JOURNAL OF CLINICAL MICROBIOLOGY, May 1991, p. 1049-1053
Vol. 29, No. 5
0095-1137/91/051049-05$02.00/0 Copyright X 1991, American Society for Microbiology
Aerococcus-Like Organism, a Newly Recognized Potential Urinary Tract Pathogen JENS J. CHRISTENSEN,1,2,3* HENRIK VIBITS,1 JAN URSING,4 AND BENT KORNER' Department of Clinical Microbiology, Bispebjerg Hospital,' and Department of Diagnostic Bacteriology and Antibiotics, Statens Seruminstitut,3 Copenhagen, and Department of Clinical Microbiology, Central Hospital, Hillerod,2 Denmark, and Department of Medical Microbiology, University of Lund, Malmo General Hospital, Malmo, Sweden4 Received 11 October 1990/Accepted 11 February 1991
During the last 10 to 15 years, Aerococcus-like organisms have been isolated from urinary tract specimens from hospitalized patients at Bispebjerg Hospital, Copenhagen, Denmark, with signs of urinary tract infection, and a single strain has been deposited at the National Collection of Type Cultures (NCTC 12142). An additional 63 isolates from the urine specimens of as many patients with suspected urinary tract infection have been identified from seven departments of clinical microbiology in Denmark. Clinical information indicates that this organism may be involved in urinary tract infection. These strains and ten previously collected strains, two of which were from blood cultures, were characterized phenotypically. By comparing the results with data on other gram-positive, catalase-negative cocci or coccobacillary organisms, the identified organisms seem to represent a separate taxon.
38°C, dysuric complaints, and pyuria), were obtained by reviewing the records of the patients. Strains. The recently collected strains of ALO (n = 63) and 10 other strains, of which eight and two strains originated from urine specimens and blood cultures, respectively, were examined. Standard cultural conditions. Strains were maintained on 5% horse blood agar and incubated at 35°C in a normal atmosphere. The standard inoculum used for liquid cultures was 1 to 2 drops of a 24-h Todd-Hewitt broth culture. Growth. Growth was tested on 0.04% tellurite agar, on 40% bile agar, on bilayered sheep blood agar (8), in 6.5% NaCl broth, and at different temperatures in trypsin broth (12). Plates and tubes were observed for 2 days. Biochemical tests. Biochemical tests were performed as described previously (2), except that they were supplemented with a test for leucine-aminopeptidase activity (Rosco Diagnostic Tablet [1]). Susceptibility testing. Antibiotic susceptibility was tested by the direct agar diffusion method by using Rosco NeoSensitabs on Danish blood agar. Strains are reported as susceptible when zone diameters were .28 mm for penicillin (5 jjtg), sulfonamides and netilmicin; >-10 and 218 mm for 5 and 70 ,ug of vancomycin, respectively; and -15 and .22 mm for 0.4 and 40 IU of bacitracin, respectively. Zone diameters less than the above-mentioned figures are reported to indicate resistance. The inhibition zone limits correlate approximately to MIC breakpoints for penicillin at 0.25 ,ug/ml, for sulfonamides at 64 ,ug/ml, for netilmicin at 2 pLg/ml, and for vancomycin (70 ,g) at 4 ,ug/ml. The bacitracin tablets and the 5 ,ug of vancomycin used are only for diagnostic purposes. Determination of G+C moles percent of DNA. The methods for isolation of DNA and estimation of G+C moles percent by the thermal denaturation method have been described earlier (13). The values given are the means of four determinations.
Infections caused by Aerococcus viridans in humans are seldom reported; cases of urinary tract infections (3, 10), bacteremia (6), endocarditis (3, 5, 10, 11, 15), and meningitis (9) have, however, been published. During the last 10 to 15 years, we have observed at Bispebjerg Hospital, Copenhagen, Denmark, Aerococcus-like organisms (ALO) in urinary tract specimens from hospitalized patients with signs of urinary tract infection. The ALO strains have been characterized phenotypically in a previous report (2), and a single urine isolate has been deposited at the National Collection of Type Cultures, Colindale, United Kingdom (NCTC 12142). ALO produce greening on 5% horse blood agar like the viridans group streptococci, form in clusters like staphylococci as confirmed by microscopy, and are catalase negative. However, the isolates cannot be easily assigned to any genus or species. We decided to investigate whether ALO also could be isolated from urinary tract specimens in other laboratories in Denmark, to further characterize ALO isolates, and to comment on the taxonomic relationship of ALO isolates. MATERIALS AND METHODS
Collection of strains and clinical features of patients. Seven departments of clinical microbiology in Denmark were asked to collect five to ten consecutive strains from urinary tract specimens with the following characteristics- gram-positive cocci, predominantly in clusters; colonies resembling alphahemolytic streptococci on 5% horse blood agar; catalase negative; susceptible to penicillin; and resistant to both sulfonamides and aminoglycosides. Patients. Predisposing conditions, together with symptoms indicative of urinary tract infection (rectal temperature* Corresponding author. 1049
1050
J. CLIN. MICROBIOL.
CHRISTENSEN ET AL.
TABLE 1. Parameters indicative of urinary tract infection in 63 patients whose urine yielded growth of ALO No. of patients Parameter(s)
Dysuria + fever + pyuria Dysuria + fever Dysuria + pyuria Fever + pyuria Dysuria Fever Pyuria Incontinence No symptoms No information
ALO
Predisposing condition
ALO in admixture
singly
Without catheter'
With catheter
Without catheter
1 1 13
1 1 1
1 1 8 1 1
2 1
6
1
1 1 10
With catheter
2b 1 3
1 4
Indwelling urinary catheter. In two patients with congenital myelomeningocele and recurrent nonsymptomatic urinary tract infection, the test for pyuria was not performed. a
b
RESULTS During the study period, 64 patients suspected of urinary tract infection due to ALO were identified (Table 1). The organism isolated from one of the patients was phenotypically different from ALO isolates. A total of i105 ALO per ml were cultured from the urine specimens of all the remaining 63 patients, except for four patients having 103 to 104 ALO per ml of urine. ALO were grown in pure culture from the urine of 36 patients, while ALO were cocultured with other bacteria from the urine of 27 patients (Table 2). From five patients with a pure culture of ALO a repeat specimen before treatment also showed i105 ALO per ml. Patients. Of the 63 patients, 34 were female and 29 were male. The median age was 74 years (range, 3 to 97 years). Forty-one patients suffered from conditions predisposing them to urinary tract infection (Table 3). Elevated serum creatinine was observed in 12 patients. Twelve patients had received sulfonamides within the last week before specimen sampling, and one patient each had received trimethoprim, nitrofurantoin, or penicillin. Description of strains. All 73 isolates were gram-positive cocci, predominantly in clusters and with a tendency to form pairs and groups of four. All strains grew well on 5% horse blood agar, producing green discoloration of the agar resemTABLE 2. Bacteria cocultured with ALO from urine of 27 patients No. of patients with: _104 _105 bacteria/ml bacteria/ml
Organism(s)
E. coli Klebsiella pneumoniae Morganella morganii Citrobacter freundii Enterobacter species Enterococcus faecalis Hemolytic streptococci (group C) Staphylococcus aureus
Corynebacterium species .3 organisms The two organisms isolated cation was not performed. a
10 2 1 1 1 1
2a
1 1 1
1 5 were
TABLE 3. Conditions predisposing to urinary tract infection found among 35 patients whose urine yielded growth of ALO'
E. coli-like, but exact species identifi-
No. of
Systemic Diabetes mellitus ............................ ................ 6 3 Parkinson's disease ............................................ Cancer (bladder, pancreas, lung, uterine cervix) ........ 5.... Multiple sclerosis ............................................ 2 2 Polyneuropathia ............................................ Local Hyperplasia of the prostate ........................................ Indwelling urinary catheter ........................................ Paresis of the bladder ............................................ Bladder papilloma ............................................ ....................... Bladder stones ..................... ....................... Ureteral reflux .....................
4 7 6 2 2 2
a SiX patients suffered from two conditions predisposing them to urinary tract infection at the same time.
bling that of viridans group streptococci. The colony diameter increased from 0.5 mm after 24 h to 1 mm after 48 h, and the colonies of all strains were convex, with a well-defined border, and were shiny and transparent. No growth was seen on tellurite agar, whereas on bile agar all strains grew scantily (after 24 h) to moderately (after 48 h). All strains grew in 6.5% NaCl broth after 2 days of incubation at 37°C. No growth occurred at 10°C, 45°C, or after the heating of inoculated tubes to 60°C for 30 min; however, when a 10-fold greater inoculum was used, strains grew at 45°C, making this test of limited value for the identification of ALO. CAMP reaction was negative for all strains. Growth characteristics, results of biochemical tests, and antibiotic susceptibilities of strains are shown in Table 4. For sulfonamides, no zone was seen at all, whereas for netilmicin a zone diameter of 20 to 22 mm was observed for most strains. The guanine-plus-cytosine content of DNA was estimated for four strains and was 44.4 + 0.5 mol% (mean ± standard deviation). DISCUSSION
This investigation confirms that strains phenotypically identical to the ALO isolates previously described (2) can be found in urinary tract specimens examined in most laboratories in Denmark. Few phenotypic characteristics (see Materials and Methods) are needed to identify these strains. Their detection is, however, dependent on whether microscopy is performed, as they otherwise may easily be mistaken for viridans group streptococci. As also seen previously, data from patient records indicate that ALO possess a certain pathogenic potential in urinary tract infection; most patients had classical signs of urinary tract infection, recurrences occurred, and the distribution of ALO in urine specimens was equal to what is seen for established pathogens such as Escherichia coli and Klebsiella spp. ALO also has the ability to cause invasive infections, i.e., urosepticemia and fatal endocarditis (unpublished data); in both cases, antibodies against ALO isolates were demonstrated in crossed immunoelectrophoretic assay. Identification of catalase-negative, gram-positive coccal and coccobacillary organisms has received renewed interest
URINARY TRACT INFECTION AND ALO
VOL. 29, 1991
1051
TABLE 4. Growth characteristics, fermentation reactions, direct enzyme test, and antibiotic susceptibility for 73 strains of ALO'
of positive Dayreaction
Reactionb
Characteristic
0 0
a
Hemolysis Production of catalase Growth on: 0.04% tellurite 40% bile Growth in 6.5% NaCl Growth at: looc
1-2 2
+
Acid production from: Arabinose Glucose (acid/gas) Maltose Lactose Sucrose Trehalose Melibiose Raffinose Salicin Esculin Melizitose Inulin Glycogen Mannitol Sorbitol Glycerol Production of:
b I
1-2
+l-
0
0 0 4 (+ day 1-4)
0
1-2
1-5
+
1-6
1 (Negative)
0 0 0 0 0 0 0 0 0
3 (Negative)
0
0 0 0 0
+(24 h), +(4 h)
Pyrrolidonyl aminopeptidase Leucine aminopeptidase Hydrolysis of: Hippurate Arginine Acetoin production Reaction with group D antiserum Susceptibility to: Penicillin G Sulfonamides Netilmicin Vancomycin (5 jig) Vancomycin (70 ,ug) Bacitracin (0.04 IU) Bacitracin (40 IU) a
0 1 (No growth)
0 0 0 0
450C Surviving 600C after 30 min CAMP reaction
P-Galactosidase P-Glucuronidase
deviating No. of strains
0 0 0 0
+
0 0 0 0 0 0 0
S
R R S S
R S
From eight departments of clinical microbiology in Denmark. S, Susceptible; R, resistant. Scanty to moderate growth.
during the last several years, partly stimulated by the emerof vancomycin-resistant strains. Genera that have to be taken into consideration, as pointed out by Facklam et al. (4), are the following: Aerococcus, Enterococcus, Lactococcus, Streptococcus, Gemella, Leuconostoc, and Pediococcus. In their report, they advise the use of the tests shown in Table 5 for differentiating between genera. We have added the phenotypic characteristics for ALO strains and also G+C moles percent of genera (14). Our strains showed little phenotypic intervariation and seem distinct from the other genera of catalase-negative, gram-positive cocci. They differ from streptococci by their microscopic appearance in that they do not form chains. Further, the following reactions are in disagreement between streptococci and ALO: for Enterococcus spp., streptococcal
gence
D antigen, bile-esculin, pyrrolidonyl aminopeptidase activity (PYRase), growth at 10 and 45°C; for Streptococcus spp., growth in 6.5% NaCl; for Lactococcus spp., growth at 10°C. However, several reactions for Streptococcus spp. and Lactococcus spp., shown in Table 5, are species intervariable, making these reactions less useful in delineating different genera. In a previous study (2) also using tests other than those included in this study, we have compared ALO with Streptococcus sanguis, Streptococcus bovis, Streptococcus group Q and Streptococcus group R, these strains being easily distinguishable from each other. By using the scheme established by Kilian et al. (7) in their extensive taxonomic revision of viridans group streptococci, ALO are also relatively easily separated from S. sanguis, Streptococcus anginosus, and Streptococcus mutans; reactions of
group
1052
J. CLIN. MICROBIOL.
CHRISTENSEN ET AL.
TABLE 5. Tests useful in differentiating catalase-negative, gram-positive cocci and coccobacillary genera of bactefiaa and G+C moles percent of genera Reaction of ALO and strains of the genusb: Gemella Pediococcus
Test Enterococcus
Morphology Cocci Coccobacilli Rods Arrangement Chains Pairs Clusters Gas from glucose Vancomycin Streptococcal antigen group Bile-esculin Aminopeptidase activity Pyrrolidonyl Leucine Growth In 6.5% NaCl At 450C At 100C Mol% (G+C)C
Streptococcus Lactococcus Aerococcus
+ +
+ +
+
+
+
-
-
-
-
+ + +
-
-
-
-
+ +
+ +
+ +
-
-
-
S D
S A-V
S N
+ +
+ +
+ +
S -
S -
R D
+
V
V
V
-
+ +
V
V
+
+
+
-
+ +
+ + +
V
V -
+
-
+
-
33.5_39d
39-46
38.6-41.5
35-40e
-
Leuconostoc Lactobacillus ALO + + +
+
+ +
+ +
+
-
+
R
V RS
+
+
V
+
-
V
+
V
V -
V V V 32-53
+ 44
-
+ -
32-35
34-42
+
+
38 44
+
+
S
a From human clinical isolates; adapted from Facklam et al. (4). b Abbreviations: V, variable; S, susceptible to vancomycin (presence of a zone of inhibition); R, resistant to vancomycin (absence of a zone of inhibition). c From Bergey's Manual of Systematic Bacteriology. d Enterococcusfaecium subsp. cassiflavum: G+C = 42 mol%. e A. viridans: G+C = 40.6 + 0.2 mol%.
special interest are esculin hydrolysis; hippurate hydrolysis; Voges-Proskauer's reaction; acid production from maltose, lactose, trehalose, inulin, and salicin; and ,B-galactosidase and ,-glucuronidase production. The type strain of A. viridans produces 3-galactosidase and PYRase but not P-glucuronidase or leucine aminopeptidase, reactions just opposite to what is seen for ALO strains. Leucine aminopeptidase is also typically produced by Gemella spp. and pediococci, with which ALO strains have major characteristics in common, although the following characteristics separate ALO from these two genera: microscopic appearance (Gram variability), PYRase, growth in 6.5% NaCl, vancomycin susceptibility, reaction with streptococcal group D antigen, bile-esculin, and growth at 45°C. Differentiating Leuconostoc spp. and Lactobacillus spp. from ALO does not pose any problem when the differences in microscopic appearance and biochemical reactions are considered. G+C moles percent determination of ALO strains suggests a possible relationship especially to Streptococcus spp., Pediococcus spp., and A. viridans (Table 5). Since there are both phenotypic similarities and major differences between these genera, DNA-DNA hybridization investigations are now being performed to clarify whether the isolates represent a separate taxon. ACKNOWLEDGMENTS We are indebted to the staffs of the participating departments of clinical microbiology at Frederiksberg Hospital, Rigshospitalet, and the Central Hospitals in Esbjerg, Hiller0d, Nwstved, and Slagelse for supplying us with the isolates and to the clinical departments at these hospitals for allowing us to study the patient records. We
thank Lene Andreassen and Tine Nedergaard for excellent technical assistance.
REFERENCES 1. Casals, J. B., and N. Pringler. 1990. Diagnostic tablets for bacterial identification, 3rd ed., p. 16. Rosco Diagnostica, Taastrup, Denmark. 2. Christensen, J. J., B. Korner, and H. Kjergaard. 1989. Aerococcus-like organism-an unnoticed urinary tract pathogen. APMIS 97:539-546. 3. Colman, G. 1967. Aerococcus-like organisms isolated from human infections. J. Clin. Pathol. 20:294-297. 4. Facklam, R., D. Hollis, and M. D. Collins. 1989. Identification of gram-positive coccal and coccobacillary vancomycin-resistant bacteria. J. Clin. Microbiol. 27:724-730. 5. Janosek, J., J. Eckert, and A. Hudac. 1980. Aerococcus viridans as a causative agent of infectious endocarditis. J. Hyg. Epidemiol. Microbiol. Immunol. 1:92-96. 6. Kern, W., and E. Vanek. 1987. Aerococcus bacteremia associated with granulocytopenia. Eur. J. Clin. Microbiol. 6:670-673. 7. Kilian, M., L. Mikkelsen, and J. Henrichsen. 1989. Taxonomic study of viridans streptococci: description of Streptococcus gordonii sp. nov. and emended descriptions of Streptococcus sanguis (White and Niven 1946), Streptococcus oralis (Bridge and Sneath 1982), and Streptococcus mitis (Andrewes and Horder 1906). Int. J. Syst. Bact. 39:471-484. 8. Lautrop, H., N. H0iby, A. Bremmelgaard, and B. Korsager. 1979. Bakteriologiske unders0gelsesmetoder. FADL's Forlag, Copenhagen. 9. Nathavitharana, K. A., S. N. Arseculeratne, H. A. Aponso, R. VUeratnam, L. Jayasena, and C. Navaratman. 1983. Acute meningitis in early childhood caused by Aerococcus viridans. Br. Med. J. 286:1248. 10. Parker, M. T., and L. C. Ball. 1976. Streptococci and aerococci associated with systemic infection in man. J. Med. Microbiol. 9:275-302.
VOL. 29, 1991 11. Pien, F. D., W. R. Wilson, K. Kunz, and J. A. Washington H. 1984. Aerococcus viridans endocarditis. Mayo Clin. Proc. 59: 47-48.
12. Pope, C. G., and F. V. Linggood. 1939. Purification of diphtheria toxoid. Br. J. Exp. Pathol. 20:297-304. 13. Sandstedt, K., J. Ursing, and M. Walder. 1983. Thermotolerant Campylobacter with no or weak catalase activity isolated from
URINARY TRACT INFECTION AND ALO
1053
dogs. Curr. Microbiol. 8:209-213. 14. Sneath, P. H. A., N. A. Mair, M. E. Sharpe, and J. G. Holt (ed.). 1986. Bergey's manual of systematic bacteriology, vol. 2. The Williams & Wilkins Co., Baltimore. 15. Untereker, W. J., and B. A. Hanna. 1976. Endocarditis and osteomyelitis caused by Aerococcus viridans. Mt. Sinai J. Med. 43:248-252.
Vol. 29, No. 5
0095-1137/91/051049-05$02.00/0 Copyright X 1991, American Society for Microbiology
Aerococcus-Like Organism, a Newly Recognized Potential Urinary Tract Pathogen JENS J. CHRISTENSEN,1,2,3* HENRIK VIBITS,1 JAN URSING,4 AND BENT KORNER' Department of Clinical Microbiology, Bispebjerg Hospital,' and Department of Diagnostic Bacteriology and Antibiotics, Statens Seruminstitut,3 Copenhagen, and Department of Clinical Microbiology, Central Hospital, Hillerod,2 Denmark, and Department of Medical Microbiology, University of Lund, Malmo General Hospital, Malmo, Sweden4 Received 11 October 1990/Accepted 11 February 1991
During the last 10 to 15 years, Aerococcus-like organisms have been isolated from urinary tract specimens from hospitalized patients at Bispebjerg Hospital, Copenhagen, Denmark, with signs of urinary tract infection, and a single strain has been deposited at the National Collection of Type Cultures (NCTC 12142). An additional 63 isolates from the urine specimens of as many patients with suspected urinary tract infection have been identified from seven departments of clinical microbiology in Denmark. Clinical information indicates that this organism may be involved in urinary tract infection. These strains and ten previously collected strains, two of which were from blood cultures, were characterized phenotypically. By comparing the results with data on other gram-positive, catalase-negative cocci or coccobacillary organisms, the identified organisms seem to represent a separate taxon.
38°C, dysuric complaints, and pyuria), were obtained by reviewing the records of the patients. Strains. The recently collected strains of ALO (n = 63) and 10 other strains, of which eight and two strains originated from urine specimens and blood cultures, respectively, were examined. Standard cultural conditions. Strains were maintained on 5% horse blood agar and incubated at 35°C in a normal atmosphere. The standard inoculum used for liquid cultures was 1 to 2 drops of a 24-h Todd-Hewitt broth culture. Growth. Growth was tested on 0.04% tellurite agar, on 40% bile agar, on bilayered sheep blood agar (8), in 6.5% NaCl broth, and at different temperatures in trypsin broth (12). Plates and tubes were observed for 2 days. Biochemical tests. Biochemical tests were performed as described previously (2), except that they were supplemented with a test for leucine-aminopeptidase activity (Rosco Diagnostic Tablet [1]). Susceptibility testing. Antibiotic susceptibility was tested by the direct agar diffusion method by using Rosco NeoSensitabs on Danish blood agar. Strains are reported as susceptible when zone diameters were .28 mm for penicillin (5 jjtg), sulfonamides and netilmicin; >-10 and 218 mm for 5 and 70 ,ug of vancomycin, respectively; and -15 and .22 mm for 0.4 and 40 IU of bacitracin, respectively. Zone diameters less than the above-mentioned figures are reported to indicate resistance. The inhibition zone limits correlate approximately to MIC breakpoints for penicillin at 0.25 ,ug/ml, for sulfonamides at 64 ,ug/ml, for netilmicin at 2 pLg/ml, and for vancomycin (70 ,g) at 4 ,ug/ml. The bacitracin tablets and the 5 ,ug of vancomycin used are only for diagnostic purposes. Determination of G+C moles percent of DNA. The methods for isolation of DNA and estimation of G+C moles percent by the thermal denaturation method have been described earlier (13). The values given are the means of four determinations.
Infections caused by Aerococcus viridans in humans are seldom reported; cases of urinary tract infections (3, 10), bacteremia (6), endocarditis (3, 5, 10, 11, 15), and meningitis (9) have, however, been published. During the last 10 to 15 years, we have observed at Bispebjerg Hospital, Copenhagen, Denmark, Aerococcus-like organisms (ALO) in urinary tract specimens from hospitalized patients with signs of urinary tract infection. The ALO strains have been characterized phenotypically in a previous report (2), and a single urine isolate has been deposited at the National Collection of Type Cultures, Colindale, United Kingdom (NCTC 12142). ALO produce greening on 5% horse blood agar like the viridans group streptococci, form in clusters like staphylococci as confirmed by microscopy, and are catalase negative. However, the isolates cannot be easily assigned to any genus or species. We decided to investigate whether ALO also could be isolated from urinary tract specimens in other laboratories in Denmark, to further characterize ALO isolates, and to comment on the taxonomic relationship of ALO isolates. MATERIALS AND METHODS
Collection of strains and clinical features of patients. Seven departments of clinical microbiology in Denmark were asked to collect five to ten consecutive strains from urinary tract specimens with the following characteristics- gram-positive cocci, predominantly in clusters; colonies resembling alphahemolytic streptococci on 5% horse blood agar; catalase negative; susceptible to penicillin; and resistant to both sulfonamides and aminoglycosides. Patients. Predisposing conditions, together with symptoms indicative of urinary tract infection (rectal temperature* Corresponding author. 1049
1050
J. CLIN. MICROBIOL.
CHRISTENSEN ET AL.
TABLE 1. Parameters indicative of urinary tract infection in 63 patients whose urine yielded growth of ALO No. of patients Parameter(s)
Dysuria + fever + pyuria Dysuria + fever Dysuria + pyuria Fever + pyuria Dysuria Fever Pyuria Incontinence No symptoms No information
ALO
Predisposing condition
ALO in admixture
singly
Without catheter'
With catheter
Without catheter
1 1 13
1 1 1
1 1 8 1 1
2 1
6
1
1 1 10
With catheter
2b 1 3
1 4
Indwelling urinary catheter. In two patients with congenital myelomeningocele and recurrent nonsymptomatic urinary tract infection, the test for pyuria was not performed. a
b
RESULTS During the study period, 64 patients suspected of urinary tract infection due to ALO were identified (Table 1). The organism isolated from one of the patients was phenotypically different from ALO isolates. A total of i105 ALO per ml were cultured from the urine specimens of all the remaining 63 patients, except for four patients having 103 to 104 ALO per ml of urine. ALO were grown in pure culture from the urine of 36 patients, while ALO were cocultured with other bacteria from the urine of 27 patients (Table 2). From five patients with a pure culture of ALO a repeat specimen before treatment also showed i105 ALO per ml. Patients. Of the 63 patients, 34 were female and 29 were male. The median age was 74 years (range, 3 to 97 years). Forty-one patients suffered from conditions predisposing them to urinary tract infection (Table 3). Elevated serum creatinine was observed in 12 patients. Twelve patients had received sulfonamides within the last week before specimen sampling, and one patient each had received trimethoprim, nitrofurantoin, or penicillin. Description of strains. All 73 isolates were gram-positive cocci, predominantly in clusters and with a tendency to form pairs and groups of four. All strains grew well on 5% horse blood agar, producing green discoloration of the agar resemTABLE 2. Bacteria cocultured with ALO from urine of 27 patients No. of patients with: _104 _105 bacteria/ml bacteria/ml
Organism(s)
E. coli Klebsiella pneumoniae Morganella morganii Citrobacter freundii Enterobacter species Enterococcus faecalis Hemolytic streptococci (group C) Staphylococcus aureus
Corynebacterium species .3 organisms The two organisms isolated cation was not performed. a
10 2 1 1 1 1
2a
1 1 1
1 5 were
TABLE 3. Conditions predisposing to urinary tract infection found among 35 patients whose urine yielded growth of ALO'
E. coli-like, but exact species identifi-
No. of
Systemic Diabetes mellitus ............................ ................ 6 3 Parkinson's disease ............................................ Cancer (bladder, pancreas, lung, uterine cervix) ........ 5.... Multiple sclerosis ............................................ 2 2 Polyneuropathia ............................................ Local Hyperplasia of the prostate ........................................ Indwelling urinary catheter ........................................ Paresis of the bladder ............................................ Bladder papilloma ............................................ ....................... Bladder stones ..................... ....................... Ureteral reflux .....................
4 7 6 2 2 2
a SiX patients suffered from two conditions predisposing them to urinary tract infection at the same time.
bling that of viridans group streptococci. The colony diameter increased from 0.5 mm after 24 h to 1 mm after 48 h, and the colonies of all strains were convex, with a well-defined border, and were shiny and transparent. No growth was seen on tellurite agar, whereas on bile agar all strains grew scantily (after 24 h) to moderately (after 48 h). All strains grew in 6.5% NaCl broth after 2 days of incubation at 37°C. No growth occurred at 10°C, 45°C, or after the heating of inoculated tubes to 60°C for 30 min; however, when a 10-fold greater inoculum was used, strains grew at 45°C, making this test of limited value for the identification of ALO. CAMP reaction was negative for all strains. Growth characteristics, results of biochemical tests, and antibiotic susceptibilities of strains are shown in Table 4. For sulfonamides, no zone was seen at all, whereas for netilmicin a zone diameter of 20 to 22 mm was observed for most strains. The guanine-plus-cytosine content of DNA was estimated for four strains and was 44.4 + 0.5 mol% (mean ± standard deviation). DISCUSSION
This investigation confirms that strains phenotypically identical to the ALO isolates previously described (2) can be found in urinary tract specimens examined in most laboratories in Denmark. Few phenotypic characteristics (see Materials and Methods) are needed to identify these strains. Their detection is, however, dependent on whether microscopy is performed, as they otherwise may easily be mistaken for viridans group streptococci. As also seen previously, data from patient records indicate that ALO possess a certain pathogenic potential in urinary tract infection; most patients had classical signs of urinary tract infection, recurrences occurred, and the distribution of ALO in urine specimens was equal to what is seen for established pathogens such as Escherichia coli and Klebsiella spp. ALO also has the ability to cause invasive infections, i.e., urosepticemia and fatal endocarditis (unpublished data); in both cases, antibodies against ALO isolates were demonstrated in crossed immunoelectrophoretic assay. Identification of catalase-negative, gram-positive coccal and coccobacillary organisms has received renewed interest
URINARY TRACT INFECTION AND ALO
VOL. 29, 1991
1051
TABLE 4. Growth characteristics, fermentation reactions, direct enzyme test, and antibiotic susceptibility for 73 strains of ALO'
of positive Dayreaction
Reactionb
Characteristic
0 0
a
Hemolysis Production of catalase Growth on: 0.04% tellurite 40% bile Growth in 6.5% NaCl Growth at: looc
1-2 2
+
Acid production from: Arabinose Glucose (acid/gas) Maltose Lactose Sucrose Trehalose Melibiose Raffinose Salicin Esculin Melizitose Inulin Glycogen Mannitol Sorbitol Glycerol Production of:
b I
1-2
+l-
0
0 0 4 (+ day 1-4)
0
1-2
1-5
+
1-6
1 (Negative)
0 0 0 0 0 0 0 0 0
3 (Negative)
0
0 0 0 0
+(24 h), +(4 h)
Pyrrolidonyl aminopeptidase Leucine aminopeptidase Hydrolysis of: Hippurate Arginine Acetoin production Reaction with group D antiserum Susceptibility to: Penicillin G Sulfonamides Netilmicin Vancomycin (5 jig) Vancomycin (70 ,ug) Bacitracin (0.04 IU) Bacitracin (40 IU) a
0 1 (No growth)
0 0 0 0
450C Surviving 600C after 30 min CAMP reaction
P-Galactosidase P-Glucuronidase
deviating No. of strains
0 0 0 0
+
0 0 0 0 0 0 0
S
R R S S
R S
From eight departments of clinical microbiology in Denmark. S, Susceptible; R, resistant. Scanty to moderate growth.
during the last several years, partly stimulated by the emerof vancomycin-resistant strains. Genera that have to be taken into consideration, as pointed out by Facklam et al. (4), are the following: Aerococcus, Enterococcus, Lactococcus, Streptococcus, Gemella, Leuconostoc, and Pediococcus. In their report, they advise the use of the tests shown in Table 5 for differentiating between genera. We have added the phenotypic characteristics for ALO strains and also G+C moles percent of genera (14). Our strains showed little phenotypic intervariation and seem distinct from the other genera of catalase-negative, gram-positive cocci. They differ from streptococci by their microscopic appearance in that they do not form chains. Further, the following reactions are in disagreement between streptococci and ALO: for Enterococcus spp., streptococcal
gence
D antigen, bile-esculin, pyrrolidonyl aminopeptidase activity (PYRase), growth at 10 and 45°C; for Streptococcus spp., growth in 6.5% NaCl; for Lactococcus spp., growth at 10°C. However, several reactions for Streptococcus spp. and Lactococcus spp., shown in Table 5, are species intervariable, making these reactions less useful in delineating different genera. In a previous study (2) also using tests other than those included in this study, we have compared ALO with Streptococcus sanguis, Streptococcus bovis, Streptococcus group Q and Streptococcus group R, these strains being easily distinguishable from each other. By using the scheme established by Kilian et al. (7) in their extensive taxonomic revision of viridans group streptococci, ALO are also relatively easily separated from S. sanguis, Streptococcus anginosus, and Streptococcus mutans; reactions of
group
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J. CLIN. MICROBIOL.
CHRISTENSEN ET AL.
TABLE 5. Tests useful in differentiating catalase-negative, gram-positive cocci and coccobacillary genera of bactefiaa and G+C moles percent of genera Reaction of ALO and strains of the genusb: Gemella Pediococcus
Test Enterococcus
Morphology Cocci Coccobacilli Rods Arrangement Chains Pairs Clusters Gas from glucose Vancomycin Streptococcal antigen group Bile-esculin Aminopeptidase activity Pyrrolidonyl Leucine Growth In 6.5% NaCl At 450C At 100C Mol% (G+C)C
Streptococcus Lactococcus Aerococcus
+ +
+ +
+
+
+
-
-
-
-
+ + +
-
-
-
-
+ +
+ +
+ +
-
-
-
S D
S A-V
S N
+ +
+ +
+ +
S -
S -
R D
+
V
V
V
-
+ +
V
V
+
+
+
-
+ +
+ + +
V
V -
+
-
+
-
33.5_39d
39-46
38.6-41.5
35-40e
-
Leuconostoc Lactobacillus ALO + + +
+
+ +
+ +
+
-
+
R
V RS
+
+
V
+
-
V
+
V
V -
V V V 32-53
+ 44
-
+ -
32-35
34-42
+
+
38 44
+
+
S
a From human clinical isolates; adapted from Facklam et al. (4). b Abbreviations: V, variable; S, susceptible to vancomycin (presence of a zone of inhibition); R, resistant to vancomycin (absence of a zone of inhibition). c From Bergey's Manual of Systematic Bacteriology. d Enterococcusfaecium subsp. cassiflavum: G+C = 42 mol%. e A. viridans: G+C = 40.6 + 0.2 mol%.
special interest are esculin hydrolysis; hippurate hydrolysis; Voges-Proskauer's reaction; acid production from maltose, lactose, trehalose, inulin, and salicin; and ,B-galactosidase and ,-glucuronidase production. The type strain of A. viridans produces 3-galactosidase and PYRase but not P-glucuronidase or leucine aminopeptidase, reactions just opposite to what is seen for ALO strains. Leucine aminopeptidase is also typically produced by Gemella spp. and pediococci, with which ALO strains have major characteristics in common, although the following characteristics separate ALO from these two genera: microscopic appearance (Gram variability), PYRase, growth in 6.5% NaCl, vancomycin susceptibility, reaction with streptococcal group D antigen, bile-esculin, and growth at 45°C. Differentiating Leuconostoc spp. and Lactobacillus spp. from ALO does not pose any problem when the differences in microscopic appearance and biochemical reactions are considered. G+C moles percent determination of ALO strains suggests a possible relationship especially to Streptococcus spp., Pediococcus spp., and A. viridans (Table 5). Since there are both phenotypic similarities and major differences between these genera, DNA-DNA hybridization investigations are now being performed to clarify whether the isolates represent a separate taxon. ACKNOWLEDGMENTS We are indebted to the staffs of the participating departments of clinical microbiology at Frederiksberg Hospital, Rigshospitalet, and the Central Hospitals in Esbjerg, Hiller0d, Nwstved, and Slagelse for supplying us with the isolates and to the clinical departments at these hospitals for allowing us to study the patient records. We
thank Lene Andreassen and Tine Nedergaard for excellent technical assistance.
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12. Pope, C. G., and F. V. Linggood. 1939. Purification of diphtheria toxoid. Br. J. Exp. Pathol. 20:297-304. 13. Sandstedt, K., J. Ursing, and M. Walder. 1983. Thermotolerant Campylobacter with no or weak catalase activity isolated from
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