Dynamic PET-facilitated modeling and high-dose rifampin regimens for Staphylococcus aureus orthopedic implant-associated infections

Abstract

Staphylococcus aureus is a major human pathogen causing serious implant–associated infections. Combination treatment with rifampin (10 to 15 mg/kg per day), which has dose-dependent activity, is recommended to treat S. aureus orthopedic implant–associated infections. Rifampin, however, has limited bone penetration. Here, dynamic ¹¹C-rifampin positron emission tomography (PET) performed in prospectively enrolled patients with confirmed S. aureus bone infection (n = 3) or without orthopedic infection (n = 12) demonstrated bone/plasma area under the concentration-time curve ratio of 0.14 (interquartile range, 0.09 to 0.19), exposures lower than previously thought. PET-based pharmacokinetic modeling predicted rifampin concentration-time profiles in bone and facilitated studies in a mouse model of S. aureus orthopedic implant infection. Administration of high-dose rifampin (human equipotent to 35 mg/kg per day) substantially increased bone concentrations (2 mg/liter versus <0.2 mg/liter with standard dosing) in mice and achieved higher bacterial killing and biofilm disruption. Treatment for 4 weeks with high-dose rifampin and vancomycin was noninferior to the recommended 6-week treatment of standard-dose rifampin with vancomycin in mice (risk difference, −6.7% favoring high-dose rifampin regimen). High-dose rifampin treatment ameliorated antimicrobial resistance (0% versus 38%; P = 0.04) and mitigated adverse bone remodeling (P < 0.01). Last, whole-genome sequencing demonstrated that administration of high-dose rifampin in mice reduced selection of bacterial mutations conferring rifampin resistance (rpoB) and mutations in genes potentially linked to persistence. These data suggest that administration of high-dose rifampin is necessary to achieve optimal bone concentrations, which could shorten and improve treatments for S. aureus orthopedic implant infections.

Fig. 1.. Rifampin bone exposures.

(A) Three prospectively enrolled patients with S. aureus bone infections underwent dynamic ¹¹C-rifampin PET for 60 to 90 min, followed by a CT scan. Frontal view of maximum intensity projection (PET). Note the implant in the left tibia (arrow). (B) ¹¹C-Rifampin PET area under the concentration-time curve (AUC) shown as a heat map overlay in the selected transverse section. (C) Representative ¹¹C-rifampin PET time-activity curve (TAC) from a S. aureus–infected patient. (D) ¹¹C-Rifampin exposure in long bones (tibia and humerus), short bones (tarsals), and spine of S. aureus–infected patients shown as the median (and range) bone-to-plasma AUC ratio (AUC_bone/plasma). NS, not significant. (E) Dynamic ¹¹C-rifampin PET in patients without an orthopedic infection (and with pulmonary tuberculosis; n = 12 patients) was used to get additional data in uninfected long bone (humerus) and spine. (F and G) Mice with a femoral orthopedic implant infected with methicillin-resistant S. aureus (SAP231) were administered rifampin (with vancomycin) corresponding to a human-equipotent rifampin dose of either 10, 35, or 45 mg/kg per day (n = 4 to 6 animals per treatment group in one experiment). Rifampin plasma (F) and bone (G) concentrations measured by mass spectrometry in postmortem samples. (H) Mice (after at least 2 weeks of antibiotic treatment) underwent dynamic ¹¹C-rifampin PET for 60 min, followed by a CT scan (n = 8 animals total in two independent experiments). Frontal view of maximum intensity projection (PET). Note the implant in the right knee (arrow). (I) Representative TAC from an S. aureus–infected mouse. (J) ¹¹C-Rifampin exposure in mouse shown as AUC_bone/plasma. *P < 0.05, ^†P < 0.01, and ^‡P < 0.001 as shown by the Kruskal-Wallis test (F and G) or by the Wilcoxon matched-pairs signed rank test (D and E), adjusted for multiple comparisons as indicated to preserve the desired false discovery rate or by the two-tailed Mann-Whitney test for comparison of infected to uninfected bone (G and J).

Fig. 2.. Pharmacokinetic model and simulations for human subjects.

(A) One-compartment model with one bone compartment, relative bioavailability, transit absorption, autoinduction, and Michaelis-Menten elimination. Plasma volume (V_c), bone volume (V_b), blood flow between plasma and bone (Q_b), partition coefficient (PC), maximal increase in bioavailability relative to 450-mg dose (relF_max), difference in dose from 450 mg at which half relF_max is achieved (relFD₅₀), bioavailability for 450-mg dose (F₄₅₀)—set to 1, number of transit compartments (NN), mean transit time (MTT), transit rate constant (k_tr), first-order rate constant for enzyme degradation (k_ENZ), maximal increase in rate of enzyme production (S_max), plasma concentration (C_p), concentration at which half S_max is achieved (SC₅₀), amount of enzyme (A_ENZ), maximal rate of elimination (V_max), and concentration at which half V_max is achieved (k_m). (B) Rifampin concentrations were simulated for 2 weeks with twice-daily dosing. Dashed red lines represent cutoffs of 1 and 2 mg/liter. The value of 1 mg/liter is the breakpoint suggested by the Clinical and Laboratory Standards Institute (CLSI).

Fig. 3.. Treatment shortening studies in mice with S. aureus implant infection.

(A) Intramuscular vancomycin (equipotent to 2 g/day intravenously in human) and oral rifampin were initiated 2 weeks after infection (designated as day 0) and continued for 6 weeks (IDSA regimen, standard-dose rifampin; n = 45 animals) or 4 weeks (high-dose rifampin; n = 45 animals). Standard- and high-dose rifampin doses were (human equipotent) 10 and 35 mg/kg per day, respectively. Monotherapy with vancomycin (equipotent to 2 g/day intravenously in humans) over 6 weeks was also performed (n = 10 animals). Data from one experiment are presented. Stable cure was determined at day 84 by assessing sterilization of the tissues and the implant. (B) Representative in vivo bioluminescence images from mice in each group. (C) Mean maximum flux (photons s⁻¹ cm⁻² sr⁻¹) (and SEM). LOD, level of detection (3 × 10³ photons s⁻¹ cm⁻² sr⁻¹). (D) Six weeks after completion of antibiotic treatment, peri-implant joint and bone tissue were homogenized, implants were sonicated, and colony-forming units (CFUs) were enumerated ex vivo. Data are presented as the mean number of CFUs (and SEM) isolated from the peri-implant bone and joint tissue. (E) To evaluate whether the antibiotic treatment eradicated infection, tissue homogenates and implants were cultured for an additional 48 hours in broth followed by overnight plate culture, and the presence or absence of bacterial growth was determined. Data are presented as the percentage of tissue samples with any bacterial growth. (F) Statistical analysis showing treatment difference in favor of high-dose rifampin and 90% confidence interval within the preset limit for noninferiority (d = 10%). *P < 0.05, ^†P < 0.01, and ^‡P < 0.001 as shown by the Kruskal-Wallis test adjusted for multiple comparisons to preserve the desired false discovery rate.

Fig. 4.. Bacterial characteristics and bone remodeling.

Twenty-one isolates, each from a unique mouse from the noninferiority study, were subjected to (A) phenotypic rifampin resistance testing (BD Phoenix), with results presented as the percentage of resistant isolates for each treatment group and (B) whole-genome sequencing (Illumina; MiGS) and compared to the parent strain SAP231. Heat maps display isolates with variants in genes related to rifampin resistance (rpoB) or to bacterial persistence (lysM, sdhB, and clfB). (C) Representative 3D reconstructed μCT images [opaque (top) and translucent (bottom) mouse femora with implants in red; scale bars, 1 mm]. (D) Mean (and SEM) volume of the distal 25% of the infected femurs (excluding the implant) (n = 5 to 15 per group in one experiment). *P < 0.05, ^†P < 0.01, and ^‡P < 0.001 as shown by the χ² test (A) or by the Kruskal-Wallis test adjusted for multiple comparisons to preserve the desired false discovery rate (D).