Fukashi Yamamichi

Does mutation in gyrA and/or parC or efflux pump expression play the main role in fluoroquinolone resistance in Escherichia coli urinary tract infections?: A statistical analysis study

There are several mechanisms of fluoroquinolone (FQ) resistance, such as mutations in the quinolone resistance-determining regions (QRDRs) of target genes and efflux pump expression. The purpose of this study was to investigate which factor plays the main role in FQ resistance in Escherichia coli causing urinary tract infections (UTIs) from a statistical analysis of our two previous works. DNA sequencing of the QRDRs of the FQ resistance-related genes gyrA and parC as well as real-time reverse transcriptase (RT)-PCR for the expression of efflux pump genes such as marA or yhiU were performed and the correlations of mutations or efflux pump gene expression with FQ minimal inhibitory concentrations (MICs) were investigated. Significant factors for high MICs of sitafloxacin (STFX), ciprofloxacin (CPFX) and levofloxacin were the mutations S83L and D87N in gyrA and S80I and E84V in parC as well as the expression of marA. Mutations in the QRDRs of gyrA or parC had a greater effect on FQ MICs than efflux pump gene expression. Based on the regression coefficient, these mutations correlated with MICs to CPFX most, and STFX had the lowest effects from these mutations among the three tested FQs. In conclusion, in E. coli causing UTIs, mutations in the QRDRs of gyrA or parC had a greater effect on FQ resistance, especially to CPFX, than efflux pump gene expression from a statistical analysis study of our two previous works. Further research into the molecular basis for FQ resistance could lead to new therapeutic strategies against FQ-resistant E. coli.

Relationship between urinary tract infection categorization and pathogens' antimicrobial susceptibilities.

Background: Urinary tract infections (UTIs) can be hard to treat and treatment plans need to include accurate categorization such as uncomplicated or complicated UTI, or catheterized or uncatheterized UTI. We investigated the antibiotic susceptibilities of representative uropathogens in UTI categories. Methods: We isolated uropathogens and analyzed their antimicrobial susceptibilities according to UTI categorization such as: (1) urology outpatients, urology inpatients, or other department inpatients; (2) uncomplicated or complicated UTIs; (3) upper or lower UTIs, and (4) non-catheterized or catheterized UTIs. Results:Escherichia coli, Enterococcus faecalis, and Pseudomonas aeruginosa were representative uropathogens. Susceptibilities to levofloxacin (LVFX) in E. coli in urology outpatients (p = 0.0179), those to ceftadizime in E. coli in other department inpatients (p = 0.0327), and those to LVFX in E. faecalis in complicated UTI (p = 0.0137) significantly decreased in these 3 years compared with the previous 3 years. Susceptibilities of upper UTI to LVFX in E. coli were significantly lower in the recent 4 years compared to lower UTI (p = 0.0452) and those of catheterized UTI to LVFX in E. faecalis were significantly lower than in non-catheterized UTI (p = 0.0153). Conclusions: Data demonstrated different tendencies of uropathogens’ antibiotic susceptibilities according to UTI categorizations and they could be useful for planning UTI treatments.

Comparison of Predictive Factors for Postoperative Incontinence of Holmium Laser Enucleation of the Prostate by the Surgeons’ Experience During Learning Curve

Purpose: To detect predictive factors for postoperative incontinence following holmium laser enucleation of the prostate (HoLEP) according to surgeon experience (beginner or experienced) and preoperative clinical data. Methods: Of 224 patients, a total of 203 with available data on incontinence were investigated. The potential predictive factors for post-HoLEP incontinence included clinical factors, such as patient age, and preoperative urodynamic study results, including detrusor overactivity (DO). We also classified the surgeons performing the procedure according to their HoLEP experience: beginner (<21 cases) and experienced (≥21 cases). Results: Our statistical data showed DO was a significant predictive factor at the super-short period (the next day of catheter removal: odds ratio [OR], 3.375; P=0.000). Additionally, patient age, surgeon mentorship (inverse correlation), and prostate volume were significant predictive factors at the 1-month interval after HoLEP (OR, 1.072; P=0.004; OR, 0.251; P=0.002; and OR, 1.008; P=0.049, respectively). With regards to surgeon experience, DO and preoperative International Prostate Symptom Score (inverse) at the super-short period, and patient age and mentorship (inverse correlation) at the 1-month interval after HoLEP (OR, 3.952; P=0.002; OR, 1.084; P=0.015; and OR,1.084; P=0.015; OR, 0.358; P=0.003, respectively) were significant predictive factors for beginners, and first desire to void (FDV) at 1 month after HoLEP (OR, 1.009; P=0.012) was a significant predictive factor for experienced surgeons in multivariate analysis. Conclusions: Preoperative DO, IPSS, patient age, and surgeon mentorship were significant predictive factors of postoperative patient incontinence for beginner surgeons, while FDV was a significant predictive factors for experienced surgeons. These findings should be taken into account by surgeons performing HoLEP to maximize the patient’s quality of life with regards to urinary continence.

Possible Role of Sonic Hedgehog and Epithelial-Mesenchymal Transition in Renal Cell Cancer Progression

Purpose Sonic hedgehog (Shh) signaling and epithelial-mesenchymal transition (EMT) are both known to relate to cancer progression. The purpose of this study was to investigate the role of Shh signaling and EMT in renal cell carcinoma (RCC). Materials and Methods Cell proliferation was assayed in RCC cell lines in the presence or absence of a Shh signaling stimulator, recombinant Shh (r-Shh) protein, or a Shh signaling inhibitor, cyclopamine. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to study the expression of EMT markers (E-cadherin, N-cadherin, and vimentin) and osteonectin. The expression of Ki-67, Gli-1, osteonectin, and EMT markers in nephrectomy specimens from RCC patients was also measured by immunohistochemical (IHC) staining. Results RCC cells showed enhanced cell proliferation by r-Shh protein, whereas cell proliferation was suppressed by the addition of cyclopamine in RenCa cells. Real-time RT-PCR showed that r-Shh suppressed the expression of E-cadherin and that this suppression was partly blocked by cyclopamine alone in RenCa cells. In the IHC results, osteonectin significantly correlated with vein sinus invasion (p=0.0218), and the expression of vimentin significantly correlated with lymphatic invasion (p=0.0392). Conclusions Shh signaling and EMT play roles in RCC progression, and the Shh signaling inhibitor cyclopamine might be a possible molecular targeted therapeutic strategy for RCC.

Prevention of infectious complication and its risk factors after urological procedures of the upper urinary tract.

Background:Invasive upper urinary tract procedures such as retrograde pyelography (RP) or single (S-J) or double J (D-J) stenting are commonly performed to assess or treat ureteral strictures. Urinary tract infection (UTI) can result after such procedures, and prophylactic antimicrobial administration (PAA) may be necessary. This study investigated infectious complications and risk factors, focusing on PAA. Methods:We studied antimicrobial prevention in 353 upper urinary tract examinations or treatments. Procedures included S-J or D-J installation or exchange, RP and percutaneous nephrostomy. We investigated PAA and the occurrence of febrile infectious complications with respect to each procedure and attempted to find the risk factors. Results:Levofloxacin was used in 149 subjects (42.2%) and cefcapene pivoxil in 114 cases (32.3%). There were 16 febrile infectious complication cases (4.5%) after procedures, and pyuria or hydronephrosis prior to examination or treatment was an independent risk factor for infectious complication (p < 0.05) as well. Conclusions:These data showed that it is necessary to evaluate the risk factors before urological procedures of the upper urinary tract and to offer a definite preventive methodology according to these risk factors for the establishment and update of guidelines.

Comparison of Postoperative Infection between Robotic-Assisted Laparoscopic Prostatectomy and Open Radical Prostatectomy

Background: Robotic-assisted laparoscopic prostatectomy (RALP) has been rapidly adapted worldwide. The purpose of this study was to investigate postoperative infection (POI) after RALP and compare it with that after open radical prostatectomy (ORP). Methods: 89 consecutive RALP cases and 105 recent ORP cases were enrolled. POIs were categorized according to CDC guideline criteria. Laboratory data relating to infection such as serum white blood cells (WBC) and C-reactive protein (CRP) were comparatively investigated before and after the surgeries. Data were collected and analyzed retrospectively. Results: There was one surgical site infection patient in the RALP cases (1/89, 1.12%). ORP cases had a comparatively higher ratio of POI (6/105, 4.77%), but the difference did not reach statistical significance (p = 0.0876). The postoperative rise of serum WBC (postoperative days 1 and 4) and CRP (postoperative days 1, 4 and 7) was significantly suppressed from pre-surgery data in RALP when compared to ORP. Conclusions: RALP tended to have a lower rate of POI than ORP even though this change was not statistically significant and the postoperative rise of serum WBC and CRP was significantly suppressed in RALP compared to ORP. A prospective study with a standardized prophylactic antibiotic administration may be necessary for further evaluation of potential guidelines for RALP.

Potential Establishment of Lung Metastatic Xenograft Model of Androgen Receptor-positive and Androgen-independent Prostate Cancer (C4-2B)

OBJECTIVE To establish a mouse xenograft model of metastatic prostate cancer (PCa) and investigate the relationship between metastasis and circulating tumor cells. METHODS Flow cytometry (FACS) was used to detect suitable PCa cells and markers for detecting circulating tumor cells in vivo. We orthotopically injected androgen receptor-positive and androgen-independent C4-2B PCa cells into 12 severe combined immunodeficiency (SCID) mouse prostates, including 1 vehicle control. We measured the serum prostate-specific antigen levels biweekly after tumor inoculation. Circulating tumor cells (CTCs) were measured qualitatively by fluorescent microscopy immediately after the mice were sacrificed. The mouse prostates and lungs were examined for tumor formation using immunohistochemistry because we found no apparent metastasis, except in the lung. RESULTS FACS analyses in vitro identified the marker, prostate-specific membrane antigen, and C4-2B cells to be appropriate for additional in vivo study. We confirmed that the serum prostate-specific antigen increase was dependent on time and prostate tumor weight in mice. Of the 11 mice, 6 could be used as the mouse PCa xenograft model. Fluorescent microscopy detected CTCs in the peripheral blood in 5 of the 6 mice constituting the PCa model. Human prostate-specific antigen expression was detected by immunohistochemistry in the prostates of all the mice and in the lung of 2 of the 6 mice, suggesting 2 mice with lung metastasis. CONCLUSION We have shown the potential establishment of a mouse lung metastatic xenograft model of androgen receptor-positive and androgen-independent C4-2B PCa tumor. However, the present model requires improvement to be a more reproducible, accurate and complete experimental model. Additional study is necessary to verify the relationship between metastasis and CTCs.

Sonic hedgehog and androgen signaling in tumor and stromal compartments drives epithelial–mesenchymal transition in prostate cancer

Abstract Objective. Sonic hedgehog (Shh) signaling, androgens and epithelial–mesenchymal transition (EMT) are related to prostate cancer (PCa) progression. The aim of this study was to investigate how Shh and androgen [dihydrotestosterone (DHT)] signaling act in prostate epithelial and stromal compartments and whether this signaling pathway drives EMT and promotes PCa progression. Material and methods. LNCaP, normal prostate fibroblast (NPF) and cancer-associated prostate fibroblast (CPF) cells were studied with DHT and/or the Shh signaling inhibitor cyclopamine. Real-time reverse transcription–polymerase chain reaction (RT-PCR) was performed to evaluate the expressions of a potential Shh target gene, osteonectin (ON) and EMT-associated markers (E-cadherin, N-cadherin and vimentin). Immunohistochemical studies using PCa prostatectomy samples were performed to assess the expression levels of ON, Gli-1, androgen receptor, Shh, E-cadherin, N-cadherin and vimentin. Results. While DHT enhanced cell proliferation in CPF more than LNCaP or NPF, cyclopamine inhibited cell proliferation enhanced by DHT in CPF. Real-time RT-PCR showed whereas both Shh and DHT induced N-cadherin and vimentin, DHT also induced the expression of osteonectin in LNCaP and cyclopamine blocked these expressions in osteonectin, N-cadherin and vimentin (p = 0.0084, 0.0002 and 0.0373, respectively). Immunohistochemistry showed that high expression of stromal, but, not epithelial, ON was significantly correlated with serum prostate-specific antigen (PSA) (p = 0.031), and high expression of Gli-1 and low expression of stromal ON with PSA recurrence (p = 0.0114 and p = 0.0005, respectively). Conclusions. Shh and androgen signaling in prostate tumor and stromal compartments drives EMT, and thus may play some role in PCa progression. Cyclopamine may be one therapeutic strategy for PCa.

Does Surgeon Experience Affect Operative Time, Adverse Events and Continence Outcomes in Holmium Laser Enucleation of the Prostate? A Review of More Than 1,000 Cases

Purpose: Holmium laser enucleation of the prostate has become an increasingly common surgical therapy for benign prostatic hyperplasia. However, the relationship between surgeon experience and surgical outcomes has not yet been fully investigated. In this study we investigated how surgeon experience with holmium laser enucleation of the prostate affected operative time, adverse events and outcomes related to urination. Materials and Methods: We gathered a total of 1,113 cases of holmium laser enucleation of the prostate from 5 hospitals in Hyogo Prefecture, Japan. Included were data on surgeon experience with the procedure, operative time, enucleation time, morcellation time, patient age, perioperative and postoperative surgery related complications, and outcomes related to urination. Results: A total of 39 surgeons were included in analysis. Statistical data showed that increasing surgical experience significantly contributed only to surgical time, enucleation time and urinary incontinence after holmium laser enucleation (p = 0.0146, 0.0216 and 0.0405, respectively). No significant changes were seen postoperatively in surgery related factors such as morcellation time, resected prostate volume, infectious or noninfectious surgery related complications, or urination related outcomes (p >0.05) Experience with at least 20 cases in particular affected surgical time (p = 0.0050), enucleation time (p = 0.0068) and urinary incontinence after holmium laser enucleation (p = 0.0021). Conclusions: Surgeon experience contributed to shortened operative time and enucleation time, and to decreased postoperative urinary incontinence but not to surgery related complications or urination related outcomes as shown by maximum urine flow and post‐void residual urine volume. We also found that experienced surgeons with 31 to 50 cases might be associated with complications after holmium laser enucleation in larger prostate cases. Based on these data further prospective studies are scheduled to establish a program for training in holmium laser enucleation of the prostate.

Prophylactic efficacy of cephamycin plus fluoroquinolones in high risk patients on inhibiting infectious complications after transrectal prostate biopsy

Brief Communications Many prophylactic antibiotic medications for use during transrectal prostate biopsy (TPBX) have been reported, but most reports did not classify the patients’ risk of infection. Since fluoroquinolones can achieve high concentrations in the prostate they have long been used for prophylactic administration, but recently fluoroquinolone-resistant Escherichia coli (FQRE) has been rapidly increasing. The resistance ratios of E. coli to levofloxacin (LVFX) have also increased from 5 to 20% in Japan in recent years. Extended beta-lactamase (ESBL) producing bacteria, especially Enterobacteriaceae, have become widespread and have been implicated in treatment failure. In updated data, 1.48% of all E. coli in Austria were ESBL-producing. Representative beta-lactamase inhibitors include Tazobactam/piperacillin (TAZ/PIPC), which is often used for urinary tract infection (UTI). To avoid the development of antibiotic-resistant bacterial strains, other narrow spectrum antibiotics are considered to be better for use. Since antibiotic susceptibilities may be different in low risk and high risk patients, selection of prophylactic antibiotic regimens should be based on the risk classifications for infectious complications. Cephamycin [cefmetazole (CMZ)] is known to have a greater effect on gram-negative bacteria and on representative anaerobic bacteria found in human intestine, such as Bacteriodes fragilis, than general cephalosporins. In this study, we focussed on high-risk patients and investigated the effect of CMZ, which generally has good activity against ESBL producing bacteria, plus LVFX, taking risk criteria for infectious complications into consideration, and compared the results for the prevention of febrile infectious complications after TPBX with our previous TAZ/PIPC regimen. We studied 142 consecutive patients with a high risk for infectious complications, who were scheduled for TPBX for suspected prostate cancer. High-risk patients are classified by factors such as severe diabetes mellitus with serum HbA1c level i8.0%, steroids dosing, a larger prostate (i75 mL), residual urine i50 mL, or pre-TPBX bacteriuria. TPBXs were performed at Kobe University Hospital from February 2013 to March 2014. Our previous regimen, TAZ/PIPC, was in use from January 2012 to January 2013. The data were analysed retrospectively. All biopsies were performed as previously described with a modification of 12 [sextant and one each far lateral on both sides from the peripheral zone (PZ) z2 cores each at base and apex on both sides from transitional zone (TZ) cores]. This study was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. As a prophylactic antibiotic, each patient received CMZ 2 g i.v. and LVFX 500 mg taken orally 30 minutes prior to TPBX. Patients continued to dose LVFX 500 mg once a day for 3 days in total. The comparative TAZ/PIPC group was given TAZ/ PIPC 4.5 g i.v. 30 minutes before TPBX and continued repeated TAZ/PIPC i.v. 3 hours after TPBX. Patients were examined for febrile infectious complications within 30 days after TPBX. Bacteriological investigations were also performed. White blood cell (WBC) counts (/mm) and C-reactive protein (CRP) (mg/mL) in plasma, pyuria, and bacteriuria (/high power field: hpf) were measured as infection Correspondence to: Katsumi Shigemura, 7-5-1 Kusunoki-Cho, Chuo-Ku, Kobe 650-0017, Japan. Email: yutoshunta@hotmail.co.jp