Five Japanese cases of antidesmoglein 1 antibody-positive and antidesmoglein 3 antibody-negative pemphigus with oral lesions.

BACKGROUND: Oral mucosal lesions develop in pemphigus vulgaris, but not in pemphigus foliaceus. This clinical phenomenon is explained by the 'desmoglein (Dsg) compensation theory'. Dsg3 and Dsg1 are major autoantigens for pemphigus vulgaris and pemphigus foliaceus, respectively. Dsg3 is overexpressed and Dsg1 is weakly expressed on the oral mucosa. Thus, on the oral mucosa, suppression of Dsg3 function by anti-Dsg3 autoantibodies is not compensated by weakly expressed Dsg1 in pemphigus vulgaris, while suppression of Dsg1 function by anti-Dsg1 autoantibodies is perfectly compensated by richly expressed Dsg3 in pemphigus foliaceus. OBJECTIVES: We present five Japanese patients with pemphigus who deviate from this theory, i.e. all patients showed oral lesions (three also had cutaneous lesions) and reacted only with Dsg1, but not with Dsg3, by enzyme-linked immunosorbent assay. METHODS: To confirm whether the unique clinical phenotypes in our patients were due to a different immunological profile from that in classical pemphigus, we examined the reactivity of the patient sera by immunoprecipitation-immunoblotting analysis using five Dsg1/Dsg2 domain-swapped molecules. RESULTS: The sera of two patients who had only oral lesions tended to react with the extracellular (EC) 5 domain of Dsg1, the domain that is considered nonpathogenic in classical pemphigus foliaceus. Sera of three patients with mucocutaneous lesions reacted with EC1 domain or with both EC1 and EC2 domains of Dsg1, like classical pemphigus foliaceus. CONCLUSIONS: These results indicate that antigenic diversity of anti-Dsg1 antibodies in these patients may cause the unique oral mucosal and cutaneous lesions, although further studies are required to elucidate the pathomechanisms.

Relationship between metabolic syndrome components and vascular properties in Japanese type 2 diabetic patients without cardiovascular disease or nephropathy.

To investigate the effect of metabolic syndrome (MS) components on early atherosclerosis markers, i.e., urinary albumin excretion rate (UAE), pulse wave velocity (PWV), and carotid intima-media thickness (IMT), we studied 536 Japanese patients with type 2 diabetes without cardiovascular disease or nephropathy. The MS definition by ATP III was employed. UAE, PWV, and IMT increased significantly with increasing the number of components even before fulfilling the diagnosis of MS. UAE was significantly influenced by high blood pressure, high triglycerides, and low HDL cholesterol. PWV was significantly increased by high blood pressure. IMT was significantly increased by high blood pressure and abdominal obesity. Multiple regression analysis, including MS components and putative risk factors, indicated that the number of MS components, age and glycosylated HbA1C were independent determinants of UAE, PWV, and IMT. LDL cholesterol and male gender were independent determinants of IMT. In conclusion, UAE, PWV, and IMT increased according to increasing the number of MS in type 2 diabetic patients without cardiovascular disease or diabetic nephropathy. The current observation considering the modifiable factors may help to identify patients who are at high risk of experiencing cardiovascular disease.

Prorenin processing and restricted endoproteolysis by mouse tissue kallikrein family enzymes (mK1, mK9, mK13, and mK22).

Four members of the tissue kallikrein family, mK1, mK9, mK13, and mK22, all of which exhibit extensive homology in amino acid sequence among themselves, were obtained from the submandibular gland of ICR mice and examined for their ability to cleave prorenin. Tissue kallikrein mK13 was confirmed to be a prorenin-converting enzyme; and mK9, which was earlier shown to be an EGF-binding protein, was found to cleave mouse Ren 2 prorenin specifically and convert it to mature renin with an activity of approximately 1/10 of that of mK13. With the same substrate, mK22 (beta-NGF endopeptidase) gave two products, renin and arginyl-renin; whereas mK1 (true tissue kallikrein) did not process it at all. The endoproteolytic activity of tissue kallikreins was examined with various peptide-MCA substrates. The substrates contained three key structures; X(Y)-Arg-Arg, X(Y)-Lys-Arg and X-Lys-Lys motifs (where X and Y are hydrophilic and hydrophobic amino acids, respectively). We found that mK1, mK9 and mK13 preferentially cleaved the former two types of substrate, except Y-Arg-Arg-MCA. The substrate X-Lys-Lys-MCA was hardly cleaved by these three tissue kallikreins but was preferentially cleaved by mK22. The four tissue kallikreins seem to have the ability to process precursor proteins containing a pair of basic amino acid residues; the specificities of three of the enzymes (mK1, mK9 and mK13) were similar to each other but were different from that of mK22.

Increased type 2 cytokine expression by both CD4+ CD45RO+ T cells and CD8+ CD45RO+ T cells in blood circulation is associated with high serum IgE but not with atopic dermatitis.

Type 2 cytokines, such as interleukin-4 (IL-4) and IL-13, are associated with immunoglobulin E (IgE) production. This association has also been observed in CD8+ T cells from patients infected with leprosy and human immunodeficiency virus (HIV). Using intracellular cytokine staining and flow cytometry, the cytokine profile [IL-2, IL-4, IL-10, IL-13, and interferon (IFN)-gamma] of both CD4+ and CD8+ memory/effector T cells circulating in atopic dermatitis (AD) patients was investigated at the single cell level. The levels of type 2 cytokines in CD4+ T cells or CD8+ T cells in AD patients with high levels of serum IgE (AD-H), low levels of serum IgE (AD-L), and healthy controls were compared. Increased production of IL-4 and IL-13 in both CD4+ CD45RO+ T cells and CD8+ CD45RO+ T cells after 4 h in vitro stimulation with phorbol 12-myristate 13-acetate and ionomycin, was more prominent in AD-H patients than in AD-L patients or healthy controls, whereas IFN-gamma-producing CD4+ CD45RO+ T cells and CD8+ CD45RO+ T cells were relatively diminished in AD-H patients. CD4+ T cells and CD8 + T cells from AD-H patients, cultured for 48 h with phorbol 12-myristate 13-acetate and ionomycin, released larger amounts of IL-4 and IL-13 but smaller amounts of IFN-gamma than both types of cells from AD-L patients or healthy controls. In addition, when stimulated with immobilized anti-CD3 monoclonal antibody (MoAb) and anti-CD28 MoAb, CD4+ CD45RO+ T cells and CD8+ CD45RO+ T cells from AD-H patients contained more IL-4-producing cells but fewer IFN-gamma-producing cells compared with healthy controls. Finally, spontaneous mRNA expression of IL-4 in blood CD8+ CD45RO+ T cells isolated from AD-H patients was increased, as determined by reverse transcriptase-polymerase chain reaction. Therefore, in AD patients with high IgE levels, type 2 cytokine (IL-4 and IL-13) expression is associated with IgE production, in both CD4+ CD45RO+ T cell and CD8+ CD45RO+ T cell subsets.

Characteristics of neutron beam generated by 500 MeV proton beam.

A neutron irradiation facility was constructed at PARMS, University of Tsukuba to produce an ultrahigh energy neutron beam with a depth dose distribution superior to an x-ray beam generated by a modern linac. This neutron beam was produced from the reaction on a thick uranium target struck by a 500 MeV proton beam from the booster synchrotron of the High Energy Physics Laboratory. The percentage depth dose of this neutron beam was nearly equivalent to that of x-rays around 20 MV and the dose rate was 15 cGy per minute. The relative biological effectiveness (RBE) of this neutron beam has been estimated using the cell inactivation effect and the HMV-I cell line. The survival curve of cells after neutron irradiation has a shoulder with n and Dq of 8 and 2.3 Gy, respectively. The RBE value at the 10(-2) survival level for the present neutron beam as compared with 137Cs gamma rays was 1.24. The results suggest that the biological effects of ultrahigh energy neutrons are not large enough to be useful, although the depth dose distribution of neutrons can be superior to that of high energy linac x-rays.

Clinical results of fractionated proton therapy.

PURPOSE: Preliminary results of a multi-site Phase I-II clinical trial investigating the efficacy of high-energy proton beams in a wide variety of human malignancies are reported. METHODS AND MATERIALS: Since 1983 proton radiotherapy using 250 MeV proton beams produced by a booster synchrotron of the National Laboratory for High Energy Physics has been carried out at Proton Medical Research Center, University of Tsukuba. As of September 1990, a total of 147 patients received a partial or full treatment with proton beams with curative intent; 92 patients (63%) were treated with proton beams alone and 55 patients (37%) with combined photon and proton beams. There were 91 males and the mean age was 61.8 years old. The follow-up observation period ranged from 10 to 97 months. With regard to a total tumor dose, nearly 80% of patients received 70 Gy or more and 53% received 80 Gy or more. While dose-fractionations used depended upon tumor sites, the large majority of patients received substantially high radiation doses in terms of larger total doses (> 70 Gy) and larger fraction sizes (> 2.5 Gy) than those traditionally used. This fractionation regimen has been used because of limited availability of the accelerator or a shortage of machine time (27-30 weeks/year, 3-3.5 hr/day), and also by the expectation that the superior dose distribution possible with protons will permit administration of high radiation doses without increasing morbidities. In connection with this, we have determined the target volume by setting margins around the tumor boundary as practically small as possible, ranging from 5 to 10 mm. RESULTS AND CONCLUSIONS: The current trial has been based on a site and dose searching program, hence a wide variety of tumor sites including the aerodigestive organs has been treated. So far, our judgment is that proton therapy has proven of potential advantage in treatment of the lung, esophageal, liver, uterine cervix, prostate, and head and neck malignancies; and of possible value in treatment of high-grade gliomas, and gastric, urinary bladder, and pediatric tumors.

Distribution of low-density lipoprotein receptors and apolipoprotein B on normal and on reconstructed human epidermis.

Immunoelectronmicroscopy was employed to investigate the ultrastructural localization of the low density lipoprotein (LDL) receptor and the protein moiety of LDL (apo B) on normal human epidermis in situ and reconstructed human epidermis in vitro. For this purpose ultrathin sections of Lowicryl K4M embedded material were incubated with monoclonal antibodies against the LDL receptor and apo B followed by a second antibody conjugated to 15 nm colloidal gold. Examination of the sections revealed a similar distribution of the LDL receptor and apo B in normal human epidermis and in reconstructed epidermis. In both systems the amount of LDL receptors decreases during keratinocyte differentiation. In contrast, apo B molecules are more abundantly expressed in the upper layers of the stratum spinosum and the stratum granulosum. The great similarities in the distribution of the LDL receptor and apo B between the in vitro reconstructed epidermis and its in vivo counterpart provide additional proof that reconstructed epidermis is an excellent tool to investigate proliferation and differentiation processes of keratinocytes in vitro.

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.

Characteristics in adherence of streptococci and Staphylococcus aureus isolated from various infective skin lesions: serum IgA decreases adherence of Streptococcus pyogenes but not Staphylococcus aureus.

We characterized adherence of streptococci and Staphylococcus aureus strains isolated from various infective skin lesions in terms of hydrophobicity, negative charge, tube adherence, slime production, and influence on adherence to coverslips by plasma and serum immunoglobulins. High hydrophobicity was more frequently observed in Streptococcus pyogenes strains than in Streptococcus agalactiae strains (P < 0.01) and S. aureus strains (P < 0.001) and slime production was more frequently observed in S. agalactiae strains than in S. pyogenes strains (P < 0.05). Serum IgA decreased adherence to coverslips of S. pyogenes strains but not that of S. aureus strains.

Calcium oxide and magnesium oxide inhibit plasma coagulation by Staphylococcus aureus cells at the lower concentration than zinc oxide.

We examined the effect of ceramic powder slurries on the coagulation of plasma by Staphylococcus aureus cells. Plasma coagulation by S. aureus strains or their cultured supernatant was inhibited in the plasma with 0.12% calcium oxide or 0.25% magnesium oxide after incubation for 24 h at 37 degrees C. Inhibition of plasma coagulation by calcium oxide and magnesium oxide was observed at the lower concentration than zinc oxide.

Adherence characteristics and susceptibility to antimicrobial agents of Staphylococcus aureus strains isolated from skin infections and atopic dermatitis.

We examined the adherence characteristics and susceptibility to various antimicrobial agents of 130 strains of Staphylococcus aureus isolated from infective skin lesions and 135 strains of S. aureus isolated from non-infective eczematous lesions of atopic dermatitis (AD) patients. The isolation rate of methicillin-resistant S. aureus (MRSA) was 27.7% in strains from clinical sources excluding AD and 31.1% in those from AD. Coagulase type II strains were most frequently observed in MRSA strains isolated from all sources excluding AD, and coagulase type III strains were most frequently observed in those isolated from AD. We proposed that antimicrobial treatment for AD patients should be carefully designed to prevent MRSA infection. Plasma coagulation ability was lowest in S. aureus strains isolated from abscesses, suggesting that the lower production of fibrin observed in abscesses may assist the infiltration of neutrophils into skin tissues and that a decrease in plasma coagulation ability may enable abscess formation. Adherence to polypropylene tubes with slime production was most evident in S. aureus strains isolated from felon and least evident in those isolated from cellulitis and lymphangitis. Tube adherence was characteristic of the S. aureus strains attached to superficial skin tissues, but not necessarily for strains that had infiltrated the deep skin tissues. Fusidic acid demonstrated significant antimicrobial activity against the MRSA strains, but rifampicin was the strongest antimicrobial agent.

The production of superantigenic exotoxins by coagulase-negative staphylococci isolated from human skin lesions.

We examined the production of superantigenic exotoxins in 136 coagulase-negative staphylococci isolated from various skin lesions in humans using a reversed passive latex agglutination test (Denka Seiken). As a control we examined the same in 50 Staphylococcus aureus strains isolated from non-infective skin ulcers in humans. Of the 136 strains of coagulase negative-staphylococci, 9 (6.6%) produced one or more identifiable exotoxins. In contrast, 21 (42%) out of the 50 S. aureus strains produced one or more identifiable exotoxins (P<0.01).

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.

Streptococci isolated from various skin lesions: the interaction with Staphylococcus aureus strains.

We isolated 73 streptococcus strains (41 from infections, and 32 from colonization) from various skin diseases between March, 1994, and June, 1998. In 29 out of 41 cases of infective origin, Staphylococcus aureus strains were simultaneously isolated. Twenty-four out of 28 patients with impetigo were suffering from atopic dermatitis. We confirmed that impetigo lesions where Streptococcus pyogenes was dominant in number always showed thick-walled pustules on an erythematous base; these skin lesions were considered to be an early manifestation of streptococcal impetigo. We further confirmed that thick-crusted lesions in streptococcal impetigo, where S. aureus exceeded S. pyogenes in number, were a late manifestation. Antimicrobial agents such as minocycline, fusidic acid, ofloxacin and tosufloxacin, were more effective against S. aureus strains than against beta-hemolytic streptococcal strains. In contrast, ampicillin, cefdinir, imipenem, erythromycin and vancomycin were more effective against beta-hemolytic streptococcal strains.

Adherence characteristics of Staphylococcus aureus and coagulase-negative staphylococci isolated from various skin lesions.

We investigated the adherence characteristics of Staphylococcus aureus and coagulase-negative staphylococci (CNS) isolated from various skin lesions by examining hydrophobicity, negative charge, tube adherence, slime production, and promotion of adherence to coverslips by blood proteins. Our results in the present study indicate that high hydrophobicity and high capacity for adhesion to tubes with slime production are much more detected in CNS from infective origin than in those from colonization origin. The results also indicate that host plasma tends to enhance adherence of S. aureus to coverslips, but it does not enhance adherence of most coagulase-negative staphylococci to coverslips.

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.

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.

Effects of various salts and irradiation with UV light on the attachment of Staphylococcus aureus strains.

We investigated the attachment of Staphylococcus aureus isolated from atopic dermatitis lesions to plastic tissue-culture coverslips. We found that attachment was weaker in (rabbit) plasma with 5 or 10% NaCl and in plasma with 5 or 10% sea salts than in the control plasma after incubation for 2 h (P < 0.01). The attachment was weaker still in plasma with 10% NaCl or 10% sea salts than in the control plasma after incubation for 24 h (P < 0.01). Plasma coagulation of four S. aureus strains isolated from atopic dermatitis lesions was not detected in plasma with 10% NaCl (pH 5.6) or 10% sea salts (containing 0.372% Mg2+) after incubation for 12, 24, 36 and 60 h. The attachment of S. aureus strain cells to the coverslip in plasma was weaker after irradiation with UVA at 25 or 50 J/cm2 (P < 0.01) and UVB at 0.5 J/cm2 (P < 0.05) both of which are covered by a black cloth, than without irradiation after incubation for 24 h. Plasma coagulation was not detected after irradiation with UVA at 25 or 50 J/cm2 with a black cloth cover (temperature reached 50 degrees C), but was detected after UVA irradiation at the same doses combined with cooling (temperature reached 22 degrees C), after incubation for 24 h. The results suggest that the attachment of S. aureus cells isolated from atopic dermatitis lesions to the coverslip is suppressed in the presence of 10% salts and irradiation with UVA and UVB, and that plasma coagulation of S. aureus cells isolated from atopic dermatitis lesions is suppressed in the presence of 10% salts, irradiation with UVA, and heating.

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.

Antimicrobial effects of acidic hot-spring water on Staphylococcus aureus strains isolated from atopic dermatitis patients.

The present study examined the antimicrobial effects of acidic hot-spring water on Staphylococcus aureus strains isolated from atopic dermatitis (AD) patients. Plasma coagulation by S. aureus cells was not detected in plasma containing acidic hot-spring water (60%, pH 5.4) or hydrochloric acid (pH 5.0) after incubation for 24 h. S. aureus cells did not grow in Mueller-Hinton broth with acidic hot-spring water (50%, pH 4.4) after 24 h incubation. The colony counts of S. aureus cells in tryptic soy broth containing acidic hot-spring water (60%, pH 3.9) were over ten times lower than those in tryptic soy broth alone after incubation for 24 h (P<0.01). A membranous structure (an immature biofilm) was formed on the coverslips of tissue culture dishes by S. aureus cells in plasma after incubation for 24 h, although the colony counts of S. aureus cells in the immature biofilms in plasma containing acidic hot-spring water (60%, pH 5.4) were about eight times lower than those in plasma alone after incubation for 24 h (P<0.01). The colony counts of S. aureus cells that attached on coverslips in plasma containing acidic hot-spring water (60%, pH 5.4) or hydrochloric acid (pH 5.4) were over 1000 times lower than those in plasma alone after incubation for 24 h. These results suggest that 50% acidic hot-spring water has a bacteriostatic effect, 60% acidic hot-spring water has a moderate bactericidal effect against floating S. aureus cells and those cells in a biofilm, and, 60% acidic hot-spring water has an inhibitory effect on plasma coagulation and attachment of S. aureus cells. Furthermore, our present results suggest that a small amount of some ions in hot-spring water such as manganese and iodide ions are very important for a bactericidal activity of hot-spring water as well as the low pH condition.