Decreased Biofilm Formation Research Articles

Inactivation of the Mla system and outer-membrane phospholipase A results in disrupted outer-membrane lipid asymmetry and hypervesiculation in Bordetella pertussis

Bordetella pertussis is the causative agent of a respiratory infection known as whooping cough. With the goal of improving the production of outer-membrane vesicles (OMVs), we studied here the mechanisms that are involved in maintaining lipid asymmetry in the outer membrane of this organism. We identified homologues of the phospholipid (PL)-transport systems Mla and Pqi and of outer-membrane phospholipase A (OMPLA). Inactivation of mlaF, encoding the ATPase of the Mla system, together with pldA, which encodes OMPLA, resulted in an accumulation of PLs at the cell surface as demonstrated by the binding of a phosphatidylethanolamine-specific fluorescent probe to intact cells of this strain. The corresponding single mutations did hardly or not affect binding of the probe. These results are consistent with a retrograde transport directionality of the Mla system in B. pertussis and indicate that PLs accumulating at the cell surface in the mlaF mutant are degraded by OMPLA. Consequently, the mlaF mutant showed a conditional growth defect due to the production of free fatty acids by OMPLA, which could be compensated by inactivation of OMPLA or by sequestration of the produced fatty acids with starch. The mlaF pldA double mutant showed markedly increased OMV production, and representative antigens were detected in these OMVs as in wild-type OMVs. Further phenotypic characterization showed that the barrier function of the outer membrane of the mlaF pldA mutant was compromised as manifested by increased susceptibility to SDS and to several antibiotics. Moreover, inactivation of mlaF alone or together with pldA resulted in increased biofilm formation, which was, however, not directly related to increased vesiculation as the addition of purified OMVs to the wild-type strain decreased biofilm formation. We conclude that the absence of MlaF together with OMPLA results in PL accumulation in the outer leaflet of the outer membrane, and the increased vesiculation of the mutant could be useful in the development of novel, OMV-based pertussis vaccines.

The Effect of Polymethylsilsesquioxane Concentration on the Liquid Foundation: Characterization of Nanostructure and Biophysical Properties

Aims: Polymethylsilsesquioxane (PMQS) is a silicone derivative that serves as a skin conditioning agent and moisturizer in cosmetics. The objective of this study is to identify the effect of different concentrations PMSQ (0.12, 0.20 and 0.35 wt %) on liquid foundation (LF) and find the optimized composition in vitro.
 Study Design: Structural and morphological features of the examined nano/micro-sized samples were investigated by different methods.
 Place and Duration of Study: Hacettepe University, Department of Physics Engineering, X-Rays Laboratory, between September 2017 and September 2019.
 Methodology: Micro/nano scale structural and morphological properties of samples were investigated by means of a multi-methodological approach based on Small‐ and/or Wide‐Angle X‐ray Scattering (SWAXS), Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy (SEM/EDX), Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) Spectroscopy and UV-vis Spectroscopy. The LF (control) and PMSQ-LF films at one-week intervals for 4 weeks, and bovine leather coated samples with LF and PMSQ-LF were also investigated by SWAXS. The effect of PMSQ on biofilm formation activity in E. coli and S. aureus were also examined.
 Results: The most significant finding of this study is that even the low concentration of polymer in LF showed differences in nano structure and it was found that a decrease in biofilm formation. Nano formation becomes more pronounced, and the number of agglomerations decreases after 4 weeks. The reason for the study over a 4-week period was the desire to take into account the independent effect of each LF use, as well as the periodic stacking and diffusing effect that will occur in the depths of the skin over time. In the UVA range (320-400 nm) lower transmittance values were found for LF and PMSQ - LF films. 
 Conclusion: The results suggest that optimum polymer concentration in liquid foundation should be considered as a main step. The smart and systematic use of polymer additives is directly manifested in the nanoscopic structure and can improve the harmful UV rays prevention properties, which are important for health as well as cosmetic effects for beauty purposes.

In vitro activities of cellulase and ceftazidime, alone and in combination against Pseudomonas aeruginosa biofilms

BackgroundBiofilms are a main pathogenicity feature of Pseudomonas aeruginosa and has a significant role in antibiotic resistance and persistent infections in humans. We investigated the in vitro activities of antibiotic ceftazidime and enzyme cellulase, either alone or in combination against biofilms of P. aeruginosa.ResultsBoth ceftazidime and cellulase significantly decreased biofilm formation in all strains in a dose-dependent manner. Combination of enzyme at concentrations of 1.25, 2.5, 5, and 10 U/mL tested with 1/16× MIC of antibiotic led to a significant reduction in biofilm biomass. Cellulase showed a significant detachment effect on biofilms at three concentrations of 10 U/mL, 5 U/mL, and 2.5 U/mL. The MIC, MBC, and MBEC values of ceftazidime were 2 to 4 µg/mL, 4 to 8 µg/mL, and 2048 to 8192 µg/mL. When combined with cellulase, the MBECs of antibiotic showed a significant decrease from 32- to 128-fold.ConclusionsCombination of the ceftazidime and the cellulase had significant anti-biofilm effects, including inhibition of biofilm formation and biofilm eradication in P. aeruginosa. These data suggest that glycoside hydrolase therapy as a novel strategy has the potential to enhance the efficacy of antibiotics and helps to resolve biofilm-associated wound infections caused by this pathogen.

Deletion of the yqeK gene leads to the accumulation of Ap4A and reduced biofilm formation in Streptococcus mutans.

Diadenosine-5',5'''-P1, P4-tetraphosphate (Ap4A) is a second messenger playing a crucial role in various life activities of bacteria. The increase of Ap4A expression is pleiotropic, resulting in an impairment in the formation of biofilm and other physiological functions in some bacteria. However, Ap4A function in Streptococcus mutans, an important pathogen related to dental caries, remains unknown. In this work, the Ap4A hydrolase, YqeK, was identified and characterized in S. mutans. Then, the effects of yqeK deletion on the growth, biofilm formation, and exopolysaccharide (EPS) quantification in S. mutans were determined by the assessment of the growth curve, crystal violet, and anthrone-sulfuric acid, respectively, and visualized by microscopy. The results showed that the in-frame deletion of the yqeK gene in S. mutans UA159 led to an increase in Ap4A levels, lag phase in the early growth, as well as decrease in biofilm formation and water-insoluble exopolysaccharide production. Global gene expression profile showed that the expression of 88 genes was changed in the yqeK mutant, and among these, 42 were upregulated and 46 were downregulated when compared with the wild-type S. mutans UA159. Upregulated genes were mainly involved in post-translational modification, protein turnover, and chaperones, while downregulated genes were mainly involved in carbohydrate transport and metabolism. Important virulence genes related to biofilms, such as gtfB, gtfC, and gbpC, were also significantly downregulated. In conclusion, these results indicated that YqeK affected the formation of biofilms and the expression of biofilm-related genes in S. mutans.

Organization, evolution and function of fengycin biosynthesis gene clusters in the Bacillus amyloliquefaciens group

The Bacillus velezensis strain PG12, belonging to the Bacillus amyloliquefaciens group, is an endophytic bacterium known for its antimicrobial activities against crop pathogens. However, our knowledge of the molecular basis underlying its biocontrol activity and the relatedness of different strains in the Bacillus amyloliquefaciens group is limited. Here, we sequenced and analyzed the genome of PG12 to test its taxonomic affiliation and identified genes involved in the biocontrol activity. The phylogenomic analysis results indicate that PG12 belongs to B. velezensis, a subgroup of the B. amyloliquefaciens group. By comparing the genomes of 22 strains in this group, we confirmed that it comprises three different phylogenetic lineages: B. amyloliquefaciens, B. velezensis and B. siamensis. Three secondary metabolism gene clusters related to the production of lipopeptides, namely fengycin, iturin and surfactin, were identified in the genomes of the B. amyloliquefaciens group. The core genome of B. velezensis is enriched in secondary metabolism genes compared with B. siamensis and B. amyloliquefaciens. Three of the five genes pertaining to the gene cluster responsible for fengycin biosynthesis (fenBCD) were found in B. velezensis and B. siamensis, but not in B. amyloliquefaciens. Phenotypic analysis showed that the ∆fenA mutant of PG12 displayed significantly decreased biofilm formation and swarming motility, which indicates that fengycin contributes to the colonization and pathogen control abilities of PG12. Our results also suggest that B. siamensis and B. velezensis have acquired the fenBCD genes from Paenibacillus spp. by horizontal gene transfer (HGT). Taken together, the results provide insights into the evolutionary pattern of the B. amyloliquefaciens group strains and will promote further researches on their taxonomy and functional genomics.

Novel Hybrid Peptide Cathelicidin 2 (1-13)-Thymopentin (TP5) and Its Derived Peptides with Effective Antibacterial, Antibiofilm, and Anti-Adhesion Activities.

The increasing numbers of infections caused by multidrug-resistant (MDR) pathogens highlight the urgent need for new alternatives to conventional antibiotics. Antimicrobial peptides have the potential to be promising alternatives to antibiotics because of their effective bactericidal activity and highly selective toxicity. The present study was conducted to investigate the antibacterial, antibiofilm, and anti-adhesion activities of different CTP peptides (CTP: the original hybrid peptide cathelicidin 2 (1-13)-thymopentin (TP5); CTP-NH2: C-terminal amidated derivative of cathelicidin 2 (1-13)-TP5; CTPQ: glutamine added at the C-terminus of cathelicidin 2 (1-13)-TP5) by determining the minimal inhibitory concentrations (MICs), minimal bactericidal concentrations (MBCs), propidium iodide uptake, and analysis by scanning electron microscopy, transmission electron microscopy, and confocal laser scanning microscopy). The results showed that CTPs had broad-spectrum antibacterial activity against different gram-positive and gram-negative bacteria, with MICs against the tested strains varying from 2 to 64 μg/mL. CTPs at the MBC (2 × MIC 64 μg/mL) showed strong bactericidal effects on a standard methicillin-resistant Staphylococcus aureus strain ATCC 43300 after co-incubation for 6 h through disruption of the bacterial membrane. In addition, CTPs at 2 × MIC also displayed effective inhibition activity of several S. aureus strains with a 40–90% decrease in biofilm formation by killing the bacteria embedded in the biofilms. CTPs had low cytotoxicity on the intestinal porcine epithelial cell line (IPEC-J2) and could significantly decrease the rate of adhesion of S. aureus ATCC 43300 on IPEC-J2 cells. The current study proved that CTPs have effective antibacterial, antibiofilm, and anti-adhesion activities. Overall, this study contributes to our understanding of the possible antibacterial and antibiofilm mechanisms of CTPs, which might be an effective anti-MDR drug candidate.

Inhibition of Biofilm Formation of Foodborne Staphylococcus aureus by the Citrus Flavonoid Naringenin.

Taking into consideration the importance of biofilms in food deterioration and the potential risks of antiseptic compounds, antimicrobial agents that naturally occurring are a more acceptable choice for preventing biofilm formation and in attempts to improve antibacterial effects and efficacy. Citrus flavonoids possess a variety of biological activities, including antimicrobial properties. Therefore, the anti-biofilm formation properties of the citrus flavonoid naringenin on the Staphylococcus aureus ATCC 6538 (S. aureus) were investigated using subminimum inhibitory concentrations (sub-MICs) of 5~60 mg/L. The results were confirmed using laser and scanning electron microscopy techniques, which revealed that the thick coating of S. aureus biofilms became thinner and finally separated into individual colonies when exposed to naringenin. The decreased biofilm formation of S. aureus cells may be due to a decrease in cell surface hydrophobicity and exopolysaccharide production, which is involved in the adherence or maturation of biofilms. Moreover, transcriptional results show that there was a downregulation in the expression of biofilm-related genes and alternative sigma factor sigB induced by naringenin. This work provides insight into the anti-biofilm mechanism of naringenin in S. aureus and suggests the possibility of naringenin being used in the industrial food industry for the prevention of biofilm formation.

Decreased biofilm formation in Proteus mirabilis after short-term exposure to a simulated microgravity environment

BackgroundMicrobes threaten human health in space exploration. Studies have shown that Proteus mirabilis has been found in human space habitats. In addition, the biological characteristics of P. mirabilis in space have been studied unconditionally. The simulated microgravity environment provides a platform for understanding the changes in the biological characteristics of P. mirabilis.ObjectiveThis study intends to explore the effect of simulated microgravity on P. mirabilis, the formation of P. mirabilis biofilm, and its related mechanism.MethodsThe strange deformable rods were cultured continuously for 14 days under microgravity simulated in high-aspect rotating vessels (HARVs). The morphology, growth rate, metabolism, and biofilm formation of the strain were measured, and the phenotypic changes of P. mirabilis were evaluated. Transcriptome sequencing was used to detect differentially expressed genes under simulated microgravity and compared with phenotype.ResultsThe growth rate, metabolic ability, and biofilm forming ability of P. mirabilis were lower than those of normal gravity culture under the condition of simulated microgravity. Further analysis showed that the decrease of growth rate, metabolic ability, and biofilm forming ability may be caused by the downregulation of related genes (pstS, sodB, and fumC).ConclusionThe simulated microgravity condition enables us to explore the potential relationship between bacterial phenotype and molecular biology, thus opening up a suitable and constructive method for medical fields that have not been explored before. It provides a certain strategy for the treatment of P. mirabilis infectious diseases in space environment by exploring the microgravity of P. mirabilis.

Effect of Natural Polysaccharide Matrix-Based Selenium Nanocomposites on Phytophthora cactorum and Rhizospheric Microorganisms.

We studied the effects of new chemically synthesized selenium (Se) nanocomposites (NCs) based on natural polysaccharide matrices arabinogalactan (AG), starch (ST), and kappa-carrageenan (CAR) on the viability of phytopathogen Phytophthora cactorum, rhizospheric bacteria, and potato productivity in the field experiment. Using transmission electron microscopy (TEM), it was shown that the nanocomposites contained nanoparticles varying from 20 to 180 nm in size depending on the type of NC. All three investigated NCs had a fungicidal effect even at the lowest tested concentrations of 50 µg/mL for Se/AG NC (3 µg/mL Se), 35 µg/mL for Se/ST NC (0.5 µg/mL Se), and 39 µg/mL for Se/CAR NC (1.4 µg/mL Se), including concentration of 0.000625% Se (6.25 µg/mL) in the final suspension, which was used to study Se NC effects on bacterial growth of the three common rhizospheric bacteria Acinetobacter guillouiae, Rhodococcus erythropolis and Pseudomonas oryzihabitans isolated from the rhizosphere of plants growing in the Irkutsk Region, Russia. The AG-based Se NC (Se/AG NC) and CAR-based Se NC (Se/CAR NC) exhibited the greatest inhibition of fungal growth up to 60% (at 300 µg/mL) and 49% (at 234 µg/mL), respectively. The safe use of Se NCs against phytopathogens requires them to be environmentally friendly without negative effects on rhizospheric microorganisms. The same concentration of 0.000625% Se (6.25 µg/mL) in the final suspension of all three Se NCs (which corresponds to 105.57 µg/mL for Se/AG NC, 428.08 µg/mL for Se/ST NC and 170.30 µg/mL for Se/CAR NC) was used to study their effect on bacterial growth (bactericidal, bacteriostatic, and biofilm formation effects) of the three rhizospheric bacteria. Based on our earlier studies this concentration had an antibacterial effect against the phytopathogenic bacterium Clavibacter sepedonicus that causes diseases of potato ring rot, but did not negatively affect the viability of potato plants at this concentration. In this study, using this concentration no bacteriostatic and bactericidal activity of all three Se NCs were found against Rhodococcus erythropolis based on the optical density of a bacterial suspension, agar diffusion, and intensity of biofilm formation, but Se/CAR and Se/AG NCs inhibited the growth of Pseudomonas oryzihabitans. The cell growth was decrease by 15–30% during the entire observation period, but the stimulation of biofilm formation by this bacterium was observed for Se/CAR NC. Se/AG NC also had bacteriostatic and antibiofilm effects on the rhizospheric bacterium Acinetobacter guillouiae. There was a 2.5-fold decrease in bacterial growth and a 30% decrease in biofilm formation, but Se/CAR NC stimulated the growth of A. guillouiae. According to the results of the preliminary field test, an increase in potato productivity by an average of 30% was revealed after the pre-planting treatment of tubers by spraying them with Se/AG and Se/CAR NCs with the same concentration of Se of 0.000625% (6.25 µg/mL) in a final suspension. The obtained and previously published results on the positive effect of natural matrix-based Se NCs on plants open up prospects for further investigation of their effects on rhizosphere bacteria and resistance of cultivated plants to stress factors.

Total phenolic content, antiradical, antimicrobial and antibiofilm properties of grape and apple vinegar

Antimicrobial resistance – the capability of microorganisms to resist antimicrobial agents – has been stated as a major concern for public health. The increase in spread of multi- and pan-resistant bacteria which are not treatable with present antimicrobials has brought the need for the use of alternative products. Vinegar is a natural product- produced from alcoholic fermentation- that has shown strong antimicrobial activity. The aim of this study was to determine the total phenolic content and antiradical activity of the commercial grape and apple vinegar as well as to evaluate their antibiofilm and antimicrobial activities against Staphylococcus aureus and Pseudomonas aeruginosa. Grape vinegar showed higher total acidity and total phenolic content, and lower antiradical activity (DPPH activity) compared to apple vinegar. The populations of S. aureus and P. aeruginosa were significantly reduced by neat grape and apple vinegar samples. The antibacterial activity of grape vinegar was superior to apple vinegar. While AV and GV samples at 50% concentration did not form a visible zone of inhibition against S. aureus, they showed an inhibitory effect against P. aeruginosa (16.24 mm for GV and 16.5 for AV). The vinegar applied at the lowest concentration (25%) did not show any antibacterial effect on either bacterium. Solutions containing 50% to 6.25% vinegar samples prevented almost 100% biofilm formation in both bacteria. However, solutions containing lower amounts of vinegar showed stronger inhibition of biofilm formation by P. aeruginosa. A positive correlation was found between the biofilm reducing ability of vinegar samples and their antibacterial activity in this study. Taken together, commercial grape and apple vinegar significantly reduced the viability of S. aureus and P. aeruginosa, thereby decreasing biofilm formation.

Algal polysaccharide's potential to combat respiratory infections caused by Klebsiella pneumoniae and Serratia marcescens biofilms.

The growth of respiratory diseases, as witnessed through the SARS and COVID-19 outbreaks, and antimicrobial-resistance together pose a serious threat to humanity. One reason for antimicrobial resistance is formation of bacterial biofilms. In this study the sulphated polysaccharides from green algae Chlamydomonas reinhardtii (Cr-SPs) is tested for its antibacterial and antibiofilm potential against Klebsiella pneumoniae and Serratia marcescens. Agar cup assay clearly indicated the antibacterial potential of Cr-SPs. Minimum inhibitory concentration (MIC50) of Cr-SPs against Klebsiella pneumoniae was found to be 850 µg/ml, and it is 800 µg/ml in Serratia marcescens. Time-kill and colony-forming ability assays suggest the concentration-dependent bactericidal potential of Cr-SPs. Cr-SPs showed 74–100% decrease in biofilm formation in a concentration-dependent manner by modifying the cell surface hydrophobic properties of these bacteria. Cr-SPs have also distorted preformed-biofilms by their ability to interact and destroy the extra polymeric substance and eDNA of the matured biofilm. Scanning electron microscopy analysis showed that Cr-SPs effectively altered the morphology of these bacterial cells and distorted the bacterial biofilms. Furthermore reduced protease, urease and prodigiosin pigment production suggest that Cr-SPs interferes the quorum sensing mechanism in these bacteria. The current study paves way towards developing Cr-SPs as a control strategy for treatment of respiratory tract infections.

The Role of graRS in Regulating Virulence and Antimicrobial Resistance in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of both community- and hospital-associated infections. The antibiotic resistance and virulence characteristics of MRSA are largely regulated by two-component signal transduction systems (TCS) including the graRS TCS. To make a relatively comprehensive insight into graRS TCS in MRSA, the bioinformatics analysis of dataset GSE26016 (a S. aureus HG001 WT strain vs. the ΔgraRS mutant) from Gene Expression Omnibus (GEO) database was performed, and a total of 563 differentially expressed genes (DEGs) were identified. GO analysis revealed that the DEGs were mainly enriched in the “de novo” IMP biosynthetic process, lysine biosynthetic process via diaminopimelate, and pathogenesis; and they were mainly enriched in purine metabolism, lysine biosynthesis, and monobactam biosynthesis in KEGG analysis. WGCNA suggested that the turquoise module was related to the blue module, and the genes in these two modules were associated with S. aureus virulence and infection. To investigate the role of graRS in bacterial virulence, a graRS knockout mutant (ΔgraRS) was constructed using MRSA USA500 2,395 strain as a parent strain. Compared to the wild-type strain, the USA500ΔgraRS showed reduced staphyloxanthin production, retarded coagulation, weaker hemolysis on blood agar plates, and a decreased biofilm formation. These altered phenotypes were restored by the complementation of a plasmid-expressed graRS. Meanwhile, an expression of the virulence-associated genes (coa, hla, hlb, agrA, and mgrA) was downregulated in the ΔgraRS mutant. Consistently, the A549 epithelial cells invasion of the ΔgraRS mutant was 4-fold lower than that of the USA500 wild-type strain. Moreover, on the Galleria mellonella infection model, the survival rate at day 5 post infection in the USA500ΔgraRS group (55%) was obviously higher than that in the USA500 group (20%), indicating graRS knockout leads to a decreased virulence in vivo. In addition, the deletion of the graRS in the MRSA USA500 strain resulted in its increased susceptibilities to ampicillin, oxacillin, vancomycin, and gentamicin. Our work suggests that the graRS TCS plays an important role in regulating S. aureus virulence in vitro and in vivo and modulate bacterial resistance to various antibiotics.

Let-7b-5p in vesicles secreted by human airway cells reduces biofilm formation and increases antibiotic sensitivity of P. aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that forms antibiotic-resistant biofilms, which facilitate chronic infections in immunocompromised hosts. We have previously shown that P. aeruginosa secretes outer-membrane vesicles that deliver a small RNA to human airway epithelial cells (AECs), in which it suppresses the innate immune response. Here, we demonstrate that interdomain communication through small RNA-containing membrane vesicles is bidirectional and that microRNAs (miRNAs) in extracellular vesicles (EVs) secreted by human AECs regulate protein expression, antibiotic sensitivity, and biofilm formation by P. aeruginosa Specifically, human EVs deliver miRNA let-7b-5p to P. aeruginosa, which systematically decreases the abundance of proteins essential for biofilm formation, including PpkA and ClpV1-3, and increases the ability of beta-lactam antibiotics to reduce biofilm formation by targeting the beta-lactamase AmpC. Let-7b-5p is bioinformatically predicted to target not only PpkA, ClpV1, and AmpC in P. aeruginosa but also the corresponding orthologs in Burkholderia cenocepacia, another notorious opportunistic lung pathogen, suggesting that the ability of let-7b-5p to reduce biofilm formation and increase beta-lactam sensitivity is not limited to P. aeruginosa Here, we provide direct evidence for transfer of miRNAs in EVs secreted by eukaryotic cells to a prokaryote, resulting in subsequent phenotypic alterations in the prokaryote as a result of this interdomain communication. Since let-7-family miRNAs are in clinical trials to reduce inflammation and because chronic P. aeruginosa lung infections are associated with a hyperinflammatory state, treatment with let-7b-5p and a beta-lactam antibiotic in nanoparticles or EVs may benefit patients with antibiotic-resistant P. aeruginosa infections.

DctR contributes to the virulence of avian pathogenic Escherichia coli through regulation of type III secretion system 2 expression

Pathogens could precisely alter their gene expression to facilitate their survival and successful infection. The LuxR family transcriptional regulator DctR (also known as YhiF) was shown to participate in the regulation of acid fitness and adhesion of enterohemorrhagic E. coli (EHEC) O157:H7. Avian pathogenic Escherichia coli (APEC) causes significant economic losses to the poultry industries and also potentially threatens human health. However, the effects of DctR on the fitness and virulence of APEC have not been investigated yet. To assess the function of DctR in APEC, the dctR gene mutant and complemented strains were constructed and biologically characterized. Our results show that inactivation of the dctR gene led to decreased biofilm formation, diminished serum resistance, reduced adherence capacity, attenuated colonization and virulence of APEC in ducks. The altered capacities of the mutant strain were restored by genetic complementation. In addition, we found that DctR positively regulates the expression of E. coli type III secretion system 2 (ETT2) core genes in APEC. The expression of the inflammatory cytokines interleukin (IL)-1β and IL-8 were decreased in HD-11 macrophages infected with the mutant strain compared with the wild-type strain. These observations indicate that regulator DctR contributes to the virulence of APEC through regulation of ETT2 expression.

The Staphylococcus aureus toxin-antitoxin system YefM-YoeB is associated with antibiotic tolerance and extracellular dependent biofilm formation.

The high antibiotic tolerance of Staphylococcus aureus biofilms is associated with challenges for treating periprosthetic joint infection. The toxin–antitoxin system, YefM–YoeB, is thought to be a regulator for antibiotic tolerance, but its physiological role is unknown. The objective of this study was to determine the biofilm and antibiotic susceptibility phenotypes associated with S. aureus yoeB homologs. We hypothesized the toxin–antitoxin yoeB homologs contribute to biofilm formation and antibiotic susceptibility. Disruption of yoeB1 and yoeB2 resulted in decreased biofilm formation in comparison to Newman and JE2 wild-type (WT) S. aureus strains. In comparison to yoeB mutants, both Newman and JE2 WT strains had higher polysaccharide intercellular adhesin (PIA) production. Treatment with sodium metaperiodate increased biofilm formation in Newman WT, indicating biofilm formation may be increased under conditions of oxidative stress. DNase I treatment decreased biofilm formation in Newman WT but not in the absence of yoeB1 or yoeB2. Additionally, WT strains had a higher extracellular DNA (eDNA) content in comparison to yoeB mutants but no differences in biofilm protein content. Moreover, loss of yoeB1 and yoeB2 decreased biofilm survival in both Newman and JE2 strains. Finally, in a neutropenic mouse abscess model, deletion of yoeB1 and yoeB2 resulted in reduced bacterial burden. In conclusion, our data suggest that yoeB1 and yoeB2 are associated with S. aureus planktonic growth, extracellular dependent biofilm formation, antibiotic tolerance, and virulence.

The Effects of Humic Substances and Humic Substance-Based Silver Nanocomposites on the Viability of Rhizospheric Microorganisms

The influence of humic substances (HSs) of coals (HS-cl), shale (HS-sl), and mud (HS-md) isolated in Mongolia and silver nanocomposites (NCs) synthesized on their basis on the viability of Rhodococcus erythropolis (gram positive bacteria) and Pseudomonas oryzihabitans (gram negative bacteria) is investigated using methods for measuring the optical density of a bacterial suspension, diffusion into agar, as well as studying the intensity of biofilm formation. It is shown that the effects of HSs and NCs on bacteria in dynamics are different in R. erythropolis and P. oryzihabitans. HS-sl stimulated the growth of R. erythropolis on the first day of observation, and in P. oryzihabitans this effect was noted later, at 2 days of the experiment. Despite the absence of bacteriostatic effects for HSs, a decrease in biofilm formation of bacteria under their influence has been revealed. HS-cl and HS-md reduced the biofilm formation of R. erythropolis, and HS-sl reduced P. oryzihabitans. The addition of HSs to HCs retains the bactericidal effect of NC HS-cl/Ag and NC HS-md/Ag with respect to R. erythropolis. A decrease in biofilm formation of gram-negative bacteria P. oryzihabitans occurred under the influence of NC HS-md/Ag. NC HS-cl/Ag, in contrast, enhanced the reproduction of P. oryzihabitans throughout the observation period. Thus, the reaction of microorganisms to the effects of HSs in the composition of NCs depends on the morphology of the bacteria themselves, as well as on the chemical structure and functional composition of the active substances. The obtained data and previously published results on the positive effect of silver NCs and HSs on plants open the prospects for further investigation of the effects of using NCs of HSs when introduced into the soil in combination with rhizospheric bacteria to increase the resistance of cultivated plants to stress factors.

Interaction between the type 4 pili machinery and a diguanylate cyclase fine-tune c-di-GMP levels during early biofilm formation

To initiate biofilm formation, it is critical for bacteria to sense a surface and respond precisely to activate downstream components of the biofilm program. Type 4 pili (T4P) and increasing levels of c-di-GMP have been shown to be important for surface sensing and biofilm formation, respectively; however, mechanisms important in modulating the levels of this dinucleotide molecule to define a precise output response are unknown. Here, using macroscopic bulk assays and single-cell tracking analyses of Pseudomonas aeruginosa, we uncover a role of the T4P alignment complex protein, PilO, in modulating the activity of the diguanylate cyclase (DGC) SadC. Two-hybrid and bimolecular fluorescence complementation assays, combined with genetic studies, are consistent with a model whereby PilO interacts with SadC and that the PilO-SadC interaction inhibits SadC's activity, resulting in decreased biofilm formation and increased motility. Using single-cell tracking, we monitor both the mean c-di-GMP and the variance of this dinucleotide in individual cells. Mutations that increase PilO-SadC interaction modestly, but significantly, decrease both the average and variance in c-di-GMP levels on a cell-by-cell basis, while mutants that disrupt PilO-SadC interaction increase the mean and variance of c-di-GMP levels. This work is consistent with a model wherein P. aeruginosa uses a component of the T4P scaffold to fine-tune the levels of this dinucleotide signal during surface commitment. Finally, given our previous findings linking SadC to the flagellar machinery, we propose that this DGC acts as a bridge to integrate T4P and flagellar-derived input signals during initial surface engagement.

Formation of Cariogenic Bacterial Biofilm on Vanillin-Incorporated Resin-Based Dental Sealant

Tooth decay or dental caries is an important oral health problem involving people of all age groups. The disease is the outcome of the demineralize process in which aciduric and acidogenic bacteria in a biofilm decompose tooth structure. Dental sealant, a resin material, which is applied on the occlusal pit and fissure surfaces of the teeth as a protective layer has been commonly used to prevent dental caries. However, the microbial effect on food residue is found to be a major cause of microleakage of sealant and secondary caries. Several types of antimicrobial agents were introduced to increase the caries preventive effect of dental sealants. Vanillin, the main component of flavoring agent vanilla, has been found to have antimicrobial property against Gram-positive and Gram-negative bacteria. The objective of this study was to investigate the antimicrobial effect of vanillin-incorporated dental sealant against biofilm formation of cariogenic bacteria. Dental sealant resin samples (Clinpro; 3M ESPE, USA) were prepared in 96-well plate in accordance with the amount of vanillin adding (0%, 0.5%, 1% and 5% vanillin). The cariogenic bacterial suspensions of Streptococcus mutans ATCC 25175 and Lactobacillus casei ATCC 334 were added to saliva-coated samples and incubated at 37°C in 5% CO2 atmosphere for 48 h to allow the biofilm formation. The quantity of vital biofilm was determined by WST Microbial Cell Counting Kit (Dojindo Molecular Technologies, USA) at 460 nm. One-way ANOVA and Tukey’s test were applied to the statistical analysis. A significant inhibitory effect against L. casei biofilm was observed in all vanillin incorporated samples (0.5%, 1% and 5% vanillin) compared with samples without vanillin. The percentage of biofilm reduction was 32-39%. For S. mutans, the suppressive effect was noticed only at >1% vanillin with 18-25% biofilm reduction. In conclusion, the incorporation of vanillin to dental sealants could decrease biofilm formation of cariogenic bacteria (S. mutans and L. casei). The use of dental sealants containing vanillin could be a promising measure to prevent dental caries due to their antibacterial biofilm formation property.

Deletion of SarX Decreases Biofilm Formation of Staphylococcus aureus in a Polysaccharide Intercellular Adhesin (PIA)-Dependent Manner by Downregulating spa.

IntroductionBiofilm formation by Staphylococcus aureus is an important virulence determinant mediated by the polysaccharide intercellular adhesin (PIA) encoded by the ica operon or mediated by surface and extracellular proteins. SarX is a 250-residue two-domain SarA homolog that activates spa transcription. Previous studies demonstrated that Staphylococcus epidermidis SarX protein regulated the transcriptional activity of the agr and ica loci and controlled the biofilm phenotype, primarily by regulating icaADBC transcription and PIA production.ResultsIn this study, biofilm formation and detachment of the clinical isolate S. aureus SA75 were significantly decreased in the sarX mutant strain. The effect of sarX mutation on S. aureus biofilm formation was related to the production of PIA and not to that of eDNA. Deletion of sarX was associated with a 1.8-fold reduction in spa transcription as determined by RT-PCR analysis, and this reduction could be restored by chromosomal complementation of sarX. Expression of Spa protein was also decreased in the S. aureus sarX mutant.ConclusionsarX promoted biofilm production of S. aureus that may primarily be mediated through increasing ica operon expression and PIA production. Furthermore, deletion of sarX reduced S. aureus biofilm formation by downregulating spa.

Proteomics analysis reveals the importance of transcriptional regulator slyA in regulation of several physiological functions in Aeromonas hydrophila

SlyA is a well-known transcription factor that plays important roles in the regulation of diverse physiological functions including virulence and stress response in various bacterial species. The biological effects of slyA have species-specific characteristics. In this study, a phenotype assay showed that slyA gene deletion in Aeromonas hydrophila (ahslyA) decreased biofilm formation capability but did not affect bacterial hemolytic activity or acid stress response. The differentially expressed proteins between ΔahslyA and wild-type strains were compared by label-free quantitative proteomics to further understand the effects of AhSlyA on biological functions. Bioinformatics assays showed that ΔahslyA may be involved in the regulation of several intracellular metabolic pathways such as galactose metabolism, arginine biosynthesis, and sulfur metabolism. A further phenotypic assay confirmed that AhSlyA plays an important role in the regulation of sulfur and phosphate metabolism. Moreover, ahslyA also directly or indirectly regulated at least eight outer membrane proteins involved in the maintenance of cell permeability. Overall, the results provide insights into the functions of ahslyA and demonstrate its importance in A. hydrophila. Biological significanceIn this study, we compared the DEPs between the transcriptional regulator slyA-deleted and the wild-type A. hydrophila strains using a label-free quantitative proteomics method. The bioinformatics analysis showed that slyA may be involved in the regulation of several metabolic pathways. Subsequent phenotype and growth assays confirmed that ΔahslyA affected sulfur and phosphate metabolism, and OM permeability. Finally, a ChIP-PCR assay further confirmed that AhSlyA directly binds to the promoters of several candidate genes, including sulfur metabolism-related genes. These results indicated that slyA plays an important regulatory role in pleiotropic physiological functions of A. hydrophila, and these functions may be different from those identified in previous reports from other bacterial species.