Introduction
Steam sterilization with an autoclave has long been established as the most dependable sterilization method for surgical instruments and equipment.1 However, many medical devices and surgical equipment are not amenable to steam sterilization because of their inability to withstand high temperatures. Chemical gas sterilization is often used as an alternative; however, safety and environmental concerns arise with the use of hydrogen peroxide and ethylene oxide for this purpose.2 Cold atmospheric plasma sterilization, which is sterilization that uses partially ionized gas (cold plasma) at room temperature (23°C) and atmospheric pressure (101.325 kPa), is an alternative method with various potential applications beyond sterilization of surgical implants and equipment3,4,5 and has shown efficacy in inactivating bacteria,6 including antimicrobial-resistant and biofilm-forming species7,8; fungi4; spores4; viruses9; and even cancer cells.10
Cold plasma produces UV light, charged particles, and RONS; these elements cause rapid destruction of microbial pathogens while simultaneously limiting any negative tissue effects (eg, ablation, scarring, and necrosis). Although UV light and charged particles produced by cold plasma may contribute to bacterial inactivation, RONS have been identified as the primary contributor of bacterial inactivation.11 Reactive oxygen and nitrogen species are components or byproducts of several essential cellular processes, and bacteria have well-established pathways for decreasing RONS before they can reach concentrations that damage the bacteria. High concentrations of RONS obtained during cold plasma sterilization can overcome the oxidative stress responses by bacteria, thus damaging bacterial lipids, proteins, and DNA and ultimately leading to cell death.12
Ozone is a neutral, relatively long-lived reactive oxygen species that has received increased interest in recent years for medical instrument sterilization. Ozone is a well-known and powerful antimicrobial agent that is produced in high concentrations with cold plasma in air,13,14 and the US FDA has recognized ozone to be generally safe for surface decontamination. In addition to ozone, cold plasma in air generates superoxide, peroxide, hydrogen peroxide, hydroxyl radical, hydroxyl ion, and nitric oxide. These contribute to the efficiency of bacterial inactivation with cold plasma and naturally return to their stable, harmless molecular states (eg, oxygen and nitrogen) when plasma is not being energized.15
Cold atmospheric plasma sterilization of medical instruments has been investigated for over 20 years, but its practical application has been limited because of the complexity of current cold plasma treatment devices and difficulty in defining appropriate and reproducible device parameters.3 A novel CAPS devicea has been developed for sterilizing medical instruments of various sizes and materials. This CAPS device only requires air and electricity to function and has a shorter cycle time and lower cost to purchase and operate than a conventional steam autoclave. Additionally, this device is compact and portable such that it can be used in field settings. Although a preliminary study16 with this device showed rapid bacterial inactivation on common substrates, the device has not yet been evaluated to sterilize instruments or implants for use with or in animals. Cold argon plasma has been evaluated as a method to modify the surface morphology and osseointegration of implants in miniature pigs17; however, little has been published regarding CAPS versus steam sterilization (with an autoclave) of implants for in vivo use and regarding subcutaneous tissue reactions associated with surgical instruments or implants sterilized with CAPS. Surgical instruments and implants are commonly made from stainless steel because it is the most corrosion-resistant metal when in direct contact with biological fluids.18 When introduced into the body with an aseptic technique, stainless steel implants are biologically inert.
The purpose of the study reported here was to determine whether a stainless steel implant sterilized with a novel CAPS devicea adversely affects local tissues in rabbits and whether CAPS was as effective as steam sterilization via an autoclave to inactivate Pasteurella multocida, a common pathogen for skin and subcutaneous infections of rabbits.19,20,21,22 We hypothesized that CAPS would be as effective (noninferior) as steam sterilization at sterilizing implants that had been contaminated with P multocida, such that the incidence of local tissue reactions and SSIs would not significantly differ between sterilization methods.
Materials and Methods
Animals
Thirty-one (22 male and 9 female) approximately 3-month-old New Zealand White rabbitsb with body weights of 2.21 ± 0.27 kg (mean ± SD) were used in the preliminary (n = 2) and investigational (29) studies. Rabbits were considered healthy, including a lack of skin disease, on the basis of physical examination, including specific examination of the skin surface after hair removal at the designated surgical sites at least 24 hours prior to implant placement. Both studies were approved by the Oklahoma State University Institutional Animal Care and Use Committee.
Preliminary study
The purpose of the preliminary study was to identify a bacterial species that would effectively cause an SSI. Each of 2 rabbits were anesthetized so stainless steel implants could be placed. On the day of surgery, each rabbit was premedicated with midazolam (0.5 mg/kg, IM) and butorphanol (0.2 mg/kg, IM).23 Ten minutes later, each rabbit was physically restrained and anesthesia was induced and maintained via face mask with isoflurane (5%) in oxygen (2 L/min).24 Anesthetic monitoring consisted of constant electro-cardiography and pulse oximetry. After all implants were placed, anesthesia was discontinued, flumazenil (0.02 mg/kg, IM) was administered to reverse the sedative effects of the midazolam,23 and meloxicam (1 mg/kg, SC)25 was administered for postoperative analgesia. Rabbits were monitored for at least 30 minutes or until they recovered from anesthesia and were subsequently returned to their cages.
One of four, 3.5-mm stainless steel keys of ameroid constrictorsc (implants) was placed subcutaneously at 1 of 4 predetermined, randomly assignedd sites (1 implant/site). These sites were 3 × 3-cm squares in both shoulder regions caudal to the triceps muscle, and both flanks caudal to the last rib and cranial to the quadriceps muscle. The sites were numbered as follows: site 1, left shoulder; site 2, left flank; site 3, right shoulder; and site 4, right flank. Before implants were placed, each implant site was aseptically prepared with gauze sponges soaked with saline (0.9% NaCl) solution followed by an iodine povacrylex (0.7% iodine) and isopropyl alcohol (74% w/w) surgical solutione according to label instructions. Implants were first steam sterilized with an autoclave at 133.4°C for 8 minutes with an 80-minute cycle, followed by contamination of 3 implants with 1 of 3 bacterial species as follows: Pseudomonas aeruginosa, P multocida, or Staphylococcus aureus. All 3 bacterial species were field isolates obtained from the Oklahoma Animal Disease Diagnostic Laboratory's archive. The fourth implant was not inoculated and served as a negative control. Staphylococcus aureus is one of the most common bacteria isolated from infected prosthetic joints in people,26,27 with P aeruginosa the most commonly isolated gram-negative bacteria from infected prosthetic joints.26 Although P multocida is not the most common organism isolated from SSIs in humans, it has been cited as a causative agent for SSIs in multiple case reports28,29,30,31 and is a known cause of subcutaneous abscess formation in rabbits.20,21,22
The steam-autoclaved implants (3.5-mm stainless steel keys of ameroid constrictorsc) were contaminated with a field isolate of P aeruginosa, P multocida, or S aureus. Five percent sheep blood agar plates were inoculated with the isolate (pure culture) and incubated at 37°C with 5% CO2 overnight. The next day, a suspension was prepared with 2 mL of PBSS and 9 colonies obtained from sheep blood agar plates in a sterile 15-mL tube. The suspension was then mixed by vortexing. Next, an implant was placed and 50 µL of this bacterial suspension was transferred with a pipette into each well of a 96-well plate, ensuring each implant was fully immersed in the suspension. The 96-well plate was then placed in a biological safety cabinet for 12 hours to dry. Next, the dried implants were carefully transferred with sterile technique to surgical instrument pouches amendable to steam sterilizationf (autoclave) or gas and other sterilization methodsg and labeled appropriately.
With aseptic technique, each implant was placed in a 12-gauge microchip hypodermic needleh by a single investigator (MRW) and handed to a blinded investigator (HKA) for implantation in 1 of the 4 randomized sites. Implants were placed subcutaneously at each site at a depth of 4 to 5 mm by advancing the stylet to deposit the implant. Tissue adhesivei was applied to the skin at the needle insertion site after removal of the needle. Investigators changed surgical gloves and instruments after each implantation to ensure sterility and minimize cross contamination.
Each rabbit and implantation site were evaluated daily by a blinded investigator (HKA). Body weight; whether a rabbit was eating, drinking, urinating, and defecating (present or normal vs absent or abnormal); and whether erythema was noted at the implantation sites were recorded. Skin thickness was measured at each site with calipers, and when a discrete swelling suggestive of subcutaneous infection or abscess was present, swelling diameter was also measured twice, with one measurement at a 90° angle to the other. Three days after surgery, each rabbit was euthanized with xylazine (5 mg/kg, IM) followed by pentobarbital (100 mg/kg, IV) 10 minutes later.32 After euthanasia, each site was cleansed with gauze sponges soaked with isopropyl alcohol and a curved incision was made through the skin and subcutaneous tissue with a No. 15 scalpel blade, such that a 1-cm margin was around each palpable implant and each could be seen. Once each implant was seen, the implant and subcutaneous tissues were swabbed with a sterile swab and placed into a sterile tube containing liquid mediumj for bacterial culture. Then, sterile mosquito hemostats were used to grasp each implant and place it in the same tube with the swab. A 2 × 2-cm section of skin and subcutaneous tissue that directly over-laid each implant site was harvested and preserved in neutral-buffered 10% formalin for histologic examination. Investigators changed surgical gloves and instruments after each sample collection to ensure sterility and minimize cross contamination.
Systemic illness was not noted but both rabbits had distinct, firm swellings suggestive of subcutaneous abscess formation within 3 days at the sites of P multocida–contaminated implants (Supplementary Figure S1, available at: avmajournals.avma.org/doi/suppl/10.2460/ajvr.82.2.118), and P multocida was isolated from each. No appreciable swelling or erythema suggestive of infection was noted at the other 3 sites. On the basis of these results, P multocida was selected as the bacteria for implant contamination for the investigational study.
Investigational study
Implant preparation—
The same steam-autoclaved implants (3.5-mm stainless steel keys of ameroid constrictorsc) used in the preliminary study were again contaminated but only with a field isolate of P multocida. As with the preliminary study, 5% sheep blood agar plates were inoculated with the isolate (pure culture) and incubated at 37°C with 5% CO2 overnight. The next day, a suspension was prepared with 2 mL of PBSS and 9 colonies of P multocida obtained from sheep blood agar plates in a sterile 15-mL tube. The suspension was then mixed by vortexing. The number of CFUs per milliliter of P multocida was estimated to be 4 × 109 in 1× PBSS on the basis of detectable growth on 5% sheep blood agar plates inoculated with serial dilutions of the suspension, after overnight incubation (37°C and 5% CO2). Next, an implant was placed and 50 µL of this bacterial suspension was transferred with a pipette into each well of a 96-well plate, ensuring each implant was fully immersed in the suspension. The 96-well plate was then placed in a biological safety cabinet for 12 hours to dry. Next, the dried implants were carefully transferred with sterile technique to surgical instrument pouches amendable to steam sterilizationf (autoclave) or gas and other sterilization methodsg and labeled appropriately.
In vitro evaluation of CAPS device—
Six of these steam-autoclaved, contaminated, and dried (AI) implants were removed from the 96-well plate and transferred to a sterile microcentrifuge tube. Fifty microliters of sterile PBSS was added to the tube, and it was vortexed for 2 minutes. The number of CFUs per milliliter in this suspension was similarly estimated as described for implant preparation. Six other AI implants were subjected to cold plasma sterilization with the novel CAPS device, and then 5% sheep blood agar plates were inoculated with these implants to determine the effectiveness of the CAPS device to sterilize these implants.
In vivo evaluation of CAPS device—
Eight rabbits were included in the first portion of the investigational study. Each rabbit was implanted with 4 implants that were randomly assignedd to 4 sites (1 implant/site). Implants were steam autoclaved (A implant); steam autoclaved and subsequently inoculated with P multocida (AI implant); steam autoclaved, subsequently inoculated with P multocida, and then reautoclaved (AIA implant); and steam autoclaved, subsequently inoculated with P multocida, and then sterilized with CAPS (AICAPS implant). Steam autoclaving of the implants was performed as described for the preliminary study.
The CAPS device worked by producing plasma within the device along the surface of a proprietary plasma sheet with a custom high-voltage, high-frequency power supply that required an input of 115 V of AC power at 50 to 60 Hz with a current draw of 4 A. The device operated at 23°C, pressure of 14.3 PSI (98.598 kPa), and 50% relative humidity. The sterilization cycle consisted of 45 minutes of plasma production16,k,l within the airtight sterilization chamber, 15 minutes of evacuation wherein the sterilant gases and RONS were pumped through activated aluminam (an adsorbent of nitric oxide species) and a manganese dioxide-copper oxide catalystn that converts ozone to oxygen, and subsequent return of altered gas into the sterilization chamber. The sterilization chamber was deemed safe to open when the concentrations of nitrogen dioxide and ozone within the chamber were < 0.1 ppm. Implants were sterilized at 7.6 cm from the plasma source.
Following the implantation surgery, each rabbit and the 4 implantation sites were evaluated daily by a blinded observer (HKA) as described for the preliminary study. All 8 rabbits had clinical signs, including fever (4 of 5 rabbits) and lethargy (8), consistent with systemic disease and 3 died within 72 hours of implantation. The first 2 rabbits that died were examined postmortem. Both had evidence of septicemia, including thrombi formation and intralesional bacteria in the liver, fibrin deposition and intralesional bacteria in the spleen, hemorrhage and intravascular bacteria in the thymus, and fibrin thrombosis in the lungs; P multocida was isolated from the liver, lungs, spleen, and all 4 implant sites.
In an attempt to minimize morbidity, the investigational study design was modified. The A and AI implants were excluded and each of the remaining 21 rabbits were implanted with 1 AIA and 3 AICAPS implants, with random assignment to the 4 sites. After implantation surgery, each rabbit and implantation site were evaluated daily for 5 days by a blinded observer (HKA) as described for the preliminary study. On day 5, rabbits were euthanized as described for the preliminary study.
Sample collection—
Following euthanasia, each implant and material obtained via swabs from each implant and associated subcutaneous tissues were cultured as described for the preliminary study. Then, a 2 × 2-cm section of skin and subcutaneous tissue that directly overlaid each implant site was harvested for histologic examination and reserved in neutral-buffered 10% formalin. A board-certified veterinary pathologist (KLB) who was blinded to sterilization method graded the degree of inflammation, hemorrhage, fibrin deposition, and degeneration or necrosis for the epidermis, dermis, subcutis, and muscle layers of each tissue sample on a scale of 0 to 5, with 0 = none, 1 = minimal, 2 = mild, 3 = moderate, 4 = marked, and 5 = severe (Supplementary Figure S2, available at: avmajournals.avma.org/doi/suppl/10.2460/ajvr.82.2.118). Scores for each layer were combined to yield an overall score.
Statistical analysis—
The 21 rabbits that were subjected to the modified investigational study design were included in the statistical analysis. A commercially available statistical software programo was used for ANOVA. Individual histologic scores for the epidermis, dermis, subcutaneous tissue, and muscle; overall histologic scores; and values for skin thickness and erythema were analyzed among AIA and AICAPS implant sites. When significance was identified, pairwise comparisons were performed. Assuming a noninferiority trial, a power analysis indicated that a difference equal to the SD for histologic scores could be detected with a probability of 0.74. Values of P < 0.05 were considered significant.
Results
In vitro evaluation of CAPS device
The mean ± SD bacterial load for 6 of the AI implants was estimated to be at least 16.7 × 103 ± 5.77 × 103 to 26.7 × 104 ± 5.77 × 104 CFUs/mL (implant). Culture of 6 evaluated AICAPS implants yielded no bacterial growth.
In vivo evaluation of CAPS device
All 21 rabbits successfully completed the modified investigational study design, allowing analysis of 84 implantation sites (21 AIA and 63 AICAPS implants). No adverse events were noted during the study period. Aerobic bacterial culture of all 84 implants and implantation sites were negative for bacterial growth. Mean maximum skin thickness was evident on days 1 and 2 for the AIA implant sites and on day 1 for the AICAPS implant sites; mean skin thickness did not significantly (P = 0.09 to 0.90) differ between AIA and AICAPS implantation sites on any day in the study period (Supplementary Table S1, available at: avmajournals.avma.org/doi/suppl/10.2460/ajvr.82.2.118). Maximum erythema was noted on day 1 at the AIA implant sites and on day 2 at the AICAPS implant sites. Implantation site erythema on any day was not significantly (P = 0.27 to > 0.99) different between the AIA and AICAPS implants. No increase in implantation site diameter (indicating formation of discrete swelling or abscess) occurred at any site; therefore, site diameter measurement data were not analyzed. Additionally, histologic scores did not significantly (P = 0.06 to 0.91) differ between AIA and AICAPS implant sites for each tissue layer (epidermis, dermis, subcutis, and muscle layers) and overall (total histologic score).
Discussion
Results of the present study supported the hypotheses that CAPS would be as effective (noninferior) as steam sterilization (autoclave) at sterilizing stainless steel keys of ameroid constrictors that had been contaminated with P multocida and implanted in rabbits and the development of local tissue reactions and SSIs would not significantly differ between sterilization methods. All rabbits implanted with AIA and AICAPS implants (investigational study) remained bright, alert, and well hydrated and had normal appetites throughout the study. This indicated that although inflammation was noted within 24 hours of implantation, inflammation was transient and no adverse effects were associated with the implantation or presence of implants, regardless of sterilization method, for 5 days in the subcutaneous tissues.
Negative bacterial culture results for all implantation sites indicated that steam sterilization and CAPS sufficiently inactivated P multocida on the implants. Likewise, in vitro evaluation of CAPS for sterilizing P multocida–contaminated (AICAPS) implants yielded the same outcome (negative bacterial growth). However, the present study did not include a PCR assay to determine whether P multocida was present but did not grow; yet, a previous study33 revealed that damage to bacterial DNA occurred secondary to CAPS, such that the DNA of P multocida may not have been detected with a PCR assay. Future studies that include a PCR assay to detect bacterial DNA on instruments, etc, after sterilization with CAPS could be considered, to better evaluate the effectiveness of CAPS.
Skin and soft tissue infections often develop when a bacterial load of approximately 1 × 105 CFUs/g of tissue is attained.34,35 Although the mean bacterial load on the implants used in the present study was < 1 × 105 CFUs/mL, the 2 rabbits of the preliminary study had infection at the implant sites with a lesser load of P multocida and the 8 rabbits used for the first portion of the investigational study rapidly developed infections at the AI implant sites or systemic disease or had died. These findings indicated that despite the bacterial load of the implants was < 1 × 105 CFUs/mL, the bacterial load was sufficient for evaluating the effectiveness of CAPS. A higher bacterial load may have resulted in higher morbidity and mortality rates. Additionally, foreign material, such as an implant and suture material, at a surgical site has been documented to require lower bacterial loads for establishing an SSI.26,36,37 Because implants were a critical component of the present study, we expected and observed the development of SSIs at a lower bacterial load.
Skin thickness measurements were greater and erythema was present at the implantation sites on the first and second days after implantation, regardless of sterilization method. Although the tissue samples associated with the implants were only evaluated at the conclusion of the study (5 days after implantation), the transient increase in skin thickness and presence of erythema in the first 2 days after implantation was likely because of an inflammatory response secondary to the implantation procedure. Compared with the 2 rabbits of the preliminary study in which a marked skin reaction and firm swelling were observed for each rabbit within 3 days of implantation of a P multocida–contaminated implant, neither AIA nor AICAPS implants induced any skin reactions or swellings at the implantation sites for the rabbits of the investigational study.
Individual histologic scores for the epidermis, dermis, subcutis, and muscle, and overall histologic score were not significantly different between the AIA and AICAPS implant sites. However, additional samples for the AIA implants or additional study subjects including additional implants from both categories may have resulted in a significant difference between groups. Additionally, extending the study duration may have resulted in hypersensitivity reactions not detected in the present study. However, a 5-day duration was chosen on the basis of the rapid development of localized infection observed in the 2 rabbits of the preliminary study.
The 8 rabbits in the first portion of the investigational study were implanted with 1 negative-control implant (A), 1 positive-control implant (AI), 1 AIA implant, and 1 AICAPS implant. Septicemia developed, presumably secondary to the SSI at the AI implant site. Therefore, the investigational study design was modified, such that the A and AI implants were not implanted. Elimination of the positive-control implant was not ideal, but its elimination was deemed necessary for the welfare of the rabbits. Successful contamination of the implants with a quantity of P multocida sufficient to induce an SSI was observed in rabbits of the preliminary study and first portion of the investigational study. Because the efficacy of steam sterilization is well established,1 culture of AIA implants did not yield bacterial growth in the present study, and adverse tissue effects have not been described, the decision was made to use only 1 AIA implant and 3 AICAPS implants/rabbit. This design provided more sites to evaluate the tissue effect of the AICAPS implants without the need for including additional rabbits.
The major limitation of the present study was that the efficacy of CAPS was only determined for implants contaminated with P multocida. A list of potential organisms to inoculate the implants was formed on the basis of bacteria reported to cause SSIs in rabbits19,20,21,22 and people.26,27,28,29,30,31,38 The preliminary study revealed that P multocida (vs P aeruginosa and S aureus) caused an SSI associated with the implants in both rabbits within a short time period. However, rabbits may have developed SSIs caused by P aeruginosa or S aureus if they had been observed for > 3 days (rabbits were euthanized 3 days after implantation of bacteria-contaminated implants). Before the CAPS device evaluated in the present study is used for sterilization of surgical instruments and implants, evidence of its efficacy for inactivating other bacteria will be necessary. Preliminary data have indicated that CAPS is effective at inactivating multiple organisms on common substrates16 and against antimicrobial-resistant bacteria, including those that produce biofilms7,8; however, additional studies that are designed similarly to that of the present study should be performed with other bacteria commonly isolated from infected skin and soft tissue plus those evaluating efficacy against bacterial biofilms on stainless steel implants.
The results of the present study indicated that CAPS was noninferior to steam-autoclave sterilization of stainless steel implants, and no important adverse tissue effects were recognized. Because the CAPS device used in the present study was compact and portable, it may facilitate onsite (ie, on a farm) sterilization of surgical instruments and equipment for veterinarians in ambulatory practice. The availability of a portable and efficient sterilization method could improve field-based veterinary health care in remote regions with limited availability of other (less portable) sterilization methods.
Acknowledgments
Supported by a grant from the Research Advisory Committee, College of Veterinary Medicine, Oklahoma State University. The cold atmospheric plasma sterilization (CAPS) device was provided by Plasma Bionics LLC.
Drs. Pai and Timmons created the CAPS device and were employees of Plasma Bionics LLC. The other authors declare that there were no conflicts of interest.
The authors thank Ian Kanda and Dr. Kailey Anderson for their technical support.
Abbreviations
| AIA | Autoclaved, inoculated, autoclaved |
| AICAPS | Autoclaved, inoculated, cold atmospheric plasma sterilization |
| CAPS | Cold atmospheric plasma sterilization |
| RONS | Reactive oxygen and nitrogen species |
| SSI | Surgical site infection |
Footnotes
PZ100 air plasma sterilizer, Plasma Bionics LLC, Stillwater, Okla.
Charles River, Senneville, QC, Canada.
Research Instruments NW Inc, Lebanon, Ore.
Research Randomizer, version 4.0, www.randomizer.org, Middletown, Conn.
Duraprep, 3M, Saint Paul, Minn.
Self-sealing autoclave pouch, Henry Schein Animal Health, Dublin, Ohio.
Tyvek Pouch, Johnson & Johnson Medical GmbH, Irvine, Calif.
Microchip ID Systems, Covington, La.
Vetbond, 3M, Saint Paul, Minn.
Copan Diagnostics Inc, Murrieta, Calif.
Pai KK. Asymmetric surface dielectric barrier discharge as a novel method for biological decontamination. PhD dissertation, College of Veterinary Medicine, Oklahoma State University, Stillwater, Okla, 2015.
Timmons C. Elucidation of the molecular mechanisms of foodborne human pathogen inactivation by cold atmospheric plasma through RNA-seq analysis. PhD dissertation, College of Veterinary Medicine, Oklahoma State University, Stillwater, Okla, 2016.
BASF F200, Delta Adsorbents, Delta Enterprises Inc, Roselle, Ill.
Carulite 200, Carus Corp, Peru, Ill.
SAS, version 9, SAS Institute, Cary, NC.
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