Penicillin plus Ceftriaxone versus Ampicillin plus Ceftriaxone Synergistic Potential against Clinical Enterococcus faecalis Blood Isolates

ABSTRACT Penicillin plus ceftriaxone is a promising alternative to ampicillin plus ceftriaxone for the treatment of Enterococcus faecalis infective endocarditis. Limited data is available supporting the utilization of penicillin plus ceftriaxone. A total of 20 E. faecalis isolates; one wild-type strain (JH2-2) and 19 clinical blood strains were assessed for penicillin plus ceftriaxone and ampicillin plus ceftriaxone synergy using a 24-h time-kill experiment. Susceptibility was determined by broth microdilution. Differences in bactericidal, bacteriostatic, or inactivity, as well as synergy between treatments were assessed by chi-square or Fisher exact test. All E. faecalis isolates were considered susceptible to ampicillin and penicillin. Ampicillin plus ceftriaxone versus penicillin plus ceftriaxone similarly demonstrated synergy. Bactericidal activity was more commonly observed for ampicillin plus ceftriaxone versus penicillin plus ceftriaxone. Among isolates with a penicillin MIC of 4 μg/mL (n = 7), synergistic activity for both combinations was less common compared to isolates with a penicillin MIC ≤ 2 μg/mL (n = 13). Ampicillin plus ceftriaxone and penicillin plus ceftriaxone demonstrate similar synergistic potential against E. faecalis clinical blood isolates, but strains with higher penicillin and ceftriaxone MICs less frequently demonstrated synergy. Further research is warranted to determine the role of the penicillin plus ceftriaxone therapy and the penicillin MIC in clinical practice. IMPORTANCE Penicillin plus ceftriaxone demonstrates similar synergistic activity against Enterococcus faecalis to ampicillin plus ceftriaxone. Isolates with a penicillin MIC of 4 mg/L and a ceftriaxone MIC of 512 or higher, lack penicillin plus ceftriaxone synergy despite the penicillin susceptibility MIC breakpoint of 8 mg/L.

parenteral antimicrobial therapy (OPAT) due to ampicillin stability. Historically, ampicillin was known to be stable (after reconstitution) for approximately 8 h at room temperature, which led to clinicians prescribing penicillin plus ceftriaxone due to improved penicillin stability (24 h at room temperature), despite limited evidence of primary support (8)(9)(10)(11). Recent data demonstrates prolonged stability of ampicillin up to 30 h at room temperature (12,13); however, ampicillin still lacks stability in an elastomeric pump, which is an advantageous outpatient intravenous medication delivery system that allows patients the freedom to travel with their medication in their pocket or a pouch without having to have the medication hung superiorly to the infusion pump (9). Penicillin maintains stability in an elastomeric pump (9), thus penicillin1ceftriaxone is a promising alternative to ampicillin1ceftriaxone.
Penicillin1ceftriaxone requires further evaluation as penicillin is known to have higher MICs compared to ampicillin against E. faecalis (14). There are also increasing reports of E. faecalis isolates with alterations in essential pbp4 that demonstrate penicillin resistance but are ampicillin susceptible (3,15). Clinical data is limited in supporting the utilization of penicillin1ceftriaxone (10,11,16,17). A comparison of penicillin1ceftriaxone in vitro synergy to ampicillin1ceftriaxone has only been assessed in checkboard assays, which are limited due to the wide variability in result interpretation (17)(18)(19). Synergy assessment via in vitro time-kill experiments have not yet been reported. We hypothesized that penicillin1ceftriaxone will have equivalent in vitro synergy to ampicillin1ceftriaxone against E. faecalis blood isolates.

RESULTS
Susceptibility testing. All isolates were considered susceptible to ampicillin and penicillin (Table 1). Wild-type isolate, JH2-2, had an ampicillin MIC of 0.5 mg/mL, which was lower than previously published MIC of 2 mg/mL (20). To confirm findings, we repeated the assay twice and obtained the same value. The penicillin MIC for JH2-2 was similar to previously published findings (15).
All ampicillin MICs were concordant with the clinical microbiology laboratory results. Penicillin MICs were within one to two, 2-fold dilutions of the reported MIC from the clinical microbiology laboratory. A total of 13 isolates had a penicillin MIC # 2 mg/mL and seven isolates had a penicillin MIC of 4 mg/mL. All ceftriaxone MICs were elevated as expected due to intrinsic resistance, except one isolate that had an MIC of 16 mg/mL (e2006). Most isolates with a penicillin MIC of 4 mg/mL had a ceftriaxone MIC $ 2048 mg/mL (n = 5/7, 71%) compared to most isolates with a penicillin MIC of 2 mg/mL had a ceftriaxone MIC # 512 mg/ mL (n = 11/13, 85%) ( Table 1). Time-kill assays. All isolates against ampicillin or penicillin monotherapy at 0.25xMIC demonstrated inactivity (Table 2). Ceftriaxone monotherapy also demonstrated inactivity against all isolates, except one isolate which was bacteriostatic (e2006; -0.44 6 0.54 log 10 CFU/mL). Ampicillin versus penicillin monotherapy at 0.5xMIC less commonly demonstrated inactivity (n = 13, 65% versus n = 19, 95%; P = 0.04). Ampicillin versus penicillin at 1xMIC similarly did not demonstrate inactivity (n = 4, 20% versus n = 2, 10%, P = 0.66), with majority of isolates demonstrating bactericidal activity against ampicillin (n = 11, 55%) and majority of isolates demonstrating bacteriostatic activity against penicillin (n = 11, 55%).
Among isolates with a penicillin MIC of 4 mg/mL (n = 7), synergistic activity for ampi-cillin1ceftriaxone and penicillin1ceftriaxone was less common compared to isolates with a penicillin MIC # 2 mg/mL (n = 13) ( Table 3). Bactericidal activity was also more common among isolates with a penicillin MIC # 2 versus a penicillin MIC of 4 mg/mL. Only one isolate (e2024) demonstrated bactericidal activity and synergy at 0.25Â and 0.5 Â MIC for both combinations and was the only penicillin MIC of 4 mg/mL isolate with a lower ceftriaxone MIC of 256 mg/mL. All other isolates with a penicillin MIC of 4 mg/mL exposed to penicillin1ceftriaxone at 0.25Â and 0.5 Â MIC were inactive and had a higher ceftriaxone MIC (range 512 to .2,048 mg/mL).

DISCUSSION
Among 20 clinical Enterococcus faecalis blood isolates, similar synergistic activity was observed for ampicillin1ceftriaxone and penicillin1ceftriaxone combinations. More frequent  a Bactericidal activity was observed, defined as $3-log 10 decrease in CFU/mL from initial inoculum. b Bacteriostatic activity was observed, defined as ,3-log 10 decrease in CFU/mL from initial inoculum.
bactericidal activity was demonstrated among isolates treated with ampicillin1ceftriaxone versus penicillin1ceftriaxone. Penicillin1ceftriaxone also demonstrated more bactericidal at higher penicillin concentrations. Ampicillin1ceftriaxone and penicillin1ceftriaxone synergy was less frequently observed among isolates with a higher penicillin MIC of 4 mg/mL as well as higher ceftriaxone MICs. The observed differences in activity may impact clinical outcomes in patients with E. faecalis IE, especially since b-lactam-based treatment failure has been shown to occur more frequently in patients infected with a penicillin-resistant ampicillin-susceptible E. faecalis (3). According to CLSI, the clinical MIC breakpoint for penicillin and E. faecalis is # 8 mg/mL, indicating our isolates were still susceptible (21). Interestingly, majority of our isolates with a penicillin MIC of 4 mg/mL had a higher reported MIC by Vitek 2 (Table 1), with two isolates having an MIC above the breakpoint (i.e., 16 mg/mL). Our findings of lower MICs by broth microdilution compared to Vitek 2 are similar to a previous report of 49 penicillin-resistant ampicillin-susceptible E. faecalis isolates, which found that 93.9% of isolate MICs by Vitek 2 were two, 2-fold dilutions higher than broth microdilution (22). While the prevalence of penicillin-resistant ampicillin-susceptible E. faecalis remains unknown in the Unites States (US), b-lactam-based treatments remain the standard of care. Ampicillin1ceftriaxone is the most commonly used first-line combination to treat E. faecalis IE due to the improved safety profile compared to aminoglycoside-based treatments as well as rising aminoglycoside resistance up to 60% (1, 6). However, due to the challenges of coordinating ampicillin therapy in the outpatient setting patients often receive penicillin1ceftriaxone (8,9). Limited clinical data is available supporting the use of penicillin1ceftriaxone in clinical practice for E. faecalis IE and has only been assessed in patients who have already received standard of care treatment. The first discussion was a retrospective review in the US that identified five patients who were discharged on penicillin1ceftriaxone after receiving 3 to 8 days of ampicillin1ceftriaxone inpatient (11). Two of the five patients were lost to follow-up and the other three achieved clinical cure with no relapse at 90 days (11). The penicillin MICs were not reported in this  a Bactericidal activity was observed, defined as $3-log 10 decrease in CFU/mL from initial inoculum. b Bacteriostatic activity was observed, defined as ,3-log 10 decrease in CFU/mL from initial inoculum. c Synergy was detected as indicated by gray shading, defined as $2-log 10 decrease in CFU/mL at 24 h from the most active single agent was observed. study (11). Another case series of four patients in the US who received penicillin1ceftriaxone, after already receiving standard of care [i.e., ampicillin1ceftriaxone (n = 3) and penicillin plus gentamicin (n = 1)] treatment for a range of 3 to 32 days, reported no recurrence in infection at 6 months (10). The authors reported penicillin MICs for three of the four patients by Etest, where two patients had an MIC of 4 mg/mL, and one had an MIC of 2 mg/mL (10). Etest methodology is found to correlate well to broth microdilution methodology, which perhaps suggests that a penicillin MIC of 4 mg/mL does not always indicate treatment failure as we observed with our isolates (22). However, one of the patients received chronic amoxicillin oral suppression and the other patient received penicillin1gentamicin for 32 days prior to transitioning to penicillin1ceftriaxone, which may falsely make penicillin1ceftriaxone appear efficacious. A larger multicenter case series in New Zealand of 41 patients with enterococcal endocarditis (E. faecalis, n = 40 and Enterococcus faecium, n = 1) compared outpatient treatment with penicillin plus gentamicin (n = 20) versus penicillin1ceftriaxone (n = 23), found no difference in recurrence (11% versus 5%, P = 0.59) but a greater incidence of side effects in patients receiving gentamicin therapy (35% versus 0%, P , 0.01) (16). Patients received a median of 15 days (interquartile range [IQR] 9-18.5 days) of inpatient penicillin, amoxicillin, amoxicillin-clavulanic acid, piperacillin, or piperacillin-tazobactam plus a synergy antibiotic (i.e., ceftriaxone or gentamicin) prior to discharge (16). The predominant synergy antibiotic received during the first 14 days of treatment was then selected for the patient on discharge (16). Penicillin susceptibility was available for 32 of the isolates, with a median MIC of 3 (IQR 2-4), but methodology was not reported (16). Similar efficacy between the two combinations may be due to more patients in the penicillin1ceftriaxone group receiving chronic amoxicillin oral suppression (5% versus 35%, P = 0.02) (16).
Most recently, a single-center retrospective cohort study in Australia identified 20 patients with an E. faecalis endovascular infection who received penicillin1ceftriaxone via OPAT from their existing OPAT database (17). Six patients (30%) experienced an unplanned readmission, one patient (5%) had a relapse in bacteremia within 6 months, and 1-year mortality was 15% (17). All isolates were considered susceptible to penicillin by Vitek 2, but MICs were not reported (17). A random six isolates were selected for testing by broth microdilution and synergy assessment by checkerboard, which revealed a median penicillin MIC of 1 mg/mL (IQR 0.5-1 mg/mL) and synergy for four isolates against ampicillin1ceftriaxone and three isolates against penicillin1ceftriaxone (17). It is important to note, however, that the interpretation of the fractional inhibitory concentration index (FICI) was not indicated (17). Overall, the clinical data is limited, and randomized controlled trials are needed to determine the equivalence of penicillin1ceftriaxone to ampicillin1ceftriaxone and the role of the E. faecalis penicillin MIC in predicting treatment success.
Only one other in vitro study has been published to date, which also compared checkboard synergy of ampicillin1ceftriaxone versus penicillin1ceftriaxone among 28 clinical E. faecalis blood isolates from Germany and one wild-type isolate (ATCC 29212) (19). The ceftriaxone concentrations utilized included the free plasma trough concentrations for a 2 g IV q12h, 4 g IV q24h, and 2 g IV q24h regimens, which were 4, 1.5, and 1 mg/L, respectively (19). Conversely, we utilized a free steady-state plasma concentration of 17.2 mg/mL based on clinical pharmacokinetic data for a 2 g IV q12h regimen, where the extrapolated trough would be 9.13 mg/mL versus the 4 mg/mL utilized by Thieme et al. (19,23). The difference in concentration is likely due to the variability in patient pharmacokinetics but should be considered when interpreting the in vitro synergy results (23)(24)(25). Additionally, the authors utilized three different definitions for the FICI, which leads to wide variability in result interpretation (18,19). When utilizing an FICI # 0.5 to indicate synergy a total of 22 (75.9%) and 16 (55.2%) isolates were synergistic against ampicillin1ceftriaxone and penicillin1ceftriaxone, respectively (19). When utilizing the median FICI of 0.8 as the synergy threshold, an additional five isolates (n = 21, 72.4%) demonstrated penicillin1ceftriaxone synergy and no additional isolates demonstrated ampicil-lin1ceftriaxone synergy (19). We observed comparable rates of synergy between the two combinations. We also observed minimal synergy for both combinations in isolates with a penicillin MIC of 4 mg/mL, whereas Thieme et al. found a strong inverse correlation indicating that the higher the penicillin MIC the lower the FICI (r s = -0.61, P = 0.001) (19). However, these results are difficult to interpret as the lower the FICI does not necessarily mean more synergy.
In addition, among our isolates with a penicillin MIC of 4 mg/mL, the ceftriaxone MICs were higher compared to isolates with a penicillin MIC # 2 mg/mL. One isolate with a penicillin MIC of 4 mg/mL (e2024) had a ceftriaxone MIC of 256 mg/mL and was also the only isolate with a penicillin MIC of 4 mg/mL that demonstrated synergy against both ampi-cillin1ceftriaxone and penicillin1ceftriaxone. Similarly, Thieme L et al.'s in vitro checkboard study found that isolates with a ceftriaxone MIC . 1024 mg/mL (n = 4) did not demonstrate synergy for either combination, and ceftriaxone concentrations required to reduce the ampicillin or penicillin were frequently unachievable or higher than physiologically achievable ceftriaxone concentrations (19). Therefore, we chose to utilize the free plasma steady-state concentrations (fCpss) of ceftriaxone to improve the clinical applicability of our results similar to previous work (26). Utilization of ceftriaxone at 0.12Â, 0.25Â, and 0.5 Â MIC in a time-kill assay in previous work with isolate JH2-2 (ceftriaxone MIC 512 mg/mL) also yielded similar synergistic results (27). The relationship of the ceftriaxone MIC along with the penicillin MIC to b-lactam synergistic potential may be related to changes in essential penicillin-binding protein-4 (PBP4). Although alterations in pbp4 have only been reported in penicillin-resistant ampicillin-susceptible isolates (15), further investigation is warranted to determine if pbp4 mutations are present in isolates with higher penicillin MICs near the breakpoint (i.e., 4 and 8 mg/mL).
The strength of our study was the inclusion of clinical blood E. faecalis strains and utilization of time-kill assays which have a clear synergy definition compared to checkboard methodology. While time-kill assays are superior to checkboard methodology, our results are limited by the static nature of these assays, which limits the applicability to determine appropriate dosing that maximizes bactericidal activity. To improve applicability to patient care we utilized physiologic concentrations of ceftriaxone, but unfortunately the physiologic steady-state plasma concentrations for ampicillin and penicillin were above the MIC and would eradicate the organism without ceftriaxone in combination. As a result, we utilized subinhibitory concentrations at 0.25Â, 0.5Â, and 1 Â MIC, which led to variability in the concentrations utilized across the isolates. Further research is warranted to determine optimal penicillin1ceftriaxone dosing and the role of the E. faecalis penicillin MIC in predicting treatment success.
Conclusion. Overall, ampicillin1ceftriaxone and penicillin1ceftriaxone demonstrates similar synergistic potential but ampicillin1ceftriaxone is more bactericidal. Strains with higher penicillin and ceftriaxone MICs less frequently demonstrated synergy with both ampicillin1ceftriaxone and penicillin1ceftriaxone. Higher penicillin concentrations were warranted to achieve bactericidal activity, but further research is warranted to determine the appropriate dose to optimize penicillin exposure.

MATERIALS AND METHODS
Bacterial isolates. A total of 20 E. faecalis isolates were included; one wild-type strain (JH2-2) and 19 clinical strains from blood. Most clinical isolates obtained were ampicillin and penicillin susceptible by the clinical microbiology laboratory (Vitek 2, bioMérieux, Inc., Durham, NC). There were two isolates that had a penicillin MIC of 16 mg/mL, which is one 2-fold dilution above the clinical breakpoint for penicillin is # 8 mg/mL (21). All isolates were stored in (CryoCare, Stamford, TX; tryptic soy broth plus glycerol) at -80°C and were subcultured once on brain heart infusion agar for 18-24 h at 35°C prior to each experiment.
Susceptibility testing. MICs were performed for ampicillin, penicillin, and ceftriaxone by broth microdilution to confirm clinical microbiology laboratory results according to CLSI (21). All MICs were performed in duplicate and were repeated to confirm any discordant MIC values between the clinical microbiology laboratory and our laboratory.
Time-kill assays. A 24-h time-kill experiment was utilized to detect synergy for ampicillin1ceftriaxone versus penicillin1ceftriaxone against all 20 isolates. Experiments were performed in duplicate in a 12-well plate with a final volume of 2 mL. Assays were repeated to confirm findings if a wide standard deviation in results was observed. The starting inoculum was 10 6 CFU/mL and plates were placed in the incubator at 35°C at 50 rotations per minute (rpm). Subinhibitory concentrations (0.25Â and 0.5 Â MIC) of ampicillin and penicillin were utilized as previously described (28), and 1xMIC was also tested. Ceftriaxone was tested at the free plasma steady-state concentration (fCpss = 17.2 mg/mL) based on population pharmacokinetic data for a 2 g IV q12h regimen (t 1/2 = 7.2 h, fC max = 28.9 mg/mL), as subinhibitory concentrations would not be physiologically achievable due to the intrinsic resistance of ceftriaxone to enterococcus (23,30). Each drug was tested as monotherapy and both ampicillin and penicillin were combined with ceftriaxone. Samples were obtained at 0, 4, and 24 h and diluted 1:10 in normal saline to obtain viable cell counts. Three 20 mcL samples of each dilution were plated onto BHIA and incubated for 18-24 h at 35°C, where the average of the three samples was taken to obtain a log 10 CFU/mL viable cell count. Samples were directly obtained from the 12well plate if bacterial growth was not visible for a lower limit of detection of 2-log 10 CFU/mL. Antimicrobial activity was defined as bacteriostatic or bactericidal, which were defined as , 3-log 10 CFU/mL or $ 3-log 10 CFU/ mL decrease from initial inoculum at 24 h. Inactivity was defined as an increase in log 10 CFU/mL from initial inoculum at 24 h. Combination therapy activity was determined to be synergistic if a $ 2-log 10 decrease in CFU/mL at 24 h from the most active single agent was observed (18).
Statistical analysis. Differences in bactericidal, bacteriostatic, or inactivity between ampicillin and penicillin monotherapies, and ampicillin1ceftriaxone and penicillin1ceftriaxone combinations were assessed by chi-square or Fisher exact test. Differences in synergy between ampicillin1ceftriaxone and penicillin1ceftriaxone, as well as between isolates with a penicillin MIC # 2 mg/mL and isolates with a penicillin MIC of 4 mg/mL for each combination were assessed by chi-square or Fisher exact test. All statistical analyses were performed using R (version 4.1.2).