Jean Barbeau

Low-temperature sterilization using gas plasmas: a review of the experiments and an analysis of the inactivation mechanisms

Utilizing an ionized gas (plasma) to achieve sterilization is an alternative to conventional sterilization means as far as sterilization of heat-sensitive materials and innocuity of sterilizing agents are concerned. The literature on plasma sterilization is reviewed. A major issue of plasma sterilization is the respective roles of UV photons and reactive species such as atomic and radicals. Insight into this matter is obtained by analyzing the survival curves of microorganisms. In contrast to classical sterilization where such plots show a unique straight line, plasma sterilization yields survival diagrams with two or three different linear segments. Three basic mechanisms are involved in the plasma inactivation of microorganisms: (A) direct destruction by UV irradiation of the genetic material of microorganisms; (B) erosion of the microorganisms atom by atom, through intrinsic photodesorption by UV irradiation to form volatile compounds combining atoms intrinsic to the microorganisms; (C) erosion of the microorganisms, atom by atom, through etching to form volatile compounds as a result of slow combustion using oxygen atoms or radicals emanating from the plasma. In some cases, etching is further activated by UV photons, increasing the elimination rate of microorganisms. These mechanisms make plasma sterilization totally different from classical sterilization techniques and suggest its use to inactivate nonconventional infectious agents such as the abnormal prions.

Plasma Sterilization : Methods and Mechanisms

Utilizing a plasma to achieve sterilization is a possible alternative to conventional sterilization means as far as sterilization of heat-sensitive materials and innocuity of sterilizing agents are concerned. A major issue of plasma sterilization is the respective roles of ultraviolet (UV) photons and reactive species such as atomic and molecular radicals. At reduced gas pressure (£10 torr) and in mixtures containing oxygen, the UV photons dominate the inactivation process, with a significant contribution of oxygen atoms as an erosion agent. Actually, as erosion of the spore progresses, the number of UV photons successfully interacting with the genetic material increases. The different physicochemical processes at play during plasma sterilization are identified and analyzed, based on the specific characteristics of the spore survival curves.

Biofilm Formation by Staphylococcus Aureus and Pseudomonas Aeruginosa is Associated with an Unfavorable Evolution after Surgery for Chronic Sinusitis and Nasal Polyposis

OBJECTIVES: To determine whether biofilm-forming capacity of bacteria demonstrated in chronic rhinosinusitis (CRS) has an impact on persistence of the disease following endoscopic sinus surgery (ESS). METHOD: Thirty-one bacterial strains recovered from 19 patients with CRS at least 1 year post-ESS. Evolution of disease was assessed by questionnaire and endoscopy as favorable or unfavorable. The bacteria were cultured on a 96-well culture plaque and a semi-quantitative method using crystal violet to quantify biofilm production was used. RESULTS: Twenty-two of 31 samples produced a biofilm thicker or equal to the positive control. Biofilm production was noted in 6/10 Pseudomonas aeruginosa isolates, 8/10 Staphylococcus aureus, and 8/11 coagulase-negative staphylococci. Biofilm formation was associated with a poor evolution for Pseudomonas aeruginosa and Staphylococcus aureus, but not coagulase-negative staphylococcus. CONCLUSION: There is a correlation between in vitro biofilm-producing capacity by Pseudomonas aeruginosa and Staphylococcus aureus and unfavorable evolution after ESS, suggesting a role for biofilm production in chronic sinusitis.

Using the flowing afterglow of a plasma to inactivate Bacillus subtilis spores: Influence of the operating conditions

The flowing afterglow of a microwave discharge can be used to efficiently inactivate bacterial spores. We have conducted a parametric study of the operating conditions of such a system, which shows that the species participating in the killing of spores are oxygen atoms and ultraviolet (UV) photons. The oxygen atoms and the excited atoms and molecules emitting the photons being carried by the flowing afterglow can be made available throughout the sterilization chamber. Typical operating conditions are: gas mixture 2%O2/98%N2, pressure range 1–7 Torr and gas flow 0.5–3 slm. Total inactivation of 106 B. subtilis spores is achieved within 40 min with 100 W absorbed microwave power, at afterglow gas temperatures not exceeding 50 °C, a feature of interest for heat sensitive medical devices. The present scheme depends on the gas flow reaching all parts of the objects to be sterilized and on the short-lived active species being transported there sufficiently rapid. Under our operating conditions, it is the UV em...

The respective roles of UV photons and oxygen atoms in plasma sterilization at reduced gas pressure: the case of N/sub 2/-O/sub 2/ mixtures

In the reduced-pressure (/spl les/10 torr) afterglow stemming from discharges in O/sub 2/- containing mixtures such as N/sub 2/-O/sub 2/, the test-reference spores are ultimately inactivated by UV photons through destruction of their genetic material (DNA). To show this, we assume the inactivation to result from a sufficiently large number of successful hits of the DNA strands by UV photons. This implies that the higher the UV intensity, the shorter the time required to reach the lethal dose. Simultaneously, the increased erosion of the spores by the oxygen atoms as time elapses reduces the incident number of photons required to meet the lethal dose. Erosion, as observed by scanning electron microscopy, also increases with the O/sub 2/ percentage in the mixture. Actually, sterilization time is found to be the shortest when the O/sub 2/ percentage in the mixture is set to maximize the UV emission intensity, which occurs at O/sub 2/ percentages typically below 2%, where erosion is low. This proves the predominant role of UV radiation over erosion as far as spore inactivation is concerned. In any case, plasma sterilization always implies some erosion of the test spores, in contrast to what happens with conventional sterilization methods.

Biofilms augment the number of free-living amoebae in dental unit waterlines

Freshwater amoebae are ubiquitous. Some species can cause infections in humans while others can ingest and protect opportunistic bacteria. Although the presence of free-living amoebae in various water sources has been reported, few studies have looked at their concentration, which may be clinically relevant, especially if they are present in healthcare devices. A simple technique was used to detect, observe, and evaluate the concentration of free-living amoebae in dental unit and tap water samples. Fifty-three water samples were collected from 35 dental units (air/water syringes) and 18 water taps. The technique was based on the ability of waterborne bacteria to create a biofilm and serve as substratum for the development of amoebae naturally present in the water samples. Laboratory-grown freshwater biofilms support the proliferation of a wide variety of free-living amoebae. All the dental unit water samples tested contained amoebae at concentrations up to 330/mL, or more than 300 times the concentration in tap water from the same source. Hartmanella, Vanella, and Vahlkampfia spp. were the most frequently encountered. Naegleria and Acanthamoeba spp. were also present in 40% of the samples. Four of the samples collected from dental units, but none from water taps, contained amoebae able to proliferate at 44 degrees C. Biofilms that form inside some dental instruments can considerably increase the concentration of free-living amoebae, some of which are potential human pathogens.

Favoring Trauma as an Etiological Factor in Denture Stomatitis

The etiology of denture stomatitis remains controversial. Trauma due to unstable dentures has been suggested as an etiological factor. Therefore, we tested the hypothesis that the prevalence of denture stomatitis is reduced when mandibular dentures are stabilized by implants. Data were collected at a one-year follow-up from 173 edentulous elders who had randomly received mandibular implant overdentures or conventional dentures. The diagnosis of denture stomatitis was determined according to the Newton classification. Elders wearing conventional dentures were almost 5 times more likely to have denture stomatitis than those wearing mandibular two-implant overdentures (P < 0.0001, Fisher’s exact test). Adjusted odds ratios showed that only the type of the prosthesis (AOR = 4.54, 95% CI 2.20 to 9.40) and nocturnal wear (AOR = 3.03, 95% CI 1.24 to 7.40) predict the frequency of denture stomatitis. Thus, implant overdentures may reduce oral mucosal trauma and control denture stomatitis.

Effectiveness of topical antibiotics on Staphylococcus aureus biofilm in vitro

Background In vitro biofilm-producing capacity in isolates of Staphylococcus aureus and Pseudomonas aeruginosa collected from the sinus cavities after endoscopic sinus surgery (ESS) are associated with a poor outcome in patients with chronic rhinosinusitis (CRS). However, conventional oral antibiotic therapy is frequently ineffective in eradicating bacteria in the biofilm form. Increasing the concentration of antibiotics may offer a means of countering this resistance. The aim of this study was to determine the in vitro activity of moxifloxacin (MOXI) against S. aureus in biofilm form (recovered from patients with CRS at least 1 year post-ESS). Method This study was performed in a research microbiology laboratory, where five isolates of S. aureus with known biofilm-forming capacity were cultured in Tryptic Soy Broth 0.5% glucose in 96-well plates at 37°C for 24 hours. After visual confirmation of biofilm formation, plates were incubated in phosphate-buffered saline (PBS) or with MOXI at concentrations of 0.1×, 1×, 100×, and 1000× minimal inhibitory concentration (MIC) for an additional 24 hours. Biofilm from 3 wells of each concentration were collected and sonicated and the number of viable bacteria was determined by serial dilution and plating. Results After incubation, the number of viable bacteria was similar for nontreated and MOXI-treated biofilms at MIC and sub-MIC levels. However, MOXI at 1000X (0.1–0.2 mg/mL) gave a 2 to 2.5 log reduction in number of viable bacteria. Conclusion In vitro results show that increased concentrations of antibiotics, easily attainable in topical solutions, are effective in killing bacteria in bacterial biofilms. This suggests a role for topical antibiotic therapies in the treatment of biofilm infections.

Clinical utilization of genomics data produced by the international Pseudomonas aeruginosa consortium

The International Pseudomonas aeruginosa Consortium is sequencing over 1000 genomes and building an analysis pipeline for the study of Pseudomonas genome evolution, antibiotic resistance and virulence genes. Metadata, including genomic and phenotypic data for each isolate of the collection, are available through the International Pseudomonas Consortium Database (http://ipcd.ibis.ulaval.ca/). Here, we present our strategy and the results that emerged from the analysis of the first 389 genomes. With as yet unmatched resolution, our results confirm that P. aeruginosa strains can be divided into three major groups that are further divided into subgroups, some not previously reported in the literature. We also provide the first snapshot of P. aeruginosa strain diversity with respect to antibiotic resistance. Our approach will allow us to draw potential links between environmental strains and those implicated in human and animal infections, understand how patients become infected and how the infection evolves over time as well as identify prognostic markers for better evidence-based decisions on patient care.

Validation of cold plasma treatment for protein inactivation: a surface plasmon resonance-based biosensor study

Gas plasma is being proposed as an interesting and promising tool to achieve sterilization. The efficacy of gas plasma to destroy bacterial spores (the most resistant living microorganisms) has been demonstrated and documented over the last ten years. In addition to causing damage to deoxyribonucleic acid by UV radiation emitted by excited species originating from the plasma, gas plasma has been shown to promote erosion of the microorganism in addition to possible oxidation reactions within the microorganism. In this work, we used lysozyme as a protein model to assess the effect of gas plasma on protein inactivation. Lysozyme samples have been subjected to the flowing afterglow of a gas discharge achieved in a nitrogen–oxygen mixture. The efficiency of this plasma treatment on lysozyme has been tested by two different assays. These are an enzyme-linked immunosorbent assay (ELISA) and a surface plasmon resonance (SPR)-based biosensor assay. The two methods showed that exposure to gas plasma can abrogate lysozyme interactions with lysozyme-specific antibodies, more likely by destroying the epitopes responsible for the interaction. More specifically, two SPR-based assays were developed since our ELISA approach did not allow us to discriminate between background and low, but still intact, quantities of lysozyme epitope after plasma treatment. Our SPR results clearly demonstrated that significant protein destruction or desorption was achieved when amounts of lysozyme less than 12.5 ng had been deposited in polystyrene 96-well ELISA plates. At higher lysozyme amounts, traces of available lysozyme epitopes were detected by SPR through indirect measurements. Finally, we demonstrated that a direct SPR approach in which biosensor-immobilized lysozyme activity is directly measured prior and after plasma treatment is more sensitive, and thus, more appropriate to define plasma treatment efficacy with more certainty.