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The Clinical Urine Culture: Enhanced Techniques Improve Detection

of Clinically Relevant Microorganisms
Travis K. Price,a Tanaka Dune,b Evann E. Hilt,a Krystal J. Thomas-White,a Stephanie Kliethermes,c Cynthia Brincat,b,d
Linda Brubaker,b,d Alan J. Wolfe,a Elizabeth R. Mueller,b,d Paul C. Schreckenbergere
Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University, Chicago, Illinois, USAa; Department of Obstetrics & Gynecology and Urology,
Loyola University Medical Center, Maywood, Illinois, USAb; Departments of Medicine and Public Health Sciences,c Department of Obstetrics & Gynecology and Urology,d
and Department of Pathology,e Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois, USA

Enhanced quantitative urine culture (EQUC) detects live microorganisms in the vast majority of urine specimens reported as
“no growth” by the standard urine culture protocol. Here, we evaluated an expanded set of EQUC conditions (expanded-spec-
trum EQUC) to identify an optimal version that provides a more complete description of uropathogens in women experiencing
urinary tract infection (UTI)-like symptoms. One hundred fifty adult urogynecology patient-participants were characterized
using a self-completed validated UTI symptom assessment (UTISA) questionnaire and asked “Do you feel you have a UTI?”
Women responding negatively were recruited into the no-UTI cohort, while women responding affirmatively were recruited into
the UTI cohort; the latter cohort was reassessed with the UTISA questionnaire 3 to 7 days later. Baseline catheterized urine sam-
ples were plated using both standard urine culture and expanded-spectrum EQUC protocols: standard urine culture inoculated
at 1 ␮l onto 2 agars incubated aerobically; expanded-spectrum EQUC inoculated at three different volumes of urine onto 7 com-
binations of agars and environments. Compared to expanded-spectrum EQUC, standard urine culture missed 67% of uropatho-
gens overall and 50% in participants with severe urinary symptoms. Thirty-six percent of participants with missed uropathogens
reported no symptom resolution after treatment by standard urine culture results. Optimal detection of uropathogens could be
achieved using the following: 100 ␮l of urine plated onto blood (blood agar plate [BAP]), colistin-nalidixic acid (CNA), and
MacConkey agars in 5% CO2 for 48 h. This streamlined EQUC protocol achieved 84% uropathogen detection relative to 33%
detection by standard urine culture. The streamlined EQUC protocol improves detection of uropathogens that are likely relevant
for symptomatic women, giving clinicians the opportunity to receive additional information not currently reported using stan-
dard urine culture techniques.

T he diagnostic gold standard for clinically relevant urinary tract

infection (UTI) continues to be questioned for both clinical
and research purposes. Since the 1950s, clinical practice has relied
toms and 75 who did not based on their yes/no response to the question
“Do you feel you have a UTI?” Participants were seeking clinical care at
Loyola University Medical Center’s Female Pelvic Medicine and Recon-
on detecting ⱖ105 CFU/ml of a known uropathogen using the structive Surgery center between June 2014 and August 2015.
standard clinical urine culture protocol (1). The standard urine Participants gave verbal and written research consent and provided
permission for abstraction of their demographic and clinical information

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culture was initially described for detection of patients at risk for
pyelonephritis (2); interpretation has been generalized to diag- from the electronic medical record. At baseline, urinary symptoms were
nose lower urinary tract infection despite studies reporting limi- characterized using a self-completed, validated UTI symptom assessment
(UTISA) questionnaire, completed by both cohorts (16). Participants
tations of the ⱖ105-CFU/ml threshold (3–6). While the clinical
were dichotomized based on their yes/no response to the question “Do
focus has centered on various cutoff thresholds, the basic uro-
you feel you have a UTI?” Those who responded affirmatively (UTI co-
pathogen detection method remains unchanged. hort) completed the UTISA questionnaire again by phone 3 to 7 days
Given emerging evidence that documents the presence of uri- postenrollment and were queried about the magnitude of symptom res-
nary microbiota in many adult women (7–15), it is clear that the
mere presence of an organism should not prompt antibiotic treat-
ment. However, clinicians are likely to benefit from a more com- Received 8 January 2016 Returned for modification 4 February 2016
plete report of organisms present within a symptomatic patient’s Accepted 1 March 2016
urine. Recent evidence reports bacteria in ⬃90% of “no-growth” Accepted manuscript posted online 9 March 2016
standard urine cultures (10, 12). We hypothesized that, in women Citation Price TK, Dune TD, Hilt EE, Thomas-White KJ, Kliethermes S, Brincat C,
experiencing UTI-like symptoms, an improved culture protocol Brubaker L, Wolfe AJ, Mueller ER, Schreckenberger PC. 2016. The clinical urine
would provide a more complete description of potentially treat- culture: enhanced techniques improve detection of clinically relevant
microorganisms. J Clin Microbiol 54:1216 –1222. doi:10.1128/JCM.00044-16.
able, clinically relevant uropathogens. This study evaluated vari-
Editor: B. A. Forbes
ous combinations of urine volumes, media, atmospheric environ-
Address correspondence to Elizabeth R. Mueller, [email protected], or
ments, and incubation durations to determine conditions that
Paul Schreckenberger, [email protected].
optimally balance uropathogen detection with feasibility.
T.K.P. and T.D. contributed equally to the manuscript.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
MATERIALS AND METHODS /JCM.00044-16.
Study design and patient population. Following institutional review Copyright © 2016, American Society for Microbiology. All Rights Reserved.
board (IRB) approval, we enrolled 75 women who reported UTI symp-

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 Improved Clinical Urine Culture Protocol

TABLE 1 Summary of urine cultivation protocols for catheterized urine specimens

Vol (␮l) of Incubation(s) (h) for Patient sample
Protocol urine Medium or media Conditions microbial identification identifiera
Standard urine culture 1 BAP, MacConkey agar Aerobic, 35°C 24 1–107
Modified urine culture 1 BAP, MacConkey agar 5% CO2, 35°C 24, 48 108–150
Expanded-spectrum EQUC 1, 10, and 100 BAP, MacConkey agar Aerobic, 35°C 24, 48 1–150
BAP, chocolate agar, 5% CO2, 35°C 24, 48
CNA agar
CDC anaerobic BAP Anaerobic, 35°C 48
CDC anaerobic BAPb Microaerophilic gas mixture 48
(5% O2, 10% CO2, 85% N),
35°C
Streamlined EQUC 100 BAP, MacConkey agar,c 5% CO2, 35°C 48 1–150d
CNA agar
a
Refers to the patient samples on which the corresponding protocol assessed the urinary microbiota. For diagnosis, the standard urine culture protocol was used on patient samples
1 to 107; the modified standard urine culture was used on patient samples 108 to 150. For research, all patient samples were assessed by expanded-spectrum EQUC.
b
The CDC anaerobic BAP microaerophilic gas mixture condition was used only for samples 10 to 150.
c
The MacConkey 5% CO2 condition was not part of the expanded-spectrum EQUC protocol.
d
The streamlined EQUC protocol was performed using a subgroup of agars/conditions from the expanded-spectrum EQUC protocol; therefore, it was used on all patient samples.

olution, if any. All clinical treatment was individually based on physician Microbial identification was determined using a matrix-assisted laser
assessment of patient symptoms and standard urine culture results. Ex- desorption ionization–time of flight mass spectrometer (MALDI-TOF
clusion criteria included age of ⬍18 years, pregnancy, catheterization (in- MS; Bruker Daltonics, Billerica, MA) as described previously (10). Only
dwelling or intermittent), or insufficient English skills to complete study clinically relevant microbes (i.e., known and emerging uropathogens)
measures. were used to calculate uropathogen detection. These uropathogens were
Sample collection and analysis. Consistent with patient care clinical selected based on previously published case reports of UTI.
protocol, urine was collected via transurethral catheter and then placed UTISA questionnaire. This questionnaire asks the participant to rate
into two BD Vacutainer Plus C&S preservative tubes: one sent to the the degree of severity and bother for seven common UTI symptoms: fre-
clinical microbiology laboratory for diagnostic purposes and one sent to quency of urination, urgency of urination, incomplete bladder emptying
the researchers for testing. (urinary retention), pain or burning during urination (dysuria), lower
Table 1 displays all culture protocols used by the clinical microbiology abdominal discomfort or pelvic pain/pressure, lower back pain, and blood
laboratory staff and the researchers. The standard urine culture protocol in the urine (hematuria). Scores for each symptom range from 0 to 3; a 0
used 1 ␮l of urine, spread quantitatively (i.e., pinwheel streak) onto 5% corresponds to no symptom present, whereas a 3 indicates highest severity
sheep blood (blood agar plate [BAP]) and MacConkey agars (BD BBL or bother. The seven symptoms can be clustered into four symptom do-
Prepared Plated Media; Cockeysville, MD) and incubated aerobically at mains: urination regularity (frequency and urgency), problems with uri-
35°C for 24 h. The modified standard urine culture used the same agars nation (incomplete bladder emptying and pain or burning), pain associ-
and temperature but changed the incubation condition to 5% CO2; if ated with the UTI (abdominal or pelvic pain and lower back pain), and
pinpoint growth was seen at 24 h, the agars were held for another 24 h blood in the urine (16).
under the same conditions. Unrelated to this study, the clinical microbi- Statistical analyses. Continuous variables were reported as means and

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ology laboratory adopted the modified standard urine culture for diagno- standard deviations (SDs) or medians and interquartile ranges (IQRs);
sis during patient recruitment for this study. Thus, diagnostic testing for categorical variables were reported as frequencies and percentages. Pear-
patients 1 to 107 was the standard urine culture, while the modified stan- son chi-square tests (or Fisher’s exact tests, when necessary) and 2-sample
dard urine culture was used for patients 108 to 150. However, this change t tests (or Wilcoxon rank sum tests, when appropriate) were used to com-
did not impact data presented in this study, as standard urine culture data pare demographics and culture results (e.g., abundance and diversity)
for patients 108 to 150 were obtained by analyzing the corresponding between cohorts. Measures of alpha diversity (diversity within a popula-
subset of expanded enhanced quantitative urine culture (EQUC) condi- tion) were represented as Shannon diversity indices and/or graphically by
tions (i.e., 1 ␮l BAP and MacConkey agars; aerobic, 35°C; 24 h). species accumulation curves (which plot accumulation of unique species
The conditions of the original enhanced quantitative urine culture per group for each additional sample included). All statistical analyses
(EQUC) protocol were described previously (10). In this study, we ex- were conducted using SAS software v9.4 (SAS Institute, Cary, NC) or
panded those conditions (i.e., expanded-spectrum EQUC protocol), us- Systat software version 13.1 (Systat Software Inc., Chicago, IL).
ing three urine volumes (1 ␮l, 10 ␮l, and 100 ␮l) and additional plating
conditions (Table 1). Each urine sample was spread quantitatively onto RESULTS
BAP, chocolate, and colistin-nalidixic acid (CNA) agars (BD BBL Pre- Participant demographics and symptoms. Table 2 describes de-
pared Plated Media) and incubated in 5% CO2 at 35°C for 48 h; BAP and mographics of the two cohorts (75 no-UTI and 75 UTI patients).
MacConkey agars were incubated aerobically at 35°C for 48 h; two CDC Most participants were white (81%) and overweight (mean body
anaerobic 5% sheep blood (anaerobic BAP) agars (BD BBL Prepared mass index [BMI] ⫽ 29.3 kg/m2). Most participants (92%, 138/
Plated Media) were incubated either in microaerophilic Campy gas mix- 150) reported at least one urinary symptom; as expected, women
ture (5% O2, 10% CO2, 85% N) or anaerobically at 35°C for 48 h. Three
in the UTI cohort had higher UTISA questionnaire scores.
sets of these conditions were used for each urine sample, each using one of
the urine volumes, for a total of 21 agars per sample. All agars were doc- Expanded-spectrum EQUC: urinary microbiota characteris-
umented for growth (i.e., for morphologies and CFU per milliliter) at 24 tics. Nearly all (93% [139/150]) catheterized urine samples grew
and 48 h, except the two anaerobic BAP conditions. Each distinct colony bacterial colonies with at least one combination of the expanded-
morphology was subcultured at 48 h to obtain pure culture for microbial spectrum EQUC protocol’s conditions (Table 1). The no-UTI
identification. and UTI cohorts had similar proportions of cultivatable urine

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Price et al.

TABLE 2 Demographic characteristics and symptoms

Entire cohort No-UTI cohort UTI cohort
Clinical variable (n ⫽ 150) (n ⫽ 75) (n ⫽ 75) P valued
Age (yr), mean (SD) 62.3 (14.9) 60.6 (12.3) 64.0 (17.1) 0.16a
BMI (kg/m2), mean (SD) 29.3 (6.3) 28.8 (5.9) 29.9 (6.6) 0.27a

Race/ethnicity, no. (%)

White 121 (81) 59 (79) 62 (83)
Hispanic 15 (10) 9 (12) 6 (8)
Black 9 (6) 5 (7) 4 (5) 0.90c
Asian 4 (3) 2 (3) 2 (3)
Other 1 (1) 0 (0) 1 (1)

No. of vaginal deliveries, median (IQR) 2 (0–11) 2 (0–6) 2 (0–11) 0.80b

Sexually active, no. (%) 58 (39) 37 (49) 21 (28) 0.01
Previous antibiotic treatment, no. (%) 45 (30) 20 (27) 25 (33) 0.37
Current anticholinergic treatment, no. (%) 26 (17) 9 (12) 17 (23) 0.08

Type of anticholinergic used, no. (%)

Oxybutynin 9 (6) 3 (4) 6 (8)
Solifenacin 8 (5) 3 (4) 5 (7)
Fesoterodine 4 (3) 2 (3) 2 (3)
Tolterodine 2 (1) 1 (1) 1 (1) 0.75c
Oxybutynin patch 1 (1) 0 (0) 1 (1)
Trospium chloride 1 (1) 0 (0) 1 (1)
Mirabegron (Myrbetriq) 1 (1) 0 (0) 1 (1)

Previous vaginal estrogen use, no. (%) 32 (21) 11 (15) 21 (28) 0.05
Current vaginal estrogen use, no. (%) 29 (19) 10 (13) 19 (25) 0.06
Prior urogynecologic surgery, no. (%) 40 (27) 13 (17) 27 (36) 0.01

Symptoms of incontinence, no. (%)

Stress urinary incontinence 17 (11) 10 (13) 7 (9) 0.44
Urgency urinary incontinence 26 (17) 11 (15) 15 (20) 0.39
Mixed urinary incontinence 42 (28) 27 (36) 15 (20) 0.03

Urgency-frequency syndrome, no. (%) 18 (12) 9 (12) 9 (12) 0.99

Myofascial pain, no. (%) 55 (37) 20 (27) 35 (47) 0.01
Painful bladder syndrome, pelvic pain, and dyspareunia, no. (%) 4 (3) 0 (0) 4 (5) 0.06c

UTISA score, mean (SD)e

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Urination regularity 6.6 (4.3) 5.4 (4.3) 7.9 (3.9) <0.001a
Problems with urination 3.4 (3.6) 2.0 (2.5) 5.4 (3.7) <0.001a
Pain associated with UTI 3.2 (3.6) 2.1 (3.1) 4.3 (3.8) <0.001a
Blood in the urine 0.2 (0.8) 0.1 (0.4) 0.4 (1.1) 0.08a
a
Independent t test.
b
Wilcoxon rank sum test.
c
Fisher’s exact test.
d
Chi-square test used unless otherwise indicated. Boldface indicates P values that are significant at ⱕ0.05.
e
UTISA scores for urinary regularity, problems with urination, and pain associated with UTI range from 0 to 12. UTISA scores for blood in the urine range from 0 to 6.

samples (89% [67/75] versus 96% [72/75]; P ⫽ 0.12), similar prevalent in the no-UTI cohort, while the genus Escherichia (P ⬍
numbers of total unique species detected per cohort (75 versus 0.001) was detected more often in the UTI cohort (see Fig. S2).
66), and similar median numbers of species detected per urine Five species, namely, Gardnerella vaginalis (P ⫽ 0.04), Streptococ-
sample (3 [IQR ⫽ 1 to 5] versus 2 [IQR ⫽ 1 to 4]; P ⫽ 0.12) (see cus mitis/oralis/pneumoniae (P ⫽ 0.01), Streptococcus parasangui-
Table S1 in the supplemental material). nis (P ⫽ 0.02), Streptococcus salivarius (P ⫽ 0.05), and Streptococ-
However, the cohorts differed in organism diversity, genus- cus sanguinis (P ⫽ 0.01), were detected more often in the no-UTI
level composition, and species-level composition. The no-UTI co- cohort; in contrast, the species Escherichia coli (P ⬍ 0.001) was
hort had more diversity with greater species richness as depicted more prevalent in the UTI cohort (see Fig. S3).
by species accumulation curves (see Fig. S1 in the supplemental Uropathogen detection. We next modeled our evaluation to
material) and greater alpha diversity as measured by the Shannon uropathogen detection by the expanded-spectrum EQUC proto-
diversity index (no-UTI ⫽ 3.89 versus UTI ⫽ 3.49). The genera col with regard to the following parameters: detection compared
Streptococcus (P ⫽ 0.003) and Gardnerella (P ⫽ 0.04) were more to standard urine culture, detection with different urine volumes,

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 Improved Clinical Urine Culture Protocol

FIG 1 Average CFU per milliliter of uropathogens between the UTI and no-UTI cohorts. Depicted are the average CFU per milliliter with which the various
uropathogens were cultured for both cohorts: UTI (blue bars) and no-UTI (red bars). Average CFU of Klebsiella pneumoniae (P ⫽ 0.04) and Streptococcus
agalactiae (P ⫽ 0.02) are significantly higher in the UTI cohort (*). Several uropathogens had substantially lower average CFU-per-milliliter values in the no-UTI
cohort than in the UTI cohort: Aerococcus urinae (P ⫽ 0.12), Enterococcus faecalis (P ⫽ 0.09), Escherichia coli (P ⫽ 0.08), Staphylococcus aureus (P ⫽ 0.06), and
Streptococcus anginosus (P ⫽ 0.06). Independent t test (*, P ⬍ 0.05). Black bars depict common UTI thresholds (ⱖ105 CFU/ml and ⱖ103 CFU/ml).

and detection using different plating conditions. With these find- standard urine culture detected 24% (16/67) of the non-E. coli
ings, we then identified an optimal subset of expanded-spectrum uropathogens detected by the expanded-spectrum EQUC protocol.

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EQUC conditions for improved detection of uropathogens, which Therefore, standard urine culture’s capacity to detect E. coli dramat-
we call the streamlined EQUC protocol. ically skewed its overall uropathogen detection value.
Expanded-spectrum EQUC versus standard urine culture. Expanded-spectrum EQUC: urine volumes. Uropathogen
The expanded-spectrum EQUC protocol detected a total of 182 detection differed greatly based on expanded-spectrum EQUC
uropathogens in all the catheterized urine samples, 110 uropatho- urine volumes: for 100 ␮l, 96% detection (174/182); for 10 ␮l,
gens in the UTI cohort urine samples, and 72 uropathogens in in 65% detection (118/182); and for 1 ␮l, 52% detection (95/182)
the non-UTI cohort urine samples. Whereas the expanded-spec- versus standard urine culture (33% [60/182]). Some uropatho-
trum EQUC did not miss any uropathogen detected by standard gens were detected equally by all volumes (e.g., E. coli and Pseu-
urine culture, the standard urine culture protocol detected 33% domonas aeruginosa); others most often required 100 ␮l for
(60/182) of all detected uropathogens, 50% (55/110) of those de- detection (e.g., Aerococcus urinae, Alloscardovia omnicolens, En-
tected in the UTI cohort, and only 7% (5/72) of those detected in terococcus faecalis, and Streptococcus anginosus) (see Fig. S4 in the
the non-UTI cohort. supplemental material).
The expanded-spectrum EQUC protocol detected all uro- Expanded-spectrum EQUC: plating conditions. Table 3 dis-
pathogens at a higher average CFU per milliliter in the UTI cohort plays uropathogens and the number of times that each was cul-
than in the no-UTI cohort (Fig. 1). This protocol detected E. coli in tured under the various expanded-spectrum EQUC plating con-
a total of 50 samples obtained from both cohorts; of these, stan- ditions. After 48 h incubation, CDC anaerobic BAP incubated
dard urine culture detected E. coli in 88% (44/50). From the UTI anaerobically detected the most uropathogens (63% [115/182]),
cohort alone, expanded-spectrum EQUC detected E. coli in 43 followed by BAP in 5% CO2 (62% [112/182]), CDC anaerobic
samples; of these, standard urine culture detected E. coli in 91% BAP incubated microaerophilically (54% [98/182]), chocolate
(39/43). In contrast, standard urine culture detected a strikingly low agar in 5% CO2 (53% [96/182]), BAP incubated aerobically (52%
fraction (12% [16/132]) of the non-E. coli uropathogens detected in [94/182]), CNA agar in 5% CO2 (43% [79/182]), and MacConkey
the two cohorts by the expanded-spectrum EQUC protocol. This agar incubated aerobically (34% [62/182]). Although the CNA
percentage was only slightly better in the UTI cohort alone, where agar condition detected fewer uropathogens, it ideally detected

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Price et al.

TABLE 3 Optimal detection of specific uropathogens by the expanded-spectrum EQUC protocola

No. of times cultured under each condition:
Chocolate agar, CNA agar, CDC anaerobic, MacConkey agar, CDC anaerobic BAP,
Uropathogen (no. of isolates) BAP, CO2 CO2 CO2 BAP anaerobic BAP, O2 O2 microaerophilic
Actinobaculum schaalii (6) 2 0 5 3 0 0 4
Aerococcus sanguinicola (1) 1 0 0 0 1 0 0
Aerococcus urinae (15) 7 7 10 6 6 0 2
Alloscardovia omnicolens (8) 4 1 5 4 0 0 1
Candida albicans (2) 1 1 2 1 1 0 1
Candida parapsilosis (4) 1 1 2 2 0 0 1
Citrobacter freundii (1) 1 1 0 1 1 1 1
Citrobacter koseri (1) 1 1 0 1 1 1 0
Corynebacterium riegelii (4) 1 1 0 2 2 0 1
Corynebacterium urealyticum (2) 1 0 1 0 1 0 0
Enterobacter aerogenes (3) 2 2 0 1 1 2 2
Enterococcus faecalis (16) 5 4 11 5 7 0 6
Escherichia coli (50) 47 46 4 49 47 47 42
Klebsiella pneumoniae (10) 7 6 0 9 8 7 7
Morganella morganii (1) 0 0 1 0 0 0 0
Oligella urethralis (1) 0 0 0 0 1 0 0
Proteus mirabilis (4) 3 3 2 4 3 3 3
Pseudomonas aeruginosa (1) 1 1 0 1 1 1 1
Serratia marcescens (1) 0 1 0 1 0 0 0
Staphylococcus aureus (7) 4 4 4 3 4 0 4
Staphylococcus lugdunensis (2) 2 2 2 2 2 0 2
Streptococcus agalactiae (10) 8 6 6 6 5 0 5
Streptococcus anginosus (32) 13 8 24 14 2 0 15

Total (182) 112 96 79 115 94 62 98

a
Listed are the uropathogens and the number of times that each was cultured under each expanded-spectrum EQUC plating condition. The condition(s) that best detected each
uropathogen is shaded.

Gram-positive uropathogens when Gram-negative bacteria over- EQUC protocol (see Table S3 in the supplemental material). Im-
whelmed other agars. For example, of the 47 samples where a portantly, all of these missed uropathogens would have also been
Gram-negative uropathogen was present at ⱖ50,000 CFU/ml, the detected using the streamlined EQUC protocol. Ten of the 24
CNA agar condition detected 27 underlying Gram-positive uro- patients who did not improve had been clinically treated with
pathogens, all of which were missed by standard urine culture (see antibiotics based on the finding of a standard urine culture-de-

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Table S2A in the supplemental material). Conversely, of the seven tected uropathogen. However, in 3 (30%) of these 10 patients, the
samples where a Gram-positive uropathogen was present at ⱖ50,000 expanded-spectrum EQUC (as well as streamlined EQUC) de-
CFU/ml, the MacConkey condition detected two underlying Gram- tected an additional Gram-positive uropathogen (Aerococcus uri-
negative uropathogens (see Table S2B). nae, Corynebacterium riegelii, or Streptococcus anginosus).
Streamlined EQUC protocol. One hundred microliters of
urine plated on a combination of BAP and CNA agars in 5% CO2 DISCUSSION
and MacConkey agar under aerobic conditions would have de- Accurate diagnosis for women with UTI symptoms is critical, both
tected 84% (152/182) of all uropathogens detected by the expand- to target appropriate therapy and to limit inappropriate antibiotic
ed-spectrum EQUC protocol. This is vastly superior to the 33% use. Our study demonstrates deficiencies in the standard urine
(60/182) uropathogen detection by standard urine culture. In the culture protocol that limit potentially important information that
UTI cohort alone, the streamlined EQUC protocol (Table 1) should be provided to clinicians. Our findings suggest that im-
would have detected 91% (100/110) of uropathogens, compared proved detection of clinically relevant urinary microbes can be
to only 52% (57/110) by standard urine culture. achieved in all diagnostic clinical laboratories using the following
Symptom resolution. Seventy-nine percent (59/75) of the par- conditions: a 100-␮l urine sample obtained by transurethral catheter
ticipants in the UTI cohort completed the follow-up UTISA ques- plated onto BAP, CNA, and MacConkey agars, with incubation of all
tionnaire. Following clinically selected treatment based on stan- agars in 5% CO2 for 48 h. While incubation of MacConkey agar in 5%
dard urine culture (or modified standard urine culture), 59% (35/ CO2 may not improve Gram-negative bacillus recovery, we recom-
59) of participants reported symptom improvement, while 41% mend that all agars be incubated in 5% CO2 for the convenience of
(24/59) reported no improvement (same or worse) (Table 4). Half using a single incubator. All detected uropathogens will grow under
(12/24) of the 24 participants who did not improve had at least one the conditions described in the streamlined EQUC protocol.
uropathogen detected only by the expanded-spectrum EQUC Our findings support the use of the streamlined EQUC proto-
protocol, and 13 (54%) had microorganisms of unknown patho- col to more fully describe uropathogens. We also recommend that
genicity, which were detected only by the expanded-spectrum 1 ␮l of the catheterized urine be plated onto BAP and MacConkey

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TABLE 4 Detection of uropathogens in UTI cohort without symptom improvementa

Uropathogen(s) detected by protocol(s):
Postenrollment questionnaire Standard urine culture and
response (sample identifier) Antibiotic prescribed expanded-spectrum EQUC Expanded-spectrum EQUC onlyd
b
Same (145) SMZ-TMP Escherichia coli Streptococcus anginosus
Same (048) Nitrofurantoin Escherichia coli Streptococcus anginosus
Same (134) SMZ-TMP Escherichia coli Aerococcus urinae, Corynebacterium riegelii
Same (033) Ciprofloxacin Klebsiella pneumoniae
Same (060) Nitrofurantoin Escherichia coli
Same (109) Nitrofurantoin Escherichia coli
Same (122) Ciprofloxacin Escherichia coli
Same (135) Nitrofurantoin Escherichia coli
Same (136) Nitrofurantoin Escherichia coli
Worse (082) Nitrofurantoin Escherichia coli
Same (140) Staphylococcus lugdunensis
Same (116) Escherichia coli
Worse (128) Escherichia coli
Same (126) Lactobacillus speciesc Staphylococcus lugdunensis, Streptococcus anginosus
Same (121) Proteus mirabilis
Same (029) Aerococcus urinae, Klebsiella pneumoniae
Same (139) Alloscardovia omnicolens, Oligella urethralis,
Morganella morganii
Same (052) Streptococcus anginosus
Same (067) Streptococcus anginosus
Worse (025) Candida albicans
Worse (112) Streptococcus agalactiae, Streptococcus anginosus
Worse (142) Escherichia coli
Same (084)
Same (108)
a
Uropathogens detected and missed by the standard urine culture in urine samples obtained by catheter from the UTI cohort patients who reported feeling the same or worse for
the postenrollment questionnaire. Antibiotics were prescribed based on the reporting of the standard urine culture results.
b
SMZ-TMP, sulfamethoxazole-trimethoprim.
c
Lactobacillus species is not considered a uropathogen, but it was detected at ⬎100,000 CFU/ml by standard urine culture.
d
All of the uropathogens detected by the expanded-spectrum EQUC protocol would have been detected using the streamlined EQUC protocol.

agars and incubated in 5% CO2 for 24 h with an option to incubate CFU per milliliter, we recommend that these testing conditions
for 48 h (modified standard urine culture). The streamlined (i.e., streamlined EQUC) be used for patients with recurrent UTIs or
EQUC protocol provides the most thorough detection of uro- patients with clear UTI-like symptoms despite a negative standard

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pathogens, while the modified standard urine culture ensures ac- urine culture result. Nonetheless, it is clear that treatment based on
curate colony count assessment and is beneficial for species detec- standard urine culture results limits diagnostic information that may
tion of underlying uropathogens when bacterial colony counts of be useful for symptom resolution. This study did not assess symptom
a predominant uropathogen exceed 105 CFU/ml. The need for relief in patients whose uropathogens were detected only with the
modified standard urine culture inclusion is apparent from the streamlined EQUC protocol. Such studies are clearly needed.
observation that the expanded-spectrum EQUC (and the stream- Compared to the expanded-spectrum EQUC, the standard
lined EQUC) protocols were not 100% sensitive. In the expanded- urine culture missed 67% (122/182) of all detected uropathogens
spectrum EQUC protocol, the use of 100 ␮l urine detected the and 88% (116/132) of non-E. coli uropathogens. Detection of uro-
most microbes and the most uropathogens. However, a small pathogens by the standard urine culture was slightly better for the
number of uropathogens were detected only with the use of a UTI cohort alone (50% total missed [55/110]; 76% non-E. coli
smaller urine volume (10 ␮l). This apparent paradox likely results missed [51/67]). This improvement may result from the higher
from microbial overcrowding in samples containing high num- average uropathogen CFU per milliliter in the UTI cohort (Fig. 1),
bers of CFU; in these circumstances, 100 ␮l was not ideal for making detection by standard urine culture more likely. The data
distinguishing morphologies. While addition of selective media in Table 1 also reveal that the use of one threshold for UTI diag-
(i.e., CNA and MacConkey agars) helped detect underlying uro- nosis is likely incorrect. Use of the ⱖ105-CFU/ml threshold would
pathogens, some samples contained both Gram-positive and result in untreated uropathogens in the UTI cohort (Fig. 1). Low-
Gram-negative bacteria at high CFU numbers, likely making the ering the threshold to ⱖ103 CFU/ml, however, creates other con-
100-␮l expanded-spectrum EQUC volume less efficient. cerns. While use of the ⱖ103-CFU/ml threshold would leave fewer
Streamlined EQUC would provide more information to clini- uropathogens in the UTI cohort untreated, it would detect some
cians who are considering the clinical need for uropathogen(s) uropathogens in the no-UTI cohort. Since individuals in the no-
treatment; many of these are currently missed by the standard UTI cohort presumably do not have an infection (i.e., no/low
urine culture protocol. Until better information is available con- severity of urinary symptoms), it is unlikely that they would ben-
cerning the relationship between clinically important UTI and efit from antibiotic use. This creates a problem in diagnosis and

May 2016 Volume 54 Number 5 Journal of Clinical Microbiology jcm.asm.org 1221

Price et al.

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conditions (i.e., streamlined EQUC) be considered both as a sup- Rohn JL, Malone-Lee J. 2013. Spectrum of bacterial colonization associ-
plemental test when individuals with UTI-like symptoms have ated with urothelial cells from patients with chronic lower urinary tract
“no growth” via standard urine culture and for use with individ- symptoms. J Clin Microbiol 51:2054 –2062. http://dx.doi.org/10.1128
uals with persistent UTI-like symptoms (i.e., recurrent UTI). /JCM.03314-12.
10. Hilt EE, McKinley K, Pearce MM, Rosenfeld AB, Zilliox MJ, Mueller
ACKNOWLEDGMENTS ER, Brubaker L, Gai X, Wolfe AJ, Schreckenberger PC. 2014. Urine is
not sterile: use of enhanced urine culture techniques to detect resident
We kindly thank Mary Tulke, Bozena Zemaitaitis, Arianna Griffin, and bacterial flora in the adult female bladder. J Clin Microbiol 52:871– 876.
Kathleen McKinley for their assistance with participant recruitment, sam- http://dx.doi.org/10.1128/JCM.02876-13.
ple collection, and clinical microbiology contributions. 11. Brubaker L, Nager C, Richter H, Visco A, Nygaard I, Barber M, Schaffer
This study was supported by NIH grant R21 DK097435 (awarded to J, Meikle S, Wallace D, Shibata N, Wolfe A. 2014. Urinary bacteria in
A.J.W. and L.B.). Our funding sources had no role in design and conduct adult women with urgency urinary incontinence. Int Urogynecol J 25:
of the study; collection, management, analysis, and interpretation of the 1179 –1184. http://dx.doi.org/10.1007/s00192-013-2325-2.
data; and preparation, review, or approval of the manuscript. 12. Pearce MM, Hilt EE, Rosenfeld AB, Zilliox MJ, Thomas-White K, Fok
A.J.W. received an investigator-initiated grant from Astellas Scientific C, Kliethermes S, Schreckenberger PC, Brubaker L, Gai X, Wolfe AJ.
2014. The female urinary microbiome: a comparison of women with and
and Medical Affairs (ASMA) for a study outside the present work. L.B.
without urgency urinary incontinence. mBio 5(4):e01283-14. http://dx
received grants from NICHD and NIDDK during conduct of a study .doi.org/10.1128/mBio.01283-14.
outside the present work. E.R.M. reports grants from ASMA for the pres- 13. Nienhouse V, Gao X, Dong Q, Nelson DE, Toh E, McKinley K,
ent work, grants and personal fees from ASMA, personal fees from Peri- Schreckenberger P, Shibata N, Fok CS, Mueller ER, Brubaker L, Wolfe
Coach, and personal fees from Allergan outside the present work. The AJ, Radek KA. 2014. Interplay between bladder microbiota and urinary
remaining authors have no conflicts to report. antimicrobial peptides: mechanisms for human urinary tract infection

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risk and symptom severity. PLoS One 9:e114185. http://dx.doi.org/10
FUNDING INFORMATION .1371/journal.pone.0114185.
This work, including the efforts of Travis K Price, Tanaka Dune, Evann E 14. Pearce MM, Zilliox MJ, Rosenfeld AB, Thomas-White KJ, Richter HE,
Hilt, Krystal Thomas-White, Stephanie Kliethermes, Cynthia Brincat, Nager CW, Visco AG, Nygaard IE, Barber MD, Schaffer J, Moalli P,
Linda Brubaker, Alan J Wolfe, Elizabeth Mueller, and Paul C Schrecken- Sung VW, Smith AL, Rogers R, Nolen TL, Wallace D, Meikle SF, Gai X,
berger, was funded by HHS | National Institutes of Health (NIH) Wolfe AJ, Brubaker L. 2015. The female urinary microbiome in urgency
(R21DK097435-01A1). This work, including the efforts of Travis K Price, urinary incontinence. Am J Obstet Gynecol 213:347.e1-11. http://dx.doi
.org/10.1016/j.ajog.2015.07.009.
Tanaka Dune, Evann E Hilt, Krystal Thomas-White, Stephanie
15. Thomas-White KJ, Hilt EE, Fok C, Pearce MM, Mueller ER,
Kliethermes, Cynthia Brincat, Linda Brubaker, Alan J Wolfe, Elizabeth Kliethermes S, Jacobs K, Zilliox MJ, Brincat C, Price TK, Kuffel G,
Mueller, and Paul C Schreckenberger, was funded by Falk Foundation Schreckenberger P, Gai X, Brubaker L, Wolfe AJ. 30 September 2015.
(LU#202567). Incontinence medication response relates to the female urinary microbi-
ota. Int Urogynecol J http://dx.doi.org/10.1007/s00192-015-2847-x.
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