Study Design. Three noncontiguous spinal implant sites in 1 rabbit were challenged with Staphylococcus aureus and local antibiotic prophylaxis was given with gentamicin in controlled-release microspheres (poly(lactic-coglycolic-acid) [PLGA]). Postoperative biomaterial centered infection on and around the titanium rods was assessed using standard bacterial quantification essays.
Objective. To assess surgical site and biomaterial-centered infection reduction with controlled release gentamicin from microspheres against S. aureus.
Summary of Background Data. A postoperative biomaterial-centered infection can be devastating after successful thoracolumbar spinal surgery and puts a high burden on patients, families, surgeons, and hospitals, endangering both our healthcare budget and our ability to perform challenging cases in patients with increasing numbers of comorbidities. Systemic antibiotics often do not reach “dead-space” hematomas where bacteria harbor after surgery, whereas local, controlled release gentamicin prophylaxis through PLGA microspheres showed favorable pharmacokinetics data to achieve local bactericidal concentrations for up to 7 days after surgery.
Methods. A well published rabbit spinal implant model with systemic cephalosporin prophylaxis was challenged to create a baseline infection of 70% in control sites. We then challenged 3 noncontiguous titanium rods inside the laminectomy defect with 10e6 colony forming units S. aureus and randomly treated 2 sites with gentamicin PLGA microspheres and 1 site with PLGA carrier only (control). Standard quantification techniques were used to assess biomaterial centered and soft tissue bacterial growth after 7 days.
Results. After establishing reliable infection rates in control sites, the therapeutic arm of the study was started. Surgical site infections were found in 75% of control sites, whereas gentamicin microspheres reduced the incidence down to 38% in the same rabbits. Biomaterial-centered infection was reduced from 58% to 23% only in all sites challenged with 10e6 S. aureus.
Conclusion. Postoperative, biomaterial-centered infection was reduced at least 50% with intraoperative gentamicin microspheres in the face of systemic cephalosporin
prophylaxis and high dose S. aureus in a laminectomy defect in rabbits. The data are statistically and clinically
significant, and further animal testing is planned to confirm these results.
Key words: postoperative infection, biomaterial centered, PLGA, gentamicin, Staphylococcus aureus, rabbit
model. Spine 2009;34:479 – 483
Surgical site infection (SSI) is the most common, potentially preventable adverse outcome of a major operation. The economic impact alone is enormous and is estimated to cost the US healthcare excess of $1.8 billion per year.1
The cost of treating a single implant-associated spinal wound infection can run in excess of $900,000 and requires substantial resource allocation on the part of hospitals and physicians who are often poorly reimbursed.2–4 As such, the burden placed on hospitals and physicians to provide care for these patients is substantial and disproportionately falls on high volume tertiarycare referral centers, where patients with an implant associated spinal infection are often referred.5,6 Thus the prevention of SSI (prophylaxis) is a first line defense in the battle against these rising healthcare costs. The cost of orthopedic SSIs to patients, in terms of loss of limb and function, goes beyond the economic impact. Infection often results in the need for multiple operations, prolonged antibiotics, and extensive rehabilitation. For patients who develop an SSI, the consequences can be severe as the average length of hospital stay and overall mortality risk are doubled.9 Interventions that decrease the risk of SSI stand to benefit the patients, their providers, the healthcare system, and society at large.
Despite improvements in surgical technique, systemic antibiotic prophylaxis, and reduced operating time, implant-associated spinal wound infections remain a serious concern.1,2,7,8 This is especially true in light of the rapid emergence of multidrug resistant pathogen strains and a large immunocompromised patient population. Though under ideal conditions the incidence of infection has been reported to be less than 1% for patients undergoing elective spinal surgery, conditions are rarely ideal. The incidence of deep infection after spinal surgery may be in excess of 10% dependant on patient- and procedure-related factors.3,7,8,10–13 Geriatric, immunocompromised, diabetic, obese, cognitively impaired, and trauma patients are all known to have greater risks of infection after spinal surgery.8,11,13,14 The purpose of the study was to investigate the use of novel local antibiotic delivery vehicle, as an adjunct to routine perioperative systemic antibiotic prophylaxis, using a spinal implant animal model. An FDA-approved biodegradable polymer (poly(lactic-co-glycolic-acid) [PLGA]) was used to create resorbable microspheres to facilitate the controlled local delivery of gentamicin to wounds and hematoma. The efficacy of these microspheres in prevention of implant-associated spinal wound infections was evaluated using a well published spinal implant model in New Zealand white Rabbits (NZW).
Materials and Methods
Animals
This investigation was approved by the Institutional Animal Care and Use Committee. Twenty-five NZW female rabbits were obtained weighing between 3.0 and 3.5 kg each. Female rabbits were used because, in the experience of the senior author, they are generally more docile and less prone to territorial marking with sprayed urine, which can potentially serve as a source of surgical site contamination.
Experimental Design
The current investigation was a randomized, prospective blinded study of the efficacy of a novel local antibiotic delivery vehicle for the prevention of implant-associated spinal wound infections, using a previously described animal infection model in the NZW rabbit.15 This multisite biomaterial-centered animal model is time tested and reliably mimics the human condition of posterior spinal surgery with instrumentation. By using 3 noncontiguous implant sites, a single animal may serve as both a treatment and internal control, thereby minimizing the number of animals needed for the study. Using an FDA-approved biodegradable polymer PLGA slurry containing 20% gentamicin, resorbable microspheres (10 m, resorption in 3–7 days) were created to facilitate a reliable, controlled release delivery system to wounds and hematoma. The pharmacokinetics of the release were studied in vitro and in vivo before application in an animal model and have been previously described. In short, the gentamicin-microspheres or powdered gentamicin was administered into the rabbit spinal defect in the absence of bacteria (500 g antibiotics per site). Animals were subsequently killed after 2, 4, 10, 24, 48, 72, 144, 168, and 208 hours. Hematoma was harvested from the implant sites and released gentamicin was determined in the supernatant after
homogenation and centrifugation. Representation of release for both the microspheres and powdered gentamicin can be seen in Figure 1. Systemic levels never rose above the detection limit of 0.05 g/mL in serum.
During the initial phase of the current study, 13 NZW rabbits were challenged at each of 3 surgical sites with varying concentrations of Staphylococcus aureus bacteria to reliably create a SSI in 70% of control sites in the absence of antibiotics local. Once an infectious dose (ID-70) was established, the second phase of the study investigated the efficacy of gentamicin microspheres (2.5 mg per site containing 500 g of gentamicin) for the prevention of implant-associated spinal wound infection. Twelve rabbits were used for the second phase of the study. Three noncontiguous surgical sites were used in each rabbit; 2 treatment sites and 1 control site, which were assigned in a random fashion. After 7 days, postoperative wound infection was assessed using standard tissue sampling and bacterial quantification techniques to study our hypothesis that the incidence of SSI and of implant-associated wound infection can be reduced using controlled, local delivery of gentamicin using microsphere technology.
Bacterial Inoculum
One day before surgery, S. aureus (ATCC 25923) was suspended in 5 mL trypticase soy broth and incubated at 37°C. After 18 hours, the culture was centrifuged (10,000 RPM) for 10 minutes, and the pellet was diluted in sterile saline. This washing process was repeated twice. Final concentrations of bacteria were obtained by making different dilutions in sterile saline. The final bacterial concentration (colony forming units (CFU) per milliliter) was estimated by using a densometric apparatus and assay (LaMotte 2020e, LaMotte, Chestertown, MD) and final determination was done by plating on Trypticase Soy Agar plates with 5% sheep blood (Fisher Scientific, Boston, MA).
Surgical Procedure
Induction of general anesthesia was performed using a combination of ketamine and xylazine, and subsequently maintained using isoflurane inhalation via nose-cone mask. All rabbits were given intravenous prophylactic ceftriaxone (20 mg/kg) before surgery to mimic preoperative prophylaxis in humans. After induction of anesthesia, the rabbits were positioned prone and each back was shaved, prepared, and draped in a sterile fashion. Three noncontiguous sites were created in each rabbit overlying the T13, L3, and L6 vertebrae. The surgical approach was identical for each site, though separate instruments and drapes were used for each surgical site to prevent cross contamination. A 1.5-cm dorsal skin incision was made longitudinally in the midline, followed by a single incision in the fascia to expose the spinous process. Using a small rongeur, the entire spinous process with surrounding musculature and ligaments was excised from the base, creating a self-contained defect, approximating a partial laminectomy defect. The ligamentum flavum was not violated, and the dura was not exposed. A 1-cm Ti90/Al6/V4
rod (2-mm diameter, Item: TI017905, Goodfellow corporation, Oakdale, PA) was implanted into the defect. Wound hemostasis was achieved with a flowable hemostatic agent (Surgifoam, Johnson and Johnson Wound Management, Somerville, NJ), mixed with either a nonantibiotic PLGA resomer (control group) or gentamicin PLGA microspheres. Bacterial inoculum (100 L) was placed into the defect using a sterile syringe needle (30 G). The fascia was closed using running sutures with biodegradable Vicryl 2/0 suture (Ethicon Inc. Piscataway, NJ). The skin was closed using a running subcutaneous suture with Vicryl 3/0 (Ethicon Inc.). During the initial phase of the study only the nonantibiotic PLGA Resomer was used and each of the 3 surgical sites was challenged with a randomly assigned bacterial load between 104 and 106 CFU to establish the ID-70. The second phase of the study started once the infectious dose was established. In this phase, 1 control site and 2 treatment sites were assigned randomly to each rabbit (using a random number generator). All wounds were challenged with 106 CFU.
After the procedure, analgesia was provided using a standard protocol, and all rabbits were permitted to drink, eat, and weight bear ad libitum. They were monitored daily, especially in regard to their wound healing, body weight, and signs of systemic infection.
Evaluation
After 7 days, postoperative wounds infections were assessed using standard tissue sampling and bacterial quantification techniques. Rabbits were killed using an intravenous injection of phenobarbital (10 mg/kg). After the skin was removed off the entire back using sterile technique, samples of the fascia, the hematoma, and the vertebral lamina were taken and the implanted metal rods were removed from all sites. A piece of the right liver lobe and an intravenous blood sample were obtained to monitor for systemic infection. Harvested tissues weighed, then immediately homogenized (PowerGen 35, Fisher Scientific, Pittsburgh, PA), and implants were sonicated (UBATH, World Precision Instruments, Sarasota, FL) for 15 minutes in cold saline to detach bacteria. Serial dilutions of all samples were created and plated on blood agar plates for 24 hours of incubation at 37°C. The final CFU was determined per gram of tissue sample and per centimeter of titanium rod. Biomaterialcentered infection was defined to occur where S. aureus was present on the implanted rods and at least 1 other tissue sample from the same site. All samples were collected by and evaluated by a member of the team blind to the treatment type at each site. 2 calculations (SigmaStat 3.5; Systat Inc. San Jose, CA) were used to determine if differences in infection incidence were statistically significant. Student t tests were performed to identify statistical differences in severity of bacterial burden, both with a P value set at 5% for significance.
Results
Two rabbits did not survive to the 7-day endpoint. One rabbit could not be resuscitated after induction of anesthesia before surgical intervention in phase 1 and another animal died unexpectedly during recovery in the
postanesthesia incubator after surgery for phase 2. None of the remaining 23 animals suffered from any systemic infection and all started to gain weight again after postoperative day 2. Phase 1 was completed with 12 animals, and results from the increasing bacterial inoculum to achieve an approximate infection incidence of 70% are listed in Table 1. Based on the results of phase 1, all spinal implant sites in phase 2 were inoculated with 106 CFU S. aureus. Eleven rabbits were evaluated after 7 days and final results for infection incidence are listed in Table 2. Both for SSI as well as implant-associated infection, incidence of infection was significantly reduced using gentamicin microspheres compared with control sites (P 0.01: 2 test; SigmaStat 3.5, Systat Software, Inc.). Severity of infection was assessed using serial plating techniques with average bacterial counts for infected samples shown in Table 3. There was no significance between the control- and treatment group for severity of bacterial growth once a site became infected.
Discussion
Despite meticulous technique, bacteria end up inside the surgical wound after long procedures.16,17 Though the routine use of systemic antibiotic prophylaxis has revolutionized the care of surgical patients, this modality
alone is insufficient for high-risk patients.17 Local hematoma harboring bacteria at the end of the procedure, combined with systemic malnutrition, tissue hypoxia, compromised skin under a stabilizing brace, and poor wound healing while patients are bedridden are important factors for the progression of these initial bacterial burdens into clinically significant infection. Even the implants themselves conspire against the surgeon physician
to decrease the body’s ability to eliminate bacteria. The use of implants enhances the formation of a surface adherent and protective “biofilm” that is difficult to eradicate despite the use of antibiotics that are highly effective
in standard in vitro susceptibility tests.18–21 Local delivery of antibiotics to spinal surgical wounds is intuitively attractive as an adjunct to systemic perioperative antibiotics. Thus, it allows for the local environment, where intravenous antibiotics cannot reach (hypoxic, devitalized tissue, dead space, pooled hematoma without vascular supply), to be sterilized. The ability to delivery antibiotics locally to wounds, primarily in the form of antibiotic powder impregnated in bone cement, is well established in the treatment of musculoskeletal infections.22–25 However, the use of bone cement for the local delivery of powdered antibiotics has many drawbacks. Foremost, the pharmokinetics of antibiotic delivery with bone cement are unpredictable and vary depending on the porosity of the cement used, the type of antibiotic, and the mixing conditions.
In general, the pharmokinetics are characterized by initial burst levels of antibiotics, which may be cytotoxic, and which rapidly decline often below therapeutic levels.23,32–35 Because bone cement is not bio-absorbable, the cement itself may serve as a nidus for infection once the antibiotics have been delivered.36,37 Bone cement also allows bacterial adhesion and growth even in the presence of antibiotics and sustained exposure to subtherapeutic antibiotic levels contributes to the further development of drugresistant bacteria. Furthermore, the bulk of the bone cement may compromise a surgeon’s ability to close the surgical wound and, because the bone cement is nonabsorbable, additional surgery is often required for its explantation.
Gentamicin microspheres described herein offer many advantages over antibiotic impregnated bone cement. The reduction of postoperative infection was statistically and clinically significant, although the spheres did not protect against the severity of the infection in cases where infections occurred. Once a site became “overcolonized” despite the presence of the gentamicin microspheres, bacterial burdens were similar as seen in infected control
sites. The explanation for that could be the “all-ornothing” phenomenon. Once S. aureus CFUs overcame the local challenge and started surviving more frequently, overcolonization of the sites occurred. There is most likely a ceiling effect, above which CFUs become nutrient deprived and therefore, severity cannot “worsen” in control sites over the sites treated prophylactically with the microspheres. The pharmokinetics of the gentamicin microspheres, which provide a controlled and sustained release of therapeutic levels of antibiotics, are clinically superior to those of powdered antibiotics.4 Additionally, because the microspheres are bioabsorbable, there is never a need for patients to undergo an additional surgical procedure for their removal nor do they serve as a nidus for infection once their antibiotics are delivered. The small size of the microspheres allows them to accommodate any existing surgical defect, even allowing them to be injected by syringe after primary fascial closure, without ever compromising the surgical wound.
Gentamicin microspheres are not just intuitively attractive; they have proven to be effective both in vitro and in vivo. The results of the current study in a well established animal model are promising and have demonstrated the ability of these microspheres to significantly decrease the incidence of implant-associated postoperative wound infections. This is in agreement with prior efficacy data for the spheres against nonimplantassociated infection.4 Most importantly, the use of gentamicin microspheres demonstrated a protective effect against SSI, in addition to that provided by systemic perioperative antibiotics, mimicking the current clinical standards. Clinical investigation of these gentamicin microspheres in postoperative spine wounds in high-risk patients is eminent.
Key Points
● Reliable biomaterial-centered infections were established in 3 spinal implant in NZW.
● Gentamicin microsphere treatments locally inside the hematoma of a laminectomy defect significantly reduced the infection incidence over control treatment in the same animal and will be investigated as an adjunct antibacterial prophylaxis in humans.