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Periodontitis is a common oral bacterial infection characterized by inflammatory responses. Its high prevalence lowers the quality of life for individuals and increases the global economic and disease burden. As microorganisms in dental plaque are responsible for this oral disease, antibacterial drug treatments are effective strategies for preventing and treating periodontitis. In this study, we investigated the inhibitory effect of nicotinamide (NAM), a vitamin B 3 derivative, on the growth and virulence of Porphyromonas gingivalis, a key member of the red complex. Our findings revealed that NAM inhibited bacterial growth and gingipain activities, which played a dominant role in protein hydrolysis and heme acquisition. NAM decreased hemagglutination and hemolysis abilities and changed hemin and hemoglobin binding capacities, controlling bacterial infection through a starvation strategy by blocking access to growth-essential nutrients from the outside and reducing bacterial virulence. Several experiments in an animal model showed the effectiveness of NAM in preventing alveolar bone loss and reducing inflammatory cell infiltration, shedding light on its potential therapeutic applicability., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Periodontitis is clinically characterized by destruction of the tooth support system and tooth loss. Porphyromonas gingivalis (Pg) plays a dominant role in periodontitis. Fractions and isolated compounds from an acetone-water extract of the roots of Limonium brasiliense (Lb) were tested in vitro for their anti-adhesive capacity against Pg on human KB buccal cells, influence on gingipains, the main virulence factors of Pg, and biofilm formation. Fractions EAF and FLB7 (50 μg/mL) reduced the bacterial adhesion of Pg to KB cells significantly (63 resp. 70%). The proanthocyanidin samarangenin A inhibited the adhesion (72%, 30 μM), samarangenin B (71%, 20 μM), and the flavan-3-ol epigallocatechin-3-O-gallate (79%, 30 μM). Fraction AQF, representing hydrophilic compounds, reduced the proteolytic activity of Arginin-specific gingipain (IC 50 12.78 μg/mL). Fractions EAF and FLB7, characterized by lipohilic constituents, inhibited Arg-gingipain (IC 50 3 μg/mL). On Lysine-specific gingipain, AQF has an IC 50 15.89, EAF 14.15, and FLB7 6 μg/mL. The reduced bacterial adhesion is due to a strong interaction of proanthocyanidins with gingipains. AQF, EAF, and FLB7 significantly inhibited biofilm formation: IC 50 11.34 (AQF), 11.66 (EAF), and 12.09 μg/mL (FLB7). In silico analysis indicated, that the polyphenols act against specific targets of Pg, not affecting mammalian cells. Therefore, Lb might be effective for prevention of periodontal disease by influencing virulence factors of Pg., Competing Interests: Declaration of competing interest This study was financially supported by CNPq, for providing a student grant to Raquel Isolani for the program “Science Without Borders” – CNPq (Grant #200173/2018–9). Residual expenses were borne by intramural annual budgets from the University of Münster to Andreas Hensel, the State University of Maringá to and project's funding agencies, CAPES, FINEP, CNPq to João Carlos Palazzo de Mello (Grant #401490/2014-9). The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Mycoplasma pneumoniae , a notable pathogen behind respiratory infections, employs specialized proteins to adhere to the respiratory epithelium, an essential process for initiating infection. The role of glycosaminoglycans, especially heparan sulfate, is critical in facilitating pathogen-host interactions, presenting a strategic target for therapeutic intervention. In this study, we assembled a glycan library comprising heparin, its oligosaccharide derivatives, and a variety of marine-derived sulfated glycans to screen the potential inhibitors for the pathogen-host interactions. By using Surface Plasmon Resonance spectroscopy, we evaluated the library's efficacy in inhibiting the interaction between M. pneumoniae adhesion proteins and heparin. Our findings offer a promising avenue for developing novel therapeutic strategies against M. pneumoniae infections.

Tentacle-like polymers decorated with several copies of peptide antigens can be interesting tools for increasing the ability to capture circulating antibodies in patient sera, using cooperative effects for stronger avidity. We previously showed that antibodies from multiple sclerosis (MS) patient sera preferentially recognize hyperglucosylated adhesin protein HMW1ct of non-typeable Haemophilus influenzae (NTHi). We selected the C-terminal HMW1ct(1347-1354) minimal epitope and prepared the diglucosylated analogue Ac-KAN(Glc)VTLN(Glc)TTG-K(N 3 )-NH 2 to graft a 40 kDa dextran scaffold modified with glycidyl-propargyl moieties to perform a copper catalyzed alkyne-azide coupling reaction (CuAAC). Quantitative NMR measurements allowed the characterization of the peptide loading (19.5 %) on the multivalent dextran conjugate. This novel polymeric structure displayed optimal capturing properties of both IgG and, more interestingly, IgM antibodies in MS sera. Specific antibodies from a representative MS serum, were successfully depleted using a Sepharose resin bearing the new glucosylated multivalent conjugate, as confirmed by ELISA. These results may offer a promising proof-of-concept for the selective purification of high affinity autoantibodies from sera of autoimmune patients, in general, and of specific high affinity antibodies against a minimally glcosylated epitope Asn(Glc) from sera of multiple sclerosis (MS) patients, in particular., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Multifunctional hydrogels that integrate stretchability, adhesion, self-healing, and antibacterial properties may find use in a variety of fields including electronic skin, wound dressings, and wearable devices; however, traditional hydrogels often exhibit short-term adhesiveness, poor mechanical properties, and a lack of antibacterial activity. Herein, a plant-inspired polyacrylamide-soybean protein isolate-pyrogallol/borax (PAM-SPI-P/B) hydrogel has been developed using a facile green method based on dynamic coordination cross-linking between pyrogallol (PG) and borax. The PG-borax dynamic bonds adjusted the network structure of the hydrogels to provide greater structural integrity to the PAM-SPI double network. This hydrogel possessed a high mechanical strength (large elongation up to 760% and compressive strength up to 1.25 MPa at 80% strain), low swelling ratio, and self-healing properties. Inspired by natural polyphenols that contain adhesive molecules, the addition of pyrogallol provided the hydrogel excellent adhesion to various hydrophilic and hydrophobic substrates. And with the inhibition of pyrogallol autoxidation due to the borax protection, the hydrogel showed repeatable and durable adhesion over 20 cycles. The obtained hydrogels also exhibited good antibacterial activities against Escherichia coli and Staphylococcus aureus because they were based on pyrogallol and borax, which have antibacterial properties. Accordingly, we envision that the PAM-SPI-P/B hydrogels have great potential for use in biomimetic tissues and biosensors.

Background/purpose: The aim of this study was to characterize the Staphylococcus aureus strains isolated from periodontal lesions of patients, to determine the expression of genes involved in cell adhesion upon their infection of human epithelial cells using an in vitro model, its biofilm formation, and its resistance to antibiotics., Methods: S. aureus was analysed by PCR, Kirby-Bauer, and pulsed-field gel electrophoresis (PFGE), measuring gene expression by real-time PCR after infection of human cells in vitro., Results: S. aureus was identified in 18.6% (50/268) of the samples. All strains (n = 50) possessed the virulence genes spa (Staphylococcal protein A), coa (coagulase), and icaAB (intercellular adhesin); 96% (n = 48) possessed clfB (clumping factor B), and 88% (n = 44) possessed ebps (elastin-binding protein) and sdrD (serine aspartate repeat protein D). All strains were resistant to methicillin, ampicillin, dicloxacillin, cefotaxime, and penicillin, and were multidrug resistant to 6-12 antibiotics. PFGE analysis showed 37 different pulsed-field types and most strains (60.4%) had a unique pulsed-field type. Twenty-four distinct combinations of virulence genes and antibiotic-resistant phenotypes were identified., Conclusion: Although S. aureus has been considered a transient member of the oral microbiota, our results indicate a high-level expression of virulence genes and multidrug resistance in the strains isolated from periodontal lesions. These strains might complicate the successful treatment of the disease., (Copyright © 2019. Published by Elsevier B.V.)

Objective: Lectin-like adhesins of enteric bacterial pathogens such as Escherichia coli are an attractive target for vaccine or drug development. Here, we have developed e-Membranome as a database of genome-wide putative adhesins in Escherichia coli (E. coli)., Methods: The outer membrane adhesins were predicted from the annotated genes of Escherichia coli strains using the PSORTb program. Further analysis was performed using Interproscan and the String database. The candidate proteins can be investigated for homology modeling of the Three-Dimensional (3D) structure (I-TASSER version 5.1), epitope region (ABCpred), and the glycan array., Results: e-Membranome is implemented using the Django (version 2.2.5) framework. The Web Application Server Apache Tomcat 6.0 is integrated into the platform on Ubuntu Linux (version 16.04). MySQL database (version 5.7) is used as a database engine. The information on homology model of the 3D structure, epitope region, and affinity information from the glycan array will be stored in the e- Membranome database. As a case study, we performed a genome-wide screening of outer membraneembedded proteins from the annotated genes of E. coli using the e-Membranome pipeline., Conclusion: This platform is expected to be a valuable resource for advancing research of outer membrane proteins for the construction of lectin-glycan interaction network of E. coli. In addition, the e- Membranome pipeline can be extended to other similar biological systems that need to address hostpathogen interactions., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Uropathogenic strains belonging to the Enterobacteriaceae family are considered one of factors for urinary tract infections, and type 1 pilus fimbrial adhesin (FimH) and beta lactamase CTX-M-15 play crucial roles in their pathogenesis and resistance. Thus, a promising approach is to explore dual-targeting therapeutic agents that act against both FimH and CTX-M-15. In the present study, active constituents of Nigella sativa were selected on the basis of significant ‎activity against UTIs. Molecular docking was used to target active constituents of Nigella sativa to the active sites of FimH and CTX-M-15; these included thymoquinone, dithymoquinone, carvacrol, p-cymene, thymol, thymohydroquinone and longifolene. Dithymoquinone was found to be the most potent dual inhibitor, with binding energy of -7.01 and -5.38kcal/mol against CTX-M-15 and FimH, respectively; In addition, Dithymoquinone exhibited superior activity compared to positive controls avibactam and heptyl α-D-mannopyranoside. Further molecular dynamic simulation studies were carried out to assess the stability of dithymoquinone-target protein complexes via RMSD, Rg, SASA, hydrogen bond number, and RMSF analysis. Both protein-ligand complexes were conserved and attained equilibrium at around 2.0 to 2.5 ns during‎10 ns runs. These results suggest that active constituents of Nigella sativa, particularly dithymoquinone, might represent a plausible therapeutic strategy against resistant uropathogenic bacteria.

Approximately 50-80% of the world population are infected with H. pylori , which is categorized as a class I carcinogen. Antiadhesive therapy is emerging as a promising alternative to antibiotics against bacterial infection. This study demonstrated that defatted wheat germ protein hydrolysates (DWGPH) effectively inhibited H. pylori adhesion to gastric epithelial cells. DWGPH prepared by pronase possessed the best activity where its inhibitory percentage at 10 mg/mL was 51.7 ± 6.8% and the minimum antiadhesive concentration was 0.31 mg/mL. The antiadhesive activity is attributable to peptides acting as receptor analogs in binding to H. pylori . Peptides with potential H. pylori -binding ability ( n = 267) were identified, and their structural characteristics were comprehensively analyzed, including net charge, Boman index, instability index, aliphatic index, molecular weight, isoelectric point, hydrophobicity, and Hmoment (α-helix and β-sheet). This work provided an array of peptide sequences for further exploration as putative ligands of H. pylori adhesins and for elucidating molecular mechanisms.

Staphylococcus aureus expresses many Microbial Surface Recognizing Adhesive Matrix Molecules (MSCRAMM's) to recognize host extracellular matrix (ECM) molecules to initiate colonization. The MSCRAMM, fibronectin binding protein A (FnBPA), is an important adhesin for S. aureus infection. FnBPA also binds with fibrinogen (Fg) by using a unique ligand binding mechanism called dock, lock and latch. Nanoparticles, especially nanosilver particles have been widely used in a variety of biomedical applications which includes disease diagnosis and treatment, drug delivery and implanted medical device coating. In a biological system, when protein molecules encounter nanoparticle, they can be absorbed onto its surface which results in the formation of protein corona. In the present study, we have analysed the fibrinogen binding ability of rFnBPA (189-512) in the presence of silver nanoparticles by employing techniques like gel shift assay, Western blot, size exclusion chromatography, enzyme-linked immunosorbent assay, bio-layer interferometry and circular dichroism spectroscopy. The results indicate that rFnBPA (189-512) is unable to bind to Fg in the presence of a nanoparticle. This could be due to the inaccessibility of the Fg binding site and conformational change in rFnBPA (189-512) . With nanoparticles, rFnBPA (189-512) undergoes significant structural changes as the β-sheet content has drastically reduced to 10% from the initial 60% at higher concentration of the nanoparticle. Pathogenic bacteria interact with its surrounding environment through their surface molecules which includes MSCRAMMs. Therefore MSCRAMMs play an important role when bacteria encounter nanoparticles. The results of the present study suggest that the orientation of the protein during the absorption on the surface of a nanoparticle as well as the concentration of the nanoparticle, will dictate the function of the absorbed protein and in this case the Fg binding property of rFnBPA (189-512) ., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The purpose of this research was to examine biofilm (icaA, icaD and bap) and adhesin (clfA, fnbA, cna) genes, and also assess the genotypic and phenotypic antimicrobial resistance patterns of Staphylococcus aureus strains taken from wound specimens in Mardin, Turkey. A total of 220 wound specimens were investigated. The biofilm forming ability and resistance pattern for eleven antimicrobial agents were investigated by conventional and multiplex PCR methods. S. aureus were taken from 112 (50.9%) of 220 wound specimens. Moreover, biofilm production was found in 79 (70.5%) of the 112 S. aureus isolates. 97 (86.6%) strains of all isolates were positive for icaA and icaD, and 15 (13.4%) for bap. The adhesin genes, cna, fnbA and clfA were detected in 98 (87.5%), 87 (77.7%), and 75 (66.9%) strains, respectively. The numbers of MSSA and MRSA bearing antimicrobial resistance genes were 19 (16.96%) and 32 (28.57%) for blaZ, 9 (8.04%) and 17 (15.18%) for tetK, 6 (5.36%) and 14 (12.5%) for ermC, 2 (1.79%) and 7 (6.25%) for tetM, 0 (0%) and 5 (4.46%) for mecA, 2 (1.79%) and 4 (3.57%) for ermA, 1 (0.89%) and 2 (1.79%) for both tetK and tetM, respectively. Our findings indicate that multiplex PCR is a suitable way for identifying biofilm and adhesin producing S. aureus. Our data also provided a country-wide oversight of the S. aureus antimicrobial resistance gene profiles for the properly therapy of patients and to control the spreading of the resistance genes., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Helicobacter pylori (H. pylori) infection is highly prevalent, and has developed antimicrobial resistance to virtually all existing antibiotics. Currently, treatment of H. pylori infection (involving proton pump inhibitors and broad-spectrum antibiotics) is suboptimal, with high failure rates. Thus, there is a pressing need to develop new anti-H. pylori therapies. Cbf-K 16 , a cathelicidin-like antimicrobial peptide, presented broad antimicrobial activity during our previous research. This study further evaluated the therapeutic potential and the mode of action underlying Cbf-K 16 against clarithromycin- and amoxicillin-resistant H. pylori SS1. The MIC and MBC of Cbf-K 16 against the tested H. pylori were 16 and 32 μg/ml, respectively, and its killing kinetics was time-dependent, reflecting the thorough elimination of drug-resistant bacteria within 24 h. This peptide also protected H. pylori-infected gastric epithelial cells (GES-1) from death by reducing the cell supernatant and intracellular bacterial counts by 1.9 and 2.9-log 10 units, respectively. These data indicated the powerful antimicrobial effects of Cbf-K 16 in vitro. Meanwhile, notable antimicrobial activity in the mouse gastritis model was observed, with decreasing bacterial counts by 3.9-log 10 units in stomach tissues and Cbf-K 16 could effectively suppress the secretion of inflammatory cytokine IL-8. For its mode of action, Cbf-K 16 not only neutralized the negative potential and increased the membrane uptake of NPN and PI by 78.5% and 85.1%, respectively, but also bound to genomic DNA, which in turn downregulated the expression of adhesion genes (alpA and alpB) and virulence gene (cagA), indicating its effective activities on membrane disruption, DNA-binding and gene expression. The data above demonstrated that Cbf-K 16 possessed effective antimicrobial and anti-inflammatory activities and downregulated the expression of adhesion- and cytotoxin-associated genes of drug-resistant H. pylori SS1, making it a potential candidate for anti-infective therapy., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Bacterial adhesion to the skin and mucosa is often a fundamental and early step in host colonization, the establishment of bacterial infections, and pathology. This process is facilitated by adhesins on the surface of the bacterial cell that recognize host cell molecules. Interfering with bacterial host cell adhesion, so-called anti-adhesive therapeutics, offers promise for the development of novel approaches to control bacterial infections. In this review, we focus on the discovery of anti-adhesives targeting the high priority pathogen Staphylococcus aureus . This organism remains a major clinical burden, and S. aureus nasal colonization is associated with poor clinical outcomes. We describe the molecular basis of nasal colonization and highlight potentially efficacious targets for the development of novel nasal decolonization strategies.

The sewage sludges represent a potential health hazard because of the quantity of different microbiota detected in sewages. Among microbiota detected in sewages, also belong representatives of the phylum Firmicutes . In the past, environmental enterococci in addition to coliforms were widely used as indicators of faecal contamination. Regarding the enterococcal strains as potential pathogenic bacteria, their pathogenicity is mainly caused by production of virulence factors. Therefore, the aim of the study was to analyse incidence of virulence factors in enterococci from cows' dung water. Species identification of 24 enterococci using MALDI-TOF MS system allotted 23 strains to the species Enterococcus faecium with highly probable species identification and E. faecalis EEV20 with a score value meaning secure genus identification/probable species identification. Enterococci were absent of cytolysin A gene, hyaluronidase gene, and element IS gene. It can be concluded that they are not invasive which is very important from safety aspect. The most frequently detected gene was adhesin E. faecium ( efaAfm , in 22 E. faecium strains and in one E. faecalis ). Adhesin efaAfs gene was detected in E. faecalis EEV20 and in two E. faecium . GelE gene was present in three strains. E. faecium EF/EC31 was absent of virulence factor genes., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2019 Andrea Lauková et al.)

Opportunistic Gram-negative Pseudomonas aeruginosa uses adhesins (e.g., LecA and LecB lectins, type VI pili and flagella) and iron to invade host cells with the formation of a biofilm, a thick barrier that protects bacteria from drugs and host immune system. Hindering iron uptake and disrupting adhesins' function could be a relevant antipseudomonal strategy. To test this hypothesis, we designed an iron-chelating glycocluster incorporating a tetrahydroxamic acid and α-l-fucose bearing linker to interfere with both iron uptake and the glycan recognition process involving the LecB lectin. Iron depletion led to increased production of the siderophore pyoverdine by P. aeruginosa to counteract the loss of iron uptake, and strong biofilm inhibition was observed not only with the α-l-fucocluster (72%), but also with its α-d- manno (84%), and α-d- gluco (92%) counterparts used as negative controls. This unprecedented finding suggests that both LecB and biofilm inhibition are closely related to the presence of hydroxamic acid groups.

Background: Bacterial adhesins play an important role in the bacterial attachment and colonization. The aim of this study was comparison of adhesin genes expression in the planktonic and biofilm mode of growth among ESBL-non-producers isolates of K. oxytoca and effect of imipenem., Materials and Methods: A total of eight extended-spectrum beta-lactamase (ESBL) non-producer K. oxytoca isolates were included from patients with hemorrhagic colitis. The adhesin genes including fimA (type 1 fimbria), mrkA (type 3 fimbria), pilQ and the capsular matB genes were adopted. Phenotypic biofilm production was assessed by microtiter tissue plate assay. Expression of adhesin genes in the planktonic and biofilm growth conditions was calculated using quantitative Real-time PCR (RT-qPCR) technique and sub-MIC (0.25 μg/ml) levels of imipenem were also added to broth culture of isolates to evaluate the gene expression., Results: The isolates produced biofilm in moderate level. The expression of pilQ, mrkA and matB but not fimA genes was significantly higher in biofilm conditions compared to the planktonic mode of growth (p = 0.002, p = 0.011 and p = 001, respectively). In addition, imipenem sub-MIC treatment led to a significant overexpression of matB (p = 0.002) and mrkA (p = 0.003) genes compared to the control group., Conclusion: Although none of isolates produced strong biofilm, biofilm conditions led to the increase in the expression of adhesin encoding genes in non-ESBL-producing K. oxytoca. Furthermore, β-lactams; and especially carbapenems possibly increase the colonization of K. oxytoca and increase the biofilm formation. Hence, accurate consumption of antibiotics must be considered., (Copyright © 2019. Published by Elsevier Ltd.)

Staphylococcus aureus is a major pathogen of subclinical bovine mastitis that usually is chronic and recurrent, which has been related to its ability to internalize into bovine mammary epithelial cells (bMECs). Previously, we reported that short and medium fatty acids and cholecalciferol reduce S. aureus internalization into pretreated-bMECs with these molecules suggesting a role as immunomodulatory agents. Hence, we assessed the role of sodium butyrate (NaB), sodium octanoate (NaO) and cholecalciferol on S. aureus adhesin expression and its internalization into bMECs. S. aureus pre-treated 2 h with 0.5 mM or 2 mM NaB showed a reduction in internalization into bMECs (∼35% and ∼55%; respectively), which coincided with a down-regulated expression of clumping factor B (ClfB). Also, the S. aureus internalization reduction by 2 mM NaB (2 h) agreed with a down-regulated expression of sdrC. Moreover, the 2 mM NaB (24 h) pre-treatment induced bacterial internalization (∼3-fold), which was related with an up-regulation of spa, clfB and sdrC genes. Also, NaO (0.25 mM and 1 mM) only reduced S. aureus internalization when bacteria were grown 2 h with this molecule but there was no relationship with adhesin expression. In addition, cholecalciferol (50 nM) reduced bacteria internalization at similar levels (∼50%) when bacteria were grown 2 and 24 h in broth supplemented with this compound, which correlated with spa and sdrC mRNA expression down-regulated at 2 h, and fnba and clfB mRNA expression decreased at 24 h. In conclusion, our data support the fact that fatty acids and cholecalciferol regulate adhesin gene expression as well as bacteria internalization in nonprofessional phagocytic cells, which may lead to development of anti-virulence agents for control of pathogens., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Staphylococcus aureus ( S. aureus ) is a human and other animal pathogen that contributes to the primary etiology of nosocomial pneumonia, a disease with high mortality rates and costs. Treatment of multidrug-resistant S. aureus infection is extremely challenging, and new therapeutic strategies beyond antibiotic treatment are needed. Anti-virulence agents that specifically target the molecular determinants of virulence may be a novel method for treating drug-resistant nosocomial infections. Sortase B (SrtB) is a crucial virulence factor in S. aureus and plays an important role during infection. In this study, we find that baicalin suppresses the activity of SrtB. Minimum inhibitory concentration and growth curve assays confirmed that baicalin has no anti- S. aureus properties. We performed live/dead, lactate dehydrogenase (LDH), adherence, and enzyme-linked immunosorbent assays to confirm that baicalin reduced human alveolar epithelial A549 cell injury caused by S. aureus , reduced the adherence of S. aureus to A549 cells, and significantly attenuated the inflammatory response of mouse macrophage J774 cells to S. aureus . Additionally, we were able to elucidate the binding mechanics and identify the interacting sites of baicalin and SrtB via a molecular dynamics simulation, site-directed mutagenesis, and fluorescence spectroscopy quenching. Finally, we confirmed that baicalin directly binds to the active center of SrtB, and the residues Asn 92 and Tyr 128 perform an important function in the interaction of SrtB and baicalin. Taken together, these data indicate that baicalin is a promising candidate to combat S. aureus infections.

Coumarin is an important heterocyclic molecular framework of bioactive molecules against broad spectrum pathological manifestations. In the present study 18 new coumarin derivatives (CDs) were synthesized and characterized for antibiofilm activity against two model bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa. It was observed that all the CDs executed significant effect in moderating activities against both planktonic and biofilm forms of these selected bacteria. Hence, to interpret the underlying probable reason of such antibiofilm effect, in-silico binding study of CDs with biofilm and motility associated proteins of these organisms were performed. All CDs have shown their propensity for occupying the native substrate binding pocket of each protein with moderate to strong binding affinities. One of the CDs such as CAMN1 showed highest binding affinity with these proteins. Interestingly, the findings of in-silico studies coincides the experimental results of antibiofilm and motility affect of CDs against both S. aureus and P. aeruginosa. Moreover, in-silico studies suggested that the antibiofilm activity of test CDs may be due to the interference of biofilm and motility associated proteins of the selected model organisms (PilT from P. aeruginosa and TarK, TarO from S. aureus). The detailed synthesis, characterization, methodology and results of biological screening along with computational studies have been reported. This study could be of greater interest in the context of the development of new anti-bacterial agent in the future.

PsaA (pneumococcal surface antigen A) is a S. pneumoniae virulence factor that belongs to the metal transport system. The Manganese PsaA binding has been associated with oxidative stress resistance becoming a pivotal element in the bacteria virulence. It has been shown that Zinc inhibits the Manganese acquisition and promotes bacteria toxicity. We have performed a PsaA conformational analysis both in the presence (Zn-rPsaA) and in the absence of Zinc (free-rPsaA). We performed experiments in the presence of different Zinc concentrations to determine the metal minimum concentration which induced a conformational change. The protein in free and Zn-binding condition was also studied in pH ranging 2.6-8.0 and in temperature ranging 25oC-85oC. pH experiments showed a decrease of fluorescence intensity only in acidic medium. Analysis of the heat-induced denaturation demonstrated that Zinc-binding promoted an increase in melting temperature from 55oC (free-rPsaA) to 78.8oC (Zn-rPsaA) according to fluorescence measurements. In addition, the rPsaA stabilization by Zinc was verified through analysis of urea and guanidine hydrochloride denaturation. Data showed that Zinc promoted an increase in the rPsaA stability and its removal by EDTA can lead to a PsaA intermediate conformation. These findings can be considered in the development of vaccines containing PsaA as antigen.

Purified plant nutraceuticals afzelin and quercetrin from an edible plant- Crotolaria tetragona was employed for the fabrication of silver nanoparticles (AgNPs) by a sunlight mediated process. From among a panel of strains tested, AgNPs displayed potent bacteriostatic and bactericidal effect against P. aeruginosa and S. Typhi. Time kill studies revealed green synthesized AgNPs displayed comparable bactericidal effect with chemically synthesized AgNPs against S. Typhi. Antibiofilm potential of AgNPs showed that they were highly effective at sub MIC concentrations in causing 50% biofilm inhibition against food borne pathogen S. Typhi implying that antibiofilm effect is independent of antibacterial effect, which was evidenced by fluorescent imaging and SEM imaging. Mechanistic studies revealed that reduced cell surface hydrophobicity, decreased surface adherence, loss of membrane potential contributed to antibiofilm potential of afzelin/quercetrin AgNPs. Green synthesized afzelin/quercetrin AgNPs were also relatively less toxic and more effective in curtailing bioburden of S. Typhi in infected zebrafish by > 3 log fold. Ability of sunlight reduced afzelin/quercetrin NPs to mitigate planktonic mode of growth in vitro and in vivo and curtail biofilm formation of S. Typhi in vitro demonstrates its potential to curtail food borne pathogen in planktonic and biofilm mode of growth., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The study aimed to investigate whether rutin affects the quorum sensing (QS) of avian pathogenic Escherichia coli (APEC). In this study, APEC-O78 was selected as the test strain. We mainly examined the effects of rutin on the AI-2 secretion by bioluminescence assay, biofilm formation through a crystal violet staining method, and expression of virulence genes of APEC by qRT-PCR. We found that rutin can significantly interfering with QS through reducing the secretion of AI-2, inhibited the biofilm formation, and reduced the expression of virulence genes of APEC. Moreover, rutin markedly decreased adhesion and damage of APEC to chicken type II pneumocytes. These results suggested rutin reduces cell damage of APEC-infected chicken type II pneumocytes through interfering with QS via decreasing AI-2 production, biofilm formation, and the expression of virulence genes. This paper may provide a new evidence for colibacillosis prevention in chicken., (Copyright © 2018. Published by Elsevier Ltd.)

Objectives: To evaluate the bonding performance, antibacterial activity, and remineralization effect on enamel of the orthodontic adhesive containing MAE-DB and NACP., Methods: Eighty non-carious human premolars were divided into 3 groups: Transbond XT (TB), PEHB + 5% MAE-DB (PD), and PEHB + 40% NACP + 5% MAE-DB (PND). Premolars were bonded with orthodontic brackets, the first subgroup (n = 10) and the second subgroup (n = 10) were subjected to shear bond strength testing after immersed in water for 1 day and in demineralization solution for 28 days respectively and then tested surface roughness, while the third subgroup (n = 6) was used for microhardness evaluation after aged in demineralization solution for 28 days. For each adhesive, fifty disk samples were prepared for antibacterial study. Specimens measuring 12 mm × 2 mm × 2 mm were fabricated for ion release test., Results: Bond strengths were in the order TB = PND > PND = PD for "1-day in water", and in the order TB = PND > PD for "28-days in pH 4 solution". No significant difference in the ARI scores for the three adhesive. Numerous bacteria adhered to TB surface, while PD and PND had minimal bacterial growth and activity. PND showed high levels of Ca and P ions release and enamel hardness. The surface roughness of enamel in PND was much lower than TB and PD and showed no significant difference with the sound, control enamel., Conclusion: PND adhesive with 5% MAE-DB and 40% NACP exhibits antibacterial and remineralizing capabilities, and did not adversely affect bond strength compared to commercial adhesive., Clinical Significance: Novel adhesive containing quaternary ammonium monomer and nano-amorphous calcium phosphate represents a promising candidate in combating enamel white spot lesions and even dental caries., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objectives: The present study was aimed to determine whether trefoil factor family (TFF) peptides which were generally considered to be resistant to proteolysis could be digested by gingipains, a major proteinases produced by Porphyromonas gingivalis., Materials and Methods: Recombinant human TFF1, TFF2, and TFF3 peptides were used as substrates. Gingipains including arginine gingipain (RgpB) and lysine gingipain (Kgp) were used as enzymes. Trypsin was used as a control protease. Matrix-assisted laser desorption/ionization with time-of-flight / time-of-flight (MALDI-TOF/TOF) and liquid chromatography mass spectrometry (LC-MS) were used for analyzing peptide mass signals and amino acid sequences of digested TFF peptides., Results: MALDI-TOF/TOF analyses demonstrated that Kgp, RgpB, and trypsin were able to cleave TFF1 and TFF2 peptides, resulting in different patterns of digested fragments. However, impurity in recombinant TFF3 peptide substrates affected the interpretations of enzymatic reaction by MALDI-TOF/TOF. LC-MS analyses demonstrated that identified fragments of TFF1, TFF2, and TFF3 from digestion by gingipains were similar to those by trypsin., Conclusions: Using MALDI-TOF/TOF and LC-MS, the present study provides new information that gingipains containing trypsin-like activities are able to digest TFF peptides., Clinical Relevance: The proteolytic effects of gingipains on TFF peptides may be responsible for reduction of salivary TFF peptides in chronic periodontitis patients. Further investigations to determine the pathological effects of gingipains on TFF peptides in saliva and periodontal tissues of patients with chronic periodontitis would be of interest.

The synthesis of eight perylenediimide-based glycoclusters was readily performed from hexa- and tetra-propargylated cores through azide-alkyne "click" conjugation. Variations in the carbohydrate epitope (Glc, Gal, Man, Fuc) and the linker arm provided molecular diversity. Interactions with LecA and LecB, two proteins involved in the adhesion of Pseudomonas aeruginosa to host tissues, were evaluated by microcalorimetry (ITC). In both cases high affinities were obtained with K d values in the nanomolar range. Further evaluation of their anti-adhesive properties using cultured epithelial cells demonstrated their potent anti-adhesive activities against Pseudomonas aeruginosa with only 30-40% residual adhesion observed. The fluorescence properties of the PDI core were then investigated by confocal microscopy on cell-bacteria cultures. However, the red fluorescence signal of the PDI-based glycocluster was too weak to provide significant data. The present study provides another type of anti-adhesive glycocluster against bacterial infection with a large aromatic PDI core.

In this study, we evaluated the inhibitory effect of a rice bran mixture extract (RBE) on Brucella abortus pathogenesis in professional (RAW 264.7) and nonprofessional (HeLa) phagocytes. We fermented the rice bran mixture and then extracted it with 50% ethanol followed by gas chromatography-mass spectrometry to identify the components in RBE. Our results clearly showed that RBE caused a significant reduction in the adherence of B. abortus in both cell lines. Furthermore, analysis of phagocytic signaling proteins by western blot assay revealed that RBE pretreatment resulted in inhibition of phosphorylation of JNK, ERK, and p38, leading to decline of internalization compared with the controls. Additionally, the intensity of F-actin observed by fluorescence microscopy and FACS was remarkably reduced in RBE-pretreated cells compared with control cells. However, the intracellular replication of B. abortus within phagocytes was not affected by RBE. Taken together, these findings suggest that the phagocytic receptor blocking and suppressive effects of RBE on the MAPK-linked phagocytic signaling pathway could negatively affect the invasion of B. abortus into phagocytes.

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading human pathogen responsible for causing chronic clinical manifestation worldwide. In addition to antibiotic resistance genes viz. mecA and vanA, biofilm formation plays a prominent role in the pathogenicity of S. aureus by enhancing its resistance to existing antibiotics. Considering the role of folk medicinal plants in the betterment of human health from the waves of multidrug resistant bacterial infections, the present study was intended to explore the effect of Vetiveria zizanioides root on the biofilm formation of MRSA and its clinical counterparts. V. zizanioides root extract (VREX) showed a concentration-dependent reduction in biofilm formation without hampering the cellular viability of the tested strains. Micrographs of scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) portrayed the devastating impact of VREX on biofilm formation. In addition to antibiofilm activity, VREX suppresses the production of biofilm related phenotypes such as exopolysaccharide, slime and α-hemolysin toxin. Furthermore, variation in FT-IR spectra evidenced the difference in cellular factors of untreated and VREX treated samples. Result of mature biofilm disruption assay and down regulation of genes like fnbA, fnbB, clfA suggested that VREX targets these adhesin genes responsible for initial adherence. GC-MS analysis revealed the presence of sesquiterpenes as a major constituent in VREX. Thus, the data of present study strengthen the ethnobotanical value of V. zizanioides and concludes that VREX contain bioactive molecules that have beneficial effect over the biofilm formation of MRSA and its clinical isolates., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Clostridium difficile infection (CDI) are the leading cause of world-wide nosocomial acquired diarrhea. The current main clinical challenge in CDI is the elevated rate of infection recurrence that may reach up to 30% of the patients, which has been associated to the formation of dormant spores during the infection. We sought to characterize the effects of oral administration of specific anti-spore IgY in mouse models of CDI and recurrent CDI. The specificity of anti-spore IgY was evaluated in vitro . In both, initiation mouse model and recurrence mouse model, we evaluated the prophylactic and therapeutic effect of anti-spore IgY, respectively. Our results demonstrate that anti-spore IgY exhibited high specificity and titers against C. difficile spores and reduced spore adherence to intestinal cells in vitro . Administration of anti-spore IgY to C57BL/6 mice prior and during CDI delayed the appearance of the diarrhea by 1.5 day, and spore adherence to the colonic mucosa by 90%. Notably, in the recurrence model, co-administration of anti-spore IgY coupled with vancomycin delayed the appearance of recurrent diarrhea by a median of 2 days. Collectively, these observations suggest that anti-spore IgY antibodies may be used as a novel prophylactic treatment for CDI, or in combination with antibiotics to treat CDI and prevent recurrence of the infection.

In this study, we investigated the protective effects of tannin-derived components, gallic acid (GA) and tannic acid (TA), in vitro and in vivo against Salmonella infection in mice. Both GA and TA showed antibacterial effects against Salmonella (S.) Typhimurium as well as inhibitory effects on the adherence, invasion, and intracellular growth of the pathogens in macrophages. Following a lethal dose of Salmonella infection in mice, reduced virulence in both GA- and TA-treated groups was observed based on reduced mortality rates. In the non-infected groups, the average weights of the spleens and livers of GA- or TA-treated mice were not significantly different with the control group. In addition, the average weights of these organs in all of the Salmonella-infected groups were not significantly different but the numbers of bacteria in the spleens and livers in both GA- and TA-treated mice were significantly reduced. The levels of cytokine production in non-infected mice revealed that GA-treated and TA-treated mice elicited an increased level of IFN-γ, and both IFN-γ and MCP-1, respectively, as compared with the PBS-treated group. These findings highlight the potential of GA and TA as alternatives for the treatment of salmonellosis and as supplements to conventional antimicrobial food additives., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Purpose: Staphylococcus aureus infections have contributed to the global healthcare burden, particularly with regard to hospital-acquired meticillin-resistant S. aureus (MRSA) infections., Methodology: This study describes the antibacterial activity of diacetylcurcumin (DAC) against meticillin-susceptible S. aureus/MRSA biofilm formation, survival, metabolic activity and structure; its ability to prevent bacterial adhesion to human cells; and toxicity in Galleria mellonella larvae., Results: DAC showed excellent antibacterial activity, with MIC ranging between 17.3 and 34.6 µmol l-1, and minimum bactericidal concentration ranging between 69 and 277 µmol l-1. It significantly reduced bacterial biofilm survival - by 22-63 % (at MIC, 10×MIC or 100×MIC) as compared to the 25-42 % reduction by vancomycin (P<0.0001) - and severely affected biofilm cell structures, leading to damaged architecture and the formation of amorphous cell clusters. Treatment with DAC (MIC/4) decreased bacterial adhesion to HaCaT keratinocytes from 1 to 5 h (P<0.0001). Finally, DAC demonstrated low toxicity in G. mellonella at its effective anti-biofilm concentrations., Conclusion: These findings open new avenues for the study of this curcumin derivative as an excellent prototype with anti-MRSA activity.

Background and Objective: Porphyromonas gingivalis is considered a major pathogen of chronic periodontitis, which also may be implicated with systemic diseases such as atherosclerosis. Secreted cysteine proteases, gingipains Rgp and Kgp, are essential for P. gingivalis virulence. Some polyphenols and flavonoids are known to inhibit gingipain activity and interfere with biofilm formation by P. gingivalis. Many bioactive compounds have been isolated from Epimedium species, but availability of these compounds on gingipains and P. gingivalis is still unclear. Therefore, the aim of this study was to evaluate natural products from medical plants to develop a new therapeutic agent against periodontal disease., Material and Methods: Prenylated flavonoids were isolated from Epimedium species plant using column chromatographies. The inhibitory effect of the prenylated flavonoids against protease activity of gingipains were examined using purified gingipains and fluorogenic substrates. Anti-P. gingivalis activity was evaluated to analyze planktonic growth and biofilm formation in brain heart infusion medium in the presence of the prenylated flavonoids., Results: We isolated 17 prenylated flavonoids (Limonianin, Epimedokoreanin B, etc.) from Epimedium species. We found that some prenylated flavonoids inhibited gingipain activity in a non-competitive manner with K i values at μm order. The prenylated flavonoids also hindered growth and biofilm formation of P. gingivalis, in a manner independent of gingipain inhibition by the compounds., Conclusion: The results indicated an inhibitory effect of the prenylated flavonoids against P. gingivalis and would provide useful information for future development of periodontitis treatment that suppresses gingipains, P. gingivalis growth and biofilm formation., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Aim: To evaluate the antibacterial and antibiofilm mechanisms of usnic acid (USN) against methicillin-resistant Staphylococcus aureus from cystic fibrosis patients., Materials & Methods: The effects exerted by USN at subinhibitory concentrations on S. aureus Sa3 strain was evaluated by proteomic, real-time PCR and electron microscopy analyses., Results & Conclusion: Proteomic analysis showed that USN caused damage in peptidoglycan synthesis, as confirmed by microscopy. Real-time PCR analysis showed that antibiofilm activity of USN is mainly due to impaired adhesion to the host matrix binding proteins, and decreasing lipase and thermonuclease expression. Our data show that USN exerts anti-staphylococcal effects through multitarget inhibitory effects, thus confirming the rationale for considering it 'lead compound' for the treatment of cystic fibrosis infections.

Background and Objective: Increasing evidence suggests that 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ), a fat-soluble secosteroid hormone, has a positive impact on periodontal health through diverse mechanisms. The present study was aimed at investigating the effect of 1,25(OH)2 D3 on the growth of and virulence factor gene expression by the periodontopathogenic bacterium Porphyromonas gingivalis. The effect of 1,25(OH)2 D3 on P. gingivalis-mediated activation of nuclear factor kappa B (NF-κB) transcription factor in monocytes was also assessed., Material and Methods: A broth microdilution assay was used to determine the antibacterial activity of 1,25(OH)2 D3 . The modulation of virulence factor gene expression in P. gingivalis was assessed by quantitative reverse transcription-polymerase chain reaction. NF-κB activation was assessed using a human monocytic cell line stably transfected with a luciferase reporter containing NF-κB binding sites., Results: Minimal inhibitory concentrations of 1,25(OH)2 D3 against P. gingivalis ranged from 3.125 to 6.25 μg/mL. Moreover, a partial synergistic effect was observed when 1,25(OH)2 D3 was used in association with metronidazole. 1,25(OH)2 D3 attenuated the virulence of P. gingivalis by reducing the expression of genes coding for important virulence factors, including adhesins (fimA, hagA and hagB) and proteinases (rgpA, rgpB and kgp). 1,25(OH)2 D3 dose-dependently prevented P. gingivalis-induced NF-κB activation in a monocyte model., Conclusion: Our study suggested that 1,25(OH)2 D3 selectively inhibits the growth of and virulence factor gene expression by P. gingivalis, in addition to attenuating NF-κB activation by this periodontopathogen. This dual action on P. gingivalis and the inflammatory response of host cells may be of particular interest with a view to developing a novel and inexpensive preventive/therapeutic strategy., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Unlabelled: Uropathogenic Escherichia coli (UPEC) is the primary cause of community-acquired urinary tract infections (UTIs). UPEC bind the bladder using type 1 pili, encoded by the fim operon in nearly all E. coli. Assembled type 1 pili terminate in the FimH adhesin, which specifically binds to mannosylated glycoproteins on the bladder epithelium. Expression of type 1 pili is regulated in part by phase-variable inversion of the genomic element containing the fimS promoter, resulting in phase ON (expressing) and OFF (nonexpressing) orientations. Type 1 pili are essential for virulence in murine models of UTI; however, studies of urine samples from human UTI patients demonstrate variable expression of type 1 pili. We provide insight into this paradox by showing that human urine specifically inhibits both expression and function of type 1 pili. Growth in urine induces the fimS phase OFF orientation, preventing fim expression. Urine also contains inhibitors of FimH function, and this inhibition leads to a further bias in fimS orientation toward the phase OFF state. The dual effect of urine on fimS regulation and FimH binding presents a potential barrier to type 1 pilus-mediated colonization and invasion of the bladder epithelium. However, FimH-mediated attachment to human bladder cells during growth in urine reverses these effects such that fim expression remains ON and/or turns ON. Interestingly, FimH inhibitors called mannosides also induce the fimS phase OFF orientation. Thus, the transduction of FimH protein attachment or inhibition into epigenetic regulation of type 1 pilus expression has important implications for the development of therapeutics targeting FimH function., Importance: Urinary tract infections (UTIs) are extremely common infections, frequently caused by uropathogenic Escherichia coli (UPEC), that are treated with antibiotics but often recur. Therefore, UTI treatment both is complicated by and contributes to bacterial antibiotic resistance. Thus, it is important to understand UTI pathogenesis to devise novel strategies and targets for prevention and treatment. Based on evidence from disease epidemiology and mouse models of infection, UPEC relies heavily on type 1 pili to attach to and invade the bladder epithelium during initial stages of UTI. Here, we demonstrate that the negative effect of planktonic growth in human urine on both the function and expression of type 1 pili is overcome by attachment to bladder epithelial cells, representing a strategy to subvert this alternative innate defense mechanism. Furthermore, this dually inhibitory action of urine is a mechanism shared with recently developed anti-type 1 pilus molecules, highlighting the idea that further development of antivirulence strategies targeting pili may be particularly effective for UPEC., (Copyright © 2015 Greene et al.)

Background: The domesticated legume, Canavalia gladiata (commonly called the sword bean), is known to contain canavanine. The fruit is used in Chinese and Japanese herbal medicine for treating the discharge of pus, but its pharmacological mechanisms are still unclear., Objectives: This study examined the effect of sword bean extract (SBE) on (i) oral bacteria and human oral epithelial cells in vitro, and (ii) the initiation and progression of experimental Porphyromonas gingivalis-induced alveolar bone resorption in rats., Material and Methods: A high-performance liquid chromatography/ultraviolet method was applied to quantitate canavanine in SBE. By assessing oral bacterial growth, we estimated the minimum inhibitory concentration and minimum bactericidal concentration of SBE, canavanine, chlorhexidine gluconate (CHX) solution. The cytotoxicity of SBE, canavanine, CHX, leupeptin and cystatin for KB cells was determined using a trypan blue assay. The effects of SBE, canavanine, leupeptin and cystatin on Arg-gingipain (Rgp) and Lys-gingipain (Kgp) were evaluated by colorimetric assay using synthetic substrates. To examine its effects on P. gingivalis-associated periodontal tissue breakdown, SBE was orally administered to P. gingivalis-infected rats., Result: Sword bean extract contained 6.4% canavanine. SBE and canavanine inhibited the growth of P. gingivalis and Fusobacterium nucleatum. The cytotoxicity of SBE, canavanine and cystatin on KB cells was significantly lower than that of CHX. Inhibition of Rgp with SBE was comparable to that with leupeptin, a known Rgp inhibitor, and inhibition of Kgp with SBE was significantly higher than that with leupeptin at 500 μg/mL ( p < 0.05). P. gingivalis-induced alveolar bone resorption was significantly suppressed by administration of SBE, with bone levels remaining comparable to non-infected animals ( p < 0.05)., Conclusion: The present study suggests that SBE might be effective against P. gingivalis-associated alveolar bone resorption., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The periodontal pathogen Porphyromonas gingivalis produces a unique class of cysteine proteinases termed gingipains that comprises Arg-gingipain (Rgp) and Lys-gingipain (Kgp). Growing evidence indicates that these 2 types of gingipains synergistically contribute to the entire virulence of the organism and increase the risk of periodontal disease (PD) by disrupting the host immune system and degrading the host tissue and plasma proteins. Therefore, a dual inhibitor of both gingipains would have attractive clinical potential for PD therapy. In this study, a novel, potent, dual inhibitor of Rgp and Kgp was developed through structure-based drug design, and its biological potency was evaluated in vitro and in vivo. This inhibitor had low nanomolar inhibitory potency (Ki=40 nM for Rgp, Ki=0.27 nM for Kgp) and good selectivity for host proteases and exhibited potent antibacterial activity against P. gingivalis by abrogating its manifold pathophysiological functions. The therapeutic potential of this inhibitor in vivo was also verified by suppressing the vascular permeability that was enhanced in guinea pigs by the organism and the gingival inflammation in beagle dog PD models. These findings suggest that a dual inhibitor of Rgp and Kgp would exhibit noteworthy anti-inflammatory activity in the treatment of PD., (© FASEB.)

In treatments of Helicobacter pylori infections, recrudescences were common because of an unfavorable bacterial eradication rate due to the ever increasing resistance to antibiotics. In this study, we chose pectin, guar gum and chitosan to synthesize their sulfates to inhibit adhesions of H. pylori and thus enhance the eradication rate. The introduction of sulfates was characterized using FT-IR and elemental analysis. Data from zeta-potential, hydrodynamic diameter, hydrolysis and rheological property demonstrated the sulfates were physicochemically stable. Inhibition assay of hemagglutination and adhesion indicated sulfates prevented H. pylori from adhering to erythrocytes and AGS cells. In binding assay, affinities of sulfates to H. pylori suggested sulfates could compete with target cells for bacteria and moderated the bacterial adhesion to hosts. A higher content of galactoses and 2,3-O-linked sulfates benefited this action. Thus polysaccharide sulfates can serve as potential adjuvants to raise the bacterial eradication rate by inhibiting adhesions of H. pylori., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Aim: To combat the resistance of Helicobacter pylori to antibiotics through the use of Garcinia mangostana extract (GME) in the form that can be localized at stomach mucosa., Materials & Methods: GME and its major active component, α-mangostin, are encapsulated into the moderately acid stable mucoadhesive nanocarriers, and tested for anti-H. pylori and antiadhesion activities in vitro and their ability to eradicate H. pylori in infected mice., Results: The two in vitro activities are observed and are enhanced when the materials are encapsulated into nanocarriers. Preliminary in vivo tests revealed the ability to combat H. pylori in mice following oral administration of the encapsulated GME, but not the unencapsulated GME., Conclusion: Nanoencapsulated GME is a potential anti-H. pylori agent.

Porphyromonas gingivalis and Tannerella forsythia are gram-negative pathogens strongly associated with periodontitis. Their abilities to interact, invade and persist within host cells are considered crucial to their pathogenicity, but the mechanisms by which they subvert host defences are not well understood. In this study, we set out to investigate whether P. gingivalis and T. forsythia directly target key signalling molecules that may modulate the host cell phenotype to favour invasion and persistence. Our data identify, for the first time, that P. gingivalis, but not T. forsythia, reduces levels of intracellular mammalian target of rapamycin (mTOR) in oral epithelial cells following invasion over a 4-h time course, via the action of gingipains. The ability of cytochalasin D to abrogate P. gingivalis-mediated mTOR degradation suggests that this effect is dependent upon cellular invasion. We also show that levels of several other proteins in the mTOR signalling pathway are modulated by gingipains, either directly or as a consequence of mTOR degradation including p-4E-BP1. Taken together, our data suggest that P. gingivalis manipulates the mTOR pathway, providing evidence for a potentially novel mechanism by which P. gingivalis mediates its effects on host cell responses to infection., (2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd)

Aim: To determine the levels of LL-37 in and its susceptibility to degradation by components of gingival crevicular fluid (GCF) in periodontal health and disease., Materials and Methods: Levels of LL-37 in GCF from periodontitis patients and periodontally healthy subjects were determined by ELISA. In addition, degradation of synthetic/exogenous LL-37 by components of GCF in the presence and absence of inhibitors was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry., Results: The concentration of native LL-37 in GCF from Porphyromonas gingivalis positive (Pg+) and P. gingivalis negative (Pg-) sites in periodontitis patients was significantly higher than in GCF from healthy subjects. When synthetic LL-37 was added to healthy GCF, the peptide was not degraded. Conversely, GCF from Pg+ sites rapidly degraded synthetic LL-37 which was prevented in the presence of Arg- and Lys- gingipain inhibitors. Synthetic LL-37 was degraded more slowly by GCF from Pg- sites., Conclusions: LL-37 is detectable in GCF in periodontal health and disease. The rapid degradation of synthetic LL-37 in periodontitis GCF, particularly in Pg+ sites, limits its role as a potential therapeutic in the gingival crevice. These results highlight the need to design stable peptide mimetics of LL-37 as future therapeutics in periodontitis., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Rhipicephalus (Boophilus) microplus is constantly challenged during its life cycle by microorganisms present in their hosts or in the environment. Tick eggs may be especially vulnerable to environmental conditions because they are exposed to a rich and diverse microflora in the soil. Despite being oviposited in such hostile sites, tick eggs remain viable, suggesting that the egg surface has defense mechanisms against opportunistic and/or pathogenic organisms. R. microplus engorged females deposit a superficial wax layer onto their eggs during oviposition. This egg wax is essential for preventing desiccation as well as acting as a barrier against attack by microorganisms. In this study, we report the detection of anti-biofilm activity of R. microplus egg wax against Pseudomonas aeruginosa PA14. Genes involved in the functions of production and maintenance of the biofilm extracellular matrix, pelA and cdrA, respectively, were markedly downregulated by a tick egg-wax extract. Moreover, this extract strongly inhibited fliC gene expression. Instead of a compact extracellular matrix, P. aeruginosa PA14 treated with egg-wax extract produces a fragile one. Also, the colony morphology of cells treated with egg-wax extract appears much paler and brownish, instead of the bright purple characteristic of normal colonies. Swarming motility was also inhibited by treatment with the egg-wax extract. The inhibition of P. aeruginosa biofilm does not seem to depend on inhibition of the quorum sensing system since mRNA levels of the 3 regulators of this system were not inhibited by egg-wax extract., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Background: Arginine-specific (RgpB and RgpA) and lysine-specific (Kgp) gingipains are secretory cysteine proteinases of Porphyromonas gingivalis that act as important virulence factors for the organism. They are translated as zymogens with both N- and C-terminal extensions, which are proteolytically cleaved during secretion. In this report, we describe and characterize inhibition of the gingipains by their N-terminal prodomains to maintain latency during their export through the cellular compartments., Methods: Recombinant forms of various prodomains (PD) were analyzed for their interaction with mature gingipains. The kinetics of their inhibition of proteolytic activity along with the formation of stable inhibitory complexes with native gingipains was studied by gel filtration, native PAGE and substrate hydrolysis., Results: PDRgpB and PDRgpA formed tight complexes with arginine-specific gingipains (Ki in the range from 6.2nM to 0.85nM). In contrast, PDKgp showed no inhibitory activity. A conserved Arg-102 residue in PDRgpB and PDRgpA was recognized as the P1 residue. Mutation of Arg-102 to Lys reduced inhibitory potency of PDRgpB by one order of magnitude while its substitutions with Ala, Gln or Gly totally abolished the PD inhibitory activity. Covalent modification of the catalytic cysteine with tosyl-l-Lys-chloromethylketone (TLCK) or H-D-Phe-Arg-chloromethylketone did not affect formation of the stable complex., Conclusion: Latency of arginine-specific progingipains is efficiently exerted by N-terminal prodomains thus protecting the periplasm from potentially damaging effect of prematurely activated gingipains., General Significance: Blocking progingipain activation may offer an attractive strategy to attenuate P. gingivalis pathogenicity., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The dominant role of biofilm-associated protein (Bap) in Staphylococcus aureus biofilm development prompted us to investigate Bap as a potential target for proteinase-mediated biofilm dispersion. Biofilm assay in microtitre plates showed that proteinase K hampered the early adhesion of cells as well as biofilm development. Proteinase K treatment of 24- and 48-h-old biofilms showed enhanced dispersion of bap-positive S. aureus biofilm; however, proteinase K did not affect the bap-negative S. aureus biofilm. When antibiotics were used in combination with proteinase K, significant enhancement in antibiotic action was noticed against bap-positive S. aureus biofilm. This study establishes that antibiotics in combination with proteinase K can be used for controlling S. aureus biofilms in whose development Bap surface protein has a major role. We propose that Bap protein could be a potential target for therapeutic control of S. aureus infections (for example, bovine mastitis).

Bacterial biofilms are involved in numerous infections of the human body, including dental caries. While conventional therapy of biofilm diseases aims at eradication and mechanical removal of the biofilms, recent therapeutic approaches target the mechanisms of biofilm formation and bacterial adhesion in particular. The effect of bovine milk osteopontin, a highly phosphorylated whey protein, on adhesion of Streptococcus mitis, Streptococcus sanguinis, and Actinomyces naeslundii, three prominent colonizers in dental biofilms, to saliva-coated surfaces was investigated. While adhesion of A. naeslundii was not affected by osteopontin, a strong, dose-dependent reduction in the number of adhering S. mitis was shown. No difference in bacterial adhesion was observed for caseinoglycomacropeptide, another phosphorylated milk protein. Osteopontin did not affect bacterial viability, but changed bacterial surface hydrophobicity, and may be suggested to prevent the adhesins of S. mitis from interacting with their salivary receptors. The antiadhesive effect of osteopontin may be useful for caries prevention.

Anti-adhesion therapies for bacterial infections offer an alternative to antibiotics, with those therapies bacteria are not killed but are prevented from causing harm to a host by inhibiting adherence to host cells and tissues, a prerequisite for the majority of infectious diseases. The mechanisms of these potential therapeutic agents include inhibition of adhesins and their host receptors, vaccination with adhesins or analogs, use of probiotics and dietary supplements that interfere with receptor-adhesin interactions, subminimal inhibitory concentrations of antibiotics and manipulation of hydrophobic interactions. Once developed, these drugs will contribute to the arsenal for fighting infectious disease in the future, potentially subverting antibiotic resistance.

The strong epidemiological association between cigarette smoke (CS) exposure and respiratory tract infections is conventionally attributed to immunosuppressive and irritant effects of CS on human cells. Since pathogenic bacteria such as Staphylococcus aureus are members of the normal microbiota and reside in close proximity to human nasopharyngeal cells, we hypothesized that bioactive components of CS might affect these organisms and potentiate their virulence. Using Staphylococcus aureus as a model organism, we observed that the presence of CS increased both biofilm formation and host cell adherence. Analysis of putative molecular pathways revealed that CS exposure decreased expression of the quorum-sensing agr system, which is involved in biofilm dispersal, and increased transcription of biofilm inducers such as sarA and rbf. CS contains bioactive compounds, including free radicals and reactive oxygen species, and we observed transcriptional induction of bacterial oxidoreductases, including superoxide dismutase, following exposure. Moreover, pretreatment of CS with an antioxidant abrogated CS-mediated enhancement of biofilms. Exposure of bacteria to hydrogen peroxide alone increased biofilm formation. These observations are consistent with the hypothesis that CS induces staphylococcal biofilm formation in an oxidant-dependent manner. CS treatment induced transcription of fnbA (encoding fibronectin binding protein A), leading to increased binding of CS-treated staphylococci to immobilized fibronectin and increased adherence to human cells. These observations indicate that the bioactive effects of CS may extend to the resident microbiota of the nasopharynx, with implications for the pathogenesis of respiratory infection in CS-exposed humans.

Background: Staphylococcus aureus is a well-armed pathogen prevalent in severe infections such as endocarditis and osteomyelitis. Fibronectin-binding proteins A and B, encoded by fnbA/B, are major pathogenesis determinants in these infections through their involvement in S. aureus adhesion to and invasion of host cells. Sub-minimum inhibitory concentrations (sub-MICs) of antibiotics, frequently occurring in vivo because of impaired drug diffusion at the infection site, can alter S. aureus phenotype. We therefore investigated their impact on S. aureus fibronectin-mediated adhesiveness and invasiveness., Methods: After in vitro challenge of S. aureus 8325-4 and clinical isolates with sub-MICs of major anti-staphylococcal agents, we explored fnbA/B transcription levels, bacterial adhesiveness to immobilised human fibronectin and human osteoblasts in culture, and bacterial invasion of human osteoblasts., Results: Oxacillin, moxifloxacin and linezolid led to the development of a hyper-adhesive phenotype in the fibronectin adhesion assay that was consistent with an increase in fnbA/B transcription. Conversely, rifampin treatment decreased fibronectin binding in all strains tested without affecting fnbA/B transcription. Gentamicin and vancomycin had no impact on fibronectin binding or fnbA/B transcription levels. Only oxacillin-treated S. aureus displayed a significantly increased adhesion to cultured osteoblasts, but its invasiveness did not differ from that of untreated controls., Conclusion: Our findings demonstrate that several antibiotics at sub-MICs modulate fibronectin binding in S. aureus in a drug-specific fashion. However, hyper- and hypo- adhesive phenotypes observed in controlled in vitro conditions were not fully confirmed in whole cell infection assays. The relevance of adhesion modulation during in vivo infections is thus still uncertain and requires further investigations.

Enteroaggregative Escherichia coli (EAEC) are bacterial pathogens that cause watery diarrhoea, which is often persistent and can be inflammatory. The antibiotic ciprofloxacin is used to treat EAEC infections, but a full understanding of the antimicrobial effects of ciprofloxacin is needed for more efficient treatment of bacterial infections. In this study, it was found that sub-minimum inhibitory concentrations (sub-MICs) of ciprofloxacin had an inhibitory effect on EAEC adhesion to glass and mammalian HEp-2 cells. It was also observed that bacterial surface properties play an important role in bacterial sensitivity to ciprofloxacin. In an EAEC mutant strain where the hydrophobic positively charged surface protein dispersin was absent, sensitivity to ciprofloxacin was reduced compared with the wild-type strain. Identified here are several antimicrobial effects of ciprofloxacin at sub-MIC concentrations indicating that bacterial surface hydrophobicity affects the response to ciprofloxacin. Investigating the effects of sub-MIC doses of antibiotics on targeted bacteria could help to further our understanding of bacterial pathogenicity and elucidate future antibiotic treatment modalities., (Copyright © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Scope: Besides flavan-3-ols, a family of N-phenylpropenoyl-L-amino acids (NPAs) has been recently identified as polyphenol/amino acid conjugates in the seeds of Theobroma cacao as well as in a variety of herbal drugs. NPAs were shown to exhibit antiadhesive activities against Helicobacter pylori., Methods and Results: For structure/activity relationship 24 homologous NPAs (2 mM) were investigated in a flow cytometric assay on potential antiadhesive effects against H. pylori adhesion to human gastric AGS cells. Dihydroxylation of the aromatic molecule part was shown to be necessary for activity; methoxylation decreases activity. High polarity of the amino acid is a prerequisite for activity. The model compound N-(E)-caffeoyl-L-glutamic acid 11 exerted a concentration-dependent inhibition of bacterial adhesion with saturation at 30% inhibition level. The antiadhesive effect was additionally confirmed by in situ adhesion assay on intact human gastric tissue. NPAs exhibited no cytotoxicity. Using immobilized ligands interaction 11 with bacterial adhesin BabA was demonstrated. RT-PCR indicated that the inhibition of BabA is not correlated with subsequent feed back regulations to express more adhesins or virulence factors (vacA, cagA, cagL, cagα, fucT, ureI, ureA, OMPs). The interaction of bacterial adhesins with the respective ligands does not automatically lead to a subsequent signal transduction towards induction of virulence processes., Conclusion: The nutritional use of NPA-containing food may justify a positive antiadhesive effect against the recurrence of H. pylori infections., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)