Effects of Piper betle Leaf Extract on Biofilm and Rhamnolipid Formation of Pseudomonas aeruginosa

High mortality rate and antimicrobial resistance are still becoming world-wide problems, due to Pseudomonas aeruginosa’s (P. aeruginosa) virulence and its ability to form biofilm. Biofilm’s formation is affected by the presence of rhamnolipid, whose production is regulated by quorum sensing systems. Piper betle (P. betle) possesses antimicrobial, antioxidant, anti-inflammatory and immunomodulatory properties. The aim of our study is to investigate the effects of P. betle leaf’s extract against biofilm formation and rhamnolipid production of P. aeruginosa. Active compounds of P. betle were identified using plate chromatography. Agar dilution method was used to determine the minimum biofilm inhibitory concentration (MBIC) of methanolic leaf extract of P. betle. A biofilm-producing P. aeruginosa isolate in the polystyrene plate adherence test was selected for confirmation of biofilm production by Scanning Electron Microscopy (SEM), after P. betle administration. Rhamnolipid detection and evaluation were performed by interpreting halo formed around the well. After administration of various concentrations of P. betle leaf extract on the microplate well, it was concluded that the MBIC of P. betle leaf extract on P. aeruginosa was 0.4%. Methanolic extract of P. betle leaf extract at concentration of 0.4% showed that P. aeruginosa could not form biofilm at all, although the bacteria could still aggregate and form a matrix. After linear regression analysis, beta-coefficient was obtained at -0.931 for P. betle leaf extract. It can be concluded that P. betle leaf extract was effective in inhibiting the growth of biofilm and formation of rhamnolipid by P. aeruginosa. The increase in concentration of P. betle leaf extract was inversely proportional to the diameter of the halo rhamnolipid formed. The higher the level of P. betel leaf extract, the smaller the diameter of the halo rhamnolipid formed.

Design of RGDS Peptide-Immobilized Self-Assembling β-Strand Peptide from Barnacle Protein

We designed three types of RGD-containing barnacle adhesive proteins using self-assembling peptides. In the present study, three types of RGD-containing peptides were synthesized by solid-phase peptide synthesis, and the secondary structures of these peptides were analyzed by CD and FT-IR spectroscopy. The mechanical properties of peptide hydrogels were characterized by a rheometer. We discuss the correlation between the peptide conformation, and cell attachment and cell spreading activity from the viewpoint of developing effective tissue engineering scaffolds. We created a peptide-coated cell culture substrate by coating peptides on a polystyrene plate. They significantly facilitated cell adhesion and spreading compared to a non-coated substrate. When the RGDS sequence was modified at N- or C-terminal of R-Y, it was found that the self-assembling ability was dependent on the strongly affects hydrogel formation and cell adhesion caused by its secondary structure.

Biomimetic Cell-Laden MeHA Hydrogels for the Regeneration of Cartilage Tissue

Methacrylated hyaluronic acid (MeHA) and chondroitin sulfate (CS)-biofunctionalized MeHA (CS-MeHA), were crosslinked in the presence of a matrix metalloproteinase 7 (MMP7)-sensitive peptide. The synthesized hydrogels were embedded with either human mesenchymal stem cells (hMSCs) or chondrocytes, at low concentrations, and subsequently cultured in a stem cell medium (SCM) or chondrogenic induction medium (CiM). The pivotal role of the synthesized hydrogels in promoting the expression of cartilage-related genes and the formation of neocartilage tissue despite the low concentration of encapsulated cells was assessed. It was found that hMSC-laden MeHA hydrogels cultured in an expansion medium exhibited a significant increase in the expression of chondrogenic markers compared to hMSCs cultured on a tissue culture polystyrene plate (TCPS). This favorable outcome was further enhanced for hMSC-laden CS-MeHA hydrogels, indicating the positive effect of the glycosaminoglycan binding peptide on the differentiation of hMSCs towards a chondrogenic phenotype. However, it was shown that an induction medium is necessary to achieve full span chondrogenesis. Finally, the histological analysis of chondrocyte-laden MeHA hydrogels cultured on an ex vivo osteochondral platform revealed the deposition of glycosaminoglycans (GAGs) and the arrangement of chondrocyte clusters in isogenous groups, which is characteristic of hyaline cartilage morphology.

Thin layer immunoassay; an economical approach to diagnose Helicobacter pylori Infection in gastroduodenal ulcer disease patients of Pakistan; a comparative analysis

Helicobacter pylori is a causative agent of gastritis, gastroduodenal ulcers and gastric adenocarcinoma. Gastric patient’s serums were screened for H. pylori infection by thin layer immunoassay. A polystyrene plate coated with H. pylori sonicate whole cell antigen (10 μg/ml). Two fold-diluted patient’s serum was allowed to react at 37 °C, incubated at 60 °C for 1 min over water bath and recorded water condensation pattern for H. pylori antibody. Gastric patient’s blood samples (62% male and 6% female) were tested positive for H. pylori, while agewise 15–25 years males (36%) and 65–75 years females (50%) showed highest number of H. pylori infection. Thin layer immunoassay showed sensitivity (72–67%), specificity (100%), accuracy (94–69%) and κ value (0.493–0.357) in comparison with wELISA, sELISA and kELISA. We conclude thin layer immunoassay was reliable, low cost, quick, simple and clinically useful method for H. pylori diagnosis in patients of Pakistan.

Contribution of Lewis blood groups molecules in biofilm formation of Pseudomonas aeruginosa isolated from atopic dermatitis patients.

Biofilm formation is a mechanism for bacterial community defense against insults including antibiotics .In this report we evaluated the potency of Pseudomonas aeruginosa(P. aeruginosa) isolates from atopic dermatitis patients skin as well as stool to colonize different Lewis types saliva , manifested by biofilm formation . The bacteria were cultured on tryptose soy broth .96-well polystyrene plate were used .Coating with heat inactivated Le (a), (b) and (c)saliva was performed. Biofilm intensity was measured using crystal violet stained films compared to non –saliva coated situation. The results showed a superior capability of most isolates to form biofilm on Le (a) followed by Le (b) saliva. The highest binding mean was for isolate ( 4). Le (a) saliva binding (mean ± SD was 0.66± 0.25 for test compared to 0.21± 0.04 for control non coated wells) , p=0.04,cl=0.041-0.864. Other isolates demonstrated variable degree of biofilm formation on this substrate .In contrast to Le (c) saliva, Le (b) saliva demonstrated weak biofilm formation . We conclude that, among atopic dermatitis patients skins, P. aeruginosa Lec (A) or Lec (B) lectins might be involved in colonization in such patients. Key Words:- Lewis blood groups – Pseudomonas aeruginosa -atopic dermatitis– Biofilm

1053. Biofilm Production and Clinical Characteristics of S. maltophilia Causing Persistent or Relapsing Bacteremia

Background This study aimed to identify clinical or microbiological factors related to persistence or recurrence of Stenotrophomonas maltophilia bacteremia in adult patients. Methods S. maltophilia isolated from blood in two tertiary hospitals between 2011 and 2017 were investigated. Persistent bacteremia was defined as the consecutive blood culture positive for ≥5 days after initiation of appropriate antibiotics therapy. Relapse was defined as isolation of S. maltophilia from blood after completion of antibiotics treatment for the first episode of bacteremia. Biofilm formation was assessed in 96-well polystyrene plate with Trypticase Soy Broth using 0.5% crystal violet staining. The presence of smf-1 gene was detected by polymerase chain reaction. Results Of total 100 patients with S. maltophilia bacteremia, 10 of persistent, 8 of relapsing, and 46 of nonpersistent, nonrelapsing cases were investigated. The presence of indwelling urinary catheter (P = 0.011), nasogastric tube (P = 0.003), mechanical ventilator treatment (P = 0.001), and previous colonization of S. maltophilia (P = 0.016) were more frequently observed in patients with persistent bacteremia compared with nonpersistent, nonrelapsing bacteremia cases. In patients with relapsing bacteremia, hematologic malignancy (P = 0.022), neutropenia (P = 0.001), and concomitant isolation of S. maltophilia in clinical samples other than blood (P = 0.041) were more common than nonpersistent, nonrelapsing bacteremia patients. Catheter-related infection (37.0%) followed by pneumonia (28.3%) was the most common primary focus of nonpersistent, nonrelapsing bacteremia whereas pneumonia was the most frequent cause of bacteremia in both of persistent and relapsing cases (40.0% and 50.0%). Most of isolates (63 of 64) were susceptible to cotrimoxazole. The resistance to levofloxacin were comparable among isolates from persistent, relapsing and nonpersistent, nonrelapsing cases (10.0% vs. 12.5% vs. 15.2%, P = 0.988). Biofilm formation ability was not significantly different between three groups (optical density at 595, mean ± SD, 0.69 ± 0.34 vs. 0.78 ± 0.33 vs. 0.70 ± 0.33, P = 0.529). The smf-1 gene was found in all isolates. Conclusion More careful treatment approaches to patients with risk factors for S.maltophilia treatment failure should be warranted. Disclosures All authors: No reported disclosures.

Enterococcus faecalis 6-Phosphogluconolactonase Is Required for Both Commensal and Pathogenic Interactions with Manduca sexta

Enterococcus faecalisis a commensal and pathogen of humans and insects. InManduca sexta,E. faecalisis an infrequent member of the commensal gut community, but its translocation to the hemocoel results in a commensal-to-pathogen switch. To investigateE. faecalisfactors required for commensalism, we identifiedE. faecalisgenes that are upregulated in the gut ofM. sextausing recombinase-basedin vivoexpression technology (RIVET). The RIVET screen produced 113 clones, from which we identified 50 genes that are more highly expressed in the insect gut than in culture. The most frequently recovered gene was locus OG1RF_11582, which encodes a 6-phosphogluconolactonase that we designatedpglA. ApglAdeletion mutant was impaired in both pathogenesis and gut persistence inM. sextaand produced enhanced biofilms compared with the wild type in anin vitropolystyrene plate assay. Mutation of four other genes identified by RIVET did not affect persistence in caterpillar guts but led to impaired pathogenesis. This is the first identification of genetic determinants forE. faecaliscommensal and pathogenic interactions withM. sexta. Bacterial factors identified in this model system may provide insight into colonization or persistence in other host-associated microbial communities and represent potential targets for interventions to preventE. faecalisinfections.

Biocompatibility of Silver-Containing Calcium-Phosphate Cements with Anti-Bacterial Properties

We have previously synthesized silver-containing hydroxyapatite (Ag-HAp) powders by an ultrasonic spray-pyrolysis (USSP) technique. On the other hand, we have successfully fabricated novel calcium-phosphate cements (CPCs) composed of mainly β-tricalcium phosphate (β-TCP) phase with anti-washout property (hereafter, β-TCP cement), which was set on the basis of chelate-bonding ability of inositol phosphate (IP6). In this study, we developed novel CPCs with both anti-bacterial and anti-washout properties by adding the Ag-HAp powder into the above β-TCP cements, and examined their anti-bacterial property and cytotoxicity. The Ag-HAp powders with Ag contents of 0, 2, and 5 mol% as a nominal composition were synthesized by an USSP technique. The raw powder for β-TCP cement was prepared by ball-milling the commercially-available β-TCP powder in the IP6 solution. The Ag-HAp/β-TCP powders were prepared by mixing Ag-HAp powder and β-TCP cement powder at a ratio of 25:75 in mass. The Ag-HAp/β-TCP cement was fabricated by mixing the above-mentioned Ag-HAp/β-TCP powder and 2.5 mass% Na2HPO4 solution at a powder/liquid ratio of 1/0.3 [g/cm3]. The anti-bacterial property of resulting cements was evaluated using Staphylococcus aureus by biofilm formation test. The Ag-HAp/β-TCP cements containing 2 and 5 mol% Ag showed strong anti-bacterial property among examined specimens. Furthermore, the cytotoxicity of Ag+ ion eluted from these cements was also examined using osteoblastic MC3T3-E1 cells and Transwell® kit. The relative cell viability cultured on each Ag-containing cement specimen was over 80 %, compared with the control (polystyrene plate). These results demonstrate that the present Ag-HAp/β-TCP cements containing 2 mol% Ag are promising one of the candidates as CPCs with both anti-bacterial property and biocompatibility.