Introduction
In dogs, Enterococcus faecalis and Enterococcus faecium commonly colonize the urinary tract and account for 9% to11% of all combined types of UTI.1, 2 However, in dogs with recurrent UTIs, enterococci account for 17% to 25% of all infections.3, 4 The reason for the 2-fold increase in the prevalence of enterococci in the urine of dogs with recurrent UTIs relative to that of all dogs with UTIs in general is unknown. Enterococci bind to fibrinogen, and that binding is integral to the development of CAUTI in humans5, 6 and may have an analogous role in the development of recurrent UTIs in dogs.
Enterococci constitutively express Ebp on the external plasma membrane.7, 8 Unlike the fimbriae of Escherichia coli that preferentially bind to mannose and glycosphingolipids,9, 10 the Ebp of enterococci bind to fibrinogen.7, 8 Urinary tract infections occur when LUT host defenses and urodynamics are compromised and allow bacteria to ascend the urethra to the urinary bladder.10 Additional requirements for the development of a UTI include a nutrient source and a means for bacteria to adhere and persist within the urinary bladder.10 The presence of fibrinogen in the urinary tract provides the latter 2 of those 3 criteria in patients with CAUTI.5
In human patients with CAUTI, the infection is initiated when the urinary catheter causes physical trauma to the urothelium, which leads to inflammation, edema, and IL-6 production.5 Once IL-6 enters the circulation, it increases fibrinogen production by the liver.11 That fibrinogen enters the circulation and then leaks into the urine through the damaged urothelium.5, 6 Subsequent binding of enterococci to fibrinogen in the urine promotes bacteria colonization and infection of the urinary tract.5 Infection-induced inflammation further increases production of IL-6, thereby creating a positive feedback loop for promotion of infection and production of inflammatory cytokines and fibrinogen.12 Biofilm formation and enterococcal CAUTI do not occur if fibrinogen is not present in the urine or Ebp is not expressed on the surface of enterococci, which indicates there is a cause-effect relationship between urine fibrinogen and enterococcal CAUTI.5, 6
In dogs, recurrent UTIs are frequently caused by LUT disorders other than CAUTI.3 However, the importance of urine fibrinogen in the development of enterococcal CAUTI in humans may provide a possible mechanistic explanation for the high prevalence of E faecalis and E faecium isolated from urine samples of dogs with recurrent UTIs.3, 4 The presence of uroliths and neoplasia and anatomic abnormalities of the LUT have been identified as risk factors for both recurrent UTI and enterococcal bacteriuria in dogs.3, 13 If urine fibrinogen contributes to enterococcal colonization of the urinary tract in dogs, than those risk factors should also be associated with uFIB. The purpose of the study reported here was to quantify uFIB and uIL-6 in dogs with risk factors for recurrent UTIs and enterococcal bacteriuria. Given the relationship between IL-6 and fibrinogen production, we hypothesized that the uFIB and uIL-6 in dogs with risk factors for recurrent UTIs and enterococcal bacteriuria would be significantly greater than those in healthy dogs.
Materials and Methods
Animals
The research protocol was approved by the University of Wisconsin-Madison Institutional Animal Care and Use Committee. Canine urine samples that were determined to have negative results on aerobic culture between May 11, 2016, and June 31, 2019, by the UW Veterinary Care Microbial Service and were subsequently stored in the service's tissue bank were considered for study inclusion. To be eligible for tissue bank storage, a urine sample had to be maintained at 4 °C prior to laboratory processing and processed within 24 hours after collection. All urine samples were collected by cystocentesis or free catch of a midstream voided sample.
The UW Veterinary Care electronic medical record for each dog with a urine sample stored in the tissue bank was reviewed. To be classified as at risk for recurrent UTI or enterococcal bacteriuria (ie, to be included in the at-risk group), a dog had to have urolithiasis or an anatomic abnormality or neoplasia of the LUT present at the time the urine sample was collected. For dogs eligible for study inclusion with multiple urine samples from multiple clinic visits in the tissue bank, only the most recent sample collected while a study qualifying–risk factor was present was evaluated. Urine samples were excluded from analysis if information in the medical record suggested that the uFIB might have been altered by something other than study-qualifying risk factors, such as growth of aerobic bacteria on a blood agar plate that was incubated at 37 °C for 5 days after inoculation with the urine sample or the dog had abnormally increased serum liver enzyme (alkaline phosphatase, alanine aminotransferase, g-glutamyltransferase) activities, azotemia (ie, elevated serum creatinine or BUN concentration), or a history of urethral catheterization.11,14–16
Banked urine samples collected from healthy dogs enrolled in an unrelated study were used as control samples. The dogs of that other study had no history of polyuria, polydipsia, pollakiuria, stranguria, or UTI and were determined to be healthy on the basis of results of a physical examination and serum biochemical analysis. Urine samples from dogs with abnormally increased serum enzyme activities, azotemia, urinary tract inflammation, or bacteriuria were excluded from the analysis. Urinary tract inflammation was defined as the presence of proteinuria or hematuria as determined on the basis of results a urine dipstick evaluation or the observation of WBCs or RBCs during cytologic evaluation of urine sediment. All urine samples evaluated in the study were obtained from dogs that underwent, at minimum, a physical examination, urinalysis, microscopic evaluation of urine sediment, aerobic culture of a urine sample, and serum biochemical profile.
Processing of banked urine samples
Each banked urine sample that was eligible for study analysis was separated into 1-mL aliquots. One aliquot of each sample was submitted to the UW Veterinary Care Clinical Pathology Laboratory for quantification of uCrea by use of an automated biochemical analyzer.a The remaining 1-mL aliquots of urine were stored frozen at–80 °C until analyzed.
Quantification of uFIB
Each urine specimen was dialyzed in reagent-grade water to reduce the nonspecific binding effects of the urine matrix. Briefly, 420 to 600 µL of urine was placed into dialysis tubingb with a molecular weight cutoff of 6 to 8 kD, and the tubing was clamped closed. The filled tubing was maintained in a circulated bath of reagent-grade water at 4 °C for 4 hours. The volume of urine placed in the dialysis tubing and the volume of urine remaining after dialysis were recorded.
Following dialysis, the uFIB was determined by means of a dog-specific fibrinogen indirect colorimetric sandwich ELISAc that was optimized for use with urine. The manufacturer's recommended reagents, reagent concentrations, and fibrinogen standard were used. The effects of the urine matrix were further mitigated by use of proprietary sampled and assayd diluents as described.17 For each urine sample analyzed, the uFIB was quantified for an undiluted sample and samples diluted 1:2 and 1:4 with sample diluent. All assays were performed in duplicate. Colorimetric detection was performed with a multimode plate reader.e Results were analyzed by use of commercially available softwaref to perform a curve fit analysis and reported as nanograms of fibrinogen per milliliter of urine. The percentage change in the volume of urine before and after dialysis was used to normalize (standardize) the effect of dialysis on uFIBs. The standard curve was accurate within a uFIB range of 12.5 to 800 ng/mL, with the lower limit of the standard curve determined by values at least 2 times the background measurements.
Quantification of uIL-6
A dog-specific IL-6 indirect chemiluminescence sandwich ELISAg that was optimized by our laboratory for use with urine samples was used to measure uIL-6. The assay optimization procedure used sampled and assayd diluents to reduce the nonspecific binding effects of the urine matrix as described.17 Because IL-6 is small and can move across the dialysis membrane,18 dialysis was not performed on the urine samples prior to undergoing the ELISA. For each urine sample analyzed, the uIL-6 was quantified for an undiluted sample and samples diluted 1:2 and 1:4 with sample diluent. All assays were performed in duplicate. Other optimized components of the ELISA included capture (concentration, 800 ng/mL) and detection (concentration, 25 ng/mL) goat anti-canine polyclonal IL-6 antibodies,g streptavidin–horseradish peroxidase,g and chemiluminescent substrate.h Recombinant canine IL-6g was used to create the standard curve. Chemiluminescent detection was performed with a multimode plate reader.e Results were analyzed by use of commercially available softwaref to perform a curve fit analysis and reported as picograms of IL-6 per milliliter of urine. The standard curve was accurate within a uIL-6 range of 15.62 to 2,000 pg/mL, with accuracy determined by values at least 2 times the background measurements and sample uIL-6 falling within the range of the standard curve.
Statistical analysis
For analysis, the at-risk group was evaluated as a whole and when divided into 3 subgroups: urolithiasis group, LUT anatomic abnormality group, and LUT neoplasia group. The urolithiasis group included urine samples obtained from dogs with radiographic or ultrasonographic evidence of cystoliths, nephroliths, or ureteroliths at the time of urine collection. The LUT anatomic abnormality group included urine samples obtained from dogs with a hooded vulva, ectopic ureters, benign prostatic hyperplasia, or persistent vaginal bands as determined by information provided in a physical examination, radiologic, or cystoscopic report. The LUT neoplasia group included urine samples obtained from dogs with a cytologic or histologic diagnosis of urothelial cell carcinoma, bladder sarcoma, or prostatic carcinoma.
Statistical analyses including data interpolation from the standard curves were performed with commercially available software.f Descriptive data were generated for each experimental group and subgroup. Samples with a uFIB or uIL-6 below the detection limit for the respective assays were assigned a value equal to the lower limit of detection for that assay (ie, 12.5 ng/mL for the uFIB ELISA and 15.62 pg/mL for the uIL-6 ELISA). The respective data distributions for continuous variables of interest (dog age, uCrea, uFIB, and uIL-6) were assessed for normality by use of the Shapiro-Wilk test. The mean (SD) was reported for normally distributed continuous variables (age), and the median (IQR) was reported for continuous variables that were not normally distributed (uCrea, uFIB, and uIL-6). A 2-sided t test was used to compare age between the at-risk and control groups.
Variation in urine concentration among analyzed samples was normalized by calculation of the uFIB:uCrea and uIL-6:uCrea ratios. Mann-Whitney U tests were used to compare the uFIB:uCrea and uIL-6:uCrea ratios between the at-risk and control groups. Kruskal-Wallis tests were used to compare those 2 ratios among the urolithiasis, LUT anatomic abnormality, LUT neoplasia, and control groups, followed by the Dunn test when multiple post hoc pairwise comparisons were warranted. Values of P ≤ 0.05 were considered significant for all analyses.
Mann-Whitney U tests were also used to perform a priori and post hoc power analyses. Those tests were performed with a commercially available software program.i
Results
Dogs
Banked urine samples from 253 dogs were considered for study inclusion, of which 27 samples were obtained from dogs with risk factors for enterococcal bacteruria and were negative for bacterial growth on aerobic culture. Those 27 urine samples comprised the at-risk group and were acquired from 8 dogs with urolithiasis, 9 dogs with LUT anatomic abnormalities, and 10 dogs with LUT neoplasia. Twenty-one urine samples from healthy dogs comprised the control group. The uFIB was determined for all 48 urine samples included in the study. However, following completion of the uFIB assays, multiple samples had an inadequate volume of urine remaining for determination of the uIL-6. Thus, the uIL-6 was determined for only 5 of the 8 dogs with urolithiasis, 8 of the 9 dogs with LUT anatomic abnormalities, 4 of the 10 dogs with LUT neoplasia, and 19 of the 21 control dogs.
The 27 dogs of the at-risk group had a mean age of 8.6 years (SD, 3.5 years) and included 4 sexually intact males, 4 neutered males, 2 sexually intact females, and 17 spayed females. The at-risk group included 5 mixed-breed dogs, 3 Labrador Retrievers, 3 Beagles, 2 Pugs, and 1 dog from each of 14 other breeds. The 8 urine samples assigned to the urolithiasis group were obtained from 6 dogs with cystoliths only, 1 dog with nephroliths only, and 1 dog with both cystoliths and nephroliths. The 9 urine samples assigned to the LUT anatomic abnormality group were obtained from 5 dogs with a hooded vulva and 4 dogs with presumptive benign prostatic hyperplasia as determined on the basis of results of ultrasonography, cytology, and rectal examination. The 10 urine samples assigned to the LUT neoplasia group were obtained from 9 dogs with urothelial cell carcinoma and 1 dog with bladder sarcoma.
The 21 control dogs had a mean age of 7.2 years (SD, 4.2 years) and included 1 sexually intact male, 7 neutered males, and 13 spayed females. The mean age of the control dogs did not differ significantly (P > 0.99) from that of the dogs in the at-risk group. The control group included 10 mixed-breed dogs, 2 Beagles, 2 Collies, and 1 dog from each of 7 other breeds.
uFIB
The median uFIB was 74.17 ng/mL (IQR, 26.22 to 458.50 ng/mL) for the urolithiasis group, 44.42 ng/mL (IQR, 24.90 to 50.71 ng/mL) for the LUT anatomic abnormality group, 791.30 ng/mL (145.60 to 1,666.00 ng/mL) for the LUT neoplasia group, and 33.90 ng/mL (IQR, 13.70 to 61.18 ng/mL) for the control group. The median uFIB:uCrea ratio for the at-risk group as a whole (0.91; IQR, 0.46 to 6.80) was significantly (P < 0.01) greater than that for the control group (0.17; IQR, 0.07 to 0.39). The median uFIB:uCrea ratio for urolithiasis group (0.72; IQR, 0.46 to 3.48; P = 0.02) and the LUT neoplasia group (6.16; IQR, 3.89 to 12.75; P < 0.01) were also significantly greater than the median uFIB:uCrea ratio for the control group. The median uFIB:uCrea ratio for the LUT anatomic abnormality group (0.48; IQR, 0.27 to 0.69) did not differ significantly (P = 0.20) from that of the control group (Figure 1).
Dot plots of the uFIB:uCrea (A) and uIL-6:uCrea (B) ratios for 21 healthy adult dogs (control) and 27 dogs with risk factors for enterococcal bacteriuria including urolithiasis (n = 8), LUT anatomic abnormalities (9), and LUT neoplasia (10). Each dot represents the results for 1 dog. Only 19 control dogs, 5 dogs with urolithiasis, 8 dogs with LUT anatomic abnormalities, and 4 dogs with LUT neoplasia are represented in the plots of panel B because there was an insufficient volume of urine available for determination of uIL-6 from some dogs. Within each plot, the middle horizontal line represents the median and the whiskers denote the IQR. *The medians for groups connected by brackets differ significantly (P ≤ 0.05).[MP1][MP2][MP3]
Citation: American Journal of Veterinary Research 82, 10; 10.2460/ajvr.82.10.846
Dot plots of the uFIB:uCrea (A) and uIL-6:uCrea (B) ratios for 21 healthy adult dogs (control) and 27 dogs with risk factors for enterococcal bacteriuria including urolithiasis (n = 8), LUT anatomic abnormalities (9), and LUT neoplasia (10). Each dot represents the results for 1 dog. Only 19 control dogs, 5 dogs with urolithiasis, 8 dogs with LUT anatomic abnormalities, and 4 dogs with LUT neoplasia are represented in the plots of panel B because there was an insufficient volume of urine available for determination of uIL-6 from some dogs. Within each plot, the middle horizontal line represents the median and the whiskers denote the IQR. *The medians for groups connected by brackets differ significantly (P ≤ 0.05).[MP1][MP2][MP3]
Citation: American Journal of Veterinary Research 82, 10; 10.2460/ajvr.82.10.846
Dot plots of the uFIB:uCrea (A) and uIL-6:uCrea (B) ratios for 21 healthy adult dogs (control) and 27 dogs with risk factors for enterococcal bacteriuria including urolithiasis (n = 8), LUT anatomic abnormalities (9), and LUT neoplasia (10). Each dot represents the results for 1 dog. Only 19 control dogs, 5 dogs with urolithiasis, 8 dogs with LUT anatomic abnormalities, and 4 dogs with LUT neoplasia are represented in the plots of panel B because there was an insufficient volume of urine available for determination of uIL-6 from some dogs. Within each plot, the middle horizontal line represents the median and the whiskers denote the IQR. *The medians for groups connected by brackets differ significantly (P ≤ 0.05).[MP1][MP2][MP3]
Citation: American Journal of Veterinary Research 82, 10; 10.2460/ajvr.82.10.846
Dot plots of the uFIB:uCrea (A) and uIL-6:uCrea (B) ratios for 21 healthy adult dogs (control) and 27 dogs with risk factors for enterococcal bacteriuria including urolithiasis (n = 8), LUT anatomic abnormalities (9), and LUT neoplasia (10). Each dot represents the results for 1 dog. Only 19 control dogs, 5 dogs with urolithiasis, 8 dogs with LUT anatomic abnormalities, and 4 dogs with LUT neoplasia are represented in the plots of panel B because there was an insufficient volume of urine available for determination of uIL-6 from some dogs. Within each plot, the middle horizontal line represents the median and the whiskers denote the IQR. *The medians for groups connected by brackets differ significantly (P ≤ 0.05).[MP1][MP2][MP3]
Citation: American Journal of Veterinary Research 82, 10; 10.2460/ajvr.82.10.846
Dot plots of the uFIB:uCrea (A) and uIL-6:uCrea (B) ratios for 21 healthy adult dogs (control) and 27 dogs with risk factors for enterococcal bacteriuria including urolithiasis (n = 8), LUT anatomic abnormalities (9), and LUT neoplasia (10). Each dot represents the results for 1 dog. Only 19 control dogs, 5 dogs with urolithiasis, 8 dogs with LUT anatomic abnormalities, and 4 dogs with LUT neoplasia are represented in the plots of panel B because there was an insufficient volume of urine available for determination of uIL-6 from some dogs. Within each plot, the middle horizontal line represents the median and the whiskers denote the IQR. *The medians for groups connected by brackets differ significantly (P ≤ 0.05).[MP1][MP2][MP3]
Citation: American Journal of Veterinary Research 82, 10; 10.2460/ajvr.82.10.846
uIL-6
All urine samples with a uIL-6 below the lower detection limit for the assay (15.62 pg/mL) were assigned that value. The median uIL-6 was 25.91 pg/mL (IQR, 15.62 to 178.30 pg/mL) for the urolithiasis group, 15.62 pg/mL (IQR, 15.62 to 39.60 pg/mL) for the LUT anatomic abnormality group, and 32.34 pg/mL (IQR, 18.49 to 48.78 pg/mL) for the LUT neoplasia group. The uIL-6 was below the detection limit of the assay for all control samples except for 1. Thus, the calculated median uIL-6 for the control group was 15.62 pg/mL (IQR, 15.62 to 15.62 pg/mL). The median uIL-6:uCrea ratio for the at-risk group as a whole (0.27; IQR, 0.17 to 0.48) was significantly (P < 0.01) greater than that for the control group (0.08; IQR, 0.06 to 0.11). The median uIL-6:uCrea ratios for the urolithiasis (0.48; IQR, 0.18 to 1.61; P < 0.01), LUT anatomic abnormalities (0.25; IQR, 0.17 to 0.33; P < 0.01), and LUT neoplasia (0.25; IQR, 0.12 to 1.01; P = 0.03) groups were likewise significantly greater than the median uIL-6:uCrea ratio for the control group (Figure 1).
Discussion
In dogs, an association between risk factors for UTI, such as inflammation and an elevated uFIB, and enterococcal colonization of the urinary tract has not been elucidated. Results of the present study provided evidence that risk factors for enterococcal bacteriuria, including the presence of uroliths and anatomic abnormalities and neoplasia of the LUT, are associated with an increase in uFIB and uIL-6. Human patients with urolithiasis and bladder cancer likewise have an elevated uIL-6.19, 20 For dogs, it is hypothesized that the inflammation associated with the described risk factors damages the urothelium, which allows fibrinogen to enter the urinary bladder in a manner similar to that observed in human patient with enterococcal CAUTI.5
In human medicine, CAUTI is the most common health care–associated infection, and enterococci organisms are a very common cultured microorganism from patients with CAUTI.21 Catheterization of the urinary tract generally leads to an increase in uIL-6 and urine interleukin-1b concentrations, which in turn promote hepatic production of fibrinogen, and localized inflammation of the urinary bladder allows fibrinogen to cross the bladder wall and increase uFIB.6 Subsequent colonization of the urinary tract by enterococci doubles the uIL-6 and urine interleukin-1b concentrations, which creates a positive feedback loop and perpetuates the inflammatory cycle and UTI.12 It is important to note that it is the presence of fibrinogen in the urine, rather than inflammation, that ultimately allows colonization of the urothelium by enterococci.5, 6 Establishing a direct cause-effect relationship between uFIB and the development of enterococcal UTI in dogs was beyond the scope of the present study. Nevertheless, the results of this study suggested a potential link between uFIB and enterococcal colonization of the urinary tract in dogs.
The potential role of uFIB as a promoter of enterococcal bacteriuria could be clinically important in the management of dogs with UTIs caused by enterococci. Enterococcal infections account for 17% to 25% of recurrent UTIs in dogs.3, 4 If the defect that promotes bacterial colonization of the urinary tract is not, or cannot, be corrected, management of dogs with recurrent UTIs frequently evolves into a frustrating cycle of infection and repeated antimicrobial administration, which merely addresses the consequence of the defect rather than the defect itself.3 Additionally, owing to inherent and acquired traits, enterococci rapidly acquire resistance to antimicrobials.1, 22 Hence, development of nonantimicrobial treatment strategies to prevent enterococcal colonization of the urinary tract in dogs without applying selection pressure for antimicrobial resistance is crucial. Because enterococci species constitutively express the Ebp pili on their surface and binding of those pili to fibrinogen within the urinary tract promotes bacterial adherence to and colonization of the urinary tract, preventing the interaction between Ebp pili and fibrinogen might confer protection against enterococcal colonization of the urinary tract.5 In mice, administration of a vaccine against EbpA decreased the incidence of enterococcal CAUTI.5 It is unclear whether vaccination of dogs against Ebp would decrease the incidence of enterococcal bacteruria, but the results of the present study suggested it should be investigated.
We were surprised that, in the present study, the uFIB:uCrea ratio (a proxy measurement for uFIB to normalize differences in concentration among urine samples) did not differ between the dogs with LUT anatomic abnormalities and healthy control dogs because anatomic abnormalities of the LUT were identified as a risk factor for enterococcal bacteriuria in dogs of another study.13 The reason uFIB did not appear to be abnormally increased for the dogs with LUT anatomic abnormalities in the present study was unclear. It is possible that another pathophysiological mechanism is responsible for enterococcal bacteriuria in dogs with LUT anatomic abnormalities or the presence of an LUT anatomic abnormality may predispose a dog to secondary disease processes, such as UTIs, that increase uFIB. In human medicine, children with anatomic abnormalities of the urinary tract appear to be predisposed to the development of enterococcal bacteriuria, although a direct cause-effect relationship between the anatomic abnormalities and enterococcal bacteriuria has yet to be established.23 Baquero et al24 suggested an antibiotic-selective environment theory, which proposes that previous antimicrobial use creates an environment within the urinary bladder that allows the growth of low levels of antimicrobial-resistant bacteria, such as enterococci, by elimination of competing bacteria that were susceptible to the antimicrobial administered. Given that dogs with congenital and acquired anatomic anomalies of the urogenital tract, such as a hooded vulva, urinary masses, vestibulovaginal stenosis, vaginal strictures, and ectopic ureters, appear to be predisposed to recurrent UTIs and often receive multiple courses of antimicrobials,25 it seems plausible that the generation of an antimicrobial-selective environment could promote enterococcal growth and colonization of the urinary tract in the absence of fibrinogen.
Alternatively, it is possible that dogs with LUT anatomic abnormalities are predisposed to secondary disease processes, and those processes rather than the anatomic abnormalities are responsible for the eventual increase in uFIB and increase in the risk for enterococcal bacteriuria. Urine IL-6 concentrations are markedly increased in women with acute pyelonephritis or asymptomatic bacteriuria.26 To date, bacteria-induced increases in uIL-6 have not been linked with increases in uFIB, although our laboratory has documented an elevated uFIB in dogs with both enterococci and E coli present in the urine (unpublished data). Thus, it could be hypothesized that anatomic abnormalities of the LUT predispose dogs to recurrent UTI, and the UTI caused an increase in uIL-6 and potentially uFIB, which facilitates enterococcal colonization of the bladder without competition owing to clearance of the E coli infection by the antimicrobial administered for the UTI. However, the fact that the dogs with LUT anatomic abnormalities analyzed in the present study had a significantly higher median uIL-6, but not uFIB, relative to the control dogs does not support that hypothesis.
We cannot explain the apparent discrepancy between the median uIL-6 and median uFIB for the dogs with LUT anatomic abnormalities relative to control dogs, but we expect it may have been caused by insufficient power owing to a small sample size. The a priori power analysis indicated that urine samples from 46 dogs would need to be analyzed for each experimental group to yield a study with 95% power. Unfortunately, urine samples from only 9 dogs with LUT anatomic abnormalities were available for analysis, and only 8 were of sufficient volume for both uFIB and uIL-6 to be determined. Results of post hoc power analyses indicated that the comparison of uIL-6 between dogs with LUT neoplasia and control dogs had a power of only 41%; to achieve a power of 95%, it would have been necessary to analyze urine samples from an additional 4 dogs with LUT neoplasia. The comparisons of uIL-6 between dogs with LUT anatomic abnormalities and control dogs and between dogs with urolithiasis and control dogs both achieved at least 95% power. Given that the uIL-6 could be determined from only a small number of dogs within each experimental group, the uIL-6 data were provided primarily as adjunctive evidence to support the uFIB data in this report.
The small sample size of the present study also prohibited analysis of the effects of the various lesions within each experimental group on the analyzed indices and likely accounted for the large variation in uFIB and uIL-6 within the experimental groups. Additionally, this study lacked sufficient power to assess the effect of urolith size, number, mineral composition, and location; neoplasia type and location; and specific LUT anatomic abnormality on uFIB. Finally, the retrospective nature of the study required us to rely on information recorded in the medical records of dogs with banked urine samples, and incomplete data in the medical records likely led to exclusion of many dogs that may otherwise have been eligible for study inclusion.
Results of the present study supported our hypothesis that the uFIB in dogs with some risk factors for recurrent UTIs and enterococcal bacteriuria (ie, urolithiasis and LUT neoplasia) were significantly greater than the uFIB in healthy control dogs. Further investigation is necessary to determine whether the presence of fibrinogen in urine is necessary for enterococcal colonization of the urinary tract. If it is, strategies to block the interaction between fibrinogen and enterococci should be investigated for the treatment of enterococcal UTIs in dogs.
Acknowledgments
Supported by a Companion Animal Fund grant provided by the University of Wisconsin-Madison School of Veterinary Medicine. Funding sources did not have any involvement in the study design, data analysis and interpretation, or writing and publication of the manuscript.
The authors declare that there were no conflicts of interest.
Abbreviations
| CAUTI | Catheter-associated urinary tract infection |
| Ebp | Endocarditis and biofilm–associated pili |
| IL-6 | Interleukin-6 |
| IQR | Interquartile (25th to 75th percentile) range |
| LUT | Lower portion of the urinary tract |
| uCrea | Urine creatinine concentration |
| uFIB | Urine fibrinogen concentration |
| uIL-6 | Urine IL-6 concentration |
| UTI | Urinary tract infection |
Footnotes
Vitros 5.1 FS, Ortho Clinical Diagnostics, Raritan, NJ.
Spectra/Por 1 Dialysis Membrane, Spectrum Laboratories Inc, Rancho Dominguez, Calif.
ELISA kit, Abcam, Cambridge, Mass.
Immunochemistry Technologies, Bloomington, Minn.
FLUOstar Omega, BMG Labtech, Cary, NC.
Prism, version 8, GraphPad Software, La Jolla, Calif.
Canine IL-6 DuoSet ELISA, R&D Systems, Minneapolis, Minn.
SuperSignal West Femto Maximum Sensitivity Substrate, ThermoFisher Scientific, Waltham, Mass.
G*Power, version 3, Heinrich Heine University, Dusseldorf, Germany.
References
- 1. ↑
Hall JL, Holmes MA, Baines SJ. Prevalence and antimicrobial resistance of canine urinary tract pathogens. Vet Rec 2013;173:549–554.
- 2. ↑
McMeekin CH, Hill KE, Gibson IR, et al. Antimicrobial resistance patterns of bacteria isolated from canine urinary samples submitted to a New Zealand veterinary diagnostic laboratory between 2005–2012. N Z Vet J 2017;65:99–104.
- 3. ↑
Seguin MA, Vaden SL, Altier C, et al. Persistent urinary tract infections and reinfections in 100 dogs (1989–1999). J Vet Intern Med 2003;17:622–631.
- 4. ↑
Wong C, Epstein SE, Westropp JL. Antimicrobial susceptibility patterns in urinary tract infections in dogs (2010–2013). J Vet Intern Med 2015;29:1045–1052.
- 5. ↑
Flores-Mireles AL, Pinkner JS, Caparon MG, et al. EbpA vaccine antibodies block binding of Enterococcus faecalis to fibrinogen to prevent catheter-associated bladder infection in mice. Sci Transl Med 2014;6:254ra127.
- 6. ↑
Guiton PS, Hannan TJ, Ford B, et al. Enterococcus faecalis overcomes foreign body-mediated inflammation to establish urinary tract infections. Infect Immun 2013;81:329–339.
- 7. ↑
Montealegre MC, Singh KV, Somarajan SR, et al. Role of the Emp pilus subunits of Enterococcus faecium in biofilm formation, adherence to host extracellular matrix components, and experimental infection. Infect Immun 2016;84:1491–1500.
- 8. ↑
Nallapareddy SR, Singh KV, Sillanpaa J, et al. Relative contributions of Ebp pili and the collagen adhesin ace to host extracellular matrix protein adherence and experimental urinary tract infection by Enterococcus faecalis OG1RF. Infect Immun 2011;79:2901–2910.
- 9. ↑
Wright KJ, Hultgren SJ. Sticky fibers and uropathogenesis: bacterial adhesins in the urinary tract. Future Microbiol 2006;1:75–87.
- 10. ↑
Flores-Mireles A, Hreha TN, Hunstad DA. Pathophysiology, treatment, and prevention of catheter-associated urinary tract infection. Top Spinal Cord Inj Rehabil 2019;25:228–240.
- 12. ↑
Guiton PS, Hung CS, Hancock LE, et al. Enterococcal biofilm formation and virulence in an optimized murine model of foreign body-associated urinary tract infections. Infect Immun 2010;78:4166–4175.
- 13. ↑
Wood MW, Lepold A, Tesfamichael D, et al. Risk factors for enterococcal bacteriuria in dogs: a retrospective study. J Vet Intern Med 2020;34:2447–2453.
- 14. ↑
Hoffmann D, Bijol V, Krishnamoorthy A, et al. Fibrinogen excretion in the urine and immunoreactivity in the kidney serves as a translational biomarker for acute kidney injury. Am J Pathol 2012;181:818–828.
- 15.
Flores-Mireles AL, Walker JN, Potretzke A, et al. Antibody-based therapy for enterococcal catheter-associated urinary tract infections. MBio 2016;7:e01653–e16.
- 16. ↑
Wang H, Zheng C, Lu Y, et al. Urinary fibrinogen as a predictor of progression of CKD. Clin J Am Soc Nephrol 2017;12:1922–1929.
- 17. ↑
Wood MW, Nordone SLK, Vaden SL, et al. Assessment of urine solute and matrix effects on the performance of an enzyme-linked immunosorbent assay for measurement of interleukin-6 in dog urine. J Vet Diagn Invest 2011;23:316–320.
- 18. ↑
Hirano T, Teranishi T, Onoue K. Human helper T cell factor(s). III. Characterization of B cell differentiation factor I (BCDF I). J Immunol 1984;132:229–234.
- 19. ↑
Seguchi T, Yokokawa K, Sugao H, et al. Interleukin-6 activity in urine and serum in patients with bladder carcinoma. J Urol 1992;148:791–794.
- 20. ↑
Rhee E, Santiago L, Park E, et al. Urinary IL-6 is elevated in patients with urolithiasis. J Urol 1998;160:2284–2288.
- 21. ↑
Ortega M, Marco F, Soriano A, et al. Epidemiology and prognostic determinants of bacteraemic catheter-acquired urinary tract infection in a single institution from 1991 to 2010. J Infect 2013;67:282–287.
- 22. ↑
Arias CA, Murray BE. The rise of the Enterococcus: beyond vancomycin resistance. Nat Rev Microbiol 2012;10:266–278.
- 23. ↑
Bitsori M, Maraki S, Raissaki M, et al. Community-acquired enterococcal urinary tract infections. Pediatr Nephrol 2005;20:1583–1586.
- 24. ↑
Baquero F, Negri MC, Morosini MI, et al. Antibiotic-selective environments. Clin Infect Dis 1998;27(suppl 1):S5–S11.
- 25. ↑
Llido M, Vachon C, Dickinson M, et al. Transurethral cystoscopy in dogs with recurrent urinary tract infections: retrospective study (2011–2018). J Vet Intern Med 2020;34:790–796.
- 26. ↑
Hedges S, Stenqvist K, Lidin-Janson G, et al. Comparison of urine and serum concentrations of interleukin-6 in women with acute pyelonephritis or asymptomatic bacteriuria. J Infect Dis 1992;166:653–656.
