Morphological study of efficacy of clarithromycin-loaded nanocarriers for treatment of biofilm infection disease.

In this study, we developed a drug delivery system (DDS) using polymeric nanocarriers for the treatment of biofilm infection disease. Clarithromycin (CAM)-encapsulated and chitosan (CS) modified polymeric nanoparticles (NPs) were prepared using a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (Soluplus®) (Sol) and poly-(DL-lactide-co-glycolide), respectively. To understand the availability of the prepared NPs, we made morphological observations of the antibacterial activity derived from the NPs toward the bacterial cells within the biofilm using scanning electron microscopy and transmission electron microscopy measurements. These results revealed different antibacterial activities for the two types of drug carriers. In the case of CAM-encapsulated + CS-modified Sol micelles treatment, NPs can exert their antibacterial activity not only by the surfactant, CAM and CS effects but also by intrusion into the bacterial cells. Thereby, CAM-encapsulated + CS-modified Sol micelles had a higher antibacterial activity. The morphological information is useful to design suitable NPs for the treatment against biofilm infections.

Scanning transmission electron microscopic analysis of nitrogen generated by 3, 3'-diaminobenzidine-besed peroxidase reaction with resin ultrathin sections of rhinoceros parotid gland acinar cells.

A 3, 3'-diaminobenzidine (DAB)-based method was used to detect the localization of endogenous peroxidase activity in Indian rhinoceros (Rhinoceros unicornis) parotid gland acinar cells. The tissue had previously been resin-embedded in gelatin capsules for routine electron microscopic observations and thus pre-incubation for endogenous peroxidase analysis was not possible. We attempted to demonstrate the relationship between secretory granules (SGs) in resin ultrathin sections of Indian rhinoceros parotid gland acinar cells and endogenous peroxidase activity. A JEM 1400 Plus scanning transmission electron microscope (STEM) was used to conduct energy dispersive X-ray spectroscopy (EDS) analysis of the presence of nitrogen generated by the DAB reaction in bipartite structural SG consisting of a dense body (or core). The mapping patterns of nitrogen were restricted to the dense body. We observed nitrogen localized in the rough endoplasmic reticulum (ER), nuclear envelope (NE) and several components of the Golgi apparatus (G) of rhinoceros parotid gland acinar cells participating in the synthetic pathway of secretory proteins. Moreover, we established a nitrogen-detection method by EDS analysis of rhinoceros parotid gland. The reliability of the method was validated by comparison of the test group (peroxidase detection in ultrathin resin sections) and the control group (ordinary peroxidase detection in semi-thin sections following glutaraldehyde pre-fixation) of rat submandibular gland. The same mapping patterns of nitrogen were detected by DAB reaction in the SG, ER, NE and G in these two groups. Hence, EDS-STEM approaches for endogenous peroxidase post-incubation analysis will prove useful for advanced cytochemical analysis for the identification of any other resin sections.

Immunocytochemical approach for surface layer proteins of freeze-substituted Tannerella forsythensis by energy-filtering transmission electron microscopy.

Tannerella forsythensis (Bacteroides forsythus), an anaerobic gram-negative potential periodontal pathogens in the progression of periodontitis. IT forsythensis has unique bacterial protein profiles containing major proteins with apparent molecular weight of more than 200-kDa shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis. It is also known to have a typical surface layer (S-layer) consisting of regularly arrayed subunits outside the outer membrane revealed by electron microscopy. On the other hand, electron microscopy showed that the best preservation of structure was obtained when cells were postfixed with OsO4, but this resulted in very low levels of gold particles labeling. Therefore, cells were applied to pieces of filter paper and freeze-substituted by plung-freezing in Liquid propane, substituted in methanol containing 0.5% uranyl acetate, and infiltrated with LR-White resin. We also examined the relation between high molecular weight proteins and S-layer in energy-filtering transmission electron microscopy (EF-TEM) to visualize 3,3'-diaminobenzidene, tetrahydrochloride (DAB) reaction. The three-window method in electron spectroscopic images (ESI) of nitrogen (N) element, reflecting the presence of DAB moieties by the DAB reaction solution, horseradish peroxidase (HRP)-conjugated secondary antibodies instead of immunogold particles obtained by the EF-TEM. The mapping patterns of net N element were restricted to the outermost cell surface.

Energy dispersive spectroscopy-scanning transmission electron microscope observations of free radical production in human polymorphonuclear leukocytes phagocytosing non-opsonized Tannerella forsythia.

We investigated the association between human polymorphonuclear leukocytes (PMNs) and non-opsonized Tannerella forsythia ATCC 43037 displaying a serum-resistant surface layer (S-layer). When PMNs were mixed with T. forsythia in suspension, the cells phagocytosed T. forsythia cells. Nitro blue tetrazolium (NBT) reduction, indicative of O2- production, was observed by light microscopy; cerium (Ce) perhydroxide deposition, indicative of H2 O2 production, was observed by electron microscopy. We examined the relationship between high-molecular-weight proteins of the S-layer and Ce reaction (for T. forsythia phagocytosis) using electron microscopic immunolabeling. Immunogold particles were localized within the PMNs and on cell surfaces, labelling at the same Ce-reacted sites where the S-layer was present. We then used energy dispersive spectroscopy (EDS)-scanning transmission electron microscope (STEM) to perform Ce and nitrogen (N) (for S-layer immunocytochemistry) elemental analysis on the phagocytosed cells. That is, the elemental mapping and analysis of N by EDS appeared to reflect the presence of the same moieties detected by the 3,3'-diaminobenzidine-tetrahydrochloride (DAB) reaction with horseradish peroxidase (HRP)-conjugated secondary antibodies, instead of immunogold labeling. We focused on the use of EDS-STEM to visualize the presence of N resulting from the DAB reaction. In a parallel set of experiments, we used EDS-STEM to perform Ce and gold (Au; from immunogold labeling of the S-layer) elemental analysis on the same phagocytosing cells.

Anchoring and length regulation of Porphyromonas gingivalis Mfa1 fimbriae by the downstream gene product Mfa2.

Porphyromonas gingivalis, a causative agent of periodontitis, has at least two types of thin, single-stranded fimbriae, termed FimA and Mfa1 (according to the names of major subunits), which can be discriminated by filament length and by the size of their major fimbrilin subunits. FimA fimbriae are long filaments that are easily detached from cells, whereas Mfa1 fimbriae are short filaments that are tightly bound to cells. However, a P. gingivalis ATCC 33277-derived mutant deficient in mfa2, a gene downstream of mfa1, produced long filaments (10 times longer than those of the parent), easily detached from the cell surface, similar to FimA fimbriae. Longer Mfa1 fimbriae contributed to stronger autoaggregation of bacterial cells. Complementation of the mutant with the wild-type mfa2 allele in trans restored the parental phenotype. Mfa2 is present in the outer membrane of P. gingivalis, but does not co-purify with the Mfa1 fimbriae. However, co-immunoprecipitation demonstrated that Mfa2 and Mfa1 are associated with each other in whole P. gingivalis cells. Furthermore, immunogold microscopy, including double labelling, confirmed that Mfa2 was located on the cell surface and likely associated with Mfa1 fimbriae. Mfa2 may therefore play a role as an anchor for the Mfa1 fimbriae and also as a regulator of Mfa1 filament length. Two additional downstream genes (pgn0289 and pgn0290) are co-transcribed with mfa1 (pgn0287) and mfa2 (pgn0288), and proteins derived from pgn0289, pgn0290 and pgn0291 appear to be accessory fimbrial components.