In vitro analysis on the adhesion potential of Lactiplantibacillus plantarum from infant faeces and its gastrointestinal localization, growth promotion, and immunomodulation in Wistar rats: a preliminary study.

Lactic acid bacteria, found in heterogenous niches, are known for their health-endorsing properties and are in demand as prospective probiotics. Hence, the scientific community around the globe is in continuous search for novel and new potential strains with extensive applicability and minimum risk. In this context, the present study evaluated the efficiency of Lactiplantibacillus plantarum (P2F2) of human origin, a highly autoaggregating and coaggregating (with pathogens) strain, for its colonization, growth promotion, and immunomodulation. Results indicated moderate hydrophobicity on adhesion to xylene and n-hexadecane and weak electron-donating properties with chloroform. The biofilm of P2F2 formed on polystyrene was strong and highly correlated to exopolysaccharide production. The autoaggregation was moderately correlated with hydrophobicity and biofilm production. It was noted that the P2F2 strain modulated the gut microbiota and increased intestinal villi length in Wistar rats. The lipid and glucose profiles remained intact. P2F2 treatment increased the activity of reactive oxygen species-generating cells in the peritoneal cavity, besides augmenting the mitogen-induced splenocyte proliferation and maintained the immunoglobulins at the normal level. Results from this study conclusively suggest that the strain P2F2 adheres to the intestine and modulates the gut ecosystem besides enhancing cell-mediated immunity without altering the serological parameters tested.

Subversion of a family of antimicrobial proteins by Salmonella enterica.

Salmonella enterica is a food-borne pathogen able to cause a wide spectrum of diseases ranging from mild gastroenteritis to systemic infections. During almost all stages of the infection process Salmonella is likely to be exposed to a wide variety of host-derived antimicrobial peptides (AMPs). AMPs are important components of the innate immune response which integrate within the bacterial membrane, thus forming pores which lead ultimately to bacterial killing. In contrast to other AMPs Bactericidal/Permeability-increasing Protein (BPI) displayed only weak bacteriostatic or bactericidal effects towards Salmonella enterica sv. Typhimurium (STM) cultures. Surprisingly, we found that sub-antimicrobial concentrations of BPI fold-containing (BPIF) superfamily members mediated adhesion of STM depending on pre-formed type 1 fimbriae. BPIF proteins directly bind to type 1 fimbriae through mannose-containing oligosaccharide modifications. Fimbriae decorated with BPIF proteins exhibit extended binding specificity, allowing for bacterial adhesion on a greater variety of abiotic and biotic surfaces likely promoting host colonization. Further, fimbriae significantly contributed to the resistance against BPI, probably through sequestration of the AMP before membrane interaction. In conclusion, functional subversion of innate immune proteins of the BPIF family through binding to fimbriae promotes Salmonella virulence by survival of host defense and promotion of host colonization.

Sweet Janus Particles: Multifunctional Inhibitors of Carbohydrate-Based Bacterial Adhesion.

Escherichia coli and other bacteria use adhesion receptors, such as FimH, to attach to carbohydrates on the cell surface as the first step of colonization and infection. Efficient inhibitors that block these interactions for infection treatment are multivalent carbohydrate-functionalized scaffolds. However, these multivalent systems often lead to the formation of large clusters of bacteria, which may pose problems for clearing bacteria from the infected site. Here, we present Man-containing Janus particles (JPs) decorated on one side with glycomacromolecules to target Man-specific adhesion receptors of E. coli. On the other side, poly(N-isopropylacrylamide) is attached to the particle hemisphere, providing temperature-dependent sterical shielding against binding and cluster formation. While homogeneously functionalized particles cluster with multiple bacteria to form large aggregates, glycofunctionalized JPs are able to form aggregates only with individual bacteria. The formation of large aggregates from the JP-decorated single bacteria can still be induced in a second step by increasing the temperature and making use of the collapse of the PNIPAM hemisphere. This is the first time that carbohydrate-functionalized JPs have been derived and used as inhibitors of bacterial adhesion. Furthermore, the developed JPs offer well-controlled single bacterial inhibition in combination with cluster formation upon an external stimulus, which is not achievable with conventional carbohydrate-functionalized particles.

Ovomucin Hydrolysates Reduce Bacterial Adhesion and Inflammation in Enterotoxigenic Escherichia coli (ETEC) K88-Challenged Intestinal Epithelial Cells.

Enterotoxigenic Escherichia coli (ETEC) K88 is the most common cause of diarrhea in neonatal and postweaning pigs. After adhering to small intestinal epithelial cells via glycoprotein receptor recognition, the pathogen can produce enterotoxins, impair intestinal integrity, trigger watery diarrhea, and induce inflammation via nuclear factor κB (NF-κB) and mitogen-activated protein kinase phosphatase (MAPK) pathways. Inhibiting ETEC K88 adhesion to cell surfaces by interfering with the receptor-fimbriae recognition provides a promising strategy to prevent the initiation and progression of infection. Ovomucin is a highly glycosylated protein in chicken egg white with diverse bioactivities. Ovomucin hydrolysates prepared by the enzymes Protex 26L (OP) and pepsin/pancreatin (OPP) were previously revealed to prevent adhesion of ETEC K88 to IPEC-J2 cells. Herein, we investigated the protective effects of ovomucin hydrolysates on ETEC K88-induced barrier integrity damage and inflammation in IPEC-J2 and Caco-2 cells. Both hydrolysates inhibited ETEC K88 adhesion to cells and protected epithelial cell integrity by restoring transepithelial electronic resistance (TEER) values. Removing sialic acids in the hydrolysates reduced their antiadhesive capacities. Ovomucin hydrolysates suppressed ETEC-induced activation of NF-κB and MAPK signaling pathways in both cell lines. The ability of ETEC K88 in activating calcium/calmodulin-dependent protein kinase 2 (CaMK II), elevating intracellular Ca2+ concentration, and inducing oxidative stress was attenuated by both hydrolysates. In conclusion, this study demonstrated the potential of ovomucin hydrolysates to prevent ETEC K88 adhesion and alleviate inflammation and oxidative stress in intestinal epithelial cells.

Nanotopography and oral bacterial adhesion on titanium surfaces: in vitro and in vivo studies.

The present study aimed to evaluate the influence of titanium surface nanotopography on the initial bacterial adhesion process by in vivo and in vitro study models. Titanium disks were produced and characterized according to their surface topography: machined (Ti-M), microtopography (Ti-Micro), and nanotopography (Ti-Nano). For the in vivo study, 18 subjects wore oral acrylic splints containing 2 disks from each group for 24 h (n = 36). After this period, the disks were removed from the splints and evaluated by microbial culture method, scanning electron microscopy (SEM), and qPCR for quantification of Streptococcus oralis, Actinomyces naeslundii, Fusobacterium nucleatum, as well as total bacteria. For the in vitro study, adhesion tests were performed with the species S. oralis and A. naeslundii for 24 h. Data were compared by ANOVA, with Tukey's post-test. Regarding the in vivo study, both the total aerobic and total anaerobic bacteria counts were similar among groups (p > 0.05). In qPCR, there was no difference among groups of bacteria adhered to the disks (p > 0.05), except for A. naeslundii, which was found in lower proportions in the Ti-Nano group (p < 0.05). In the SEM analysis, the groups had a similar bacterial distribution, with a predominance of cocci and few bacilli. In the in vitro study, there was no difference in the adhesion profile for S. oralis and A. naeslundii after 24 h of biofilm formation (p > 0.05). Thus, we conclude that micro- and nanotopography do not affect bacterial adhesion, considering an initial period of biofilm formation.

Antimicrobial Peptide Assembly on Zwitterionic Polymer Films to Slow Down Biofilm Formation.

Formation of biofilms on equipment used in various fields, such as medicine, domestic sanitation, and marine transportation, can cause serious problems. The use of antibiofouling and bactericidal modifications is a promising strategy for inhibiting bacterial adhesion and biofilm formation. To further enhance the antibiofilm properties of a surface, various combinations of bactericidal modifications alongside antibiofouling modifications have been developed. Optimization of the arrangements of antimicrobial peptides on the antibiofouling surface would allow us to design longer-life antibiofilm surface modifications. In this study, a postmodification was conducted with different design using the antimicrobial peptide KR12 on an antibiofouling copolymer film consisting of 2-methacryloyloxyethyl phosphorylcholine, 3-methacryloxypropyl trimethoxysilane, and 3-(methacryloyloxy) propyl-tris(trimethylsilyloxy) silane. The distance of KR12 from the film was adjusted by combining different lengths of poly(ethylene glycol) (PEG) spacers (molecular weights are 2000 and 5000). The density of KR12 was ranged from 0.06 to 0.22 nm-2. When these modified surfaces were exposed to a nutrient-rich TSB suspension, the bacterial area formed by E. coli covered 5-127% of the original copolymer film. We found that a significant distance between the bactericidal and antibiofouling modifications, along with a higher density of bactericidal modifications, slows down the biofilm formation.

Adhesion Inhibition Assay for Helicobacter pylori to Mucin by Lactobacillus.

The ability of Lactobacillus to adhere to mucin is a parameter for evaluating the effectiveness of probiotics. In particular, a competitive inhibition assay of pathogenic bacteria using mucin-adherent lactobacilli is useful for identifying Lactobacillus strains capable of preventing mucus from being colonized by pathogens. Here, we describe an adhesion inhibition assay method for Helicobacter pylori to porcine gastric mucin by Limosilactobacillus reuteri.

Pathogens in Crohn's Disease: The Role of Adherent Invasive Escherichia coli.

In Crohn's disease (CD), gut dysbiosis is marked by the prevalence of pathogenic bacterial species. Although several microbes have been reported as risk factors or causative agents of CD, it is not yet clear which is the real trigger of the disease. Thirty years ago, a new pathovar of Escherichia coli strain was isolated in the ileal mucosa of CD patients. This strain, called adherent invasive E. coli (AIEC), for its ability to invade the intestinal mucosa, could represent the causative agent of the disease. Several authors studied the mechanisms by which the AIEC penetrate and replicate within macrophages, and release inflammatory cytokines sustaining inflammation. In this review we will discuss about the role of AIEC in the pathogenesis of CD, the virulence factors mediating adhesion and invasion of AIEC in mucosal tissue, the environmental conditions improving AIEC survival and replication within macrophages. Finally, we will also give an overview of the new strategies developed to limit AIEC overgrowth.

In Vitro and In Vivo Assessment of the Infection Resistance and Biocompatibility of Small-Molecule-Modified Polyurethane Biomaterials.

Bacterial intracellular nucleotide second messenger signaling is involved in biofilm formation and regulates biofilm development. Interference with the bacterial nucleotide second messenger signaling provides a novel approach to control biofilm formation and limit microbial infection in medical devices. In this study, we tethered small-molecule derivatives of 4-arylazo-3,5-diamino-1H-pyrazole on polyurethane biomaterial surfaces and measured the biofilm resistance and initial biocompatibility of modified biomaterials in in vitro and in vivo settings. Results showed that small-molecule-modified surfaces significantly reduced the Staphylococcal epidermidis biofilm formation compared to unmodified surfaces and decreased the nucleotide levels of c-di-AMP in biofilm cells, suggesting that the tethered small molecules interfere with intracellular nucleotide signaling and inhibit biofilm formation. The hemocompatibility assay showed that the modified polyurethane films did not induce platelet activation or red blood cell hemolysis but significantly reduced plasma coagulation and platelet adhesion. The cytocompatibility assay with fibroblast cells showed that small-molecule-modified surfaces were noncytotoxic and cells appeared to be proliferating and growing on modified surfaces. In a 7-day subcutaneous infection rat model, the polymer samples were implanted in Wistar rats and inoculated with bacteria or PBS. Results show that modified polyurethane significantly reduced bacteria by ∼2.5 log units over unmodified films, and the modified polymers did not lead to additional irritation/toxicity to the animal tissues. Taken together, the results demonstrated that small molecules tethered on polymer surfaces remain active, and the modified polymers are biocompatible and resistant to microbial infection in vitro and in vivo.

Surface Degradation of Ion-releasing Restorative Materials With Cariogenic Challenge.

This study examined the influence of cariogenic environments on the surface roughness of ion-releasing restorative materials (IRMs). Custom-made stainless steel molds with holes of 5 mm × 2mm were used to fabricate 60 disc-shaped specimens of each of the following materials: Activa Bioactive (AV), Beautifil Bulk Restorative (BB), Cention N (Bulk-fill) (CN), and Filtek Z350XT (FZ) (Control). Baseline surface roughness (Ra) measurements were obtained using an optical 3D measurement machine (Alicona Imaging GmbH, Graz, Austria). The specimens were then randomly divided into five subgroups (n=12) and exposed to 10 ml of the following mediums at 37°C: distilled water (DW), demineralization solution (DM), remineralization solution (RM), pH cycling (PC) and air (AR) (control). Ra measurements were again recorded after one week and one month, followed by statistical evaluations with two-way analysis of variance (ANOVA) to determine interactions between materials and mediums. One-way ANOVA and post hoc Games Howell tests were performed for intergroup comparisons at a significance level of 0.05. Mean Ra values ranged from 0.085 ± 0.004 (µm) to 0.198 ± 0.001 µm for the various material-medium combinations. All IRMs showed significant differences in Ra values after exposure to the aqueous mediums. The smoothest surfaces were observed in the AR for all materials. When comparing materials, AV presented the roughest surfaces for all mediums. All IRM materials showed increased surface roughness over time in all cariogenic environments but were below the threshold value for bacterial adhesion, except for AV 1-month post immersion with pH cycling. Therefore, besides AV, the surface roughness of IRMs did not deteriorate to an extent that it is clinically relevant.

Bacterial adhesion inhibition by microalgal EPSs from Cylindrotheca closterium and Tetraselmis suecica biofilms.

In the food industry, successful bacterial pathogen colonization and persistence begin with their adhesion to a surface, followed by the spatial development of mature biofilm of public health concerns. Compromising bacterial settlement with natural inhibitors is a promising alternative to conventional anti-fouling treatments typically based on chemical biocides that contribute to the growing burden of antimicrobial resistance. In this study, three extracellular polymeric substance (EPS) fractions extracted from microalgae biofilms of Cylindrotheca closterium (fraction C) and Tetraselmis suecica (fraction Ta rich in insoluble scale structure and fraction Tb rich in soluble EPS) were screened for their anti-adhesive properties, against eight human food-borne pathogens belonging to Escherichia coli, Staphylococcus aureus, Salmonella enterica subsp. enterica, and Listeria monocytogenes species. The results showed that the fraction Ta was the most effective inducing statistically significant reduction for three strains of E. coli, S. aureus, and L. monocytogenes. Overall, EPSs coating on polystyrene surfaces of the different fractions increased the hydrophilic character of the support. Differences in bacterial adhesion on the different coated surfaces could be explained by several dissimilarities in the structural and physicochemical EPS compositions, according to HPLC and ATR-FTIR analysis. Interestingly, while fractions Ta and Tb were extracted from the same microalgal culture, distinct adhesion patterns were observed, highlighting the importance of the extraction process. Overall, the findings showed that EPS extracted from microalgal photosynthetic biofilms can exhibit anti-adhesive effects against food-borne pathogens and could help develop sustainable and non-toxic anti-adhesive surfaces for the food industry. KEY POINTS: •EPSs from a biofilm-based culture of C. closterium/T. suecica were characterized. •Microalgal EPS extracted from T. suecica biofilms showed bacterial anti-adhesive effects. •The anti-adhesive effect is strain-specific and affects both Gram - and Gram + bacteria.

In Vitro Assessment of Bacterial Adhesion and Biofilm Formation on Novel Bioactive, Biodegradable Electrospun Fiber Meshes Intended to Support Tendon Rupture Repair.

The surgical repair of a ruptured tendon faces two major problems: specifically increased fibrous adhesion to the surrounding tissue and inferior mechanical properties of the scar tissue compared to the native tissue. Bacterial attachment to implant materials is an additional problem as it might lead to severe infections and impaired recovery. To counteract adhesion formation, two novel implant materials were fabricated by electrospinning, namely, a random fiber mesh containing hyaluronic acid (HA) and poly(ethylene oxide) (PEO) in a ratio of 1:1 (HA/PEO 1:1) and 1:4 (HA/PEO 1:4), respectively. Electrospun DegraPol (DP) treated with silver nanoparticles (DP-Ag) was developed to counteract the bacterial attachment. The three novel materials were compared to the previously described DP and DP with incorporated insulin-like growth factor-1 (DP-IGF-1), two implant materials that were also designed to improve tendon repair. To test whether the materials are prone to bacterial adhesion and biofilm formation, we assessed 10 strains of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Enterococcus faecalis, known for causing nosocomial infections. Fiber diameter, pore size, and water contact angle, reflecting different degrees of hydrophobicity, were used to characterize all materials. Generally, we observed higher biofilm formation on the more hydrophobic DP as compared to the more hydrophilic DP-IGF-1 and a trend toward reduced biofilm formation for DP treated with silver nanoparticles. For the two HA/PEO implants, a similar biofilm formation was observed. All tested materials were highly prone to bacterial adherence and biofilm formation, pointing toward the need of further material development, including the optimized incorporation of antibacterial agents such as silver nanoparticles or antibiotics.

Nanotextured titanium inhibits bacterial activity and supports cell growth on 2D and 3D substrate: A co-culture study.

Medical implant-associated infections pose a significant challenge to modern medicine, with aseptic loosening and bacterial infiltration being the primary causes of implant failure. While nanostructured surfaces have demonstrated promising antibacterial properties, the translation of their efficacy from 2D to 3D substrates remains a challenge. Here, we used scalable alkaline etching to fabricate nanospike and nanonetwork topologies on 2D and laser powder-bed fusion printed 3D titanium. The fabricated surfaces were compared with regard to their antibacterial properties against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, and mesenchymal stromal cell responses with and without the presence of bacteria. Finite elemental analysis assessed the mechanical properties and permeability of the 3D substrate. Our findings suggest that 3D nanostructured surfaces have potential to both prevent implant infections and allow host cell integration. This work represents a significant step towards developing effective and scalable fabrication methods on 3D substrates with consistent and reproducible antibacterial activity, with important implications for the future of medical implant technology.

Preventing Static Biofilm Formation of Staphylococcus aureus on Different Types of Surfaces Using Microbubbles.

Bacterial fouling and biofilm formation on surfaces have been ongoing problems in real life as well as in the medical field. Different approaches have been taken to tackle the issues, from costly surface modification to antibiotic-delivering strategies. In this study, we examined the potential of using stabilized microbubbles (MBs) to shield against bacterial adhesion. Three types of surfaces were tested: hydrophilic glass (hydrophilic surface), neutral hydrophobic polystyrene (PS)-coated surfaces, and negatively charged hydrophobic octadecyltrichlorosilane (OTS)-coated surfaces. By evaluating the colony-forming unit (CFU) values from each surface, MBs stabilized by 0.05 mM SDS were shown to only produce significant reduction of Staphylococcus aureus adhesion on PS surfaces, up to 60.29 and 82.32% compared to no-MB PS surfaces, and no-MB uncoated surfaces, correspondingly, due to the appropriate size, stability, and negative charges of the MB shielding layer. On the other hand, OTS coatings had an intrinsic antiadhesion effect (69.83% compared to uncoated surface), given that the negatively charged OTS-aqueous interface or surface porosity nature of the coating prohibited the attachment of MBs, leading to the elimination of the antifouling effect of MBs. Ultimately, MBs gave better shielding results than surface modification when compared to uncoated surfaces and hence can be applied more widely.

Molecular Adhesion of a Pilus-Derived Peptide Involved in Pseudomonas aeruginosa Biofilm Formation on Non-Polar ZnO-Surfaces.

Bacterial colonization and biofilm formation on abiotic surfaces are initiated by the adhesion of peptides and proteins. Understanding the adhesion of such peptides and proteins at a molecular level thus represents an important step toward controlling and suppressing biofilm formation on technological and medical materials. This study investigates the molecular adhesion of a pilus-derived peptide that facilitates biofilm formation of Pseudomonas aeruginosa, a multidrug-resistant opportunistic pathogen frequently encountered in healthcare settings. Single-molecule force spectroscopy (SMFS) was performed on chemically etched ZnO 11 2 ‾ 0 ${\left(11\bar{2}0\right)}$ surfaces to gather insights about peptide adsorption force and its kinetics. Metal-free click chemistry for the fabrication of peptide-terminated SMFS cantilevers was performed on amine-terminated gold cantilevers and verified by X-ray photoelectron spectroscopy (XPS) and polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). Atomic force microscopy (AFM) and XPS analyses reveal stable topographies and surface chemistries of the substrates that are not affected by SMFS. Rupture events described by the worm-like chain model (WLC) up to 600 pN were detected for the non-polar ZnO surfaces. The dissociation barrier energy at zero force ΔG(0), the transition state distance xb and bound-unbound dissociation rate at zero force koff (0) for the single crystalline substrate indicate that coordination and hydrogen bonds dominate the peptide/surface interaction.

Biomaterials coated with zwitterionic polymer brush demonstrated significant resistance to bacterial adhesion and biofilm formation in comparison to brush coatings incorporated with antibiotics.

A critical problem with the use of biomaterial implants is associated with bacterial adhesion on the surface of implants and in turn the biofilm formation. Among different strategies that have been reported to resolve this dilemma, surface design combined with both antiadhesive and antimicrobial properties has proven to be highly effective. Physiochemical properties of polymer brush coatings possess non-adhesive capability against bacterial adhesion and create a niche for further functionalization. The current study aims to evaluate the effect of antibiotics incorporated into the polymer brush on bacterial adhesion and biofilm formation. Brushes made of zwitterionic polymers were synthesized, functionalized with vancomycin via both physical and chemical conjugation, and grafted onto the silicon rubber surfaces. Antibacterial and antiadhesive measurements of designed coated biomaterials were mediated through the use of a parallel plate flow chamber against biofilm growth developed by Staphylococcus aureus and Escherichia coli over a period of 24 h. The analysis of biofilm growth on designed coated biomaterials showed that the pristine coated zwitterionic brushes are significantly resistant to bacterial adhesion and biofilm formation but not in the polymer brush coating incorporated with antibiotics.

Serine-rich repeat proteins: well-known yet little-understood bacterial adhesins.

Serine-rich-repeat proteins (SRRPs) are large mucin-like glycoprotein adhesins expressed by a plethora of pathogenic and symbiotic Gram-positive bacteria. SRRPs play major functional roles in bacterial-host interactions, like adhesion, aggregation, biofilm formation, virulence, and pathogenesis. Through their functional roles, SRRPs aid in the development of host microbiomes but also diseases like infective endocarditis, otitis media, meningitis, and pneumonia. SRRPs comprise shared domains across different species, including two or more heavily O-glycosylated long stretches of serine-rich repeat regions. With loci that can be as large as ~40 kb and can encode up to 10 distinct glycosyltransferases that specifically facilitate SRRP glycosylation, the SRRP loci makes up a significant portion of the bacterial genome. The significance of SRRPs and their glycans in host-microbe communications is becoming increasingly evident. Studies are beginning to reveal the glycosylation pathways and mature O-glycans presented by SRRPs. Here we review the glycosylation machinery of SRRPs across species and discuss the functional roles and clinical manifestations of SRRP glycosylation.

Impact of nanoscale silicon dioxide coating of stainless-steel surfaces on Listeria monocytogenes.

High resistance to environmental factors as well as the ability to form biofilms allow Listeria monocytogenes to persist for a long time in difficult-to-reach places in food-producing plants. L. monocytogenes enters final products from contaminated surfaces in different areas of plants and poses a health risk to consumer. Modified surfaces are already used in the food industry to prevent cross-contamination. In this study, stainless-steel surfaces were coated with nanoscale silicon dioxide and the effects on attachment, bacterial growth and detachment of L. monocytogenes were evaluated. Attachment was considered for three different ways of application to simulate different scenarios of contamination. Bacterial growth of L. monocytogenes on the surface was recorded over a period of up to 8 h. Detachment was tested after cleaning inoculated stainless-steel surfaces with heated distilled water or detergent. Coating stainless-steel surfaces with nanoscale silica tends to reduce adherence and increased detachment and does not influence the bacterial growth of L. monocytogenes. Further modifications of the coating are necessary for a targeted use in the reduction of L. monocytogenes in food-processing plants.

The IbeA protein from adherent invasive Escherichia coli is a flavoprotein sharing structural homology with FAD-dependent oxidoreductases.

Invasion of brain endothelium protein A (IbeA) is a virulence factor specific to pathogenic Escherichia coli. Originally identified in the K1 strain causing neonatal meningitis, it was more recently found in avian pathogenic Escherichia coli (APEC) and adherent invasive Escherichia coli (AIEC). In these bacteria, IbeA facilitates host cell invasion and intracellular survival, in particular, under harsh conditions like oxidative stress. Furthermore, IbeA from AIEC contributes to intramacrophage survival and replication, thus enhancing the inflammatory response within the intestine. Therefore, this factor is a promising drug target for anti-AIEC strategies in the context of Crohn's disease. Despite such an important role, the biological function of IbeA remains largely unknown. In particular, its exact nature and cellular localization, i.e., membrane-bound invasin versus cytosolic factor, are still of debate. Here, we developed an efficient protocol for recombinant expression of IbeA under native conditions and demonstrated that IbeA from AIEC is a soluble, homodimeric flavoprotein. Using mass spectrometry and tryptophan fluorescence measurements, we further showed that IbeA preferentially binds flavin adenine dinucleotide (FAD), with an affinity in the one-hundred nanomolar range and optimal binding under reducing conditions. 3D-modeling with AlphaFold revealed that IbeA shares strong structural homology with FAD-dependent oxidoreductases. Finally, we used ligand docking, mutational analyses, and molecular dynamics simulations to identify the FAD binding pocket within IbeA and characterize possible conformational changes occurring upon ligand binding. Overall, we suggest that the role of IbeA in the survival of AIEC within host cells, notably macrophages, is linked to modulation of redox processes.

The adhesion capability of Staphylococcus aureus cells is heterogeneously distributed over the cell envelope.

Understanding and controlling microbial adhesion is a critical challenge in biomedical research, given the profound impact of bacterial infections on global health. Many facets of bacterial adhesion, including the distribution of adhesion forces across the cell wall, remain poorly understood. While a recent 'patchy colloid' model has shed light on adhesion in Gram-negative Escherichia coli cells, a corresponding model for Gram-positive cells has been elusive. In this study, we employ single cell force spectroscopy to investigate the adhesion force of Staphylococcus aureus. Normally, only one contact point of the entire bacterial surface is measured. However, by using a sine-shaped surface and recording force-distance curves along a path perpendicular to the rippled structures, we can characterize almost a hemisphere of one and the same bacterium. This unique approach allows us to study a greater number of contact points between the bacterium and the surface compared to conventional flat substrata. Distributed over the bacterial surface, we identify sites of higher and lower adhesion, which we call 'patchy adhesion', reminiscent of the patchy colloid model. The experimental results show that only some cells exhibit particularly strong adhesion at certain locations. To gain a better understanding of these locations, a geometric model of the bacterial cell surface was created. The experimental results were best reproduced by a model that features a few (5-6) particularly strong adhesion sites (diameter about 250 nm) that are widely distributed over the cell surface. Within the simulated patches, the number of molecules or their individual adhesive strength is increased. A more detailed comparison shows that simple geometric considerations for interacting molecules are not sufficient, but rather strong angle-dependent molecule-substratum interactions are required. We discuss the implications of our results for the development of new materials and the design and analysis of future studies.