Inactivation of Dried Bacteria and Bacterial Spores by Means of Gamma Irradiation at High Temperatures

Dried preparations with Streptococcus faecium, strain A(2)1, and spores of Bacillus sphaericus, strain C(I)A, normally used for control of the microbiological efficiency of radiation sterilization plants and preparations with spores of Bacillus subtilis, normally used for control of sterilization by dry heat, formalin, and ethylene oxide, as well as similar preparations with Micrococcus radiodurans, strain R(1), and spores of Bacillus globigii (B. subtilis, var. niger) were gamma irradiated with dose rates from 16 to 70 krad/h at temperatures from 60 to 100 C. At 80 C the radiation response of the spore preparations was the same as at room temperature, whereas the radiation resistance of the preparations with the two vegetative strains was reduced. At 100 C the radiation response of preparations with spores of B. subtilis was unaffected by the high temperature, whereas at 16 and and 25 krad/h the radiation resistance of the radiation-resistant sporeformer B. sphaericus, strain C(I)A, was reduced to the level of radiation resistance of preparations with spores of B. subtilis. It is concluded that combinations of heat and gamma irradiation at the temperatures and dose rates tested may have very few practical applications in sterilization of medical equipment.     

Clinico-laboratory effectiveness of modern ointments with a polyethylene glycol base in the treatment of purulent wounds]

The 25-year experience with the underbandage treatment of soft tissue purulent wounds of various location and genesis with modern ointments with polyethylene glycol as the basis was analyzed. Levocin, levomecole, dioxycole, 5-percent dioxydinic, 1-percent iodopyronic, 0.5-percent quinifuryl and furagel ointments proved to preserve their high activity against aerobic grampositive and gramnegative flora. 10-percent mafenide acetate ointment had a high selective effect on Pseudomonas aeruginosa. The new ointments nitacid and streptonitole containing nitazole and white streptocide were highly active against both aerobic and anaerobic infections. The use of the ointments with the polyethylene glycol as the basis made it possible to decrease 2 times the period of the patient hospitalization in surgical units and to shorten the terms of the systemic antibacterial therapy. The marked therapeutic effect of such ointments due to their high dehydrating capacity and broad antibacterial spectrum enabled to consider them as the drugs of choice in the local treatment of purulent wounds during the 1st phase of the wound process, trophic and decubic ulcers, infected burns, diabetic and atherosclerotic gangrene, furuncles, carbuncles, mastitis, etc. The ointments in the water soluble vehicle can be as well used with success for the prophylactic treatment of infected wounds after the suture. The multitarget effect of the ointments in the water soluble vehicle and their ability to prevent severe purulent complications permitted to consider them as the 1st order drugs in cases of emergency.     

吴茱萸次碱软膏对小鼠尾部鳞片银屑病模型的治疗作用

目的：吴茱萸次碱（Rutaecarpa RUT）是芸香科植物吴茱萸的主要成分,它可激活辣椒素受体促进降钙素基因相关肽（CGRP） 等神经递质的释放来发挥药理作用。本实验的目的为观察吴茱萸次碱软膏对小鼠银屑病模型治疗作用。方法：通过药剂学方法制 成了不同浓度（2%, 5%, 10%）RUT 软膏,在小鼠尾部与背部银屑病模型给药14 天,观察小鼠尾部颗粒层形成的变化,给药结束后 取小鼠背部皮肤组织匀浆后采用放射免疫方法检测CGRP水平变化,并且取小鼠血浆检测CGRP水平变化。结果：不同浓度的吴 茱萸次碱软膏均能促进小鼠尾部颗粒层形成（P<0.05）,并且浓度为5%与10%的RUT 软膏能显著降低小鼠背部CGRP 含量（P<0. 05）,但是其对小鼠血浆的CGRP 没有影响。结论：吴茱萸次碱外用对小鼠银屑病模型有一定的治疗作用,这种作用与其促进小鼠 皮肤CGRP 释放而导致其含量降低有关。     

Copper-induced change in the ESR signal of hemoglobin nitrosyl complexes in wound by the action of copper nanoparticles

The results concerning changes in the ESR signal of hemoglobin nitrosyl complexes in wound tissues in the course of healing by the action of ointments with copper nanoparticles (patent N2460532, Russia) are presented. It is shown that the wound healing process modified by the influence of copper nanoparticles demonstrates the increase in the ESR signal amplitude for hemoglobin nitrosyl complexes as compared with controls (the ointment base without nanoparticles). Planimetric measurements of wound area through reparation course indicate an active process of wound healing for injuries treated with copper nanoparticles in the ointment, resulting in lessening half-reparation time up to 5.0 times as compared with controls (treatment with the ointment base). The paper discusses the role of copper nanoparticles, NO and their potential synergistic effect on the skin wound regeneration.     

Quantitative assessment of sterilization efficiency using lyophilized calcium alginate biological indicators

Biological indicators were prepared using either Bacillus stearothermophilus or Bacillus subtilis incorporated into lyophilized calcium alginate beads. These were subjected to a variety of moist heat and dry heat treatments as appropriate and then dissolved in buffer to allow enumeration of survivors. The utility of these spore carriers as biological indicators for the quantitative assessment of heat sterilization processes is discussed.     

Microcount Method for Petrolatum-Based Topical Ointments Containing Waxes

A practical solvent system for the detection of microorganisms in topical ointments has been developed. The method involves dissolving 0.5 g of topical ointment in 50 ml of a solvent mixture (92 parts isopropyl myristate, 6 parts carbon disulfide, and 2 parts xylene) and filtering it through a 0.45-mum membrane filter. Residual solvent is then washed from the filter pad with 200 ml of sterile 0.5% Brain Heart Infusion broth containing 0.1% Tween 80. The filter pad is then removed and placed on a petri plate containing Trypticase Soy Agar medium. The petri plates thus prepared are then incubated at 37 C for 7 days, and the colonies produced are then counted. The toxicity of the solvent mixture was determined against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella newington, and spores of Bacillus subtilis and was found to be less toxic than the heat-sterilized isopropyl myristate and comparable to the filter-sterilized isopropyl myristate.     

Increased Radiation Resistance of Vegetative Bacillus pumilus

A 4.5-fold increase in vegetative cell radiation resistance of Bacillus pumilus E601, the internationally recognized biological standard for irradiation sterilization, was obtained by the repeated passage of resistant survivors through successive sublethal doses of (60)Co irradiation. This increase in resistance was accompanied by a corresponding increase in spore resistance through the seventh irradiation passage. By the fifteenth passage, the ability for spore formation was lost. Other effects noted by the successive irradiation dosages included loss of motility and pellicle formation, and changes in the Gram reaction, cell morphology, and colonial morphology. Increased resistance was also accompanied by an increased nutritional requirement for specific amino acids. Radiation resistance was not transferred from vegetative cells to spores.     

Resistance of Bacillus subtilis var. niger Spores Occluded in Water-insoluble Crystals to Three Sterilization Agents

The resistance to destruction of spores of Bacillus subtilis var. niger occluded in crystals of calcium carbonate and exposed to ethylene oxide and moist and dry heat was determined and compared with the destruction of unoccluded spores. Occluded spores could not be inactivated with ethylene oxide. Resistance to inactivation was approximately 900 and 9 times higher for occluded than for unoccluded spores subjected to moist and dry heat, respectively, at 121 C. The protective effect may be due either to the unavailability of oxygen for destruction by oxidation or to inhibition of the loss of essential cell constituents by vaporization. Evidence also implicates the crystal structure as a thermal conductivity barrier. Occluded spores retained viability over a 3-year period compared with unoccluded spores which decreased over 90% during this period. Occluded spores in insoluble materials are seldom encountered in the technology of sterilization, but could be the most critical factor in the sterilization of interplanetary vehicles. Entrapped spores in insoluble materials are usually difficult to detect, and are very stable as well as extremely resistant to destruction by heat and ethylene oxide.     

Studies on the Dose Requirement for the Radiation Sterilization of Medical Equipment. II. A Comparison between Continuous and Fractionated Doses

S ummary : The lethal effect of gamma radiation on spores of Bacillus pumilus air dried on to a glass surface was measured by constructing dose-survivor curves. In one set of experiments the doses used were delivered without interruption, and in another set they were fractionated in various ways with storage intervals between fractions of up to one week. The data presented confirms the view that the efficiency of sterilization by irradiation would not be affected by interruption of dose delivery as might occur in plant operation. A situation where effects might be observed is discussed.     

Anti-biofilm properties of a mupirocin spray formulation against Escherichia coli wound infections

Mupirocin ointment is a widely used topical drug for the treatment of bacterial skin infections. However, ointments have some limitations which motivated the development of a film forming spray of mupirocin. Mupirocin spray (2%) was formulated with Eudragit E100 as a film forming agent and tested for its antibacterial and anti-biofilm activities against Escherichia coli, a skin pathogen causing wound and surgical site infections. Treatment with mupirocin spray resulted in significant antibacterial and anti-biofilm activities (inhibition and disruption) with single spray and sub-actual dose concentrations at par with the commercial ointment concentration. The spray formulation was found to be non-toxic to fibroblast cells and greatly resisted removal from the site of application upon washing, in contrast to the ointment which was significantly removed after a single wash. This is the first study to develop and evaluate a spray formulation for mupirocin that forms a stable thin film for sustained release of the drug.     

Destruction of Bacillus anthracis strain Sterne 34F2 spores in postal envelopes by exposure to electron beam irradiation

AIMS: To determine the irradiation dose necessary to reduce the populations of Bacillus anthracis spores in a dry medium in postal envelopes. METHODS AND RESULTS: Bacillus anthracis Sterne 34F2 spores were dispersed in non-fat dry milk and then placed into standard business postal envelopes. The spores were treated with a sequence of irradiation doses to determine the decimal reduction value (D10) in kiloGrays (kGy). The average D10 value was 3.35 +/- 0.02 kGy. CONCLUSIONS: An irradiation dose of 40.2 kGy would be required to result in a process equivalent to the thermal canning process (12 D10 reduction) to eliminate Clostridium botulinum spores. SIGNIFICANCE AND IMPACT OF THE STUDY: Irradiation is an effective means of reducing or eliminating B. anthracis spores in a dry medium in postal envelopes.     

Pharmacokinetics of protegentin, a combined preparation

Protegentin is a combined preparation in the form of ointment containing 0.1 per cent of gentamicin, 0.25 per cent of erythromycin and 0.1 per cent of protease C. Pharmacokinetic studies on the preparation were conducted. Protegentin and gentamicin ointment, currently manufactured in this country, were applied to the surface of experimental pure cutaneous wounds in guinea pigs in a dose of 1 g. It was shown that inspite of the same contents of gentamicin in the ointments, the mean maximum concentration of the antibiotic in the underlying muscular tissue after the protegentin application was somewhat higher than that after the use of the gentamicin ointment. The differences in the drug concentration maintained during the whole observation period of 24 hours. However, they were not statistically significant. The gentamicin concentrations in serum after the use of protegentin were also somewhat higher than those after application of the gentamicin ointment (the differences were not statistically significant). Still, in no case the concentrations reached the potentially toxic ones. The erythromycin concentrations in the muscular tissue were much higher than those in the blood.     

Microbiological Evaluation of a Large-Volume Air Incinerator

Two semiportable metal air incinerators, each with a capacity of 1,000 to 2,200 standard ft(3) of air per min, were constructed to sterilize infectious aerosols created for investigative work in a microbiological laboratory. Each unit has about the same air-handling capacity as a conventional air incinerator with a brick stack but costs only about one-third as much. The units are unique in that the burner housing and combustion chamber are air-tight and utilize a portion of the contaminated air stream to support combustion of fuel oil. Operation is continuous. Aerosols of liquid and dry suspensions of Bacillus subtilis var. niger spores and dry vegetative cells of Serratia marcescens were disseminated into the two incinerators to determine the conditions required for sterilization of contaminated air. With the latter organisms (concentration 2.03 x 10(7) cells/ft(3) of air), a temperature of 525 F (274 C), measured at the firebox in front of the heat exchanger, was sufficient for sterilization. To sterilize 1.74 x 10(7) and 1.74 x 10(9) wet spores of B. subtilis per ft(3), the required temperature ranged from 525 to 675 F (274 to 357 C) and 625 to 700 F (329 to 371 C), respectively. Air-sterilization temperature varied with each incinerator. This was because of innate differences of fabrication, different spore concentrations, and use of one or two burners With dry B. subtilis spores (1.86 x 10(8)/ft(3)), a temperature of 700 F was required for sterilization. With dry spores, no difference was noted in the sterilization temperature for the two incinerators.     

Cold atmospheric air plasma sterilization against spores and other microorganisms of clinical interest

Physical cold atmospheric surface microdischarge (SMD) plasma operating in ambient air has promising properties for the sterilization of sensitive medical devices where conventional methods are not applicable. Furthermore, SMD plasma could revolutionize the field of disinfection at health care facilities. The antimicrobial effects on Gram-negative and Gram-positive bacteria of clinical relevance, as well as the fungus Candida albicans, were tested. Thirty seconds of plasma treatment led to a 4 to 6 log(10) CFU reduction on agar plates. C. albicans was the hardest to inactivate. The sterilizing effect on standard bioindicators (bacterial endospores) was evaluated on dry test specimens that were wrapped in Tyvek coupons. The experimental D(23)(°)(C) values for Bacillus subtilis, Bacillus pumilus, Bacillus atrophaeus, and Geobacillus stearothermophilus were determined as 0.3 min, 0.5 min, 0.6 min, and 0.9 min, respectively. These decimal reduction times (D values) are distinctly lower than D values obtained with other reference methods. Importantly, the high inactivation rate was independent of the material of the test specimen. Possible inactivation mechanisms for relevant microorganisms are briefly discussed, emphasizing the important role of neutral reactive plasma species and pointing to recent diagnostic methods that will contribute to a better understanding of the strong biocidal effect of SMD air plasma.     

A comparative multi-centre study of the efficacy of propolis, acyclovir and placebo in the treatment of genital herpes (HSV).

Ninety men and women with recurrent genital HSV type 2 participated in a randomized, single-blind, masked investigator, controlled multi-centre study comparing the efficacy of ointment of Canadian propolis containing natural flavonoids with ointments of acyclovir and placebo (vehicle) on healing ability and capacity to remedy symptoms. Thirty individuals were randomized to each group. Treatment was intended to start in the blister phase. All participants had HSV type 2 isolated, confirmed by serum immunoglobulin levels. The participants were examined on the 3rd, 7th and 10th days of treatment by gynaecologists, dermatovenerologists or urologists at seven different medical centres. Apart from clinical symptoms the number and size of the herpetic lesions were noted. At each examination the lesions were classified into four stages: vesicular, ulcerated, crusted and healed. The study ointments were applied to affected areas four times daily. In women with vaginal or cervical lesions a tampon with the appropriate ointment was inserted four times daily for 10 days. Endpoint variables were healing time and time until loss of symptoms. RESULTS: On Day 10, 24 out of 30 individuals in the propolis group had healed. In the acyclovir group 14 out of 30 and in the placebo group 12 out of 30 had healed. (p = 0.0015). The healing process appeared to be faster in the propolis group. In the propolis group 15 individuals had crusted lesions on Day 3 compared to 8 individuals in the acyclovir group and none in the placebo group (p = 0.0006). On Day 7, 10 participants in the propolis group, 4 in the acyclovir group and 3 in the placebo group had healed. At the initial examination all patients had local symptoms and 28% general symptoms. At Day 3, 3 patients in the propolis group had local symptoms compared to 8 and 9 in the acyclovir and placebo groups respectively. Of the women, 66% had vaginal superinfections of microbial pathogens at the initial examination. In the acyclovir and placebo groups no change in the vaginal flora was found following treatment whereas in the propolis group the incidence of superinfection was reduced by 55%. (p = 0.10 n.s.). CONCLUSION: An ointment containing flavonoids appeared to be more effective than both acyclovir and placebo ointments in healing genital herpetic lesions, and in reducing local symptoms.     

Validation of the sterilization procedure of allogeneic avital bone transplants using peracetic acid-ethanol.

Different procedures are available to inactivate bacteria and fungi, including their spores, as well as viruses in human bone transplants. The most efficient methods are considered to be gamma irradiation and thermal inactivation as well as chemical sterilization methods like the peracetic acid-ethanol treatment (PES). Following national and international standards or draft standards, the antimicrobial effectiveness of this procedure was evaluated. Due to the standardizable size as well as the clinical relevance, defatted human spongiosa cuboids (15x15x15 mm) served as model system. After treatment with PES for 2 and 4 hours, respectively, the titre of living micro-organisms was determined in the supernatant and the cuboid. A reduction in the titre of viable micro-organisms below the detection level (reduction factor >5 log10) was already achieved after an incubation time of 2 hours (Staphylococcus aureus, Enterococcus faecium, Pseudomonas aeruginosa, Bacillus subtilis, Clostridium sporogenes, Mycobacterium terrae, Candida albicans as well as spores of Bacillus subtilis). No viable micro-organisms could be detected in any of the PES-treated test cuboids. Spores of Aspergillus niger were also completely inactivated. The PES procedure proved to be a reliable method for the sterilization of human bone transplants derived from spongiosa.     

The Effect of Sterilization Methods on the Physical Properties of Silk Sericin Scaffolds

Protein-based biomaterials respond differently to sterilization methods. Since protein is a complex structure, heat, or irradiation may result in the loss of its physical or biological properties. Recent investigations have shown that sericin, a degumming silk protein, can be successfully formed into a 3-D scaffolds after mixing with other polymers which can be applied in skin tissue engineering. The objective of this study was to investigate the effectiveness of ethanol, ethylene oxide (EtO) and gamma irradiation on the sterilization of sericin scaffolds. The influence of these sterilization methods on the physical properties such as pore size, scaffold dimensions, swelling and mechanical properties, as well as the amount of sericin released from sericin/polyvinyl alcohol/glycerin scaffolds, were also investigated. Ethanol treatment was ineffective for sericin scaffold sterilization whereas gamma irradiation was the most effective technique for scaffold sterilization. Moreover, ethanol also caused significant changes in pore size resulting from shrinkage of the scaffold. Gamma-irradiated samples exhibited the highest swelling property, but they also lost the greatest amount of weight after immersion for 24 h compared with scaffolds obtained from other sterilization methods. The results of the maximum stress test and Young’s modulus showed that gamma-irradiated and ethanol-treated scaffolds are more flexible than the EtO-treated and untreated scaffolds. The amount of sericin released, which was related to its collagen promoting effect, was highest from the gamma-irradiated scaffold. The results of this study indicate that gamma irradiation should have the greatest potential for sterilizing sericin scaffolds for skin tissue engineering.     

The effect of ribose pre-treatment of cortical bone on γ-irradiation sterilization effectiveness

Reconstruction of large skeletal defects is a significant and challenging issue. Tissue banks often use γ-irradiation (15–35 kGy) to sterilize bone allografts, which, however, drastically impairs the post-yield mechanical properties. In previous studies, we reported the development of a method that protects human bone collagen connectivity through ribose crosslinking while still undergoing γ-irradiation. Given these promising results, the next step was to determine if the presence of ribose within the bone tissue would interfere with the effectiveness of the γ-irradiation sterilization against bacteria. This study had two stages. The aim of the first stage was to assess the protective effect of ribose in solution using a Bacillus pumilus spore strip model. The aim of the second stage was to assess the protective effect of ribose (R) on spores within a human cortical bone model in comparison to conventionally irradiated bone (I). Treatment of B. pumilus spore strips with ribose in solution led to temperature-dependent effects on spore viability versus spore strips treated with PBS alone. Ribose solution at 60 °C led to a notable two logs decrease in spore count relative to PBS at 60 °C. In the human bone model, the number of spores in the I and R groups were greatly decreased in comparison to the non-irradiated N group. No spore colonies were detected in the R group (n = 4) whereas two of the four plates of group I formed colonies. This study provides evidence that the method of pre-treating bone with ribose crosslinking prior to irradiation sterilization, while improving irradiation sterilized bone allograft quality, also may improve the effectiveness of the sterilization process.     

A microwave-powered sterilizable interface for aseptic access to bioreactors that are vulnerable to microbial contamination

Novel methods and apparatus that employ the rapid heating characteristics of microwave irradiation to facilitate the aseptic transfer of nutrients, products, and other materials between microbially sensitive systems and the external environment are described. The microwave-sterilizable access port (MSAP) consists of a 600-W magnetron emitting at a frequency of 2.45 GHz, a sterilization chamber with inlet and outlet flow lines, and a specimen transfer interface. Energy is routed to the sterilization chamber via a coaxial transmission line where small quantities of water couple strongly with the incident radiation to produce a superheated vapor phase. The efficiency of energy transfer is enhanced through the use of microwave susceptors within the sterilization chamber. Mating surfaces are thermally sterilized through direct contact with the hot gas. Efficacy has been demonstrated using the thermophile Bacillus stearothermophilus.     

Sterility Test Method for Petrolatum-Based Ophthalmic Ointments

A sensitive sterility testing procedure for the detection of microbial contamination in petrolatum-based ointments is described. The method involves dissolving the ointment in filter-sterilized isopropyl myristate and filtering through a membrane filter. Improved sensitivity is obtained by blending the membrane in Trypticase Soy Broth before incubation. Filter-sterilized isopropyl myristate is shown to be less toxic to microorganisms than heat-sterilized isopropyl myristate. The isopropyl myristate method is more sensitive than the polyethylene glycol-ether method for the detection of microbial contamination.