Hiroko Kanzaki

Effects of zinc oxide on the attachment of Staphylococcus aureus strains

We examined the attachment of Staphylococcus aureus to plastic tissue-culture coverslips after incubation for 24 h. The attachment to coverslips was weaker in rabbit plasma with 5% zinc oxide (ZnO) than in the control rabbit plasma without ZnO (P < 0.01). Plasma coagulation by S. aureus strains was not detected in plasma with 5% ZnO after incubation for 24 h. The membranous structure (an immature biofilm) was formed on the coverslips by S. aureus cells in plasma after incubation for 24 h. The colony counts of S. aureus cells on the membranous structures were lower in plasma with 5% ZnO, plasma with 0.2% hinokitiol, plasma with 5% ZnO + 0.2% hinokitiol, plasma with cefdinir at 4 minimum inhibitory concentration (MIC) and plasma with levofloxacin at 4 MIC, than in the control plasma after incubation for 24 h (P < 0.01). The colonies on the membranous structures completely disappeared in the case of plasma with 5% ZnO and 0.2% hinokitiol. The colony counts on membranous structures were lower in plasma with cefdinir at 4 MIC or levofloxacin at 4 MIC containing 5% ZnO than in plasma with cefdinir at 4 MIC or levofloxacin at 4 MIC only, (P < 0.05). The MICs of hinokitiol against S. aureus strains peaked at an MIC distribution of 16-32 micrograms/ml. The peak shifted to below 1 microgram/ml by adding 5% ZnO in agar plate method. The results suggest that the attachment of S. aureus cells to the coverslips is suppressed in the presence of 5% ZnO and that antistaphylococcal activities of cefdinir, levofloxacin and hinokitiol increase in the presence of 5% ZnO.

Staphylococcus aureus infection on cut wounds in the mouse skin : experimental staphylococcal botryomycosis

Staphylococcus aureus cells were inoculated on the cut wounds in the skin of cyclophosphamide-treated mice. Biopsy specimens were taken from three mice at 1, 3, 6, 12, 24, 36, 48 and 60 h after the inoculation and were examined by light and electron microscopies. One hour after the inoculation Staphylococcus aureus cells were seen around the cut wound and deeper into the subcutaneous tissue. By 6 h after the inoculation, Staphylococcus aureus cells formed clusters of bacterial colonies. By 36 h after the inoculation inflammatory cells, mainly polymorphonuclear leukocytes and macrophages, were seen around the clusters. Electron microscopic examination revealed fibril-like structures around the Staphylococcus aureus cells at 1 h. The Staphylococcus aureus cells were enclosed in membrane-like structures at 3 h. The membrane-like structures and the fibril-like structures were positive for Ruthenium red. By 12 h after the inoculation, the membrane-like structures increased in thickness and in electron density. Inflammatory cells were seen around but outside of the membrane-like structures at 24, 36 and 48 h. At 60 h the tissues around the membrane-like structures were degenerated and almost necrotic. These results suggest that Staphylococcus aureus cells may form biofilm in dermal or subcutaneous tissues in a neutropenic condition.

Biofilm formation of Staphylococcus aureus strains isolated from impetigo and furuncle: role of fibrinogen and fibrin

The formation of membranous structure (thickness from the plastic tissue-culture coverslip (hematoxylin-eosin) > 1 mm; periodic acid-Schiff-positive) was more prominent with Staphylococcus aureus (S. aureus) strains isolated from impetigo (coagulase types I.V origin) than with S. aureus strains isolated from furuncle (coagulase type IV origin) (P < 0.05) in the plastic tissue-culture coverslip in human plasma after 72 h. Attachment of S. aureus cells to a plastic tissue-culture coverslip was more marked in 0-3% fibrinogen/tryptic soy broth (TSB) than in plasma (P < 0.05). The formation of the membranous structure was observed on the plastic tissue-culture coverslip with 0.3% fibrinogen/human serum but not with 0.3% fibrinogen + 5% glucose/TSB. Electron microscopy revealed abundant fibrin around S. aureus cells at 4 h and Ruthenium red-positive materials increased at 24 and 72 h in plasma. Staphylococcus aureus cell attachment to the plastic tissue-culture coverslip in plasma decreased by addition of levofloxacin (LVFX) at 1/2 minimum inhibitory concentration (MIC) and clarithromycin (CAM) at 1/4 MIC. Polysaccharide production of S. aureus cells on the plastic tissue-culture coverslip in plasma decreased with the addition of CAM at 1/4 MIC. Fibrinogen is closely related to initiation of infection but biofilm formation requires the conversion of fibrinogen to fibrin. Thus, attachment of S. aureus cells to the plastic tissue-culture coverslip, conversion of fibrinogen to fibrin by coagulase-prothrombin complex, and production of abundant glycocalyx by S. aureus cells are at least required for the production of biofilm in staphylococcal skin infection.

Nasal and nasal-type natural killer/ T–cell lymphoma☆☆☆

Nasal and nasal-type natural killer (NK)/T-cell lymphomas follow an aggressive course and have a poor prognosis. Recent pathologic studies suggest that the disease is a malignant proliferation of NK cells, which often express CD56. An association with the Epstein-Barr virus has also been reported. Skin involvement occurred in each of the 3 patients studied. Radiation therapy provided some benefit to the patients in the early stages. Conventional chemotherapies were not effective. To overcome this multiple-drug resistance of the tumor cells, cyclosporine and high-dose chemotherapy was combined with peripheral-blood stem-cell transplantation. The average life span from the onset of the disease for our patients was 9.6 months. Further improvement in the management of nasal and nasal-type NK/T-cell lymphomas is necessary.

Changes in Staphylococcus aureus density and lesion severity after topical application of povidone-iodine in cases of atopic dermatitis

A case-control study was performed to examine the efficacy of 10% povidone-iodine solution applied to atopic dermatitis patients. The density of Staphylococcus aureus on the eczematous lesions and the lesion severity before and after the topical application of povidone-iodine were compared. We found a 10-100-fold decrease in the density of S. aureus after povidone-iodine treatment in patients colonizing S. aureus at an initial density of > 1000 CFU/10 cm2. Erythema and exudation also decreased after povidone-iodine treatment in patients colonizing S. aureus at an initial density of > 1000 CFU/10 cm2. The 10% povidone-iodine solution disinfected S. aureus cells when added immediately after the cells were mixed in human plasma; however, 10% povidone-iodine solution only reduced the density of S. aureus cells by 10-100-fold when S. aureus cells were harvested after a 24 h incubation in human plasma. Staphylococcus aureus cells harvested after 24 h incubation in human plasma were often surrounded by fibrin bundles and cells circumscribed by fibrin bundles could not be disinfected with 10% povidone-iodine solution. We suggest that S. aureus cells may produce biofilm-like structures in atopic dermatitis patients and that these structures may help S. aureus cells resist the 10% povidone-iodine treatment.

Antiepiligrin cicatricial pemphigoid: The first case report from Japan☆

We describe a Japanese man with antiepiligrin cicatricial pemphigoid and typical clinical features, including ocular involvement. Direct immunofluorescence showed IgG deposition at the basement membrane zone. Indirect immunofluorescence of 1M sodium chloride-split skin showed circulating antibasement membrane zone antibodies of IgG class reactive with the dermal side of the split. Immunoblotting of human epidermal and dermal extracts, as well as a bacterial fusion protein of BP180 NC16a domain, showed no specific reactivity. In contrast, with immunoprecipitation of either culture medium or cell lysate from normal keratinocytes, the patients serum clearly reacted with the protein epiligrin, a laminin isoform present in the lamina lucida of the human epidermal basement membrane zone. This is the first confirmed case of a Japanese patient with this disease entity.

Staphylococcus aureus infection on experimental croton oil-inflamed skin in mice

Staphylococcus aureus cells were inoculated on the surface of skin inflamed by application of croton oil in cyclophosphamide-treated mice. Skin specimens were taken at 1, 3, 6, 12, and 24 h inoculation and each specimen was examined by microscopy. The S. aureus cells which attached to the surface of the skin immediately after inoculation had invaded the horny layer within 1 h. The cells gradually penetrated deeper into the epidermis. Electron microscopy revealed fibril-like structures around the S. aureus cells and the cells which adhered to the horny layer and fibrin by means of Ruthenium red-positive, fibril-like structures. A combined application of 0.1% gentamicin ointment, 2% fusidic acid ointment, and clobetasol propionate ointment was more effective in decreasing the number of S. aureus cells in the lesions than was an application of clobetasol propionate ointment alone. However, a combined application of 0.1% gentamicin ointment and 2% fusidic acid ointment without clobetasol propionate ointment showed almost the same efficacy as that with clobetasol propionate ointment. Although povidone iodine killed S. aureus in vitro at a concentration of 0.01% (100 micrograms/ml) in 40 s, its in vivo efficacy was limited.

Comparison of the severity of atopic dermatitis lesions and the density of Staphylococcus aureus on the lesions after antistaphylococcal treatment

We examined the relationship between atopic dermatitis (AD) andStaphylococcus aureus by comparing changes in AD lesions and the bacterial density on the lesions after antimicrobial treatment with cefdinir. We found that there was a greater density ofS. aureus on red erythemas and exudative lesions than in light/dark red erythemas and non-exudative lesions of AD. Forty-one of 59 cases (69%) showed a decrease in colony count following antimicrobial treatment. In 28 of 39 cases (72%) there was a decrease of erythema, and in 18 of 22 cases (82%) there was a decrease in the amount of exudate both associated with a decrease in colony density following antimicrobial treatment. Because acute phases of atopic dermatitis, such as red erythemas and exudative lesions, were closely related to the colonization ofS. aureus, dense colonization withS. aureus may be an important factor in the exacerbation of AD. We believe that staphylococcal products such as α-toxin, various enzymes, coagulase, and superantigenic exotoxins affect some aspect of the inflammatory process, resulting in exacerbation of AD.

Coagulase-Negative Staphylococci Isolated from Various Skin Lesions

We isolated 162 coagulase‐negative staphylococci (48: from infection, 114: from colonization) from various skin diseases between January, 1995, and January, 1998. From eighteen infected cysts, 10 Staphylococcus epidermidis strains, 3 S. capitis strains, 2 S. hominis strains, 2 S. auricularis strains, and one S. saprophyticus strain were individually detected. Similarly, from ten folliculitis lesions, 6 S. epidermidis strains, 2 S. capitis strains, and 2 S. hominis strains, and from five furuncle lesions, 3 S. lugdunensis strains, one S. epidermidis strains, and one S. hominis strain were detected, respectively. Four abscesses with mild inflammatory signs were localized on the scalp; S. epidermidis strains alone were detected from them. From two felons, one S. lugdunensis strain and one S. haemolyticus strain were detected, respectively. Staphylococus epidermidis and S. lugdunensis strains seems to be more frequently associated with skin suppurative lesions than other strains. Staphylococcus hominis strains and S. capitis strains were suggested to be potential pathogens in the initiation of suppuration in various purulent skin lesions. Among the 28 S. epidermidis strains, 13 (46.4%) were methicillin‐resistant (oxacillin. minimum inhibitory concentration ≥4 μg/ml). Twelve (29.3%) out of the other 41 coagulase‐negative staphylococci were methicillin‐resisitant. Coagulase‐positive and ‐negative staphylococci showed no differences in susceptibility tests against various antistaphylococcal agents.

Initial Recruitment of Interferon-γ-Producing CD8+ Effector Cells, Followed by Infiltration of CD4+ Cells in 2,4,6-Trinitro-1-Chlorobenzene (TNCB)-Induced Murine Contact Hypersensitivity Reactions

Contact hypersensitivity (CHS) is an antigen‐specific, T‐cell‐mediated skin reaction in sensitized individuals. Recent studies have demonstrated that the murine CHS reaction to 2,4‐dinitrofluorobenzene (DNCB) is mediated by CD8+ T cells and down‐regulated by CD4+ T cells. We studied cellular events and functions of infiltrating cells in CHS reactions to 2,4,6‐trinitro‐1‐chlorobenzene (TNCB) in the skin and draining lymph nodes (LN) of BALB/c mice and compared them with the CHS reaction to a house dust mite antigen, Dermatophagoides farinae (Df). Mice were sensitized with TNCB or Df antigens, and CHS reactions elicited with the corresponding antigens were examined immunohistologically. Cytokines produced by individual cells isolated from the CHS reactions were analyzed by flow cytometry, and the expression of cytokine mRNA was assayed by reverse transcription‐polymerase chain reaction (RT‐PCR). Results demonstrated that the intensity of TNCB‐elicited CHS skin reactions reached its peak at 24 hr after elicitation and decreased gradually. Flow cytometric analysis of isolated cells from the TNCB‐elicited CHS reactions demonstrated that the infiltrating cells were composed of approximately 25% CD4+ and CD8+ cells at 12, 24, and 36 hr after challenge, although infiltrating cells became dense at 36 hr by histological observation. The percentage of IFN‐γ‐producing CD8+ cells (Tc1) in the cell fractions reached its peak at 12 hr and decreased gradually. The peak infiltration of IFN‐γ‐producing CD4+ cells (Th1) was observed at 24 hr. IL‐4‐producing cells, however, were always below 5% in the cell fractions. The RT‐PCR method demonstrated that IFN‐γ mRNA was detected in the TNCB‐elicited skin reactions at 12, 18 and 24 hr after elicitation, became weak at 48 hr, and disappeared at 72 hr. No IL‐4 mRNA was detected from 12 to 72 hr. In the draining LN cells, however, the percentages of both IFN‐γ‐producing CD8+ (Tc1) and CD4+ cells (Th1) decreased 12 to 36 hr after TNCB elicitation. CHS reactions of Df antigens were predominantly composed of infiltrations of CD4+ cells in to the skin, associated with the expression of IL‐4 mRNA from 12 to 48 hr after elicitaion. The expression of IFN‐γ mRNA was detected at 48 hr or later. Our findings indicate that the CHS skin reaction to TNCB is induced by early recruitment of IFN‐γ‐producing CD8+ effector cells (Tc1), followed by infiltration of IFN‐γ‐producing CD4+ cells (Th1), whereas IL‐4‐producing T cells (Th2) induce the early CHS response to Df antigens.