Comparison of physical properties and drug-releasing characteristics of white petrolatums.

White petrolatums of Japanese Pharmacopoeia grade and Sun white marketed as a cosmetic were characterized by measuring their physical properties and drug-releasing characteristics. White petrolatums of Japanese Pharmacopoeia grade available commercially in Japan were Perfecta, White 1S, Ultima, Snow, Snow V and Regent (Propeto). Penetrating stress, shear stress and spreading properties were measured as physical properties of the white petrolatums. The physical properties of white petrolatums varied, and Regent was the softest and the most spreadable ointment base. In vitro release test was performed using flow-through Franz diffusion cells. Fluorescein isothiocyanate and tetracycline hydrochloride were used as drug models. Their release characteristics varied among the tested white petrolatums, and Regent had the best release properties. Among the white petrolatums, with the exception of Regent, the release properties should depend on the distribution of drugs between white petrolatum and the receiver solution. Considerations of usability and characteristics of theprincipal agent are needed when choosing white petrolatums.

Change in environmental bacterial flora in a new hospital building.

Microbial surveillance of environmental bacteria was performed in order to study the microbial changes in a newly established hospital building. Airborne bacteria and surface-associated bacteria on floors and sinks were systematically collected between 2002 and 2005. The number of isolates obtained from frequently used floors was significantly higher than that obtained from those floors used less often. A significant increase in Staphylococcus aureus, the appearance of Pseudomonas aeruginosa, and changes among species of Gram-negative bacilli were observed 8-11 months after the new building had been opened. Furthermore, pulsed-field gel electrophoresis (PFGE) typing of meticillin-resistant S. aureus (MRSA) and P. aeruginosa showed that strains of the same PFGE groups were isolated from different sinks, floors and the adjoining old buildings. The number of MRSA isolates obtained from the new building increased as time passed. The sinks from which P. aeruginosa strains of the same PFGE type were isolated are connected by the same drainage pipe. Human movement has considerable effects on bacterial flora and their subsequent spread.

Vascular allografts are resistant to methicillin-resistant Staphylococcus aureus through indoleamine 2,3-dioxygenase in a murine model.

OBJECTIVE: Surgical results have shown the superiority of human heart valve and vascular allografts over artificial prostheses when used for the treatment of infectious cardiovascular diseases. However, the mechanism of infection resistance in these allografts has not been determined. In this study the contribution of the inflammatory response after allogeneic transplantation to the antimicrobial mechanism was assessed, focusing on the induction of indoleamine 2,3-dioxygenase, a tryptophan-metabolizing enzyme. METHODS: Aortic transplantation was performed with inbred rats, and aortic allografts, isografts, and control grafts were obtained for the following analyses. The extent of inflammatory-related and indoleamine 2,3-dioxygenase gene expression was measured by means of quantitative reverse transcriptase-polymerase chain reaction, and tryptophan metabolite production in the graft was measured by means of liquid chromatographic/tandem mass spectrometric analysis. The bacteriostatic effect of each graft and tryptophan metabolites was determined by using the methicillin-resistant Staphylococcus aureus proliferation assay. RESULTS: The inflammatory response, including interferon gamma, tumor necrosis factor alpha, and indoleamine 2,3-dioxygenase gene expression, was significant in the allografts but minimal in the isografts and control grafts. Methicillin-resistant S. aureus proliferation was remarkably suppressed when cultured with the allografts but not with the control grafts. Among tryptophan metabolites, the bacteriostatic effect against methicillin-resistant S. aureus was remarkable with 3-hydroxykynurenine, with a minimum inhibitory concentration of 32 mg/L. The 3-hydroxykynurenine level in the allografts was 9-fold greater than that in the control grafts. CONCLUSION: The bacteriostatic effect of the allografts was acquired by inducing indoleamine 2,3-dioxygenase, which resulted in local production of 3-hydroxykynurenine as an antimicrobial agent. This is the first report to document a mechanism of the allograft's infection-resistant property against methicillin-resistant S. aureus growth.

Susceptibilities to clarithromycin, amoxycillin and metronidazole of Helicobacter pylori isolates from the antrum and corpus in Tokyo, Japan, 1995-2001.

The aim of this study was to determine the susceptibilities to clarithromycin, amoxycillin and metronidazole of Helicobacter pylori isolates from the antrum and corpus of Japanese patients examined during the period 1995-2001. There was an increase, from 6.2% in 1995 to 22.1% in 2000-2001, in the proportion of patients infected with clarithromycin-resistant H. pylori. Of patients infected with clarithromycin-resistant H. pylori, 39.1% were infected with both clarithromycin-susceptible and -resistant H. pylori. Furthermore, the MIC90 of clarithromycin for H. pylori rose from < 1 mg/L in 1995-1998 to 8 mg/L in 1999. In contrast, the MIC90s of amoxycillin and metronidazole were < or = 0.125 and 4 mg/L, respectively, throughout the study period. The results showed that, while most H. pylori isolates were susceptible to amoxycillin and metronidazole, resistance to clarithromycin among H. pylori isolates increased markedly in Japan during 1995-2001. The results also indicated a need to test the susceptibility of H. pylori isolates from more than two samples obtained from two different sites in the stomach of a single patient in order to diagnose the presence of clarithromycin-resistant H. pylori correctly.

Comparison of the effects in vitro of tea tree oil and plaunotol on methicillin-susceptible and methicillin-resistant strains of Staphylococcus aureus.

The effects in vitro of tea tree oil (TTO) and plaunotol were examined by monitoring the growth of a standard strain of Staphylococcus aureus FDA 209P and of fourteen methicillin-susceptible strains of S. aureus (MSSA), together with twenty methicillin-resistant strains (MRSA). The minimum inhibitory concentrations (MIC) and the doses for 50% inhibition of growth (ID50) were determined by the micro-broth dilution (MD) method, and the broth dilution with shaking (BDS) method, respectively. The MIC of plaunotol for 50 and 90% of the MSSA and MRSA were assessed by the MD method, as 16 microg/ml and > or = 1,024 microg/ml, respectively. No antibacterial effects of TTO on MSSA and MRSA were detected by the MD method. The growth-inhibitory effects of TTO on S. aureus by the BDS method were examined, and it appeared that TTO was effective over a lower range of concentrations than previously reported. It seems that TTO is very effective in vitro against MSSA and MRSA at high concentrations but less effective below 40 microg/ml of TTO.

Inhibition by the essential oils of peppermint and spearmint of the growth of pathogenic bacteria.

The effects of the, essential oils of peppermint (Mentha piperita L.), spearmint Mentha spicata L.) and Japanese mint (Mentha, arvensis L.), of four major constituents of the esssential oil of peppermint, and of three major constituents of the essential oil of spearmint, on the proliferation of Helicobacter pylori, Salmonella enteritidis, Escherichia coli O157:H7, methicillin-resistant Staphylococcus aureus (MRSA) and methicillin sensitive Staphylococccus aureus (MSSA) were examined. The essential oils and the various constituents inhibited the proliferation of each strain in liquid culture in a dose-dependent manner. In addition, they exhibited bactericidal activity in phosphate-buffered saline. The antibacterial activities varied among the bacterial species tested but were almost the same against antibiotic-resistant and antibiotic-sensitive strains of Helicobacter pylori and S. aureus. Thus, the essential oils and their constituents may be useful as potential antibacterial agents for inhibition of the growth of pathogens.

DNA topology on an increase in positive writhing number of DNA: conformation changes in the time course of cis-diamminedichloroplatinum(II)-DNA adducts.

We show that the topological significance of the gel mobility of cis-diamminedichloroplatinum(II) (DDP)-closed circular DNA (ccDNA) adducts decreases with reaction time, until a point at which it joins relaxed DNA, and that the mobility of the adducts increases again. There is no relationship between the relative length of the adducts and the gel mobility. Although the significance of the decrease of gel mobility is due to the unwinding of cis-DDP-DNA (or trans-DDP-DNA) adducts, the conformational significance of the subsequent increase in mobility is unclear. To elucidate the conformational significance for unwinding of the adducts, we measured the writhing number (Wk) of the adducts using electron microscopy and analyzed the topological states of cis-DDP (or trans-DDP) adducts based on the White rule, Lk=Wk+Tk. Where, Lk and Tk represent the linking and twisting number in the ring, respectively. From the data, we found that the Wk of cis-DDP-ccDNA adducts in comparison with trans-DDP-ccDNA adducts increases from a negative to a positive number with time. This suggests that cis-DDP plays a role in the change of the topological state of ccDNA. In the abstraction of platinum from the adducts with CN- ion, the differences in both topological states may explain why Pt in trans-DDP is abstracted more easily than in cis-DDP. To explain the abstraction of Pt ion, we also discuss the findings based on the thermodynamic cycle in a intermolecular crosslink model Pt(NH3)2(guanine)2(2+)-->Pt(CN)4(2-) using the Pt parametrized PM3 method.

Activities of bismuth thiols against staphylococci and staphylococcal biofilms.

Indwelling medical devices are associated with infectious complications. Incorporating antimicrobials into indwelling materials may reduce bacterial colonization. Bismuth thiols are antibiofilm agents with up to 1,000-fold-greater antibacterial activity than other bismuth salts. Staphylococci are particularly sensitive, as determined by agar diffusion and broth dilution susceptibility testing. Bismuth-ethanedithiol inhibited 10 methicillin-resistant Staphylococcus epidermidis strains at 0.9 to 1.8, Staphylococcus aureus ATCC 25923 at 2.4, and S. epidermidis ATCC 12228 at 0.1 microM Bi(3+). Antiseptic-resistant S. aureus was sensitive to bismuth-2-3-dimercaptopropanol (BisBAL) at < or = 7 microM Bi(3+). Hydrogel-coated polyurethane rods soaked in BisBAL inhibited S. epidermidis for 39 days (inhibitory zone diameter in agar, > or = 30 mm for > 25 days). Slime from 16 slime-producing S. epidermidis strains was inhibited significantly by bismuth-3,4-dimercaptotoluene (BisTOL), but not by AgNO3, at subinhibitory concentrations. In conclusion, bismuth-thiols are bacteriostatic and bactericidal against staphylococci, including resistant organisms, but are also inhibitors of slime at subinhibitory concentrations. At subinhibitory concentrations, BisTOL may be useful in preventing the colonization and infection of indwelling intravascular lines, since staphylococci are important pathogens in this setting.

Identification of the tetracycline resistance gene, tet(M), in Erysipelothrix rhusiopathiae.

This is the first report to demonstrate the presence of tet(M) in naturally occurring isolates of tetracycline-resistant Erysipelothrix rbusiopathiae, which causes swine erysipelas. The tet(M) gene was isolated from E. rhusiopathiae strain KY5-42. The nucleotide and the deduced amino acid sequence were 99% identical to the tet(M) gene from Enterococcus faecalis. The gene was necessary and sufficient for the expression of tetracycline resistance in Escherichia coli. The presence of the tet(M) gene in the 114 tetracycline-resistant E. rhusiopathiae isolates from diseased pigs was detected by the polymerase chain reaction assay. The specific amplified DNA fragment was obtained from all 114 tetracycline-resistant strains. It was suggested that the tet(M) gene was widely present in the field isolates of E. rhusiopathiae resistant to tetracycline.

A transposon carrying the gene mphB for macrolide 2'-phosphotransferase II.

The mphB gene for macrolide 2'-phosphotransferase II is located on two plasmids, pTZ3721 and pTZ3723, in Escherichia coli BM2506. We examined translocation of mphB that originated from pTZ3721. The transposable element carrying mphB is 39 kb long and has a Tn21-like transposition module at one end and a Tn1721-like transposition module at other. The structure of the transposition modules of this element resembles that of Tn2610. However, the gene arrangement of the internal region on the transposon carrying mphB was reverse to that of Tn2610. The nucleotide sequences of both terminal regions suggested that the inversion of the DNA fragment occurred between the res sites by resolvase-mediated recombination.

Regulation of transcription of the mph(A) gene for macrolide 2'-phosphotransferase I in Escherichia coli: characterization of the regulatory gene mphR(A).

The synthesis of macrolide 2'-phosphotransferase I [Mph(A)], which inactivates erythromycin, is inducible by erythromycin. The expression of high-level resistance to erythromycin requires the mph(A) and mrx genes, which encode Mph(A) and an unidentified protein, respectively. We have studied the mphR(A) gene, which regulates the inducible expression of mph(A). An analysis of the synthesis of Mph(A) in minicells and results of a complementation test indicated that mphR(A) is located downstream from mrx and that its product, MphR(A), represses the production of Mph(A). DNA sequencing indicated that the mph(A), mrx, and mphR(A) genes exist as a cluster that begins with mph(A) and that the deduced amino acid sequence of MphR(A) can adopt an alpha-helix-turn-alpha-helix structure. To study the regulation of gene expression by MphR(A), we performed Northern blotting and primer extension. A transcript of 2. 9 kb that corresponded to the transcript of mph(A) through mphR(A) was detected, and its level was elevated upon exposure of cells to erythromycin. Gel mobility shift assays and DNase I footprinting indicated that MphR(A) binds specifically to the promoter region of mph(A), and the amount of DNA shifted as a results of the binding of MphR(A) decreased as the concentration of erythromycin was increased. These results indicate that transcription of the mph(A)-mrx-mphR(A) operon is negatively regulated by the binding of a repressor protein, MphR(A), to the promoter of the mph(A) gene and is activated upon inhibition of binding of MphR(A) to the promoter in the presence of erythromycin.

Disinfectant effects of hot water, ultraviolet light, silver ions and chlorine on strains of Legionella and nontuberculous mycobacteria.

The disinfectant effects on Legionella and nontuberculous mycobacteria of hot water, ultraviolet light, silver ions and chlorine, were evaluated. The bacterial strains Legionella pneumophila ATCC33152 and Mycobacterium avium ATCC25291 and strains of L. pneumophila and M. avium which had been isolated from a 24 h bath, were examined for their resistance to treatments. All strains were killed within 3 min on exposure to hot water at 70 degrees C and exposure to ultraviolet light at 90 mW.s/cm2. The strains of L. pneumophila tested were killed within 6 h on exposure to a solution of silver ions at 50 micrograms/l. The number of viable cells of strains of M. avium fell from 10(5) CFU/ml to 10(3) CFU/ml after exposure to an aqueous solution of silver ions at 100 micrograms/l for 24 h. Chlorine effectively killed strains of Legionella which were exposed to an aqueous solution of chlorine at 2 mg/l within 3 min, but strains of Mycobacterium survived exposure to chlorine at 4 mg/l for more than 60 min.

A new factor from Bacillus mesentericus which promotes the growth of Bifidobacterium.

It was reported previously that supernatants of cultures of Bacillus mesentericus TO-A promote the growth of Bifidobacterium species. In this study, a new growth-promoting factor, BM-1, was purified from the supernatant of such a culture and its chemical structure was determined. BM-1 was identified as 3,3-dihydroxyazetidine, and it promoted the growth of several strains of Bifidobacterium.

Variations in the gene for vacuolating toxin, vacA, in Japanese isolates of Helicobacter pylori.

Each of 284 strains of Helicobacter pylori which had been isolated in Japan was shown, by use of the polymerase chain reaction (PCR), to be positive for the vacA genes. The amplified vacA genes generated by PCR were classified into six classes (five for the clinical isolates, and one which corresponded to the standard strains). Endoscopic analysis revealed that cases of gastritis were most likely to be associated with class D, while none were associated with class A. The patterns of products of PCR obtained from the Japanese isolates were compared with theoretical patterns derived from sequences of vacA which had been reported previously. The nucleotide sequences of amplified fragments of vacA from representative strains in each class were determined and compared with those of previously reported vacA genes.

Characterization of the antiseptic-resistance gene qacE delta 1 isolated from clinical and environmental isolates of Vibrio parahaemolyticus and Vibrio cholerae non-O1.

The nucleotide sequence and mechanism of action were examined on the antiseptic-resistance gene qacE delta 1 that had been isolated from Pseudomonas aeruginosa, Vibrio parahaemolyticus and Vibrio cholerae non-O1. The nucleotide sequences of qacE delta 1 genes isolated from environmental isolates of V. cholerae non-O1 and V. parahaemolyticus differed by one base from that of the gene from P. aeruginosa. Escherichia coli C600 that harbored qacE delta 1 genes from several strains of Vibrio spp. exhibited low-level resistance to intercalating dyes. The resistance of E. coli cells with these genes to intercalating dyes, such as ethidium bromide, was mediated by an efflux system. Moreover, the activity of QacE delta 1 was inhibited in the presence of calcium channel blockers but not of calmodulin inhibitors. These results indicate that the qacE delta 1 gene can be function in E. coli and that the gene mediates resistance in a similar manner to the antiseptic-resistance gene smr.

Antiseptic susceptibility and distribution of antiseptic-resistance genes in methicillin-resistant Staphylococcus aureus.

We examined the antiseptic susceptibilities and distribution of antiseptic-resistance genes qacA and smr in 98 isolates of methicillin-resistant Staphylococcus aureus obtained in 1992. Seventy-one strains were resistant to antiseptics. The qacA and smr genes were detected in 10 and 20 strains, respectively. The remaining 41 strains without qacA and smr were divided into two groups that exhibited low-level (n = 22) and high-level (n = 19) resistance to acriflavin. DNA cloning and sequencing suggested that norfloxacin-resistance gene norA was responsible for the high-level resistance to acriflavin. Our results indicated that four or more antiseptic-resistance genes exist in methicillin-resistant S. aureus and that antiseptic-resistant methicillin-resistant S. aureus strains without qacA and smr are widely spread in Japan.

Biological activities of 1,1,6-trisubstituted indanes: beyond magainin 2.

MSI-78 is a peptide analog of naturally occurring magainin 2 isolated from the skin of Xenopus laevis. The peptide is known to have one of the strongest antibacterial activities in magainin 2 analogs against methicillin-resistant Staphylococcus aureus (MRSA). To find novel compounds superior to MSI-78, we have further designed, synthesizing 1,1-di(4-aminobutyl)-6-benzylindane (PM4) and 1,1-dibenzyl-6-(4-aminobutyl) indane (PM5), and tested their inhibitory ability of the growth of S. aureus. In an in vitro assay, PM4 showed the same antibacterial activity against the bacterium as MSI-78, and non-hemolytic activity against human red blood cells (RBCs) at the MIC (minimum inhibitory concentration) value, in contrast to the latter. On the other hand, PM5 showed stronger antibacterial activity than MSI-78, but being still accompanied with hemolysis at the MIC value. Otherwise, stronger decarboxylase activity for oxaloacetate was observed in PM5, rather than magainin 2 analogs or Oxaldie 1 as a control peptide, but not in PM4.

Distribution of the antiseptic-resistance gene qacE delta 1 in gram-positive bacteria.

The distribution of the antiseptic-resistance genes qacE and qacE delta 1, originally isolated from Gram-negative bacteria, was studied in a large number of Gram-positive bacteria by a method that included the polymerase chain reaction. A total of 151 strains of Staphylococcus and Enterococcus, isolated from clinical sources and obtained from the Japanese Collection of Microorganisms, was used in this analysis. We found the qacE delta 1 gene in 36 of 103 strains of Staphylococcus and in nine of 48 strains of Enterococcus. All of the strains in which we detected the qacE delta 1 gene were clinical isolates. The qacE gene was not detected in any of the strains examined in this study. The nucleotide sequences of the qacE delta 1 genes from the strains of Staphylococcus and Enterococcus were identical to that of the gene located on integron InC in Pseudomonas aeruginosa. These results indicate that the antiseptic-resistance gene qacE delta 1 is present in Gram-positive, as well as Gram-negative, bacteria.

Isolation of an Escherichia coli mutant which permits the high-level expression of a streptomycin-resistance gene derived from Pseudomonas aeruginosa.

Pretreatment with streptomycin at a low concentration influenced the susceptibility to streptomycin of a strain of Escherichia coli carrying a streptomycin-resistance plasmid, pSA1700, derived from Pseudomonas aeruginosa. This phenomenon was due to a mutation that occurred at about 10(-8)-10(-10) of frequency in a regulatory gene involved in gene expression on the chromosome of E. coli. A product encoded by the regulatory gene on the chromosome of E. coli might normally repress gene expression by binding to part of the promoter region of the streptomycin-resistance gene derived from P. aeruginosa.