Intrawound vancomycin powder eradicates surgical wound contamination: An in vivo rabbit study

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1 Washington University School of Meicine Digital Open Access Publications 2014 Intrawoun vancomycin power eraicates surgical woun contamination: An in vivo rabbit stuy Lukas P. Zebala Tapanut Chuntarapas Michael P. Kelly Michael Talcott Suellen Greco See next page for aitional authors Follow this an aitional works at: Recommene Citation Zebala, Lukas P.; Chuntarapas, Tapanut; Kelly, Michael P.; Talcott, Michael; Greco, Suellen; an Riew, K. Daniel,,"Intrawoun vancomycin power eraicates surgical woun contamination: An in vivo rabbit stuy." The Journal of Bone an Joint Surgery.96, (2014). This Open Access Publication is brought to you for free an open access by Digital It has been accepte for inclusion in Open Access Publications by an authorize aministrator of Digital For more information, please contact

2 Authors Lukas P. Zebala, Tapanut Chuntarapas, Michael P. Kelly, Michael Talcott, Suellen Greco, an K. Daniel Riew This open access publication is available at Digital

3 46 COPYRIGHT Ó 2014 BY THE JOURNAL OF BONE AND JOINT SURGERY, INCORPORATED Intrawoun Vancomycin Power Eraicates Surgical Woun Contamination An in Vivo Rabbit Stuy Lukas P. Zebala, MD, Tapanut Chuntarapas, MD, Michael P. Kelly, MD, Michael Talcott, DVM, Suellen Greco, DVM, an K. Daniel Riew, MD Investigation performe at the Departments of Orthopaeic Surgery an Comparative Meicine, Washington University in St. Louis, St. Louis, Missouri Backgroun: Surgical site infection remains a complication of spine surgery espite routine use of prophylactic antibiotics. Retrospective clinical stuies of intrawoun vancomycin use have ocumente a ecrease prevalence of surgical site infection after spine surgery. The purpose of the present stuy was to assess the efficacy of intrawoun vancomycin power in terms of eraicating a known bacterial surgical site contamination in a rabbit spine surgery moel. Methos: Twenty New Zealan White rabbits unerwent lumbar partial laminectomy an wire implantation. The surgical sites were inoculate, prior to closure, by injecting 100 ml of cefazolin-sensitive an vancomycin-sensitive Staphylococcus aureus (S. aureus) ( colony-forming units [CFU]/mL) into the woun. Preoperative cefazolin was aministere to all rabbits, an vancomycin power (100 mg) was place into the woun of ten rabbits prior to closure. The rabbits were kille on postoperative ay four, an tissue an wire samples were obtaine for bacteriologic assessment. An inepenent samples t test was use to assess mean group ifferences, an a Fisher exact test was use to assess ifferences in categorical variables. Results: The vancomycin-treate an the control rabbits were similar in weight (mean [an stanareviation], 4.1 ± 0.5 kg an 4.0 ± 0.4 kg, respectively; p = 0.60) an sex istribution an ha similar urations of surgery (21.7 ± 7.7 minutes an 16.9 ± 6.7 minutes; p = 0.15). The bacterial cultures of the surgical site tissues were negative for all ten vancomycin-treate rabbits an positive for all ten control rabbits (p < ). Bacterial growth occurre in thirty-nine of forty samples from the control group but in zero of forty samples from the vancomycin group (p < ). All bloo an liver samples were sterile. No rabbit ha evience of sepsis or vancomycin toxicity. Gross examination of the surgical sites showe no ifferences between the groups. Conclusions: In a rabbit spine-infection moel, intrawoun vancomycin power in combination with preoperative cefazolin eliminate S. aureus surgical site contamination. All rabbits that were manage with only prophylactic cefazolin ha persistent S. aureus contamination. Clinical Relevance: This animal stuy supports the finings in prior clinical reports that intrawoun vancomycin power helps reuce the risk of surgical site infections. Peer Review: This article was reviewe by the Eitor-in-Chief an one Deputy Eitor, an it unerwent bline review by two or more outsie experts. It was also reviewe byan expert in methoologyan statistics. The Deputy Eitor reviewe each revision of the article, an it unerwent a final reviewbythe Eitor-in-Chief prior to publication. Final corrections an clarifications occurreuring one or more exchanges between the author(s) an copyeitors. Surgical site infection is a known complication of spine surgery. The prevalence of postoperative spine infection varies with the type of surgery, ranging from 1% to 3% after anterior cervical an lumbar ecompression surgery to approximately 10% to 15% after fusion to treat spine trauma or neuromuscular scoliosis The risk of eveloping a surgical site infection is multifactorial. Authors of prior stuies have escribe both patient risk factors, such as obesity, iabetes, an an immunocompromise state, an operative risk factors, such as multilevel surgery, use of Disclosure: None of the authors receive payments or services, either irectly or inirectly (i.e., via his or her institution), from a thir party in support of any aspect of this work. One or more of the authors, or his or her institution, has ha a financial relationship, in the thirty-six months prior to submission of this work, with an entity in the biomeical arena that coul be perceive to influence or have the potential to influence what is written in this work. No author has ha any other relationships, or has engage in any other activities, that coul be perceive to influence or have the potential to influence what is written in this work. The complete Disclosures of Potential Conflicts of Interest submitte by authors are always provie with the online version of the article. J Bone Joint Surg Am. 2014;96:

4 47 TABLE I Rabbit an Surgical Descriptive Characteristics Vancomycin Rabbits Control Rabbits P Value Sex (M:F) 6:4 2: Weight*(kg) 4.1 ± ± Duration of surgery* (min) 21.7 ± ± Initial S. aureus concentration (CFU) *The values are given as the mean an stanareviation. instrumentation, revision surgery, an large intraoperative bloo loss The use of preoperative prophylactic intravenous antibiotics reuces the rate of surgical site infections an has become routine practice Unfortunately, surgical site infection can still evelop. Local aministration of antibiotics is an alternative treatment strategy to prevent surgical site infection. The avantages of locally aministere antibiotics are that they are introuceirectly into the surgical site, eliminate the nee for iffusion into the woun that is associate with systemic antibiotics, an offer the potential to obtain a high antibiotic concentration within the surgical site. At high concentration, an antibiotic may become bactericial even to resistant bacteria 21. Local antibiotic aministration has been stuie with ifferent elivery methos an antibiotics, with positive results The use of intrawoun vancomycin power in ault spine surgery has been escribe in several retrospective clinical series In these series, the authors foun a significantly lower postoperative infection rate with use of intrawoun vancomycin power for iverse surgical populations, incluing those treate for ault egenerative isease 27,29, eformity 30,or spine trauma 28 an those treate with cervical spine surgery 31. The purpose of the current stuy was to assess the efficacy of intrawoun vancomycin power with regar to reucing surgical site contamination in a rabbit spine-infection moel. Our hypothesis was that rabbits manage with intrawoun vancomycin power woul have less bacterial colonization an tissue infection as emonstrate by microbiological testing. Materials an Methos Bacterial Preparation Staphylococcus aureus (S. aureus) was utilize for the surgical site infections because it is the most common bacterial cause of postoperative infections 3,31,32. The S. aureus (American Type Culture Collection [ATCC] number 25923) isolate use for inoculation was obtaine from a repository maintaine at our hospital clinical microbiology laboratory. This is a methicillin-sensitive strain that has been use in prior animal stuies on intrawoun gentamicin application 24,25. This bacterial strain was confirme to be sensitive to both cefazolin an vancomycin. The ientity of this isolate was confirme on the basis of morphologic characteristics an biochemical analysis (VITEK 2; biomérieux, Durham, North Carolina). Organisms use for this experiment were grown on 5% sheep bloo agar culture (Northeast Laboratory Services, Waterville, Maine) for twenty-four hours at 37 C. Bacteria were collecte with use of a sterile swab an then were suspene in sterile phosphate-buffere saline solution to the esire concentration. Colony counts an the presence of the organisms in the pure culture were verifie by plating serial ilutions of the bacterial suspension on 5% sheep bloo agar. In an initial phase, New Zealan White rabbits were challenge with varying concentrations of S. aureus to reliably create surgical site contamination. On the basis of this preliminary work an a prior report 23,a colony forming units [CFU]/mL concentration of S. aureus was use for bacterial surgical site inoculation because it reliably prouce a contaminate surgical site. Stuy Design The stuy protocol was approve by our Institutional Biosafety Committee an Institutional Animal Care an Use Committee. The stuy esign was base on a prior rabbit spine-surgery infection moel that reliably mimics human posterior spine surgery 33. Twenty New Zealan White rabbits were ivie into two groups of ten rabbits each. Ten control rabbits receive one preoperative ose of cefazolin, an ten experimental rabbits receive preoperative cefazolin as well as intrawoun vancomycin power. The preoperative 30-mg/kg cefazolin ose was base on a prior prophylactic antibiotic rabbit spine-infection stuy 34. Postoperative cefazolin was not aministere because a prior stuy ha shown that a single preoperative cefazolin ose was as effective as a combination of preoperative an postoperative cefazolin in preventing surgical site infection in a rabbit-spine moel 35. Surgical Proceure In the presurgical holing area, the rabbits were anesthetize with a ketamine an xylazine cocktail (20 mg/kg an 2.5 mg/kg, respectively, given intramuscularly) an maintaine with 3% to 5% isoflurane gas. The rabbits were intubate, an the posterior aspect of the thoracic an lumbar spine was shave. The preoperative cefazolin was aministere fifteen minutes before the skin incision was mae. The posterior thoracic an lumbar regions of the trunk were prepare with a Betaine (povione-ioine) scrub, an alcohol swab, an Betaine paint, an the surgical site was sterilely rape. A 1.5-cm incision centere on approximately L3 was mae, an sharp issection through the subcutaneous tissue an fascia own to the lumbar spinous process was performe. Retractors were place to reflect the superficial tissue an expose the spinous process, an a rongeur was use to remove the entire spinous process an surrouning musculature, creating a efect mimicking a partial laminectomy. The ligamentum flavum an the ura were not expose. A 5-mm Kirschner wire was implante into the transverse process to mimic posterior instrumentation. A sterile pipette was use to inoculate the woun with 100 ml of CFU/mL of S. aureus. The experimental rabbits were treate with 100 mg of vancomycin power placeirectly within the woun prior to closure. A 100-mg vancomycin ose in a 4-kg rabbit is equivalent to a 2-goseinan80-kghumanault,whichhasbeenpreviouslyshowntobe safe an effective in humans 27. The eep muscle an the fascia were close with PROLENE (polypropylene) sutures in orer to contain the vancomycin power within the surgical site, an the skin was close with subcuticular PROLENE sutures an DERMABOND ahesive (Ethicon, Somerville, New Jersey). Postoperative analgesia inclue Buprenex (buprenorphine hyrochlorie; 0.02 mg/kg subcutaneously) an a 25-mg/hr fentanyl patch. The animals were monitoreaily for signs of pain, infection, an other complications.

5 48 TABLE II Difference in Rates of Positive Cultures Between Vancomycin-Treate an Control Rabbits on Final Bacteriologic Culture Analysis Fascia Muscle Bone Wire Bloo Liver No. positive cultures/total no. Vancomycin-treate 0/10 0/10 0/10 0/10 0/10 0/10 Control 10/10 10/10 10/10 9/10 0/10 0/10 P value < < < < Bacterial Evaluation Four ays postoperatively, the rabbits were reseate for bloo collection an were then kille via an intravenous pentobarbital overose. The surgical incision was prepare with a Betaine scrub, the surgical site was sterilely rape, an the skin an fascial incisions were reopene uner sterile conitions in orer to expose the laminectomy efect. Sterile instruments were use to harvest woun tissue, incluing the fascia, muscle, lamina, an transverse process, an the surgical wire an the tissues were place into sterile test tubes. A 2 2-cm liver biopsy specimen was also collecte uner sterile conitions. The collecte specimens were ivie for bacteriologic analysis. The tissues use for bacteriologic culture were iniviually weighe an then place with a specifie volume of sterile saline solution in a sterile tissue griner (Fisherbran Close Ultra Tissue Griner System; Thermo Fisher Scientific, Houston, Texas). Samples were groun with use of this system to achieve isruption of tissues an the release of bacterial colonies, resulting in a clouy supernatant. A specifie volume of this supernatant consisting of the groun tissue an saline solution was plate on 5% sheep bloo agar an incubate for a minimum of twenty-four hours at 37 C. The retrieve implante wire was weighe an was place in a known volume of sterile saline solution. The wire an saline solution were sonicate (Ultrasonic FS-14; Fisher Scientific, Houston, Texas) at room temperature for ten minutes to facilitate release of bacterial colonies from the wire. A specifie aliquot of this sonicate saline solution was plate on 5% sheep bloo agar an incubate for a minimum of twenty-four hours at 37 C. The bloo collecte just prior to killing the rabbits was place in a bloo culture tube (Bac T/ALERT FA; biomérieux), accoring to the manufacturer s recommenations, an was incubate at 37 C for seven ays, after which it was plate on 5% sheep bloo agar an then incubate at 37 C for a minimum of twenty-four hours. After the incubation perio, the colonies grown were counte an were ientifie on the basis of morphologic characteristics an biochemical analysis (VITEK 2; biomérieux). Antibiotic sensitivity tests were performe on the bacteria isolate from each animal. Statistical Analysis An inepenent samples t test was use to assess mean group ifferences an a Fisher exact test was use to assess ifferences in categorical variables (SPSS; IBM, Armonk, New York). Significance was set at p < Source of Funing No external funing was receive to support the present stuy. Results The vancomycin-treate an control rabbits were similar in weight (vancomycin: 4.1 ± 0.5 kg [mean an stanar eviation], control: 4.0 ± 0.4 kg; p = 0.6) an sex istribution (six vancomycin-treate male rabbits, two control male rabbits; p = 0.17), an ha similar urations of surgery (vancomycin: 21.7 ± 7.7 minutes, control: 16.9 ± 6.7 minutes; p = 0.15) (Table I). There were no postoperative woun complications, eaths, or evience of systemic illness. The surgical wouns of the ten vancomycin-treate rabbits i not isplay any evience of local infection, an there were no apparent systemic effects from the vancomycin aministration. One control rabbit ha erythema an a small woun ehiscence, whereas the other nine control rabbits ha subjectively unremarkable incisions. No rabbit ha woun rainage. TABLE III Results of Final Bacteriologic Culture Analysis of Control Rabbit Infections Bacterial Colony Count (CFU/g) Control Rabbit Fascia Muscle Bone Wire * * * * * * * * * * * * * * Negative culture * * * * *Too many bacteria to quantify.

6 49 Bacteriologic Culture Bacteriologic cultures confirme that all surgical sites were inoculate with CFU/mL of S. aureus. All ten control rabbits ha positive bacteriologic woun cultures on postoperative ay four, whereas none of the ten vancomycin-treate rabbits ha a positive culture. This ifference was significant (p < ) (Table II). None of the forty samples (tissue or wire) from the vancomycin-treate rabbits an thirty-nine of the forty from the control rabbits ha bacterial growth (p < ) (Table II). For the control rabbits, bacteriologic colony counts typically range from 10 3 to 10 5 CFU/g tissue (Table III). In some tissues, bacterial colonies were too numerous to count (Table III), which preclue a etermination of recovere bacterial concentrations. All organisms grown on culture were S. aureus. There were no polymicrobial infections. The S. aureus grown on culture was sensitive to cefazolin an vancomycin. None of the bloo or liver samples in either rabbit group showe bacterial growth. Discussion Using a rabbit spine surgical-woun-infection moel, we foun that intrawoun vancomycin was substantially more effective at eliminating surgical site contamination than intravenous cefazolin alone. In fact, all surgical sites in the vancomycin group were sterile, whereas all control-rabbit wouns ha evience of persistent contamination on bacteriologic culture. Systemically aministere antibiotics, such as cefazolin, rely on iffusion into the surgical woun for their treatment effect against surgical site infection. The concentration of these antibiotics within the woun is expecte to be low. In contrast, locally aministere antibiotics achieve high concentrations at the surgical site, with safe systemic concentrations 22,36. Locally aministere vancomycin can reach levels twenty times the toxic serum levels while maintaining a safe systemic concentration, which is likely ue in part to the large molecule size of vancomycin preventing systemic absorption 36. The ability to achieve increase antibiotic concentrations with intrawoun aministration is important because bacteria resistant to a particular antibiotic at low concentration may be susceptible to the antibiotic at a higher concentration 21. In aition, the sprea of bacterial resistance may be less with local elivery because fewer bacteria are expose to the antibiotic than with systemic aministration. Our ata suggest that the intrawoun vancomycin concentration was sufficiently high to overcome a known S. aureus woun contamination in the experimental rabbits. Our results for the elimination of acute surgical site bacterial contamination with use of a locally aministere antibiotic are similar to those in a stuy by Stall et al., who reporte a significantly reuce surgical site infection rate in rabbits manage with local gentamicin microspheres 12. The two stuies were similar in esign an use the same S. aureus strain in a New Zealan White rabbit spine-infection moel. However, 38% of the surgical sites in the stuy by Stall et al. ha persistent positive bacteriologic cultures with gentamicin microspheres. The possible explanations for this iscrepancy are that the microspheres acte as both an antibiotic-elivery platform an a foreign boy for bacterial ahesion. In aition, local antibiotic-elivery systems were utilize to allow for a more graual an sustaine elution of the antibiotic over time. The consequence may have been that the initial local antibiotic concentration i not reach a level high enough to overwhelm the bacterial loa an allowe some bacteria to survive. Using a rat infection moel, Yarboro et al. foun a significantly reuce infection rate for surgical sites treate with locally injecte aqueous gentamicin compare with those treate with locally aministere calcium-sulfate flakes with gentamicin or treate with systemic gentamicin 24. They propose that this ifference might have arisen because the calcium sulfate acte as a harbor for bacterial aherence. In aition, gentamicin peak concentrations may have iffere between locally aministere gentamicin an gentamicin-laen calcium sulfate. In a follow-up stuy, Cavanaugh et al. reporte that the use of a combination of systemic cefazolin an local gentamicin was more effective at ecreasing surgical site bacterial count than the use of systemic cefazolin or local gentamicin alone 25. Our results support this fining, as the use of systemic cefazolin an local vancomycin was superior to the use of systemic cefazolin alone. A combination of systemic an local antibiotics may have a synergistic effect on the reuction of a surgical bacterial loa. The current stanar of care for preoperative antibiotic prophylaxis for spine surgery is the use of an intravenous cephalosporin, most commonly 1 to 2 g of cefazolin (2 g for patients weighing >80 kg) 37. We chose to use 30 mg/kg of cefazolin in the present stuy because this ose ha been use for prior rabbit spine-infection stuies an was recommene by our veterinarian (M.T.) 34,35. Clinical reports on intrawoun vancomycin power use for spinal surgery have escribe the application of 1 to 2 g, an in the largest clinical series of which we are aware (911 patients) 2 g of vancomycin power was place subfascially without systemic toxicity 27. On the basis of an average weight of 4 kg for the New Zealan White rabbits an surgical site area of 3.38 cm 3, application of 100 mg of intrawoun vancomycin power to the rabbit woul be equivalent to 2 g of vancomycin for an 80-kg human patient. There is concern that high concentrations of vancomycin power applie locally to a woun may be cytotoxic to osteoblasts. Authors of prior stuies have reporte that a vancomycin concentration below 1000 mg/ml ha little or no effect on osteoblast growth, whereas 10,000 mg/ml cause cell eath 38,39.We place 100 mg of vancomycin into a surgical site of 3.38 cm 3,for a local vancomycin concentration of approximately 30 mg/ml. This vancomycin concentration falls well below the critical threshol for reuce cell activity aneath. It is important to recognize that, in these in vitro stuies, irect contact between the high-concentration vancomycin an the osteoblast cells was maintaine for twenty-four to seventy-two hours. In contrast, a high concentration of local vancomycin in a surgical site is transient, an is often significantly reuce by twenty-four hours post-aministration to below-cytotoxic concentration levels 27,40.

7 50 Other investigators also have use the methicillinsensitive S. aureus strain that we utilize in the current stuy This bacterial strain has cause the eath of untreate control rats, as reporte by Yarboro et al. 24,butnotof treate rats or New Zealan White rabbits 23,25. This ifference may be associate with the iffering immune systems of the ifferent species an the ability of each species to combat local an systemic infections. In the current investigation, there was no associate mortality in either treatment group. In aition, bloo an liver samples were sterile, inicating that a systemic infection i not occur. As we i not inclue acontrolgroupthatwasnotmanagewithcefazolinor vancomycin, we o not know whether untreate rabbits woul have a higher bacterial inoculation or mortality rate. Also, caution shoul be use when extrapolating these results to infections cause by organisms other than methicillinsensitive S. aureus. We acknowlege several stuy limitations. First, we use a strain of methicillin-sensitive S. aureus. This bacterium was chosen because, in prior animal experiments, it ha been shown to reliably reprouce in vivo infections. Our results may not extrapolate to other bacteria, which will be a focus of future work, but, to our knowlege, these results provie the first basic-science ata that support prior clinical reports of iminishe rates of surgical site infections with use of intrawoun vancomycin. Secon, we chose the fourth postoperative ay en point to evaluate for tissue bacterial infection because the local tissue concentration of vancomycin rops steaily after the thir postoperative ay 27. Thus, we are unable to offer evience regaring the effectiveness of local vancomycin in preventing chronic, elaye infections. The fact that no bacteria were foun in the vancomycin group supports the notion that the contaminant ha been eliminate an that any future, eep infection woul require hematogenous seeing. The primary aim of the present stuy was to assess whether a local vancomycin concentration coul substantially eliminate a known bacterial woun contaminant, an this was shown by our results. Although our stuy was aequately powere to achieve our primary aim, it may have been unerpowere to accurately assess the risk of complications relate to highconcentration, local vancomycin. The use of a 100-mg vancomycin local ose was chosen to mimic that use in the largest clinical series of which we aware that of Sweet et al., who use 2 g of vancomycin power in 911 patients without reporte systemic toxicity 27. None of the vancomycin-treate rabbits ha any apparent clinical vancomycin toxicity effects within the four postoperative ays, but we were unable to reliably measure serum vancomycin levels in these rabbits. In conclusion, the use of intrawoun vancomycin power eliminate S. aureus bacterial contamination in rabbit spine surgical wouns, whereas the use of systemic cefazolin alone i not. 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