Evaluation of Steam Penetration and Sterilization of Natural Latex Wraps Matteo Rossanese, DVM, James Gasson, Colin Barker, BSc, PhD, PGCMP, and Kelly Bowlt, BVM&S, Diplomate ECVS Animal Health Trust, Newmarket, United Kingdom
Corresponding Author Matteo Rossanese, DVM, The Old Golf House, 4 High Street Thetford, Norfolk, IP25 6XE, United Kingdom. E‐mail: [email protected] Submitted May 2013 Accepted December 2013 DOI:10.1111/j.1532-950X.2014.12168.x
Objective: To evaluate the efficacy of steam and ethylene oxide (EtO) sterilization of VetrapTM bandages. Study Design: Prospective experimental study. Sample Population: VetrapTM bandages (n ¼ 70; 35 as supplied by the manufacturer, 35 unwound and tightly rewound). Methods: VetrapTM bandage rolls (n ¼ 60) marked with a 1 cm square were inoculated with 0.1 mL Geobacillus stearothermophilus spores, packaged in a pouch together with independent sterilization indicators and assigned into 3 sub‐groups for sterilizer type: dynamic air removal, gravity displacement, and bench‐top pre‐vacuum and further sub‐ divided into 2 sterilization temperatures. VetrapTM bandages rolls (n ¼ 10) were inoculated with 0.1 mL Bacillus atrophaeus spores in the same manner and underwent EtO sterilization. After sterilization, the 1 cm marked square was aseptically resected to the level of the cardboard tube and enriched in a flask containing 10 mL tryptic soy broth for 24 hours at 60°C for G. stearothermophilus and 37°C for B. atrophaeus. Aliquots were subsequently plated on a Petri dish of tryptic soy agar and incubated at 60°C for G. stearothermophilus and 37°C for B. atrophaeus for 24 hours. Samples were scored positive if colonies of indicator organism were present on the nutrient agar after 24 hours. Results: Three VetrapTM bandages yielded post‐sterilization growth of G. stearothermophilus: 2 from the dynamic air removal sterilizer at 134°C for 3.5 minutes, and 1 from the bench‐top pre‐vacuum sterilizer at 121°C for 15 minutes. After EtO sterilization, no positive samples were detected. Conclusions: Steam sterilization may be incomplete for VetrapTM bandages whereas EtO showed complete destruction of resistant bacterial spores.
Surgical site infections (SSI) represent the most common nosocomial infection in people1,2 and may lead to prolonged hospitalization, additional surgery, poor cosmesis, and increased morbidity and mortality.2 Bacterial contamination occurs during surgery and subsequent infection depends on the number and virulence of the bacteria, the immune competence of the host, and tissue damage and dead space produced. 3,4 We are unaware of detailed reports of nosocomial infections in veterinary surgery, but SSI has been described as a complication in 0.8–18.1% of small animal surgical procedures, with substantial variation based on the type of surgery performed.2,5 Although SSI cannot be completely avoided preventive strategies can be used to reduce occurrence.2,6 Few preventive measures have been validated in small animal surgery, but many have been extrapolated from human medicine based on levels of evidence.1,2,7 Aseptic preparation of animal limbs is challenging because of regional anatomy and contamination. To reduce skin exposure during surgical
procedures of the proximal aspect of the limb, the distal part is typically wrapped with a non‐sterile cohesive bandage, followed by a sterile dressing (e.g., glove or stockinette) and eventually covered with a sterile cohesive bandage.3,4,6,8,9 A sterile cohesive dressing (CobanTM; 3 M Healthcare, St Paul, MN), available only in the US, and sterilized using gamma radiation is typically used in people. In the absence of sterile commercial veterinary bandages and because of the increased cost of CobanTM compared with other non‐sterile cohesive bandages (e.g., VetrapTM, 3 M Healthcare), it is usual practice to sterilize cohesive bandage in‐house. In our experience in a variety of referral and first opinion practices within the UK, this is performed with steam sterilization. The bandage is sterilized directly from the manufacturer’s pack, or remnants may be rolled and autoclaved. We are unaware of studies that have evaluated effective steam penetration of cohesive bandages. Because VetrapTM is a self‐adherent, porous latex bandage material,10 and it is unknown if handling VetrapTM alters pore size, we speculated that the presence of
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pores is a critical element for sterilization to allow complete, full thickness steam penetration. Thus our purpose was to evaluate the efficacy of steam and ethylene oxide (EtO) sterilization of VetrapTM bandages. We hypothesized that sterilization of VetrapTM is adequate using a pre‐vacuum autoclave or EtO sterilizer, regardless of whether the bandage is tightly rolled or sterilized directly after removal from the manufacturer’s packaging. To test these hypotheses, VetrapTM bandage rolls were injected with a commercial suspension of resistant bacterial spores and subjected to different sterilization protocols and subsequently cultured to determine efficacy.
MATERIALS AND METHODS Isolation of Bacteria and Growth Conditions Recovery and germination of spores resistant to each of the sterilization processes was chosen as a measure of sterilization efficacy. Geobacillus stearothermophilus spores were chosen as an indicator of steam sterilization efficacy and Bacillus atrophaeus for ethylene oxide sterilization. To demonstrate that spores could be recovered and germinated in non‐treated rolls of VetrapTM bandage (4 inches 5 yards, 3 M Health Care), 100 mL G. stearothermophilus spore suspension (3.3 106 CFU/0.1 mL, D121Value 1.9 minutes, Mesa Laboratories, Inc., Omaha, NE) was injected into a 1 cm marked square in the central portion of the bandage (Fig 1). The sample was air dried overnight at room temperature (21°C). After 24 hours the marked square to the depth of the cardboard roll (1 cm3) was resected in a sterile manner and added to 10 mL tryptic soy broth (TSB, PN T8907, Sigma‐Aldrich; St. Louis, MO). As a positive control, 10 mL TSB was directly inoculated with 100 mL G. stearothermophilus spore suspension, with a non‐inoculated broth as a negative control. Samples were incubated for 24 hours at 60°C before plating 100 mL of the sample on nutrient agar plates (NA, PN N9405, Sigma‐Aldrich). The inoculated plates were incubated for a further 24 hours at 60°C and scored positive for bacterial growth if visible colonies of the indicator organisms were observed. Colonies were Gram stained and compared
Figure 1
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with the positive control. This experiment was repeated for B. atrophaeus (3.3 106 CFU/0.1 mL, DEtOValue 3.6 minutes, Mesa Laboratories) with incubations at 37°C. Assessment of Sterilization Efficacy To determine the efficacy of different sterilization methods, rolls of VetrapTM bandage were inoculated as described above and sterilized under 14 different conditions (n ¼ 5 replicates; Table 1). To simulate the re‐use of VetrapTM remnants, samples was made by completely unwinding bandage from the original packaging and then incrementally stretching 20–30 cm segments as much as possible and tightly rewinding onto the cardboard roll until all the entire roll had been rewound. These samples were then tested for each of the sterilization conditions. Each inoculated VetrapTM bandage roll was sterilized in a 20 33 cm self‐seal sterilization pouch (Vis‐U‐All, Steris Corporation1, Leicester, UK) together with a Class 6 cycle verification chemical indicator (TST controlTM, Albert Browne Ltd, Leicester, UK) and a self‐contained biological indicator (SCBI) containing a mixed population of G. stearothermophilus and B. atrophaeus (VerifyTM SCBI, Steris Corporation1; B. atrophaeus 3.3 106 CFU; G. stearothermophilus 1.9 105 CFU; EtO: D‐value 3.3, survival time 15 minutes, Kill Time 60 minutes; Steam 121°C: D‐value 2.3, survival time 7.5 minutes, Kill Time 21.3 minutes; Steam 132°C: D‐value 0.8, survival time 2.6 minutes, Kill Time 7.4 minutes; Z‐value 13.6°C). To maximize air removal and steam penetration, all pouches were stacked on their side and orientated paper‐to‐ plastic in a single layer. For ethylene oxide (EtO) sterilization, the Class 6 indicators were replaced with a Class 5 integrating chemical indicator (Ethylene Oxide integrating indicator, Albert Browne Ltd). Humidification of the load before sterilization was not performed. Positive controls were included for both rewound and “as supplied” VetrapTM (n ¼ 1 for each indicator organism). Positive controls were injected with indicator organism and placed into TSB without sterilization. The controls were then incubated and cultured with the other samples (60°C for G. stearothermophilus, 37°C for B. atrophaeus).
Injection method: (A) 1 cm marked square; (B) depth of the injection; (C) injection in the most central portion of the VetrapTM.
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Table 1
Sterilization of Natural Latex Wraps
Sterilization Conditions
Group 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Sterilization Method
Protocol
Steam: dynamic air removal pre‐vacuum
121°C, 15 minutes 134°C, 3.5 minutes
Steam: gravity displacement
121°C, 15 minutes 134°C, 3.5 minutes
Steam: bench‐top pre‐vacuum
121°C, 15 minutes 134°C, 3.5 minutes
Ethylene oxide
22°C, 12 hours
VetrapTM Winding As supplied Tightly rewound As supplied Tightly rewound As supplied Tightly rewound As supplied Tightly rewound As supplied Tightly rewound As supplied Tightly rewound As supplied Tightly rewound
Indicator Organism G. stearothermophilus
B. atrophaeus
Dynamic air removal pre‐vacuum sterilizer (ESTS ACE Upgrade), gravity displacement steam sterilizer (Instaclave, Burton Autoclave Series 3, Model 2530 Serial N°016258), bench‐top pre‐vacuum sterilizer (Eschmann SES 2000), ethylene oxide sterilizer (Anprolene1, Andersen Sterilizers, Inc.).
Two VetrapTM samples (1 cm3) were inoculated into TSB without injection of indicator organism as negative controls; 1 was incubated at 60°C, the other at 37°C. Samples were scored positive if colonies of indicator organism were present on the nutrient agar after 24 hours incubation. Samples were scored negative if no growth was observed.
Bench‐Top Pre‐Vacuum Sterilizer One specimen (rewound; 121°C, 15 minutes) yielded G. stearothermophilus growth. EtO Sterilization. No bacterial growth was detected.
DISCUSSION RESULTS The steam sterilizers’ recorded data demonstrated that the critical parameters for sterilization within the autoclave chamber had been achieved and all chemical and SCBI independently verified that the conditions within the pouch were satisfactory for achieving sterilization of the VetrapTM bandage roll. For EtO sterilization, a chemical indicator (CI) integrating the effects of time, temperature and concentration (Dosimeter1 Andersen Sterilizers, Inc.) verified that a minimum of 2000 mg/ L‐hours of exposure to EtO was reached during the 12‐hour sterilization cycle at 22°C, confirming adequate conditions for sterilization. None of the negative controls had bacterial growth whereas all positive controls had growth. Dynamic Air Removal (Pre‐Vacuum) Sterilizer All samples sterilized at 121°C for 15 minutes were negative. Two group 3 (“as supplied”; 134°C, 3.5 minutes) specimens yielded growth of G. stearothermophilus, but none of the rewound specimens under these conditions. Gravity Displacement Steam Sterilizer There was no bacterial growth from any of the samples.
Microbial contamination of the surgical site is the precursor to SSI1 and adequate preoperative patient, surgeon and facility preparation is critical to reduce wound contamination. VetrapTM bandages are often used for surgical procedures involving the extremities: the surgeon and assistant constantly touch the wrap during the procedures and to minimize bacterial strike through, an impervious layer should be incorporated into the dressing.6 3 M Healthcare states that VetrapTM is not sterile but biologically clean10; however, we are unaware of studies that validate this claim and further investigations should be performed to evaluate the bioburden of the bandage, particularly if the bandages have been stored in a hospital environment for a prolonged period, potentially allowing bacterial colonization. The manufacturer does not mention intraoperative applications of VetrapTM bandage and recommends only ethylene oxide sterilization.10 Despite this recommendation, and because sterilization methods other than steam are usually only available in large referral facilities within the UK, most first opinion veterinary practices steam sterilize the bandage, although we are unaware of evidence that validates this technique to ensure a sterile bandage. Of 70 specimens tested (5 replicates of 14 test conditions; Table 1), 3 samples (2 dynamic air removal sterilization at 134°C for 3 minutes and 1 bench‐top pre‐vacuum sterilization at 121°C for 15 minutes) yielded spore growth after
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sterilization. The colonies were Gram stained and compared with the positive control: a Gram positive, rod‐shape, endospore‐forming organism was detected and considered compatible with G. stearothermophilus.11,12 No bacterial growth was detected from samples after gravity displacement sterilization; however, this may reflect the small sample size (n ¼ 5) we used and a larger number of bandage samples may yield different results. In the human literature, 3 studies have assessed the efficacy of steam sterilization (using a dynamic air removal autoclave13 or at 134–136°C at 2.3 bars for 3 minutes14,15) of bandages with similar results: bandages remained contaminated in 8–10% of samples.13–15 Although steam is not suitable for use with some materials, it is economical, non‐toxic, and considered the most efficient form of sterilization. Using the correct combination of temperature, pressure and exposure time, steam sterilization destroys microbes by coagulating and denaturing cellular protein.3,4,13,16 Air removal is a prerequisite for successful steam sterilization of porous materials. Air entrapment is a major risk for gravity displacement sterilization; however, for pre‐vacuum sterilization the presence of a pre‐vacuum pump ensures complete air removal and instantaneous steam penetration even into porous items.17 Steam sterilization is considered to an overkill process; the steam exposure time required to kill a known population of heat resistant spores is multiplied to ensure a margin of safety and allow for a theoretical sterility assurance level of 106 and therefore recovery of spores represents a major failure of sterilization.18 In addition to sterilization time, the heat up and cool down periods also contribute to lethality.18 We do not know why, even in a pre‐vacuum sterilizer, that steam did not penetrate the entire thickness of the bandage. To maximize air removal and steam penetration, all bandages were arranged so that all surfaces were directly exposed to the sterilizing agent. Load configuration should ensure adequate air removal, penetration of steam (or another sterilant) into each package and steam evacuation.17 In our experience, steam sterilization does not appear to alter the adhesive properties of the VetrapTM bandage when used clinically. We found no positive cultures after EtO sterilization. Previous studies confirm that EtO penetrates through the complete bandage roll without altering cohesive qualities of the material.10 EtO is a mixed gas, that kills microbes by altering their RNA and DNA through alkylation, and is used to sterilize delicate instruments sensitive to temperatures >60°C (e.g., endoscopes, cameras, plastics). It penetrates items easily but some plastics, like nylon or polyester are impermeable to EtO.3,4,16 To predetermine and minimize procedural errors and monitor the lethality of the sterilization process, chemical and biological indicators (BI) were included in every pack to confirm that each individual package was completely sterilized. The use of BI within the United Kingdom and other parts of Europe is restricted to the validation of processes rather than routine monitoring. To be meaningful, any indicator should be placed in the area of the load considered least accessible to the sterilant, usually the center of a porous item. At the end of a sterilization cycle a positive BI represents
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a major failure in achieving the parameters critical for sterilization.1,17 Chemical indicators (CI) are an alternative to biological indicators for routine “in‐pack” and load monitoring. Chemical changes are brought about by the sterilization process and are typically identified by a color change or elongation of a bar within a reference range. These may yield more information in the event of failure compared to a BI.17 A class 5 chemical indicator used for steam sterilization mimics the performance of a stated BI at a minimum of 3 reference temperatures along the thermal death curve of the organism as stated by the manufacturer and can be used to release loads for immediate use. Class 6 CI for steam are cycle specific and verify the conditions required for sterilization have been achieved. Class 6 indicators represent the only group of useful in pack monitoring for extended cycles as specified by some equipment manufacturers. CI are appropriately positioned within the least accessible part of the pack or load.1,17 After sterilization, we handled all specimens in a sterile manner and within batches, consecutive samples did not yield bacterial growth. This finding rules out any possible direct and indirect contamination by the operator, because otherwise all samples subsequent to the positive bandage would likewise have yielded bacterial growth. All negative controls in our study had no bacterial growth, whereas all positive controls had growth. No differences in efficacy of steam sterilization were noticed between VetrapTM bandages removed directly from the package or samples that were unrolled and tightly rewound before sterilization. Inclusion of the rewound bandage was selected to represent clinical scenarios where bandage remnants, rather than intact and newly opened bandages may be sterilized for intraoperative use. Vince et al. demonstrated with electron microscopy that the pore size of a CobanTM bandage ranged from 50–175 mm and likely increased with stretching. Bacteria range from 0.2–10 mm in size so that the dimensions of the bandage pores are sufficient to allow bacteria to pass through the material. We concluded that steam sterilization is not a reliable method of sterilization for VetrapTM bandages and its use should be discouraged. Our study confirms that ethylene oxide is suitable for in‐house sterilization of VetrapTM bandages.
DISCLOSURE The authors report no financial or other conflicts related to this report.
REFERENCES 1. Mangram AJ, Horan TC, Pearson ML, et al: Guideline for prevention of surgical site infection, 1999. Hospital Infection Control Practices Advisory Commitee. Infect Control Hosp Epidemiol 1999;20:250–278 2. Nelson L: Surgical site infection in small animal surgery. Vet Clin North Am Small Anim Pract 2011;41:1041–1056
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3. Brown DC, Conzemius MG, Shofer F, et al: Epidemiologic evaluation of postoperative wound infections in dogs and cats. J Am Vet Med Assoc 1997;210:1302–1306 4. Eugster S, Schawalder P, Gaschen F, et al: A prospective study of postoperative surgical site infections in dogs and cats. Vet Surg 2004;33:542–550 5. Dohmen PM, Konertz W: A review of current strategies to reduce intraoperative bacterial contamination of surgical wounds. GMS Krankenhhyg Interdiszip 2007;2:1–7 6. Vince KJ, Lascelles BDX, Mathews KG, et al: Evaluation of wraps covering the distal aspect of pelvic limbs for prevention of bacterial strike‐through in an ex vivo canine model. Vet Surg 2008;37:406–411 7. Evans LKM, Knowles TG, Werrett G, et al: The efficacy of chlorhexidine gluconate in canine skin preparation—practice survey and clinical trials. J Small Anim Pract 2009;50:458–465 8. Hutchinson T, Aseptic technique, in Lipscomb V, Hutchinson T, Baines S (eds): BSAVA manual of canine, feline surgical principles: a foundation manual. Gloucester, UK, BSAVA, 2012, pp 198–209 9. Vince KJ, Lascelles BDX, Mathews KG, et al: Evaluation of a sterile bandage foot wrap in the prevention of bacterial strike through in the canine surgical patient. Proc 34th Annual Conference of the Veterinary Orthopedic Society, 2007, Sun Valley, pp 3–10 10. 3M HC: Animal care solution: FAQ: PET: wound management, VetrapTM bandaging tape. Accessed February 14, 2013. http:// www.3m.com/us/healthcare/professionals/animalcare/pdfs/ pet_wmanagement_faq.pdf 11. Nazina TN, Tourova TP, Poltaraus AB, et al: Taxanomic study of aerobic thermophilic bacilli: description of Geobacillus
Sterilization of Natural Latex Wraps
subterraneus gen. nov., sp. nov., and Geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Baciluus thermoglucosidasius and Bacillus thermodenitrificans to Geobacillus as the new combinations G. stearothermophilus, G. thermocatenulatus, G. thermoleovorans, G. kaustophilus, G. thermoglucosidasius and G. thermodenitrificans. Int J Syst Evol Microbiol 2001;51:433–446 12. Obeidat M, Khyami‐Horani H, Al‐Zoubi A, et al: Isolation, characterization, and hydrolytic activities of Geobacillus species from Jordanian hot springs. Afr J Biotech 2012;11:6763–6768 13. Wood EV, Fenwick H, Manning MP, et al: The sterility of hospital‐ prepared Soffban bandages. J Hosp Infect 2005;61:248–250 14. Gaines E, Johnson R, Manning MP: The efficacy of sterilizing Esmarch bandages. J Bone Joint Surg 1987;69:671–672 15. O’Hara JN, Coleman M, Hutton RM: A simple and effective method of sterilizing Esmarch bandages. J Arthroplasty 1991;6:95–96 16. Hamilton MH: Sterilisation and disinfection, in Lipscomb V, Hutchinson T, Baines S (eds): BSAVA manual of canine and feline surgical principles: a foundation manual. Gloucester, UK, BSAVA, 2012, pp 8–16 17. Rutala WA, Weber DJ: HICPAC: Guideline for disinfection and sterilization in healthcare facilities. Department of health and human service, USA; CDC, 2008. Accessd February 14, 2013, http://www.cdc.gov/hicpac/pdf/guidelines/ disinfection_nov_2008.pdf 18. Boca BM, Pretorius E, Gochin R, et al: An overview of the validation approach for moist heat sterilization, part I. Pharm Techonol 2002;26:62–70
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Corresponding Author Matteo Rossanese, DVM, The Old Golf House, 4 High Street Thetford, Norfolk, IP25 6XE, United Kingdom. E‐mail: [email protected] Submitted May 2013 Accepted December 2013 DOI:10.1111/j.1532-950X.2014.12168.x
Objective: To evaluate the efficacy of steam and ethylene oxide (EtO) sterilization of VetrapTM bandages. Study Design: Prospective experimental study. Sample Population: VetrapTM bandages (n ¼ 70; 35 as supplied by the manufacturer, 35 unwound and tightly rewound). Methods: VetrapTM bandage rolls (n ¼ 60) marked with a 1 cm square were inoculated with 0.1 mL Geobacillus stearothermophilus spores, packaged in a pouch together with independent sterilization indicators and assigned into 3 sub‐groups for sterilizer type: dynamic air removal, gravity displacement, and bench‐top pre‐vacuum and further sub‐ divided into 2 sterilization temperatures. VetrapTM bandages rolls (n ¼ 10) were inoculated with 0.1 mL Bacillus atrophaeus spores in the same manner and underwent EtO sterilization. After sterilization, the 1 cm marked square was aseptically resected to the level of the cardboard tube and enriched in a flask containing 10 mL tryptic soy broth for 24 hours at 60°C for G. stearothermophilus and 37°C for B. atrophaeus. Aliquots were subsequently plated on a Petri dish of tryptic soy agar and incubated at 60°C for G. stearothermophilus and 37°C for B. atrophaeus for 24 hours. Samples were scored positive if colonies of indicator organism were present on the nutrient agar after 24 hours. Results: Three VetrapTM bandages yielded post‐sterilization growth of G. stearothermophilus: 2 from the dynamic air removal sterilizer at 134°C for 3.5 minutes, and 1 from the bench‐top pre‐vacuum sterilizer at 121°C for 15 minutes. After EtO sterilization, no positive samples were detected. Conclusions: Steam sterilization may be incomplete for VetrapTM bandages whereas EtO showed complete destruction of resistant bacterial spores.
Surgical site infections (SSI) represent the most common nosocomial infection in people1,2 and may lead to prolonged hospitalization, additional surgery, poor cosmesis, and increased morbidity and mortality.2 Bacterial contamination occurs during surgery and subsequent infection depends on the number and virulence of the bacteria, the immune competence of the host, and tissue damage and dead space produced. 3,4 We are unaware of detailed reports of nosocomial infections in veterinary surgery, but SSI has been described as a complication in 0.8–18.1% of small animal surgical procedures, with substantial variation based on the type of surgery performed.2,5 Although SSI cannot be completely avoided preventive strategies can be used to reduce occurrence.2,6 Few preventive measures have been validated in small animal surgery, but many have been extrapolated from human medicine based on levels of evidence.1,2,7 Aseptic preparation of animal limbs is challenging because of regional anatomy and contamination. To reduce skin exposure during surgical
procedures of the proximal aspect of the limb, the distal part is typically wrapped with a non‐sterile cohesive bandage, followed by a sterile dressing (e.g., glove or stockinette) and eventually covered with a sterile cohesive bandage.3,4,6,8,9 A sterile cohesive dressing (CobanTM; 3 M Healthcare, St Paul, MN), available only in the US, and sterilized using gamma radiation is typically used in people. In the absence of sterile commercial veterinary bandages and because of the increased cost of CobanTM compared with other non‐sterile cohesive bandages (e.g., VetrapTM, 3 M Healthcare), it is usual practice to sterilize cohesive bandage in‐house. In our experience in a variety of referral and first opinion practices within the UK, this is performed with steam sterilization. The bandage is sterilized directly from the manufacturer’s pack, or remnants may be rolled and autoclaved. We are unaware of studies that have evaluated effective steam penetration of cohesive bandages. Because VetrapTM is a self‐adherent, porous latex bandage material,10 and it is unknown if handling VetrapTM alters pore size, we speculated that the presence of
Veterinary Surgery 43 (2014) 1009–1013 © Copyright 2014 by The American College of Veterinary Surgeons
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Rossanese et al.
pores is a critical element for sterilization to allow complete, full thickness steam penetration. Thus our purpose was to evaluate the efficacy of steam and ethylene oxide (EtO) sterilization of VetrapTM bandages. We hypothesized that sterilization of VetrapTM is adequate using a pre‐vacuum autoclave or EtO sterilizer, regardless of whether the bandage is tightly rolled or sterilized directly after removal from the manufacturer’s packaging. To test these hypotheses, VetrapTM bandage rolls were injected with a commercial suspension of resistant bacterial spores and subjected to different sterilization protocols and subsequently cultured to determine efficacy.
MATERIALS AND METHODS Isolation of Bacteria and Growth Conditions Recovery and germination of spores resistant to each of the sterilization processes was chosen as a measure of sterilization efficacy. Geobacillus stearothermophilus spores were chosen as an indicator of steam sterilization efficacy and Bacillus atrophaeus for ethylene oxide sterilization. To demonstrate that spores could be recovered and germinated in non‐treated rolls of VetrapTM bandage (4 inches 5 yards, 3 M Health Care), 100 mL G. stearothermophilus spore suspension (3.3 106 CFU/0.1 mL, D121Value 1.9 minutes, Mesa Laboratories, Inc., Omaha, NE) was injected into a 1 cm marked square in the central portion of the bandage (Fig 1). The sample was air dried overnight at room temperature (21°C). After 24 hours the marked square to the depth of the cardboard roll (1 cm3) was resected in a sterile manner and added to 10 mL tryptic soy broth (TSB, PN T8907, Sigma‐Aldrich; St. Louis, MO). As a positive control, 10 mL TSB was directly inoculated with 100 mL G. stearothermophilus spore suspension, with a non‐inoculated broth as a negative control. Samples were incubated for 24 hours at 60°C before plating 100 mL of the sample on nutrient agar plates (NA, PN N9405, Sigma‐Aldrich). The inoculated plates were incubated for a further 24 hours at 60°C and scored positive for bacterial growth if visible colonies of the indicator organisms were observed. Colonies were Gram stained and compared
Figure 1
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with the positive control. This experiment was repeated for B. atrophaeus (3.3 106 CFU/0.1 mL, DEtOValue 3.6 minutes, Mesa Laboratories) with incubations at 37°C. Assessment of Sterilization Efficacy To determine the efficacy of different sterilization methods, rolls of VetrapTM bandage were inoculated as described above and sterilized under 14 different conditions (n ¼ 5 replicates; Table 1). To simulate the re‐use of VetrapTM remnants, samples was made by completely unwinding bandage from the original packaging and then incrementally stretching 20–30 cm segments as much as possible and tightly rewinding onto the cardboard roll until all the entire roll had been rewound. These samples were then tested for each of the sterilization conditions. Each inoculated VetrapTM bandage roll was sterilized in a 20 33 cm self‐seal sterilization pouch (Vis‐U‐All, Steris Corporation1, Leicester, UK) together with a Class 6 cycle verification chemical indicator (TST controlTM, Albert Browne Ltd, Leicester, UK) and a self‐contained biological indicator (SCBI) containing a mixed population of G. stearothermophilus and B. atrophaeus (VerifyTM SCBI, Steris Corporation1; B. atrophaeus 3.3 106 CFU; G. stearothermophilus 1.9 105 CFU; EtO: D‐value 3.3, survival time 15 minutes, Kill Time 60 minutes; Steam 121°C: D‐value 2.3, survival time 7.5 minutes, Kill Time 21.3 minutes; Steam 132°C: D‐value 0.8, survival time 2.6 minutes, Kill Time 7.4 minutes; Z‐value 13.6°C). To maximize air removal and steam penetration, all pouches were stacked on their side and orientated paper‐to‐ plastic in a single layer. For ethylene oxide (EtO) sterilization, the Class 6 indicators were replaced with a Class 5 integrating chemical indicator (Ethylene Oxide integrating indicator, Albert Browne Ltd). Humidification of the load before sterilization was not performed. Positive controls were included for both rewound and “as supplied” VetrapTM (n ¼ 1 for each indicator organism). Positive controls were injected with indicator organism and placed into TSB without sterilization. The controls were then incubated and cultured with the other samples (60°C for G. stearothermophilus, 37°C for B. atrophaeus).
Injection method: (A) 1 cm marked square; (B) depth of the injection; (C) injection in the most central portion of the VetrapTM.
Veterinary Surgery 43 (2014) 1009–1013 © Copyright 2014 by The American College of Veterinary Surgeons
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Table 1
Sterilization of Natural Latex Wraps
Sterilization Conditions
Group 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Sterilization Method
Protocol
Steam: dynamic air removal pre‐vacuum
121°C, 15 minutes 134°C, 3.5 minutes
Steam: gravity displacement
121°C, 15 minutes 134°C, 3.5 minutes
Steam: bench‐top pre‐vacuum
121°C, 15 minutes 134°C, 3.5 minutes
Ethylene oxide
22°C, 12 hours
VetrapTM Winding As supplied Tightly rewound As supplied Tightly rewound As supplied Tightly rewound As supplied Tightly rewound As supplied Tightly rewound As supplied Tightly rewound As supplied Tightly rewound
Indicator Organism G. stearothermophilus
B. atrophaeus
Dynamic air removal pre‐vacuum sterilizer (ESTS ACE Upgrade), gravity displacement steam sterilizer (Instaclave, Burton Autoclave Series 3, Model 2530 Serial N°016258), bench‐top pre‐vacuum sterilizer (Eschmann SES 2000), ethylene oxide sterilizer (Anprolene1, Andersen Sterilizers, Inc.).
Two VetrapTM samples (1 cm3) were inoculated into TSB without injection of indicator organism as negative controls; 1 was incubated at 60°C, the other at 37°C. Samples were scored positive if colonies of indicator organism were present on the nutrient agar after 24 hours incubation. Samples were scored negative if no growth was observed.
Bench‐Top Pre‐Vacuum Sterilizer One specimen (rewound; 121°C, 15 minutes) yielded G. stearothermophilus growth. EtO Sterilization. No bacterial growth was detected.
DISCUSSION RESULTS The steam sterilizers’ recorded data demonstrated that the critical parameters for sterilization within the autoclave chamber had been achieved and all chemical and SCBI independently verified that the conditions within the pouch were satisfactory for achieving sterilization of the VetrapTM bandage roll. For EtO sterilization, a chemical indicator (CI) integrating the effects of time, temperature and concentration (Dosimeter1 Andersen Sterilizers, Inc.) verified that a minimum of 2000 mg/ L‐hours of exposure to EtO was reached during the 12‐hour sterilization cycle at 22°C, confirming adequate conditions for sterilization. None of the negative controls had bacterial growth whereas all positive controls had growth. Dynamic Air Removal (Pre‐Vacuum) Sterilizer All samples sterilized at 121°C for 15 minutes were negative. Two group 3 (“as supplied”; 134°C, 3.5 minutes) specimens yielded growth of G. stearothermophilus, but none of the rewound specimens under these conditions. Gravity Displacement Steam Sterilizer There was no bacterial growth from any of the samples.
Microbial contamination of the surgical site is the precursor to SSI1 and adequate preoperative patient, surgeon and facility preparation is critical to reduce wound contamination. VetrapTM bandages are often used for surgical procedures involving the extremities: the surgeon and assistant constantly touch the wrap during the procedures and to minimize bacterial strike through, an impervious layer should be incorporated into the dressing.6 3 M Healthcare states that VetrapTM is not sterile but biologically clean10; however, we are unaware of studies that validate this claim and further investigations should be performed to evaluate the bioburden of the bandage, particularly if the bandages have been stored in a hospital environment for a prolonged period, potentially allowing bacterial colonization. The manufacturer does not mention intraoperative applications of VetrapTM bandage and recommends only ethylene oxide sterilization.10 Despite this recommendation, and because sterilization methods other than steam are usually only available in large referral facilities within the UK, most first opinion veterinary practices steam sterilize the bandage, although we are unaware of evidence that validates this technique to ensure a sterile bandage. Of 70 specimens tested (5 replicates of 14 test conditions; Table 1), 3 samples (2 dynamic air removal sterilization at 134°C for 3 minutes and 1 bench‐top pre‐vacuum sterilization at 121°C for 15 minutes) yielded spore growth after
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sterilization. The colonies were Gram stained and compared with the positive control: a Gram positive, rod‐shape, endospore‐forming organism was detected and considered compatible with G. stearothermophilus.11,12 No bacterial growth was detected from samples after gravity displacement sterilization; however, this may reflect the small sample size (n ¼ 5) we used and a larger number of bandage samples may yield different results. In the human literature, 3 studies have assessed the efficacy of steam sterilization (using a dynamic air removal autoclave13 or at 134–136°C at 2.3 bars for 3 minutes14,15) of bandages with similar results: bandages remained contaminated in 8–10% of samples.13–15 Although steam is not suitable for use with some materials, it is economical, non‐toxic, and considered the most efficient form of sterilization. Using the correct combination of temperature, pressure and exposure time, steam sterilization destroys microbes by coagulating and denaturing cellular protein.3,4,13,16 Air removal is a prerequisite for successful steam sterilization of porous materials. Air entrapment is a major risk for gravity displacement sterilization; however, for pre‐vacuum sterilization the presence of a pre‐vacuum pump ensures complete air removal and instantaneous steam penetration even into porous items.17 Steam sterilization is considered to an overkill process; the steam exposure time required to kill a known population of heat resistant spores is multiplied to ensure a margin of safety and allow for a theoretical sterility assurance level of 106 and therefore recovery of spores represents a major failure of sterilization.18 In addition to sterilization time, the heat up and cool down periods also contribute to lethality.18 We do not know why, even in a pre‐vacuum sterilizer, that steam did not penetrate the entire thickness of the bandage. To maximize air removal and steam penetration, all bandages were arranged so that all surfaces were directly exposed to the sterilizing agent. Load configuration should ensure adequate air removal, penetration of steam (or another sterilant) into each package and steam evacuation.17 In our experience, steam sterilization does not appear to alter the adhesive properties of the VetrapTM bandage when used clinically. We found no positive cultures after EtO sterilization. Previous studies confirm that EtO penetrates through the complete bandage roll without altering cohesive qualities of the material.10 EtO is a mixed gas, that kills microbes by altering their RNA and DNA through alkylation, and is used to sterilize delicate instruments sensitive to temperatures >60°C (e.g., endoscopes, cameras, plastics). It penetrates items easily but some plastics, like nylon or polyester are impermeable to EtO.3,4,16 To predetermine and minimize procedural errors and monitor the lethality of the sterilization process, chemical and biological indicators (BI) were included in every pack to confirm that each individual package was completely sterilized. The use of BI within the United Kingdom and other parts of Europe is restricted to the validation of processes rather than routine monitoring. To be meaningful, any indicator should be placed in the area of the load considered least accessible to the sterilant, usually the center of a porous item. At the end of a sterilization cycle a positive BI represents
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a major failure in achieving the parameters critical for sterilization.1,17 Chemical indicators (CI) are an alternative to biological indicators for routine “in‐pack” and load monitoring. Chemical changes are brought about by the sterilization process and are typically identified by a color change or elongation of a bar within a reference range. These may yield more information in the event of failure compared to a BI.17 A class 5 chemical indicator used for steam sterilization mimics the performance of a stated BI at a minimum of 3 reference temperatures along the thermal death curve of the organism as stated by the manufacturer and can be used to release loads for immediate use. Class 6 CI for steam are cycle specific and verify the conditions required for sterilization have been achieved. Class 6 indicators represent the only group of useful in pack monitoring for extended cycles as specified by some equipment manufacturers. CI are appropriately positioned within the least accessible part of the pack or load.1,17 After sterilization, we handled all specimens in a sterile manner and within batches, consecutive samples did not yield bacterial growth. This finding rules out any possible direct and indirect contamination by the operator, because otherwise all samples subsequent to the positive bandage would likewise have yielded bacterial growth. All negative controls in our study had no bacterial growth, whereas all positive controls had growth. No differences in efficacy of steam sterilization were noticed between VetrapTM bandages removed directly from the package or samples that were unrolled and tightly rewound before sterilization. Inclusion of the rewound bandage was selected to represent clinical scenarios where bandage remnants, rather than intact and newly opened bandages may be sterilized for intraoperative use. Vince et al. demonstrated with electron microscopy that the pore size of a CobanTM bandage ranged from 50–175 mm and likely increased with stretching. Bacteria range from 0.2–10 mm in size so that the dimensions of the bandage pores are sufficient to allow bacteria to pass through the material. We concluded that steam sterilization is not a reliable method of sterilization for VetrapTM bandages and its use should be discouraged. Our study confirms that ethylene oxide is suitable for in‐house sterilization of VetrapTM bandages.
DISCLOSURE The authors report no financial or other conflicts related to this report.
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3. Brown DC, Conzemius MG, Shofer F, et al: Epidemiologic evaluation of postoperative wound infections in dogs and cats. J Am Vet Med Assoc 1997;210:1302–1306 4. Eugster S, Schawalder P, Gaschen F, et al: A prospective study of postoperative surgical site infections in dogs and cats. Vet Surg 2004;33:542–550 5. Dohmen PM, Konertz W: A review of current strategies to reduce intraoperative bacterial contamination of surgical wounds. GMS Krankenhhyg Interdiszip 2007;2:1–7 6. Vince KJ, Lascelles BDX, Mathews KG, et al: Evaluation of wraps covering the distal aspect of pelvic limbs for prevention of bacterial strike‐through in an ex vivo canine model. Vet Surg 2008;37:406–411 7. Evans LKM, Knowles TG, Werrett G, et al: The efficacy of chlorhexidine gluconate in canine skin preparation—practice survey and clinical trials. J Small Anim Pract 2009;50:458–465 8. Hutchinson T, Aseptic technique, in Lipscomb V, Hutchinson T, Baines S (eds): BSAVA manual of canine, feline surgical principles: a foundation manual. Gloucester, UK, BSAVA, 2012, pp 198–209 9. Vince KJ, Lascelles BDX, Mathews KG, et al: Evaluation of a sterile bandage foot wrap in the prevention of bacterial strike through in the canine surgical patient. Proc 34th Annual Conference of the Veterinary Orthopedic Society, 2007, Sun Valley, pp 3–10 10. 3M HC: Animal care solution: FAQ: PET: wound management, VetrapTM bandaging tape. Accessed February 14, 2013. http:// www.3m.com/us/healthcare/professionals/animalcare/pdfs/ pet_wmanagement_faq.pdf 11. Nazina TN, Tourova TP, Poltaraus AB, et al: Taxanomic study of aerobic thermophilic bacilli: description of Geobacillus
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subterraneus gen. nov., sp. nov., and Geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Baciluus thermoglucosidasius and Bacillus thermodenitrificans to Geobacillus as the new combinations G. stearothermophilus, G. thermocatenulatus, G. thermoleovorans, G. kaustophilus, G. thermoglucosidasius and G. thermodenitrificans. Int J Syst Evol Microbiol 2001;51:433–446 12. Obeidat M, Khyami‐Horani H, Al‐Zoubi A, et al: Isolation, characterization, and hydrolytic activities of Geobacillus species from Jordanian hot springs. Afr J Biotech 2012;11:6763–6768 13. Wood EV, Fenwick H, Manning MP, et al: The sterility of hospital‐ prepared Soffban bandages. J Hosp Infect 2005;61:248–250 14. Gaines E, Johnson R, Manning MP: The efficacy of sterilizing Esmarch bandages. J Bone Joint Surg 1987;69:671–672 15. O’Hara JN, Coleman M, Hutton RM: A simple and effective method of sterilizing Esmarch bandages. J Arthroplasty 1991;6:95–96 16. Hamilton MH: Sterilisation and disinfection, in Lipscomb V, Hutchinson T, Baines S (eds): BSAVA manual of canine and feline surgical principles: a foundation manual. Gloucester, UK, BSAVA, 2012, pp 8–16 17. Rutala WA, Weber DJ: HICPAC: Guideline for disinfection and sterilization in healthcare facilities. Department of health and human service, USA; CDC, 2008. Accessd February 14, 2013, http://www.cdc.gov/hicpac/pdf/guidelines/ disinfection_nov_2008.pdf 18. Boca BM, Pretorius E, Gochin R, et al: An overview of the validation approach for moist heat sterilization, part I. Pharm Techonol 2002;26:62–70
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