INTRODUCTION

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Enterococci are increasingly implicated as a cause of serious systemic infections, especially in debilitated hosts with lowered defense mechanisms (4, 21). The problem is complicated by the inherent drug resistance of these species as well as by the resistance which has recently developed to previously active drugs (1, 3-7, 10, 12, 18-21). Enterococcus faecalis, the most commonly occurring species in this group, has developed ampicillin resistance (both chromosomal and plasmid mediated), high-level aminoglycoside resistance, and (less commonly) glycopeptide resistance. Enterococcus faecium, the second most commonly occurring enterococcal species, is inherently more resistant than E. faecalis, with higher rates of glycopeptide resistance (1, 3-7, 10, 12, 18-21).

Previously published large multicenter surveys have documented good activities of piperacillin and piperacillin-tazobactam against randomly isolated E. faecalis strains compared to the activities of ticarcillin and ticarcillin-clavulanate (2, 8, 11, 13). The present study extends these studies by (i) examining the susceptibilities of 212 E. faecalis strains to piperacillin, piperacillin-tazobactam, ticarcillin, ticarcillin-clavulanate, ampicillin, ampicillin-sulbactam, vancomycin, and teicoplanin and (ii) testing the activities of the above compounds against selected E. faecalis strains by time-kill analysis.

	MATERIALS AND METHODS

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Bacteria and antimicrobial agents. The -Lactamase-negative strains used in this study were recent clinical isolates from the Hershey Medical Center and the University Hospitals of Cleveland, Ohio. -Lactamase-producing strains were obtained from L. Rice (Cleveland Veterans Administration Hospital, Cleveland, Ohio) and G. Eliopoulos (New England Deaconess Hospital, Boston, Mass.). Identification of strains was by standard methodology. Strains were stored at 70°C in double-strength litmus milk (Difco Laboratories, Detroit, Mich.) before being tested. Antimicrobial agents were obtained as follows: piperacillin and tazobactam, Wyeth Ayerst Laboratories, Philadelphia, Pa.; ticarcillin and clavulanate, SmithKline Beecham Laboratories, Philadelphia, Pa.; ampicillin and sulbactam, Pfizer, Inc., New York, N.Y.; vancomycin, Eli Lilly & Co., Indianapolis, Ind.; and teicoplanin, Marion Merrell Dow, Gerenzano, Italy.

Agar dilution MICs. For the testing of 212 strains, agar dilution by standard methodology (14) was performed with Mueller-Hinton agar. Tazobactam was added to piperacillin at a fixed ratio of 1:8 and a fixed concentration of 4 µg/ml, clavulanate was added to ticarcillin at a fixed concentration of 2 µg/ml, and sulbactam was added to ampicillin at a 1:2 ratio. Inocula were prepared by suspending growth from overnight cultures in sterile saline to a turbidity of approximately 0.5 McFarland standard. Final inocula contained 10⁴ CFU/spot. Plates were incubated overnight for all antibiotics except vancomycin; vancomycin plates were incubated for a full 24 h and inspected carefully for evidence of faint growth (14). The lowest concentration of antibiotic showing no growth was read as the MIC. Standard quality control strains were included in each run. -Lactamase testing was by the nitrocefin disk method (Cefinase; BBL Microbiology Systems, Cockeysville, Md.). For -lactamase-producing strains, -lactam agar dilution MIC determinations were repeated with inocula of 10⁶ CFU/spot (15). Breakpoints were those approved by the National Committee for Clinical Laboratory Standards (14), i.e., 8.0 for ampicillin and teicoplanin and 4.0 µg/ml for vancomycin; in the cases of piperacillin, piperacillin-tazobactam, ticarcillin, and ticarcillin-clavulanate, for which no approved breakpoints are available, 8.0 µg/ml was empirically chosen. For ampicillin-sulbactam, for which no approved breakpoint is available either, the approved ampicillin breakpoint was used.

Microdilution MICs. For 15 randomly selected strains (14 -lactamase negative and 1 -lactamase positive) examined by time-kill analysis, determination of microbroth dilution MICs was performed by NCCLS methodology (14) with cation-adjusted Mueller-Hinton broth (Difco). Suspensions (prepared as described above) were further diluted 1:10 to obtain final inocula of 5 × 10⁵ CFU/ml; for the one -lactamase-producing strain, an inoculum of 10⁷ CFU/ml was used. MICs were read after overnight incubation except for vancomycin, for which the MIC was read after 24 h. Quality controls were included for each run.

Time-kill studies. Time-kill studies were performed as described previously (17) with cation-adjusted Mueller-Hinton broth. Viability counts were performed at 0, 3, 6, 12, and 24 h. Data were analyzed by determining the number of strains which yielded a log₁₀ CFU/ml reduction of 1, 2, or 3 compared to counts at time zero, for all compounds at all time periods. Antimicrobial agents were considered bactericidal at the lowest concentration which reduced the original inoculum by 3 log₁₀ CFU/ml (99.9%) and were considered bacteriostatic if the inoculum was reduced by 0 to 3 log₁₀ CFU/ml. Antibiotic carryover was minimized by dilution, as described previously (17). All strains were tested with final inocula of 5 × 10⁵ to 5 × 10⁶ CFU/ml; additionally, one -lactamase-positive strain was tested at an inoculum of 1 × 10⁷ to 5 × 10⁷ CFU/ml (15). Piperacillin-tazobactam at a fixed inhibitor concentration of 4 µg/ml and ampicillin-sulbactam were tested only against one -lactamase-negative and one -lactamase-positive strain.

	RESULTS

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Of the 212 strains tested, 9 were -lactamase positive. MICs of the various drugs by the agar dilution method with the standard inoculum of 10⁴ CFU/spot are listed in Table 1. The MICs at which 50 and 90% of the isolates were inhibited (MIC₅₀s and MIC₉₀s, respectively) were as follows (µg/ml): piperacillin, 4.0 and 8.0; piperacillin-tazobactam, 4.0 and 8.0; ticarcillin, 64.0 and 128.0; ticarcillin-clavulanate, 64.0 and 128.0; ampicillin, 2.0 and 2.0; ampicillin-sulbactam, 1.0 and 2.0; vancomycin, 1.0 and 4.0; and teicoplanin, 0.5 and 1.0. Addition of tazobactam to piperacillin at a fixed concentration of 4.0 µg/ml yielded MICs which were slightly lower than those obtained with a 1:8 ratio. One strain had a reduced susceptibility to vancomycin, requiring an MIC of 16.0 µg/ml. MICs of other agents for this strain were as follows (µg/ml): piperacillin, 8.0; piperacillin-tazobactam, 8.0; ticarcillin, 256.0; ticarcillin-clavulanate, 256.0; ampicillin, 4.0; ampicillin-sulbactam, 4.0; and teicoplanin, 0.5. MICs of the -lactamase-negative strains which required raised piperacillin and piperacillin-tazobactam MICs (16.0 to 64.0 µg/ml) were as follows (µg/ml) ticarcillin ± clavulanate, 64.0 to >256.0 µg/ml; ampicillin ± sulbactam, 2.0 to 8.0 µg/ml, vancomycin, 1.0 to 4.0 µg/ml; teicoplanin, 0.5 to 1.0 µg/ml. When -lactamase-producing strains were tested using inocula of 10⁶ CFU/spot (Table 2), -lactam MICs were higher than those obtained with 10⁴ CFU/spot (piperacillin, 64 to >256 µg/ml; ticarcillin, 128 to >256 µg/ml; and ampicillin, 16 to 128 µg/ml); however, addition of -lactamase inhibitors to each of the three -lactams reduced their MICs to within 2 dilutions of those obtained at the lower inoculum (Table 2). Addition of tazobactam to piperacillin at a fixed concentration of 4 µg/ml gave MICs either identical or 1 to 2 dilutions lower than those obtained with the 1:8 ratio for -lactamase-positive strains.

View this table: [in this window] [in a new window]   TABLE 2.   -Lactam agar dilution MICs for nine -lactamase-positive strains

Using current NCCLS breakpoints with standard inocula of 10⁴ CFU/spot the susceptibilities of the strains were 100% to ampicillin with or without sulbactam, 99.5% to vancomycin, 100% to teicoplanin, 97.0% to piperacillin with or without tazobactam (8:1), 99.0% to piperacillin-tazobactam (4.0 µg/ml) and 0% to ticarcillin with or without clavulanate. All -lactamase-positive strains were resistant to piperacillin, ticarcillin, and ampicillin at the above breakpoints when the higher inoculum was used but were susceptible upon addition of inhibitors.

Microdilution MICs of drugs for strains tested by time-kill analysis are listed in Table 3. Microdilution MICs of selected strains were all within 1 dilution of those obtained by agar dilution. The results of time-kill analyses are presented in Table 4. Enterococci did not survive at higher numbers in the presence of concentrations above the MIC than they did at concentrations below the MIC. As can be seen, piperacillin-tazobactam was bactericidal (99.9% killing) for 14 of 15 organisms (including the -lactamase strain) after 24 h at four times the MIC and resulted in 90% killing of all 15 strains at two times the MIC. After 12 and 6 h, 90% killing of 14 and 13 strains, respectively, was found at two times the MIC. For the 14 -lactamase-negative strains, results for piperacillin-tazobactam were similar to those for piperacillin. Ampicillin resulted in 99.9% killing of 14 strains after 24 h at eight times the MIC, with 90% killing of all 15 strains at two times the MIC. After 12 and 6 h, 90% killing of 14 and 13 strains, respectively, was found at two times the MIC. For the 14 -lactamase-negative strains, results for piperacillin-tazobactam were similar to those for piperacillin. Ampicillin resulted in 99.9% killing of 14 strains after 24 h at eight times the MIC, with 90% killing of all 15 strains at two times the MIC. After 12 and 6 h, 90% killing of 14 and 13 strains, respectively, was found at two times the MIC. Killing by ticarcillin-clavulanate was slower than that observed for piperacillin-tazobactam, relative to the MIC, at all time periods: 99.9% killing of 12 strains was found at eight times the MIC after 24 h, with 90% killing of 14 strains at four times the MIC. Ninety percent killing of 14 strains was found after 12 h at four times the MIC, with slower killing at earlier time periods. By comparison, kill kinetics of vancomycin and teioplanin were significantly slower than those of the -lactams, with bactericidal activity against only 6 strains after 24 h at eight times the MIC and 90% killing of 12 and 14 strains, respectively, at four times the MIC (Table 4).

Addition of tazobactam to piperacillin at a fixed concentration of 4 µg/ml and addition of sulbactam to ampicillin did not affect the kill kinetics of the one -lactamase-negative strain tested (Table 3, strain 15). By contrast, addition of tazobactam to piperacillin at a 1:8 ratio and at a fixed concentration of 4 µg/ml and of clavulanate and sulbactam to ticarcillin and ampicillin, respectively, resulted in bactericidal activity against -lactamase-positive strain 4 (Table 3) at two times the MIC after 24 h with the higher initial inoculum.

	DISCUSSION

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Drug-resistant enterococci present a major therapeutic problem, especially in immunosuppressed patients (4, 21). High-level aminoglycoside resistance, when present, makes strains refractory to synergistic effects with penicillins and aminoglycosides. Ampicillin-resistant, -lactamase-negative strains have been reported, especially for E. faecium and E. raffinosus (1, 3-7, 12, 16, 18-21). -Lactamase-producing E. faecalis strains are currently rare in the United States but have the capacity for rapid nosocomial spread (12, 21). Murray and coworkers have described the clonal spread of a single strain of a -lactamase-producing E. faecalis to six hospitals in five states (12), and Rhinehart et al. (20) have reported the rapid spread of a -lactamase-producing aminoglycoside-resistant E. faecalis among patients and staff in an infant-toddler surgical unit.

Enterococcal glycopeptide resistance comprises four groups. (i) Highly resistant vanA strains pose the greatest threat among E. faecium strains but may occur less commonly in E. faecalis and other enterococcal species. Such strains are also usually resistant to -lactams. (ii) vanB strains are susceptible to teicoplanin but resistant to vancomycin. This phenotype is seen in strains of both E. faecalis and E. faecium. (iii) The vanC phenotype (low-level glycopeptide resistance) is only found in strains of E. gallinarum. (iv) A fourth group (vanC2 genotype), also with low-level glycopeptide resistance, is observed in strains of E. casseliflavus and E. gallinarum (4, 10, 21).

The results of our susceptibility studies reflect those reported previously by other workers (2, 8, 11, 13). Piperacillin was more active than ticarcillin against all strains. At the conservative breakpoint of 8 µg/ml and with inocula of 10⁴ CFU/spot, 97 to 99% of strains were susceptible to piperacillin with or without tazobactam, depending on the concentration at which the inhibitor was added, compared to 0% to ticarcillin with or without clavulanate and 100% to ampicillin with or without sulbactam. -Lactamase-producing strains were similarly susceptible by MIC to piperacillin-tazobactam and ampicillin-sulbactam with a higher inoculum. Our time-kill data showed that of the drugs tested, piperacillin with or without tazobactam and ampicillin gave the best kill kinetics. Although not tested against all 15 strains in our study, it can reasonably be deduced that kill kinetics of ampicillin-sulbactam against E. faecalis will be the same as those of ampicillin. Although on the surface reasonable kill kinetics were obtained with ticarcillin, its high MICs against E. faecalis compared with those of piperacillin and ampicillin precludes its use. For the one -lactamase-producing strain, addition of inhibitors to -lactams at the higher inoculum resulted in good kill kinetics at lower MICs, similar to those observed in enzyme-negative strains for all three combinations and with both piperacillin-tazobactam formulations. Murray and coworkers (15), using similar techniques, have reported time-kill results similar to ours for -lactamase-producing E. faecalis strains. The slow-kill kinetics of both glycopeptides tested should be considered together with the fact that glycopeptide-resistant E. faecalis strains are currently very rare when planning treatment strategies for infections caused by these organisms. We have no explanation as to why the "paradoxical" survival of enterococci at higher numbers in the presence of higher versus lower drug concentrations was not observed in this study.

The majority of clinically encountered E. faecalis strains in the United States are susceptible to vancomycin, with various susceptibilities to aminoglycosides (4, 21). In such cases, therapy with a combination of a -lactam and an aminoglycoside is appropriate. Results of the present study suggest that piperacillin with or without tazobactam with (where possible) an aminoglycoside is an alternate to established therapy with ampicillin with or without sulbactam plus an aminoglycoside. Monotherapy with piperacillin alone may also be possible. Klepser and coworkers (9) have recently demonstrated with human volunteers that intravenous administration of 3.375 g of piperacillin-tazobactam every 6 h, 4.5 g of piperacillin-tazobactam every 8 h, and 3.0 g of ampicillin-sulbactam every 6 h resulted in bactericidal activity for <50% of the dosing interval for E. faecalis strains. This suggests that use of shorter dosing intervals or continuous infusion regimens should be considered in combination with an aminoglycoside to improve the bactericidal profiles of these combinations against E. faecalis. Clinical studies will be necessary in order to find out how these in vitro and pharmacokinetic data translate into therapeutic regimens.