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Synergistic effect of gentamicin plus ampicillin on enterococci with differing sensitivity to gentamicin:
ANTIMICROBIAL SUSCEPTIBILITY STUDIES
Synergistic Effect of Gentamicin Plus Ampicillin on Enterococci with Differing Sensitivity to Gentamicin: A Phenotypic Assessment of NCCLS Guidelines Dana C. Dressel, Michelle A. Tornatore-Reuscher, Cynthia R. Boschman, Valentina Stosor, Teresa Zembower, Michael J. Postelnick, Gary A. Noskin, and Lance R. Peterson
Between December 1, 1993, and December 1, 1996, we tested 4,411 isolates of Enterococcus sp. at gentamicin concentrations of 500 g/mL and 2000 g/mL using agar dilution to phenotypically categorize them into 3 groups: those with a MIC ⱕ500 g/mL (n ⫽ 3,132; 71%); a MIC ⬎500, but ⱕ2000 g/mL (n ⫽ 441; 10%); and those with a MIC ⬎2000 g/mL (n ⫽ 838; 19%). Ten unique strains of each phenotype were tested to determine which gentamicin concentration was the best in vitro predictor of synergy with ampicillin. Testing was done by a time-kill method using clinically achievable levels of ampicillin and gentamicin. We found that for the gentamicin MIC ⱕ500 g/mL group, 7 of 10 isolates demonstrated synergy with ampicillin as manifested by a ⱖ2 log10 increase in killing versus the effect of ampicillin alone (at 1⁄2 the MIC for ampicillin). In the group sensitive to a gentamicin MIC range
between ⬎500 and ⱕ2,000 g/mL, none of the 10 isolates demonstrated synergy. Absence of synergy was also found in the group resistant to 2,000 g/mL of gentamicin. Assessment of eight additional enterococcal isolates with reduced sensitivity to ampicillin (MIC from 32–256 g/mL) found no correlation between gentamicin sensitivity at 500 g/mL and any in vitro test for synergy, nor with clinical therapeutic outcome. Gentamicin at 2g/mL combined with ampicillin was as effective in enhancing killing as a higher level of 4 g/mL. These findings validate the current NCCLS guideline for predicting synergistic activity against enterococci in strains with usual susceptibility to ampicillin, and suggest that a therapeutic level less than maximal recommended dosing is sufficient when using gentamicin in this setting. © 1999 Elsevier Science Inc.
Northwestern Memorial Hospital and Northwestern University supported this work. Presented, in part, at the 97th General Meeting of the American Society for Microbiology, May 4–8, 1997, Miami Beach, FL. Abstract C147. From the Clinical Microbiology Division, Northwestern Memorial Hospital (DCD, CRB, VS, TZ, GAN, LRP); Department of Pathology, Northwestern University Medical School (MAT-R, CRB, VS, TZ, GAN, LRP); Infectious Disease Division, Northwestern University Medical School (VS, TZ, GAN, LRP); and Pharmacy Department, Northwestern Memorial Hospital (MJP), Chicago, Illinois 60611 USA. Address reprint requests to: Dr. Lance R. Peterson, Clinical Microbiology, Wesley Pavilion, Room 565, Northwestern Memorial Hospital, 250 E. Superior Street, Chicago, IL 60611.
INTRODUCTION
DIAGN MICROBIOL INFECT DIS 1999;35:219 –225 © 1999 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
It has long been a standard of care that bactericidal therapy consisting of an aminoglycoside in combination with a cell-wall active agent such as ampicillin is required for effective treatment of serious infections with enterococci (Caputo et al. 1993; Lopardo et al. 1995; Moellering 1995; Simmons et al. 1998). For more than 20 years it was recognized that demonstrating susceptibility of enterococci to high concentrations of an aminoglycoside, usually ⱕ2000 g/
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D.C. Dressel et al.
220 mL, could be used to predict in vitro synergy (Murray 1990). Resistance to a higher level than this is an indication that an aminoglycoside-inactivating enzyme is likely present within the enterococcal cell. The synergistic action is distinct from a direct effect of gentamicin alone on enterococci (Fasola et al. 1994). However, there has been considerable discordance as to which in vitro concentration of gentamicin, 500 g/mL or 2000 g/mL, best predicts synergistic killing (i.e., absence of an inactivating enzyme) useful in clinical therapy. Most recently, Swenson et al., correlated molecular characterization for aminoglycoside susceptibility with MICs, and determined that the best screening method for detecting highlevel resistance was gentamicin in agar or broth at a concentration of 500 g/mL (1995a). The purpose of our study was to correlate kill curve results for synergy with gentamicin MICs to phenotypically confirm which cut-off, 500 g/mL or 2000 g/mL of gentamicin, best predicts synergy. Additionally, we sought to determine if low level “synergistic” or high level “therapeutic” clinical dosing is necessary to demonstrate in vitro synergy using time-kill studies.
MATERIALS AND METHODS Organisms The organisms tested were unique clinical isolates recovered by the microbiology laboratory at Northwestern Memorial Hospital, Chicago, IL, between December 1, 1993, and December 1, 1996. Enterococci were identified to species level based on colonial morphology, Gram-stain reaction, catalase, growth in 6.5% NaCl, ability to grow in bile and hydrolyze esculin, and ability to ferment the sugars arabinose, raffinose, sorbitol and mannitol. All isolates were saved frozen at ⫺85°C in Mueller Hinton Broth with glycerol. They were removed from freezer storage and subcultured to blood agar twice prior to testing. Enterococcus faecalis ATCC 29212 was used as a susceptible (synergistic) control isolate, and a strain known to be resistant to high-level (MIC ⬎2000 g/mL for gentamicin and streptomycin) aminoglycoside concentrations was chosen as a resistant control strain. They were included in every test run. After completion of the initial study, we tested eight additional E. faecium strains from patients with persistent bacteremia (Stosor et al. 1996). These strains were selected for time-kill analysis because collectively they had reduced susceptibility to ampicillin, variable sensitivity to gentamicin, and a known outcome of therapy using ampicillin or ampicillin/sulbactam, either agent given with gentamicin (Stosor et al. 1996).
Inoculum preparation For each isolate tested, the inoculum was prepared from 3 to 5 distinct, 18 –24 h old colonies grown in 5 mL aliquots of Trypticase Soy Broth. These were incubated for 3–5 h to achieve a turbid suspension. Just prior to testing, the broth was adjusted to a 0.5-McFarland turbidity standard to give approximately 1 ⫻ 108 CFU/mL. Then, 0.1 mL of the prepared inoculum was added to macrobroth dilution tubes as described below.
Antimicrobial agents Ampicillin trihydrate was generously provided by the Bristol-Myers Squibb Company (Evansville, IN), and gentamicin sulfate was purchased from U.S. Biochemical Corporation (Cleveland, OH).
Phase 1: Phenotype categorization Isolates selected for testing were chosen based upon results from prior agar dilution done following the NCCLS standard method (National Committee for Clinical Laboratory Standards 1997). This testing was done using a Steers’ replicator to spot inoculum onto agar containing either 500 or 2000 g/mL of gentamicin. Plates were incubated for 22 to 24 h and observed for visible growth. Isolates were then separated into three groups based on susceptibility to specific concentrations of gentamicin: ⱕ500 g/mL; ⬎500, but ⱕ2000 g/mL; and ⬎2000 g/mL. Ten isolates of each susceptibility phenotype were selected for synergy testing.
Phase 2: Individual drug time-kill studies All 30 isolates plus the two control strains were tested against ampicillin and gentamicin alone. The ampicillin concentrations tested were 1⁄2 the MIC, the MIC and twice the MIC, based on the results obtained from the agar dilution screening of phase 1. The concentrations of gentamicin tested were 2 and 4 g/mL. These levels were selected to represent clinically achievable mean serum or tissue concentrations when using low (synergistic—1 mg/kg/dose) and usual (therapeutic—1.5 to 2.0 mg/kg/dose) gentamicin given by a standard dosing schedule for use in combination with ampicillin when treating serious enterococcal infections. A time-kill method was used, modeled after the approach for bactericidal testing described by Peterson and Shanholtzer (Peterson and Shanholtzer 1992; Swenson et al. 1995b). Dilutions of antibiotic stock solutions were prepared in cation supplemented Mueller Hinton Broth, leaving a final volume in each tube of 2 mL (Swenson et al. 1999). Next, the organ-
Aminoglycoside Synergy Determination isms were adjusted to match a 0.5 McFarland turbidity standard, and 0.1 mL of this inoculum was added to each tube with a calibrated pipette, just below the surface to produce a recommended density of 5 ⫻ 106–1 ⫻ 107 CFU/mL (Haden et al. 1994; MederskiSamoraj and Murray 1983). Gentle mixing was achieved by flushing 4 –5 times using the inoculating pipette. A positive growth control tube and a negative sterility control tube were run with each isolate. All tubes were incubated at 35°C for a full 24 h, without agitation.
Phase 3: Synergism time-kill studies The 30 isolates plus controls were tested against combinations of ampicillin and gentamicin. The concentrations run for ampicillin were 1⁄2 the MIC and 1⁄4 the MIC (Knapp and Moody 1992). Ampicillin was chosen at these concentrations since some strains were reduced ⬎3 log10 by ampicillin alone when tested at or twice the MIC in phase 2. The concentrations of gentamicin tested were again 2 and 4 g/mL. Here, 1 mL of ampicillin containing broth was combined with 1 mL of gentamicin containing broth in the various concentration combinations tested. The final volume in each tube was 2 mL.
Sampling Baseline (0 h) colony counts were determined by sampling the growth control tube at the time of inoculation for each isolate. For time-kill determinations, sampling of all drug-containing tubes was performed at 3, 6, and 24 h by removing a 0.1 mL aliquot from each tube and plating it to a single blood agar plate (Peterson and Shanholtzer 1992). The blood agar plates were lawned, and incubated for 20 –24 h (Shanholtzer et al. 1984). After incubation, colony counts were made and results were recorded.
MIC/MBC determination Testing was done using a standard broth macrodilution technique used in our laboratory (Hacek et al. 1999). At the end of 24 h, all tubes were inspected for the MIC, which was defined as the first tube in the series to show no trace of visible growth. The colony count cutoff for bactericidal activity determination (24 h minimum bactericidal concentration; MBC) in the testing was done using the formula n ⫹ 2公n, recommended by Anhalt et al. (1980). This determined a quantitative endpoint that includes 95% confidence limits for 99.9% killing. In this calculation, n is 0.1% of the preincubation colony count and n ⫹ 2公n is the corrected MBC cut-off point. The first dilution that grows less than or equal to the corrected
221 MBC cut-off point represents a 99.9% colony count reduction indicating bactericidal activity. All testing was performed in duplicate.
RESULTS Between December 1, 1993 and December 1, 1996, we tested 4,411 enterococcal strains against gentamicin at 500 g/mL and 2,000 g/mL. Of these, a total of 3,132 (71%) were inhibited by 500 g/mL of gentamicin, and 441 (10%) of the isolates had gentamicin MICs over 500, but less than 2,000 g/mL. A total of 838 (19%) isolates were resistant to 2,000 g/mL of gentamicin. Table 1 details the MICs of the individual agents, and the synergy data of the drugs combined for our initial 30 test organisms. Figure 1 depicts the kill curve synergy results when ampicillin and gentamicin (at 2 g/mL) were tested alone and in combination. Each isolate was resistant to gentamicin at 4 g/mL when it was tested as an individual agent. In the gentamicin ⱕ500 g/mL group, we found that 7 of 10 strains demonstrated synergy as manifested by a ⱖ2 log10 increase in killing versus the effect of ampicillin alone, when tested at 1⁄2 MIC for ampicillin (Table 1, Figure 1a). In the gentamicin 500 –2000 g/mL group, none of the 10 isolates demonstrated synergy (Figure 1b), with the same results found in the gentamicin ⬎2000 g/mL group (Table 1). We also found that gentamicin at 2 g/mL was equivalent to a higher level of 4 g/mL for enhancing in vitro killing by ampicillin. For the gentamicin, ⱕ500 g/mL group, the same seven isolates that demonstrated synergy at 2 g/mL showed synergy at 4 g/mL. The group of eight strains with reduced sensitivity to ampicillin interestingly had different, unpredictable results. These are summarized in Table 2. The seven clinically evaluable patients all received gentamicin combined with ampicillin or ampicillin/sulbactam, with the ampicillin or ampicillin/sulbactam given at very high dose (20 to 30 g per day by continuous infusion) to achieve serum ampicillin levels approaching 200 g/mL. Of these, 50% of the six patients with a successful outcome had strains of E. faecium sensitive to 500 g/mL gentamicin, and 50% had strains resistant to that concentration. The single treatment failure was attributable to infection with a strain sensitive to gentamicin at the level of 500 g/mL. None of the eight isolates showed susceptibility to ampicillin/gentamicin synergy as tested by time-kill curves, whereas all eight demonstrated this phenomenon when assessed by a ⱖfourfold reduction in MBC (Table 2).
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TABLE 1 Strain Susceptibility to Ampicillin and Gentamicin, Alone, and in Combination Isolate Designation
Ampicillin MIC (in g/mL)
Susceptible Control 32 33 34 35 36 37 38 39 40 42 EF599 F255 2 3 4 5 7 8 9 10 16 17 19 20 21 22 23 26 51 52 Resistant Control
1 2 2 1 2 1 1 1 1 2 2 64 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 1 2 128 1 32
Gentamicin Sensitivity Phenotype ⱕ500 (MIC ⫽ 8 g/mL) ⱕ500 (MIC ⫽ 8 g/mL) ⱕ500 (MIC ⫽ 16 g/mL) ⱕ500 (MIC ⫽ 8 g/mL) ⱕ500 (MIC ⫽ 8 g/mL) ⱕ500 (MIC ⫽ 16 g/mL) ⱕ500 (MIC ⱖ 16 g/mL) ⱕ500 (MIC ⫽ 8 g/mL) ⱕ500 (MIC ⫽ 8 g/mL) ⱕ500 (MIC ⱖ 16 g/mL) ⱕ500 (MIC ⱖ 16 g/mL) ⬎500 ⬍2000 ⬎500 ⬍2000 ⬎500 ⬍2000 ⬎500 ⬍2000 ⬎500 ⬍2000 ⬎500 ⬍2000 ⬎500 ⬍2000 ⬎500 ⬍2000 ⬎500 ⬍2000 ⬎500 ⬍2000 ⬎2000 ⬎2000 ⬎2000 ⬎2000 ⬎2000 ⬎2000 ⬎2000 ⬎2000 ⬎2000 ⬎2000 ⬎2000
DISCUSSION The accurate detection of enterococcal strains that are susceptible to the synergistic activity of aminoglycosides is important because it guides clinical treatment and helps ensure that only those patients who can benefit from such therapy will receive it. The mechanism of synergy for this use of gentamicin is unique. Aminoglycosides alone are inherently poorly active against enterococci, thought largely attributable to limited penetration into the bacterial cell. When an aminoglycoside is combined with ampicillin, the -lactam is thought to injure the cell wall, facilitating aminoglycoside uptake and leading to cell death (Zembower et al. 1998). In the report by Swenson et al., well done phenotypic synergy data, based on classic in vitro testing for bactericidal activity, was presented for streptomycin (1995a), so we did not repeat that work. However, they performed limited bactericidal testing with gentamicin because of the good correlation found between molecular
Effect of Gentamicin
Demonstration of Synergy
ⱖ3 log102 ⱖ3 log102 ⱖ3 log102 1 log102 ⱖ3 log102 no change no change 2 log102 ⱖ3 log102 ⱖ3 log102 ⱖ3 log102 1 log102 no change no change no change no change no change no change no change no change no change no change no change no change no change no change no change no change no change no change no change no change
yes yes yes no yes no no yes yes yes yes no no no no no no no no no no no no no no no no no no no no no
characterization and agar plate screening. We undertook this current study to validate their conclusions for gentamicin by examining results from traditional kill curves on enterococcal strains, including those with gentamicin MICs ⬎500 and ⱕ2,000 g/mL. The results support the recommended screening breakpoint of 500 g/mL for gentamicin as a predictor of synergy with a cell-wall active agent such as ampicillin, in enterococci with usual ampicillin susceptibility, because no synergy was seen in any of the 20 isolates that were not susceptible to gentamicin at that level. Based on our clinical laboratory screening data, using this lower gentamicin concentration eliminates the administration of gentamicin to some 10% of patients who would not likely benefit from such therapy, but may receive it if 2,000 g/mL is used as a breakpoint for determining synergy. Our tests done on strains of E. faecium with reduced sensitivity to ampicillin do not support the application of high level (500 g/mL) gentamicin
Aminoglycoside Synergy Determination
223
FIGURE 1. a. Kill curve synergy results for the gentamicin ⱕ500 g/mL group when ampicillin (at 1⁄2 MIC) and gentamicin (at 2 g/mL) were tested alone and in combination. b. Kill curve synergy results for the gentamicin ⬎500 and ⱕ2000 g/mL group when ampicillin (at 1⁄2 MIC) and gentamicin (at 2 g/mL) were tested alone and in combination.
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224
TABLE 2 Susceptibility Data on Eight Strains of E. faecium with Reduced Sensitivity to Ampicillin and Known Clinical Outcome Isolate Designation
Ampicillin MIC/MBC (in g/mL)
Gentamicin Sensitivity Phenotype
EF1 EF2 EF3 EF4 EF5 EF6 EF7 EF8
128/⬎256 32/⬎256 128/⬎256 256/⬎256 128/⬎256 128/⬎256 128/⬎256 128/⬎256
ⱕ500 ⬎500 ⱕ500 ⬎500 ⬎500 ⱕ500 ⬎500 ⱕ500
Time-kill Synergya no no no no no no no no
change change change change change change change change
MBC Reduction Synergyb
Clinical Outcomec
yes yes yes yes yes yes yes yes
Cured Cured Failed Unevaluable Cured Cured Cured Cured
a Synergy defined in text as a ⱖ2 log10 reduction in colony counts from ampicillin alone using an inoculum density of 5 ⫻ 106 to 1 ⫻ 107 CFU/mL. b Synergy defined as ⱖ4-fold reduction (ⱖ2 doubling dilutions) in MBC for ampicillin plus gentamicin (at 2 g/mL) compared to ampicillin alone using an inoculum density of 1–5 ⫻ 105 CFU/mL. c Cure defined as clinical and microbiologic eradication; failure defined as ongoing bacteremia; unevaluable defined as death with less than 48 h of therapy.
screening as an indication for synergistic action on such isolates. Using a battery of eight enterococci, whose clinical therapy outcome was known (Stosor et al. 1996), demonstrated that screening at 500 g gentamicin/mL agar, time-kill analysis for synergy, or a fourfold reduction in MBC activity had no correlation with therapeutic outcome. Furthermore, none of the three tests assessed to predict the utility of a synergistic combination of ampicillin and gentamicin correlated with each other (Table 2). Therefore, any of the commonly used tests to predict synergy in enterococcal isolates that would be considered resistant to ampicillin using current NCCLS criteria cannot be recommended.
The results also showed that a gentamicin concentration of 2 g/mL was equivalent to one of 4 g/ mL, when combined with ampicillin in strains where screening for high level aminoglycoside sensitivity is appropriate. This supports the concept that lower (less toxic) “synergistic” doses of gentamicin are as effective as higher “therapeutic” dose therapy when treating enterococcal infections. Overall, these results validate the NCCLS currently recommended breakpoint for gentamicin of 500 g/mL as a predictor of synergy with a cell-wall active agent for ampicillin susceptible enterococci, and suggest that lower ‘synergistic’ doses of gentamicin are sufficient in clinical practice.
REFERENCES Anhalt JP, Sabath LD, Barry AL (1980) Special tests: bactericidal activity, activity of antimicrobics in combination, and detection of -lactamase production. In: Manual of Clinical Microbiology, 3rd ed. Eds, Lennette EH, Ballows A, and Hausler, WJ, Jr. Washington, DC: The American Society for Microbiology, p. 478. Caputo GM, Singer M, White S, Weitekamp MR (1993) Infections due to antibiotic-resistant gram-positive cocci. J Gen Int Med 8:626–634. Fasola EL, Moody JA, Shanholtzer CJ, Peterson LR (1994) Bactericidal action of gentamicin against enterococci that are sensitive, or exhibit low- or high-level resistance to gentamicin. Diagn Microbiol Infect Dis 19:57–60. Hacek DM, Dressel DC, Peterson LR (1999) Highly reproducible bactericidal activity test results using a modified National Committee for Clinical Laboratory Standards broth macrodilution technique. J Clin Microbiol 37:1881–1884. Haden MK, Koenig GI, Trenholme GM (1994) Bactericidal
activities of antibiotics against vancomycin-resistant Enterococcus faecium blood isolates and synergistic activities of combinations. Antimicrob Agents Chemother 38: 1225–1229. Knapp C, Moody JA (1992) Tests to assess bactericidal activity. In: Clinical Microbiology Procedures Handbook. Ed, Isenberg HD. Washington, DC: American Society for Microbiology, pp. 5.16.1–5.16.33. Lopardo HA, Venuta ME, Rubeglio EA (1995) Penicillin resistance and aminoglycoside-penicillin synergy in enterococci. Chemotherapy 41:165–171. Mederski-Samoraj BD, Murray BE (1983) High level resistance to gentamicin in clinical isolates of enterococci. J Infect Dis 147:751–757. Moellering, Jr RC (1995) Enterococcus species, Streptococcus bovis, and Leuconostoc species. In: Principles and Practice of Infectious Diseases, 4th ed. Eds, Mandell GL, Bennett JE, and Dolin R. New York: Churchill Livingston, pp. 1826– 1835.
Aminoglycoside Synergy Determination
Murray BE (1990) The life and times of enterococcus. Clin Microbiol Rev 3:46–65. National Committee for Clinical Laboratory Standards (1997) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically—Approved standard. Document M7–A4. National Committee for Clinical Laboratory Standards, Villanova, PA. Peterson LR, Shanholtzer CJ (1992) Tests for bactericidal effects of antimicrobial agents: Technical performance and clinical relevance. Clin Microbiol Rev 5:420–432. Shanholtzer CJ, Peterson LR, Mohn ML, Moody JA, Gerding DN (1984) MBCs for Staphylococcus aureus as determined by macrodilution and microdilution techniques. Antimicrob Agents Chemother 26:214–219. Simmons NA, Ball AP, Eykyn SJ, Littler WA, McGowan DA, Oakley CM, Shanson D (1998) Antibiotic treatment of streptococcal, enterococcal, and staphylococcal endocarditis. Heart 79:207–210. Stosor V, Peterson LR, Postelnick M, Noskin GA (1996) Therapy of vancomycin resistant Enterococcus faecium sepsis: A novel strategy. In: Program and Abstracts. Thirty-fourth Annual Meeting of the Infectious Diseases Society of America, New Orleans, LA, September 18–20, 1996. Abstract 170.
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Swenson JM, Ferraro MJ, Sahm DF, Clark NC, Culver DH, Tenover FC, the National Committee for Clinical Laboratory Standards Study Group on Enterococci (1995a) Multilaboratory evaluation of screening methods for detection of high-level aminoglycoside resistance in enterococci. J Clin Microbiol 33:3008– 3018. Swenson JM, Hindler J, Peterson LR (1995b) Special tests for detecting antibacterial resistance. In: Manual of Clinical Microbiology, 6th ed. Eds, Murray PR, Baron EJ, Pfaller MA, Tenover FC, and Yolken RH. Washington, DC: American Society for Microbiology, pp. 1356– 1367. Swenson JM, Hindler J, Peterson LR (1999) Special phenotypic methods for detecting antibacterial resistance. In: Manual of Clinical Microbiology, 7th ed. Eds, Murray PR, Baron EJ, Pfaller MA, Tenover FC, and Yolken RH. Washington, DC: American Society for Microbiology, pp. 1563–1577. Zembower TR, Noskin GA, Postelnick MJ, Nguyen C, Peterson LR (1998) The utility of aminoglycosides in an era of emerging drug resistance: A review. Internat J Antimicrob Agents 10:95–105.
Synergistic Effect of Gentamicin Plus Ampicillin on Enterococci with Differing Sensitivity to Gentamicin: A Phenotypic Assessment of NCCLS Guidelines Dana C. Dressel, Michelle A. Tornatore-Reuscher, Cynthia R. Boschman, Valentina Stosor, Teresa Zembower, Michael J. Postelnick, Gary A. Noskin, and Lance R. Peterson
Between December 1, 1993, and December 1, 1996, we tested 4,411 isolates of Enterococcus sp. at gentamicin concentrations of 500 g/mL and 2000 g/mL using agar dilution to phenotypically categorize them into 3 groups: those with a MIC ⱕ500 g/mL (n ⫽ 3,132; 71%); a MIC ⬎500, but ⱕ2000 g/mL (n ⫽ 441; 10%); and those with a MIC ⬎2000 g/mL (n ⫽ 838; 19%). Ten unique strains of each phenotype were tested to determine which gentamicin concentration was the best in vitro predictor of synergy with ampicillin. Testing was done by a time-kill method using clinically achievable levels of ampicillin and gentamicin. We found that for the gentamicin MIC ⱕ500 g/mL group, 7 of 10 isolates demonstrated synergy with ampicillin as manifested by a ⱖ2 log10 increase in killing versus the effect of ampicillin alone (at 1⁄2 the MIC for ampicillin). In the group sensitive to a gentamicin MIC range
between ⬎500 and ⱕ2,000 g/mL, none of the 10 isolates demonstrated synergy. Absence of synergy was also found in the group resistant to 2,000 g/mL of gentamicin. Assessment of eight additional enterococcal isolates with reduced sensitivity to ampicillin (MIC from 32–256 g/mL) found no correlation between gentamicin sensitivity at 500 g/mL and any in vitro test for synergy, nor with clinical therapeutic outcome. Gentamicin at 2g/mL combined with ampicillin was as effective in enhancing killing as a higher level of 4 g/mL. These findings validate the current NCCLS guideline for predicting synergistic activity against enterococci in strains with usual susceptibility to ampicillin, and suggest that a therapeutic level less than maximal recommended dosing is sufficient when using gentamicin in this setting. © 1999 Elsevier Science Inc.
Northwestern Memorial Hospital and Northwestern University supported this work. Presented, in part, at the 97th General Meeting of the American Society for Microbiology, May 4–8, 1997, Miami Beach, FL. Abstract C147. From the Clinical Microbiology Division, Northwestern Memorial Hospital (DCD, CRB, VS, TZ, GAN, LRP); Department of Pathology, Northwestern University Medical School (MAT-R, CRB, VS, TZ, GAN, LRP); Infectious Disease Division, Northwestern University Medical School (VS, TZ, GAN, LRP); and Pharmacy Department, Northwestern Memorial Hospital (MJP), Chicago, Illinois 60611 USA. Address reprint requests to: Dr. Lance R. Peterson, Clinical Microbiology, Wesley Pavilion, Room 565, Northwestern Memorial Hospital, 250 E. Superior Street, Chicago, IL 60611.
INTRODUCTION
DIAGN MICROBIOL INFECT DIS 1999;35:219 –225 © 1999 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
It has long been a standard of care that bactericidal therapy consisting of an aminoglycoside in combination with a cell-wall active agent such as ampicillin is required for effective treatment of serious infections with enterococci (Caputo et al. 1993; Lopardo et al. 1995; Moellering 1995; Simmons et al. 1998). For more than 20 years it was recognized that demonstrating susceptibility of enterococci to high concentrations of an aminoglycoside, usually ⱕ2000 g/
0732-8893/99/$–see front matter PII S0732-8893(99)00088-7
D.C. Dressel et al.
220 mL, could be used to predict in vitro synergy (Murray 1990). Resistance to a higher level than this is an indication that an aminoglycoside-inactivating enzyme is likely present within the enterococcal cell. The synergistic action is distinct from a direct effect of gentamicin alone on enterococci (Fasola et al. 1994). However, there has been considerable discordance as to which in vitro concentration of gentamicin, 500 g/mL or 2000 g/mL, best predicts synergistic killing (i.e., absence of an inactivating enzyme) useful in clinical therapy. Most recently, Swenson et al., correlated molecular characterization for aminoglycoside susceptibility with MICs, and determined that the best screening method for detecting highlevel resistance was gentamicin in agar or broth at a concentration of 500 g/mL (1995a). The purpose of our study was to correlate kill curve results for synergy with gentamicin MICs to phenotypically confirm which cut-off, 500 g/mL or 2000 g/mL of gentamicin, best predicts synergy. Additionally, we sought to determine if low level “synergistic” or high level “therapeutic” clinical dosing is necessary to demonstrate in vitro synergy using time-kill studies.
MATERIALS AND METHODS Organisms The organisms tested were unique clinical isolates recovered by the microbiology laboratory at Northwestern Memorial Hospital, Chicago, IL, between December 1, 1993, and December 1, 1996. Enterococci were identified to species level based on colonial morphology, Gram-stain reaction, catalase, growth in 6.5% NaCl, ability to grow in bile and hydrolyze esculin, and ability to ferment the sugars arabinose, raffinose, sorbitol and mannitol. All isolates were saved frozen at ⫺85°C in Mueller Hinton Broth with glycerol. They were removed from freezer storage and subcultured to blood agar twice prior to testing. Enterococcus faecalis ATCC 29212 was used as a susceptible (synergistic) control isolate, and a strain known to be resistant to high-level (MIC ⬎2000 g/mL for gentamicin and streptomycin) aminoglycoside concentrations was chosen as a resistant control strain. They were included in every test run. After completion of the initial study, we tested eight additional E. faecium strains from patients with persistent bacteremia (Stosor et al. 1996). These strains were selected for time-kill analysis because collectively they had reduced susceptibility to ampicillin, variable sensitivity to gentamicin, and a known outcome of therapy using ampicillin or ampicillin/sulbactam, either agent given with gentamicin (Stosor et al. 1996).
Inoculum preparation For each isolate tested, the inoculum was prepared from 3 to 5 distinct, 18 –24 h old colonies grown in 5 mL aliquots of Trypticase Soy Broth. These were incubated for 3–5 h to achieve a turbid suspension. Just prior to testing, the broth was adjusted to a 0.5-McFarland turbidity standard to give approximately 1 ⫻ 108 CFU/mL. Then, 0.1 mL of the prepared inoculum was added to macrobroth dilution tubes as described below.
Antimicrobial agents Ampicillin trihydrate was generously provided by the Bristol-Myers Squibb Company (Evansville, IN), and gentamicin sulfate was purchased from U.S. Biochemical Corporation (Cleveland, OH).
Phase 1: Phenotype categorization Isolates selected for testing were chosen based upon results from prior agar dilution done following the NCCLS standard method (National Committee for Clinical Laboratory Standards 1997). This testing was done using a Steers’ replicator to spot inoculum onto agar containing either 500 or 2000 g/mL of gentamicin. Plates were incubated for 22 to 24 h and observed for visible growth. Isolates were then separated into three groups based on susceptibility to specific concentrations of gentamicin: ⱕ500 g/mL; ⬎500, but ⱕ2000 g/mL; and ⬎2000 g/mL. Ten isolates of each susceptibility phenotype were selected for synergy testing.
Phase 2: Individual drug time-kill studies All 30 isolates plus the two control strains were tested against ampicillin and gentamicin alone. The ampicillin concentrations tested were 1⁄2 the MIC, the MIC and twice the MIC, based on the results obtained from the agar dilution screening of phase 1. The concentrations of gentamicin tested were 2 and 4 g/mL. These levels were selected to represent clinically achievable mean serum or tissue concentrations when using low (synergistic—1 mg/kg/dose) and usual (therapeutic—1.5 to 2.0 mg/kg/dose) gentamicin given by a standard dosing schedule for use in combination with ampicillin when treating serious enterococcal infections. A time-kill method was used, modeled after the approach for bactericidal testing described by Peterson and Shanholtzer (Peterson and Shanholtzer 1992; Swenson et al. 1995b). Dilutions of antibiotic stock solutions were prepared in cation supplemented Mueller Hinton Broth, leaving a final volume in each tube of 2 mL (Swenson et al. 1999). Next, the organ-
Aminoglycoside Synergy Determination isms were adjusted to match a 0.5 McFarland turbidity standard, and 0.1 mL of this inoculum was added to each tube with a calibrated pipette, just below the surface to produce a recommended density of 5 ⫻ 106–1 ⫻ 107 CFU/mL (Haden et al. 1994; MederskiSamoraj and Murray 1983). Gentle mixing was achieved by flushing 4 –5 times using the inoculating pipette. A positive growth control tube and a negative sterility control tube were run with each isolate. All tubes were incubated at 35°C for a full 24 h, without agitation.
Phase 3: Synergism time-kill studies The 30 isolates plus controls were tested against combinations of ampicillin and gentamicin. The concentrations run for ampicillin were 1⁄2 the MIC and 1⁄4 the MIC (Knapp and Moody 1992). Ampicillin was chosen at these concentrations since some strains were reduced ⬎3 log10 by ampicillin alone when tested at or twice the MIC in phase 2. The concentrations of gentamicin tested were again 2 and 4 g/mL. Here, 1 mL of ampicillin containing broth was combined with 1 mL of gentamicin containing broth in the various concentration combinations tested. The final volume in each tube was 2 mL.
Sampling Baseline (0 h) colony counts were determined by sampling the growth control tube at the time of inoculation for each isolate. For time-kill determinations, sampling of all drug-containing tubes was performed at 3, 6, and 24 h by removing a 0.1 mL aliquot from each tube and plating it to a single blood agar plate (Peterson and Shanholtzer 1992). The blood agar plates were lawned, and incubated for 20 –24 h (Shanholtzer et al. 1984). After incubation, colony counts were made and results were recorded.
MIC/MBC determination Testing was done using a standard broth macrodilution technique used in our laboratory (Hacek et al. 1999). At the end of 24 h, all tubes were inspected for the MIC, which was defined as the first tube in the series to show no trace of visible growth. The colony count cutoff for bactericidal activity determination (24 h minimum bactericidal concentration; MBC) in the testing was done using the formula n ⫹ 2公n, recommended by Anhalt et al. (1980). This determined a quantitative endpoint that includes 95% confidence limits for 99.9% killing. In this calculation, n is 0.1% of the preincubation colony count and n ⫹ 2公n is the corrected MBC cut-off point. The first dilution that grows less than or equal to the corrected
221 MBC cut-off point represents a 99.9% colony count reduction indicating bactericidal activity. All testing was performed in duplicate.
RESULTS Between December 1, 1993 and December 1, 1996, we tested 4,411 enterococcal strains against gentamicin at 500 g/mL and 2,000 g/mL. Of these, a total of 3,132 (71%) were inhibited by 500 g/mL of gentamicin, and 441 (10%) of the isolates had gentamicin MICs over 500, but less than 2,000 g/mL. A total of 838 (19%) isolates were resistant to 2,000 g/mL of gentamicin. Table 1 details the MICs of the individual agents, and the synergy data of the drugs combined for our initial 30 test organisms. Figure 1 depicts the kill curve synergy results when ampicillin and gentamicin (at 2 g/mL) were tested alone and in combination. Each isolate was resistant to gentamicin at 4 g/mL when it was tested as an individual agent. In the gentamicin ⱕ500 g/mL group, we found that 7 of 10 strains demonstrated synergy as manifested by a ⱖ2 log10 increase in killing versus the effect of ampicillin alone, when tested at 1⁄2 MIC for ampicillin (Table 1, Figure 1a). In the gentamicin 500 –2000 g/mL group, none of the 10 isolates demonstrated synergy (Figure 1b), with the same results found in the gentamicin ⬎2000 g/mL group (Table 1). We also found that gentamicin at 2 g/mL was equivalent to a higher level of 4 g/mL for enhancing in vitro killing by ampicillin. For the gentamicin, ⱕ500 g/mL group, the same seven isolates that demonstrated synergy at 2 g/mL showed synergy at 4 g/mL. The group of eight strains with reduced sensitivity to ampicillin interestingly had different, unpredictable results. These are summarized in Table 2. The seven clinically evaluable patients all received gentamicin combined with ampicillin or ampicillin/sulbactam, with the ampicillin or ampicillin/sulbactam given at very high dose (20 to 30 g per day by continuous infusion) to achieve serum ampicillin levels approaching 200 g/mL. Of these, 50% of the six patients with a successful outcome had strains of E. faecium sensitive to 500 g/mL gentamicin, and 50% had strains resistant to that concentration. The single treatment failure was attributable to infection with a strain sensitive to gentamicin at the level of 500 g/mL. None of the eight isolates showed susceptibility to ampicillin/gentamicin synergy as tested by time-kill curves, whereas all eight demonstrated this phenomenon when assessed by a ⱖfourfold reduction in MBC (Table 2).
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TABLE 1 Strain Susceptibility to Ampicillin and Gentamicin, Alone, and in Combination Isolate Designation
Ampicillin MIC (in g/mL)
Susceptible Control 32 33 34 35 36 37 38 39 40 42 EF599 F255 2 3 4 5 7 8 9 10 16 17 19 20 21 22 23 26 51 52 Resistant Control
1 2 2 1 2 1 1 1 1 2 2 64 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 1 2 128 1 32
Gentamicin Sensitivity Phenotype ⱕ500 (MIC ⫽ 8 g/mL) ⱕ500 (MIC ⫽ 8 g/mL) ⱕ500 (MIC ⫽ 16 g/mL) ⱕ500 (MIC ⫽ 8 g/mL) ⱕ500 (MIC ⫽ 8 g/mL) ⱕ500 (MIC ⫽ 16 g/mL) ⱕ500 (MIC ⱖ 16 g/mL) ⱕ500 (MIC ⫽ 8 g/mL) ⱕ500 (MIC ⫽ 8 g/mL) ⱕ500 (MIC ⱖ 16 g/mL) ⱕ500 (MIC ⱖ 16 g/mL) ⬎500 ⬍2000 ⬎500 ⬍2000 ⬎500 ⬍2000 ⬎500 ⬍2000 ⬎500 ⬍2000 ⬎500 ⬍2000 ⬎500 ⬍2000 ⬎500 ⬍2000 ⬎500 ⬍2000 ⬎500 ⬍2000 ⬎2000 ⬎2000 ⬎2000 ⬎2000 ⬎2000 ⬎2000 ⬎2000 ⬎2000 ⬎2000 ⬎2000 ⬎2000
DISCUSSION The accurate detection of enterococcal strains that are susceptible to the synergistic activity of aminoglycosides is important because it guides clinical treatment and helps ensure that only those patients who can benefit from such therapy will receive it. The mechanism of synergy for this use of gentamicin is unique. Aminoglycosides alone are inherently poorly active against enterococci, thought largely attributable to limited penetration into the bacterial cell. When an aminoglycoside is combined with ampicillin, the -lactam is thought to injure the cell wall, facilitating aminoglycoside uptake and leading to cell death (Zembower et al. 1998). In the report by Swenson et al., well done phenotypic synergy data, based on classic in vitro testing for bactericidal activity, was presented for streptomycin (1995a), so we did not repeat that work. However, they performed limited bactericidal testing with gentamicin because of the good correlation found between molecular
Effect of Gentamicin
Demonstration of Synergy
ⱖ3 log102 ⱖ3 log102 ⱖ3 log102 1 log102 ⱖ3 log102 no change no change 2 log102 ⱖ3 log102 ⱖ3 log102 ⱖ3 log102 1 log102 no change no change no change no change no change no change no change no change no change no change no change no change no change no change no change no change no change no change no change no change
yes yes yes no yes no no yes yes yes yes no no no no no no no no no no no no no no no no no no no no no
characterization and agar plate screening. We undertook this current study to validate their conclusions for gentamicin by examining results from traditional kill curves on enterococcal strains, including those with gentamicin MICs ⬎500 and ⱕ2,000 g/mL. The results support the recommended screening breakpoint of 500 g/mL for gentamicin as a predictor of synergy with a cell-wall active agent such as ampicillin, in enterococci with usual ampicillin susceptibility, because no synergy was seen in any of the 20 isolates that were not susceptible to gentamicin at that level. Based on our clinical laboratory screening data, using this lower gentamicin concentration eliminates the administration of gentamicin to some 10% of patients who would not likely benefit from such therapy, but may receive it if 2,000 g/mL is used as a breakpoint for determining synergy. Our tests done on strains of E. faecium with reduced sensitivity to ampicillin do not support the application of high level (500 g/mL) gentamicin
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FIGURE 1. a. Kill curve synergy results for the gentamicin ⱕ500 g/mL group when ampicillin (at 1⁄2 MIC) and gentamicin (at 2 g/mL) were tested alone and in combination. b. Kill curve synergy results for the gentamicin ⬎500 and ⱕ2000 g/mL group when ampicillin (at 1⁄2 MIC) and gentamicin (at 2 g/mL) were tested alone and in combination.
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TABLE 2 Susceptibility Data on Eight Strains of E. faecium with Reduced Sensitivity to Ampicillin and Known Clinical Outcome Isolate Designation
Ampicillin MIC/MBC (in g/mL)
Gentamicin Sensitivity Phenotype
EF1 EF2 EF3 EF4 EF5 EF6 EF7 EF8
128/⬎256 32/⬎256 128/⬎256 256/⬎256 128/⬎256 128/⬎256 128/⬎256 128/⬎256
ⱕ500 ⬎500 ⱕ500 ⬎500 ⬎500 ⱕ500 ⬎500 ⱕ500
Time-kill Synergya no no no no no no no no
change change change change change change change change
MBC Reduction Synergyb
Clinical Outcomec
yes yes yes yes yes yes yes yes
Cured Cured Failed Unevaluable Cured Cured Cured Cured
a Synergy defined in text as a ⱖ2 log10 reduction in colony counts from ampicillin alone using an inoculum density of 5 ⫻ 106 to 1 ⫻ 107 CFU/mL. b Synergy defined as ⱖ4-fold reduction (ⱖ2 doubling dilutions) in MBC for ampicillin plus gentamicin (at 2 g/mL) compared to ampicillin alone using an inoculum density of 1–5 ⫻ 105 CFU/mL. c Cure defined as clinical and microbiologic eradication; failure defined as ongoing bacteremia; unevaluable defined as death with less than 48 h of therapy.
screening as an indication for synergistic action on such isolates. Using a battery of eight enterococci, whose clinical therapy outcome was known (Stosor et al. 1996), demonstrated that screening at 500 g gentamicin/mL agar, time-kill analysis for synergy, or a fourfold reduction in MBC activity had no correlation with therapeutic outcome. Furthermore, none of the three tests assessed to predict the utility of a synergistic combination of ampicillin and gentamicin correlated with each other (Table 2). Therefore, any of the commonly used tests to predict synergy in enterococcal isolates that would be considered resistant to ampicillin using current NCCLS criteria cannot be recommended.
The results also showed that a gentamicin concentration of 2 g/mL was equivalent to one of 4 g/ mL, when combined with ampicillin in strains where screening for high level aminoglycoside sensitivity is appropriate. This supports the concept that lower (less toxic) “synergistic” doses of gentamicin are as effective as higher “therapeutic” dose therapy when treating enterococcal infections. Overall, these results validate the NCCLS currently recommended breakpoint for gentamicin of 500 g/mL as a predictor of synergy with a cell-wall active agent for ampicillin susceptible enterococci, and suggest that lower ‘synergistic’ doses of gentamicin are sufficient in clinical practice.
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Murray BE (1990) The life and times of enterococcus. Clin Microbiol Rev 3:46–65. National Committee for Clinical Laboratory Standards (1997) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically—Approved standard. Document M7–A4. National Committee for Clinical Laboratory Standards, Villanova, PA. Peterson LR, Shanholtzer CJ (1992) Tests for bactericidal effects of antimicrobial agents: Technical performance and clinical relevance. Clin Microbiol Rev 5:420–432. Shanholtzer CJ, Peterson LR, Mohn ML, Moody JA, Gerding DN (1984) MBCs for Staphylococcus aureus as determined by macrodilution and microdilution techniques. Antimicrob Agents Chemother 26:214–219. Simmons NA, Ball AP, Eykyn SJ, Littler WA, McGowan DA, Oakley CM, Shanson D (1998) Antibiotic treatment of streptococcal, enterococcal, and staphylococcal endocarditis. Heart 79:207–210. Stosor V, Peterson LR, Postelnick M, Noskin GA (1996) Therapy of vancomycin resistant Enterococcus faecium sepsis: A novel strategy. In: Program and Abstracts. Thirty-fourth Annual Meeting of the Infectious Diseases Society of America, New Orleans, LA, September 18–20, 1996. Abstract 170.
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Swenson JM, Ferraro MJ, Sahm DF, Clark NC, Culver DH, Tenover FC, the National Committee for Clinical Laboratory Standards Study Group on Enterococci (1995a) Multilaboratory evaluation of screening methods for detection of high-level aminoglycoside resistance in enterococci. J Clin Microbiol 33:3008– 3018. Swenson JM, Hindler J, Peterson LR (1995b) Special tests for detecting antibacterial resistance. In: Manual of Clinical Microbiology, 6th ed. Eds, Murray PR, Baron EJ, Pfaller MA, Tenover FC, and Yolken RH. Washington, DC: American Society for Microbiology, pp. 1356– 1367. Swenson JM, Hindler J, Peterson LR (1999) Special phenotypic methods for detecting antibacterial resistance. In: Manual of Clinical Microbiology, 7th ed. Eds, Murray PR, Baron EJ, Pfaller MA, Tenover FC, and Yolken RH. Washington, DC: American Society for Microbiology, pp. 1563–1577. Zembower TR, Noskin GA, Postelnick MJ, Nguyen C, Peterson LR (1998) The utility of aminoglycosides in an era of emerging drug resistance: A review. Internat J Antimicrob Agents 10:95–105.