In vitro identification of underutilized β-lactam combinations against methicillin-resistant Staphylococcus aureus bacteremia isolates

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is a serious clinical challenge with high mortality rates. Antibiotic combination therapy is currently used in cases of persistent infection; however, the limited development of new antibiotics will likely increase the need for combination therapy, and better methods are needed for identifying effective combinations for treating persistent bacteremia. To identify pairwise combinations with the most consistent potential for benefit compared to monotherapy with a primary anti-MRSA agent, we conducted a systematic study with an in vitro high-throughput methodology. We tested daptomycin and vancomycin each in combination with gentamicin, rifampicin, cefazolin, and oxacillin, and ceftaroline with daptomycin, gentamicin, and rifampicin. Combining cefazolin with daptomycin lowered the daptomycin concentration required to reach 95% growth inhibition (IC₉₅) for all isolates tested and lowered daptomycin IC₉₅ below the sensitivity breakpoint for five out of six isolates that had daptomycin minimum inhibitory concentrations at or above the sensitivity breakpoint. Similarly, vancomycin IC₉₅s were decreased when vancomycin was combined with cefazolin for 86.7% of the isolates tested. This was a higher percentage than was achieved by adding any other secondary antibiotic to vancomycin. Adding rifampicin to daptomycin or vancomycin did not always reduce IC₉₅s and failed to produce synergistic interaction in any of the isolates tested; the addition of rifampicin to ceftaroline was frequently synergistic and always lowered the amount of ceftaroline required to reach the IC₉₅. These analyses rationalize further in vivo evaluation of three drug pairs for MRSA bacteremia: daptomycin+cefazolin, vancomycin+cefazolin, and ceftaroline+rifampicin.IMPORTANCEBloodstream infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have a high mortality rate despite the availability of vancomycin, daptomycin, and newer antibiotics including ceftaroline. With the slow output of the antibiotic pipeline and the serious clinical challenge posed by persistent MRSA infections, better strategies for utilizing combination therapy are becoming increasingly necessary. We demonstrated the value of a systematic high-throughput approach, adapted from prior work testing antibiotic combinations against tuberculosis and other mycobacteria, by using this approach to test antibiotic pairs against a panel of MRSA isolates with diverse patterns of antibiotic susceptibility. We identified three antibiotic pairs-daptomycin+cefazolin, vancomycin+cefazolin, and ceftaroline+rifampicin-where the addition of the second antibiotic improved the potency of the first antibiotic across all or most isolates tested. Our results indicate that these pairs warrant further evaluation in the clinical setting.

Keywords: MRSA; bacteremia; cefazolin; ceftaroline; combination therapy; rifampicin.

Fig 1

DiaMOND dosing schematics and metrics: combination interaction does not always indicate the greatest decrease in primary antibiotic required to reach the IC₉₅ in combination. (A) DiaMOND dosing schematic for all combinations not involving oxacillin or cefazolin and (B) DiaMOND dosing schematic for all combinations involving oxacillin or cefazolin. See Materials and Methods for further description. (C) Dose–response curves showing % growth inhibition versus microgram per milliliter antibiotic for daptomycin alone (black circles with dashed line), ceftaroline alone (light aqua triangles with dashed line), daptomycin when used in combination with ceftaroline (dark aqua circles with solid line), and ceftaroline when used in combination with daptomycin (dark aqua triangles with solid line) against isolate TR399 (grown in CAMHB+Ca²⁺). (D) Dose–response curve fits showing % growth inhibition versus microgram per milliliter antibiotic for daptomycin alone (black circles with dashed line), gentamicin alone (red diamonds with dashed line), daptomycin when used in combination with gentamicin (dark red circles with solid line), and gentamicin when used in combination with daptomycin (dark red diamonds with solid line) against isolate TR399 (grown in CAMHB+Ca²⁺). Antibiotic abbreviations: DAP, daptomycin; VAN, vancomycin; CRF, ceftaroline; GEN, gentamicin; RIF, rifampicin; CZN, cefazolin; OXA, oxacillin.

Fig 2

Adding cefazolin lowers the amount of daptomycin required to reach the IC₉₅, particularly as daptomycin susceptibility decreases over the course of treatment. This figure shows two series of MRSA bacteremia isolates (isolates are shown in the order that they were taken) from two different Tufts Medical Center patients during their courses of treatment. For patient A, daptomycin alone was started at 6 mg/kg between the second positive culture and the third positive culture. The third positive culture was obtained approximately 5 days after daptomycin was started. The fourth positive culture was obtained after approximately 12 days of daptomycin and 5 days of gentamicin. No antibiotic administration data were available for patient B. Both series of isolates showed a decrease in daptomycin susceptibility for the third isolate in the series, as seen in the daptomycin alone IC₉₅ (µg/mL) presented on the x-axis of the graph for each isolate. The graphs for each isolate show the fold decrease in daptomycin required to reach the IC₉₅ when cefazolin (blue), gentamicin (red), ceftaroline (light aqua), or rifampicin (green) are added to daptomycin. The third isolate from patient B is resistant to rifampicin, so it was not tested with daptomycin+rifampicin. See Table S1 for isolate and MIC info.

Fig 3

Cefazolin performs relatively well compared to other antibiotics in terms of decreasing but not increasing the amount of daptomycin or vancomycin required to reach the IC₉₅. (A) For the 30 MRSA bacteremia isolates tested with all DAP combinations (TR258 and the 29 non-serial isolates), this graph shows the percentage of isolates, specified on the y-axis, that achieved at least an x-Fold Change in Primary antibiotic (FCP), specified on the x-axis. For this graph, daptomycin is always the primary antibiotic, and percentages are indicated for the combination of daptomycin with ceftaroline (light aqua line), gentamicin (red line), cefazolin (blue line), rifampicin (green line), or oxacillin (brown line). Negative values on the x-axis indicate a fold decrease in daptomycin required to reach the IC₉₅ when the secondary antibiotic is added, and positive values on the x-axis indicate a fold increase in daptomycin required to reach the IC₉₅ when the secondary antibiotic is added. (B) This graph is the same style as the graph in (A), except this graph shows the results for the vancomycin-containing combinations.

Fig 4

Adding rifampicin to ceftaroline always lowers the amount of ceftaroline required to reach the IC₉₅ and vice versa, and the combination is often synergistic or at least additive, unlike when rifampicin is added to daptomycin or vancomycin. (A) This graph is the same style as the graphs in Fig. 3, except this graph shows the results for ceftaroline+gentamicin, ceftaroline+rifampicin, and daptomycin+ceftaroline, where ceftaroline is treated as the primary antibiotic in the fold change calculations. (B) This graph shows the fold change in ceftaroline required to reach the IC₉₅ when rifampicin is added (y-axis) vs the fold change in rifampicin required to reach the IC₉₅ when ceftaroline is added (x-axis). (C) This graph shows the fold change in daptomycin required to reach the IC₉₅ when rifampicin is added (y-axis) vs the fold change in rifampicin required to reach the IC₉₅ when daptomycin is added (x-axis). (D) This graph shows the fold change in vancomycin required to reach the IC₉₅ when rifampicin is added (y-axis) vs the fold change in rifampicin required to reach the IC₉₅ when vancomycin is added (x-axis). For the graphs in (B) through (D), the points representing each isolate are colored by the log₂FIC₉₅ score of ceftaroline+rifampicin (B), daptomycin+rifampicin (C), or vancomycin+rifampicin (D) for that isolate.