Molecular epidemiology and antimicrobial susceptibility of Clostridium difficile isolated from a university teaching hospital in Japan.

Clostridium difficile infection control strategies require an understanding of its epidemiology. In this study, we analysed the toxin genotypes of 130 non-duplicate clinical isolates of C. difficile from a university hospital in Tokyo, Japan. Multilocus sequence typing (MLST) and eBURST analysis were performed for these isolates and nine strains previously analysed by polymerase chain reaction (PCR) ribotyping. Minimum inhibitory concentrations (MICs) were determined for six antibiotics, and the bacterial resistance mechanisms were investigated. Ninety-five toxigenic strains (73%), including seven tcdA-negative, tcdB-positive and cdtA/cdtB-negative strains (A(-)B(+)CDT(-)) and three A(+)B(+)CDT(+) strains, and 35 (27%) non-toxigenic strains, were classified into 23 and 12 sequence types, respectively. Of these, sequence type (ST)17 (21.8%) was the most predominant. MLST and eBURST analysis showed that 139 strains belonged to seven groups and singletons, and most A(+)B(+)CDT(-) strains (98%, 89/91) were classified into group 1. All isolates were susceptible to metronidazole, vancomycin and meropenem; the ceftriaxone, clindamycin and ciprofloxacin resistance rates were 49, 59 and 99%, respectively. Resistance rates to ceftriaxone and clindamycin were higher in toxigenic strains than in non-toxigenic strains (P < 0.001). All ST17 and ST81 strains were resistant to these antibiotics. The clindamycin- and fluoroquinolone-resistant strains carried erm(B) and mutations in GyrA and/or GyrB, respectively. To our knowledge, this is the first MLST-based study of the molecular epidemiology of toxigenic and non-toxigenic strains in Japan, providing evidence that non-toxigenic and toxigenic strains exhibit high genetic diversity and that toxigenic strains are more likely than non-toxigenic strains to exhibit multidrug resistance.

Riverine environmental characteristics and seasonal habitat use by adult Sakhalin taimen Hucho perryi.

The study identified seasonal habitat use by endangered adult Sakhalin taimen Hucho perryi and the environmental characteristics of their habitat (water depth, amount of riparian forest and sinuosity). Fifteen adult H. perryi with acoustic tags were tracked by towing an acoustic receiver with a canoe in the Bekanbeushi River system in eastern Hokkaido Island, Japan, during each month from late April to late November 2008. Individuals mainly used midstream (shallower than downstream) habitats in all seasons. These locations were generally characterized by relatively dense riparian forests and high sinuosity, indicating the presence of pools. In spring, individuals used habitats with less riparian forest cover compared to mean value of the river channel. From spring to autumn, adult H. perryi selected limnologically complex habitats with meandering channels. From summer to autumn, individuals selected habitats with more riparian forest cover. The inverse relationship between H. perryi detection and riparian forest area in spring was a result of seasonal defoliation in deciduous riparian forests.

Molecular analysis of Clostridium difficile at a university teaching hospital in Japan: a shift in the predominant type over a five-year period.

Clostridium difficile isolates recovered from patients admitted to a teaching hospital in Japan over a 5-year period were analyzed. Two molecular typing systems, PCR ribotyping and pulsed-field gel electrophoresis (PFGE) analysis, were used. Twenty-six PCR ribotypes were found among the 148 isolates. The predominant type at our hospital appeared to shift during the study period, from PCR ribotype a in 2000 (15/33, 45%) to PCR ribotype f in 2004 (18/28, 64%). By using PFGE with thiourea added to both the gel and running buffer, all 148 Clostridium difficile isolates were successfully classified into 37 types and 61 subtypes. The PCR ribotype f isolates were further classified into four types and 11 subtypes by PFGE. The PFGE patterns of the 11 subtypes differed from each other by only 1 to 4 bands, suggesting that these differences might reflect genetic changes during patient-to-patient transmission over the 5-year period analyzed, and that PCR ribotype f isolates might be outbreak-related. In addition, the PCR ribotype f was identical to the PCR ribotype designated smz, which is reported to have caused multiple outbreaks in Japan. These results confirmed that PCR ribotype f (type smz) has specific virulence or survival factors that make it more likely to cause nosocomial outbreaks at Japanese hospitals. PCR ribotype 027, which has been reported to have caused recent outbreaks in North America and Europe, was recovered from one patient in this study; however, this strain was community-acquired. Our findings emphasize the importance of monitoring specific strains to control and prevent nosocomial infection.

Cyclic and linear peptides derived from alpha-amylase inhibitory protein tendamistat.

Tendamistat is a strong inhibitory protein of porcine pancreatic alpha-amylase (PPA) with a K(i) value of 0.2 nM. To develop potent alpha-amylase inhibitors, we synthesized six odd-length cyclic peptides (5-15 residues) and four even-length cyclic peptides (10 and 12 residues) having the inhibitory sequence of tendamistat. Their PPA inhibitory activities were evaluated, and, among them, the 11-residue cyclic peptide Ten(15-23) (K(i) = 0.27 microM) exhibited the strongest inhibitory activity (K(i) = 0.27-1.41 microM). To examine the effect of cyclic structure on PPA inhibition, ten linear peptides corresponding to the cyclic peptides were also synthesized, and their PPA inhibitory activities were evaluated (K(i) = 0.28-1.00 microM). Interestingly, the 11-residue linear peptide Ten(15-23) exhibited almost the same inhibitory activity (K(i) = 0.28 microM) as that of cyclic Ten(15-23). The results of a circular dichroism study indicated that stabilization of the beta-hairpin structure occurred only for cyclic Ten(15-23). Also, the results of proteolytic digestion experiments of the cyclic and linear Ten(15-23) peptides by trypsin and chymotrypsin suggested no differences in protease resistance between the cyclic and linear structures. Therefore, we demonstrated that both cyclic and linear peptides containing the inhibitory sequence of tendamistat exhibit potent PPA inhibitory activity.

Monocyte proliferation induced by modified serum is associated with endogenous M-CSF production: evidence for involvement of a signalling pathway via scavenger receptors.

It was found that human serum stored for 2 months at 4 degrees C (modified serum) induced monocyte proliferation and simultaneous macrophage colony stimulating factor (M-CSF) production by these cells in vitro. Cell number, estimated by DNA content, doubled after 10 days in culture in the presence of modified serum, while it decreased in culture with freshly thawed control serum. As the addition of more than 2.5 ng/ml of recombinant M-CSF significantly supported monocyte survival/proliferation, cells were cultured for 10 days in medium supplemented with control serum, and endogenous M-CSF production was investigated by enzyme-linked immunosorbent assay. M-CSF concentration in the supernatants was 15-30 ng/ml after 10 day in culture with modified serum, a level that might be sufficient for monocyte proliferation. The modified serum induced M-CSF from freshly isolated monocytes, while M-CSF was hardly detected in cultures supplemented with control serum. Assay for peroxidized lipid and agarose gel electrophoresis demonstrated that the modified serum contained more oxidized low density lipoproteins (LDL) than the control serum. Ligands of scavenger receptors, which are receptors for oxidized LDL, such as dextran sulphate, polyinosinic acid, heparin and acetylated LDL also significantly induced M-CSF production from human monocytes, although this was at levels below 2 ng/ml. These results indicate that serum modified by oxidation stimulates monocytes to produce M-CSF resulting in their proliferation, and that signalling via scavenger receptors is one of the mechanisms responsible for this induction of M-CSF.

Effects of macrophage colony-stimulating factor (M-CSF) on protease production from monocyte, macrophage and foam cell in vitro: a possible mechanism for anti-atherosclerotic effect of M-CSF.

M-CSF is a growth factor that stimulates proliferation and differentiation of monocyte/macrophage-lineage cells. In our previous studies, M-CSF regresses atherosclerotic lesions preformed in aorta of high cholesterol-fed rabbit. Immunohistochemical analysis indicated that extracellular matrix (ECM), such as collagen, was especially eliminated in the intima of atherosclerotic lesion. To define the collagen-lowering potential of M-CSF, we have studied the effects of M-CSF on production of collagen-degrading proteases, such as MMP-1, -9 and urokinase in vitro. Monocytes freshly isolated from human peripheral blood produced MMP-9, but not urokinase, and M-CSF enhanced MMP-9 production. Macrophages were prepared by culturing monocytes for 10 days in the presence or absence of M-CSF, and protease production was assayed. M-CSF augmented production of MMP-9 and urokinase in a dose-dependent manner. M-CSF also enhanced MMP-1 production of macrophages, but not significantly. Foam cells were prepared by culturing macrophages in the presence of acetyl LDL, and protease production from these cells were also elevated by M-CSF. These results suggest that M-CSF exogenously administered in atherosclerotic rabbits might regress the thickened intima by activating macrophages to degrade collagen accumulated in the lesion.

Differential expression of CD44 splice variants in malignant and benign pleural effusions.

Expression of CD44 isoform that contains variant exons (v1-v10) has been implicated in tumor progression and metastasis. Especially, CD44 isoform containing v6 (CD44v6) and that containing v7 (CD44v7) were shown to confer full metastatic behavior on tumor cells. We examined the expression of CD44v6 and CD44v7 in malignant pleural effusions (13 lung cancers and 4 non-lung cancers) and in benign pleural effusions (7 tuberculosis and 3 pneumonia) with Southern blot analysis of reverse transcription (RT)-polymerase chain reaction (PCR) products. CD44v6 was expressed not only in malignant pleural effusions (12 of 13 lung cancers and 4 of 4 non-lung cancers), but also in benign pleural effusions(9/10). In contrast, although expression of CD44v7 was found in most malignant pleural effusions (12 of 13 lung cancers and 4 of 4 non-lung cancers), it was found in only a few cases of benign pleural effusions. These results suggest that the expression of CD44v7 may be correlated with a tumor-specific event such as metastasis or dissemination in malignant pleural effusion, while no such correlation can be found with CD44v6.

Integration of bacteriophage Mx8 into the Myxococcus xanthus chromosome causes a structural alteration at the C-terminal region of the IntP protein.

Mx8 is a generalized transducing phage that infects Myxococcus xanthus cells. This phage is lysogenized in M. xanthus cells by the integration of its DNA into the host chromosome through site-specific recombination. Here, we characterize the mechanism of Mx8 integration into the M. xanthus chromosome. The Mx8 attachment site, attP, the M. xanthus chromosome attachment site, attB, and two phage-host junctions, attL and attR, were cloned and sequenced. Sequence alignments of attP, attB, attL, and attR sites revealed a 29-bp segment that is absolutely conserved in all four sequences. The intP gene of Mx8 was found to encode a basic protein that has 533 amino acids and that carries two domains conserved in site-specific recombinases of the integrase family. Surprisingly, the attP site was located within the coding sequence of the intP gene. Hence, the integration of Mx8 into the M. xanthus chromosome results in the conversion of the intP gene to a new gene designated intR. As a result of this conversion, the 112-residue C-terminal sequence of the intP protein is replaced with a 13-residue sequence. A 3-base deletion within the C-terminal region had no effect on Mx8 integration into the chromosome, while a frameshift mutation with the addition of 1 base at the same site blocked integration activity. This result indicates that the C-terminal region is required for the enzymatic function of the intP product.

[Effects of a prosthetic dental device used to treat obstructive sleep apnea syndrome, and compliance of patients using the device].

We studied the short-term effects and complications of the use of a dental device (prosthetic mandibular advancement: PMA) in 72 patients with obstructive sleep apnea syndrome (OSAS), and the compliance of these patients with this treatment. In 61 (84.5%) of the 72 patients, the apnea index decreased by more than 50%. The lowest SaO2 and symptom scores also improved significantly. The severity of OSAS was not related to the percent reduction in apnea index. Complications of PMA use were observed in 22 patients (30.6%), but no severe adverse effects were observed. Sixty-two of the 72 patients continued using the PMA throughout the entire study period (overall compliance rate, 86.1%). In addition, the long-term compliance rate (more than 5 years) was 61.5%. We conclude that the effects of PMA in patients with OSAS are clinically significant, that there are no severe complications, and that compliance with treatment is good.

[A case of alveolar soft part sarcoma found by pulmonary metastasis].

A 34-year-old housewife presented to a hospital because of dry cough. Her chest radiograph showed bilateral multiple nodular lesions. Smaller but similar lesions had been seen on the chest radiograph 2 years earlier. Because the tissue taken during a trans bronchial biopsy was non-diagnostic, open lung biopsy was done and the diagnosis was pulmonary metastasis of alveolar soft part sarcoma. The primary tumor was found in her left calf by MRI. Malignant tumors are important for differential diagnosis of slow-growing multiple pulmonary nodules, and in some cases MRI is useful for finding the primary site.

Protein, cell, and LTB4 concentrations of lung edema fluid produced by high capillary pressures in rabbit.

We previously demonstrated disruptions of the pulmonary capillary endothelium and alveolar epithelium at transmural pressures (Ptm) of 52.5 cmH2O in rabbit by electron microscopy. In the present study, we determined the characteristics of the alveolar edema fluid in this condition by carrying out bronchoalveolar lavage after blood perfusion for 10 min at Ptm of 12.5 (low), 32.5 (intermediate), and 52.5 cmH2O (high). At low Ptm, where our previous studies showed no ultrastructural changes, the volume of alveolar fluid obtained by urea dilution was very small, and the concentrations of proteins, cells, and leukotriene B4 (LTB4) in the bronchoalveolar lavage fluid (BALF) were low. However, at high Ptm the volume of alveolar fluid and the concentrations of total protein and cells in the BALF were greatly increased. The amount of LTB4 in the BALF also increased substantially from 6.0 to 49.5 micrograms (P < 0.001). Intermediate changes were seen at intermediate Ptm. We concluded that exposing pulmonary capillaries to high Ptm results in a high-permeability form of edema. In addition, the presence of LTB4 suggests that chemical mediators are released, possibly as the result of exposure of the reactive capillary endothelial basement membrane, as demonstrated by electron microscopy.

The lonD gene is homologous to the lon gene encoding an ATP-dependent protease and is essential for the development of Myxococcus xanthus.

Myxococcus xanthus contains two genes (lonV and lonD) homologous to the Escherichia coli lon gene for an ATP-dependent protease. We found that the lonD gene encodes a 90-kDa protein consisting of 827 amino acid residues. The lonD gene product shows 49, 48, and 52% sequence identity to the products of the M. xanthus lonV, E. coli lon, and Bacillus brevis lon genes, respectively. When a lonD-lacZ fusion was used, lonD was expressed during both vegetative growth and development. However, while lonD-disrupted strains were able to grow normally vegetatively, the development of M. xanthus was found to be arrested at an early stage in these strains. The mutant strains were able to form neither fruiting bodies nor myxospores.

Cloning and nucleotide sequence of the Myxococcus xanthus lon gene: indispensability of lon for vegetative growth.

The lon gene of Escherichia coli is known to encode protease La, an ATP-dependent protease associated with cellular protein degradation. A lon gene homolog from Myxococcus xanthus, a soil bacterium which differentiates to form fruiting bodies upon nutrient starvation, was cloned and characterized by use of the lon gene of E. coli as a probe. The nucleotide sequence of the M. xanthus lon gene was determined. It contains an open reading frame that encodes a 92-kDa protein consisting of 817 amino acid residues. The deduced amino acid sequence of the M. xanthus lon gene product showed 60 and 56% identity with those of the E. coli and Bacillus brevis lon gene products, respectively. Analysis of an M. xanthus strain carrying a lon-lacZ operon fusion suggested that the lon gene is similarly expressed during vegetative growth and development in M. xanthus. In contrast to that of E. coli, the M. xanthus lon gene was shown to be essential for cell growth, since a null mutant could not be isolated.

Changes of circulating atrial natriuretic peptide and antidiuretic hormone in obstructive sleep apnea syndrome.

Patients with obstructive sleep apnea (OSA) syndrome are known to exhibit nocturnal natriuresis/diuresis. We studied plasma and urinary levels of atrial natriuretic peptide (ANP), a potent natriuretic hormone released from the heart, and plasma antidiuretic hormone (ADH) levels in patients with OSA during awake and sleeping periods, to compare with those of normal subjects. Seven patients with OSA and 6 normal subjects were studied. Arterial blood samples were drawn during the awake and the sleeping period, while in patients with OSA, blood samples were obtained during the apneic period. Urine samples were collected over two 12-hour periods (9 a.m.-9 p.m. and 9 p.m.-9 a.m.) In patients with OSA, plasma ANP as well as urinary ANP excretion increased during the apneic period compared with the awake period. There was a significant negative correlation between plasma levels of ANP and ADH in patients with OSA. On the other hand, normal subjects had no apparent differences in plasma and urinary ANP levels between the two periods. It is suggested that nocturnal increase in ANP and decrease in ADH are responsible for the nocturnal diuresis and natriuresis associated with OSA.

Vitiligo and chronic photosensitivity in human immunodeficiency virus infection.

A 56-year-old man was admitted with hemiparesis and shortness of breath. He was positive to human immunodeficiency virus (HIV) antibody and was diagnosed as acquired immunodeficiency syndrome (AIDS) with Kaposi's sarcoma and pneumocystis carinii pneumonia. He developed chronic photosensitivity and vitiligo preceding the onset of the AIDS-related complex (ARC). Association of the two skin lesions with HIV infection is very rare. Although the role of HIV infection in these skin lesions is not significant, the immunological responses in the early course of HIV infection may have contributed to the development of both of these skin lesions.

Ventilatory response and arterial potassium concentration during incremental exercise in patients with chronic airways obstruction.

The relationship of ventilation response (VE) to arterial potassium concentration (K+) during ramp incremental exercise was assessed in nine patients with chronic obstructive pulmonary disease (COPD), and in 10 healthy subjects. For COPD patients the maximum oxygen uptake (VOmax) was 19.6 +/- 3.8 ml kg-1 min-1 (+/- SD), and percentage of forced expired volume at 1 s (% FEV1) was 47.8 +/- 10.4%. In healthy subjects, VO2max was 44.4 +/- 7.0 ml kg-1 min-1 and FEV1 was 89.7 +/- 7.4%. Breath-by-breath determinations for VE, oxygen uptake (VO2) and carbon dioxide output (VCO2), as well as determinations for K+, partial pressure of oxygen (PO2), partial pressure of carbon dioxide (PCO2), pH and lactate in arterial blood were performed during a workout on an exercise bicycle at a ramp function work rate of 20 W min-1, preceded by a 40 min warm-up period. The major findings in the present study are: (1) that there is a linear relation between ventilation and arterial K+ concentration during ramp exercise in both healthy subjects and COPD patients; (2) that the slope of the VE-K+ relationship is significantly lower in COPD patients (16.2 +/- 7.3 l min-1 mM-1) than in normal subjects (37.4 +/- 6.9 l min-1 mM-1, P less than 0.01); and, (3) that the slope of the VE-K+ relationship is significantly related to the ability to ventilate during maximal exercise in both healthy subjects and COPD patients (P less than 0.05). It is thought that the significantly reduced slope of the VE-K+ relationship in the COPD patients could be interpreted as a reduced sensitivity to the stimulus and/or as a mechanical impairment of the ventilation.