide (EPS) using glucosyltransferases (Gtfs), resulting in biofilm formation on the tooth surface. Bacterial cells in the biofilms become strongly resistant to a harsh environment, such as antibiotics and host defense mechanisms, making biofilm-based infections difficult to eliminate. Discovering novel antibiofilm agents, especially from natural products, helps to develop effective strategies against this kind of diseases. The present study investigated the inhibitory effect of shikimic acid (SA), one abundant compound derived from Illicium verum extract, on the biofilm formation of S. mutans. We found SA can reduce the EPS synthesized by this oral pathogen and modulate the transcription of biofilm formation related genes, leading to fewer bacterial cells in its biofilm. SA also interacted with cell membrane and membrane proteins, causing damage to bacterial cells. Ex vivo testing of biofilm formation on bovine teeth showed SA strongly decreased the number of S. mutans cells and the number of EPS accumulated on dental enamel surfaces. Moreover, SA exhibits almost no toxicity to human oral cells evaluated by in vitro biocompatibility assay. In conclusion, shikimic acid exhibits remarkable antibiofilm activity against S. mutans and has the potential to be further developed as a novel anticaries agent.IMPORTANCE Natural products are an important and cost-effective source for screening antimicrobial agents. Here, we identified one compound, shikimic acid, from Illicium verum extract, exhibiting antimicrobial activity against S. mutans proliferation. It also inhibits biofilm formation of this bacteria through decreasing Gtf expression and EPS synthesis. Furthermore, this compound exhibits no significant cytotoxicity at its MIC against S. mutans, providing evidence for its clinical application.

Comparative transcriptome analysis of Monascus purpureus at different fermentation times revealed candidate genes involved in exopolysaccharide biosynthesis

Exopolysaccharides (EPS), metabolites of the medicinal edible fungus Monascus purpureus, have antioxidant, immunomodulatory, and anti-inflammatory effects. However, the biosynthetic mechanism of EPS from M. purpureus is still unclear, which hinders its utilization. In this study, the fermentation conditions of M. purpureus were optimized and comparative transcriptomic analysis was performed to understand the mechanisms and effects of fermentation on EPS synthesis. The optimal medium composition was 40 g/L mannose, 4 g/L yeast powder, 1 g/L MgSO4·7H2O, 0.8 g/L KH2PO4, 1.6 g/L K2HPO4·3H2O, and 2 mL/L Tween 80, and the optimal cultivation conditions were an inoculum of 7 %, culture temperature 30 °C, initial pH 6.0, and 180 rpm for 4 d. A total of 8095 unigenes were obtained, and 17 key enzymes for EPS synthesis were identified. Interestingly, 12 carbohydrate metabolism subcategories were enriched in the group with 4 days of fermentation compared to 2 days, with most of the differentially expressed genes (DEGs) being upregulated, but only nine carbohydrate metabolism subcategories were enriched with longer fermentation time, with all DEGs being downregulated. This study provides a theoretical basis for enhancing the EPS content and reveals the dynamics of EPS synthesis in M. purpureus, providing important targets for future EPS molecular modifications and gene knockdown studies.

Transcriptional Response and Plant Growth Promoting Activity of Pseudomonas fluorescens DR397 under Drought Stress Conditions.

ABSTRACTDrought is one of the most vulnerable factors that affect crop productivity. Little is known about plant-associated microbiomes and their functional roles in assisting plant growth under drought. We investigated the genetic and transcriptomic characteristics of opportunistic beneficial microorganisms that selectively alleviate stress through plant-bacteria interactions under drought. Pseudomonas fluorescens DR397 was isolated from the drought-prone rhizospheric soil of soybean and showed high metabolic activity at −1.25 Mpa. The genome of DR397 possesses several genes related to the synthesis of compatible solutes (choline and glycine-betaine), exopolysaccharides (alginate and cellulose), and secretion systems (type II, III, IV, and VI), as well as genes related to plant growth promotion (indole-3-acetic acid, transketolase, and thiamine phosphate synthesis). The expression of these genes was significantly upregulated (8- to 263-fold change) only under drought conditions with plant root exudate treatment, whereas subtle transcriptomic changes were observed under solely root exudate treatment. When DR397 was placed on both legume cultivars (Pisum sativum and Phaseolus vulgaris), growth was hardly affected under well-watered conditions, but the shoot and root growths were increased by up from 62.0% to 149.1% compared with the control group under drought conditions. These results provide fundamental insight on the plant-bacterial interactions that alleviate plant stress as an important ecological strategy for improving drought tolerance.IMPORTANCE Drought is a serious abiotic stress on plants as wells as the microbes that coexist with plants, which significantly lowers their fitness. The plant-bacterial interaction is an important strategy to enhance their fitness under drought. However, many knowledge gaps still exist in our understanding of transcriptomic features of bacteria interacting with plant under drought. Here, by investigating the transcriptomic profiles and pot cultivation with legume, we show that the interactions of Pseudomonas fluorescens DR397 with plants change with drought. We, therefore, provide a fundamental evidence of a hidden hero in the soil that promote plant fitness from external stress.

Distinct Agents Induce Streptococcus mutans Cells with Altered Biofilm Formation Capacity.

ABSTRACTDental caries is a multifactorial biofilm- and sugar-dependent disease. This study investigated the influence of different agents on the induction of surviving Streptococcus mutans cells after successive treatment cycles and characterized the biofilms formed by these cells recovered posttreatment. The agents (with their main targets listed in parentheses) were compound 1771 (lipoteichoic acids), 4′ hydroxychalcone (exopolysaccharides), myricetin (exopolysaccharides), tt-farnesol (cytoplasmatic membrane), sodium fluoride (enolase—glycolysis), chlorhexidine (antimicrobial), and vehicle. Recovered cells from biofilms were generated from exposure to each agent during 10 cycles of consecutive treatments (modeled on a polystyrene plate bottom). The recovered cell counting was different for each agent. The recovered cells from each group were grown as biofilms on saliva-coated hydroxyapatite discs (culture medium with sucrose/starch). In S. mutans biofilms formed by cells recovered from biofilms previously exposed to compound 1771, 4′ hydroxychalcone, or myricetin, cells presented higher expression of the 16S rRNA, gyrA (DNA replication and transcription), gtfB (insoluble exopolysaccharides), and eno (enolase—glycolysis) genes and lower quantities of insoluble dry weight and insoluble exopolysaccharides than those derived from other agents. These findings were confirmed by the smaller biovolume of bacteria and/or exopolysaccharides and the biofilm distribution (coverage area). Moreover, preexposure to chlorhexidine increased exopolysaccharide production. Therefore, agents with different targets induce cells with distinct biofilm formation capacities, which is critical for developing formulations for biofilm control.IMPORTANCE This article addresses the effect of distinct agents with distinct targets in the bacterial cell (cytoplasmatic membrane and glycolysis), the cell’s extracellular synthesis of exopolysaccharides that are important for cariogenic extracellular matrix construction and biofilm buildup in the generation of cells that persisted after treatment, and how these cells form biofilms in vitro. For example, if preexposure to an agent augments the production of virulence determinants, such as exopolysaccharides, its clinical value may be inadequate. Modification of biofilm formation capacity after exposure to agents is critical for the development of formulations for biofilm control to prevent caries, a ubiquitous disease associated with biofilm and diet.

Silicon Controls Bacterial Wilt Disease in Tomato Plants and Inhibits the Virulence-Related Gene Expression of Ralstonia solanacearum.

Silicon (Si) has a multifunctional role in improving plant growth and enhancing plant disease resistance, but its mechanisms are not fully understood. In this study, we investigated the impacts of silicon application on the control of bacterial wilt and elucidated the molecular mechanisms using transcriptome sequencing. Compared to non-Si treatment, Si application (0.5–2 mM) significantly reduces tomato bacterial wilt index by 46.31–72.23%. However, Si does not influence the growth of R. solanacearum. Si application negatively influences R. solanacearum exopolysaccharide (EPS) synthesis and biofilm formation. Transcriptome analysis showed that Si treatment significantly downregulates the expression of virulence genes’ transcriptional regulator (xpsR), EPS synthesis-related genes (epsD and tek), and type III effectors (HrpB2, SpaO, and EscR) in R. solanacearum. In addition, Si remarkably upregulates the expression of twitch motor-related genes (pilE2, pilE, fimT, and PilX). These findings suggest that silicon-suppressed tomato wilt incidence may be due to the regulation of the virulence-related genes of R. solanacearum by Si. Our research adds new knowledge to the application of Si in the field of disease control.

CRISPR/dCas9-based metabolic pathway engineering for the systematic optimization of exopolysaccharide biosynthesis in Streptococcus thermophilus

Streptococcus thermophilus is used extensively in the dairy industry and has shown great promise as a chassis cell for the biosynthesis of high-value metabolites. However, metabolic engineering in S. thermophilus lacks effective genetic modification tools to modulate gene expression to relieve metabolic burden and maximize the production of desired compounds. Here, we developed a clustered regularly interspaced short palindromic repeats interference (CRISPRi) system for efficient gene transcriptional modulation in S. thermophilus. Our CRISPRi system typically achieved 66 to 98% knockdown of single or multiple gene expression. We used CRISPRi for the biosynthesis of a new exopolysaccharide (EPS) as a paradigm model. Repression of galK at module of uridine diphosphate glucose sugar metabolism and overexpression of epsA and epsE at EPS synthesis module resulted in an approximately 2-fold increase in EPS titer (277 mg/L) when compared with a control strain. This study demonstrated the effectiveness of CRISPRi as a powerful metabolic engineering tool and synthetic biology strategy for S. thermophilus.

Boron nutrition affects growth, adaptation to stressful environments, and exopolysaccharide synthesis of Ensifer meliloti

Boron (B) is a micronutrient required for the development of symbiotic legume nodules but is apparently not essential for rhizobia, although it has been related to bacteria cell surface formation and adaptation to stress responses. In this paper, we explore whether B nutrition can influence the growth of several strains of Ensifer meliloti (formerly Sinorhizobium meliloti), including mutants defective in exopolysaccharide (EPS) production. All the strains were able to grow in B-deficient (-B) media, except for the non-nodulating Sm11605 mutant, which was unable to produce EPS. The addition of boric acid before the exponential phase of growth (in all strains, including Sm11605) showed that 50 μM H3BO3 (50 B) resulted in the highest growth rate. Moreover, the addition of B increased the tolerance of E. meliloti to salinity (up to 0.5 mM NaCl), to the cell surface stressor SDS (up to 100 μg mL−1 SDS), and to 0.5 mM H2O2. Furthermore, 50 B increased survival rate after 90 min of exposure to 1 mM H2O2, which can be crucial to overcoming transient oxidative burst due to ROS production by root legumes during the initial phases of infection. Given that rhizobia exopolysaccharides (EPSs) are involved in the responses to stress and in the adaptation to and endophytic environment, we hypothesized that B affects EPS production and it can be crucial for growth and survival under stressful environments. Indeed, the amount of EPS extracted from –B cultures was 4–5 times reduced. Gene expression analysis by using qRT-PCR revealed that the genes mucR (a positive regulator of EPS-I but negative regulator of EPS-II synthesis), and emmA (a negative regulator of EPS-I), were overexpressed in –B cells. Meanwhile, expG (a positive regulator of EPS-II), and the genes exoY and expA1 (involved in the synthesis of EPS-I and EPS-II, respectively) were downregulated. These results are consistent with a reduction of both EPS-I and EPS-II synthesis under B deficiency supporting our hypothesis.

Diaryl Urea Derivative Molecule Inhibits Cariogenic Streptococcus mutans by Affecting Exopolysaccharide Synthesis, Stress Response, and Nitrogen Metabolism.

Different small molecules have been developed to target cariogenic bacteria Streptococcus mutans. Based on target-based designing and in silico screening, a novel diaryl urea derivative, 1,3-bis[3,5-bis(trifluoromethyl)phenyl]urea (BPU), has previously been found effective in inhibiting the growth of S. mutans. However, the exact mechanism remains unclear. This current study aimed to explore the antimicrobial and antibiofilm effects of BPU on S. mutans and locate key enzymes and biological processes affected by the molecule via in silico molecular docking analysis and transcriptomic profile. Our in vitro results confirmed that BPU was capable of inhibiting planktonic growth as well as biofilm formation of S. mutans. The virtual binding analysis predicted that the molecule had strong binding potentials with vital enzymes (3AIC and 2ZID) involved in extracellular exopolysaccharide (EPS) synthesis. The predicted inhibitive binding was further confirmed by in vitro quantification of EPS, which found a decreased amount of EPS in the biofilms. The transcriptomic profile also found differential expression of genes involved in EPS synthesis. Moreover, the transcriptomic profile implied alterations in stress response and nitrogen metabolism in S. mutans treated with BPU. Examination of differentially expressed genes involved in these biological processes revealed that altered gene expression could contribute to impaired growth, biofilm formation, and competitiveness of S. mutans. In conclusion, the novel diaryl urea derivative BPU can inhibit the virulence of S. mutans by affecting different biological processes and serves as a potent anti-caries agent.

Whole Genome Sequence Analysis of a Novel Apilactobacillus Species from Giant Honeybee (Apis dorsata) Gut Reveals Occurrence of Genetic Elements Coding Prebiotic and Probiotic Traits.

Apilactobacillus spp. are classified as obligate fructophilic lactic acid bacteria (FLAB) that inhabit fructose-rich niches such as honeybee gut. Lactic acid bacteria are an important component of the gut microbiome and play a crucial role in maintaining gut health. In this study, a new FLAB strain HBW1, capable of producing glucan-type exopolysaccharide, was isolated from giant honeybee (Apis dorsata) gut and subjected to whole genome sequencing (WHS) to determine its health-beneficial traits. The genome size of the isolate was 1.49 Mb with a GC content of 37.2%. For species level identity, 16S rDNA sequence similarity, genome to genome distance calculator (dDDH), and average nucleotide identity (ANI) values were calculated. Phylogenetic analysis showed that the isolate HBW1 belongs to the Apilactobacillus genus. The dDDH and ANI values in comparison with closely clustered Apilactobacillus kunkeei species were 52% and 93.10%, respectively. Based on these values, we concluded that HBW1 is a novel species of Apilactobacillus, and we propose the name Apilactobacillus waqarii HBW1 for it. Further, WHS data mining of HBW1 revealed that it harbors two glucosyltransferase genes for prebiotic glucan-type exopolysaccharide synthesis. Moreover, chaperon (clp) and methionine sulfoxide reductase (msrA, msrB, and msrC) genes as well as nutritional marker genes for folic acid (folD) and riboflavin biosynthesis (rib operon), important for conferring probiotic properties, were also detected. Occurrence of these genetic traits make HBW1 an excellent candidate for application to improve gut function.

Intracellular glycosyl hydrolase PslG shapes bacterial cell fate, signaling, and the biofilm development of Pseudomonas aeruginosa.

Biofilm formation is one of most important causes leading to persistent infections. Exopolysaccharides are usually a main component of biofilm matrix. Genes encoding glycosyl hydrolases are often found in gene clusters that are involved in the exopolysaccharide synthesis. It remains elusive about the functions of intracellular glycosyl hydrolase and why a polysaccharide synthesis gene cluster requires a glycosyl hydrolase-encoding gene. Here, we systematically studied the physiologically relevant role of intracellular PslG, a glycosyl hydrolase whose encoding gene is co-transcribed with 15 psl genes, which is responsible for the synthesis of exopolysaccharide PSL, a key biofilm matrix polysaccharide in opportunistic pathogen Pseudomonas aeruginosa. We showed that lack of PslG or its hydrolytic activity in this opportunistic pathogen enhances the signaling function of PSL, changes the relative level of cyclic-di-GMP within daughter cells during cell division and shapes the localization of PSL on bacterial periphery, thus results in long chains of bacterial cells, fast-forming biofilm microcolonies. Our results reveal the important roles of intracellular PslG on the cell fate and biofilm development.

Exopolysaccharide synthesis repressor genes (exoR and exoX) related to curdlan biosynthesis by Agrobacterium sp.

Curdlan is a neutral, water-insoluble, unbranched, linear β-(1,3)-glucan. This study explored the roles of exoR and exoX in curdlan biosynthesis in Agrobacterium sp. ATCC 31749. The microcapsule biosynthesis of ΔexoR strain was reduced, and the motility of this strain increased remarkably compared with the wild-type (WT) strain during the cell growth phase. The curdlan yields of ΔexoR and ΔexoX strains enhanced by 19% and 17%, and the glucose utilization increased by 12% and 11%, respectively, compared with the WT strain during batch fermentation. By contrast, the curdlan yields of exoR and exoX overexpression strains decreased by 28% and 33%, respectively. The gel strength produced by ΔexoR and exoX overexpression strains decreased compared with the WT strain. RT-qPCR analysis at the transcriptional level revealed that key genes in exopolysaccharide synthesis and central metabolic pathways were up-regulated in ΔexoX and ΔexoR strains during gel production. Metabolomics analysis of ΔexoR and ΔexoX mutants proved the rates of central metabolic and electron transport chain were accelerated.

Nicotinamide could reduce growth and cariogenic virulence of Streptococcus mutans

ABSTRACT Dental caries is among the most prevalent chronic oral infectious diseases. Streptococcus mutans, a major cariogenic bacterial species, possesses several cariogenicity-associated characteristics, including exopolysaccharides (EPS) synthesis, biofilm formation, acidogenicity, and aciduricity. Nicotinamide (NAM), a form of vitamin B3, is a non-toxic, orally available, and inexpensive compound. The present study investigated the inhibitory effects of NAM on the cariogenic virulence factors of S. mutans in vitro and in vivo. NAM inhibited the growth of S. mutans UA159 and the clinical isolates. In addition, there was a decrease in the acid production and acid tolerance ability, as well as biofilm formation and EPS production of S. mutans after NAM treatment. Global gene expression profiling showed that 128 and 58 genes were significantly downregulated and upregulated, respectively, in NAM-treated S. mutans strains. The differentially expressed genes were mainly associated with carbohydrate transport and metabolism, glycolysis, acid tolerance. Moreover, in a rat caries model, NAM significantly reduced the incidence and severity of smooth and sulcal-surface caries in vivo. NAM exhibited good antimicrobial properties against S. mutans, indicating its potential value for antibiofilm and anti-caries applications.

YfiB: An Outer Membrane Protein Involved in the Virulence of Shigella flexneri.

The intracellular pathogen Shigella flexneri, which is the causative agent of bacillary dysentery, significantly influences the worldwide implication of diarrheal infections, consequentially causing about 1.1 million deaths each year. Due to a nonavailability of an authorized vaccine and the upsurge of multidrug resistance amongst Shigella strains, there has been a huge demand for further genetic analyses which could help in the advancement of new/improved drugs, and finding vaccine candidates against the pathogen. The present study aims to illustrate the role of the yfiB gene in Shigella virulence, part of the periplasmic YfiBNR tripartite signalling system. This system is involved in the regulation of cyclic-di-GMP levels inside the bacterial cells, a vital messenger molecule impacting varied cellular processes such as biofilm formation, cytotoxicity, motility, synthesis of exopolysaccharide, and other virulence mechanisms such as adhesion and invasion of the bacteria. Through a combination of genetic, biochemical, and virulence assays, we show how knocking out the yfiB gene can disrupt the entire YfiBNR system and affect the native c-di-GMP levels. We found that this subsequently causes a negative effect on the biofilm formation, bacterial invasion, host–surface attachment, and the overall virulence of Shigella. This study also carried out a structural and functional assessment of the YfiB protein and determined critical amino acid residues, essential for proper functioning of this signalling system. The present work improves our understanding of the in vivo persistence and survival of Shigella, brings light to the c-di-GMP led regulation of Shigella virulence, and provides a prospective new target to design anti-infection drugs and vaccines against S. flexneri and other bacterial pathogens.

Experimental Polymorphism Survey in Intergenic Regions of the icaADBCR Locus in Staphylococcus aureus Isolates from Periprosthetic Joint Infections.

Staphylococcus aureus is a leading cause of prosthetic joint infections (PJI) characterized by bacterial biofilm formation and recalcitrance to immune-mediated clearance and antibiotics. The molecular events behind PJI infection are yet to be unraveled. In this sense, identification of polymorphisms in bacterial genomes may help to establish associations between sequence variants and the ability of S. aureus to cause PJI. Here, we report an experimental nucleotide-level survey specifically aimed at the intergenic regions (IGRs) of the icaADBCR locus, which is responsible for the synthesis of the biofilm exopolysaccharide PIA/PNAG, in a collection of strains sampled from PJI and wounds. IGRs of the icaADBCR locus were highly conserved and no PJI-specific SNPs were found. Moreover, polymorphisms in these IGRs did not significantly affect transcription of the icaADBC operon under in vitro laboratory conditions. In contrast, an SNP within the icaR coding region, resulting in a V176E change in the transcriptional repressor IcaR, led to a significant increase in icaADBC operon transcription and PIA/PNAG production and a reduction in S. aureus virulence in a Galleria mellonella infection model. In conclusion, SNPs in icaADBCR IGRs of S. aureus isolates from PJI are not associated with icaADBC expression, PIA/PNAG production and adaptation to PJI.

Antisense vicR-Loaded Dendritic Mesoporous Silica Nanoparticles Regulate the Biofilm Organization and Cariogenicity of Streptococcus mutans.

PurposeVicR is the essential response regulator related to the synthesis of exopolysaccharide (EPS) – one of the main cariogenic factors of S. mutans. An antisense vicR RNA (ASvicR) could bind to vicR mRNA, hindering the transcription and translation of the vicR gene. We had constructed a recombinant plasmid containing the ASvicR sequence (plasmid-ASvicR) and proved that it could reduce EPS synthesis, biofilm formation, and cariogenicity. However, the recombinant plasmids are supposed to be protected from enzymatic degradation and possess higher transformation efficiency. The principal objective of the present research was to construct an appropriate vector that can carry and protect the plasmid-ASvicR and investigate the effects of the carried plasmids on the cariogenicity of the S. mutans.MethodsAminated dendritic mesoporous silica nanoparticles (DMSNs-NH2) were synthesized and characterized. The ability of DMSNs-NH2 to carry and preserve the plasmid-ASvicR (DMSNs-NH2-ASvicR) was proved by the loading curve, agarose electrophoresis, DNase I digestion assays, and energy-dispersive spectrometry (EDS) mapping. Transformation assays demonstrated whether the plasmid could enter S. mutans. The effect of DMSNs-NH2-ASvicR on the 12-hour and 24-hour biofilms of S. mutans was evaluated by biofilm formation experiments and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The cytotoxicity of DMSNs-NH2-ASvicR was assessed by CCK-8 and live/dead staining assays. The regulation of DMSNs-NH2-ASvicR on the cariogenicity of S. mutans was also evaluated in vivo.ResultsDMSNs-NH2 could load approximately 92% of plasmid-ASvicR at a mass ratio of 80 and protect most of plasmid-ASvicR from degradation by DNase I. The plasmid-ASvicR loaded on DMSNs-NH2 could be transformed into S. mutans, which down-regulated the expression of the vicR gene, reducing EPS synthesis and biofilm organization of S. mutans. DMSNs-NH2-ASvicR exhibited favorable biocompatibility, laying a foundation for its subsequent biomedical application. In addition, DMSNs-NH2-ASvicR led to decreased caries in vivo.ConclusionDMSNs-NH2 is a suitable vector of plasmid-ASvicR, and DMSNs-NH2-ASvicR can inhibit biofilm formation, reducing the cariogenicity of S. mutans. These findings reveal that DMSNs-NH2-ASvicR is a promising agent for preventing and treating dental caries.

Inhibitory Effects of Nicotinamide on Streptococcus mutans Growth and Biofilm Formation

To explore the effects of nicotinamide (NAM) on the growth, biofilm formation and exopolysaccharides (EPS) production of Streptococcus mutans. The minimum inhibitory concentration (MIC) of NAM on S. mutanswas determined by the planktonic bacterial susceptibility assay. The NAM mass concentrations were set as 1/2 MIC, 1/4 MIC and 1/8 MIC for hree separate treatment groups. Culture medium without NAM was used in the negative control group and culture medium containing 0.1 mg/mL NaF was used for the positive control group (except for the scanning electron microscopy). The growth curves of S. mutans under different NAM concentrations were drawn. Crystal violet assay and anthrone-sulfuric acid method were used to explore the effects of NAM on S. mutans biofilm formation and water-insoluble EPS production, respectively. The morphology and structure of S. mutansplanktons and biofilms after NAM treatment were observed by scanning electron microscopy. The MIC of NAM on S. mutans was 32 μg/μL. After 16 μg/μL (1/2 MIC), 8 μg/μL (1/4 MIC) and 4 μg/μL (1/8 MIC) NAM treatments, S. mutans growth and biofilm formation were inhibited, with the 16 μg/μL NAM group displaying the most significant inhibitory effects. The synthesis of EPS decreased significantly in the 16 μg/μL and 8 μg/μL NAM groups in comparison with that of the negative control group (P<0.05). Under scanning electron microscope, the cell length of S. mutans was shortened, the cell width was extended, and the length/width ratio was decreased, showing significant difference when comparing the 16 μg/μL and 8 μg/μL NAM groups with the negative control group (P<0.05). Under the influence of NAM at certain concenrations, the growth, biofilm formation, and EPS synthesis of S. mutanswere inhibited.

High throughput virtual screening and molecular dynamics simulation analysis of phytomolecules against BfmR of Acinetobacter baumannii: anti-virulent drug development campaign

Acinetobacter baumannii is a notorious multidrug resistant bacterium responsible for several hospital acquired infections assisted by its capacity to develop biofilms. A. baumannii BfmR (RstA), a response regulator from the BfmR/S two-component signal transduction system, is the major controller of A. baumannii biofilm development and formation. As a result, BfmR represents a novel target for anti-biofilm treatment against A. baumannii. The discovery of the high-resolution crystal structure of BfmR provides a good chance for computational screening of its probable inhibitors. Therefore, in this study we aim to search new, less toxic, and natural BfmR inhibitors from 8450 phytomolecules available in the Indian Medicinal Plants, Phytochemistry and Therapeutic (IMPPAT) database by analyzing molecular docking against BfmR (PDB ID: 6BR7). Out of these 8450 phytomolecules 6742 molecules were successfully docked with BfmR with the docking score range −6.305 kcal/mol to +5.120 kcal/mol. Structure based-molecular docking (SB-MD) and ADMET (absorption, distribution, metabolism, excretion, & toxicity) profile examination revealed that Norepinephrine, Australine, Calystegine B3, 7,7 A-Diepialexine, and Alpha-Methylnoradrenaline phytocompounds strongly binds to the active site residues of BfmR. Furthermore, molecular dynamics simulation (MDS) studies for 100 ns and the binding free energy (MM/GBSA) analysis elucidated the binding mechanism of Calystegine B3, 7,7 A-Diepialexine, and Alpha-Methylnoradrenaline to BfmR. In summary, these phytocompounds seems to have the promising molecules against BfmR, and thus necessitates further verification by both in vitro and in vivo experiments. Highlights BfmR plays a key role in biofilm development and exopolysaccharide (EPS) synthesis in A. baumannii. Computational approach to search for promising BfmR inhibitors from IMPAAT database. The lead phytomolecules such as Calystegine B3, 7,7 A-Diepialexine, and Alpha-Methylnoradrenaline displayed significant binding with BfmR active site. The outcome of BfmR binding phytomolecules has broadened the scope of hit molecules validation. Communicated by Ramaswamy H. Sarma