In vitro antimicrobial susceptibility of Aerococcus urinae to 14 antibiotics, and time–kill curves for penicillin, gentamicin and vancomycin

Robert Skova,*, Jens Jørgen Christensenb, Bent Kornerc, Niels Frimodt-Møllerb and Frank Espersena

a Departments of Research and Development and b Clinical Microbiology, Statens Serum Institut, Copenhagen; c Department of Clinical Microbiology, Bispebjerg Hospital, Copenhagen, Denmark


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aerococcus urinae is a newcomer in clinical and microbiological practice, causing urinary tract infections, bacteraemia/septicaemia and/or endocarditis. This study presents for the first time an evaluation of the activity of a representative panel of antibiotics against a large number of A. urinae isolates. The in vitro susceptibilities (MICs) of 56 isolates of A. urinae to 14 antibiotics were determined by agar dilution. In general, A. urinae isolates showed little inter-isolate variability, and had low MICs of penicillin, amoxicillin, piperacillin, cefepime, vancomycin and rifampicin. High-level aminoglycoside resistance was not found for any of the isolates. Moderate to good activity was seen with quinolones, erythromycin and tetracycline. Isolates from two patients with endocarditis were studied with time–kill curves for penicillin, gentamicin and vancomycin. Penicillin and vancomycin alone exhibited slow or no bactericidal activity against the two strains. When combining either penicillin or vancomycin with gentamicin, rapid bactericidal activity was obtained for both strains with both combinations. The treatment options for A. urinae seem to include penicillins for less severe cases. In severe cases, i.e. endocarditis, the time–kill investigations suggest a beneficial effect of combination with gentamicin. In the penicillin-allergic patient vancomycin in combination with gentamicin represents the most obvious alternative.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
During the past decade the clinical relevance of various catalase-negative, Gram-positive cocci has received much attention.1 Among these, Aerococcus urinae is a newcomer in clinical and microbiological practice, first reported in 19892 and designated in 1992.3A. urinae isolates were originally recognized by their cell morphology (Staphylococcus-like in Gram's stain) and growth characteristics (resembling {alpha}-haemolytic streptococci on blood agar), a negative catalase reaction, and a very constant antibiogram demonstrating susceptibility to penicillin and resistance to sulphonamides and aminoglycosides.2–4 In two reports A. urinae strains have been isolated from 0.4–0.8% of urine specimens examined.2,5 Most patients from whom A. urinae was isolated were elderly with predisposing conditions and signs of urinary tract infection.2,4,5 Strains causing urinary tract infections have been recognized in Denmark, Sweden, The Netherlands, France, the USA, Canada and South American countries.6A. urinae has also been isolated from blood from patients suffering from urogenic bacteraemia/septicaemia with or without endocarditis.7–11 A nationwide survey in Denmark from 1987 to 1995 identified 26 patients with A. urinae isolated from blood,6 corresponding to 0.5 patients/year/1 million inhabitants, and 0.8% of infective endocarditis episodes.8 Bacteraemic/ septicaemic episodes responded well to treatment with ß-lactams alone or in combination with an aminoglyco-side, unless bacteraemia was complicated with endocarditis, in which cases five of six patients died. In one patient A. urinae could, at autopsy, be grown from the valves, despite apparently relevant antibiotic treatment. Therefore, the optimal treatment of endocarditis with A. urinae has to be determined.

Testing of A. urinae isolates by tablet/disc diffusion methods has revealed susceptibility to a wide range of antimicrobial agents including both ß-lactam and non- ß-lactam antibiotics.5,6,12 As A. urinae isolates have primarily been associated with urinary tract infections, it is noteworthy that the strains have been found resistant to sulphonamides and other antimicrobials used for treating urinary tract infections, including co-trimoxazole, trimethoprim, nalidixic acid and polymyxins.2,5,6,12 Recently, the susceptibility of two A. urinae isolates from endocarditis patients have been investigated by MICs of seven antibiotics, and time–kill curves for penicillin in combination with gentamicin or netilmicin were performed.11 The present study further characterizes the susceptibility pattern of 56 A. urinae isolates of various geographical origins. MICs of 14 antibiotics were determined, and for two isolates time–kill curve experiments with penicillin, gentamicin and vancomycin, alone or in combination, were performed in order to obtain more information on optimal treatment regimens of serious infections caused by A. urinae.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Strains

Fifty-six epidemiologically unrelated clinical isolates of A. urinae were studied, comprising 42 Danish isolates (17 blood isolates and 25 urine isolates) and 14 non-Danish urine isolates: one from The Netherlands (kindly provided by P. J. G. M. Rietra), four from Sweden (kindly provided by E. Falsen, Culture Collection, University of Göteborg) and seven North American isolates (kindly provided by R. R. Facklam, The Streptococcus Laboratory, CDC, Atlanta, GA, USA). The following reference strains were used: Staphylococcus aureus ATCC 29213, S. aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATTC 25922 and Enterococcus faecalis ATCC 29212.

Antimicrobial agents

The following antimicrobial agents were used: benzylpenicillin (Leo, Ballerup, Denmark); cefepime (Bristol-Myers Squibb, Wallingford, CT, USA); amoxicillin, ceftriaxone, erythromycin, amikacin, gentamicin, netilmicin, rifampicin, vancomycin (Sigma, St Louis, MO, USA); oxytetracycline (Rosco, Taastrup, Denmark), piperacillin (Lederle, Pearl River, NY, USA), ciprofloxacin (Bayer, Leverkusen, Germany); and sparfloxacin (Rhône Poulenc Rorer, Vitry, France).

MIC determination

Owing to the fastidious growth of A. urinae the test conditions had to be modified from the standard recommendations in order to perform MIC determinations (i.e. 5% blood supplementation of the agar and incubation in 5% CO2 atmosphere for 48 h).13–15 The MICs were determined by the agar dilution method. Two-fold serial dilutions of antimicrobial agents were prepared in Mueller–Hinton agar (Difco Laboratories GmbH, Augsburg, Germany) supplemented with 5% lysed horse blood. Antibiotics were tested from 0.008 to 512 mg/L, except for amoxicillin (0.032–256 mg/L), rifampicin (0.008–32 mg/L) and oxytetracycline (0.008–2 mg/L). The inocula of 107 cfu/mL (104 cfu/spot) were prepared from overnight agar cultures and suspended in 0.9% NaCl, and were applied with a 21 point multiple inoculator (Denley Instruments Ltd, Billinghurst, UK). Results were read after incubation for 2 days at 37°C in 5% CO2. The MIC was read as the lowest concentration showing growth of a maximum of three colonies.

Time–kill curve investigations

Two of the tested blood isolates (B1 and B8), both from patients with endocarditis, were investigated. The strains were tested against penicillin (MIC 0.125 and 0.6 mg/L, respectively), gentamicin (MIC 256 and 64 mg/L, respectively), and vancomycin (MIC 0.5 and 1 mg/L, respectively) in the following clinically obtainable concentrations: penicillin 2 x and 10 x MIC (P2 and P10, respectively); vancomycin 4 x and 10 x MIC (V4 and V10, respectively); gentamicin 10 mg/L (G10); and the combinations penicillin + gentamicin and vancomycin + gentamicin in the following concentrations: P2G10, P10G10, V4G10 and V10G10, respectively. At t = –1 h fresh overnight-grown colonies were suspended to c. 105 cfu/mL in tubes with prewarmed Todd–Hewitt broth. The tubes were placed in a water bath at 37°C. At t = 0 h antibiotics, prepared immediately before the assay, were added to the tubes in duplicate. Three tubes without antibiotics were used as growth control. Samples in duplicate were taken at 0, 3, 6 and 24 h from each tube. The samples were serially diluted and plated. The numbers of surviving colonies were counted after overnight incubation at 37°C in ambient air in plastic bags together with wet tissue paper [a pilot study had shown that the bacteria were able to grow satisfactory in these conditions (data not shown)]. Using 20 µL samples the minimal countable number of bacteria was 50 cfu/mL. Carry-over effect was eliminated for the penicillin–gentamicin experiments by adding 100 µL 100 000 U/mL penicillinase to each plate. For the vancomycin–gentamicin experiments carry-over effect was eliminated by dilution. A pilot investigation demonstrated carry-over effect in the 100 sample but not in the 10–1 sample, cfu were therefore counted using 200 µL of the 10–1 samples still giving a 50 cfu/mL detection limit. All experiments were performed at least twice on separate days. Bactericidal activity was defined as a reduction of the inoculum by at least 99.9% within 24 h. Synergy (S), additive effect (A) and antagonism were defined as: >2 log more kill (S), >1 and <2 log more kill (A) and >2 log decreased killing, respectively, for the combination as compared with the best of the individual antibiotics.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MIC determination

The MICs obtained for the 56 isolates are shown in the TableGo. Using the breakpoints for streptococci recommended by the NCCLS14,15 or by the Swedish Reference Group of Antibiotics (SRGA),16,17 the isolates were susceptible to the penicillins, whereas reduced susceptibility was found for the cephalosporins tested. The MICs for the aminoglycosides indicated reduced susceptibility for almost all isolates. However, high-level aminoglycoside resistance (i.e. gentamicin > 1000 mg/L) was not found for any of the isolates. All isolates were susceptible to vancomycin. Rifampicin was the most active drug tested with an MIC50 of 0.031 mg/L (range <0.004–0.064 mg/L), except for one isolate with an MIC > 32 mg/L (repeated testing). The activities of the quinolones ciprofloxacin and sparfloxacin were found to be equally good.


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Table. MICs of 14 antimicrobial agents ofr A. urinae (n = 56)

 
Killing curve examinations

Average time–kill curves for each experiment are presented in the FigureGo. Vancomycin alone (both 4 x and 10 x MIC) exhibited bactericidal activity against strain B1. For penicillin, bactericidal activity was found only for 10 x MIC against B8. For both antibiotics the bactericidal effect was slow, i.e. first appeared after 24 h. Gentamicin was not bactericidal—in most experiments growth was seen. Penicillin in combination with gentamicin was synergic and was rapidly bactericidal for strain B8 in both P2G10 and P10G10. For strain B1, however, only additive and slow bactericidal effect was found. When combining vancomycin with gentamicin a rapid bactericidal effect was found for both strains. A synergic effect was detectable after 3 h and 6 h for strain B8. At 24 h, however, the killing activity of vancomycin was so pronounced that it was not possible to detect a synergic effect. For B1 only additive effect was found. No antagonistic effects were recorded in any of the experiments.



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Figure. Time–kill curves of two A. urinae blood-culture isolates (B1 and B8) (average of two or three experiments). (a) Isolate B1, penicillin + gentamicin; (b) isolate B8, penicillin + gentamicin; (c) isolate B1, vancomycin + gentamicin; (d) isolate B8, vancomycin + gentamicin. Error bars are standard deviation. A solid line marks the detection limit. Symbols for (a) and (b): x, P2G10; {blacktriangleup}, P10G10; {blacktriangledown}, P2; {diamondsuit}, P10; •, G10; *, control. Symbols for (c) and (d): x, V4G10; {blacktriangleup}, V10G10; {blacktriangledown}, V4; {diamondsuit}, V10; •, G10; *, control.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study presents for the first time an evaluation of the MICs of a representative panel of antibiotics against a large number of A. urinae isolates obtained from patients with urinary tract infection, bacteraemia/septicaemia and/or endocarditis. In order to evaluate the use of antibiograms for taxonomic purposes, MICs of 15 antibiotics for 10 A. urinae strains have been examined previously,12 but detailed data have not been presented.

A. urinae are fastidious organisms requiring supplementation of blood in the media and sometimes incubation in a CO2-enriched atmosphere and/or a prolonged incubation period in order to obtain sufficient growth for testing antibiotic susceptibility. Therefore, the strains could not be tested using standard conditions as described by the BSAC or NCCLS.13–15 The need to modify culture conditions is, however, a common problem for fastidious Grampositive bacteria.18,19 A. urinae isolates exhibited an antimicrobial susceptibility pattern similar to that of {alpha}-haemo-lytic streptococci with only small inter-isolate susceptibility variations. MICs of ß-lactams for the isolates were typic-ally within four two-fold dilution steps, except for the cephalosporins ceftriaxone and cefepime, which showed a greater variation in MIC (TableGo). Owing to the possible influence of CO2 incubation (lowering of pH), caution should be taken when judging the susceptibility to tetracycline (overestimated susceptibility) and erythromycin (underestimated susceptibility). Among the A. urinae isolates examined, no high-level resistance was identified against the tested aminoglycosides leaving open the possibility of a synergic effect when combined with either a ß-lactam or vancomycin (vide infra). As judged by MICs several treatment alternatives do exist for serious infections since the isolates were susceptible to penicillins, vancomycin and rifampicin (except one isolate). The quinolones selected, ciprofloxacin and sparfloxacin, also showed good activity. Although a fastidious organism, the results obtained by disc diffusion methods2,5,20 have been in accordance with the MICs presented in this study, suggesting that a reliable result may be obtained using these routine susceptibility methods.

Guidelines for treatment of serious infections such as septicaemia with or without endocarditis should optimally be based on clinical experience in carefully designed studies, but this is difficult when dealing with rare bacteria. Therefore, in vitro studies including killing curve techniques may supply important information for the clinician in these cases.

Antibiotic concentrations achievable in vivo were chosen for the experiments. For gentamicin a 10 mg/L dose was chosen, corresponding to the concentration obtained by once daily dosing of 240 mg (equivalent to 3–4 mg/kg).21 Furthermore, although several papers advocate twice daily dosing for endocarditis, in Denmark once daily gentamicin is commonly used, owing to the reduction of nephrotoxicity and the fact that the synergic effect with ß-lactam antibiotics increases with increasing aminoglycoside concentrations.22 The data obtained in our in vitro study of killing curves for penicillin and vancomycin showed that neither penicillin nor vancomycin was bactericidal against either of the two isolates tested from patients with infective endocarditis. When combined with gentamicin, a bactericidal effect was obtained against both isolates for both penicillin and vancomycin. Fortunately, these regimens are recommended for treating infective endocarditis of unknown aetiology throughout the world.23 The killing rates of the two strains were different, indicating that interstrain variations may exist. Despite in vitro susceptibility to the antibiotics given, we found that the prognosis was poor for elderly patients suffering from infective endocarditis caused by A. urinae.8 The present results for penicillin, gentamicin and netilmicin (MIC) are in accordance with those in the recent Swiss report by Zbinden et al.11 for two A. urinae isolates from endocarditis patients.

The treatment options for A. urinae seem to include penicillins for less severe cases. In severe cases, i.e. endocarditis, the time–kill investigations suggest a benefit of combining penicillin or vancomycin with gentamicin. In the penicillin-allergic patient vancomycin represents the most obvious alternative.


    Notes
 
* Correspondence address. Department of Research and Development, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark. Tel: +45-3268-3535; Fax: +45-3268-3873; E-mail: rsk{at}ssi.dk Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Facklam, R. R. & Elliott, J. A. (1995). Identification, classification, and clinical relevance of catalase negative, gram-positive cocci, excluding the streptococci and enterococci. Clinical Microbiological Reviews 8, 479–95.

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15 . National Committee for Clinical Laboratory Standards. (1999). Performance Standards for Antimicrobial Disk Susceptibility Testing: Approved Standard M2-A7. NCCLS, Wayne, PA.

16 . Swedish Reference Group of Antibiotics. (2000). ß-lactam antibiotics – pharmacological and species-related breakpoints. [On-line.] http://www.srga.org/MICTAB/MIC1.htm (3 January 2001, date last accessed).

17 . Swedish Reference Group of Antibiotics. (2000). Non-ß-lactam antibiotics – pharmacological and species-related breakpoints. [On-line.] http://www.srga.org/MICTAB/MICTAB2.htm (3 January 2001, date last accessed).

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20 . Jensen, K. T., Schønheyder, H., Pers, C. & Thomsen, V. F. (1992). In vitro activity of teicoplanin and vancomycin against gram-positive bacteria from human clinical and veterinary sources. Acta Pathologica Microbiologica et Immunologica Scandinavica 100, 543–52.

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Received 8 September 2000; returned 24 November 2000; revised 10 January 2001; accepted 14 September 2001





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