Failure of Urinary Beta-Glucuronidase Activity to Localize the Site of Urinary Tract Infection

The differentiation of renal from bladder bacteriuria is difficult on clinical grounds alone. To evaluate the correlation between site of infection and urinary beta-glucuronidase activity, 46 patients with well documented recurrent bacteriuria were studied by bilateral ureteral catheterization. Urinary beta-glucuronidase activity was also determined in 46 control subjects. In general, asymptomatic patients with renal bacteriuria, either unilateral or bilateral, had levels of enzyme activity in their urine comparable to patients with infection confined to the bladder and to normals. Only 4 of 25 patients with renal bacteriuria had significant elevations of urinary beta-glucuronidase. After localization of infection, 9 of 10 patients treated with kanamycin, a potentially nephrotoxic drug, developed significant elevations of urinary beta-glucuronidase. The results of these studies indicate that determination of beta-glucuronidase activity in urine is not useful in predicting the site of infection in patients with bacteriuria but may find a role in screening for early nephrotoxicity.     

A Comparative Study of the Triphenyltetrazolium Chloride (Uroscreen) Test and Conventional Methods for the Detection of Bacteriuria

Acute urinary tract infection may be preceded by and active pyelonephritis may be associated with asymptomatic bacteriuria. Treatment of asymptomatic bacteriuria may prevent or arrest active, chronic pyelonephritis and its sequelae. Consequently, there is a need for a reliable and simple screening procedure to detect asymptomatic bacteriuria in large segments of the population.The reliability and practicability of tests advocated for the detection of bacteriuria, including the new chemical triphenyltetrazolium chloride (T.T.C.) (Uroscreen) test, were evaluated. Reliability was assessed by correlating results of these tests with bacterial counts of tested urines. Significant bacteriuria is defined as the presence of 100,000 or more organisms per ml. of urine.The T.T.C. (Uroscreen) test was positive in 92.5% of cases of bacteriuria; there were 7.5% false-negative and 2.8% false-positive results. Bacteria on Gram-stained smear were found in 95.5% of the cases of bacteriuria and in 14.6% of those with non-infected urine; pyuria (more than three leukocytes per high-power field), in 60% of those with bacteriuria and in 15.9% of those with presumably non-infected urine. Bacteria were conspicuous in the urinary sediment in 91.1% of cases of bacteriuria and in 3.7% of presumably non-infected urines.The T.T.C. (Uroscreen) test fulfilled the criteria for a reliable and simple screening procedure. It should be used concomitantly with other screening tests when the urine is examined routinely.     

Detection of bacteriuria by bioluminescence: effect of pre-analysis centrifugation of specimens

N ichols , W.W., C urtis , G.D.W. & J ohnston , H.H. 1984. Detection of bacteriuria by bioluminescence: effect of pre-analysis centrifugation of specimens. Journal of Applied Bacteriology 56 , 247–257.
Three bioluminescence-based, rapid methods of detecting significant bacteriuria were applied in parallel to 514 urine specimens. The results were compared with those of a quantitative pour plate viable count method, defined as positive if ≥ 10⁵ c.f.u./ml of urine were observed. When adjusted to yield 21% falsely positive results the three rapid methods yielded 24%, 21% and 19% falsely negative results. If specimens with evidence of urethral or vaginal contamination were excluded (237 specimens remaining) the three methods yielded respectively 14%, 8% and 13% falsely negative results. A major source of disagreement between the bioluminescence-based methods and quantitative culture thus appeared to be contaminated urine specimens.     

Simple Disposable Method for Quantitative Cultures of Urine

A disposable kit was tested as a means of detecting significant bacteriuria by quantitative culture of urine. The total error in 3,563 specimens tested by five investigators was less than 1%. The method was very effective in differentiating significant bacteriuria, i.e., more than 100,000 bacteria per ml of urine from uninfected urine. In specimens from patients with urinary tract abnormalities who had mixed bacterial flora, the absolute numbers obtained with the dip-inoculum method had a 10% variation when compared to results obtained by calibrated loop or dilution pour plate methods. Therefore, the main utility of the kit is for screening and following patients after therapy. A significant delay in time between inoculation of the medium in the kit with the freshly voided urine and incubation of the kit to promote growth did not affect the reliability of the kit as a method of doing quantitative urine cultures to detect bacteriuria.     

The inhibition of bacterial growth by hypochlorous acid. Possible role in the bactericidal activity of phagocytes.

The 'respiratory burst' of phagocytes such as neutrophils generates superoxide which forms H2O2 by dismutation. H2O2 and Cl- ions serve as substrates for the enzyme myeloperoxidase to generate hypochlorous acid (HOCl). HOCl is thought to play an important role in bacterial killing, but its mechanism of action is not well characterized. Furthermore, although many studies in vitro have shown HOCl to be a damaging oxidant with little or no specificity (particularly at high concentrations), bacteria which have been ingested by phagocytes appear to experience a rapid and selective inhibition of cell division. Bacterial membrane disruption, protein degradation, and inhibition of protein synthesis, do not seem to occur in the early phases of phagocyte action. We have now found that low concentrations of HOCl exert a rapid and selective inhibition of bacterial growth and cell division, which can be blocked by taurine or amino acids. Only 20 microM-HOCl was required for 50% inhibition of bacterial growth (5 x 10(8) Escherichia coli/ml), and 50 microM-HOCl completely inhibited cell division (colony formation). These effects were apparent within 5 min of HOCl exposure, and were not reversed by extensive washings. DNA synthesis (incorporation of [3H]-thymidine) was significantly affected by even a 1 min exposure to 50 microM-HOCl, and decreased by as much as 96% after 5 min. In contrast, bacterial membrane disruption and extensive protein degradation/fragmentation (release of acid-soluble counts from [3H]leucine-labelled cells) were not observed at concentrations below 5 mM-HOCl. Protein synthesis (incorporation of [3H]leucine) was only inhibited by 10-30% following 5 min exposure to 50 microM-HOCl, although longer exposure produced more marked reductions (80% after 30 min). Neutrophils deficient in myeloperoxidase cannot convert H2O2 to HOCl, yet can kill bacteria. We have found that H2O2 is only 6% as effective as HOCl in inhibiting E. coli growth and cell division (0.34 mM-H2O2 required for 50% inhibition of colony formation), and taurine or amino acids do not block this effect. Our results are consistent with a rapid and selective inhibition of bacterial cell division by HOCl in phagocytes. H2O2 may substitute for HOCl in myeloperoxidase deficiency, but by a different mechanism and at a greater metabolic cost.     

Urinary bladder, ionic composition of seminal fluid and urine with characterization of sperm motility in tench (Tinca tinca L.)

A small urinary bladder attached to the seminal duct in caudal part of the abdominal cavity was registered for the first time in dissected males of tench. The urinary bladder wall was of whitish color and the bladder contained 0.5–2 ml of urine. When collected in the experiment, the tench sperm was white‐colored. Spermatozoa density is highly variable due to contamination by urine, and the latter additionally activates spontaneous motility of the spermatozoa. Seminal fluid contains ions such as Na⁺ (18.4 ± 1.3 mm ), K⁺ (1.9 ± 0.6 mm ), Ca²⁺ (0.6 ± 0.2 mm ) and Mg²⁺ (0.5 ± 0.1 mm ), leading to osmolality of 230 ± 82 mOsmol kg^?1 depending on the dilution by urine. Urea was detected in urine samples uncontaminated by sperm with an osmolality of 85 ± 58 mOsmol kg^?1. Urine also contained high concentrations of ions such as Na⁺ (30.9 ± 8.9 mm ), K⁺ (4.3 ± 2.9 mm ), Ca²⁺ (0.9 ± 0.5 mm ) and Mg²⁺ (0.6 ± 0.2 mm ). The spontaneous sperm activation by urine was up to 100%, but could be prevented by collection in an immobilizing solution. Motility was observed for 90–100% spermatozoa just after their transfer to distilled water or in a swimming medium (SM, 30–45 mm KCl) with a velocity of 120–140 μm s^?1. A flagellar beat frequency of 60–70 Hz and forward motility lasted up to 80 s in distilled water, and up to 180 s in SM at room temperature.     

Suprapubic aspiration under ultrasound guidance in children with fever of undiagnosed cause.

OBJECTIVES--To assess the ease of use of suprapubic aspiration of urine under ultrasound guidance in babies with fever of uncertain cause and to assess the importance of bacterial counts and pyuria in relation to abnormalities of the urinary tract and the importance of pyuria in the absence of bacteriuria. DESIGN--Analysis of urine samples obtained by suprapubic aspiration in babies and children from July 1991 to June 1992. The clinical records of the children with bacteriuria and sterile pyuria were examined retrospectively. SETTING--Neonatal and paediatric wards of a district general hospital. SUBJECTS--508 babies and children who had fever of uncertain cause or were seriously ill. RESULTS--No difficulties arose in the collection of 545 specimens. Bacteria were isolated from the specimens of 44 children, 24 of whom had abnormalities of the urinary tract. The bacterial count was < 10(8)/l in 18 of the children with bacteriuria, 10 of whom had abnormalities. No white cells were seen in 22 of the 46 bacteriuric specimens; nine of the children with no pyuria had vesicoureteric reflux. 439 of the 499 non-bacteriuric specimens showed no white cells. 60 children had pyuria without bacteriuria. CONCLUSIONS--The use of ultrasound guidance simplifies suprapubic aspiration of urine in babies. Low bacterial counts may be associated with abnormalities of the urinary tract. Laboratory techniques capable of detecting such counts reliably should be used. Pyuria is absent in half of babies and very young children with bacteriuria. It rarely occurs without bacteriuria, and if it does an explanation should be sought.     

Molecular hydrogen from water radiolysis as an energy source for bacterial growth in a basin containing irradiating waste

Although being deionized, filtered and therefore normally deeply oligotrophic, the water from a basin containing irradiating waste presented relatively high bacterial concentrations (ca 10(5) cfu ml(-1)) and biofilm development at its surface and on the walls. This water was characterized by a high concentration of molecular H2 due to water radiolysis, while its electrochemical potential was around +400 mV due the presence of dissolved O2 and active oxygen compounds. This combination of H2 availability and of an oxidant environment is completely original and not described in nature. From surface and wall biofilms, we enumerated the autotrophic populations ( approximately 10(5) bacteria ml(-1)) able to grow in presence of H2 as energy source and CO2 as carbon source, and we isolated the most abundant ones among cultivable bacteria. They efficiently grew on a mineral medium, in the presence of H2, O2 and CO2, the presence of the three gases being indispensable. Two strains were selected and identified using their rrs gene sequence as Ralstonia sp. GGLH002 and Burkholderia sp. GGLH005. In pure culture and using isotope exchange between hydrogen and deuterium, we demonstrated that these strains are able to oxidize hydrogen as energy source, using oxygen as an electron acceptor, and to use carbon dioxide as carbon source. These chemoautotroph hydrogen-oxidizing bacteria probably represent the pioneer bacterial populations in this basin and could be primary producers in the bacterial community.     

Suprapubic Bladder Aspiration in Diagnosis of Urinary Tract Infection

Suprapubic aspiration of the bladder has proved a safe and reliable method of collecting urine for culture. It avoids contamination of the specimen and dispenses with the need for colony counts, because the isolation of any organism indicates bladder bacteriuria. In addition, urine collected in this way can be kept at 4°C. or at room temperature for up to 10 days before it is cultured.     

Site of Action of Nitrofurantoin in Experimental Urinary Tract Infection

The effectiveness of nitrofurantoin in suppressing bacterial growth in the urinary tract was evaluated by using two different experimental models. Pyelonephritis was produced in rats by direct inoculation of 10(4)Escherichia coli in the medulla of left kidney. Ascending urinary tract infection was induced by inoculation into the urinary bladder of 10(7)Proteus mirabilis, after a partial cystectomy. Nitrofurantoin was shown to be effective in suppressing bladder bacteriuria, in preventing ascending pyelonephritis, and also in preventing bacterial multiplication in kidney tissue following direct inoculation.     

Effects of urine from females in oestrus on puberty in female mice

A series of 9 experiments was conducted to examine various characteristics of the urinary chemosignal found in the urine of oestrous female mice that accelerates the sexual development of conspecific females. This urinary chemosignal was effective in doses as small as 0.001 ml/day, was present in excreted and bladder urine, required 3 days of treatment starting before Day 29 of age to effect an acceleration of puberty, required a minimum daily exposure of 2 h, and was relatively nonvolatile. In addition the chemosignal from oestrous females was effective in summer but not in winter months, was significantly more effective when collected at the middle or end of the dark portion of the daily cycle than at the beginning of the dark phase or middle of the light phase, and was not affected by food deprivation or shortened photoperiod. Simultaneous treatment of test subjects with urine from oestrous females and grouped females resulted in delays in puberty and simultaneous treatment with urine from oestrous females and urine from males or pregnant or lactating females did not result in any enhanced acceleration of puberty.     

Systems for Maintenance of Urinary Sterility After Prostatectomy

Closed drainage is recommended for all patients after prostatectomy where hemostasis has been adequate. Although closed drainage can maintain sterility of the bladder, thereby fostering healing and reducing infectious complications, such drainage is not insisted upon at most hospitals because of the inconveniences associated with it. However, when closed drainage was used in 25 consecutive cases of transurethral resection, infection was reduced to 25 per cent (in contrast to the 85 to 100 per cent encountered with open drainage).The ideal closed system should incorporate:1. Fixed tubing to prevent contamination where the catheter joins the tubing and where the tubing is attached to the container;2. An aseptic method of emptying;3. A device to prevent reflux of the potentially contaminated urine in the container into the bladder;4. Free urinary flow from bladder to container; and5. Portability for the patient and convenience for the staff.A system is proposed that incorporates these features. Particularly effective are a fixed drip chamber with vents at the site of attachment of the tubing to the bag and a protected spigot for emptying.     

Use of Pyromark Q96 ID pyrosequencing system in identifying bacterial pathogen directly with urine specimens for diagnosis of urinary tract infections

Detection and identification of bacterial etiology in urine is critical for accurate diagnosis and subsequent rational treatment of urinary tract infections (UTIs). Urine culture followed by a series of biochemical reactions is currently the standard method for detecting and distinguishing microorganisms associated with UTIs. The whole procedure commonly takes more than 24 h. Here we developed a new system combining 16S rRNA gene broad-range PCR with pyrosequencing technology that allows for bacteria detection and identification in urine in 5 h. To evaluate this system for rapid diagnosis of bacteriuria, 768 urine specimens were collected from patients with suspected UTIs and were tested side-by-side using standard urine culture-based identification method and the pyrosequencing method. The results from pyrosequencing correlated well with those from traditional culture-based identification method. The overall agreement between these two methods reached 98.0% (753/768). In addition, we tested the sensitivity of pyrosequencing method and determined that urine bacterial numbers as low as 10⁴ cfu/ml could be accurately detected and identified. In conclusion, compared with traditional biochemical method, the PCR-pyrosequencing system significantly improved the detection and identification of bacteriuria with shorter time, higher accuracy, and higher throughput, thus allowing earlier pathogen-adapted antibiotic therapy for patients.     

A Real-Time PCR-Based Semi-Quantitative Breakpoint to Aid in Molecular Identification of Urinary Tract Infections

This study presents a novel approach to aid in diagnosis of urinary tract infections (UTIs). A real-time PCR assay was used to screen for culture-positive urinary specimens and to identify the causative uropathogen. Semi-quantitative breakpoints were used to screen for significant bacteriuria (presence of ≥10⁵ CFU/ml of uropathogens) or low-level bacteriuria (containing between 10³ and 10⁴ CFU/ml of uropathogens). The 16S rDNA-based assay could identify the most prevalent uropathogens using probes for Escherichia coli, Pseudomonas species, Pseudomonas aeruginosa, Staphylococcus species, Staphylococcus aureus, Enterococcus species and Streptococcus species. 330 urinary specimens were analysed and results were compared with conventional urine culture. Using a PCR Ct value of 25 as semi-quantitative breakpoint for significant bacteriuria resulted in a sensitivity and specificity of 97% and 80%, respectively. In 78% of the samples with monomicrobial infections the assay contained probes to detect the bacteria present in the urine specimens and 99% of these uropathogens was correctly identified. Concluding, this proof-of-concept approach demonstrates that the assay can distinguish bacteriuria from no bacteriuria as well as detect the involved uropathogen within 4 hours after sampling, allowing adequate therapy decisions within the same day as well as drastically reduce consequent urine culturing.     

Sodium ascorbate and basic fibroblast growth factor protect muscle-derived cells from H2O2-induced oxidative stress

Engraftment of muscle-derived cells (MDCs) into the urethral sphincter may cure urinary incontinence. However, poor cell survival after injection prompted us to evaluate whether 24-h preincubation with sodium ascorbate (ASC) or basic fibroblast growth factor (bFGF) prior to cell transfer improves the survival of MDCs facing oxidative stress in vitro. We examined MDCs isolated from female rats for the presence of myogenic markers and for the ability to differentiate and respond to growth factors. Isolated cells were positive for desmin, M-cadherin, and myogenin. The fusion index exceeded 29%, and Akt kinase was phosphorylated at Ser473 residue upon exposure to insulin-like growth factor 1 (100 ng/ml). We then autologously transplanted MDCs transfected with lacZ marker gene into urethral wall of the rats; 2 wk later, the urethra and caudal wall of the urinary bladder were harvested from these animals. Serial cryosections revealed that transplanted cells formed multinuclear clusters at injection sites. In addition, we found that the viability of MDCs exposed to a cytotoxic concentration of H2O2 was higher after preincubation with 0.1 mM ASC (2.6-fold), 10 ng/ml bFGF (2.9-fold), or 20 ng/ml bFGF (3.5-fold) than that after exposure to H2O2 only. We conclude that preincubation with ASC or bFGF increases the resistance of MDCs to oxidative stress in vitro. Pretreatment with either agent might be used to enhance survival of MDCs after transplantation.     

Bacterial profile and drug susceptibility pattern of urinary tract infection in pregnant women at University of Gondar Teaching Hospital, Northwest Ethiopia

ABSTRACT: BACKGROUND: Urinary tract infection (UTI) is a common health problem among pregnant women. Proper investigation and prompt treatment are needed to prevent serious life threatening condition and morbidity due to urinary tract infection that can occur in pregnant women. Recent report in Addis Ababa, Ethiopia indicated the prevalence of UTI in pregnant women was 11.6 % and Gram negative bacteria was the predominant isolates and showed multi drug resistance. This study aimed to assess bacterial profile that causes urinary tract infection and their antimicrobial susceptibility pattern among pregnant women visiting antenatal clinic at University of Gondar Teaching Hospital, Northwest Ethiopia. METHODS: A cross-sectional study was conducted at University of Gondar Teaching Hospital from March 22 to April 30, 2011. Mid stream urine samples were collected and inoculated into Cystine Lactose Electrolyte Deficient medium (CLED). Colony counts yielding bacterial growth of 105/ml of urine or more of pure isolates were regarded as significant bacteriuria for infection. Colony from CLED was sub cultured onto MacConkey agar and blood agar plates. Identification was done using cultural characteristics and a series of biochemical tests. A standard method of agar disc diffusion susceptibility testing method was used to determine susceptibility patterns of the isolates. RESULTS: The overall prevalence of UTI in pregnant women was 10.4 %. The predominant bacterial pathogens were Escherichia coli 47.5 % followed by coagulase-negative staphylococci 22.5 %, Staphylococcus aureus 10 %, and Klebsiella pneumoniae 10 %. Gram negative isolates were resulted low susceptibility to co-trimoxazole (51.9 %) and tetracycline (40.7 %) whereas Gram positive showed susceptibility to ceftriaxon (84.6 %) and amoxicillin-clavulanic acid (92.3 %). Multiple drug resistance (resistance to two or more drugs) was observed in 95 % of the isolates. CONCLUSION: Significant bacteriuria was observed in asymptomatic pregnant women. Periodic studies are recommended to check the outcome of asymptomatic bacteriuria and also monitor any changes in the susceptibility patterns of urinary tract pathogens in pregnant women.     

Free DNA in urine: a new marker for bladder cancer? Preliminary data

The aim of the present preliminary study was to investigate the presence of free DNA (FDNA) in urine as a possible marker for the diagnosis of bladder cancer. Naturally voided morning urine specimens were collected from 57 patients with suspected bladder cancer before cystoscopy. A standard urine test was performed; the specimens were then processed in order to obtain a quantitative evaluation of the presence of free DNA in the urine. Twenty-two patients were excluded from the study because they had leukocyturia and/or bacteriuria. Free DNA concentrations higher than 250 ng/mL were found in all 16 patients showing bladder cancer at cystoscopy and in seven (36.8%) of the 19 patients with negative cystoscopy. Urinary FDNA seems to have an excellent sensitivity: we observed no false negative cases and 36.8% false positive cases. By contrast, only 6.25% of the bladder cancer patients had positive urine cytology. Our results seem promising, although further studies and larger numbers are needed to define urinary free DNA as a reliable marker of bladder cancer.     

Factors contributing to hydrogen peroxide resistance in Streptococcus pneumoniae include pyruvate oxidase (SpxB) and avoidance of the toxic effects of the fenton reaction

Aerobic growth of Streptococcus pneumoniae results in production of amounts of hydrogen peroxide (H(2)O(2)) that may exceed 1 mM in the surrounding media. H(2)O(2) production by S. pneumoniae has been shown to kill or inhibit the growth of other respiratory tract flora, as well as to have cytotoxic effects on host cells and tissue. The mechanisms allowing S. pneumoniae, a catalase-deficient species, to survive endogenously generated concentrations of H(2)O(2) that are sufficient to kill other bacterial species is unknown. In the present study, pyruvate oxidase (SpxB), the enzyme responsible for endogenous H(2)O(2) production, was required for survival during exposure to high levels (20 mM) of exogenously added H(2)O(2). Pretreatment with H(2)O(2) did not increase H(2)O(2) resistance in the mutant, suggesting that SpxB activity itself is required, rather than an H(2)O(2)-inducible pathway. SpxB mutants synthesized 85% less acetyl-phosphate, a potential source of ATP. During H(2)O(2) exposure, ATP levels decreased more rapidly in spxB mutants than in wild-type cells, suggesting that the increased killing of spxB mutants was due to more rapid ATP depletion. Together, these data support the hypothesis that S. pneumoniae SpxB contributes to an H(2)O(2)-resistant energy source that maintains viability during oxidative stress. Thus, SpxB is required for resistance to the toxic by-product of its own activity. Although H(2)O(2)-dependent hydroxyl radical production and the intracellular concentration of free iron were similar to that of Escherichia coli, killing by H(2)O(2) was unaffected by iron chelators, suggesting that S. pneumoniae has a novel mechanism to avoid the toxic effects of the Fenton reaction.     

Limitations of the direct immunofluorescence test for antibody-coated bacteria in determining the site of urinary tract infections in children.

The results of the direct immunofluorescence test for antibody-coated bacteria to determine the site of a urinary tract infection do not always correlate with the clinical data. When this test was performed on urine specimens from 282 children with significant bacteriuria, false-negative and false-positive results were observed in 20% (19/94) and 52% (19/188) of the specimens. Contamination of the specimen during collection and the presence of Fc receptors (receptors for the crystallizable fragment of the immunoglobulin molecule) on the surface of some strains of Staphylococcus aureus yielded false-positive results, and stools and vaginal secretions were shown to be potential sources of antibody-coated bacteria. It is suggested that for children this test be run on urine collected by bladder puncture. The use of conjugated anti-IgG antiserum containing only F(ab'')2 (the antigen-binding fragments of the IgG molecule) is also recommended to eliminate false-positive results due to the presence of Fc receptors on the bacterial surface.     

Helicobacter pylori Induces Inflammation in Mouse Urinary Bladder and Pelvis

Helicobacter pylori was transurethrally inoculated into the mouse urinary tract. The organism established infection and induced inflammation in the urinary bladder and pelvis. During the infection, urinary pH was elevated, probably due to the production of NH3 by bacterial urease. H. pylori was recovered from the urinary bladder, kidney and urine of the infected mice. Histopathologically, severe neutrophil infiltration was observed in the mucosal layer of both organs. H. pylori was detected on the surface of the epithelial cells. These results indicate that low pH and bacterial flora were not essential factors in establishing the mucosal infection with H. pylori. This experimental system is useful to investigate the pathogenicity of H. pylori in mucosal organs.