Proteus mirabilis fimbriae- and urease-dependent clusters assemble in an extracellular niche to initiate bladder stone formation.

The catheter-associated uropathogenProteus mirabilisfrequently causes urinary stones, but little has been known about the initial stages of bladder colonization and stone formation. We found thatP. mirabilisrapidly invades the bladder urothelium, but generally fails to establish an intracellular niche. Instead, it forms extracellular clusters in the bladder lumen, which form foci of mineral deposition consistent with development of urinary stones. These clusters elicit a robust neutrophil response, and we present evidence of neutrophil extracellular trap generation during experimental urinary tract infection. We identified two virulence factors required for cluster development: urease, which is required for urolithiasis, and mannose-resistantProteus-like fimbriae. The extracellular cluster formation byP. mirabilisstands in direct contrast to uropathogenicEscherichia coli, which readily formed intracellular bacterial communities but not luminal clusters or urinary stones. We propose that extracellular clusters are a key mechanism ofP. mirabilissurvival and virulence in the bladder.

Bladder stones in catheterized spinal cord-injured patients in Nigeria.

OBJECTIVE: The objective was to determine the incidence of bladder stones in patients with spinal cord injury (SCI) and to assess if catheter encrustation or positive urinary culture of Proteus mirabilis is predictive of bladder stones. BACKGROUND: Bladder stones are common urological complication in those with SCI managed with indwelling urinary catheter. Detection and removal of bladder stones are important to prevent possible further complications. DESIGN: This was a prospective cohort study. MATERIALS AND METHODS: Ultrasound scan was performed in persons with SCI seen from 1st January to 31st December 2009 who had indwelling urethral catheter for at least 3-month post-injury. Indwelling urethral catheters were examined for encrustation at the time of removal, urine culture taken specifically for P. mirabilis and ultrasound scan done to detect bladder stones. RESULTS: There were 89 patients with spinal cord injury and 68 (76.4%) patients were evaluated during the review period. Twenty-nine (42.6%) patients had bladder stones and 22 (32.3%) patients had catheter encrustation. Of the 22 patients with catheter encrustation, 19 (86.3%) also had bladder stones. Forty-six (67.6%) patients had no catheter encrustation. Of these, 7 (14.7%) were found to have bladder stones. Thirty-seven (38.2%) urine cultures were positive for P. mirabilis. Of these 37 (54.4%) patients, 27 also had bladder stones. Catheter encrustation (P = 0.004) and a positive urine culture of P. mirabilis (P = 0.007) in patients with indwelling urinary catheter is highly predictive of the presence of bladder stone. CONCLUSIONS: This study shows that a large number of SCI patients have an indwelling urethral catheter and suggests that ultrasound scan for the presence of stone should be schedule in a catheterized SCI patient if catheter encrustation or a positive urine culture of P. mirabilis is noted.

[A modified rat model for infection-induced stone formation].

OBJECTIVE: To employ a newly modified rat model for infection-induced bladder stone formation. METHODS: 24 adult male Sprague Dawley rats were divided into 3 groups, model group (n=12), sham operation group (n=8) and control group (n=4). The surgical procedures were performed aseptically under anesthesia (25% Ultane 1.0 g/kg). The bladder in model group was exposed through a short lower midline abdominal incision, the puncture needle (G18) with guideline was inserted aseptically into bladder, a metal wire, which have been contaminated by the Proteus mirabilis, was put into the puncture canal, then implanted into the bladder lumen through the guideline. In the sham operated group the puncture needle (G18) with guideline was inserted into bladder without metal wire implanted into the bladder. There was no any operation in control group. The rats were sacrificed by excessive anesthesia at 21 days post challenge. The bladder were removed aseptically and inspected for evidence of urolithiasis. RESULTS: On Day 2 after surgery, two rats died in model group, no rats died in other groups. Twenty-one days after surgery, all of rats in model group developed various-sized bladder stones. There was no stone formation in sham operation group and control group. All stones were verified by infrared spectroscopy and optical crystallography. These stone were struvite stone. CONCLUSION: This model has a less trauma, faster recovery and excellent stone formation so that it may be used for the study of infection stone.

Detection by light microscopy of Candida in thin sections of bladder stone.

We detected fungi morphologically resembling Candida albicans in an infected bladder stone by light microscopy of thin sections. The fungi were found in the layers precipitated with oxalate crystals and were invading the interstices surrounded with apatite or struvite crystals as in tissue infection. This presumably represents a superimposed infection due to changes in flora following treatment with antibiotics.

Relationship of bacterial infection in urine and calculi to canine urolithiasis.

In 26 dogs treated surgically for urolithiasis, bacteriological examination of the urine and the interior of calculi showed that infection was present in both materials in 14 cases. Infection with phosphate calculi, present in 13 of these 14 dogs, was associated with a variety of bacteria including Staphylococcus aureus, Staph epidermidis, Streptococcus faecalis, Escherichia coli and Proteus spp. In a follow-up examination of 16 dogs, organisms different from the original isolates were recovered from some cases. The significance of the persistence of viable bacteria within canine bladder calculi is discussed.

[Formation of experimental rat bladder calculus and adherence of Pseudomonas aeruginosa to the calculus].

The formation of experimental bladder calculus was studied. The calculus was formed by the uptake of ethylene glycolwater (1%) and retaining the silk thread in rat bladder with high frequency. The components of the calculus were calcium oxalate and calcium phosphate from the results of the electron prove micro analysis (EPMA) and ion chromatography. On the 7th day after the beginning of experiment, Pseudomonas aeruginosa was inoculated to the rat bladder via the urethra. Seven days after the infection, P. aeruginosa adhered to the surface of the calculus such as an aspect of a biofilm. It was considered that this experimental model was useful to study the adherence of bacteria, biofilm formation and its chemotherapy by antibacterial agents.

[Experimental bladder stone production by Ureaplasma urealyticum].

We examined the relationship between struvite stone formation and Ureaplasma urealyticum in male Wister rats by inoculating U. urealyticum and implanting a zinc disc into the bladder surgically. Cultures of U. urealyticum and pH measurements in urine obtained by forced urination were done on days 2 and 6 after operation. Half of the rats were killed 7 days after the operation and the remaining half 14 days after the operation. A total of 46 rats, consisting of 10 rats in the control group and 36 rats inoculated with U. urealyticum, were studied. U. urealyticum was weakly virulent, because it was not detected in the urine culture on day 2 in 10 of the 36 rats inoculated with the organism, and showed a tendency towards spontaneous elimination in the other rats. Urinary pH was elevated slightly by the presence of U. urealyticum. Struvite stones were formed in 52% of the rats by the inoculation on of U. urealyticum, and 69% and 71% of the positive rats in urine culture for U. urealyticum on day 2 and day 6 respectively. The stones tended to be larger the longer U. urealyticum was detected in urine. U. urealyticum may play a role in struvite stone formation, but not a major role.

[Experimental bladder stone production by human uropathogenic bacteria].

To investigate the formation of infection stones by 5 bacterial species isolated from patients with urinary calculi, such as Proteus mirabilis, Staphylococcus saprophyticus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli, a zinc disc was implanted and 10(6) bacteria were surgically inoculated at the same time into the bladder of male Wistar rats (10 rats per group). Urinary pH was measured on days 1, 3, 5 and 7. The rats were killed on day 7 and the bladder examined for the formation of infection stones. Infection stones were formed in all rats inoculated with P. mirabilis (mean stone weight 29.7 mg), and in 60% of those inoculated with S. saprophyticus (9.7 mg). Small bladder stones were formed in 30% of the rats inoculated with K. pneumoniae and 25% of those inoculated with P. aeruginosa. No bladder stones were formed in the rats inoculated with E. coli. Urinary pH was increased by P. mirabilis and S. saprophyticus but was unaffected by the other species. In conclusion, P. mirabilis and S. saprophyticus play a significant role in the formation of infection stones.

[Observation on fungi within urinary stones].

We examined the fungi within the urinary stones of two cases by light microscopy and scanning electron microscopy. In one case, fungal elements, hyphae and spores, were found in some layers within a bladder stone. This patient probably had a history of fungal urinary infection. In another case brown hyphae suggesting dematiaceous fungi were found in the surface and peripheral layers of a kidney stone. This maybe due to contamination of the fungi after removal of the stones. These fungi invaded into interstices of the apatite crystals within the stone as in the tissue.

[An experimental study of infection stone formation by Corynebacterium species].

The relationship between infection stone and Corynebacterium species was investigated in vitro and in vivo. Urease activity of urease-splitting Corynebacterium species was evaluated by 2 methods; an increase in pH of human urine after inoculation of Corynebacterium species and direct measurement of urease activity of 10(7) CFU organisms from amounts of ammonia by indophenol method. Formation of infection bladder stone was induced in male Wistar rats by implanting a zinc disc and inoculating 10(6) CFU organisms surgically into the bladder. Urine was alkalinized by the inoculation of Corynebacterium renale, C. pilosum and group D2 Corynebacterium. C. renale and C. pilosum had strong urease activity, and group D2 Corynebacterium had moderate activity. C. pseudodiphtheriticum did not produce the elevation of urinary pH and had little urease activity. Infection stones were formed in 100% of rats by inoculation of C. renale and C. pilosum and 88% of rats by group D2. Urinary pH was elevated in all inoculated rats. In conclusion, C. renale, C. pilosum and group D2 Corynebacterium may play a role in formation of infection stones.

[A study on bacteria within stones in urolithiasis].

The bacteria in 37 stones obtained from 37 patients with urinary stone diseases, that is, 11 renal stones (containing 2 staghorn calculi), 21 ureteral stones, 4 bladder stones and 1 urethral stone, were studied, according to the Nemoy & Stamey's method. The stones were collected by partial nephrectomy (1 case), nephrolithotomy (1 case), pyelolithotomy (1 case), percutaneous nephrolithotripsy (PNL) (6 cases), 12 ureterolithotomies, transurethral ureterolithotripsy (2 cases), cystolithotripsies (4 cases) and spontaneous deliveries (10 cases). According to a stone analysis by infrared spectrophotometer revealed 30 were noninfection stones (81.1%) and 7 infection stones (18.9%). Of these 37 stones, 5 stones (13.5% of 11 stones) had bacteria within them. These stones consisted of 4 infection stones (57.1% of all infection stones) and 1 noninfection stone (3.3% of all noninfection stones). Out of 4 patients having bacteria within their stones, urine culture of whom were carried out before stones were collected, only one patient (25%) had the same species of bacterium (E. coli) both within the stone and in urine. The E. coli within the stone and that in urine, however, showed quite different reactions to some antibiotics. The fact that all organisms within stones cannot be detected by urine culture before collecting stones of the patients in our present study, suggests that patients undergoing endourological surgery, such as extracorporeal shock wave lithotripsy and PNL, may have a risk of complications, such as severe urinary tract infection and urosepsis, if the possibility of the presence of organisms within stones is not taken into consideration.

[Urinary tract infection caused by Corynebacterium sp.--a case report and an experimental study].

We repose of chronic cystitis associated with alkali urine, struvite stones and a subsequent vesicorectal fistula caused by Corynebacterium sp, probably Corynebacterium group D2. We also studied in vitro and in vivo stone formation caused by Corynebacterium renale isolated clinically. C. renale inoculated into normal human urine increased urine pH and formed struvite crystals after a 24-hr incubation. Bladder stones were also formed in rats experimentally infected by C. renale as well as P. mirabilis. Some urea splitting species of Corynebacterium such as C. group D2 and C. renale may play a role in the formation of human struvite stone.

The effect of preoperative antimicrobial administration on culture results in dogs undergoing cystotomy.

OBJECTIVE: To evaluate the effect of preoperative antimicrobial administration on culture results in dogs undergoing cystotomy as a treatment for urolithiasis. DESIGN: Prospective controlled study. Animals-41 dogs undergoing cystotomy for cystic calculi removal. PROCEDURES: Urine samples were collected at time of anesthetic induction and during surgery prior to cystotomy, and a mucosal biopsy and culturette swab was collected during surgery from a control group, which received antimicrobials only after surgical culture sample collection, and from an experimental group, which received antimicrobials at the time of anesthetic induction. RESULTS: 17 of 41 patients had positive culture results at anesthetic induction. Twenty of 41 patients had positive results of cultures for the surgical sample. No dogs that had positive results before antimicrobial administration had negative results after antimicrobial administration. There were no significant changes to urinalysis results regardless of group. Calcium monohydrate uroliths were the most common stone removed (24/41), followed by magnesium ammonium phosphate uroliths (11/41). CONCLUSIONS AND CLINICAL RELEVANCE: There was no difference in culture results (positivity and bacterial type) when antimicrobials were given at anesthetic induction versus after surgical culture sample collection for dogs undergoing cystotomy for cystic calculi removal.