Tsuguru Usui

Permeation of Antimicrobial Agents through Pseudomonas aeruginosa Biofilms: A Simple Method

In this study, we evaluated the permeation of piperacillin (PIPC), imipenem (IPM), amikacin (AKM), gentamicin (GM), ofloxacin (OFLX), levofloxacin (LVFX), ciprofloxacin (CPFX) and sparfloxacin (SPFX) through Pseudomonas aeruginosa biofilm with a simple new method. Bacteria used were a leucine-requiring mucoid mutant. Bacteria were grown on the membrane of a cell culture insert in chemically defined medium and incubated at 37 degrees C for 5 days. At days 0, 1, 3 and 5, the penetration rates through the biofilms were measured. PIPC and IPM demonstrated relatively high permeation both with penetration rates at day 5 of 50%, whereas AMK and GM, which are aminoglycosides, showed low permeation both with penetration rates after day 1 of less than 25%. Among the 4 fluoroquinolones, LVFX and SPFX demonstrated excellent permeation with penetration rates that reached 100% from day 0 to 5, while OFLX and CPFX showed almost the same permeation as IPM. This method of measuring penetration rates of antimicrobial agents through biofilm is very simple and useful for the evaluation of antibiotics against biofilm-forming bacteria.

Usefulness and Limitations of Methotrexate, Vinblastine, Doxorubicin And Cisplatin for the Treatment of Advanced Urothelial Cancer

Methotrexate, vinblastine, doxorubicin and cisplatin were used to treat 66 patients with advanced urothelial cancer. Of these 66 patients 58 could be evaluated for response. A total of 84 sites was evaluated in these patients. Response rates were 73% in the bladder, 67% in the renal pelvis, 50% in the ureter, 60% in the lung, 68% in the lymph nodes, 14% in the liver and 25% in the bone. Ten patients (17%) had a complete response and 23 (40%) had a partial response, with an over-all response rate of 57% (the 95% confidence limits are 44 to 69%). The mean durations of response were 10.1 months for complete response patients and 6.2 months for partial response patients. The most prominent toxicity was severe myelosuppression that resulted in 2 septic deaths. While this chemotherapy regimen provided an excellent over-all response rate, the matters of concern were the short duration of response and low effectiveness in the liver and bone.

Nocturnal enuresis and overactive bladder in children: An epidemiological study

Aims:  To investigate the prevalence and characteristics of nocturnal enuresis (NE) and to examine the prevalence of overactive bladder (OAB) symptoms in primary schoolchildren.

Increased prevalence and clonal dissemination of multidrug-resistant Pseudomonas aeruginosa with the blaIMP-1 gene cassette in Hiroshima

OBJECTIVES The aim of this study was to evaluate the dissemination of metallo-beta-lactamase (MBL)-encoding genes among multidrug-resistant (MDR) Pseudomonas aeruginosa isolates recovered from major hospitals in the Hiroshima region. METHODS During July to December from 2004 to 2006, a surveillance of eight major hospitals in the Hiroshima region identified 387 non-duplicate isolates resistant to imipenem (MIC >or= 16 mg/L). They were screened for resistance to amikacin (MIC >or= 64 mg/L) and ciprofloxacin (MIC >or= 4 mg/L) and MBL-encoding genes. The structure of the variable regions of the integrons was determined using PCR mapping. Clonality was assessed using PFGE and multilocus sequence typing (MLST). RESULTS The frequency of MBL-positive isolates in MDR P. aeruginosa isolates significantly increased from 42.3% in 2004 to 81.4% in 2006. Most of the MBL-positive isolates produced IMP-1 followed by VIM-2. The bla(IMP-1) and bla(VIM-2) genes were present in class 1 integrons. Characterization of the variable regions of the integron showed the presence of six different gene cassette arrays in bla(IMP-1) cassettes and a single array in bla(VIM-2) cassettes. The IMP-1 producers belonged to two clonal lineages using PFGE and MLST analyses and the integron variations correlated well with the clonal complexes. Among them, strains positive for a newly identified In113-derived bla(IMP-1) gene cassette array were most widely distributed in Hiroshima. CONCLUSIONS This study shows a dramatic increase in MBL genes, primarily bla(IMP-1), in MDR P. aeruginosa isolates in Hiroshima during these 3 years. In addition, MDR P. aeruginosa with the newly discovered In113-derived bla(IMP-1) gene cassette array appears to be clonally expanding.

EXPRESSION OF MATRIX METALLOPROTEINASE-7 AND TISSUE INHIBITOR OF METALLOPROTEINASE-1 IN HUMAN PROSTATE

PURPOSE Matrix metalloproteinase-7 (MMP-7), one of the extracellular matrix-degrading metalloproteinases, plays an important role in carcinoma invasion and metastasis. Tissue inhibitor metalloproteinase-1 (TIMP-1), one of the inhibitors of MMP-7, regulates extracellular matrix turnover. MATERIALS AND METHODS Gene expression levels of MMP-7 and TIMP-1 were examined in 20 prostate carcinomas after hormonal therapy and 12 benign prostate hyperplasias (BPH) by Northern blot analysis. Enzymatic activities of MMP-7 were examined in 7 prostate carcinomas and 1 BPH in the above prostate tissues by the method of caseinolytic zymography. These data were compared with the clinicopathological features. RESULTS There were significant correlations between levels of MMP-7 mRNA or the ratio of MMP-7 mRNA/TIMP-1 mRNA and pathological stage (p <0.01), lymph node metastasis (p <0.05), histological differentiation (p <0.05), vascular invasion (p <0.05), and lymphatic invasion (p <0.05). Levels of MMP-7 mRNA and the ratio of MMP-7 mRNA/TIMP-1 mRNA were significantly increased in prostate carcinomas from patients with high levels of serum prostate specific antigen (PSA) (>10 ng./ml.) after hormonal therapy (p <0.05). The activation ratio of pro MMP-7 was elevated in the cases with advanced prostate carcinoma compared with those of organ-confined prostate carcinoma and BPH. CONCLUSION These results suggest that MMP-7 may play an important role for invasion and metastasis in prostate carcinomas, and the balance between MMP-7 and TIMP-1 expression may relate to an invasive ability of prostate carcinomas.

Effect of the growth rate of Pseudomonas aeruginosa biofilms on the susceptibility to antimicrobial agents : β-lactams and fluoroquinolones

The growth rate of biofilm bacteria of a leucine-requiring mutant Pseudomonas aeruginosa HU1 was regulated by the leucine concentration in a chemically-defined medium. The semiquantitative measurement of glycocalyx and scanning electron microscopy revealed that the kinetics of HU1-biofilm formation were dependent on the incubation time and the leucine concentration in the medium. The effect of the growth rate of biofilm cells on their susceptibility to antimicrobial agents, three β-lactams and four fluoroquinolones, was evaluated. β-Lactams showed weak bactericidal activity to biofilm cells; the activity was greater in younger biofilm cells growing in high concentrations of leucine. Fluoroquinolones revealed strong bactericidal activity to biofilm bacteria regardless of the growth rate. The following is suggested: the bactericidal action of β-lactams against biofilm cells is affected by the cell growth rate, while that of fluoroquinolones is considerably greater and independent on the growth rate.

Neoadjuvant Chemotherapy for Locally Advanced Urothelial Cancer of the Upper Urinary Tract

Cisplatin-based multiple-drug chemotherapy is currently considered the most effective treatment for advanced and metastatic urothelial cancers. We treated 15 patients with locally advanced urothelial cancers of the upper urinary tract using the cisplatin-based multiple-drug regimen in a neoadjuvant setting. The regimens administered were: M-VAC (methotrexate, vinblastine, doxorubicin and cisplatin); MEC (methotrexate, etoposide and cisplatin), or M-VEC (methotrexate, vinblastine, epirubicin and cisplatin). Total nephroureterectomy was performed in all patients and response was evaluated pathologically Of 15 patients 2 (13%) achieved a pathological complete response, 6 (40%) a pathological partial response, for an overall response rate of 53% (95% confidence limits 29-77%). The median durations of response were 54 months for patients with a pathological complete response and 15.5 months for patients with a pathological partial response. One of six patients with a pathological partial response and 4 of 7 with no remission died of cancer. While a positive relationship between the pathological response and prognosis was observed, adequate follow-up is needed to assess the ability of neoadjuvant chemotherapy to improve the prognosis of patients with locally advanced urothelial cancer of the upper urinary tract.

Simple Method for Measuring the Antibiotic Concentration Required to Kill Adherent Bacteria

A simple method was developed for measuring the antibiotic susceptibility of bacteria adherent to plastic surfaces. Staphylococcus aureus cells adhered to the bottom of a 96-well plastic tissue culture plate were incubated in serially diluted antibiotic solutions. After a 24-hour-incubation the solutions were removed, and fresh medium without antibiotics was added to each well. The viability of the cells was judged by their growth after a further 24-hour-incubation. The antibiotic concentration required to kill adherent bacteria was far higher than that required for planktonic cells, although we used bactericidal drugs; beta-lactam, quinolone, and aminoglycoside antibiotics. The tolerance demonstrated by adherent cells is likely to play a role in the difficulties encountered in the chemotherapy of biofilm infections.

Expression of the insulin-like growth factor system and cancer progression in hormone-treated prostate cancer patients

Abstract Background : The insulin‐like growth factor (IGF) system, which is composed of two ligands (IGF‐I and IGF‐II), two receptors (IGFR‐I and IGFR‐II) and six binding proteins (IGFBP‐1 to ‐6), plays an important role in cell biology. To identify the main components in the IGF system that contribute to human prostate cancer progression after hormone therapy, mRNA expression of the IGF system in human prostate cancer tissue was systematically examined.

Reg IV is an independent prognostic factor for relapse in patients with clinically localized prostate cancer

Regenerating islet‐derived family, member 4 (REG4, which encodes Reg IV) is a candidate marker for cancer and inflammatory bowel disease. We investigated the potential prognostic role of Reg IV immunostaining in clinically localized prostate cancer (PCa) after radical prostatectomy. Immunohistochemical staining of Reg IV was performed in 98 clinically localized PCa tumors obtained during curative radical prostatectomy. Intestinal and neuroendocrine differentiation was investigated by MUC2 and chromogranin A immunostaining, respectively. The prognostic significance of immunohistochemical staining for these factors on prostate‐specific antigen (PSA)‐associated recurrence was assessed by Kaplan–Meier analysis and a Cox regression model. Phosphorylation of the epidermal growth factor receptor (EGFR) by Reg IV was analyzed by Western blot. In total, 14 (14%) of the 98 PCa cases were positive for Reg IV staining. Reg IV positivity was observed frequently in association with MUC2 (P = 0.0182) and chromogranin A positivity (P = 0.0012). Univariate analysis revealed that Reg IV staining (P = 0.0004), chromogranin A staining (P = 0.0494), Gleason score (P < 0.0001) and preoperative PSA concentration (P = 0.0167) were significant prognostic factors for relapse‐free survival. Multivariate analysis indicated that Reg IV staining (P = 0.0312), Gleason score (P = 0.0014) and preoperative PSA concentration (P = 0.0357) were independent predictors of relapse‐free survival. In the LNCaP cell line, EGFR phosphorylation was induced by the addition of Reg IV‐conditioned medium. These results suggest that Reg IV expression is an independent prognostic indicator of relapse after radical prostatectomy. (Cancer Sci 2008; 99: 1570–1577)