Biological Actions of Peronema canescens Jack on the Properties of Alginate and Virulence of Staphylococcus aureus

Klebsiella pneumoniae (K. pneumoniae) is a major human nosocomial infectious agent and an important veterinary pathogen, frequently resistant to various antibiotics. It causes diseases such as pneumonia, urinary tract infections, surgical wound infections and septicemia. Biofilm formation of K. pneumoniae promotes persistent infection and contributes to resistance against antimicrobial agents. The objective of this study was to comparatively evaluate the effect of selected AMPs on the formation, metabolic activity and viability of Klebsiella pneumoniae biofilms of veterinary and human origin. Biofilm formation of three K. pneumoniae strains was quantified using the crystal violet assay and visualized by scanning electron microscopy (SEM). The inhibitory effects of eight different AMPs on the formation and metabolic activity of K. pneumoniae biofilms, as well as on planktonic growth, were examined using crystal violet, resazurin and broth microdilution assays, respectively. The effect on living and dead bacteria in mature biofilms was investigated using the fluorescent dyes SYTO™ 9 and propidium iodide. In addition, the distribution of rhodamine B-labeled peptide DJK-5 in mature biofilms of strain 17349 was visualized by confocal laser scanning microscopy (CLSM). Biofilm formation was confirmed for all three K. pneumoniae strains. Depending on the strain, we found that planktonic growth was affected by the AMPs DJK-5, DJK-6, Onc72, and Onc112. Biofilm formation of all three strains was inhibited by hbD3, LL-37, DJK-5, and DJK-6, with biofilm mass reduced to less than 40% of the untreated control. In addition to the inhibition of biofilm formation, a reduction in the metabolic activity of the biofilm-associated bacteria was also observed. These four AMPs also showed an effect on mature biofilms by reducing the number of both viable and dead bacteria in 22 h-old biofilms. Rhodamine B-labeled DJK-5 took 7 h to visibly accumulate in the planktonic bacteria. Multi-layered biofilm aggregations were mainly negative for rhodamine B-labeled DJK-5, even 44 h after AMP treatment, indicating that certain parts of mature K. pneumoniae biofilms are not accessible for this AMP. In conclusion, we found differences in the effect of AMPs on biofilms including both increases and decreases in biofilm mass and viability.

Event Abstract Back to Event Inhibition of bacterial adhesion and biofilm formation by antimicrobial functionalized textured biomaterial surfaces Lichong Xu1, Yaqi Wo2, Mark E. Mayerhoffb2, Zhicheng Tian3, Harry Allcock3 and Christopher A. Siedlecki1, 4 1 Penn State College of Medicine, Department of Surgery, United States 2 University of Michigan, Department of Chemistry, United States 3 The Pennsylvania State University, Department of Chemistry, United States 4 Penn State College of Medicine, Department of Bioengineering, United States Introduction: Infection due to bacterial adhesion and biofilm formation represetns a major drawback to the use of blood-contacting devices. Previously we have developed textured polyurethane biomaterials for reducing bacterial adhesion and inhibiting biofilm formation. In this work, we applied the texturing pattern to functionalized polymers bearing antimicrobial properties, and show a combination of antimicrobial functionalization and physical approaches can produce additive effects on biological responses and enhance the hemocompatibility of biomaterials. Material and Methods: Carbosil polyurethane (PU, 2080A) and phosphazene polymers (trifluoroethoxy (TFE), crosslinakble trifluoroethoxy (XLTFE), and octafluoropentoxy (OFP)) were textured with ordered arrays of pillars using a soft lithography two-stage replication molding technique[1]. The Carbosil polyurethane material was doped with S-nitroso-N-acetylpenicillamine (SNAP) with different contents of SNAP in PU (5, 10, and 15 wt%) for nitric oxide (NO) release[2]. Antimicrobial properties of materials were evaluated by the optical density (OD600) of microbial cultures incubated with materials and strain S. epidermidis RP62A. Bacterial adhesion and biofilm formation on material surfaces were carried out in a multiwell plate or rotating disk system (RDS)[1] with same strain. Bacterial adhesion was counted under a fluorescent optical microscope. To observe biofilm formation, samples were in incubated in multiwell plates under static condition for 2 days or RDS system under shear for 8 days. Samples were stained with FTIC conjugated wheat germ agglutinin, and biofilm observed by fluorescence. Results and Discussion: Characterization of biomaterial surfaces: AFM images showed material surfaces textured with ordered arrays of pillars, as expected, except for TFE and XLTFE materials that were difficult to texture.Water contact angle measurements show surface hydrophobicity increased after texturing due to entrapped air. Antimicrobial properties of materials:The optical densities of culture media indicate the growth of bacteria in the presence of a variety of polymers (Fig. 1). Carbosil PU 0% SNAP and PUU films show the similar optical density as that of control (no polymers). Significant inhibition of bacterial growth was observed in the cultures for Carbosil 10% and 15% SNAP polymers, indicating NO release inhibits the growth of bacteria. A slight decrease of OD600 was observed for all phosphazene polymers. Fig. 1 Optical density of culture media indicating antimicrobial properties of polymers. Bacterial adhesion on functionalized textured polymer surfaces: Higher bacterial adhesion was observed on smooth surfaces without functionalization (Fig. 2). Surface texturing with pattern decreased bacterial adhesion on all surfaces, except for TFE and XLTFE polymers, indicating the importance of surface texturing in inhibiting adhesion. Furthermore, a significant decrease in adhesion was observed on textured PU surfaces containing 10% or 15% SNAP as well as on OFP textured surfaces, suggesting antimicrobial functionalization and texturing produce a synergistic effect on inhibiting bacterial adhesion, as the OFP polymer alone inhibited bacterial growth only slightly. Fig. 2 Bacterial adhesion on polymer surface in PBS for 1 hr at 37°C under static condition. Biofilm formation: Under static condition, biofilms were observed on Carbosil PU surfaces containing 0% SNAP and 5% SNAP after 2 days, while no biofilms were observed on surfaces containing 10% and 15% SNAP. Under shear stresses, no biofilms were observed on smooth or textured PU surfaces containing 15% SNAP after 8 days. Long term exposure experiments for biofilm formation on functionalized textured surfaces are on-going. Fig. 3 Biofilm on textured 500/500 nm patterned surfaces containing (a) 0% SNAP, (b) 15% SNAP under static condition for 2 days, and (c) 15% SNAP under shear for 8 days. Conclusion: A combination of antimicrobial functionalization of polymers and surface texturing provided an improved approach to inhibiting bacterial adhesion and biofilm formation, and thus to potentially preventing biomaterial related infections.

Ultraviolet C (UVC) light is a physical method proposed for disinfecting dental impression materials and preventing cross-infections in clinical practice. The investigations have focused on the UVC disinfection potential, but little is known about the consequences on dental materials' properties. This scoping review's objective is to evaluate information about the effect of UVC light on the dimensional stability of dental impression materials. An electronic search of dental literature in the Medline (via PubMed), Scopus, and Embase databases were systematically searched until July 31 of 2024 following the PRISMA-ScR guidelines. The search strategy was carried out considering three groups of words with indexing terms and Boolean operators. Two reviewers selected the titles and analyzed the abstracts according to the inclusion and exclusion criteria. A total of six articles were included through electronic database searches. Four studies evaluated the dimensional stability by measuring dental casts made from an impression and two studies measured distances on discs made from stainless steel dies. The studies reported the use of polyether, addition and condensation silicones, alginate, and zinc oxide eugenol in the protocols followed. Three of the six included studies compared the effect of UVC light against glutaraldehyde 2% and sodium hypochlorite (1% or 5.25%), one study compared the UVC light against quaternary ammonium salts, phenoxyethanol, alcohol, and ozone, meanwhile another study compared the effect of UVC light against peracetic acid 0.2%, natural polymer of glucosamine and ozonated water. Regarding measuring devices, only one study reported the use of a measuring software, the majority used traveling microscope. Based on the findings, UVC light showed no significant dimensional changes in polyether, addition, and condensation silicones. Key words:Disinfection, dental impression materials, ultraviolet light.

The effects of anodization and instrumentation on titanium abutment surface characteristics and biofilm formation.

The aim of this study was to investigate the effects of genomic DNA purified from Candida albicans and pneumonia-related pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, on in vitro biofilm formation and morphological change of 3 Candida species (C. albicans, C. glabrata, and C. tropicalis). Biofilm formation was evaluated by the crystal violet assay and colony-forming unit counts. Morphological characteristics of biofilms were evaluated by scanning electron microscopy and fluorescence microscopy. Addition of DNA at a low concentration (<1·0 μg ml(-1)) significantly increased biofilm mass of all three Candida species. In contrast, the addition of DNA at a high concentration (10 μg ml(-1)) decreased the biofilm mass. Interestingly, the formation of hyphae in a dense network of yeast cells was observed in C. albicans biofilms exposed to a low concentration of DNA (<1·0 μg ml(-1)). These findings demonstrated that extracellular DNA (eDNA) plays a crucial role in Candida biofilm formation and suggested that eDNA may induce the morphological transition from yeast to hyphal growth form during C. albicans biofilm development. A novel therapy targeting eDNA may be applicable for Candida infection to decrease biofilm formation and hyphal formation.

Most bacteria form biofilms, which are thick multicellular communities covered in extracellular matrix. Biofilms can become thick enough to be even observed by the naked eye, and biofilm formation is a tightly regulated process. Paracoccus denitrificans is a non-motile, Gram-negative bacterium that forms a very thin, unique biofilm. A key factor in the biofilm formed by this bacterium is a large surface protein named biofilm-associated protein A (BapA), which was recently reported to be regulated by cyclic diguanosine monophosphate (cyclic-di-GMP or c-di-GMP). Cyclic-di-GMP is a major second messenger involved in biofilm formation in many bacteria. Though cyclic-di-GMP is generally reported as a positive regulatory factor in biofilm formation, it represses biofilm formation in P. denitrificans. Furthermore, quorum sensing (QS) represses biofilm formation in this bacterium, which is also reported as a positive regulator of biofilm formation in most bacteria. The QS signal used in P. denitrificans is hydrophobic and is delivered through membrane vesicles. Studies on QS show that P. denitrificans can potentially form a thick biofilm but maintains a thin biofilm under normal growth conditions. In this review, we discuss the peculiarities of biofilm formation by P. denitrificans with the aim of deepening the overall understanding of bacterial biofilm formation and functions.

The biofilm formation has been widely recognized as one of the main mechanisms of antimicrobial resistance development in microorganisms. However, few studies are focusing on this phenomenon in Candida spp. in clinical settings, especially on immuno-compromised patients. In this study, both the rate of biofilm formation in those patients and its drug susceptibility in initial and mature biofilm were assessed using crystal violet assay and dilution method. The results demonstrated that the biofilm formation rate was similar between albicans and non-albicans Candida. However, the biofilm formation capacity was more pronounced in non-albicans Candida, especially, C. glabrata. As expected, there was a significant relationship between biofilm formation and drug resistance. In addition, our study reconfirmed that the age of high concentration of antifungal agents only affected Candida before its biofilm formation regardless of its biofilm formation capacity. In the contrary, once the biofilm was formed even elevated drug concentrations did not show sufficient efficacy, highlighting a need for high dosage at the early stage of treatment for those patients. The results of this study highlighted the importance of using appropriate antifungal agents for Candida treatment before the formation of biofilm.

The irresponsible overuse of antibiotics has increased the occurrence of resistant bacterial strains, which represents one of the biggest patient safety risks today. Due to antibiotic resistance and biofilm formation in bacteria, it is becoming increasingly difficult to suppress the bacterial strains responsible for various chronic infections. Honey was proven to inhibit bacterial growth and biofilm development, offering an alternative solution in the treatment of resistant infections and chronic wounds. Our studies included chestnut honey, valued for its high antibacterial activity, and the bacteria Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and S. epidermidis, known to form multi-species biofilm communities. Minimum inhibitory concentrations (MIC) of chestnut honey were determined for each bacterial strain. Afterwards, the mixed bacterial biofilms were treated with chestnut honey at different stages of maturity (incubation times: 2, 4, 6, 12, 24 h). The extent of biofilm inhibition was measured with a crystal violet assay and demonstrated by scanning electron microscopy (SEM). As the incubation time increased and the biofilm became more mature, inhibition rates decreased gradually. The most sensitive biofilm was the combination MRSA-S. epidermidis, with a 93.5% inhibition rate after 2 h of incubation. Our results revealed that chestnut honey is suitable for suppressing the initial and moderately mature stages of mixed biofilms.

Background: Staphylococcus aureus is the main pathogen infect chronic rhinosinusitis (CRS) and the biofilms play an essential role in the pathophysiology of infection and treatment of patients. However, research on clinical conditions is still limited. This study aimed to evaluate the relationship between S. aureus biofilms with severity and infection degree in CRS patients. Methods: A post-test-only study was conducted at Dr Zaionel Abidin Hospital in Banda Aceh, Indonesia from January to April 2022. Thirty-five CRS patients, classified according to the disease severity and infection degree, were included and followed in the study. A mucosal swab on each patient was collected and biofilm formation analyzed quantitatively and qualitatively using a spectrophotometer and scanning electron microscope (SEM), respectively. The relationships between the bacterial growth, biofilm formation and mass of biofilms with the disease severity and degree of infection were assessed statistically. Results: More than half of the patients (54.3%) were aged younger than 40 years, and 51.4% were male. Nasal congestion and rhinorrhoea were the most common reported obligate symptoms (78.2%) and 82.9% patients have additional symptom on facial pain. There was the relationship between bacterial growth and disease severity (p=0.046) and infection degree of CSR cases (p=0.031). Our data showed a significant association between biofilm formation and the disease severity (p=0.022) and infection degree of CSR patients (p=0.020). The mass of the biofilm was also associated significantly with disease severity (p=0.029) and degree of infection (p=0.018). Conclusions: Our study shows that the formation and mass of biofilm associated with the disease severity and degree of infection in CSR. Biofilms may contribute to the clinical condition and also be considered during management of CRS patients.

Serine-rich Repeat Adhesin Gordonii Surface Protein B is Important for Streptococcus gordonii Biofilm Formation

When laser beam interacts with dental hard tissues, the results can be affected by the parameters of laser, such as power, water spray, air spray etc. The purpose of this study was to compare the influences of various parameters on the efficiency of dentin ablation. Thirty extracted human molars without caries or restorations were cut into dentin slices of 2 mm in thickness using Isomet. The dentin slices were stored in 4oC distilled water. The specimens were randomly divided into 3 groups to evaluate the influences of different power settings, water spray, and air spray on the ablation rate of human dentin. Each group contained 10 dentin slices. The specimens were observed under light microscope (LM) and scanning electron microscope (SEM) after 30 s of Er;Cr:YSGG laser irradiation. In order to evaluate the topography of laser-induced crater on each specimen, we used dental impression material to take negative impression of crater and measured the depth and area of the impression under LM. The results showed that the morphological changes of craters created by different power settings, water spray, and air spray were similar except for the melted and charred dentin surfaces when the water spray was less than 20%. The power settings and air spray were positively correlated with the cutting efficiency of human dentin while the water spray had an opposite effect. Although lower water spray could cut dentin more efficiently, the dentin appeared melted and charred if the amount of water spray was too low. From this study the following conclusions could be addressed: 1. Different power settings, air spray, and water spray cause similar morphological changes of dentin. 2. If the amount of water spray is below 20%, melted and charred dentin are produced. 3. The cutting efficiency of dentin is elevated as the power settings and air spray is increased. However, the more water spray, the lower the cutting efficiency of dentin.

Registration of fingertip pattern with dental impression materials: A scanning electron microscope study

Platelet concentrates (PCs) are stored at 20–24˚C in a biologically favorable environment that may support bacterial growth. Staphylococcus epidermidis, a typical contaminant, can form biofilms in PCs, complicating detection and increasing the risk of transfusion-transmitted bacterial infections. The material composition and surface texture of PC storage bags may influence biofilm formation. The impact of different PC storage bag materials on S. epidermidis biofilm formation was evaluated using the ISO 4768:2023(E) crystal violet (CV) assay. Four surface conditions were tested: polyvinyl chloride (PVC) plasticized with n-butyryl-tri(n-hexyl)-citrate (BTHC) – both smooth and rough sides, PVC plasticized with tri-(2-ethylhexyl)-trimellitate (TEHTM) and ethylene-vinyl acetate (EVA). Coupons and bags made from each material were used in the experiments. Biofilm-positive S. epidermidis was cultured in tryptic soy broth (TSB), PCs and plasma and added on plastic coupons under static conditions or directly in the bags with agitation. Bacterial enumeration and CV assay were performed on days 2, 5, and 7. In TSB, EVA coupons significantly formed more biofilm than the smooth side of PVC-BTHC or TEHTM over seven days. In PCs, more biofilm formed on the rough side of PVC-BTHC coupons than the smooth side, with no other differences between plastics, suggesting similar biofilm amount across PC bag materials in the presence of platelets. No biofilm was detected on coupons in plasma. Under continuous agitation and reduced oxygen levels, only the rough side of PVC-BTHC showed significant biofilm formation in TSB in PC storage bags over seven days. These findings highlight the need for standardized biofilm testing and suggest that some plastics are more conducive to biofilm formation under static conditions. However, during blood bank storage (i.e., continuous agitation and reduced oxygen levels), biofilm formation is limited, regardless of the platelet bag material, thereby reducing the risk of undetected bacterial contamination.

Platelet concentrates (PCs) are stored at 20-24˚C in a biologically favorable environment that may support bacterial growth. Staphylococcus epidermidis, a typical contaminant, can form biofilms in PCs, complicating detection and increasing the risk of transfusion-transmitted bacterial infections. The material composition and surface texture of PC storage bags may influence biofilm formation. The impact of different PC storage bag materials on S. epidermidis biofilm formation was evaluated using the ISO 4768:2023(E) crystal violet (CV) assay. Four surface conditions were tested: polyvinyl chloride (PVC) plasticized with n-butyryl-tri(n-hexyl)-citrate (BTHC) - both smooth and rough sides, PVC plasticized with tri-(2-ethylhexyl)-trimellitate (TEHTM) and ethylene-vinyl acetate (EVA). Coupons and bags made from each material were used in the experiments. Biofilm-positive S. epidermidis was cultured in tryptic soy broth (TSB), PCs and plasma and added on plastic coupons under static conditions or directly in the bags with agitation. Bacterial enumeration and CV assay were performed on days 2, 5, and 7. In TSB, EVA coupons significantly formed more biofilm than the smooth side of PVC-BTHC or TEHTM over seven days. In PCs, more biofilm formed on the rough side of PVC-BTHC coupons than the smooth side, with no other differences between plastics, suggesting similar biofilm amount across PC bag materials in the presence of platelets. No biofilm was detected on coupons in plasma. Under continuous agitation and reduced oxygen levels, only the rough side of PVC-BTHC showed significant biofilm formation in TSB in PC storage bags over seven days. These findings highlight the need for standardized biofilm testing and suggest that some plastics are more conducive to biofilm formation under static conditions. However, during blood bank storage (i.e., continuous agitation and reduced oxygen levels), biofilm formation is limited, regardless of the platelet bag material, thereby reducing the risk of undetected bacterial contamination.

Escherichia coli can cause diarrheal and extraintestinal illnesses in humans. Diarrheagenic E. coli can be transmit to human through consumption of contaminated food, including vegetables. Biofilm produced by E. coli during food processing plays a role in development of foodborne illnesses. Vegetables have often been involved in diarrheal E. coli infections. A total of 40 E. coli isolates from vegetables were tested to determine biofilm formation at 12°C, 25°C and 37°C by the crystal violet and MTT assays. All isolates were performed for the production of curli fimbriae and cellulose associated with biofilm formation on Congo red agar. Biofilm formation at 37°C, 25°C and 12°C was detected in 87.5%, 70% and 70% of the isolates, respectively. Biofilm formation among the E. coli isolates using the crystal violet and MTT assays showed a statistically significant difference between 12°C and 25°C as well as 12°C and 37°C (p &lt; 0.05). However, no significant difference between 25 and 37°C (p &gt; 0.05) was obtained. Three different morhotypes (bdar, pdar and saw) were identified based on the expression of curli fimbriae and cellulose. The incidence of the bdar morhotype was 27.5% and 50% at 25°C and 37°C, respectively. Prevalence of the pdar morphotype was 50% and 70% at 25°C and 37°C, respectively. At 25°C, only one isolate (2.5%) showed the saw morphotype. All isolates tested expressed curli fimbriae or cellulose, only three of which were non-biofilm producer using the crystal violet assay. This study demonstrated that the presence of biofilm forming E.coli isolates in vegetables may cause a risk to human health and food safety.