Sterilizing Bacillus pumilus spores using supercritical carbon dioxide

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Abstract

Supercritical carbon dioxide (SC CO2) has been evaluated as a new sterilization technology. Results are presented on killing of B. pumilus spores using SC CO2 containing trace levels of additives. Complete killing was achieved with 200 part per million (ppm) hydrogen peroxide in SC CO2 at 60 °C, 27.5 MPa. Addition of water to SC CO2 resulted in greater than three-log killing, but this is insufficient to claim sterilization. Neither ethanol nor isopropanol when added to SC CO2 affected killing.

Introduction

Rapid development of novel surgical or implantable devices and biomaterials presents a challenge to existing medical sterilization technologies (Moisan et al., 2001). SC CO2 is receiving interest as a potential new sterilization technology because of drawbacks of known sterilization methods. Standard sterilization methods include steam autoclaving, gamma-irradiation, and ethylene oxide (Matthews et al., 2001, Dempsey and Thirucote, 1989). Steam autoclaving damages heat-sensitive materials (Dempsey and Thirucote, 1989) and may deposit an oxide layer onto metallic surfaces (Lausmaa et al., 1985). Also, autoclaving may wet the surface, damaging electronic devices and hydrolyzing polymers. Dry sterilization is preferred for these applications. Gamma irradiation reduces shear and tensile strength, elastic modulus, and transparency of medical polymers by breaking polymer chains and generating active free radicals (Dillow et al., 1999, Premnath et al., 1996). Ethylene oxide not only degrades certain polymers, but also is flammable and toxic (Dempsey and Thirucote, 1989).

This work concerns the effectiveness of CO2-based technology in killing B. pumilus spores. B. pumilus ATCC 27142 was selected because it is one of three species of spores commonly used to validate commercial sterilizers, and because no data exist on the effects of SC CO2 on this organism (Spilimbergo and Bertucco, 2003).

Supercritical CO2 is of interest as a potential sterilization medium because it is nontoxic, nonflammable, chemically inert, and physiologically safe, and because the critical temperature (31 °C) is low enough to qualify as a ‘cold sterilization’ technology (Elvassore et al., 2000, Enomoto et al., 1997b, Hong et al., 1999). The chief hazard is asphyxiation. CO2 exists in the supercritical state at a temperature higher than its critical temperature (Tc = 31.1 °C) and at a pressure higher than its critical pressure (Pc = 7.38 MPa). SC CO2 has a liquid-like density (0.9–1.0 × 103 kg m 3 ) (Span and Wagner, 1996), gas-like diffusivity (10 7–10 8 m2 s 1 ) and viscosity (3–7 × 10 5 N s m 2), and zero surface tension (McHugh and Krukonis, 1993), which allows SC CO2 to easily penetrate complex structures. A SC CO2 sterilization process is a tantalizing sterilization option for heat-sensitive and/or porous medical devices and biomaterials.

CO2 kills many vegetative bacteria, but its effect on bacterial spores has received limited attention (Fraser, 1951, Kamihira et al., 1987, Haas et al., 1989, Dillow et al., 1999, Spilimbergo and Bertucco, 2003). Of more than twenty-four species of vegetative bacteria, only one, Salmonella senftenberg, has not been completely deactivated with CO2 (Haas et al., 1989). However, bacterial spores are difficult to destroy using pure CO2 because of their deeply dehydrated state, unique chemical composition, and thick envelope (Madigan et al., 2002, Driks, 1999). In order to demonstrate sterilization for potential commercial applications (e.g. for FDA approval of new sterilization technology) it is essential to demonstrate at least 6-log reduction of bacterial spores.

No data on killing of B. pumilus spore have been reported. Eight other species of bacterial spores have been treated with SC CO2 include Bacillus atrophaeus (Spilimbergo et al., 2003), Bacillus cereus (Dillow et al., 1999, Ishikawa et al., 1997), Bacillus coagulans (Ishikawa et al., 1997), Bacillus megaterium (Enomoto et al., 1997b, Enomoto et al., 1997a, Ishikawa et al., 1997), Bacillus polymyxa (Ishikawa et al., 1997), Geobacillus stearothermophilus (Kamihira et al., 1987, Roskey and Sikes, 1994, Sikes and Martin, 1995), Bacillus subtilis (Ballestra and Cuq, 1998, Hata et al., 1996, Ishikawa et al., 1997, Kamihira et al., 1987), and Clostridium sporogenes (Haas et al., 1989). Because spores are highly resistant, combinations of elevated pressures up to 30 MPa (Ishikawa et al., 1997), high temperatures up to 100 °C (Sikes and Martin, 1995), or extended treatment times up to 100 h (Sikes and Martin, 1995) were required to achieve significant killing. These harsh conditions may damage heat-sensitive devices and increase costs. Therefore, it is desirable to operate at the lowest possible temperature and pressure and for the shortest period of time. Presented here are the effects of SC CO2 containing various additives on killing of B. pumilus ATCC 27142 spores at temperatures between 40 and 80 °C, pressures 10.3 and 27.5 MPa, and for 1–6 h treatment.

Section snippets

Materials

B. pumilus ATCC 27142 spore strips were purchased from Raven Biological Laboratories, Inc., Omaha, NE (LOT #: 716663, mean recovery: 3.5 × 106 cfu/strip). Difco™ tryptic soy agar (Becton, Dickinson and Company, Sparks, MD) and 30% hydrogen peroxide (H2O2) aqueous solution (Fisher Scientific, Fair Lawn, NJ) were obtained from Fisher Scientific. Anhydrous CO2 (purity > 99.8%) was obtained from National Specialty Gases (Durham, NC).

Supercritical CO2 sterilization procedure

B. pumilus spore strips were exposed to SC CO2 under controlled

Experimental results

Table 1 shows the results of the control experiments. Dry heat (60 °C) did not give significant log reduction. Dilute aqueous H2O2 resulted in 2.8 log reduction at 60 °C, but was not significant at 40 °C. Pure CO2 has been shown to be ineffective in killing dry spores (Kamihira et al., 1987), and our unpublished results with B. atrophaeus spores confirm this. Therefore, only the results on the use of additives in SC CO2 is presented.

As will be shown, substantial killing can be obtained using

Discussion

We begin by distinguishing the individual sporicidal effects of temperature and H2O2. B. pumilus is resistant to high temperature, as shown by (Gibriel and Abd-El Al, 1973, Janstova and Lukasova, 2001, Ruiz et al., 2002). These studies used temperatures up to 100 °C and short treatment times (less than 1 h). Our control experiments at 60 °C with 4-h exposure are consistent with these studies, showing only 0.41 log reduction (Table 1).

No specific data on susceptibility of B. pumilus spores to H2O2

Acknowledgement

This work was supported by NIH/National Institute of Biomedical Imaging and Bioengineering under a Bioengineering Research Partnership grant (R01 EB55201).

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