Dynamics of the urinary microbiome in pregnancy and the coincidental predictive value of the microbiota for IVF/IVF-ICSI outcome.

RESEARCH QUESTION: What is the impact of clinical pregnancy on the composition of the urinary microbiota? DESIGN: Eighty-five women receiving IVF, without or with intracytoplasmic sperm injection (ICSI) treatment were enrolled in a prospective observational study performed in 2008. Approximately 14 weeks before the start of hormonal treatment and embryo transfer, a midstream urine sample was obtained, followed by an additional sample 16 weeks after embryo transfer. The microbial composition was determined by polymerase chain reaction of the V1-V3 regions of the 16S rRNA bacterial gene. Clinical pregnancy data were collected after the first IVF/IVF-ICSI cycle and 1 year later. RESULTS: A significant decrease in the abundance of Lactobacillus species as well as a significant increase in that of Staphylococcus species was observed in women who became pregnant after IVF/IVF-ICSI treatment (both P < 0.0001). In addition, based on the composition of the pretreatment microbiome it was possible to identify women with a lower likelihood of achieving clinical pregnancy after IVF/IVF-ICSI treatment. The resulting prediction model was validated in another 27 women who did not become pregnant during the first cycle and received additional IVF/IVF-ICSI cycle(s) or frozen embryo transfer(s). The model predicted the women with no clinical pregnancy after IVF/IVF-ICSI treatment with a sensitivity of 0.42 and a specificity of 1.00. CONCLUSIONS: The data primarily showed that clinical pregnancy results in significant changes in the abundance and diversity of the urinary microbiota. Coincidentally, it was discovered that the urinary microbiome composition before IVF/IVF-ICSI treatment can potentially be used as a predictor of clinical pregnancy.

Urine and stone analysis for the investigation of the renal stone former: a consensus conference.

The Consensus Group deliberated on a number of questions concerning urine and stone analysis over a period of months, and then met to develop consensus. The Group concluded that analyses of urine and stones should be routine in the diagnosis and treatment of urinary stone diseases. At present, the 24-h urine is the most useful type of urine collection, and accepted methods for analysis are described. Patient education is also important for obtaining a proper urine sample. Graphical methods for reporting urine analysis results can be helpful both for the physician and for educating the patient as to proper dietary changes that could be beneficial. Proper analysis of stones is also essential for diagnosis and management of patients. The Consensus Group also agreed that research has shown that evaluation of urinary crystals could be very valuable, but the Group also recognizes that existing methods for assessment of crystalluria do not allow this to be part of stone treatment in many places.

PME and Other ESBL-Positive Multiresistant Pseudomonas aeruginosa Isolated from Hospitalized Patients in the Region of Kurdistan, Iraq.

Nosocomial infections occur worldwide and also in the Kurdistan region. Frequently patients colonized with multiresistant Pseudomonas aeruginosa isolates are encountered in many hospitals. As information is lacking with respect to the mechanisms of resistance responsible for the multiresistant character of the P. aeruginosa isolates and their genetic relationship, isolates were prospectively collected and characterized with respect to their mechanism of resistance. During 2012 and 2013, 81 P. aeruginosa isolates were collected from three teaching hospitals in the city of Erbil, Iraq. Susceptibility testing was performed using the VITEK-2 system. Isolates were screened for the presence of extended-spectrum ß-lactamases (ESBLs) and for the presence of metallo ß-lactamases (MBLs). The presence of serine carbapenemases was detected by PCR. The genetic relationship of the isolates was demonstrated by amplified fragment length polymorphism (AFLP). Susceptibility results revealed high rates of resistance against all classes of antibiotics except polymyxins. Genetic characterization demonstrated the presence of ESBL-genes, that is, blaVEB (30%) and blaPER (17%), also ESBL blaPME was detected in four isolates. AFLP typing revealed clonal spread of blaVEB, blaPER, and three clusters of blaOXA-10-positive isolates. Only one isolate was MBL (blaVIM) positive. Of a selected number of isolates (n = 11), whole-genome sequencing analysis revealed that these isolates belonged to "high-risk" MLSTs ST244, ST235, ST308, and ST654. This study reveals the presence and clonal spread of widely resistant high-risk clones of P. aeruginosa in Iraqi Kurdistan. As far as we are aware, this is the first report of multiple, polyclonal, PME producing P. aeruginosa outside the Arabian Peninsula.

OXA-Carbapenemases Present in Clinical Acinetobacter baumannii-calcoaceticus Complex Isolates from Patients in Kurdistan Region, Iraq.

In addition to intrinsic resistance in Acinetobacter baumannii, many different types of acquired resistance mechanisms have been reported, including the presence of VIM and IMP metallo ß-lactamases and also of blaOXA-23-like and blaOXA-58-like enzymes. In the Kurdistan region of Iraq, the multiresistant A. baumannii-calcoaceticus complex is prevalent. We characterized the different mechanisms of resistance present in clinical isolates collected from different wards and different hospitals from the Kurdistan region. One hundred twenty clinical nonduplicate A. baumannii-calcoaceticus complex isolates were collected from four hospitals between January 2012 and October 2013. The identification of the isolates was confirmed by MALDI-TOF. The susceptibility to different antibiotics was determined by disk diffusion and analyzed in accordance to EUCAST guidelines. By PCR, the presence of blaOXA-51-like, blaOXA-23-like, blaOXA-24-like, and blaOXA-58-like genes was determined as well as the presence of the insertion element ISAba1. Clonal diversity was analyzed by pulsed-field gel electrophoresis (PFGE) using the restriction enzyme ApaI and, in addition, multilocus sequence typing (MLST) was performed on a selected subset of 15 isolates. All 120 A. baumannii isolates harbored blaOXA-51-like genes. One hundred one out of 110 (92%) imipenem (IMP)-resistant A. baumannii-calcoaceticus complex isolates additionally carried the blaOXA-23-like gene and four isolates (3%) were positive for blaOXA-24-like. All 101 blaOXA-23-like-positive isolates had the ISAba1 insertion sequence, 1,600 bp upstream of the blaOXA-23-like gene. The blaOXA-58-like gene was not detected in any of the 110 IMP-resistant strains. Eight different PFGE clusters were identified and distributed over the different hospitals. MLST analysis performed on a subset of 15 representative isolates revealed the presence of the international clone ST2 (Pasteur). Besides ST2 (Pasteur), also many other STs (Pasteur) were encountered such as ST136, ST94, ST623, ST792, and ST793, all carrying the blaOXA-23 gene. In clinical A. baumannii-calcoaceticus complex isolates from Kurdistan-Iraq, the blaOXA-23 gene in combination with the upstream ISAba1 insertion element is largely responsible for carbapenem resistance. Several small clusters of identical genotypes were found from patients admitted to the same ward and during overlapping time periods, suggesting transmission within the hospital. Identification of source(s) and limiting the transmission of these strains to patients needs to be prioritized.

Changes in the penile arteries of the rat after fractionated irradiation of the prostate: a pilot study.

INTRODUCTION: External beam radiotherapy for prostate cancer leads to erectile dysfunction in 36%-43% of patients. The underlying mechanism is largely unknown, although some clinical studies suggest that the arterial supply to the corpora cavernosa is responsible. Two animal experimental studies reported on the effects of a single fraction of prostate irradiation on the penile structures. However, irradiation in multiple fractions is more representative of the actual clinical treatment. AIM: The present prospective, controlled study was initiated to investigate the effect of fractionated prostate irradiation on the arteries of the corpora cavernosa. MAIN OUTCOME MEASURES: Histological evaluation of the penile tissue in comparison with control rats at 2, 4, and 9 weeks after irradiation. METHODS: The prostate of twelve rats was treated with external beam radiation in 5 daily fractions of 7.4 gray. Three control rats were treated with sham irradiation. Prostatic and penile tissue was evaluated for general histology (hematoxylin-eosin). The penile tissue was further evaluated after combined staining for collagen (resorcin fuchsin) and alpha-smooth muscle actin (SMA) (Biogenex). RESULTS: The prostate showed adequate irradiation with fibrosis occurring at 9 weeks after irradiation. The corpora cavernosa showed arteries that had developed loss of smooth muscle cells expressing SMA, thickening of the intima, and occlusions. All the control rats maintained normal anatomy. CONCLUSION: This is the first animal experimental study that demonstrates changes in the arteries of the corpora cavernosa after fractionated irradiation to the prostatic area. The preliminary data suggests that erectile dysfunction after radiotherapy might be caused by radiation damage to the arterial supply of the corpora cavernosa.

Influence of body temperature on indinavir crystallization under loop of Henle conditions.

OBJECTIVES: Indinavir is a protease inhibitor used in the therapy of HIV-1+ patients. It causes indinavir stone formation. It has been shown to precipitate in the loop of Henle (LH) at plasma concentrations (conc[P]) of approximately 8 mg/L. Those experiments were performed at room temperature. Given the influence of temperature on crystallization in general, and solubility of indinavir in particular, we repeated the experiments under physiological (body) temperature conditions. METHODS: Test solutions contained indinavir concentrations of 100-750 mg/L at ionic strengths varying from 0 to 800 mM simulating conditions in the proximal tubule and the LH. Solutions were titrated with base (NaOH) to find the pH value where nucleation is initiated. Experiments were conducted at room temperature (20 degrees C) and repeated under constantly monitored (body) temperature (37 degrees C). RESULTS: Experiments at 20 degrees C confirmed our previous results. At 37 degrees C, the relationship between pH and indinavir concentration remained inversely proportional. Again, the LH was confirmed as the most likely localization of crystallization. However, at 37 degrees C precipitation occurred at a lower urinary concentration (100 versus 125 mg/L) and within a lower pH range (6.67-7.26 versus 7.23-7.44). This lower urinary concentration corresponds to a lower conc[P] [critical value (CV)] of 6.41 mg/L, as compared with 8.01 mg/L at 20 degrees C. CONCLUSIONS: The CV is even lower at 37 degrees C than previously assumed. Plasma peak concentration above the CV of 6.4 mg/L will induce crystallization in the LH and should be avoided.

Is the impaired flow after hypospadias correction due to increased urethral stiffness?

PURPOSE: A low flow rate without clinical symptoms is commonly found in boys after hypospadias correction. Urethral calibration usually shows no abnormalities. We investigated whether this impairment might be caused by increased neo-urethral wall stiffness. METHODS: From polyvinyl alcohol cryogel two models of the urethra were made, hypospadias and control; both had a constant and equal inner diameter and equal compliance. The hypospadias model had a less compliant distal segment mimicking the distal neo-urethra after hypospadias correction. In both models, flow rate was recorded as a function of bladder pressure. To test whether the length of the less compliant segment had an effect on the flow rate, both models were shortened by cutting off 1-cm segments. RESULTS: In a physiological range of bladder pressures (10-130 cmH(2)O) the mean flow rate (+/-1 SEM) in the hypospadias model was 2.8+/-0.3 ml/s, significantly lower (P<0.05) than in the control model (5.4+/-0.6 ml/s). Shortening of the hypospadias model showed some increase in flow rate, but this was not statistically significant. In the control model there was also no significant variation in flow rate. CONCLUSION: A low-compliant segment of a urethral model reduced the flow rate. Extrapolating these results to asymptomatic boys with a low urinary flow rate after hypospadias repair might justify a watchful waiting policy.

Pathological and therapeutic significance of cellular invasion by Proteus mirabilis in an enterocystoplasty infection stone model.

Proteus mirabilis infection often leads to stone formation. We evaluated how bacterium-mucin adhesion, invasion, and intracellular crystal formation are related to antibiotic sensitivity and may cause frequent stone formation in enterocystoplasties. Five intestinal (Caco-2, HT29, HT29-18N2, HT29-FU, and HT29-MTX) and one ureter cell line (SV-HUC-1) were incubated in artificial urine with five Proteus mirabilis strains. Fluorescence-activated cell sorting (FACS), laser scanning microscopy, and electron microscopy evaluated cellular adhesion and/or invasion, pathologic changes to mitochondria, and P. mirabilis-mucin colocalization (MUC2 and MUC5AC). An MTT (thiazolyl blue tetrazolium bromide) assay and FACS analysis of caspase-3 evaluated the cellular response. Infected cells were incubated with antibiotics at dosages representing the expected urinary concentrations in a 10-year-old, 30-kg child to evaluate bacterial invasion and survival. All cell lines showed colocalization of P. mirabilis with human colonic mucin (i.e., MUC2) and human gastric mucin (i.e., MUC5AC). The correlation between membrane mucin expression and invasion was significant and opposite for SV-HUC-1 and HT29-MTX. Microscopically, invasion by P. mirabilis with intracellular crystal formation and mitochondrial damage was found. Double membranes surrounded bacteria in intestinal cells. Relative resistance to cotrimoxazole and augmentin was found in the presence of epithelial cells. Ciprofloxacin and gentamicin remained effective. Membrane mucin expression was correlated with relative antibiotic resistance. Cell invasion by P. mirabilis and mucin- and cell type-related distribution and response differences indicate bacterial tropism that affects crystal formation and mucosal presence. Bacterial invasion seems to have cell type-dependent mechanisms and prolong bacterial survival in antibiotic therapy, giving a new target for therapeutic optimalization of antibiotic treatment.