Antiadhesive Compounds Research Articles

Molecular epidemiology, antibiotic resistance profile and frequency of integron 1 and 2 in adherent-invasive Escherichia coli isolates of colorectal cancer patients

This study explores the prevalence of adherent-invasive Escherichia coli (AIEC) in colorectal cancer (CRC) patients and investigates the potential of effective intracellular antibiotics as a therapeutic strategy for CRC patients with AIEC infections. Considering the pivotal role of integrons in bacterial antibiotic resistance, the frequency of class 1 and 2 integrons in AIEC isolated from CRC patients, in one of the referenced 3 gastroenterology clinics in Isfahan, Iran was examined. AIEC strains were isolated from the colorectal biopsies and their antimicrobial sensitivity was assessed using the disc diffusion method. Polymerase chain reaction (PCR) was employed to detect intl1 and intl2. The multilocus sequence typing (MLST) method was utilized to type 10 selected isolates. Of the 150 samples, 24 were identified as AIEC, with the highest number isolated from CRC2 (33.4%) and CRC1 (29.16%), and the least from the FH group (8.3%) and control group (12.5%). int1 in 79.2% and int2 in 45.8% of AIEC strains were found and 41.6% of strains had both integrons. AIEC isolates with int1 exhibited the highest sensitivity to trimethoprim-sulfamethoxazole (57.9%), while those with int2 showed the highest sensitivity to ciprofloxacin (63.6%). A significant association between resistance to rifampin and integron 2 presence in AIEC isolates was observed. Furthermore, a significant correlation between integron 1 presence, invasion, survival, and replication within macrophages in AIEC strains was identified. MLST analysis revealed ST131 from CC131 with integron 1 as the most common sequence type (ST). The emergence of such strains in CRC populations poses a serious public health threat. The distribution pattern of STs varied among studied groups, with pandemic STs highlighting the importance of examining and treating patients infected with these isolates. Comprehensive prospective clinical investigations are warranted to assess the prognostic value of detecting this pathovar in CRC and to evaluate therapeutic techniques targeting drug-resistant AIECs, such as phage therapy, bacteriocins, and anti-adhesion compounds, for CRC prevention and treatment.

Awareness of Anti-adhesive Barriers Among Surgeons in Saudi Arabia.

Introduction Post-operative adhesions present a number of difficulties, including intestinal obstruction and infertility, and they frequently require readmission due to adhesion-related problems. Notwithstanding these ramifications, there are surprisingly few thorough national surveys that address surgeons' awareness of adhesives. By assessing Saudi surgeons' knowledge of post-operative adhesions and their use of anti-adhesive medications, this study aims to close this knowledge gap. Methods This study is a cross-sectional observational research study aimed at Saudi Arabian surgeons utilizing a self-administered, semi-structured online questionnaire. The questionnaire was distributed to participants via social media and in-person email using basic random selection. It included adhesion morbidity and prevalence, pre-operative informed consent issues, adhesion preventive viewpoints, and anti-adhesive chemical use. Results There were 111 participants in total, of 41% were experienced surgeons with more than five years of experience. According to the survey, the majority of surgeons occasionally employed anti-adhesive compounds, especially during laparotomies (28%), and38%never used them during laparoscopies. The study found that participants varied in what they informed patients regarding adhesion complications: 25% of participants informed 5%-10% of the patients about the possible adhesion complications in laparotomy procedures, whereas 26% of participants informed 10-25% of the patients in laparoscopic procedures. Compared with their specialist peers, general surgeons agreed more on the clinical significance of adhesions and prevention. Notably, three-quarters of participants were unclear about when to use anti-adhesive compounds. Conclusion While acknowledging the clinical significance of post-operative adhesions and recognizing the potential for prevention, most surveyed surgeons did not include adhesions as a post-operative complication in informed consent. The study underscores a belief in the efficacy of anti-adhesives yet reveals a widespread lack of clarity regarding specific indications for their use. Recommendations include implementing educational sessions during surgical training to heighten awareness of adhesions as a major post-operative complication and to encourage the appropriate utilization of available barriers and pharmacological anti-adhesive products.

High molecular/low acetylated chitosans reduce adhesion of Campylobacter jejuni to host cells by blocking JlpA

Infections caused by Campylobacter spp. are a major cause of severe enteritis worldwide. Multifactorial prevention strategies are necessary to reduce the prevalence of Campylobacter. In particular, antiadhesive strategies with specific inhibitors of early host–pathogen interaction are promising approaches to reduce the bacterial load. An in vitro flow cytometric adhesion assay was established to study the influence of carbohydrates on the adhesion of C. jejuni to Caco-2 cells. Chitosans with a high degree of polymerization and low degree of acetylation were identified as potent antiadhesive compounds, exerting significant reduction of C. jejuni adhesion to Caco-2 cells at non-toxic concentrations. Antiadhesive and also anti-invasive effects were verified by confocal laser scanning microscopy. For target identification, C. jejuni adhesins FlpA and JlpA were expressed in Escherichia coli ArcticExpress, and the influence of chitosan on binding to fibronectin and HSP90α, respectively, was investigated. While no effects on FlpA binding were found, a strong inhibition of JlpA-HSP90α binding was observed. To simulate real-life conditions, chicken meat was inoculated with C. jejuni, treated with antiadhesive chitosan, and the bacterial load was quantified. A strong reduction of C. jejuni load was observed. Atomic force microscopy revealed morphological changes of C. jejuni after 2 h of chitosan treatment, indicating disturbance of the cell wall and sacculi formation by electrostatic interaction of positively charged chitosan with the negatively charged cell surface. In conclusion, our data indicate promising antiadhesive and anti-invasive potential of high molecular weight, strongly de-acetylated chitosans for reducing C. jejuni load in livestock and food production.Key points• Antiadhesive effects of chitosan with high DP/low DA against C. jejuni to host cells• Specific targeting of JlpA/Hsp90α interaction by chitosan• Meat treatment with chitosan reduces C. jejuni load

Surface proteins of Shiga toxin-producing Escherichia coli mediate association with milk fat globules in raw milk.

By adhering to host cells and colonizing tissues, bacterial pathogens can successfully establish infection. Adhesion is considered the first step of the infection process and bacterial adhesion to anti-adhesive compounds is now seen as a promising strategy to prevent infectious diseases. Among the natural sources of anti-adhesive molecules, the membrane of milk fat globules (MFGs) is of interest because of its compositional diversity of proteins and glycoconjugates. However, few studies have focused on the bacterial molecules involved in MFG- mediated inhibition of bacterial adhesion to enterocytes. We used three pathogenic Shiga toxin-producing Escherichia coli (STEC) strains (O26:H11 str. 21765, O157:H7 str. EDL933, and O103:H3 str. PMK5) as models to evaluate whether STEC surface proteins are involved in the affinity of STEC for MFG membrane proteins (MFGMPs). The affinity of STEC for MFGMPs was assessed both indirectly by a natural raw milk creaming test and directly by an adhesion test. Mass spectrometry was used to identify enriched STEC proteins within the protein fraction of MFGMs. Bacterial mutants were constructed and their affinity to MFGs were measured to confirm the role of the identified proteins. We found that free STEC surface proteins inhibit the concentration of the pathogen in the MFG-enriched cream in a strain-dependent manner. Moreover, the OmpA and FliC proteins were identified within the protein fraction of MFGMs. Our results suggest that FliC protein participates in STEC adhesion to MFGMPs but other STEC molecules may also participate. For the first time, this study highlighted, the involvement of STEC surface proteins in the affinity for MFGs. The mechanism of STEC-MFG association is still not fully understood but our results confirm the existence of receptor/ligand type interactions between the bacteria and MFGs. Further studies are needed to identify and specify the molecules involved in this interaction. These studies should consider the likely involvement of several factors, including adhesion molecules, and the diversity of each STEC strain.

Design of an electrospun tubular construct combining a mechanical and biological approach to improve tendon repair

Hand tendon injuries represent a major clinical problem and might dramatically diminish a patient’s life quality. In this study, a targeted solution for flexor tendon repair was developed by combining a mechanical and biological approach. To this end, a novel acrylate-endcapped urethane-based polymer (AUP) was synthesized and its physico-chemical properties were characterized. Next, tubular repair constructs were developed using electrospinning of the AUP material with incorporated naproxen and hyaluronic acid (i.e. anti-inflammatory and anti-adhesion compounds, respectively), and with a tubular braid as mechanical reinforcement. Tensile testing of the repair constructs using ex vivo sheep tendons showed that the developed repair constructs fulfilled the required mechanical properties for tendon repair (i.e. minimal ultimate stress of 4 MPa), with an ultimate stress of 6.4 ± 0.6 MPa. Moreover, in vitro biological assays showed that the developed repair tubes and the incorporated bioactive components were non-cytotoxic. In addition, when equine tenocytes and mesenchymal stem cells were co-cultured with the repair tubes, an increased production of collagen and non-collagenous proteins was observed. In conclusion, this novel construct in which a mechanical approach (fulfilling the required mechanical properties) was combined with a biological approach (incorporation of bioactive compounds), shows potential as flexor tendon repair application.Graphical abstract

Neutron crystallography reveals mechanisms used by Pseudomonas aeruginosa for host-cell binding

The opportunistic pathogen Pseudomonas aeruginosa, a major cause of nosocomial infections, uses carbohydrate-binding proteins (lectins) as part of its binding to host cells. The fucose-binding lectin, LecB, displays a unique carbohydrate-binding site that incorporates two closely located calcium ions bridging between the ligand and protein, providing specificity and unusually high affinity. Here, we investigate the mechanisms involved in binding based on neutron crystallography studies of a fully deuterated LecB/fucose/calcium complex. The neutron structure, which includes the positions of all the hydrogen atoms, reveals that the high affinity of binding may be related to the occurrence of a low-barrier hydrogen bond induced by the proximity of the two calcium ions, the presence of coordination rings between the sugar, calcium and LecB, and the dynamic behaviour of bridging water molecules at room temperature. These key structural details may assist in the design of anti-adhesive compounds to combat multi-resistance bacterial infections.

Etarska ulja kao antimikrobna i antiadhezivna sredstva protiv bakterija Salmonella Typhimurium i Staphylococcus aureus, i kvasaca Candida albicans i Saccharomyces cerevisiae

A serious global problem with the increasing resistance of microorganisms to currently used antimicrobials has opened up the promotional research in the identification of new, more effective drugs with a broad spectrum of antimicrobial activities. Plant essential oils, due to the large biological and structural diversity of their components, are known to have many potential benefits. This study aimed to evaluate the antimicrobial and anti-adhesion activity of fifteen essential oils and their compounds against two bacterial and two yeast species responsible for food spoilage and infectious diseases. Antimicrobial activity was determined by testing the minimum inhibitory concentration (MIC), the minimum bactericidal concentration (MBC) and the minimum fungicidal concentration (MFC) of essential oils and compounds. The essential oils of Cinnamomum zeylanicum and Eugenia caryophyllus showed the highest antimicrobial activity with MICs ranging from 0.078 to 1.25 mg/mL, and 0.039 to 1.25 mg/mL, respectively. On the other hand, essential oils of Eucalypti aetheroleum and Salvia officinalis had significantly weaker antimicrobial properties than the others. Further, MICs were used to assess the inhibition of adhesion of bacteria Salmonella Typhimurium ATCC 25923 and Staphylococcus aureus ATCC 14208, and yeasts Candida albicans ATCC 10231 and Saccharomyces cerevisiae ATCC 9763 in a microtiter plate using the crystal violet staining method. Based on the percentage of adhesion inhibition, yeast S. cerevisiae ATCC 9763 showed a high level of antimicrobial resistance. E. caryophyllus had the strongest effect with inhibition up to 73%. Consistent with the antimicrobial susceptibility results, the most active anti-adhesion compounds were carvacrol and thymol. Considering the role of biofilm in food spoilage and clinical diseases, inhibition of the initial phase of biofilm formation by natural antimicrobial agents may be an alternative to commonly used synthetic ones.

Etarska ulja kao antimikrobna i antiadhezivna sredstva protiv bakterija Salmonella Typhimurium i Staphylococcus aureus, i kvasaca Candida albicans i Saccharomyces cerevisiae

A serious global problem with the increasing resistance of microorganisms to currently used antimicrobials has opened up the promotional research in the identification of new, more effective drugs with a broad spectrum of antimicrobial activities. Plant essential oils, due to the large biological and structural diversity of their components, are known to have many potential benefits. This study aimed to evaluate the antimicrobial and anti-adhesion activity of fifteen essential oils and their compounds against two bacterial and two yeast species responsible for food spoilage and infectious diseases. Antimicrobial activity was determined by testing the minimum inhibitory concentration (MIC), the minimum bactericidal concentration (MBC) and the minimum fungicidal concentration (MFC) of essential oils and compounds. The essential oils of Cinnamomum zeylanicum and Eugenia caryophyllus showed the highest antimicrobial activity with MICs ranging from 0.078 to 1.25 mg/mL, and 0.039 to 1.25 mg/mL, respectively. On the other hand, essential oils of Eucalypti aetheroleum and Salvia officinalis had significantly weaker antimicrobial properties than the others. Further, MICs were used to assess the inhibition of adhesion of bacteria Salmonella Typhimurium ATCC 25923 and Staphylococcus aureus ATCC 14208, and yeasts Candida albicans ATCC 10231 and Saccharomyces cerevisiae ATCC 9763 in a microtiter plate using the crystal violet staining method. Based on the percentage of adhesion inhibition, yeast S. cerevisiae ATCC 9763 showed a high level of antimicrobial resistance. E. caryophyllus had the strongest effect with inhibition up to 73%. Consistent with the antimicrobial susceptibility results, the most active anti-adhesion compounds were carvacrol and thymol. Considering the role of biofilm in food spoilage and clinical diseases, inhibition of the initial phase of biofilm formation by natural antimicrobial agents may be an alternative to commonly used synthetic ones.

Structural characterization, antioxidant, and antibiofilm activities of Coffea canephora green seeds.

In order to explore Coffea canephora green seeds as natural extract for application in the functional-food industry, we focused this study to the evaluation of the antioxidant and the antiadhesion effect of C. canephora green seeds extracts. The analysis of C. canephora green seeds extracts was carried out by RP-HPLC-PDA. These extracts were screened for antioxidant activities by ABTS and phenanthroline assays. The antibacterial activity was determined by microdilution method against three reference bacteria. The inhibition of bacterial adhesion at 1/8 MIC was carried out against three reference bacteria. The RP-HPLC-PDA revealed the presence of gallic acid, vanillin, quercetin, chlorogenic acid, and P-coumaric acid. The n-buatnol extract have the highest activity ABTS assays (3.96±0.08μg/mL). For this extract, the A0.5 was 1.90±0.05μg/mL for phenanthroline assay. The n-butanol extract and the methanolic extract have the higher antibacterial activity against Staphylococcus aureus ATCC 25923 (40µg/mL).At MIC/8, the extracts of C. canephora showed 70% higher antidhesive activity against S. aureus ATCC 25923. Our finding provides an effective and specific new approach to the search of antioxidant and antiadhesive compounds for different uses.

Antiadhesive natural products against uropathogenic E. coli: What can we learn from cranberry extract?

Ethnopharmacological relevanceExtracts from Cranberry fruits (Vaccinium macrocarpon) are traditionally used against urinary tract infections, mainly due to antiadhesive activity against uropathogenic E. coli (UPEC), but the exact mode of action and compounds, responsible for the activity, are unknown. Aim of the studyi. To investigate if cranberry extract acts only by a single component or must be assessed as a multi-active-compound preparation; ii to screen isolated cranberry-related natural products under in vitro conditions to pinpoint natural products with antiadhesive effects against UPEC, followed by in silico calculations (QSAR) to predict potential antiadhesive compounds; iii. investigations by using urine samples from cranberry treated volunteers for evaluation on the bacterial transcriptome and the mannose-binding side of FimH, iv. to investigate if besides Tamm Horsfall Protein induction in the kidney, the extract acts also directly against UPEC. Material and methodsAntiadhesive activity of 105 compounds was determined by flow cytometric adhesion assay (UPEC UTI89 on T24 bladder cells). Urine samples from 16 volunteers treated with cranberry extract (p.o., 7 days, 900 mg/day) were used for ex vivo testing concerning influence on the bacterial transcriptome (Illumina RNA-seq) and interaction with the mannose binding domain of type-1 fimbriae. Resultsi. The antiadhesive effect of cranberry extract cannot be attributed to a single compound or to a single fraction. ii. Unglycosylated flavones and flavonols with bulky substitution of the B ring contribute to the antiadhesive activity. 3′-8″-biflavones and flavolignans (not related to cranberry fruits) were identified as potent antiadhesive compounds against UPEC. iii. QSAR yielded a model with good statistical performance and sufficient internal and external predictive ability. iv. Urine samples from male cranberry-treated volunteers indicated significant interaction with the mannose binding domain of type-1 fimbriae, which correlated with the amount of Tamm-Horsfall Protein in the test samples. v Cranberry extract did not influence the UPEC transcriptome; gene expression of bacterial adhesins (P-, S-fimbrae, curli) was not influenced by the urine samples, while a slight, but non-significant upregulation of type 1 fimbriae was observed. ConclusionsB-ring substituted flavones and flavonols from cranberry contribute to the antiadhesive activity against UPEC by inhibition of the FimH-mediated interaction with the host cell bladder epithelium.

Marine Microalgae Biomolecules and Their Adhesion Capacity to Salmonella enterica sv. Typhimurium

Different molecules have been tested as analog receptors due to their capacity to bind bacteria and prevent cell adhesion. By using in vitro assays, the present study characterized the aqueous and alkaline extracts from microalgae Pavlova lutheri and Pavlova gyrans and evaluated the capacity of these extracts to adhere to enterobacteria (Salmonella Typhimurium). The aqueous and alkaline extracts of both species were fractionated via freeze-thawing, giving rise to soluble and insoluble (precipitate) fractions in cold water. The obtained fractions were studied using thermogravimetric, methylation analyses, and using 1D and 2D NMR techniques. The cold-water-soluble fractions obtained from the aqueous extracts were mainly composed of highly branched (1→3),(1→6)-β-glucans, whereas the cold-water-precipitate fractions were constituted by (1→3)-β-glucans. The alkaline extract fractions showed similar compositions with a high protein content, and the presence of glycosides (sulfoquinovosylglycerol (SQG), digalactosylglycerol (DGG)), and free fatty acids. The linear (1→3)-β-glucans and the alkaline extract fractions showed an adhesion capacity toward Salmonella. The chemical composition of the active fractions suggested that the presence of three-linked β-glucose units, as well as microalgal proteins and glycosides, could be important in the adhesion process. Therefore, these microalgal species possess a high potential to serve as a source of anti-adhesive compounds.

BabA and LPS inhibitors against Helicobacter pylori: pectins and pectin-like rhamnogalacturonans as adhesion blockers.

The first step in the development of Helicobacter pylori pathogenicity is receptor-mediated adhesion to gastric epithelium. Adhesins of H. pylori not only enable colonisation of the epithelium, with BabA interacting with Lewisb, but also interaction of lipopolysaccharide (LPS) with galectin-3 contributes to attachment of H. pylori to the host cells. Anti-adhesive compounds against H. pylori have been described, but specific analytical assays for pinpointing the interaction with BabA are limited. LPS-galectin-3 inhibitors have not been described until now. A sandwich ELISA with recombinant BabA547-6K was developed to investigate the interaction of BabA with Lewisb-HSA. Isothermal titration calorimetry gave thermodynamic information on the interaction between BabA, Lewisb-HSA and anti-adhesive compounds. A highly esterified rhamnogalacturonan from Abelmoschus esculentus inhibited the adhesion of H. pylori to adherent gastric adenocarcinoma (AGS) cells (IC50 550 μg/mL) and interacted with BabA (IC50 17 μg/mL). Pectins with similar rhamnogalacturonan structure showed weak anti-adhesive activity. Highly branched rhamnogalacturonans with low uronic acid content and high degree of esterification are potent BabA inhibitors. BabA represents a promising target for the development of anti-adhesive drugs against H. pylori. The rhamnogalacturonan influenced also the binding affinity of H. pylori to recombinant galectin-3 in a concentration-dependent manner with an IC50 of 222 μg/mL. Similar effects were obtained with pectin from apple fruits, while pectins from other sources were inactive.

Real-time interaction analysis of Shiga toxins and membrane microdomains of primary human brain microvascular endothelial cells.

Infections of the human intestinal tract with enterohemorrhagic Escherichia coli (EHEC) result in massive extraintestinal complications due to translocation of EHEC-released Shiga toxins (Stxs) from the gut into the circulation. Stx-mediated damage of the cerebral microvasculature raises serious brain dysfunction being the most frequent cause of acute mortality in patients suffering from severe EHEC infections. Stx2a and Stx2e are associated with heavy and mild course of infection, respectively. Stx2a preferentially binds to globotriaosylceramide (Gb3Cer, Galα1-4Galβ1-4Glcβ1-1Cer), while Stx2e prefers globotetraosylceramide (Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1Cer). Both glycosphingolipids (GSLs) were detected in detergent-resistant membranes (DRMs) of primary human brain microvascular endothelial cells (pHBMECs) resembling microdomains of the plasma membrane. In this study, we show that Gb3Cer and Gb4Cer of pHBMECs with saturated C16:0, C22:0, and C24:0 fatty acids dominated in DRMs, corresponding to the liquid-ordered membrane phase, whereas lipoforms carrying unsaturated C24:1 and C24:2 fatty acids prevailed in the non-DRM fractions, which correspond to the liquid-disordered membrane phase. Similarly, a shift of the phospholipids from saturated lipoforms in the DRM to unsaturated species in the non-DRM fractions was observed. Real-time biomolecular interaction analysis using affinity-purified Stx2a and Stx2e, recorded with a surface acoustic wave (SAW) biosensor, evidenced high binding strength of both toxins toward DRMs and failure in interaction with non-DRMs. These results support the hypothesis of preferential binding of Stxs toward microdomains harboring GSL receptors carrying saturated fatty acids in their lipid anchors. Collectively, unraveling the precise mechanisms of Stx-microdomain interaction may help to develop antiadhesive compounds to combat Stx-mediated cellular injury.

Polymethoxylated flavones from Orthosiphon stamineus leaves as antiadhesive compounds against uropathogenic E. coli

Aqueous and acetone extracts of O. stamineus leaves reduce the adhesion of uropathogenic E. coli (UPEC, strain UTI89) to T24 bladder cells significantly (IC25 ~ 524 mg/mL, resp. 40 μg/mL). The acteonic extract had no cytotoxic effects against UPEC in concentrations that inhibited the bacterial adhesion. The extract significantly reduced the gene expression of fimH, fimC, fimD, csgA and focG, which are strongly involved in the formation of bacterial adhesins. The antiadhesive effect was due to the presence of polymethoxylated flavones, enriched in the acetonic extract. Five flavones have been isolated by fast centrifugal partition chromatography, followed by preparative HPLC. Eupatorin, ladanein, salvigenin, sinensetin, 5,6,7,4′-tetramethoxyflavone and 5-hydroxy-6,7,3′,4′-tetramethoxyflavone were identified as the main polymethoxylated flavones. With the exception of eupatorin, all of these flavones reduced the bacterial adhesion in a concentration depending manner, indicating that B-ring hydroxylation and methoxylation seems to have a major impact on the antiadhesive activity. In addition, this was confirmed by investigation of the flavones chrysoeriol and diosmetin, which had only very weak antiadhesive activity. From these data, Orthosiphon extracts can be assessed to have a pronounced antiadhesive activity against UPEC, based on a variety of polymethoxylated flavones.

Antiadhesive phthalides from Apium graveolens fruits against uropathogenic E. coli

Ethnopharmacological relevanceFruits of Apium graveolens (celery) are used traditionally in Persian and European medicine for the treatment of uncomplicated urinary tract infections.Aim of the study: The study aimed at identifying potential antiadhesive compounds from celery extracts to provide strategies for improved standardization of the herbal material. Materials and methodsDecoction, hydroalcoholic and acetone extracts were prepared from celery fruits. Bioassay-guided fractionation was performed by Fast Centrifugal Partition Chromatography and preparative HPLC, followed by LC-MS and NMR investigations for structure elucidation. The antiadhesive activity of extracts, fractions and purified compounds was assessed by flow cytometry, evaluating the adhesion of fluorescent-labelled uropathogenic bacteria (UPEC NU14) to T24 bladder cells; mannose served as positive control. Influence of the extract on gene expression of selected adhesins and fitness genes was monitored by qPCR. ResultsConcentration-dependent antiadhesive activity was found for the hydroalcoholic and even more for the acetone extract AE (IC50 85 μg/mL) from celery fruits. Bioassay-guided fractionation revealed the presence of the phthalides senkyunolide (1, inactive) and sedanenolide (2, IC50 790 μM). 2 is assessed as the main antiadhesive compound, which accounts for 4.0% in the water extract, for 18% in the hydroethanolic extract and for 71% in AE. Additionally a similar phthalide, Z-ligustilide (5), was shown to exert an IC50 of 611 μM. Furthermore, AE caused a significant upregulation of fimH and sfaG in free floating, non-attached UPEC and significantly down-regulated these genes in adherent bacteria. ConclusionsPhthalides were identified as the main active compounds in polar and semi-polar extracts, which exert strong antiadhesive activity against uropathogenic E. coli. The current findings support the traditional use in phytotherapy for urinary tract infections and provide a base for standardization of the herbal material.

Development of an in vitro system to study oral biofilms in real time through impedance technology: validation and potential applications

ABSTRACTBackground and objectives: We have developed a standardized, easy-to-use in vitro model to study single- and multiple-species oral biofilms in real time through impedance technology, which elucidates the kinetics of biofilm formation in 96-well plates, without the requirement for any further manipulation.Design and Results: Using this system, biofilms of Streptococcus mutans appear to be sugar-dependent and highly resistant to amoxicilin, an antibiotic to which this oral pathogen is highly sensitive in a planktonic state. Saliva, tongue and dental plaque samples were also used as inocula to form multiple-species biofilms. DNA isolation and Illumina sequencing of the biofilms showed that the multi-species biofilms were formed by tens or hundreds of species, had a similar composition to the original inoculum, and included fastidious microorganisms which are important for oral health and disease. As an example of the potential applications of the model, we show that oral biofilms can be inhibited by amoxicilin, but in some cases they are induced by the antibiotic, suggesting the existence of responders and non-responders to a given antibiotic.Conclusions: We therefore propose the system as a valid in vitro model to study oral biofilm dynamics, including their susceptibility to antibiotics, antiseptics or anti-adhesive compounds.

Red pepper Capsicum baccatum: source of antiadhesive and antibiofilm compounds against nosocomial bacteria

Currently, there is a great interest in the search for new natural bioactive compounds against bacterial biofilms, ubiquitous in natural, clinical and industrial environments, and responsible for the high resistance of bacteria to antimicrobial agents. Several species of Capsicum red peppers are used worldwide in a number of preparations to treat a variety of illnesses, including infections. The aim of this study was to investigate the antiadhesive and antibiofilm activity of Capsicum baccatum, after sequential extraction, against important bacteria. Antimicrobial and antibiofilm activities against Staphylococcus epidermidis and Pseudomonas aeruginosa were performed by the OD600 absorbance and crystal violet method, respectively, and using scanning electron microscopy (SEM). The most active extract of C. baccatum was incorporated in Permanox™ slides by spin-coated technique in order to produce an anti-infective surface. In addition, an in vivo toxicological evaluation was performed using the alternative host model of Galleria mellonella larvae. The residual aqueous extract from C. baccatum seeds (RAqS) was the most active extract, able to inhibit up to 80% and 60% of the S. epidermidis and P. aeruginosa biofilm, respectively, without inhibiting the planktonic bacterial growth, neither promoting acute toxicity in G. mellonella. The surface coating with the RAqS modified the highly hydrophobic feature of Permanox™ slides and prevented the bacterial adhesion and biofilm development. SEM analysis showed the presence of only small clusters or individual cells without the presence of matrix on the RAqS treated samples, even when RAqS was in solution or as coating. The phytochemical evaluation of RAqS indicated the main presence of tannins and amino acids/proteins. The study presented herein demonstrated, for the first time, the ability of C. baccatum to prevent bacterial adhesion and biofilm formation of two significant pathogens. This property of C. baccatum evidences its potential as a promising source of natural compounds for the development of novel strategies, targeting anti-virulence factors, for combating antibiotic-resistant bacteria, and controlling clinical and industrial problems related to biofilms.

Direct Evaluation of Live Uropathogenic Escherichia coli Adhesion and Efficiency of Antiadhesive Compounds Using a Simple Microarray Approach

Many pathogens use host glycans as docking points for adhesion. Therefore, the use of compounds blocking carbohydrate-binding adhesins is a promising strategy for fighting infections. In this work, we describe a simple and rapid microarray approach for assessing the bacterial adhesion and efficiency of antiadhesive compounds targeting uropathogenic Escherichia coli UTI89, which displays mannose-specific adhesin FimH at the tip of fimbriae. The approach consisted in direct detection of live fluorescently labeled bacteria bound to mannan printed onto microarray slides. The utility of the arrays for binding/inhibition assays was first validated by comparing array-derived results for the model mannose-binding lectin concanavalin A with data obtained by isothermal titration calorimetry. Growth phase-dependent binding of UTI89 to the arrays was observed, proving the usefulness of the setup for detecting differences in FimH expression. Importantly, bacteria labeling and binding assays entailed minimal manipulation, helping to preserve the integrity of fimbriae. The efficiency of three different dodecamannosylated fullerenes as FimH-targeted antiadhesives was next evaluated in competition assays. The results revealed a superior activity of the mannofullerenes (5- to 18-fold per mannose residue) over methyl α-d-mannopyranoside. Moreover, differences in activity were detected for mannofullerenes differing in the structure/length of the spacer used for grafting mannose onto the fullerene core, further demonstrating the sensitivity of the assay. Overall, the approach combines straightforward and time-saving protocols for microarray preparation, bacteria labeling, and binding assays, and it can be easily tailored to other bacteria bearing carbohydrate-binding adhesins.

Do raspberry extracts and fractions have antifungal or anti-adherent potential against Candida spp.?

Candida spp., especially Candida albicans, is one of the main colonisers of the oral cavity. Due to its ability to form biofilms, it can be implicated in dental caries, periodontal disease and denture stomatitis. Microbial cells in biofilms are minimally impacted by conventional drugs. The aim of this study was to find new substances able to inhibit the adhesion of Candida spp. in order to prevent biofilm formation in the oral cavity. This study focused on the red raspberry (Rubus idaeus) fruit, known for its richness in potentially antimicrobial tannins. Extraction with a polarity gradient was performed on acetone extracts from frozen ripe and unripe fruits, resulting in eight extracts. The antifungal and anti-adhesion effects of the extracts were determined using broth microdilution and XTT methods, respectively, against C. albicans, Candida glabrata and Candida parapsilosis strains. Interestingly, four extracts (hexane and ethyl acetate) displayed anti-adhesion activity against C. albicans at low concentrations [50% inhibitory concentration (IC50) 15.6-62.5 µg/mL]. Bioassay-guided fractionation by chromatographic methods of the most active extract obtained from ripe fruit (ethyl acetate extract) led to two subfractions enriched in anti-adhesion compounds, identified by mass spectrometry analysis as hydrolysable and condensed tannins. Their activities were dose-dependent with maximum inhibition at 80% (IC50 = 25 µg/mL and 12.5 µg/mL). Regarding antifungal activity, no extract was active against planktonic cells of the tested strains. This work highlights for the first time the potential of raspberries to prevent oral C. albicans biofilms.

Bacterial Sexuality at the Nanoscale.

Understanding the basic mechanisms of bacterial sexuality is an important topic in current microbiology and biotechnology. While classical methods used to study gene transfer provide information on whole cell populations, nanotechnologies offer new opportunities for analyzing the behavior of individual mating partners. We introduce an innovative atomic force microscopy (AFM) platform to study and mechanically control DNA transfer between single bacteria, focusing on the large conjugative pXO16 plasmid of the Gram-positive bacterium Bacillus thuringiensis. We demonstrate that the adhesion forces between single donor and recipient cells are very strong (∼2 nN). Using a mutant plasmid, we find that these high forces are mediated by a pXO16 aggregation locus that contains two large surface protein genes. Notably, we also show that AFM can be used to mechanically induce plasmid transfer between single partners, revealing that transfer is very fast (<15 min) and triggers major cell surface changes in transconjugant cells. We anticipate that the single-cell technology developed here will enable researchers to mechanically control gene transfer among a wide range of Gram-positive and Gram-negative bacterial species and to understand the molecular forces involved. Also, the method could be useful in nanomedicine for the design of antiadhesion compounds capable of preventing intimate cell-cell contacts, therefore providing a means to control the resistance and virulence of bacterial pathogens.