Clinical differences in enterococcal bacteriuria compared with other bacteriuria in cats

Abstract

Objectives

The clinical differences between Enterococcus species bacteriuria compared with other bacteria has been reported in a small number of cats. The objective of this study was to compare a large number of cats with Enterococcus species bacteriuria to cats with other bacteriuria and determine the clinical differences. It was hypothesized that enterococcal bacteriuria would be associated with subclinical bacteriuria and polymicrobial infections more than other bacteriuria, and that when local or systemic comorbidities were present, enterococcal bacteriuria would be more common.

Methods

This retrospective case-control study compared case cats with enterococcal bacteriuria to control cats with other bacteriuria. Cats with enterococcal bacteriuria were age, year and weight matched with 1–2 control cats with any other bacteriuria.

Results

Lower urinary tract clinical signs were statistically significantly more common in controls (n = 38/77 [49%]) compared with Enterococcus cases (n = 12/47 [25%]; P = 0.01). Specifically, control cats (n = 20/77 [26%]) were statistically significantly more likely to have gross hematuria compared with Enterococcus case cats (n = 3/47 [6%]; P = 0.01). Enterococcus cases were statistically significantly more likely to have a polymicrobial infection compared with controls (odds ratio 5.84, 95% confidence interval 1.33–34.70; P = 0.01).

Conclusions and relevance

Enterococcus species are associated with subclinical bacteriuria and polymicrobial urinary tract infections in cats vs other bacteriuria.

Introduction

Enterococcus species have been identified as important pathogens both in bacterial cystitis and subclinical bacteriuria (SBU) in cats. Enterococci are often the second or third most common cultured organisms from the urinary tract, accounting for 5–27% of isolates.^1–4 A large study of 150 cats with 169 urine cultures identified SBU in 55% and bacterial cystitis in 38%. In this study, Enterococcus species were isolated in 15.1% of cultures and was significantly associated with SBU: 55% of cats had no lower urinary tract signs, whereas 39% of cats did have signs. ² SBU has been investigated in healthy older cats, as well as cats with systemic comorbidities such as chronic kidney disease (CKD), diabetes mellitus and hyperthyroidism.^4–12 Enterococcus species were identified as the second-most common isolate in these studies, ranging from 5% to 55%. Studies of cats with bacterial cystitis have identified Enterococcus species less commonly, in 11–27% of cats studied.^1,6 A small study investigating enterococcal bacteriuria in 11 cats identified predisposing factors such as urolithiasis, cystostomy tube placement, urethral catheterization, spinal dysfunction, CKD and/or hyperthyroidism. ⁵ Lower urinary tract signs were present in 5/11 of these cats. Another recent study in dogs identified risk factors for enterococcal bacteriuria as the presence of a lower urinary tract anatomic abnormality (ie, recessed vulva), urolithiasis or lower urinary tract neoplasia. ¹³

Previous veterinary studies have identified Enterococcus species as a common co-isolate in polymicrobial urinary tract infections (UTIs), often with other uropathogens such as Escherichia coli and Proteus mirabilis.^1,13 The prevalence of polymicrobial UTIs in cats has been previously reported to be 13–23%.^1,6,13,14 In these clinical scenarios, it is unclear if, or to what degree, the Enterococcus species isolate contributes to clinical signs when compared with the other organism. Anecdotally, some clinicians will target treatment of the non-enterococcal organism with an assumption that treatment of the more pathogenic bacteria may lead to resolution of clinical signs or bacteriuria. ¹⁵

The primary goals of this study were to evaluate whether the absence of lower urinary tract clinical signs was more commonly associated with enterococcal bacteriuria compared with other pathogens, and whether enterococcal bacteriuria is more common in cats with systemic comorbidities compared with other urinary pathogens. A secondary goal was to determine the frequency with which Enterococcus species are detected in polymicrobial UTIs in cats, compared with other bacteria. We hypothesized that enterococcal bacteriuria would be associated with SBU and polymicrobial infections more than other bacteriuria, and that when local or systemic comorbidities were present, enterococcal bacteriuria would be more common.

Materials and methods

Case selection

The Matthew J Ryan Veterinary Hospital of the University of Pennsylvania’s clinical microbiology laboratory database was searched for positive aerobic urine cultures from cats examined from April 2017 to December 2019 and used to identify cats with urine cultures positive for Enterococcus species. Cats were excluded if the medical record was missing or incomplete, or if the aerobic urine culture did not yield significant growth. Significant growth was defined as >10³ colony-forming units (CFU)/ml for urine collected via cystocentesis, >10⁴ CFU/ml for urine collected via catheterization and ⩾10⁵ CFU/ml for urine collected via free-catch.

Data collected from medical records included signalment, past medical history, physical examination (including body weight, body condition score [BCS] and anatomic urinary abnormalities), lower urinary tract clinical signs (including hematuria, pollakiuria, stranguria and/or dysuria) and urine collection method. Records were evaluated for a historical or current diagnosis of hyperthyroidism, neurologic disease, gastrointestinal disease, CKD (based on International Renal Interest Society staging), immunosuppressive medication administration, urinary calculi, urethral obstruction or urinary tract neoplasia.

Control selection

Controls were selected from the same database containing all positive aerobic urine cultures. Cats were age, weight and year matched. Cats were matched by age and weight because older cats and those with a lower body weight have been shown to be at increased risk for UTIs.^1,16 Matching by year was performed to account for any changes in antimicrobial susceptibility patterns and bacterial populations over the study period. ¹⁷ Cultures were excluded from analysis if they did not meet the definition for significant growth defined for the Enterococcus species cases. For every aerobic urine culture positive for enterococcal growth, 1–2 (depending on case availability) aerobic urine cultures positive for any other bacterial species were selected as controls. Missing or incomplete medical records were excluded. Data collected from control medical records were the same as for cases.

Bacterial culture procedures

Urine specimens were submitted to the clinical microbiology laboratory with the following handling requirements: specimens were submitted within 72 h of collection, specimens must have been collected in a sterile container and refrigerated if not immediately submitted. Following submission to the clinical microbiology laboratory, a 1 µl loop was used to streak urine specimens to tryptic soy agar with 5% sheep’s blood, MacConkey agar and colistin–naladixic acid agar plates (Remel) in a quantitative fashion. Plates were incubated overnight for at least 16 h at 37°C. ¹⁸ Plates were evaluated for growth and growth enumerated into one of the following four categories: <10,000 CFU/ml; 11,000–50,000 CFU/ml; 50–100,000 CFU/ml; and >100,000 CFU/ml. Significant growth was defined as >10³ CFU/ml for urine collected via cystocentesis, >10⁴ CFU/ml for urine collected via catheterization and ⩾10⁵ CFU/ml for urine collected via free-catch or unknown method. Subculture for purity was performed if required. Pure isolates were identified, and antimicrobial susceptibility testing was performed using the Vitek 2 automated system (Biomerieux) according to the Clinical and Laboratory Standards Institute. ¹⁹

Data and statistical analyses

Most continuous variables were not normally distributed as determined visually and by the skewness and kurtosis tests for normality. Therefore, results are reported as median (range) and the non-parametric two-sample Wilcoxon rank-sum (Mann–Whitney) test was used for comparisons of continuous variables in cases and controls. The Fisher’s exact test or χ ² test were employed to examine the relationship between two categorical variables, depending on the number of cells with a frequency of five or less. A P value <0.05 was considered to be statistically significant for all tests. All statistical evaluations were performed using a statistical software package (Stata 14.0 for Mac; Stata Corp.).

Results

Enterococcus species-positive urine cultures and controls

Of 160 positive aerobic feline urine cultures during the study period, 47/160 (29%) cases of Enterococcus species bacteriuria were identified. Seventy-seven control cases were selected from the same population. A retrospective review of collection and processing dates confirmed that all urine specimens were processed within 24 h. Information on the method of urine collection was available for 43 cases and 68 controls. Samples were collected via cystocentesis (n = 78/111; 70.2%), free-catch (n = 18/111; 16.2%), urinary catheterization (n = 6/111; 5.4%) or not reported (n = 9/111; 8.1%). The collection method between cases and controls was not statistically different.

Urine culture results

Isolates from the enterococcal bacteriuria cases were E faecalis (n = 35/47 [75%]) and E faecium (n = 12/47 [25%]). Eleven organisms were isolated from the control group, including E coli (n = 57/77 [74%]), Staphylococcus pseudintermedius (n = 8/77; 10.3%), Staphylococcus felis (n = 3/77; 3.9%), two (2.6%) of the following: Pasteurella multocida, Staphylococcus warneri and one (1.3%) of each of the following: P mirabilis, Pseudomonas aeruginosa, Staphylococcus schleiferi, Serratia marcescens and Morganella morganii. The majority of urine cultures (n = 112/124 [90%]) had monomicrobial growth. From the case population of 47 cultures, 38 were monomicrobial (80.8%) and nine had two bacterial isolates (19.1%). The control population with 77 urine cultures had 74 monomicrobial infections (96.1%) and three polymicrobial infections with two bacterial isolates (3.9%). Cases were statistically significantly more likely to have a polymicrobial infection compared with controls (odds ratio [OR] 5.84, 95% confidence interval [CI] 1.33– 34.70; P = 0.01). Coisolates from the Enterococcus species group included E coli (n = 5/9 [55.6%]) and one each (n = 1/9 [11.1%]) of P mirabilis, P aeruginosa and Klebsiella pneumonia. Within the polymicrobial infections from the control population, coisolates included S felis (n = 1/3 [33.3%]), S pseudintermedius (n = 1/3 [33.3%]) and P aeruginosa (n = 1/3 [33.3%]). No difference was detected when comparing coisolates between cases and controls.

Signalment and BCS

For case cats, the median age was 13 years (range 4 months to 19 years) and the median weight was 4.4 kg (range 2.13–9.7). In cats in the control group, the median age was 12 years (range 1–19) and the median weight was 4.2 kg (range 2.13–8.25). BCS was available for 43/47 (91%) cases and 52/77 (67%) controls. The median BCS for cases was 4 (range 2–9) and was not statistically different than the median BCS of 5 (range 2–9) for the controls. In the Enterococcus species case population, there were 37 female spayed (79%) and 10 male castrated (21%) cats. Within the control group, 60 were spayed females (78%) and 17 were castrated males (22%).

Presence of clinical signs

Fewer case cats (n = 12/47 [25%]) showed lower urinary tract signs than controls (n = 38/77 [49%]). Specifically, case cats (n = 3/47 [6%]) were less likely to have gross hematuria than control cats (n = 20/77 [26%]; OR 0.19, 95% CI 0.04–0.73).

Systemic comorbidities

Twenty-six (55.3%) case cats and 33 (42.8%) control cats had CKD. Vomiting was noted in 12/47 cases (25.5%) and 22/77 controls (28.6%). Eight case cats (17.0%) and seven (9%) control cats were diabetic. There were seven (14.9%) cats with hyperthyroidism in the case group and 12 (15.6%) in the control group. Diarrhea was recorded in 2/47 cases (4.3%) and 1/77 controls (1.3%). Two control cats (2.6%) and no case cats had neurologic disease. There was no statistically significant difference in the frequency of CKD, gastrointestinal signs (vomiting and diarrhea), diabetes mellitus, hyperthyroidism, CKD, neurologic disease or diabetes mellitus between the groups. Similarly, no difference was found in the number of cases compared with controls that were receiving corticosteroids (n = 5/47 cases [10.6%]; n = 15/77 controls [19.5%]), intravenous chemotherapy (four control cats [5.2%]) or other immunosuppressive medications (one control cat [1.3%]).

Presence of additional urinary tract disease

Urinary calculi were present in 5/47 (10.6%) case cats and 4/77 (5.2%) control cats (no statistical difference). Four case cats (8.5%) and seven control cats (9.0%) had a urethral obstruction, which was not different between groups. A single control cat had urinary neoplasia (transitional cell carcinoma).

Discussion

This study identified important clinical differences in cats with enterococcal bacteriuria compared with other bacteriuria. This study demonstrates that while enterococci can act as agents of UTIs in some cats, they are also commonly isolated from cats with SBU. Previous studies have identified Enterococcus species as the causative agent in cats with bacterial cystitis, which is consistent with the 26% of cats with enterococcal bacteriuria and lower urinary tract signs in our study.^1,2 It is also possible that in cats with clinical signs the Enterococcus species could have colonized the urinary bladders of cats with feline idiopathic cystitis, which is thought to be a multifactorial sterile inflammatory condition. The role of Enterococcus species in this complicated disease process requires further investigation. Owing to its retrospective nature, this study did not evaluate follow-up urine cultures to determine persistent colonization with Enterococcus species or other bacteria. Further prospective studies are needed to determine patient factors that may lead to a Enterococcus species role in causing clinical signs or as a colonizing bacteria. These studies should include microbiologic and molecular characterization of Enterococcus species isolates associated with SBU or UTIs to determine if strain-level differences exist in their propensity to colonize and/or lead to clinical signs in cats.

Previous studies have evaluated SBU in older healthy cats and cats with obesity, hyperthyroidism, diabetes mellitus and CKD.^4–12 In these studies, E coli was the most common bacteria isolated, with Enterococcus bacteriuria rates varying from 5% to 55%. In our study, the prevalence of enterococcal bacteriuria compared with other bacteriuria was not different for patients with these diseases. Along with our data, the high rates of Enterococcus species in cats with SBU support the idea that Enterococcus species colonize rather than infect the lower urinary tract in most cats. Alternatively, it is possible that enterococci lead to more mild lower urinary signs, which may go unnoticed by owners and lead to bias in detecting an association between bacteriuria and clinical signs. For example, this study showed a difference in the presence of gross hematuria between cats with enterococcal or other bacteriuria, the presence of which may prompt an owner to seek veterinary care sooner than if pollakiuria or dysuria are observed alone.

This study found that enterococci are significantly more likely to be associated with polymicrobial growth than other bacteria in the urine of cats. Previous studies investigating SBU and bacterial cystitis in cats have had low numbers of polymicrobial infections.^1,6,14,18 There are multiple proposed mechanisms for the role of Enterococcus species in polymicrobial infections. Enterococcus species can colonize the lower urinary tract after another bacterial infection causes inflammation. ²⁰ Alternatively, colonization with Enterococcus species may cause local, innate immune suppression, which could allow for proliferation of less virulent bacteria. ²¹ Further studies of polymicrobial Enterococcus species UTIs are needed to assess whether the practice of targeting the non-enterococcal bacteria during therapy is sound.

The limitations of this study are its retrospective nature and the inability to standardize and assess treatments and outcome. The study had broad inclusion and minimal exclusion criteria, which led to a heterogeneous study population, which may introduce bias. However, a heterogeneous study population also improves the generalizability of the study findings to the true heterogeneous population of cats observed in the clinic. A major limitation is that the study was not designed to investigate prior antibiotic use in cases or controls. Many commonly administered antibiotics such as cephalosporins and fluoroquinolones are not active against Enterococcus species. Therefore, prior exposure to these antibiotics could select for Enterococcus species as a persistent pathogen in the urine. ²² It is possible that some case cats had prior antibiotic treatment that increased their risk for Enterococcus species bacteriuria. Further studies should evaluate prior antibiotic use as a risk factor for enterococcal bacteriuria in cats. Some of the study findings, such as a lack of a difference in comorbidities between cases and controls might be due to a small sample size and a type II statistical error, and the findings should be interpreted with this limitation in mind. Larger future studies are needed to confirm or refute these findings. An unequal number of cats were used as controls, specifically in 2017, due the limited availability of cats matching the control requirements. Owing to the retrospective nature of this study, information on the urine collection method and resolution of clinical signs were not available for all patients. Follow-up cultures to ensure microbiological resolution of bacteriuria were not assessed.

Conclusions

This study contributes to the existing literature on the importance of Enterococcus species in the urinary tract, both as a uropathogen and an agent of SBU in cats. Cats with Enterococcus species bacteriuria were less likely to show clinical signs than cats with other bacteriuria, which suggests an important role of Enterococcus species bacteria in SBU. It also identified the importance of Enterococcus species in polymicrobial infections, as polymicrobial growth was identified significantly more often in cats with enterococcal bacteriuria than in the control population. Both of these findings have important implications toward antimicrobial stewardship and the controversy surrounding treatment of enterococcal bacteriuria. While our study is suggestive of a limited role of enterococci in causing clinical signs in cats, further research is needed to clarify if therapy targeting Enterococcus species in cases where multiple organisms are present is necessary, especially given the inherent resistance of Enterococcus species to commonly used antimicrobials.