Publications by authors named "Masanori Sasatsu"

48 Publications

Development of effective antimicrobial cocktails to prevent bacterial contamination of allograft tissues under low temperature conditions.

Interact Cardiovasc Thorac Surg 2019 01;28(1):128-136

Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan.

Objectives: Prevention of bacterial transmission in recipient patients via allograft decontamination with an antimicrobial cocktail consisting of cefmetazole (cefoxitin), vancomycin, lincomycin and polymyxin B is an important procedure commonly practised in tissue banks. However, some allografts are lost due to the failure of decontamination under low temperature conditions. Here, we aimed to develop new antimicrobial cocktails that exert a high bactericidal activity at 4°C.

Methods: Bacterial species used in this study were selected as major causative pathogens of allograft tissue contamination. The efficacy of the combination of 2 antimicrobial agents was determined by the checkerboard titration method. The bactericidal effects of the new antimicrobial cocktails were evaluated under the same conditions as those used for the storage and preservation of allograft tissues.

Results: Among the selected antimicrobial agents, daptomycin exhibited the highest bactericidal activity against methicillin-resistant Staphylococcus aureus under low temperature conditions. The combination of daptomycin + gentamicin and daptomycin + levofloxacin showed a synergistic or additive effect against various bacterial species. The antimicrobial cocktail containing 200 μg/ml of daptomycin, gentamicin and levofloxacin could eradicate ≤104 colony-forming units/ml of methicillin-resistant S. aureus and Enterococcus faecalis, which exhibit a low susceptibility to antimicrobial agents at 4°C for 24 h.

Conclusions: We have developed a new formula for an antimicrobial cocktail to effectively and sufficiently prevent bacterial contamination of allograft tissues under low temperature conditions in vitro.
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http://dx.doi.org/10.1093/icvts/ivy209DOI Listing
January 2019

Specific clones of Staphylococcus lugdunensis may be associated with colon carcinoma.

J Infect Public Health 2018 Jan - Feb;11(1):39-42. Epub 2017 May 11.

Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.

Staphylococcus lugdunensis produces a tannase with activity that may be associated with the onset of colon carcinoma. To clarify this feature of colon carcinoma-associated S. lugdunensis, we obtained isolates from healthy subjects and patients with colon adenomas and carcinomas and analyzed their genetic backgrounds. In total, 40 S. lugdunensis isolates from 288 rectal swabs collected between 2002 and 2008 were used. These isolates were classified into four groups according to the diseases of the subjects: healthy (n=13), colon carcinoma (n=13), colon adenoma (n=9), and unknown (n=5). The isolates were also classified by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing. In addition, an antimicrobial susceptibility test and detection of resistance genes were performed for all isolates. According to the PFGE analysis, 40 isolates could be classified into five groups. Among the groups, carcinoma and colon adenoma patients were significantly more frequently (40.9%) classified into group D (p<0.05), whereas healthy subjects were more frequently (38.5%) classified into group A. All isolates in group D were typed as ST27, which was clearly different than isolates in the other groups. All isolates were susceptible to the antimicrobial agents tested, including β-lactams, although seven strains produced β-lactamase. Our data suggest that a specific clone of S. lugdunensis might be associated with colon carcinoma and colon adenoma. This clone showed high susceptibility to many antimicrobial agents. Therefore, eradication therapy may lead to a decreased risk of colon carcinoma.
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http://dx.doi.org/10.1016/j.jiph.2017.03.012DOI Listing
July 2018

[Status and problem of first line eradication of H. pylori infection].

Nihon Rinsho 2013 Aug;71(8):1394-8

Endoscopy Centre, Tokyo Medical University Hospital.

This year eradication of H. pylori was applied for not only peptic ulcer but also chronic gastritis on National insurance system. However recently decrease in first line eradication rate of H. pylori using PPI/AC regimen. Certainly eradication rate after 2000 decreased in intention to treat (ITT) and per protocol(PP) compared to that before 2000. This tendency was induced by increase in CAM-resistant H. pylori. But after 2007 eradication rate decreased only in ITT, eradication rate didn't decrease in PP. That tendency was induced by bad compliance and evaluation of the eradication.
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August 2013

PCR detection of Helicobacter pylori in clinical samples.

Methods Mol Biol 2013 ;943:279-87

Department of Bacteriology II, National Institute of Infectious Diseases, Tokyo, USA.

Helicobacter pylori is an important pathogen whose primary niche is the human stomach. H. pylori is etiologically associated with gastric inflammation (gastritis), peptic ulcer disease, and gastric cancer. Both noninvasive (e.g., urea breath and stool antigen tests) and invasive (gastric biopsy for histology, culture, or PCR) tests are used for diagnosis. PCR detection of H. pylori has been reported using a variety of clinical samples including gastric biopsy, gastric juice, saliva, dental plaque, and stools as well as environmental samples. Whenever possibly, noninvasive tests are preferred over invasive tests. H. pylori are excreted in the stool. Culture from stool is variable whereas stool antigen testing is widely used. Stool consists of a complicated mixture of commensal bacteria and chemicals and often includes inhibitors of PCR. Nevertheless, simple extraction methods are available to efficiently extract DNA from human stools and nested-PCR targeting the 23S rRNA gene have proven to be highly sensitive for the detection of H. pylori. Detection of clarithromycin susceptibility/resistance is important clinically and the mutation of the 23S rRNA gene responsible for resistance can also be detected using stool. This described method can be modified for other clinical samples such as gastric juice or biopsy material.
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http://dx.doi.org/10.1007/978-1-60327-353-4_19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3973746PMC
March 2013

Novel anti-acne actions of nadifloxacin and clindamycin that inhibit the production of sebum, prostaglandin E(2) and promatrix metalloproteinase-2 in hamster sebocytes.

J Dermatol 2012 Sep 6;39(9):774-80. Epub 2012 Mar 6.

Departments of Biochemistry and Molecular Biology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan.

Acne vulgaris is characteristic of excess sebum production and the induction of inflammatory reactions, for example, the augmentation of cytokine, prostaglandin (PG) and matrix metalloproteinase (MMP) production in sebaceous glands and pilosebaceous units. As Propionibacterium acnes is considered to be involved in the aggravation of acne vulgaris, antimicrobial agents have been found to be effective for treating acne leading to the remission of inflammation. However, it is not fully understood whether antimicrobial agents influence sebum production and/or the inflammatory reactions in sebaceous gland cells (sebocytes). In the present study, topical antimicrobial agents such as nadifloxacin (NDFX) and clindamycin (CLDM) decreased the production of triacylglycerols (TG), which are a major component of sebum in insulin-differentiated hamster sebocytes. These antibiotics also suppressed insulin-augmented gene expression and the production of perilipin, by which intracellular lipid droplet formation was concomitantly inhibited. On the other hand, peptidoglycan (PGN) from Gram-positive bacteria dose-dependently increased TG production in hamster sebocytes. The augmented TG production was decreased by treating NDFX or CLDM. Furthermore, NDFX and CLDM inhibited the PGN-augmented PGE(2) production in the sebocytes. Moreover, NDFX, but not CLDM, suppressed the PGN-augmented gene expression and production of pro-MMP-2/progelatinase A in hamster sebocytes. Therefore, these results provide novel evidence that NDFX and CLDM exhibit anti-lipogenesis and anti-inflammatory activities against insulin- or PGN-activated sebocytes which at least partly mimic acne pathology in vitro. Moreover, NDFX for acne therapy is likely to be effective in not only inhibiting microbial proliferation but also in preventing the onset of acne scar formation.
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http://dx.doi.org/10.1111/j.1346-8138.2012.01525.xDOI Listing
September 2012

Fluoroquinolone resistance in Helicobacter pylori: role of mutations at position 87 and 91 of GyrA on the level of resistance and identification of a resistance conferring mutation in GyrB.

Helicobacter 2012 Feb;17(1):36-42

Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Horinouchi, Tokyo, Japan.

Background And Aims: Fluoroquinolone-containing regimens have been suggested as an alternate to standard triple therapy for the treatment of Helicobacter pylori infections. To determine the relationship between fluoroquinolone resistance and mutations of GyrA and GyrB in H. pylori, we exchanged the mutations at positions 87 and 91 of GyrA among fluoroquinolone-resistant clinical isolates. GyrB of a strain with no mutations in GyrA was also analyzed to identify mechanisms of resistance to norfloxacin.

Materials & Methods: Natural transformation was performed using the amplified fragment of the gyrA and gyrB gene as donor DNA. The amino acid sequences of GyrA and GyrB were determined by DNA sequencing of the gyrA and gyrB genes.

Results: Norfloxacin-resistant strains which had mutations at position 87 and 91 became susceptible when the mutations were converted to the wild type. When the mutation from Asp to Asn at position 91 was exchanged to the mutation from Asn to Lys at position 87, the MIC to levofloxacin, gatifloxacin, and sitafloxacin increased. Norfloxacin-resistant strain TS132 with no mutations in GyrA but had a mutation at position 463 in GyrB. Transformants obtained by natural transformation using gyrB DNA of TS132 had a mutation at position 463 of GyrB and revealed resistant to norfloxacin and levofloxacin.

Conclusion: Mutation from Asn to Lys at position 87 of GyrA confers higher resistance to levofloxacin and gatifloxacin than does mutation from Asp to Asn at position 91. We propose that mutation at position 463 in GyrB as a novel mechanism of fluoroquinolone resistance in H. pylori.
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http://dx.doi.org/10.1111/j.1523-5378.2011.00912.xDOI Listing
February 2012

Effect of pretreatment with Lactobacillus gasseri OLL2716 on first-line Helicobacter pylori eradication therapy.

J Gastroenterol Hepatol 2012 May;27(5):888-92

Gastroenterology, Tokai University School of Medicine, Isehara, Kanagawa, Japan.

Background And Aim: Helicobacter pylori eradication clearly decreases peptic ulcer recurrence rates. H. pylori eradication is achieved in 70-90% of cases, but treatment failures due to poor patient compliance and resistant organisms do occur. Lactobacillus gasseri can suppress both clarithromycin-susceptible and -resistant strains of H. pylori in vitro. The aim of this study was to determine the effect of pretreatment with L. gasseri- containing yogurt on H. pylori eradication. We conducted a randomized, controlled clinical trial in patients with H. pylori infection.

Methods: A total of 229 patients were randomized into either a 1-week triple therapy of rabeprazole (10 mg bid), amoxicillin (750 mg bid), and clarithromycin (200 mg bid) or triple therapy plus L. gasseri-containing yogurt. In the yogurt-plus-triple therapy groups, yogurt containing L. gasseri OLL2716 (112 g) was given twice daily for 4 weeks (3 weeks pretreatment and also 1 week during eradication therapy). Clarithromycin resistance was determined by the detection of a mutation in 23S rRNA using nested polymerase chain reaction and the direct sequencing of DNA from pretreatment feces. H. pylori eradication was diagnosed based on the urea breath test and a stool antigen test after 8 weeks of eradication.

Results: The status of H. pylori susceptibility to clarithromycin was successively determined in 188 out of 229 samples. The rate of infection with clarithromycin-resistant strains of H. pylori was 27.1%. Overall eradication (intention to treat/per protocol) was 69.3/74.5% for the triple-only group, and 82.6/85.6% for the yogurt-plus-triple group (P = 0.018/P = 0.041). Eradication of primary clarithromycin-resistant strains tended to be higher for yogurt-plus-triple therapy than triple-only therapy (38.5 vs 28.0%, respectively, P = 0.458).

Conclusion: This study confirmed that the major cause of treatment failure is resistance to clarithromycin. A 4-week treatment with L. gasseri-containing yogurt improves the efficacy of triple therapy in patients with H. pylori infection.
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http://dx.doi.org/10.1111/j.1440-1746.2011.06985.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3504346PMC
May 2012

Susceptibility of Propionibacterium acnes isolated from patients with acne vulgaris to zinc ascorbate and antibiotics.

Clin Cosmet Investig Dermatol 2011 5;4:161-5. Epub 2011 Oct 5.

BML General Laboratory, Matoba, Kawagoe, Saitama.

Purpose: The in vitro antimicrobial activity of ascorbic acid derivatives against Propionibacterium acnes was tested either alone or in combination with a variety of antimicrobial agents, and their fractional inhibitory concentration index was determined using checkerboard tests. The antimicrobial effectiveness of zinc ascorbate in the treatment of acne vulgaris, either alone or in combination with antibiotics such as clindamycin that are commonly used in Japan for the treatment of acne vulgaris, was therefore examined.

Materials And Methods: The antimicrobial susceptibility of 41 strains of clindamycin-sensitive and/or clindamycin-resistant P. acnes isolated from acne vulgaris patients was tested, in comparison with a type strain of P. acnes.

Results: Zinc ascorbate showed antimicrobial activity against a type strain of P. acnes and its concentration (0.064%) was sufficiently lower than the normal dose (5%) of other ascorbic acid derivatives. Combinations of zinc ascorbate with clindamycin, erythromycin, and chloramphenicol showed an additive effect, and zinc ascorbate alone effectively inhibited the growth of all P. acnes including clindamycin-resistant strains.

Conclusion: The results provide novel evidence that the combination of zinc ascorbate and clindamycin is effective for acne vulgaris treatment.
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http://dx.doi.org/10.2147/CCID.S23840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3208449PMC
November 2011

[Antimicrobial activity and frequency of spontaneous gentamicin-resistant mutants in bacteria related skin infections].

Yakugaku Zasshi 2011 ;131(11):1653-9

Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan.

Gentamicin is used in an ointment form for the treatment of skin infections. To investigate the effect of gentamicin used as an ointment, the antimicrobial susceptibilities against Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus pyogenes, and Pseudomonas aeruginosa isolated from community and medical settings were studied and compared with other antibacterial agents such as fradiomycin, chloramphenicol, and bacitracin used as active ingredient for each ointment. Gentamicin showed antibacterial activities for all standard bacteria tested, but fradiomycin and chloramphenicol showed no such activities for St. pyogenes and P. aeruginosa, respectively. Bacitracin showed activity for St. pyogenes only. The strains of staphylococci isolated from healthy people were highly susceptible to gentamicin, while 49.3% of the isolates from the patients with skin infections were resistant to gentamicin and 96.4% of the gentamicin-resistant staphylococci carried the aminoglycoside-resistance gene aacA-aphD. The growths of all strains tested, except for two strains of P. aeruginosa, were inhibited by close below 128 µg/ml of gentamicin. Furthermore, the frequencies of spontaneous mutants resistant to gentamicin, fradiomycin, and chloramphenicol were each investigated using S. aureus, S. epidermidis, St. pyogenes, and P. aeruginosa. At doses of more than 32 µg/ml of gentamicin, no resistant mutants in any of bacteria strains tested were obtained. The concentration of gentamicin on the skin was calculated at approximately 895 µg/ml at least when the commercially used 0.1% gentamicin ointment was applied to the skin. Therefore, our study strongly indicates that the gentamicin ointment used has a potency of sufficiently inhibiting the growth of bacteria, including gentamicin-resistant strains, which cause skin infections in the community.
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http://dx.doi.org/10.1248/yakushi.131.1653DOI Listing
February 2012

Characterization of enterococcus strains contained in probiotic products.

Biol Pharm Bull 2011 ;34(9):1469-73

Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan.

Probiotics are additives containing live microbes that beneficially affect a host by improving the properties of the host intestinal microflora. Recently, advances in medical treatments have led to increased numbers of immunocompromised patients; some patients contract opportunistic infections of Enterococcus species, which are considered non-pathogenic bacteria. To evaluate the safety of probiotics containing Enterococcus strains, we isolated Enterococcus from six probiotic products and compared the pathogenic genes and antimicrobial susceptibility of the probiotic strains to those of clinical isolates. Our study showed that all Enterococcus strains contained in probiotic products were E. faecium, and no vancomycin-resistant strains were found. In addition, no pathogenic genes, such as ace, agg, gelE, cylM, cylB, cylA, cpd, cob, ccf, efaA(fs), efaA(fm), esp(fs), or esp(fm), were found in the probiotic strains. Pulsed-field gel electrophoresis (PFGE) analysis showed obvious genetic differences between the probiotic strains and the clinical isolates. The data suggested that the probiotic Enterococcus strains were not transmitted to hospitalized patients. Therefore, our results strongly suggest that probiotic products are unlikely agents for causing opportunistic infections.
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http://dx.doi.org/10.1248/bpb.34.1469DOI Listing
December 2011

Augmentation of gene expression and production of promatrix metalloproteinase 2 by Propionibacterium acnes-derived factors in hamster sebocytes and dermal fibroblasts: a possible mechanism for acne scarring.

Biol Pharm Bull 2011 ;34(2):295-9

Department of Biochemistry and Molecular Biology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432–1 Horinouchi, Hachioji, Tokyo 192–0392, Japan.

Aberrant extracellular matrix (ECM) remodeling in sebaceous glands and pilosebaceous units in the skin is associated with scar formation under acne conditions. To investigate the involvement of Propionibacterium acnes (P. acnes), a Gram-positive anaerobic microbial species, in ECM remodeling in sebaceous glands and pilosebaceous units, we examined the effects of P. acnes culture media, formalin-fixed P. acnes, and peptidoglycan (PGN) from Gram-positive bacteria walls on the production of promatrix metalloproteinase 2 (proMMP-2)/progelatinase A in hamster sebocytes and dermal fibroblasts. When hamster sebocytes (1.8×10(5) cells) and dermal fibroblasts (1×10(5) cells) were treated with P. acnes culture media and formalin-fixed P. acnes (corresponding to 1×10(6) and 1×10(7) bacterial cells), the production of proMMP-2 was augmented. In addition, PGN (5-50 µg/ml) dose-dependently augmented the production of proMMP-2 in both cells. Furthermore, the PGN (50 µg/ml)-augmented proMMP-2 production was resulted from an increase of its transcript. In contrast, there were no changes in cell proliferative activity in either the P. acnes or PGN-treated sebocytes and dermal fibroblasts, indicating that the augmented proMMP-2 production was not due to an increase in cell numbers. Therefore, these results provide novel evidence that PGN transcriptionally up-regulates the production of proMMP-2 in hamster sebocytes and dermal fibroblasts. Given an increase in the quantity of Gram-positive bacteria, including P. acnes in acne lesions, the aberrant ECM degradation may progress in sebaceous glands and pilosebaceous units, which is associated with acne scar formation.
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http://dx.doi.org/10.1248/bpb.34.295DOI Listing
November 2011

Fluoroquinolone efflux by the plasmid-mediated multidrug efflux pump QacB variant QacBIII in Staphylococcus aureus.

Antimicrob Agents Chemother 2010 Oct 26;54(10):4107-11. Epub 2010 Jul 26.

Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.

Plasmids that carry the multidrug efflux genes qacA and qacB are widely distributed in methicillin-resistant Staphylococcus aureus (MRSA). Although the QacA and QacB proteins are similar to each other, their respective substrate specificities may differ. We investigated the variability and structure-function relationships of QacA and QacB in MRSA isolates. The amino acid sequences of 7 QacA and 25 QacB proteins showed that QacB was present in three variants, designated QacBII, QacBIII, and QacBIV, that were different from the prototypic QacB variant encoded by plasmid pSK23, which was named QacBI, while QacA was present in two variants. When cloned and expressed in S. aureus, the strain carrying qacBIII exhibited higher susceptibility to dyes and decreased susceptibility to norfloxacin and ciprofloxacin compared to strains carrying the other QacB variants. Site-directed mutagenesis experiments revealed that the residue at position 320 in QacB plays an important role in the resistance phenotypes to dyes and fluoroquinolones. Furthermore, the accumulation of norfloxacin and ciprofloxacin in the strain carrying qacBIII was significantly decreased. Our data demonstrate that the plasmid-mediated multidrug efflux pump QacB variant QacBIII confers the capability for fluoroquinolone efflux on S. aureus.
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http://dx.doi.org/10.1128/AAC.01065-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2944592PMC
October 2010

Analysis of clarithromycin resistance and CagA status in Helicobacter pylori by use of feces from children in Thailand.

J Clin Microbiol 2009 Dec 30;47(12):4144-5. Epub 2009 Sep 30.

Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan.

The clarithromycin resistance and CagA status of Helicobacter pylori in Thai children were investigated using fecal samples. Of the 284 samples, H. pylori was detected in 120 samples, and the clarithromycin resistance rate was 29.2%. The cagA gene was detected in 59 samples, and only 6.8% of these samples contained the East Asian CagA type.
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http://dx.doi.org/10.1128/JCM.00786-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2786625PMC
December 2009

Using the tannase gene to rapidly and simply identify Staphylococcus lugdunensis.

Diagn Microbiol Infect Dis 2010 Jan 15;66(1):120-3. Epub 2009 May 15.

Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan.

The coagulase-negative Staphylococcus lugdunensis, a bacterium similar to Staphylococcus aureus, produces tannase that degrades tannin. We developed a polymerase chain reaction-based method to rapidly and simply identify this species by detecting the tanA gene for S. lugdunensis tannase.
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http://dx.doi.org/10.1016/j.diagmicrobio.2009.03.028DOI Listing
January 2010

Involvement of Propionibacterium acnes in the augmentation of lipogenesis in hamster sebaceous glands in vivo and in vitro.

J Invest Dermatol 2009 Sep 12;129(9):2113-9. Epub 2009 Mar 12.

Department of Biochemistry and Molecular Biology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan.

Propionibacterium acnes is considered to be involved in the aggravation of acne vulgaris, but it remains unclear whether P. acnes directly influences lipogenesis in sebaceous glands. In this study, we showed that a culture medium of P. acnes (acnes-CM) and formalin-killed P. acnes (F-acnes) prepared from P. acnes strains, JCM6473 and JCM6425, intracellularly augmented lipid droplet formation and triacylglycerol (TG) synthesis in undifferentiated and insulin-differentiated hamster sebocytes. Acnes-CM and F-acnes prepared from four clinical P. acnes strains elicited the same lipogenesis augmentation. The augmented TG production resulted from an increase in the diacylglycerol acyltransferase activity. Topical application of acnes-CM to the skin of hamster auricles every day for 4 weeks revealed that sebum accumulation was augmented in sebaceous glands and ducts. Furthermore, both acnes-CM and F-acnes increased the production of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a cytochrome P450 (CYP)-linked sebaceous lipogenic factor, in differentiated sebocytes. A CYP inhibitor, SKF-525A, decreased the acnes-CM- and F-acnes-augmented production of TG and 15d-PGJ(2). Thus, to our knowledge these results provide previously unreported evidence that P. acnes directly participates in the augmentation of sebaceous lipogenesis through a proposed mechanism in which an increase of 15d-PGJ(2) production through the CYP pathway is closely associated with the enhancement of TG production.
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http://dx.doi.org/10.1038/jid.2009.46DOI Listing
September 2009

Isolation and identification of a potent antimalarial and antibacterial polyacetylene from Bidens pilosa.

Planta Med 2009 May 4;75(6):624-8. Epub 2009 Mar 4.

Showa Pharmaceutical University, Higashi-tamagawagakuen, Machida, Tokyo 194-8543, Japan.

Diseases caused by malaria parasites and pathogenic bacteria were thought to be on the brink of eradication in the 1950-1960s, but they have once again become a serious threat to mankind as a result of the appearance of multidrug resistant strains. The spread of these multidrug resistant organisms has prompted a worldwide search for new classes of effective antimalarial and antibacterial drugs. Natural products have been recognized as highly important candidates for this purpose. Our attention has focused on the herbal plant Bidens pilosa, a weed common throughout the world, as one of the target plants in the search for new active compounds, owing to its empirical use in the treatment of infectious diseases and to pharmaco-chemical studies of its crude extract. We report the isolation of two new compounds of B. pilosa, the linear polyacetylenic diol 1 and its glucoside 2 which have previously been isolated from different plants. Compound 1 exhibited highly potent antimalarial and antibacterial properties in vitro as well as potent antimalarial activity by way of intravenous injection in vivo, thereby representing a promising new class of drugs potentially effective in the treatment of malarial and bacterial diseases. We suspect that discovery of these compounds in B. pilosa in appreciable quantity is because the Fijian tradition of using the fresh plant for extraction rather than the Asian tradition of using dried plants (1 is unstable in the dried state) was followed.
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http://dx.doi.org/10.1055/s-0029-1185377DOI Listing
May 2009

Tailored eradication therapy based on fecal Helicobacter pylori clarithromycin sensitivities.

J Gastroenterol Hepatol 2008 Dec;23 Suppl 2:S171-4

Endoscopy Center, Tokyo Medical University, Tokyo, Japan.

Background And Aim: Helicobacter pylori (H. pylori) eradication rates using the PPI/AC regimen (proton pump inhibitor + amoxicillin + clarithromycin) are declining. We trialed tailoring eradication regimens according to clarithromycin (CAM) susceptibility.

Methods: The subjects were 70 H. pylori positive adults. They were randomly allocated to a tailored group and a control group. In the tailored group, subjects with CAM-sensitive strains were given PPI/AC eradication therapy, and those with CAM-resistant strains were given PPI/AM (metronidazole instead of clarithromycin) therapy. The control group were all given PPI/AC therapy. CAM sensitivity was measured by collecting fecal specimens, and extracting the DNA. The 23S rRNA domain, associated with CAM susceptibility in H. pylori, was amplified using a nested polymerase chain reaction (PCR), and DNA sequencing was used to detect point mutations at A2143G and A2144G.

Results: Eradication rates were 94.3% in the tailored group and 71.4% in the control group. In particular, the eradication rate was 100% for CAM-resistant strains in the tailored group.

Conclusions: In Japan, where CAM-resistant H. pylori strains are expected to continue to increase, tailored eradication therapy according to CAM sensitivity will be of benefit.
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http://dx.doi.org/10.1111/j.1440-1746.2008.05408.xDOI Listing
December 2008

Anti-infectious activity of tryptophan metabolites in the L-tryptophan-L-kynurenine pathway.

Biol Pharm Bull 2009 Jan;32(1):41-4

Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Japan.

To study the anti-infectious effect of a vascular allograft, the antimicrobial activity of tryptophan metabolites mediated by indoleamine 2,3-dioxygenase was determined. The growth of methicillin-resistant Staphylococcus aureus (MRSA) over 10 h in extracts from post-transplantation vascular allograft was significantly slower than that of extracts from non-transplantation vascular allograft regardless of the presence of tryptophan (p<0.05). When the antimicrobial activity of the tryptophan metabolites in the L-tryptophan-L-kynurenine pathway was examined, 3-hydroxy-DL-kynurenine and alpha-picolinic acid had strong antibacterial activity against MRSA, S. epidermidis, Escherichia coli, and multidrug-resistant Pseudomonas aeruginosa, although antimicrobial activities of anthranilic acid, 3-hydroxyanthranilic acid, and quinolinic acid against them were low. The results showed that, of the tested tryptophan metabolites, 3-hydroxy-DL-kynurenine and alpha-picolinic acid contributed to the anti-infectious effects of the allograft by inhibiting of the growth of microorganisms.
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http://dx.doi.org/10.1248/bpb.32.41DOI Listing
January 2009

Antimicrobial susceptibilities of Propionibacterium acnes isolated from patients with acne vulgaris.

Microbiol Immunol 2008 Dec;52(12):621-4

Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo, Japan.

Antibiotic susceptibilities of Propionibacterium acnes in Japan were determined. Erythromycin-resistance was found in 10.4% (5/48) of the strains, and four of these were cross-resistance to clindamycin. Although the erythromycin ribosome methylase gene erm(X) was looked for, no strain carrying erm(X) was found. Sequencing analysis revealed that all of the erythromycin-resistant strains had a mutation in the peptidyl transferase region of the 23S rRNA gene: G2057A, A2058G, or A2059G. Consequently, our results show that P. acnes resistance to macrolides is caused by a mutation in the 23S rRNA gene, and has been increasing in Japan.
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http://dx.doi.org/10.1111/j.1348-0421.2008.00081.xDOI Listing
December 2008

Molecular epidemiology and antimicrobial susceptibilities of 273 exfoliative toxin-encoding-gene-positive Staphylococcus aureus isolates from patients with impetigo in Japan.

J Med Microbiol 2008 Oct;57(Pt 10):1251-1258

Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.

The molecular epidemiology and antimicrobial susceptibilities of 273 Staphylococcus aureus isolates positive for the exfoliative toxin-encoding gene obtained from patients with impetigo in Japan in 2006 were studied. The mecA gene was detected in 74 meticillin-resistant S. aureus (MRSA) and 23 meticillin-susceptible S. aureus (MSSA) isolates. All isolates with the staphylococcal cassette chromosome (SCC) mec were classified into type IV (92.8%, 90/97) or V (7.2%, 7/97). The ET-encoding gene etb was found primarily in strains with mecA (87.7%, 71/81), whilst eta (86.6%, 161/186) was detected mainly in strains without mecA. The chromosomal enterotoxin-encoding gene cluster egc was found in 83.0% of strains with eta, whilst no enterotoxin-encoding gene was detected in strains with only etb. PFGE showed that each strain carrying eta, etb and etd could be classified into distinct groups. The susceptibility profiles of MRSA to antimicrobial agents excluding beta-lactams were similar to those of MSSA. Gentamicin- and clarithromycin-resistant strains were frequently found for both MRSA and MSSA. The aminoglycoside-resistance gene aacA-aphD was detected in 97.3% of MRSA and 85.4% of MSSA. Additionally, the macrolide-resistance gene ermA or ermC was detected in 67.6% of MRSA and 71.4% of MSSA. Therefore, these results suggest that SCCmec types IV or V have spread, particularly in MSSA carrying etb in the community.
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http://dx.doi.org/10.1099/jmm.0.2008/002824-0DOI Listing
October 2008

A study of the relationship between Helicobacter pylori microbial susceptibility, 13C-urea breath test values.

Hepatogastroenterology 2008 Mar-Apr;55(82-83):786-90

Endoscopy Center, Tokyo Medical University Hospital, Tokyo, Japan.

Background/aims: Diagnostic methods for Helicobacter pylori (H. pylori) infection can be divided into invasive endoscopic methods and non-invasive methods. A typical and widely used non-invasive method is the 13C urea breath test (UBT). In this study, the possibility of a correlation between pre-treatment UBT values with H. pylori antimicrobial resistance is investigated.

Methodology: The subjects were 119 consecutive patients who attended this hospital for H. pylori testing. Average age was 47.5 +/- 13.2 years, with a male:female ratio of 2.05:1. The diagnosis was gastric ulcer in 43 subjects, duodenal ulcer in 27, gastroduodenal ulcer in 21 and chronic gastritis in 28. Subjects underwent UBT as well as upper gastrointestinal endoscopy (UGITE). The diagnosis of H. pylori infection was examined by the results of culture, histological examination and the rapid urease test (RUT). The mean inhibitory concentration (MIC) was determined for each antimicrobial agent in the bacterial isolates that could be cultured.

Results: In this study, the sensitivity and specificity were excellent at 97.0% and 100% with a cut-off point of 3.5 per thousand for UBT respectively. Clarithromycin resistance was more common in the group with high UBT values. No correlation at all was seen between UBT values and metronidazole, sparafloxacin, cefaclor and amoxicillin susceptibility.

Conclusions: It is possible that UBT values also tend to be higher in cases of CAM resistance.
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December 2008

Anti-infectious effect of S-benzylisothiourea compound A22, which inhibits the actin-like protein, MreB, in Shigella flexneri.

Biol Pharm Bull 2008 Jul;31(7):1327-32

Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Tokyo, Japan.

S-Benzylisothiourea compound A22 induces coccoid forms in Escherichia coli by inhibiting the function of the actin-like cytoskeletal protein, MreB. The minimum inhibitory concentration of A22 and the minimum concentration to induce coccoid forms for various pathogenic bacteria were determined. At 10 microg/ml, A22 induced coccoid forms in Shigella flexneri but did not inhibit the growth. No alteration of coccoid forms in the Gram-positive bacteria and anaerobic bacteria tested were observed following treatment with A22. To study the relationship between pathogenicity and alterations in bacterial shape, the infectious capacity of A22-induced coccoid S. flexneri was examined using CHO-K1 cells. Invasion of the coccoid cells was significantly reduced, however, no changes in adherence were observed. Using a mutant defective in the type III secretion apparatus, which delivers effectors to the host, we examined the secretion of effectors by A22-induced coccoid S. flexneri. The amount of secreted effectors in the coccoid cells was clearly decreased compared to rod-shaped cells. These results showed that the maintenance of rod-shaped cells by MreB in bacteria was essential for the secretion of effectors via the type III secretion system. Therefore, our results suggest that A22 is a useful lead compound for a novel anti-infectious agent without bactericidal activity and MreB is a candidate target site for development of new anti-infectious agents.
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http://dx.doi.org/10.1248/bpb.31.1327DOI Listing
July 2008

Characterization of the pTZ2162 encoding multidrug efflux gene qacB from Staphylococcus aureus.

Plasmid 2008 Sep 9;60(2):108-17. Epub 2008 Jun 9.

Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.

The plasmid-borne multidrug efflux gene qacB is widely distributed in methicillin-resistant Staphylococcus aureus (MRSA). We analyzed the complete nucleotide sequence of the plasmid pTZ2162 (35.4 kb) encoding qacB. The plasmid pTZ2162 contains 47 ORFs and four copies of IS257 (designated IS257A to D). The 24.7-kb region of pTZ2162, which excluding the region flanked by IS257A and IS257D, is 99.9% identical to pN315 carried by MRSA N315. However, the repA-like region of pTZ2162 was divided into two ORFs, ORF46 and ORF47. Functional analysis with the pUC19-based vector pTZN03 showed that both ORF46 and ORF47 were essential for the replication of pTZ2162 and ORF1 is required for the stable maintenance of pTZ2162 in S. aureus. When pTZ2162 was searched for evidence of mobile elements, an 8-bp duplicated sequence (GATAAAGA) was existed at the left boundary of IS257A and the right boundary of IS257D. Therefore, the 10.7-kb region between IS257A and IS257D in pTZ2162 has the potential to act as a transposon. In addition to qacB, the pTZ2162 transposon-like element contains a novel fosfomycin resistance determinant fosD and an aminoglycoside resistance determinant aacA-aphD. This transposon-like element appears to have translocated into the beta-lactamase gene blaZ. Our data suggest that qacB is transferred between MRSA as a multiple antibiotic resistance transposon.
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http://dx.doi.org/10.1016/j.plasmid.2008.04.003DOI Listing
September 2008

Mutations in penicillin-binding proteins 1, 2 and 3 are responsible for amoxicillin resistance in Helicobacter pylori.

J Antimicrob Chemother 2008 May 14;61(5):995-8. Epub 2008 Feb 14.

Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji-shi, Tokyo 192-0392, Japan.

Objectives: To elucidate the relationship between the mutations of penicillin-binding protein (PBP)1, PBP2 and PBP3 and amoxicillin resistance in Helicobacter pylori.

Methods: The mutations detected only in clinical amoxicillin-resistant strains were determined by comparison of the deduced amino acid sequences of PBP1(HP0597), PBP2(HP1556) and PBP3(HP1565) encoded by the pbp1, ftsI and pbp2 genes, respectively, in 13 clinical H. pylori strains and three ATCC strains. The contribution of the mutations in PBPs was analysed by the natural transformation of the amoxicillin-susceptible strain ATCC 700392 with various combinations of the pbp1, ftsI and pbp2 genes from the amoxicillin-resistant strain TH743 (MIC of amoxicillin: 8 mg/L).

Results: We initially identified six, four and two mutations of PBP1, PBP2 and PBP3, respectively, which were detected only in amoxicillin-resistant strains. By the natural transformation of an amoxicillin-susceptible strain ATCC 700392, we found that mutations in PBP1 and PBP3 conferred higher resistance to amoxicillin than mutations in PBP1 and PBP2, or mutations only in PBP1. Furthermore, mutations in PBP1, PBP2 and PBP3 conferred a 256-fold higher amoxicillin resistance when compared with ATCC 700392.

Conclusions: Multiple mutations in PBP2 and PBP3, in addition to mutations in PBP1, confer higher amoxicillin resistance in H. pylori.
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http://dx.doi.org/10.1093/jac/dkn051DOI Listing
May 2008

Correlation between substitutions in penicillin-binding protein 1 and amoxicillin resistance in Helicobacter pylori.

Microbiol Immunol 2007 ;51(10):939-44

Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji-shi, Tokyo 192-0392, Japan..

The correlation between the substitutions of penicillin-binding protein 1 (PBP1) and amoxicillin resistance was studied for the determination of the substitutions in PBP1 which confer amoxicillin resistance in Helicobacter pylori. By the comparison of the amino acid sequences of PBP1 in the amoxicillinresistant (n=3), low-susceptible (n=3), and susceptible (n=13) H. pylori isolates, the substitution Asn562-->Tyr, which is adjacent to KTG motif (555-557), was common and specific to amoxicillin-resistant H. pylori. Additionally, all amoxicillin-resistant isolates had multiple substitutions such as Ser414-->Arg in the transpeptidase region of PBP1 of H. pylori. Furthermore all transformants obtained by the natural transformation using the pbp1 genes of amoxicillin-resistant H. pylori isolates had multiple substitutions including Asn562-->Tyr. These results suggest that multiple amino acid substitutions in the transpeptidase region of PBP1 are closely related to amoxicillin resistance in H. pylori.
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http://dx.doi.org/10.1111/j.1348-0421.2007.tb03990.xDOI Listing
January 2008

Detection of mixed clarithromycin-resistant and -susceptible Helicobacter pylori using nested PCR and direct sequencing of DNA extracted from faeces.

J Med Microbiol 2007 Sep;56(Pt 9):1174-1180

Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.

The major cause of chemotherapy failure in patients with chronic gastritis and peptic ulcers caused by Helicobacter pylori is clarithromycin (CAM) resistance due to a mutation in the 23S rRNA gene. This study describes a non-invasive and accurate method for the detection of mixed CAM-resistant and -susceptible H. pylori by sequencing of the H. pylori 23S rRNA gene. Faeces were crushed with beads and the 23S rRNA gene was amplified using a nested PCR on the extracted DNA. Mutation analysis of this gene using this method showed that 20.4 % of patients carried mixed CAM-susceptible (wild type) and -resistant (A2142G or A2143G mutant) H. pylori. Furthermore, it was found that 66.6 % of patients who had been treated unsuccessfully carried one of these mutations in the 23S rRNA gene (including the mixed type), whilst standard culture detected CAM-resistant isolates in only 22.2 % of patients with unsuccessful treatment. These data suggest that, for successful therapy, the diagnosis method described here would more accurately detect CAM-resistant H. pylori, including mixed infections.
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http://dx.doi.org/10.1099/jmm.0.47302-0DOI Listing
September 2007

Transduction of the plasmid encoding antiseptic resistance gene qacB in Staphylococcus aureus.

Biol Pharm Bull 2007 Aug;30(8):1412-5

Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.

The plasmid-borne qacA and qacB genes encode a multidrug efflux protein. The proteins encoded by qacA and qacB mediate efflux of cationic antiseptic agents such as quaternary ammonium compounds. In methicillin-resistant Staphylococcus aureus (MRSA), qacA and qacB are widely prevalent and decrease antiseptic susceptibility. However, it is difficult to find the plasmids encoding qacA or qacB in community-associated MRSA (C-MRSA) isolated from patients with impetigo. Most MRSA, the strains causative of impetigo, carry the plasmid-borne exfoliative toxin-producing gene etb. To find the reason for the paucity of qacA or qacB in MRSA isolated from patients with impetigo, we performed transfer experiments of the plasmid pTZ2162qacB encoding qacB. The pTZ2162qacB was transferred to S. aureus strain RN4220 by transduction, although no pTZ2162qacB was transferred by conjugation. Additionally, pTZ2162qacB was transduced to MRSA carrying etb, and was coexistence with the plasmid encoding etb. Our results showed that pTZ2162qacB was horizontally transferred by transduction and was compatible with the plasmid encoding etb. Consequently, there will be risk of the emergence of C-MRSA with decreased antiseptic susceptibility among patients with impetigo.
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http://dx.doi.org/10.1248/bpb.30.1412DOI Listing
August 2007
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