Sterilization effect of atmospheric plasma on Escherichia coli and Bacillus subtilis endospores

Aims:  Escherichia coli and Bacillus subtilis spores were treated with an atmospheric plasma mixture created by the ionization of helium and oxygen to investigate the inactivation efficiency of a low-temperature plasma below 70°C.
Methods and results:  An electrical discharge plasma was produced at a radio frequency (RF) of 13·56 MHz, connected to a perforated circular electrode with a discharge spacing of 1–15 mm. The discharge gas was helium with 0–2% oxygen. For the plasma treatment, a dried E. coli cell or B. subtilis endospore suspension on a cover-glass was exposed to oxygen downstream of the plasma from holes in an RF-powered electrode. The sterilization effect of the RF plasma was highest with 0·2% oxygen, corresponding to the maximum production of oxygen radicals.
Conclusions:  Oxygen radicals generated by RF plasma are effective for the destruction of bacterial cells and endospores.
Significance and Impact of the study:  Low-temperature atmospheric plasma can be used for the disinfection of diverse objects, especially for the inactivation of bacterial endospores.     

Survival of microbial films in the microwave oven.

Air-dried films of Escherichia coli, Saccharomyces cerevisiae, and Bacillus subtilis spores on membrane filters, exposed to 10 min full power (650 W, 2450 MHz) irradiation in a Toshiba model ER776BT microwave oven, showed a 5-, 2-, and 0-log reduction of viable organisms respectively. Suspensions of cells or spores in phosphate buffer treated under similar conditions showed 8 logs of killing within 30 s (S. cerevisiae), 45 s(E. coli), and by 10 min (B. subtilis spores) of exposure.     

Decomposition of biological macromolecules by plasma generated with helium and oxygen

In this study, we attempted to characterize the biomolecular effects of an atmospheric pressure cold plasma (APCP) system which utilizes helium/oxygen (He/O(2)). APCP using He/O(2) generates a low level of UV while generating reactive oxygen radicals which probably serve as the primary factor in sterilization; these reactive oxygen radicals have the advantage of being capable to access the interiors of the structures of microbial cells. The damaging effects of plasma exposure on polypeptides, DNA, and enzyme proteins in the cell were assessed using biochemical methods.     

The antimicrobial activity of essential oil from Dracocephalum foetidum against pathogenic microorganisms

A number of essential oils from Mongolian aromatic plants are claimed to have antimicrobial activities. The essential oil of Dracocephalum foetidum, a popular essential oil used in Mongolian traditional medicine, was examined for its antimicrobial activity. Eight human pathogenic microorganisms including B. subtilis, S. aureus, M. lutens, E. hirae, S. mutans, E. coli, C. albicans, and S. cerevisiae were examined. The essential oil of Dracocephalum foetidum exhibited strong antimicrobial activity against most of the pathogenic bacteria and yeast strains that were tested; by both the agar diffusion method and the minimum inhibitory concentration (MIC) assay (MIC range was 26-2592 microg/ml). Interestingly, Dracocephalum foetidum even showed antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) strains. We also analyzed the chemical composition of the oil by GC-MS and identified several major components, including n-Mentha-1,8-dien-10-al, limonene, geranial, and neral.     

Yeast centromeric plasmids as shuttle vectors between Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae

A number of hybrid plasmids which can autonomously replicate in E. coli, B. subtilis and S. cerevisiae was constructed. Replication of these plasmids both in yeast and in B. subtilis starts on a sequences originating from Staphylococcus aureus plasmids pC194 and pE194. In yeast these hybrids are unstable like those yeast vectors which contain eukaryotic ARSs, but their stability has been increased by addition of yeast centromeric sequence. Both pC194 and pE194 DNAs contain sequences which reveal strong similarities with the yeast ARS consensus. Nevertheless the replication efficiences of these plasmids in yeast are different.     

Effect of heat-generated product from uronic acids on the physiological activities of microbial cells and its application

Filtered samples of monogalacturonic (GA) and monoglucuronic acids (GL) that were prepared using millipore filter (pore size=0.2 microm) slightly inhibited the growth of Escherichia coli while the autoclaved (at 121 degrees C for 20 min) samples of GA and GL completely inhibited the growth of E. coli. The most effective substance generated upon autoclave treatment was isolated and characterized as trans-4,5-dihydroxy-2-cyclopenten-1-one (DHCP). The optimal conditions for DHCP generation were also established by autoclaving GA (pH 2.3) at 121 degrees C for 3h. DHCP completely inhibited the growth of E. coli. However, the growth of E. coli was restored when superoxide dismutase and catalase were added to the culture broth that contained DHCP. It was thought that DHCP might have induced the release of active oxygen, which resulted in the inhibition of microbial growth. In the case of gram-positive bacteria (Bacillus cereus, Bacillus subtilis and Staphylococcus aureus) and yeast (Saccharomyces cerevisiae and Candida brassicae), DHCP inhibited the cell growth. Based on our results, methods for preparation of food preservatives that contained pectin degraded products (oligo-galacturonic acid and monogalacturonic acid) and DHCP were developed. The preservatives were very effective in inhibiting the growth of E. coli and S. cerevisiae.     

Synthesis, structural characterization and antimicrobial activities of 4- and 6-coordinate nickel(II) complexes with three thiosemicarbazones and semicarbazone ligands

To investigate the relationship between antimicrobial activities and the molecular structures of nickel(II) complexes with thiosemicarbazone and semicarbazone ligands, nickel(II) complexes with ligands Hmtsc, Hatsc, Hasc and H2dmtsc, were prepared and characterized by elemental analysis, FT-IR, 1H and 13C NMR spectroscopies, magnetic susceptibility measurements, UV-Vis absorption spectra, TG/DTA and single-crystal X-ray analysis. Their antimicrobial activities were evaluated by the MIC against four bacteria (B. subtilis, S. aureus, E. coli and P. aeruginosa), two yeasts (C. albicans and S. cerevisiae) and two molds (A. niger and P. citrinum). The 4-coordinate, diamagnetic nickel(II) complexes showed antimicrobial activities which were different from those of free ligands or the starting nickel(II) compounds; [Ni(mtsc)(OAc)] 1 showed selective and effective antimicrobial activities against two Gram-positive bacteria (B. subtilis and S. aureus) and modest activities against a yeast (S. cerevisiae), [Ni(mtsc)Cl] 3 exhibited moderate activities against a Gram-positive bacterium (S. aureus), and [Ni(atsc)(OAc)] 5 showed modest activities against two Gram-positive bacteria (B. subtilis and S. aureus). On the other hand, the 6-coordinate, paramagnetic nickel(II) complexes with two protonated or deprotonated ligands ([Ni(mtsc)2] 2, [Ni(atsc)(mtsc)] 4, [Ni(atsc)2] 6, [Ni(Hatsc)2](NO3)(2)7, [Ni(Hatsc)2]Cl(2)8 and [Ni(Hasc)2](OAc)(2)9) and the sterically crowded 4-coordinate, diamagnetic nickel(II) complex ([Ni(dmtsc)] 10) did not inhibit the growth of the test organisms. The structure-activity correlation in this series of nickel(II) complexes was discussed based on their ligand-replacement abilities.     

Cloning, sequencing, and expression in Escherichia coli of the Bacillus subtilis gene for phosphatidylserine synthase.

The Bacillus subtilis pss gene encoding phosphatidylserine synthase was cloned by its complementation of the temperature sensitivity of an Escherichia coli pssA1 mutant. Nucleotide sequencing of the clone indicated that the pss gene encodes a polypeptide of 177 amino acid residues (deduced molecular weight of 19,613). This value agreed with the molecular weight of approximately 18,000 observed for the maxicell product. The B. subtilis phosphatidylserine synthase showed 35% amino acid sequence homology to the yeast Saccharomyces cerevisiae phosphatidylserine synthase and had a region with a high degree of local homology to the conserved segments in some phospholipid synthases and amino alcohol phosphotransferases of E. coli and S. cerevisiae, whereas no homology was found with that of the E. coli counterpart. A hydropathy analysis revealed that the B. subtilis synthase is very hydrophobic, in contrast to the hydrophilic E. coli counterpart, consisting of several strongly hydrophobic segments that would span the membrane. A manganese-dependent phosphatidylserine synthase activity, a characteristic of the B. subtilis enzyme, was found exclusively in the membrane fraction of E. coli (pssA1) cells harboring a B. subtilis pss plasmid. Overproduction of the B. subtilis synthase in E. coli cells by a lac promoter system resulted in an unusual increase of phosphatidylethanolamine (up to 93% of the total phospholipids), in contrast to gratuitous overproduction of the E. coli counterpart. This finding suggested that the unusual cytoplasmic localization of the E. coli phosphatidylserine synthase plays a role in the regulation of the phospholipid polar headgroup composition in this organism.     

Glutaraldehyde: its uptake by sporing and non-sporing bacteria, rubber, plastic and an endoscope

Uptake of glutaraldehyde to bacterial spores, germinating and outgrowing spores, vegetative cells (sporing and non-sporing bacteria), various types of rubber, plastic and an endoscope was investigated. Escherichia coli NCTC 10418 exhibited greatest uptake, followed by Bacillus subtilis NCTC 8236 vegetative cells and Staphylococcus aureus NCTC 6571. Germinated and outgrowing B. subtilis spores adsorbed more glutaraldehyde than resting spores, but less than vegetative cells. Low concentrations of alkaline and acid glutaraldehyde increased the surface hydrophobicity and inhibited the germination of bacterial spores, the alkaline solution to a greater extent in both cases. Rubbers exhibited varying degrees of uptake and are listed in decreasing order of uptake: red rubber, fluorinated rubber (Vinescol), silicone rubber (Silescol), butyl rubber (Butyl XX). Polypropylene, the only plastic examined, was found not to adsorb any glutaraldehyde. The endoscope adsorbed more glutaraldehyde (per gram) than fluorinated rubber but less than red rubber. No damage was observed.     

Glutaraldehyde: its uptake by sporing and non-sporing bacteria, rubber, plastic and an endoscope

Uptake of glutaraldehyde to bacterial spores, germinating and outgrowing spores, vegetative cells (sporing and non-sporing bacteria), various types of rubber, plastic and an endoscope was investigated. Escherichia coli NCTC 10418 exhibited greatest uptake, followed by Bacillus subtilis NCTC 8236 vegetative cells and Staphylococcus aureus NCTC 6571. Germinated and outgrowing B. subtilis spores adsorbed more glutaraldehyde than resting spores, but less than vegetative cells. Low concentrations of alkaline and acid glutaraldehyde increased the surface hydrophobicity and inhibited the germination of bacterial spores, the alkaline solution to a greater extent in both cases.
Rubbers exhibited varying degrees of uptake and are listed in decreasing order of uptake: red rubber, fluorinated rubber (Vinescol), silicone rubber (Silescol), butyl rubber (Butyl XX). Polypropylene, the only plastic examined, was found not to adsorb any glutaraldehyde. The endoscope adsorbed more glutaraldehyde (per gram) than fluorinated rubber but less than red rubber. No damage was observed.     

黔西南薏苡内生真菌多样性及其抗菌活性研究

为进一步了解贵州黔西南薏苡(Coix lacryma-Jobi)可培养内生真菌的多样性组成及其抗菌活性。通过组织块分离法,选取薏苡根、叶和种仁为分离对象,进行内生真菌分离,并通过形态学观察,分子生物学特征对所分离内生真菌进行鉴定。采用平板菌块对峙法对薏苡内生真菌进行抗菌活性筛选。从薏苡根、叶和种仁中共分离纯化获得76株薏苡内生真菌,根据形态和分子生物学特征,它们属于10个目、14个科、26个属和1个不明属以及未知菌;以大肠杆菌(Escherichia coli)、金黄色葡萄球菌(Staphyloccocus aureus)、枯草芽胞杆菌(Bacillus subtilis)、酿酒酵母(Saccharomyces cerevisiae)为指示菌株,对薏苡内生真菌进行抗菌活性筛选。其中有25株对大肠杆菌具有抗菌活性,占分离菌株的比例为32. 9%;有34株对金黄色葡萄球菌具有抗菌活性,其比例为44. 7%;有24株对枯草芽胞杆菌具有抗菌活性,比例是31. 6%;有23株对酿酒酵母具有抗菌活性,占比例为30. 3%;对4种标准菌株都有抗菌活性的有13株,占总株数的17. 1%。薏苡的不同组织结构中存在丰富的内生真菌资源,部分内生真菌具有抑制其他微生物生长的活性,具有产生天然活性产物成分的潜力,为工业应用奠定了资源基础,具有进一步发掘和研究的价值。     

Factors Affecting the Antimicrobial Activity of Vitamin K5

Pure cultures of Escherichia coli, Bacillus subtilis, Proteus vulgaris, Staphylococcus aureus, and Pseudomonas fluorescens were used in this investigation. The bactericidal concentrations of vitamin K(5) required for E. coli, B. subtilis, P. vulgaris, S. aureus, and P. fluorescens; the effect of an absence of oxygen; the effect of contact time with E. coli and S. aureus; and the effect of initial counts per milliliter of E. coli were studied. The bactericidal concentrations ranged from 60 ppm of K(5) for S. aureus to 220 ppm for E. coli, with an initial count of 160,000 to 200,000 cells per milliliter and a contact time of 12 hr in nutrient broth. The gram-positive bacteria tested were more susceptible to the antimicrobial activity of vitamin K(5) than the gram-negative bacteria. In the studies conducted under nitrogen atmosphere, the per cent inhibition showed an inverse relationship to the bactericidal concentrations required for complete inhibition in studies conducted under air atmosphere. This finding suggested that there might be different factors responsible for inhibition depending on the species of bacteria being tested, and it also might help explain the difference in concentrations necessary for inhibition. Cells of E. coli and S. aureus were not inhibited immediately on coming into contact with vitamin K(5); 50% inhibition occurred after 25 and 32 min, respectively. A rapid inhibition rate was maintained until approximately 90% inhibition occurred, after whch a rapid decrease in the rate was noted.     

过氧化氢干雾对生物安全柜微环境表面消毒效果的研究

生物安全实验室微环境消毒是控制实验室污染的重要环节。过氧化氢广泛用于病原微生物实验室微环境消毒,但其对不同病原微生物的消毒效果有待研究。本文研究了过氧化氢干雾（粒径％10μm）以不同消毒程序对生物安全柜表面常见微生物的消毒效果。结果显示,在生物安全柜内采用优化的消毒程序（发散循环8次,每次1min,达60ppm后,静置消毒2h）,过氧化氢干雾可完全杀灭1×106CFU枯草芽胞杆菌、嗜热脂肪芽胞杆菌、金黄色葡萄球菌、表皮葡萄球菌、耻垢分枝杆菌,以及1×106CFU大肠埃希菌。然而,当金黄色葡萄球菌、表皮葡萄球菌、耻垢分枝杆菌浓度达1×107CFU时,过氧化氢干雾无法完全杀灭。因此,建议在进行过氧化氢干雾消毒时,应先用消毒剂处理,以期彻底杀灭生物安全柜微环境中污染的病原微生物。     

江西迷迭香精油的成分分析及抗氧化、抑菌活性研究

为了研究江西迷迭香精油的化学成分及抗氧化、抑菌活性,采用水蒸气蒸馏法提取迷迭香精油,利用气相色谱-质谱联用法(GC-MS)对迷迭香精油成分进行分析,通过对DPPH自由基、羟基自由基的清除活性和还原力来研究迷迭香精油的体外抗氧化活性;通过以枯草芽孢杆菌、金黄色葡萄球菌和大肠杆菌为供试菌,测定抑菌圈大小和最低抑菌浓度(MIC)来研究迷迭香精油的抑菌活性。实验结果表明,从迷迭香精油中鉴定出40种化学成分,占精油总量的99.46%,其主要化学成分有α-蒎烯(39.05%)和1,8-桉叶素(16.86%),其次是莰烯(4.22%)、D-柠檬烯(3.87%)、龙脑(3.74%)、β-石竹烯(3.11%)等,α-蒎烯的含量高于国内其他产地;迷迭香精油对DPPH、羟基自由基和还原力的半数清除率IC₅₀值分别为76.42、51.40和49.15μL/mL;精油对枯草芽孢杆菌、金黄色葡萄球菌和大肠杆菌的抑菌圈大小分别为14.40±0.66、11.41±0.19、11.70±0.27 mm,最低抑菌浓度(MIC)分别为2.50、10.00、10.00μL/mL,对枯草芽孢杆菌的抑制作用明显强于金黄色葡萄球菌和大肠杆菌。结果表明迷迭香精油具有较好的抗氧化、抑菌活性。     

Effect of Various Gas Atmospheres on Destruction of Microorganisms in Dry Heat

The heat resistance of dry bacterial spores was tested in various gases at temperatures ranging from 121.1 to 160 C (250 to 320 F). Spores of Clostridium sporogenes (PA 3679) were heated in air, carbon dioxide, and helium; spores of Bacillus subtilis 5230 were heated in these gases and also in oxygen and in nitrogen. The surrounding gas influenced the heat resistance, but the differences among gases were small. D values were about 7 min at 148.9 C (300 F); z values were about 18.3 C (33 F) for B. subtilis, and about 21.7 C (39 F) for C. sporogenes. The resistance of B. subtilis in carbon dioxide was about the same as in air, but lower than in all other gases; resistance in helium and nitrogen was about the same, and was higher than in all other gases. C. sporogenes had the least resistance in air; the resistance was about the same in carbon dioxide and helium. For B. subtilis, the gases in order of increasing heat resistance were carbon dioxide, air, oxygen, helium, and nitrogen, and for C. sporogenes, air, carbon dioxide, and helium. Neither oxygen content nor molecular weight of the gas appeared to have a marked influence on dry-heat resistance of the spores, whereas the more inert gases seemed to yield larger D values.     

Gas discharge plasmas are effective in inactivating <Emphasis Type="Italic">Bacillus</Emphasis> and <Emphasis Type="Italic">Clostridium</Emphasis> spores

Bacterial spores are the most resistant form of life and have been a major threat to public health and food safety. Nonthermal atmospheric gas discharge plasma is a novel sterilization method that leaves no chemical residue. In our study, a helium radio-frequency cold plasma jet was used to examine its sporicidal effect on selected strains of Bacillus and Clostridium. The species tested included Bacillus subtilis, Bacillus stearothermophilus, Clostridium sporogenes, Clostridium perfringens, Clostridium difficile, and Clostridium botulinum type A and type E. The plasmas were effective in inactivating selected Bacillus and Clostridia spores with D values (decimal reduction time) ranging from 2 to 8 min. Among all spores tested, C. botulinum type A and C. sporogenes were significantly more resistant to plasma inactivation than other species. Observations by phase contrast microscopy showed that B. subtilis spores were severely damaged by plasmas and the majority of the treated spores were unable to initiate the germination process. There was no detectable fragmentation of the DNA when the spores were treated for up to 20 min. The release of dipicolinic acid was observed almost immediately after the plasma treatment, indicating the spore envelope damage could occur quickly resulting in dipicolinic acid release and the reduction of spore resistance.     

Virulent spores of Bacillus anthracis and other Bacillus species deposited on solid surfaces have similar sensitivity to chemical decontaminants

AIMS: To compare the relative sensitivity of Bacillus anthracis and spores of other Bacillus spp. deposited on different solid surfaces to inactivation by liquid chemical disinfecting agents. METHODS AND RESULTS: We prepared under similar conditions spores from five different virulent and three attenuated strains of B. anthracis, as well as spores of Bacillus subtilis, Bacillus atrophaeus (previously known as Bacillus globigii), Bacillus cereus, Bacillus thuringiensis and Bacillus megaterium. As spore-surface interactions may bias inactivation experiments, we evaluated the relative binding of different spores to carrier materials. The survival of spores deposited on glass, metallic or polymeric surfaces were quantitatively measured by ASTM standard method E-2414-05 which recovers spores from surfaces by increasing stringency. The number of spores inactivated by each decontaminant was similar and generally within 1 log among the 12 different Bacillus strains tested. This similarity among Bacillus strains and species was observed through a range of sporicidal efficacy on spores deposited on painted metal, polymeric rubber or glass. CONCLUSIONS: The data obtained indicate that the sensitivity of common simulants (B. atrophaeus and B. subtilis), as well as spores of B. cereus, B. thuringiensis, and B. megaterium, to inactivation by products that contain either: peroxide, chlorine or oxidants is similar to that shown by spores from all eight B. anthracis strains studied. SIGNIFICANCE AND IMPACT OF THE STUDY: The comparative results of the present study suggest that decontamination and sterilization data obtained with simulants can be safely extrapolated to virulent spores of B. anthracis. Thus, valid conclusions on sporicidal efficacy could be drawn from safer and less costly experiments employing non-pathogenic spore simulants.     

Absence in Bacillus subtilis and Staphylococcus aureus of the sequence-specific deoxyribonucleic acid methylation that is conferred in Escherichia coli K-12 by the dam and dcm enzymes

Restriction analysis of plasmid pHV14 deoxyribonucleic acid isolated from Escherichia coli K-12, Bacillus subtilis, and staphylococcus aureus with restriction endonucleases MboI, Sau3AI, and EcoRII was used to study the methylation of those nucleotide sequences which in E. coli contain the major portions of N6-methyladenine and 5-methylcytosine. The results showed that neither B. subtilis nor S. aureus methylates deoxyribonucleic acid at the same sites and nucleotides which are recognized and methylated by dam and dcm enzymes in E. coli K-12.