Takashi Oono

Assessment of Streptococcus pyogenes microcolony formation in infected skin by confocal laser scanning microscopy

BACKGROUND Streptococcus pyogenes and Staphylococcus aureus are often simultaneously detected from many cases of non-bullous impetigo with atopic dermatitis. OBJECTIVES Using confocal laser scanning microscopy (CLSM), to investigate formation of S. pyogenes microcolonies in skin lesions. METHODS The S. pyogenes cells in the stationary growth phase alone were strongly stained with fluorescein isothiocyanate-concanavalin A (FITC-ConA), and this staining was reduced by pretreatment with amylase. Although the components of sugars in glycocalyx produced by S. pyogenes cells are unknown, we suggested that the materials stained by FITC-ConA were consistent with the presence of ConA-reactive sugars in glycocalyx produced by S. pyogenes cells. RESULTS S. pyogenes cells associated with streptococcal impetigo skin and croton-oil inflamed mouse skin formed microcolonies encircled by materials (glycocalyx) that stained strongly with FITC-ConA, and these findings were consistent with those in biofilms. In croton-oil inflamed mouse skin, polymorphonuclear leukocytes (PMNs) infiltrated to just below the epidermis in the cefdinir-treated group but only to the middle dermis in the cefdinir-non-treated group. In this case S. pyogenes and S. aureus cells formed separate microcolonies and existed independently in the outer walls of pustule lesions of streptococcal impetigo. CONCLUSION In skin infections, S. pyogenes and S. aureus formed aggregates of microcolonies (similar to that in biofilms) encircled by glycocalyx, which can make the infection hard to eradicate using an antimicrobial agent alone. The effect of conventional antimicrobial agents against biofilm is mainly due to the increase of the invasion of PMNs into the biofilm.

Confocal laser scanning microscopic observation of glycocalyx production by Staphylococcus aureus in mouse skin: does S. aureus generally produce a biofilm on damaged skin?

Summary Background Bacteria that adhere to damaged tissues encase themselves in a hydrated matrix of polysaccharides, forming a slimy layer known as a biofilm. This is the first report of detection of glycocalyx production by Staphylococcus aureus using confocal laser scanning microscopy (CLSM) on damaged skin tissues.

Confocal laser scanning microscopic observation of glycocalyx production by Staphylococcus aureus in skin lesions of bullous impetigo, atopic dermatitis and pemphigus foliaceus

Summary Background Glycocalyx collapses during dehydration to produce electron‐dense accretions. Confocal laser scanning microscopy (CLSM) may be used to visualize fully hydrated microbial biofilms.

Dynamic alteration of human β-defensin 2 localization from cytoplasm to intercellular space in psoriatic skin

Abstract. Defensins are cationic antimicrobial peptides with a broad spectrum. Recently human β-defensin 2 (hBD-2) has been isolated from psoriatic skin; however, its exact localization and fate have not been fully understood. We studied the distribution pattern of hBD-2 in skin tissues of psoriasis and other inflammatory skin diseases. In the upper spinous and granular layer of psoriasis vulgaris hBD-2 was present in the cytoplasm. In the horny layer the positive signals were in a basket-weave pattern, indicating possible accumulation of hBD-2 in the intercellular space. The similar pattern of hBD-2 distribution was observed in the lesions of nummular eczema and atopic dermatitis. hBD-2 was not detected in the section of normal elbow and knee skin. When isolated psoriatic scales were stained, hBD-2 was detected in a wrapping paper-like distribution pattern surrounding the corneocytes. In horny layer of psoriatic skin hBD-2 was closely associated or colocalized with elafin, which is known to be in extracellular space, as demonstrated by double staining. Western blot analysis using cultured human keratinocytes detected hBD-2 with an expected size in the conditioned medium and in the cell lysates when stimulated with 5% FCS or IL-α. These results indicate that hBD-2 was synthesized and remained in cytoplasm in the upper spinous and granular layer, and then secreted into intercellular space in the horny layer. This dynamic change in hBD-2 distribution in epidermis is certainly relevant to function as an innate host defense mechanism against invading micro-organisms.

Assessment of cadexomer iodine against Staphylococcus aureus biofilm in vivo and in vitro using confocal laser scanning microscopy.

Cadexomer iodine releases iodine (0.9% weight/weight) slowly from beads of dextrin and epichlorhydrin. This preparation is an effective debridement and antiseptic agent for chronic exdudative wounds. The purpose of the present study is to examine the influence of cadexomer iodine against glycocalyx production of Staphylococcus aureus isolated from furuncle lesions on cut wounds in mice using confocal laser scanning microscope (CLSM), and the increase in and glycocalyx production of S. aureus in vitro. In the present study, distinct S. aureus cells and glycocalyx were not detected in the dermis around the cadexomer iodine beads or within those beads, while S. aureus cells encircled by glycocalyx were soaked up by the cadexomer beads and were detected within them in vivo and in vitro. We suggest that cadexomer iodine soaks up S. aureus cells encircled by glycocalyx, directly destroys biofilm structures, and collapses glycocalyx during dehydration, and further, that iodine can subsequently kill S. aureus cells within biofilm. Cadexomer iodine is a promising treatment to clear S. aureus cells within biofilm from skin lesions of exudative or infectious wounds and to prevent wound exacerbation.

The role of CD4 and CD8 cytotoxic T lymphocytes in the formation of viral vesicles.

Background  Herpetic vesicles caused by herpes simplex virus and varicella zoster virus, and hydroa vacciniforme (HV) are characterized by umbilicated vesicule formation.

Effects of human neutrophil peptide-1 on the expression of interstitial collagenase and type I collagen in human dermal fibroblasts.

Human neutrophil peptide-1 (HNP-1), a defensin with antimicrobial properties, is also thought to promote wound healing. To elucidate the mechanism by which wound healing is facilitated by this factor, we investigated the effect of HNP-1 on the expression of interstitial collagenase (matrix metalloproteinase 1, MMP-1), collagen types I and III, and tissue inhibitor of metalloproteinase 1 (TIMP-1) by cultured fibroblasts by means of RT-PCR and ELISA. Our results showed that synthetic HNP-1 increased the expression of proα1(I) collagen mRNA and protein. In contrast, the expression of MMP-1 was decreased at both the mRNA and protein levels. Our observations suggest that HNP-1 may promote wound repair by enhancing extracellular matrix deposition and by controlling its degradation.

Actions of Farnesol and Xylitol against Staphylococcus aureus

Background: Heavy colonization of atopic dermatitis (AD) with Staphylococcus aureus is well documented. The isolation rate of methicillin-resistant S. aureus is high in strains from AD in Japan. Our objective in the present study was to investigate the actions of farnesol and xylitol against S. aureus for the control of AD skin lesion-colonizing S. aureus.Methods: We examined the actions of farnesol on plasma coagulation and superantigenic exotoxin production by S. aureus, the antimicrobial activity of β-lactam antibiotics combined with farnesol at concentrations below the minimal inhibitory concentration (MIC) and the effect of xylitol on glycocalyx production. Results: Coagulation by S. aureus cells was inhibited in plasma containing farnesol at a concentration of 1/12 of the MIC (100 µg/ml) after incubation for 24 h. The production of superantigenic exotoxins by S. aureus cells with farnesol (100 µg/ml) was about 10 times lower than that by S. aureus cells alone. The MICs of ampicillin and cefdinir against S. aureus were reduced to ≤0.06 µg/ml in Mueller-Hinton agar plates with farnesol (100 µg/ml). We suggest that farnesol at concentrations above the MIC had a suppressive effect against S. aureus cells in the exponential and stationary phase and acted on the cell wall of S. aureus cells in both phases. Conclusions: Farnesol is a promising adjuvant agent against S. aureus skin infections treated with β-lactam antibiotics. Further, 5% xylitol inhibited glycocalyx production by S. aureus cells and consequently had a suppressive effect on the colonization of S. aureus on the horny cells of AD lesions.

Confocal laser microscopic observation of glycocalyx production by Staphylococcus aureus in vitro

We used a scanning confocal laser microscope to study the effects of various agents on sugar production by Staphylococcus aureus in vitro. S. aureus cells attached to coverslips in Pl-TSB (plasma:tryptic soy broth=1:1) were stained with fluorescein isothiocyanate-conjugated concanavalin A (FITC-conA) and were more strongly stained over time. We considered that the materials that stained positive for FITC-conA consistent with S. aureus cells were sugars, probably glycocalyx, produced by the S. aureus cells. Since the cells in the stationary growth phase alone were strongly stained with FITC-conA, all S. aureus cells attached to the coverslips in Pl-TSB were considered to be in this phase (low growth rate). The positive staining for FITC-conA was markedly reduced when fibrin was not formed in Pl-TSB with plasmin and sucrose, and was also markedly reduced when the fibrin in Pl-TSB was destroyed with plasmin. In conclusion, the results of the present study indicate that the existence of fibrin is essential for glycocalyx production and biofilm formation of S. aureus cells to aid in the attachment of S. aureus cells in vitro, because S. aureus cells attached on coverslips and fibrin alone produce glycocalyx. Of the antimicrobial agents tested, sulfadiazine silver most strongly inhibited the production of FITC-conA-positive materials by S. aureus cells at a sub-MIC concentration. Plasmin, sucrose, and sulfadiazine silver may be useful topical applications for use on clinical dermatology for the prevention and the treatment of staphylococcal biofilms. We consider that this simple method is very useful for the detection of S. aureus glycocalyx on dermatology field.

Pemphigus vegetans with IgG and IgA antidesmoglein 3 antibodies

SIR, Pemphigus vegetans is an uncommon variant of pemphigus that is characterized by vegetating erosions on the intertriginous area. Direct immunofluorescence (DIF) reveals IgG deposition on keratinocyte cell surfaces in the epidermis in all cases, usually associated with C3 deposition. A few case reports of pemphigus vegetans demonstrated IgA deposition on keratinocyte cell surfaces, although the nature of the IgAclass antibodies remains to be determined. We describe a patient with pemphigus vegetans who had both IgG and IgA antidesmoglein (Dsg) 3 antibodies. A 67-year-old Japanese man visited our hospital in August 2003 because of a 2-month history of pustules and erosions appearing on his face, axillae and groins. The skin lesions extended to the abdomen, buttocks and scalp. He had had a gastric ulcer and cerebral infarction at the age of 58 and 61 years, respectively, and he was treated with an antihypertensive drug. Cutaneous examination revealed pustules and erosions on the verrucous plaques in the above-mentioned areas (Fig. 1). Aphthae and white plaques were seen in the oral mucosa and soft palate. Laboratory tests showed anaemia and hypoproteinaemia. The patient’s C-reactive protein level was 3Æ3 mg dL (normal < 0Æ3), and his serum IgA level was 411 mg dL (normal 110–424). Other routine tests were within normal limits. Skin biopsy from the inguinal lesion showed marked acanthosis, suprabasal acantholysis in focal areas and large intraepidermal abscesses composed of many eosinophils (Fig. 2). A dense infiltrate with mixed cells of eosinophils, neutrophils and lymphocytes was observed in the upper dermis. DIF revealed the deposition of IgG, IgA and C3 on keratinocyte cell surfaces in the epidermis. Indirect immunofluorescence for IgG, IgA, IgM and C3 was negative in each case. Immunoblotting using human epidermal extracts showed no specific binding of IgG or IgA from the patient’s serum. The IgG enzyme-linked immunosorbent assay (ELISA) index values of anti-Dsg1 and anti-Dsg3 antibodies were 5Æ2 and 51Æ2 (cut-off value 20), respectively, determined by MESACUP desmoglein test ‘Dsg1’ and ‘Dsg3’ (MBL, Nagoya, Japan). We further performed ELISA for detecting IgA anti-Dsg1 or anti-Dsg3 antibodies using the same plates as described above and horseradish peroxidase-conjugated rabbit polyclonal antihuman IgA antibody (Dako, Glostrup, Denmark). We prepared 20 serum samples from normal individuals (age 59 ± 16Æ6 years) following informed consent to set a cut-off value for the ELISA. The mean ± SD of the normal control samples on ELISA for IgA anti-Dsg1 and anti-Dsg3 antibodies was 0Æ095 ± 0Æ014 and 0Æ106 ± 0Æ029, respectively. To exclude the background levels of normal sera, we set the cutoff value of 0Æ200 for Dsg1 and 0Æ250 for Dsg3. The values in the patient’s sera before therapy were 0Æ123 (negative) for Dsg1 and 0Æ900 (positive) for Dsg3. We also performed a novel ELISA using baculovirusexpressed human desmocollins 1–3, as reported previously. Neither IgG nor IgA was detected in the desmocollin ELISAs. The patient was hospitalized, and treated with oral prednisolone 20 mg daily, but no improvement was observed after 10 days of treatment. Therefore, treatment with a combination of oral prednisolone 30 mg daily and dapsone 75 mg daily was started. This regimen resulted in healing of the verrucous lesions within 3 weeks. The doses of prednisolone and dapsone were tapered to 20 mg and 50 mg daily, A