Evidence of Bisphosphonate-Conjugated Sitafloxacin Eradication of Established Methicillin-Resistant S. aureus Infection with Osseointegration in Murine Models of Implant-Associated Osteomyelitis

Eradication of MRSA osteomyelitis requires elimination of distinct biofilms. To overcome this, we developed bisphosphonate-conjugated sitafloxacin (BCS, BV600072) and hydroxybisphosphonate-conjugate sitafloxacin (HBCS, BV63072), which achieve “target-and-release” drug delivery proximal to the bone infection and have prophylactic efficacy against MRSA static biofilm in vitro and in vivo. Here we evaluated their therapeutic efficacy in a murine 1-stage exchange femoral plate model with bioluminescent MRSA (USA300LAC::lux). Osteomyelitis was confirmed by CFU on the explants and longitudinal bioluminescent imaging (BLI) after debridement and implant exchange surgery on day 7, and mice were randomized into seven groups: 1) Baseline (harvested at day 7, no treatment); 2) HPBP (bisphosphonate control for BCS) + vancomycin; 3) HPHBP (bisphosphonate control for HBCS) + vancomycin; 4) vancomycin; 5) sitafloxacin; 6) BCS + vancomycin; and 7) HBCS + vancomycin. BLI confirmed infection persisted in all groups except for mice treated with BCS or HBCS + vancomycin. Radiology revealed catastrophic femur fractures in all groups except mice treated with BCS or HBCS + vancomycin, which also displayed decreases in peri-implant bone loss, osteoclast numbers, and biofilm. To confirm this, we assessed the efficacy of vancomycin, sitafloxacin, and HBCS monotherapy in a transtibial implant model. The results showed complete lack of vancomycin efficacy, while all mice treated with HBCS had evidence of infection control, and some had evidence of osseous integrated septic implants, suggestive of biofilm eradication. Taken together these studies demonstrate that HBCS adjuvant with standard of care debridement and vancomycin therapy has the potential to eradicate MRSA osteomyelitis.


INTRODUCTION
Chronic bone infections from Staphylococcus aureus (S. aureus) present a major challenge for orthopaedics, as clinical studies have shown that standard of care (SOC) surgery with antibiotic therapy only results in a ~ 40% cure rate and outcomes from methicillin-resistant S. aureus (MRSA) are more severe. 1 These clinical failures are largely due to recalcitrant bio lm bacteria, which are now known to exist in four distinct forms: 1) intracellular bacteria, 2) bacteria within glycocalyx on the implant and necrotic tissue, 3) Staphylococcus abscess communities (SACs) and 4) bacteria within the osteocytelacuno canalicular network (OLCN) of cortical bone. [2][3][4] While SOC debridement, irrigation, and conventional antibiotic therapy are effective against planktonic bacteria and some forms of bio lm, animal model studies demonstrated that this intervention has no e cacy against SACs and bacteria within the OLCN. 5,6 It has also been demonstrated that the combination of high dose local and systemic antibiotic therapy fails to achieve the minimum bio lm eradication concentration (MBEC) within the OLCN compartment, likely due to unfavorable pharmacokinetics, which resulting in persistent infection of susceptible bacterial strains. 7 To overcome the pharmacokinetic limitations of SOC antibiotics, we pioneered bisphosphonateconjugated antibiotics (BCA), which utilize a "target and release" mode of drug delivery to sites of bone infection. [8][9][10] In this strategy non-nitrogen-containing bisphosphonates that possess a high a nity to hydroxyapatite at resorbing bone surfaces, but have negligible effects on bone cells, are conjugated to SOC antibiotics via a semi-labile chemical linkage that is stable in the bloodstream. In this way, antibiotics with potency against bio lm bacteria can be speci cally targeted to the bone-bacteria interface and kill the pathogens following acid and/or enzymatic cleavage of the linkage that releases active antibiotic at the diseased bone surface. 9 In support of this concept, we previously demonstrated that systemic delivery of a uorescent bisphosphonate probe binds to the bone surface adjacent to bacteria in a murine model of S. aureus osteomyelitis, con rming bone targeting. 9 To identify an ideal candidate antibiotic to conjugate with a bisphosphonate, we conducted a large screen of available FDA-approved drugs. 11 These antibiotics were tested for their e cacy against S. aureus bio lm bacteria and small colony variants, which model the bacteria within the OLCN of infected bone. These experiments demonstrated that sita oxacin was the most potent among these bactericidal drugs against both methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) in vitro and in vivo. 11 The results also showed that bisphosphonate-conjugated sita oxacin (BCS) and hydroxybisphosphonate-conjugated sita oxacin (HBCS) kill S. aureus in vitro and in vivo, as evidenced by permeabilized cell wall morphology imaged by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). 9,10 As SOC antibiotics are highly effective as prophylaxis for elective surgery (e.g. infection rates for primary total joint replacement are < 2% 12 ), and conservative antibiotic stewardship posits that adjuvant antimicrobials should only be used for challenging established infections, 13 the open question for HBCS and BCS is if it can eradicate chronic MRSA osteomyelitis. Here, we tested this hypothesis in two rigorous murine models: i) the 1-stage revision septic femoral plate, and ii) the septic transtibial pin.

RESULTS
Adjuvant therapy effects of BCA on established implant-associated osteomyelitis following 1-stage revision of a femoral plate in mice.
To assess the adjuvant therapy effects of BCA on top of vancomycin therapy, we utilized an established murine femoral plate 1-stage revision model, 14 in which mice were infected with bioluminescent MRSA for 7 days to establish chronic osteomyelitis prior to 1-stage revision and randomization into 6 treatment groups based on BLI as described in Fig. 1. Of note is that there were 5 independent control groups: 1) mice euthanized at day 7 to serve as peri-implant osteolysis controls, 2) parenteral vancomycin monotherapy, 3) parenteral sita oxacin monotherapy, 4) the bone-targeting moiety of BCS, hydroxyphenylethanebisphosphonate (HPHBP) plus vancomycin, and 5) the bone-targeting moiety of HBCS, hydroxyphenylethane hydroxybisphosphonate (HPHBP) plus vancomycin. Equivocal implantassociated osteomyelitis was con rmed in all groups via CFU assays of the explanted screws and plates ( Fig. 2A-C). We also recorded the weekly body weight for each mouse, which is presented as the % pre-op body weight for each mouse and the group in contiguous line (Fig. 2D) and bar (Fig. 2E) graph format.
Interestingly, mice treated with HBCS plus vancomycin was the only group that demonstrated signi cant increase over their pre-op weight, and had greater weights than mice treated with vancomycin monotherapy at day 21.
Longitudinal in vivo BLI was performed to assess BCA therapy on MRSA planktonic growth following revision surgery, and the data are presented in Fig. 3. While the results demonstrated that parenteral sita oxacin, BCS and HBCS reduced BLI at 12 days, only the HBCS treated mice demonstrated a signi cant decrease in BLI versus vancomycin monotherapy controls 2-weeks post-revision surgery.
As catastrophic femur fracture secondary to peri-implant osteolysis is the most severe adverse event observed in our 1-stage revision model, we assessed this negative outcome via longitudinal X-rays and ex vivo micro-CT (Fig. 4). Assessment of the in vivo day 21 plain lms revealed evidence of septic screw hole radiolucency and catastrophic femur fracture originating from this site (#4 screw hole) in all treatment groups except BCS and HBCS treated mice. Remarkably, while none of the vancomycin treated mice demonstrated a signi cant increase in catastrophic fractures prior to implant removal, all parental sita oxacin treated mice had radiographic evidence of catastrophic femur fracture on day 21 (Fig. 4B), suggesting that this non-bone-targeted antibiotic has no e cacy against MRSA bone infection. Quanti cation of the screw hole volumes via ex vivo micro-CT following implant removal con rmed the lack of parenteral sita oxacin e cacy, and the signi cant therapeutic effect of HBCS adjuvant therapy on peri-implant osteolysis ( Fig. 4C-G).
Although the poor tissue quality of the fractured femurs prohibited rigorous histologic analyses to con rm the antimicrobial and bone protective effects of BCA, we performed a histomorphometry screen for TRAP-stained osteoclasts ( To overcome the challenges of performing rigorous histologic assessments on fractured femurs, and to directly assess the e cacy of our lead BCA candidate vs. SOC antibiotics, we performed a focused study of HBCS vs. parenteral vancomycin and sita oxacin monotherapy in a well-established septic transtibial pin model in which the mice were infected with a MRSA contaminated pin for 7 days prior to 14 days of treatment as described in Fig. 7. Although this experiment was designed with n = 10, some mice were lost to anesthesia and poor tissue quality, which left n = 8 in the vancomycin group, n = 7 in the sita oxacin group, and n = 8 in the HBCS group (Table 1).
Consistent with their lack of e cacy in the femur model, plain radiographs of the infected tibiae prior to sacri ce in mice treated with parenteral vancomycin and sita oxacin demonstrated extensive periimplant osteolysis and dislocation of the MRSA contaminated pin (Fig. 8A, B). While similar radiographs were obtained from 5 of the HBCS treated mice, 3 of the animals in this group display remarkable osseointegration of the implant as evidenced by radiodensity around the pin with concomitant reactive cortical bone formation from the initial infection, suggesting that the treatment irradicated the bone infection (Fig. 8C). For comparison purposes, we present an example of aseptic loosening of a transtibial pin from a prior study (Fig. 8D), which illustrates the superior outcome achieved in some MRSA infected mice treated with HBCS versus some mice that receive a sterile transtibial pin.  (Table 1). In contrast, only 5 out of 7 sita oxacin and all HBCS treated mice had histologic evidence of infection control by these criteria (Table 1). Most remarkable was the frank osseointegration of the implant, as evidenced by new cortical bone completely surrounding the pin hole, whose dimensions are the same as the circumference of the at wire implant (Fig. 8G).
Brown and Brenn-stained histology and TEM assessment of SACs within the bone marrow con rmed the lack of e cacy for vancomycin, as no effects on the brin ring or the bacteria within were noted ( Fig. 9A-E). In contrast, both sita oxacin and HBCS treated tibiae contained SACs with degenerated brin rings with necrotic bacteria cores ( Fig. 9F-O). In some of the HBCS treated tibiae we observed complete disintegration of the brin ring with only a few viable bacteria within (Fig. 9O). These results demonstrate the unique potency of sita oxacin to eradicate SACs and are consistent with the remarkable MSSA and MRSA killing we previously observed with in vitro bio lms. We also used this Brown and Brenn-stained histology to perform TEM assessment of bacteria within the OLCN of MRSA colonized bone fragments (Fig. 10). The results con rmed the decreased incidence of chronic osteomyelitis detected in HBCS treated mice (Table 1). Interestingly, two mice treated with HBCS contained MRSA colonized bone fragments in their tibiae, and no morphological difference of the bacteria within the OLCN of these samples could be detected versus TEM images of tibiae treated with vancomycin ( Fig. 10). Thus, HBCS e cacy against this unique bio lm appears to be an all or none phenomena. However, osseointegration of the septic implant could still be observed in an infected tibia with a colonized necrotic bone fragment ( Fig. 10F), which is an unexplained result to be followed up in future investigations.

DISCUSSION
Implant-associated osteomyelitis remains a major healthcare problem, and current clinical outcomes remain similar to the 77% of 5-year success (survival) rates obtained with SOC implant removal, debridement, and antibiotic therapy rst developed in the 1970s. 15 Moreover, data from the 2018 International Consensus Meeting (ICM) on Musculoskeletal Infections (MSKI) reported no changes in bone infection rates, the primary pathogen, treatment algorithm, and poor outcomes since the SOC treatment algorithm for implant-associated osteomyelitis was established half a century ago. [16][17][18] However, research in this eld has produced several major advances that have refocused our thinking about microbial pathogenesis and the pharmacokinetic and biodistribution requirements of antibiotic therapy for chronic bone infection. Most notable are the discoveries of SACs 19,20 and S. aureus invasion and colonization of the OLCN during chronic osteomyelitis 21,22 , and that SOC antibiotics (i.e. vancomycin and gentamycin) have little to no effects on these bio lms. 6,7,14,23 Thus, drug development efforts are needed towards bone-targeted antibiotics that can kill bacteria within mature SACs and OLCN.
Our approach toward overcoming the biodistribution limits of SOC antibiotics for chronic osteomyelitis therapy is to target the drugs directly to the resorbing surface at the bone-bacteria interface via chemical conjugation to bisphosphonate. The motivation for this came from the robust history of safe and effective bisphosphonate therapies, which have proven bone targeting properties with great speci city and minimal side effects on soft tissues. 24 We have previously demonstrated the utility of this bonetargeting platform with several different types of FDA-approved drugs (e.g. bortezomib 25,26 ), and the subject has been extensively reviewed. 24,27−29 From that success, we aimed to develop a bone-targeted antibiotic conjugate that could achieve sustained concentrations of drug well above the MBEC at the site of bone infection. As an initial proof of concept, we demonstrated that systemic administration of a BPconjugated uorophore (AF647-ZOL) speci cally labels the cortical surface of the bone in immediate proximity to S. aureus bacteria during chronic osteomyelitis, 9 and that bisphosphonate-conjugated cipro oxacin has clinical potential for oral indications. 8 However, since uoroquinolones are known to have reduced activity against established bio lms, which is particularly true for virulent strains of S.
aureus, 30,31 we synthesized a sita oxacin BCA based on our prior drug library screens for bactericidal activity against S. aureus static bio lm and small colony variants. 11,32 Following our studies demonstrating BCS and HBCS killing of MRSA within bio lms in vitro 9 and in a prophylaxis model in vivo, 10 in this work we evaluated their therapeutic e cacy versus SOC antibiotics and non-antibiotic conjugated bisphosphonate controls in a 1-stage revision model.
As longitudinal in vivo BLI of bioluminescent S. aureus in mice is known to be a biomarker of planktonic growth during the establishment of implant-associated osteomyelitis, 33 and a tool to demonstrate BCA "target and release" pharmacokinetics in the septic transtibial pin model, 10 we utilized this approach to assess BCA adjuvant therapy effects on MRSA reactivation following 1-stage revision as previously described in the femoral plate model. 14 Consistently, we observed post-op spikes in BLI on days 7 and 12 in most of the mice (Figs. 3A and B), con rming MRSA reactivation. While this signal was retained in the control groups, HBCS treated mice displayed a signi cant decrease in BLI by day 14 versus vancomycin control (Fig. 3C), which was consistent with the signi cant increase in body weight observed at day 21 (Fig. 2E). Although BCS treated mice displayed a similar trend in e cacy, the results versus vancomycin monotherapy were not signi cant.
From both a scienti c and potential clinical translation standpoint, the most dramatic effect of BCA adjuvant therapy was its complete protection against catastrophic fracture (Fig. 4A, B). For HBCS treated mice, this e cacy was associated with signi cant decreases in peri-implant osteolysis ( Fig. 4C-G), a decrease in osteoclasts proximal to the screw holes (Fig. 5), and a bio lm decrease on necrotic bone fragments proximal to the screw holes versus vancomycin controls (Fig. 6). BCS also demonstrated signi cant e cacy over vancomycin monotherapy for some of these outcomes, but not as many as HBCS. Thus, although no signi cant differences were observed between these BCA, we concluded that HBCS was superior to BCS in this 1-stage revision model, likely due to greater binding a nity to bone of the hydroxybisphosphonate component of the HBCS conjugate vs the bisphosphonate component of BCS. 34 Although prior studies have demonstrated sita oxacin's remarkable e cacy against MRSA bio lm in vitro and in vivo, [9][10][11] rigorous head-to-head assessment of its superiority to eradicate established SACs vs. vancomycin has not been reported. Thus, our histologic nding that parental vancomycin has no effects, while parenteral sita oxacin eradicates SACs via degeneration of the brin ring and killing the bacteria within is important (Fig. 9). It is equally remarkable that despite its ability to eradicate SACs, parenteral sita oxacin had no other e cacy against MRSA as assessed by in vivo BLI, radiology, and histology. However, our nding that the bone-targeting of this drug results in a signi cantly greater e cacy against reactivation of infection (post-op BLI), greater prevention of catastrophic fractures, and greater eradication of SACs and colonized bone fragments is consistent with our hypothesis that bacteria within the OLCN are not affected by untargeted SOC antibiotics due to biodistribution constraints.
There are several noteworthy limitations to this study, the most apparent being the small number of animals per group in the 1-stage revision model as designed for the screen to identify our lead BCA candidate. Despite these small numbers, we succeeded in demonstrating signi cally e cacy with our lead conjugat HBCS, warranting expanded studies with HBCS to facilitate its eventual clinical use. Another important limitation is that due to the established infections prior to BCA treatment, these studies could not formally demonstrate that BCS and HBCS reduction of peri-implant osteolysis is secondary to their antimicrobial activity, and not due to direct inhibition of osteoclasts that might be predicted for nitrogen-containing bisphosphonates. 35 Since BCA comprises a non-nitrogen-containing bisphosphonates known to have minimal effects on bone cells, 9 it should have no effects on osteoclasts at these concentrations, which is consistent with what we observed in the growth plate of the proximal tibiae adjacent to the infected transtibial pin (Supplemental Fig. 1). However, since the orientation of these histology sections was optimized for the pin hole, additional studies are needed to further assess the direct effects of HBCS on osteoclasts at sites of bone modeling, remodeling, and repair.
There are also intriguing ndings that invite further investigation. Most notably, the osseointegration of MRSA contaminated transtibial implants in HBCS treated mice (Fig. 8C, G & K), suggesting that this drug can irradicate implant-associated bone infection with implant retention. More puzzling is our nding of septic implant osseointegration proximal to a MRSA colonized necrotic bone fragment (Fig. 10E-H), suggesting that HBCS treatment allows for a unique sequestrum isolating the infection from adjacent bone formation around the implant. It is also unclear, why the HBCS treatment failed to kill the bacteria within this isolated necrotic bone de es explanation at this time.

CONCLUSIONS
Here we describe initial evidence of HBCS therapeutic e cacy in murine models of implant-associated osteomyelitis and this drug's unique ability to prevent catastrophic fractures, eradicate SACs, and mediate osseointegration of septic implants. Follow up studies to con rm these ndings in large animals and establish preclinical safety and toxicology are warranted for clinical trial evaluation as a novel drug to treat patients with chronic osteomyelitis.

MRSA strain, in vitro culture, and colony forming units (CFU)
The most prevalent community-acquired MRSA strain is USA300. Thus, we used USA300 LAC::Lux, which has a chromosomal insertion of the bacterial luciferase lux gene that allows for in vivo bioluminescent imaging (BLI). 36 This bioluminescent construct constitutively emits a blue-green light with a maximal emission wavelength of 490 nm only in live and metabolically active bacteria. 37 The USA300 LAC::Lux bacteria were cultured in tryptic soy broth (TSB) media at 37 0 C overnight as previously described. 9,38 CFU assays of overnight cultures and sonicated implants were performed as previously described. 14,39 Animal surgeries and antibiotics treatments All animal experiments were conducted under IACUC-approved protocols. Female BALB/c mice that were 10 weeks old were purchased from Jackson Research Labs (Bar Harbor, ME) and acclimated to a standard microisolator environment for one week prior to surgeries, as previously described 14,40 . Brie y, the mice were anesthetized with xylazine (20 mg/kg) and ketamine (100 mg/kg) administered intraperitoneally, and the right femur or tibia was exposed in accordance with animal surgery guidelines to follow either the septic femoral plate 1-stage revision model or septic transtibial pin model, respectively. Experiment 1: The septic femoral plate 1-stage revision model. A total of 28 mice underwent septic femoral plate surgery as described in Fig. 1. The right femur was implanted with a 6-hole femoral titanium plate and screws (RISystem, Davos, Switzerland) that was secured by a sterile screw in hole # 3 and a screw containing ~ 10 5 CFU of USA300LAC::Lux in hole # 4. The presence of osteomyelitis was con rmed via X-ray, BLI, and CFU assays on day 7 as previously described. 14 Also on day 7, all implants were removed with debridement and irrigation, and a sterile plate was implanted and secured by sterile screws in holes # 1 and # 6 with the initiation of antibiotic therapy (Fig. 1A, B). To verify the e cacy of SOC with bisphosphonate-conjugated sita oxacin (BCS) and hydroxybisphosphonate-conjugated sita oxacin (HBCS), the mice were randomized into seven treatment groups: 1) Baseline (day 7 post-op to serve as a cross-section control for peri-implant osteolysis) (n = 4); 2) Hydroxyphenylethanebisphosphonate (HPBP, BV6001) + Vancomycin (n = 4); 3) Hydroxyphenylethanehydroxybisphosphonate (HPHBP, BV6301) + Vancomycin(n = 4); 4) Vancomycin (n = 4); 5) Sita oxacin(n = 4); 6) BCS (BV600072) + Vancomycin (n = 4); 7) HBCS (BV63072) + Vancomycin (n = 4). The parent sita oxacin was used to assess the effects of non-bone targeted antibiotics, while HPHBP and HPBP were used as control for non-nitrogen-containing bisphosphonate effects on bone, and the mice were euthanized on day 21 (Fig. 1C). The chemical structures and commercial product numbers of these compounds are shown in Fig. 1D.
Experiment 2: Septic transtibial pin model. To further assess the antimicrobial e cacy of HBCS and its mechanism of action, a transtibial pin model was employed as previously described 40 . Brie y, the right tibia was shaved, and the skin was cleansed with 70% ethanol. Infection was initiated by placing a stainless steel L-shape pin contaminated with ~ 10 5 CFU of USA300LAC::Lux transcortical through the tibia via medical to lateral implantation which remained untreated for 1 week to establish chronic osteomyelitis (Fig. 7A). On day 7, the mice were randomized into three monotherapy treatment groups (n = 10/group): 1) Vancomycin (110mg/kg/24hr); 2) Sita oxacin (2.5mg/kg/24hr); and 3) HBCS (BV63072) (3mg/kg/24hr). The infected mice were treated for another 14 days, euthanized via heart perfusion with 4% paraformaldehyde/2.5% glutaraldehyde on day 21, and the infected tibiae were processed for histology and TEM (Fig. 7B).

Bioluminescence Imaging
For mice in Experiment 1, in vivo BLI was performed on days 0, 1, 3, 5, 7, 7 (postoperative), 8, 10, 12 and 14 as indicated in Fig. 1. Prior to performing the BLI, the mice were anesthetized with ketamine/xylazine (100mg/20mg/kg) and the femur with implant was included in the region of interest (ROI) for imaging and analysis. The BLI data are presented on a color-scale overlapped with a grayscale photograph of the femur and quanti ed as total ux (photons/s) within a standardized circular ROI using Living Image software (Caliper). 41 Of note is that absence of BLI signal on day 1 is an exclusion criteria for this outcome measure, as the data at later timepoints are normalized to this value. 14 Thus, as 7 mice had no BLI signal on day 1 post-op, BLI analyses for all treatment groups were reduced to n = 3. Following randomization into treatment groups, one mouse in Group 2 and one mouse in Group 3 died due to accidental overdose of anesthesia. Thus these Groups ended up with n = 2 mice and were excluded from statistical analysis of the BLI on Day 14 (Fig. 3C).

Radiographic and Micro-CT
Infected femurs were harvested on day 21 in experiment 1, peri-implant osteolysis and osseointegration was assessed radiographically using a Faxitron Cabinet X-ray system (Faxitron, Wheeling, IL, USA) as previously described. 42 Micro-CT was performed to quantify screw hole volume as previously described. 41,43 Histology Infected long bones were dissected to remove the implants, decalci ed, and embedded in para n. At the mid-point of the femur or tibia on the sagittal cuts, ve 5 µm tissue sections were obtained from three levels and mounted onto slides. One slide per orientation of cut was stained with H&E, Brown-Brenn, or for tartrate-resistant acid phosphatase (TRAP) as previously described. 10 Semi-automated histomorphometry to quantify bio lm area and TRAP + area was performed with Visiopharm software as previously described. 10 Transmission Electron microscopy (TEM) For mice in Experiment 2, regions of interest (ROI) within serial sectioned para n blocks of infected tibia samples were identi ed from the Brown-Brenn stained sections, and adjacent unstained slides were reprocessed for TEM using the "pop-off" technique, as previously described. 44 Slides were depara nized in 3 changes xylene and then rehydrated through a graded series of ethanol back to ddH 2 O. Rehydrated sections on slides were post xed overnight at 4 o C in 0.1M phosphate buffered 2.5% glutaraldehyde, then 1% OsO 4 for 20 minutes at room temperature. The slides were washed in ddH 2 O, dehydrated in a graded series ethanol to 100% (x3), in ltrated with a 1:1 mixture of 100% ethanol and Spurr's resin, then 100% Spurr's resin overnight at room temperature. The ROI (etched with a diamond pen on the back side of the slides) was re-identi ed after the slides were drained of excess resin. A size 3 BEEM capsule lled with fresh resin was placed over the para n section's ROI and polymerized 24 hours at 65˚C. BEEM capsules, with entrapped ROIs, were popped off slides by dipping them 5-10 seconds rapidly in liquid nitrogen. The popped off block was trimmed to the ROI, thin sectioned at ~ 70 nm, and placed onto formvar carbon coated nickel slot grids for imaging using a Gatan Erlangshen 11-megapixel digital camera (Pleasanton, CA) and a Hitachi 7650 TEM (Pleasanton, CA).

Statistics
CFU data were quanti ed for each implant to determine the mean +/-SD for the group, and statistical signi cance was assessed by 1-way ANOVA. Percent of body weight from pre-op day 0 was quanti ed for each mouse to determine the mean +/-SD for the group, and statistical signi cance was assessed by 2way ANOVA. Longitudinal BLI data are presented as the % BLI normalized to day 1 for each group and for each mouse with mean +/-SD for the group, and statistical signi cance was assessed by Wilcoxon matched-pairs signed rank test. Boolean assessment of catastrophic fractures was determined from Xray prior to removal of the implants by Fisher's exact test. Micro-CT assessment of screw hole volume was determined for each mouse with mean +/-SD for the group, and statistical signi cance was assessed by 1-way ANOVA. Histomorphometric quanti cation of bio lm and TRAP + area was determined with ImageJ for each mouse with mean +/-SD for the group, and statistical signi cance was assessed by 1-way ANOVA. Boolean assessments of ongoing infection (presence of necrotic marrow, viable SACs, Gram positive bone fragments, and endosteal ring of osteoclasts) and infection control (presence of bridging new bone and osseous integration of the implant) were quanti ed as the percentage of animals per group and signi cant differences from vancomycin control were assessed by Fisher's exact test.  parental sita oxacin to assess effects of non-bone targeted antibiotic, and the bone targeting moieties 4) hydroxyphenylethanebisphosphonate (HPHBP, BV6301) and 5) hydroxyphenylethanehydroxybisphosphonate (HPBP, BV6001) to control for non-nitrogen containing bisphosphonate effects on bone.

Figure 2
Con rmation of implant-associated osteomyelitis and post-op body weight recovery. The original screws and femoral plate were removed from the mice on day 7 post-implantation, and the sonicate from each was assessed for bacterial load via CFU assay. Data for each implant is presented with the mean +/-SD.
No differences between any groups (3rd, 4th screws and the primary plate) were detected) (A-C). The body weight for each mouse was determined weekly and reported as the % pre-op body weight for the group (D, mean over time), and for each mouse with mean +/-SD for the group (E; *p<0.05 2-way ANOVA).

Figure 3
Combination bisphosphonate-conjugated sita oxacin and vancomycin therapy eradicate planktonic MRSA following 1-stage revision surgery. Longitudinal in vivo BLI was performed on the indicated days following the original septic implant surgery on day 0, and representative BLI images are shown (A), with the longitudinal data presented as the % BLI normalized to day 1 for each group (B, mean over time).
Unfortunately, one mouse of each group of HPBP+ Vancomycin and HPHBP+Vancomycin was lost on day 14 due to overdose of anesthesia. BLI data for each mouse on day 7 and 14 is presented with mean +/-SD for the group (C, *p<0.05 Wilcoxon matched-pairs signed rank test).

Figure 4
Combination bisphosphonate-conjugated sita oxacin and vancomycin therapy prevents catastrophic fractures and peri-implant osteolysis following 1-stage revision surgery. Longitudinal X-rays were obtained on the indicated days following the original septic implant surgery on day 0, and micro-CT was performed on the femurs after harvest on day 21. Representative in vivo X-rays are shown (A) to illustrate the progression of peri-implant osteolysis with Boolean assessment of catastrophic fractures indicated by yellow arrows on day 21 (B; *p<0.05 vs. Baseline via Fisher's exact test). The implants were removed for ex vivo volumetric micro-CT of the femurs on day 21, and representative 3D renderings are shown to illustrate the osteolysis area at each screw hole and catastrophic fractures (yellow arrows) observed in the groups that did not receive bisphosphonate-conjugated sita oxacin(C). The volume of each screw hole for each mouse is presented with mean +/-SD for the group (D-G, *p<0.05 1-way ANOVA).

Figure 5
Combination bisphosphonate-conjugated sita oxacin and vancomycin therapy reduces peri-implant osteoclast numbers at the site of the screw holes following 1-stage revision surgery. The infected femurs (n=3 or 4) described in Figure 4 were processed for TRAP-stained histology, and semi-automated histomorphometry was performed to quantify the TRAP+ area proximal to the revision screw hole #1, and the original MRSA-infected screw hole #4. Representative 1x images (A-G) with 40x region of interest (H-N) are shown to illustrate the number of osteoclasts at the site of bone infection. The TRAP-stained tissue area proximal to screw holes #1 (O) and #4 (P) was quanti ed with ImageJ, and the data for each femur is presented with the mean +/-SD for the group (*p<0.05 1-way ANOVA).

Figure 6
Combination bisphosphonate-conjugated sita oxacin and vancomycin therapy reduces SACs and Gram+ bio lm following 1-stage revision surgery. The infected femurs (n=3 or 4) described in Figure 4 were processed for Brown and Brenn-stained histology, and semi-automated histomorphometry was performed to quantify the Gram+ area proximal to screw holes # 1 and # 4. Representative 1x images (A-G) with 5x region of interest (H-N) are shown to illustrate the SACs (yellow arrows) and bacterial bio lm on bone (red arrows) at the site of bone infection. The Gram-stained tissue area proximal to screw hole # 1 (O) and #4 (P) was quanti ed with ImageJ, and the data for each femur is presented with the mean +/-SD for the group (*p<0.05 1-way ANOVA).

Figure 7
Schematic of transtibial implant model of S. aureusimplant-associated osteomyelitis with antibiotic therapy. L-shaped wires contaminated with ~105 CFU of USA300LAC:Lux were transcortical implanted through the right tibia of 30 mice (A), which remained untreated for 1 week to establish chronic osteomyelitis. On day 7, the mice were randomized into three treatment groups (n=10): Group 1= Vancomycin (110mg/kg/24hr); Group 2 =Sita oxacin (2.5mg/kg/24hr); and Group 3= BV63072 (3mg/kg/24hr). The infected mice were treated for 14 days and euthanized via perfusion with 4% paraformaldehyde/2.5% glutaraldehyde on day 21 post-op, and the infected tibiae were processed for histology and TEM (B).  Sita oxacin kills MRSA within SACs with evidence of brin capsule disintegration. To assess antibiotic effects on intramedullary SAC eradication and effects on bacteria morphology, Brown and Brenn-stained histology with subsequent TEM were performed on the infected tibiae described in Figure 7.    (Table 1). TEM assessment of this bio lm at x5,000 con rmed MRSA colonization of the OLCN (C, G), and x15,000 high-power images of the boxed regions (D, H) failed to detect any remarkable differences in bacteria morphology between groups. However, while the marrow necrosis (#) with peri-implant osteolysis as typically seen in this model is observed around the pinhole in the Vancomycin treated tibiae (* in A), the pinhole in the HBCS treated tibiae is surrounded by new bone (* in E), which may suggest infection control despite the infected necrotic bone fragment in the adjacent marrow space.

Supplementary Files
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