Evaluation of Oritavancin Combinations with Rifampin, Gentamicin, or Linezolid against Prosthetic Joint Infection-Associated Methicillin-Resistant Staphylococcus aureus Biofilms by Time-Kill Assays

TEXT

Prosthetic joint infection (PJI) is a serious complication of total joint arthroplasty (1). Staphylococcus aureus is a one of the most common organisms causing PJI (1, 2). S. aureus can attach to the surfaces of implanted materials and form dense biofilms that decrease susceptibility to antimicrobial agents (1, 3). Methicillin-resistant S. aureus (MRSA) PJI is particularly challenging to treat and has been associated with higher treatment failure rates than methicillin-susceptible S. aureus PJI (4, 5). Rifampin-based combination therapy is recommended by the Infectious Disease Society of America (IDSA) guidelines for patients with staphylococcal PJI managed with debridement, antimicrobial agents, and implant retention (DAIR) (6). Rifampin is considered to have antibiofilm activity, but resistance to it is easily selected; the second antimicrobial agent is traditionally considered important not so much for its antibiofilm activity but to prevent emergence of rifampin resistance. If, however, the companion drugs to rifampin (or other drug combinations) were shown to have antibiofilm activity themselves, they may be considered for treating biofilm-associated infections, such as PJI.

Oritavancin is a semisynthetic lipoglycopeptide with activity against Gram-positive bacteria; it inhibits bacterial cell wall biosynthesis and disrupts bacterial membrane integrity (7). Oritavancin has demonstrated in vitro activity against staphylococcal, including S. aureus, biofilms (8–11). Previous studies have shown that oritavancin in combination with rifampin, gentamicin, moxifloxacin, linezolid, and some β-lactams has in vitro synergistic activity against planktonic S. aureus (12–14). However, the in vitro activity of oritavancin combination therapy against MRSA biofilms has not been evaluated. In the present study, we used novel biofilm time-kill assays to investigate the in vitro activity of oritavancin in combination with rifampin, gentamicin, or linezolid against PJI-associated MRSA biofilms.

Ten MRSA isolates recovered from patients with PJI between 2000 and 2016 at Mayo Clinic (Rochester, MN) were studied. MICs of oritavancin, rifampin, gentamicin, and linezolid (Sigma-Aldrich, St. Louis, MO) were determined by broth microdilution following Clinical and Laboratory Standards Institute (CLSI) guidelines (15). Supplemental 0.002% polysorbate 80 (Sigma-Aldrich, St. Louis, MO) was added to oritavancin solutions throughout experimentation to prevent oritavancin binding to plastic surfaces (16). The minimum biofilm bactericidal concentration (MBBC) of each antimicrobial agent was determined with a Calgary biofilm device-based method after the biofilms were grown on pegs for 24 h (17). MIC and MBBC values are shown in Table 1. All 10 isolates were susceptible to the four tested antimicrobial agents according to CLSI and European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines (18, 19).

Biofilm time-kill assays were performed to investigate the antibiofilm activity of antimicrobial agents alone and in combination against the 10 MRSA isolates. In brief, sterile Teflon (polytetrafluoroethylene) coupons (10 by 9 by 1 mm) were placed in tryptic soy broth (TSB) inoculated with ∼10⁶ CFU/ml of the bacterial isolate and incubated at 37°C on an orbital shaker (120 rpm) for biofilm formation. After 24 h, Teflon coupons were rinsed with saline and incubated in oritavancin, rifampin, gentamicin, or linezolid in cation-adjusted Mueller-Hinton broth solutions alone or in combinations of oritavancin with rifampin, gentamicin, or linezolid at 37°C. The antimicrobial concentrations were equal to 1× MBBC values or the free peak concentration (fC_max) of the antimicrobial (i.e., 16 μg/ml for oritavancin [20], 4 μg/ml for rifampin [21], 12 μg/ml for gentamicin [22], and 18 μg/ml for linezolid [23]) when the MBBC values were greater than fC_max. Biofilm bacterial densities on Teflon coupons were measured before (time zero) and at 8 and 24 h after incubation in the antimicrobial solutions. To determine biofilm bacterial densities, Teflon coupons were rinsed with saline to remove the planktonic bacteria and placed in tubes containing 2 ml saline, followed by 30 s of vortexing and 5 min of sonication (40 ± 2 kHz), followed by an additional 30 s of vortexing. Then, suspensions were serially diluted in saline, and 100 μl of each dilution was placed on blood agar plates. After incubation at 37°C for 24 h, viable colonies were counted and reported as log₁₀ CFU/cm². For suspensions without growth on blood agar, 8 ml TSB was added and incubated for 24 h at 37°C and subcultured on blood agar for evidence of growth. The limit of detection was 0.1 log₁₀ CFU/cm² (24). Biofilm time-kill assays were performed in triplicate. Bactericidal activity was defined as a ≥3-log₁₀ CFU/cm² reduction at 24 h compared to the starting biofilm bacterial density at time zero (25). Synergy was defined as a ≥2-log₁₀ CFU/cm² decrease at 24 h for the antimicrobial combination compared with that of the most active single antimicrobial (25). The combination of oritavancin with rifampin or gentamicin demonstrated bactericidal activity against all 10 isolates, whereas bactericidal activity was not achieved by either oritavancin or rifampin alone; interestingly, however, gentamicin alone demonstrated bactericidal activity against 80% (8/10) of the isolates (Fig. 1). Reductions of biofilm bacterial density on Teflon coupons after 8 and 24 h of incubation with single antimicrobials were compared with those of antimicrobial combinations by one-way analysis of variance with Tukey's post hoc test (GraphPad Prism, version 7.0 GraphPad Software, San Diego, CA). A P value of ≤0.05 was considered significant. At 24 h, oritavancin combined with rifampin demonstrated a significant reduction in biofilm bacterial density compared with that of either antimicrobial alone against all 10 isolates (P ≤ 0.001). Oritavancin combined with gentamicin exhibited significant reductions in biofilm density compared with those of the antimicrobials alone for 30% (3/10) of the isolates (P ≤ 0.001), and oritavancin combined with linezolid showed significant reductions in biofilm density compared with those of the antimicrobials alone for 80% (8/10) of the isolates (P ≤ 0.01) (Fig. 2). Based on the synergy definition of a ≥2-log₁₀ CFU/cm² decrease at 24 h for the antimicrobial combination compared with the most active single antimicrobial, synergy was observed for 80% (8/10) of the isolates with oritavancin in combination with rifampin, 20% (2/10) of the isolates with oritavancin in combination with gentamicin, and 30% (3/10) of the isolates with oritavancin in combination with linezolid. Detailed data of bacterial concentrations and reductions in log₁₀ CFU/cm² at 8 and 24 h in biofilm time-kill assays can be found in Table S1 in the supplemental material.

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FIG 1

Biofilm time-kill curves against 10 MRSA isolates. (A) IDRL-6169, oritavancin, rifampin, and gentamicin at 1× MBBC. (B) IDRL-7126, oritavancin and gentamicin at 1× MBBC, rifampin at fC_max. (C) IDRL-7680, oritavancin and gentamicin at fC_max, rifampin at 1× MBBC. (D) IDRL-8302, oritavancin and rifampin at 1× MBBC, gentamicin at fC_max. (E) IDRL-8454, oritavancin, rifampin, and gentamicin at 1× MBBC. (F) IDRL-8459, oritavancin at 1× MBBC, gentamicin and rifampin at fC_max. (G) IDRL-8508, oritavancin, rifampin, and gentamicin at 1× MBBC. (H) IDRL-9121, oritavancin and rifampin at 1× MBBC, gentamicin at fC_max. (I) IDRL-9337, oritavancin, rifampin, and gentamicin at 1× MBBC. (J) IDRL-11468, oritavancin and gentamicin at 1× MBBC, rifampin at fC_max. All isolates were tested with linezolid at fC_max. ORI, oritavancin; RIF, rifampin; GEN, gentamicin; LZD, linezolid. Each isolate was tested in triplicate, and data are presented as means of biofilm densities.

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FIG 2

Reductions in biofilm bacterial densities at 8 and 24 h after exposure to antimicrobial alone or in combination. (A) IDRL-6169, oritavancin combined with rifampin, gentamicin, or linezolid reduced bacterial densities compared with single antimicrobials (P < 0.001). (B) IDRL-7126, oritavancin combined with rifampin reduced bacterial densities compared with single antimicrobials (P < 0.001). (C) IDRL-7680, oritavancin combined with rifampin or linezolid reduced bacterial densities compared with single antimicrobials (P ≤ 0.001). (D) IDRL-8302, oritavancin combined with rifampin, gentamicin, or linezolid reduced bacterial densities compared with single antimicrobials (P < 0.001). (E) IDRL-8454, oritavancin combined with rifampin or linezolid reduced bacterial densities compared with single antimicrobials (P < 0.001). (F) IDRL-8459, oritavancin combined with rifampin or linezolid reduced bacterial densities compared with single antimicrobials (P < 0.01). (G) IDRL-8508, oritavancin combined with rifampin, gentamicin, or linezolid reduced bacterial densities compared with single antimicrobials (P < 0.0001). (H) IDRL-9121, oritavancin combined with rifampin reduced bacterial densities compared with single antimicrobials (P = 0.001). (I) IDRL-9337, oritavancin combined with rifampin or linezolid reduced bacterial densities compared with single antimicrobials (P ≤ 0.01). (J) IDRL-11468, oritavancin combined with rifampin or linezolid reduced biofilm densities compared with single antimicrobials (P < 0.0001). P values are calculated for reduction at 24 h and presented as the number to cover all P values applied. See details in Table S1 in the supplemental material. ORI, oritavancin; RIF, rifampin; GEN, gentamicin; LZD, linezolid. Data presented are means ± SD (n = 3).

Synergy between oritavancin and rifampin may be explained by oritavancin's ability to inhibit cell wall synthesis and disrupt bacterial membrane integrity (7), facilitating rifampin's entry into bacterial cells. In addition, oritavancin has been reported to inhibit RNA synthesis itself (26), which, when combined with rifampin's RNA polymerase inhibiting activity, may enhance synergistic activity between them (12). Time-kill assays have been widely used to assess in vitro synergy between antimicrobial agents; antibiotic concentration selection in time-kill assays is debatable. Some studies have selected antibiotic concentrations approximating the fC_max (21, 22), whereas others have chosen antibiotic concentrations based on MICs (for planktonic studies) or MBBCs (for biofilm studies) (25). We selected concentrations representing MBBCs if they were lower than the fC_max. We acknowledge the limitation that the fC_max may not reflect bone concentrations. Of note, oritavancin is administered as a single 1,200-mg intravenous infusion; in this study, the oritavancin non-protein-bound plasma concentration at 24 h after dosing was used as the fC_max (20). Although oritavancin has been approved by the U.S. Food and Drug Administration only for treating adult patients with acute bacterial skin and skin structure infections, our study suggests that the combination of oritavancin and rifampin deserves further study as an option for treating PJIs caused by MRSA.