Anticoagulant Carryover May Influence Clot Formation in Direct Tube Coagulase Tests from Blood Cultures

The tube coagulase test (TCT) performed directly from positive blood culture bottles has been used to reduce the turnaround time for identifying Staphylococcus aureus. Most reports have shown the test to be specific but often lacking sufficient sensitivity to be useful. In a prospective study of blood culture bottles (BCB) signaling positive, with a Gram-stained smear showing gram-positive cocci resembling staphylococci, the sensitivity of the direct TCT was improved by diluting the BCB broth 1:10 in saline before inoculating 0.1 ml into 1.0 ml of 10% pooled human plasma. It was hypothesized that the improved sensitivity might be explained by reduced carryover of the anticoagulant sodium polyanetholesulfonate (SPS) used in blood culture media. By titrating the inoculum size and the concentration of SPS in an in vitro checkerboard assay, it was shown that concentrations of SPS >0.0008% prevented plasma coagulation. The 1:10 dilution of blood culture broth reduced the amount of residual SPS carried over to the TCT to a level (0.0005%) that did not impair plasma coagulation. The direct TCT inoculated with a 1:10 saline dilution of blood culture broth achieved 100% specificity and sensitivity within 4 h of inoculation without reducing the quality or quantity of coagulum

Low-count bacteriuria in refractory idiopathic detrusor overactivity versus controls

Background. Previous studies suggest an association between idiopathic detrusor overactivity (IDO) and high-count bacteriuria (>105 CFU/mL). Recently, the importance of low-count bacteriuria (103–105 CFU/mL) in dysuric women with has been recognised. However, the optimal microbiological threshold for women with overactive bladder (OAB) symptoms remains unclear. Design and Methods A 2-year prospective cohort study, to examine the incidence of low-count and high-count bacteriuria in women with refractory IDO (RIDO) versus a control group without OAB symptoms. Women with dysuria, voiding dysfunction or recent urethral instrumentation were excluded. Mid-stream urine (MSU) specimens were collected during acute symptomatic exacerbation in the IDO group and cultured at the 103 CFU/mL threshold. Statistical analysis was performed using Statsdirect statistical package 2.7.2. Results. Between November 2007 and November 2009, 218 study MSU specimens were collected. Both the overall incidence of any significant bacteriuria ≥103 CFU/mL (P<0.0001) and the incidence of low-count bacteriuria (P=0.0091) were significantly higher in the RIDO women than the controls. There were no baseline differences in age, menopausal status or prior continence surgery between the groups. In the RIDO group, specimens positive for low-count bacteriuria only were less likely to be associated with significant pyuria P<0.0001) and were cultured from younger women (P=0.0009), than specimens positive for high-count bacteriuria. Conclusions The prevalence of bacteriuria in women with RIDO during symptomatic exacerbation is 40%, which is significantly higher than the incidence in similarly-aged women without OAB. One-third of positive cultures show low-count bacteriuria only. The management of women with RIDO should include a search for bacteriuria, including low-count bacteriuria

Virulence Mechanisms of Common Uropathogens and Their Intracellular Localisation within Urothelial Cells

A recurrent urinary tract infection (UTI) is a common debilitating condition whereby uropathogens are able to survive within the urinary tract. In this study, we aimed to determine if the common uropathogens Escherichia coli, Enterococcus faecalis, and Group B Streptococcus possessed virulence mechanisms that enable the invasion of urothelial cells. Urothelial cells were isolated from women with detrusor overactivity and recurrent UTIs; the intracellular localisation of the uropathogens was determined by confocal microscopy. Uropathogens were also isolated from women with acute UTIs and their intracellular localisation and virulence mechanisms were examined (yeast agglutination, biofilm formation, and haemolysis). Fluorescent staining and imaging of urothelial cells isolated from women with refractory detrusor overactivity and recurrent UTIs demonstrated that all three uropathogens were capable of intracellular colonisation. Similarly, the bacterial isolates from women with acute UTIs were also seen to intracellularly localise using an in vitro model. All Enterococcus and Streptococcus isolates possessed a haemolytic capacity and displayed a strong biofilm formation whilst yeast cell agglutination was unique to Escherichia coli. The expression of virulence mechanisms by these uropathogenic species was observed to correlate with successful urothelial cell invasion. Invasion into the bladder urothelium was seen to be a common characteristic of uropathogens, suggesting that bacterial reservoirs within the bladder contribute to the incidence of recurrent UTIs

Decreased intravesical adenosine triphosphate in patients with refractory detrusor overactivity and bacteriuria

Purpose Although several studies have examined the relationship between ATP release from the urothelium and bladder sensations including painful filling and urgency, the association between bacteriuria and urothelial ATP release is not well studied. Our aim was to evaluate women with “refractory” detrusor overactivity (DO), who were experiencing an acute exacerbation of their DO symptoms, including frequency, urgency, nocturia (and/or urge incontinence); we wished to measure changes in intravesical ATP levels in these women with and without bacteriuria. Methods A prospective cohort study of women with “refractory” DO, were invited to attend our unit during acute symptomatic exacerbation. On presentation, a catheter urine specimen (CSU) was collected and 50ml normal saline was instilled into the bladder to evoke gentle stretch and removed after 5 minutes; ATP concentrations were determined on fresh washings using a bioluminescence assay. Results The incidence of bacteriuria ≥103 CFU/ml was 27% (15/56 specimens) over the 16-month study period. ATP concentrations were lower during bacteriuric episodes in both the overall cohort (p=0.0013) and paired samples from individual patients (p=0.031), compared to episodes of sterile urine. Conclusions For the first time, we have demonstrated a striking difference between ATP levels measured in the presence of, and absence of, bacteriuria in this patient group

Bacterial filaments are more common in detrusor overactivity than controls: evidence for intracellular bacterial invasion of urothelial cells

Abstract of Oral Presentations at the 2014 AUGS-IUGA Scientific Meeting, July 22-26, 2014, Washington DC

The urinary microbiome in patients with refractory urge incontinence and recurrent urinary tract infection

Urinary urge incontinence is a chronic, debilitating condition that is difficult to treat. Patients refractory to standard antimuscarinic therapy often experience recurrent urinary tract infections (rUTIs). The microbiota of these refractory patients with rUTI remains unexplored.A midstream urine (MSU) sample was collected from patients with refractory urge incontinence and coexistent rUTI during acute symptomatic episodes. Culture-based diagnosis was performed using routine microbiological methods. Culture-independent profiling was performed using bacterial 16S RNA profiling. E. coli strain typing was performed by amplicon pyrosequencing of the fimH gene.Over 2\ua0years, 39 patients with refractory urge incontinence and coexistent rUTI were studied, yielding 9 severely affected cases. These 9 patients were carefully monitored for a further 2\ua0years, resulting in the collection of 102 MSU samples, 70 of which were diagnosed as UTI (median of 8 UTIs/woman). Culture-independent analysis of 38 of these samples revealed the existence of a diverse urinary microbiota. Strain typing of E. coli identified instances of rUTI caused by the same persisting strain and by new infecting strains.Patients with refractory urge incontinence and coexistent rUTI possess a diverse urinary microbiota, suggesting that persistent bladder colonisation might augment the pathology of their chronic condition

Detection of intracellular bacteria in exfoliated urothelial cells from women with urge incontinence

The role of subclinical infection in patients with urge incontinence has been largely ignored. The aim of this study was to test for the presence of intracellular bacteria in exfoliated urothelial cells obtained from the urine of patients with detrusor overactivity or mixed incontinence +/- a history of UTI, and compare this to a control group of patients with stress incontinence and no history of infection. Bacterial cystitis was assessed by routine microbiology and compared to microscopic analysis of urine by Wright staining. Subsequent analysis of urothelial cells by confocal microscopy was performed to determine the existence of intracellular bacteria. Bacterial cystitis was seen in 13% of patients based on routine microbiology. Wright staining of concentrated urothelial cells demonstrated the presence of bacteria in 72% of samples. Filamentous bacterial cells were observed in 51% of patients and were significantly more common in patients with detrusor overactivity. Intracellular Escherichia coli were observed by confocal microscopy. This study supports the possibility that a subset of patients with urge incontinence may have unrecognised chronic bacterial colonisation, maintained via an intracellular reservoir. In patients with negative routine microbiology, application of the techniques used in this study revealed evidence of infection, providing further insights into the aetiology of urge incontinence