Aerococcus urinae in Urinary Tract Infections

Abstract

Aerococcus urinae is a rarely reported pathogen, possibly due to difficulties in the identification of the organism. A. urinae is a gram-positive coccus that grows in pairs and clusters, produces alpha-hemolysis on blood agar, and is negative for catalase and pyrrolidonyl aminopeptidase. Some of these characteristics and its being absent from the databases of most commercial identification systems could allow A. urinae to be misidentified as a streptococcus, enterococcus, or staphylococcus. We report two cases of urinary tract infection (UTI) caused by A. urinae and characterize these isolates by morphology, biochemical testing, whole-cell fatty acid analysis, 16S rRNA gene sequencing, and antibiotic susceptibilities. Most patients infected with A. urinae are elderly males with predisposing conditions who present initially with UTI. Because A. urinae is resistant to sulfonamides, treatment could be inappropriate, with infections resulting in serious complications, including death. It is important for the clinician and the microbiologist to consider A. urinae a potential pathogen and proceed with thorough microbiological identification.

CASE REPORTS

Case 1.

An 80-year-old male with preexisting coronary artery disease, left-sided hemiparesis (the residual effect of a stroke), and multiinfarct dementia was admitted to the hospital for altered mental status. The patient developed slowly worsening urinary symptoms, including nocturia and frequency, over the 3 weeks prior to admission.

On admission the patient was afebrile with normal vital signs. The physical examination was unremarkable except that a marked disorientation to place and time and a left-sided hemiparesis (the residual effect of a previous stroke) were observed. Urinalysis showed white blood cells (WBCs) too numerous to count, 8 red blood cells/high power field (HPF), many bacteria, a specific gravity of 1.017, and a pH of 5.0, with negative results for protein, ketones, and bilirubin. The patient had a serum WBC count of 6.6 (64% neutrophils, 19% lymphocytes, 9% monocytes, 7% eosinophils, and 1% basophils) and a hemoglobin and a hematocrit of 10.6 g/dl and 31.5%, respectively. Serum chemistries including electrolytes, glucose, liver, renal, and thyroid function tests were all reported as normal. Chest X-ray was normal except for an elevated right hemidiaphragm. Computed tomogram of the head revealed a previous infarct in the distribution of the right middle cerebral artery as well as hypodensity consistent with diffuse small vessel disease. The patient was treated empirically with intravenous ciprofloxacin for presumed urinary tract infection (UTI). Urine cultures grew >10⁵ CFU of Aerococcus urinae, the only isolate, per ml. His urinary symptoms resolved and his mental status improved by hospital day 3. The patient was discharged, having had oral ciprofloxacin prescribed. Results of repeated urinalysis after discharge were normal.

Case 2.

A 58-year-old white male with adequately treated hypothyroidism presented at the Veterans Affairs Medical Center outpatient clinic with several days of dysuria, increased urinary frequency, and nocturia. He was afebrile at the time, with normal vital signs and a large, tender prostate on physical exam. Urinalysis revealed 4 to 6 WBCs/HPF, no bacteria, trace amounts of leukocyte esterase, and negative results for nitrites, protein, ketones, bilirubin, and blood. He was diagnosed clinically with prostatitis and treated with tetracycline due to a history of ciprofloxacin allergy. The patient developed a rash and discontinued the antibiotic after 2 days of treatment. He returned to the clinic 2 weeks later, still complaining of urinary symptoms, and was febrile at 100.1°F. A second urinalysis demonstrated 20 to 30 WBCs/HPF, 1 to 3 red blood cells/HPF, a moderate level of leukocyte esterase, and negative urine chemistries. The urine culture on the second urine sample later grew >10⁵ CFU of A. urinae, the only isolate, per ml. The patient was lost to follow-up.

Microbiology. We compared two strains of A. urinae from the present cases and two blood isolates of Aerococcus viridans from our stock for growth on Trypticase soy agar with 5% sheep blood, with incubation at 35°C in three different atmospheres. At 24 h, the diameters of colonies of A. urinae grown in ambient air, air plus 8% CO₂, or anaerobic conditions were 0.1, 0.5, or 0.5 mm, respectively. At 48 h all colonies were larger, but at 24 h, A. viridans grew to 0.7- and 1-mm diameters in air and in air plus 8% CO₂, respectively, and showed no growth under anaerobic conditions. A. viridans growth in anaerobic conditions was barely visible at 48 h. The colonies of both species produce alpha-hemolytic reactions on blood agar.

The two A. urinae strains are catalase negative and pyrrolidonyl aminopeptidase (PYR) test negative. Biochemical identification was performed by the API 20 Strep system (bioMeriéux, Marcy l'Etoile, France), the Rapid CB Plus system (Remel, Lenexa, Kans.), and an automated identification system (Vitek 120; bioMeriéux, Hazelwood, Mo.) according to the manufacturers' instructions. The results shown in Table 1 are consistent with the results reported by Christensen et al. (4). The API biotype number (3442300) is appropriate for A. urinae but is not in the code book. The Vitek gave a unique code number (52025410000 or 50025410200) that is also not in the code book. The whole-cell fatty acid (CFA) analysis was performed on isolates grown on blood agar plates for 48 h at 35°C in 8% CO₂ (Hewlett-Packard HP 5890 II microbial identification system [MIDI, Inc., Newark, Del.]), which gave no match or called the isolate Streptococcus equinus at a 0.291 similarity index. The major CFAs were hexadecanoic acid (16:0) (24%), octadecenoic acid (18:1 omega 9 cis) (21%), octadecanoic acid (18:0) (17.5%), hexadecenoic acid (16:1 omega 9 cis) (12%). However, A. viridans was correctly named by all these biochemical identification systems. The nucleotide sequences of 16S rRNA were determined (MicroSeq 16S rRNA gene kit; Perkin-Elmer Applied Biosystems, Foster City, Calif.). The two isolates were identified as A. urinae by 16S rRNA gene sequencing, with a 0.2 to 0.4% difference from the type strain. A. urinae demonstrated a 7 to 8% difference from A. viridans, which indicates a genetic diversity similar to that found among other well-described genera and does not support Facklam and Elliott's assertion that these organisms, which they call Aerococcus-like organisms, belong in a separate genus (6).

TABLE 1.

Biochemical results of the present clinical isolates compared with the literature values for A. urinae and A. viridans (adapted from references 1, 2, 4, 5, 6, and 7)

Test	Result fora:
	Present clinical isolates	A. urinae	A. viridans
Hydrolysis of:
Hippurate	+	+	NA
Arginine	−	−	NA
Acid production from:
Glucose	+	+	NA
Sucrose	+	+	+
Maltose	−	−	+
Sorbitol	+	+	−
Lactose	−	−	+
Trehalose	−	−	+
Mannitol	+	+	V
Inulin	−	−	NA
Starch	−	NA	NA
Glycogen	−	−	NA
Ribose	+b	+b	V
l-Arabinose	−	−	NA
Enzyme production
β-Glucosidase	−	−	NA
β-Glucuronidase	+	+	−
β-Galactosidase	−	−	+
α-Galactosidase	−	−	NA
Alkaline phosphatase	−	−	NA
Leucine arylamidase	+	+	−
Pyrrolidonyl aminopeptidase	−	−	+
Urease	−	NA	NA
Nitrite reductase	−	−	NA

^aNA, not available; V, variable; +, positive; −, negative. 

^bAcid production was slow. 

Antibiotic susceptibility was tested by the disk diffusion method using Mueller-Hinton blood agar, and the MICs were determined by E-test. National Committee for Clinical Laboratory Standards guidelines for Staphylococcus spp. were used for susceptibility testing (11). Our strains of A. urinae are resistant to trimethoprim-sulfamethoxazole and gentamicin but susceptible to penicillin (Table 2).

TABLE 2.

Antibiotic susceptibilities of present isolates, together with those of A. urinae and A. viridans (adapted from references 3, 4, and 10)^a

Antibiotic	Result for:
	Present isolates		A. urinae	A. viridans
	Disk diffusion testb	E-testc
Penicillin G	S (40, 40)	S (0.016, 0.064)	S	R
Trimethoprim-sulfamethoxazole	R (0, 0)	R (0, 0)	R	S
Vancomycin	S (26, 25)	S (0.75, 0.75)	S	S
Ciprofloxacin	S (30, 30)	S (0.25, 0.25)	NA	NA
Gentamicin	R (0, 0)	ND	R	S
Tetracycline	S (32, 32)	S (0.25, 0.5)	S	S

^aS, susceptible; R, resistant; NA, not available; ND, not done. 

^bStandard disks recommended by National Committee for Clinical Laboratory Standards guidelines were used. Values in parentheses are inhibitory zone sizes (in millimeters) for the isolates from cases 1 and 2 (in that order). 

^cValues in parentheses are MICs (in micrograms per milliliter) of the respective drugs for the isolates from cases 1 and 2 (in that order). 

Discussion.

There are currently three described species of the genus Aerococcus: A. viridans, Aerococcus christensenii, and A. urinae. A. viridans is more commonly isolated from blood and has been associated with granulocytic bacteremia (9) and endocarditis (12). The recently characterized species A. christensenii (5) has not been reported as a human pathogen. A. urinae, previously known as Aerococcus-like organism, is an uncommon pathogen. Previous reports from European countries indicate that it is associated with UTI (4), bacteremia (3), sepsis (2), and potentially fatal endocarditis (2, 7, 10, 13). The main features of our two cases and previously reported cases are summarized in Table 3. One report described a total of 63 cases of A. urinae infection; however, little clinical information was available (4). The single case reports are summarized. Similar to our patients, the other patients are elderly males with a median age of 73 and have underlying predisposing conditions, such as prostatic diseases, diabetes mellitus, and alcoholism. All patients had confirmed UTIs or symptoms typical of UTI. When these patients were treated with antibiotics effective against A. urinae, as in cases 1, 6, and 8, the infection resolved and the patients recovered fully. On the other hand, as in cases 3, 4, 5, and 7, when A. urinae was not recognized or correct antibiotic treatment was delayed, simple UTI progressed to systemic infection, including endocarditis and sepsis. Thus, it is important to consider A. urinae a potential pathogen in UTIs especially for patients with predisposing factors who are unresponsive to sulfonamides. If not recognized by the clinician and microbiologist, A. urinae infection can cause serious complications.

TABLE 3.

Summary of clinical features of our two cases and previously reported cases^a

Case no. (reference)	Patient age (yr)	Sex	Underlying disease(s)	Main symptoms and signs	Urine culture resultc	Blood culture result	Initial treatment	Result of treatment
1 (PR)	80	M	Coronary artery disease, cerebrovascular disease	Nocturia, urinary frequency, altered mental status	>105	No sign of sepsis	CIP	Recovered
2 (PR)	58	M	Hypothyroidism	Dysuria and large, tender prostate	>105	Not septic	TET	Did not return for follow-up
3 (10)	78	M	Meningioma	Fever, heart murmur	>105	Positive	CXM	Endocarditis, death
4 (7)	43	M	Alcoholism, hepatic cirrhosis, chronic pancreatitis	Antecedent UTI, heart murmur	Normal	Positive	CIP	Septic shock, endocarditis, death
5 (2)	81	M	Prostate cancer	Fever, hematuria, dysuria	>105	2 of 2 positive	TMP	Sepsis, endocarditis, death
6 (2)	63	M	Coronary artery disease, alcoholism	Sepsis, fever, urinary incontinence, pyuria	>105	Positive	AMP, GEN	Recovered
7 (13)	81	M	Not identified	Fever, symptoms of a UTI, heart murmur	Negative	2 of 2 positive at 3 days	SULFA	Endocarditis, death
8 (8)	80	M	Prostate cancer	Dysuria, testicular pain, septicemia	Negative	Positive	AMP, GEN	Recovered
9 (4)b	74	M, F	Diabetes mellitus, prostatic disease, indwelling catheter	Fever, dysuria, pyuria, incontinence	>105	Positive for 17 patients	Unknown	Unknown

^aPR, present report; M, male; F, female; CIP, ciprofloxacin; TET, tetracycline; TMP, trimethoprim; AMP, ampicillin; GEN, gentamicin; CXM, cefuroxime; SULFA, sulfamethizole. 

^bCase 9 was a study of 63 patients (34 female patients and 29 male patients). The age listed is the median age (range, 3 to 97 years). 

^cValues given are in CFU per milliliter. The result for case 4 was obtained with a dipstick. 

The laboratory diagnosis of A. urinae can be difficult. The presumptive identification of aerobic gram-positive alpha-hemolytic cocci and the decision on whether to more fully identify the isolates are frequently based on Gram stain, colony appearance, and catalase reaction. Because A. urinae is catalase negative, it could be mistaken for alpha-hemolytic streptococci or enterococci that are more common urine isolates. At 24 h the colony morphology resembles that of an alpha-hemolytic streptococcus or lactobacillus; at 48 h it is similar to that of an enterococcus. The Gram stain should be differential, as A. urinae forms pairs, tetrads, and clusters. However, since A. urinae shows smaller cocci and fewer tetrads than A. viridans does, it could be confused with pediococci or densely packed streptococci or enterococci.

We characterized our isolates by macroscopic and microscopic appearance, biochemical profiles, CFA analysis, 16S rRNA gene sequencing analysis, and antibiotic susceptibilities.

However, according to our study and previous reports (3, 4, 5, 6), the most important routine tests are detection of leucine arylamidase, β-glucuronidase, PYR, hydrolysis of hippurate (Table 1), and antibiotic susceptibility patterns (Table 2). Rapid PYR testing is useful for distinguishing between A. viridans or enterococci (both PYR positive) and A. urinae (PYR negative). A Gram stain should be carefully examined for the characteristic arrangement in clusters and tetrads to rule out lactobacillus and other streptococcus. Pediococci are PYR negative and have a Gram stain morphology similar to that of A. urinae; however, they differ in their resistance to vancomycin and in their positive bile esculin test result. Other newly described genera that are rarely encountered in the clinical laboratory can be differentiated by the characteristics listed in Table 1 (6).