Functional and ultrastructural changes in Pseudomonas aeruginosa and Staphylococcus aureus cells induced by Cinnamomum verum essential oil

Aims: To study cellular damage induced by Cinnamomum verum essential oil in Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213. Methods and Results: The effect of cinnamon bark essential oil on these two strains was evaluated by plate counts, potassium leakage, flow cytometry and transmission electron microscopy (TEM). Exposure to this oil induced alterations in the bacterial membrane of Ps. aeruginosa, which led to the collapse of membrane potential, as demonstrated by bis‐oxonol staining, and loss of membrane‐selective permeability, as indicated by efflux of K⁺ and propidium iodide accumulation. Thus, respiratory activity was inhibited, leading to cell death. In Staph. aureus, cells treated with the oil entered a viable but noncultivable (VNC) state. The oil initially caused a considerable decrease in the metabolic activity and in the replication capacity of these bacterial cells. The loss of membrane integrity appeared later, as indicated by bis‐oxonol and Propidium iodide (PI) staining. Data provided by TEM showed various structural effects in response to cinnamon essential oil. In Ps. aeruginosa cells, coagulated cytoplasmic material was observed, and intracellular material was seen in the surrounding environment, while oil‐treated Staph. aureus showed fibres extending from the cell surface. Conclusions: Cinnamon essential oil damages the cellular membrane of Ps. aeruginosa, which leads to cell death. There is evidence of VNC Staph. aureus after exposure to the oil. Significance and Impact of the Study: Cinnamon essential oil shows effective antimicrobial activity and health benefits and is therefore considered a potential food additive. To use this oil as a natural food preservative, especially in combination with other preservation methods, a thorough understanding of the mechanism through which this oil exerts its antibacterial action is required.     

Preservative properties of Calamintha officinalis essential oil with and without EDTA

AIMS: This study was focused on the preserving properties of Calamintha officinalis essential oil, a plant known for its diaphoretic, expectorant and aromatic properties. METHODS AND RESULTS: The commercial aerial parts of C. officinalis Moench were hydrodistilled and the essential oil analysed by Gas chromatography/Electron impact mass spectrometry (GC/EIMS). The inhibition efficacy of this essence, alone (0.5 and 1.0% v/v) and in combination with 2.0 mM EDTA, was assayed, in culture medium and in cetomacrogol cream, using preservative efficacy testing against standard microrganisms (E. coli ATCC 25922, Ps. aeruginosa ATCC 9027, Staph. aureus ATCC 6538P, C. albicans ATCC 10231 and A. niger ATCC 16404). C. officinalis essential oil in cetomacrogol cream with EDTA showed long-lasting antimicrobial activity, satisfying the European Pharmacopoeia Commission (E. P.) criteria. CONCLUSION: C. officinalis essential oil could have a potential for a future use as a cosmetic preservative. IMPACT OF THE STUDY: To find natural compounds with antimicrobial activity which could be alternatives to the synthetic chemical preservatives.     

Cellular effects of monohydrochloride of L-arginine, N-lauroyl ethylester (LAE) on exposure to Salmonella typhimurium and Staphylococcus aureus

AIMS: Here we study the effect of monohydrochloride of L-arginine, N(alpha)-lauroyl ethylester (LAE), a cationic preservative derived from lauric acid and arginine, on the cell envelopes of Salmonella typhimurium and Staphylococcus aureus at sub-lethal concentration such as their respective minimal inhibitory concentrations, 32 and 8 microg ml(-1), respectively. METHODS AND RESULTS: Bacterial populations were studied by using transmission electron and fluorescence microscopy (TEM and FM), flow cytometry (FC) and ion-flux across the cellular membrane. Cell integrity was altered mainly in the outer membrane of S. typhimurium, but there was no significant change in the cytoplasm. However, in Staph. aureus, clear zones, abnormal septation and mesosome-like structures were observed in the cytoplasm. Bacterial populations were double-stained with propidium iodide (PI) and SYTO-13 for FC analysis. In S. typhimurium the proportion of damaged cells after 24 h was 97% and in Staph. aureus 56.3%. LAE induced transmembrane ion flux, the increase of potassium leakage after 30 min of contact was 7.7 and 3.34 microg ml(-1) for Staph. aureus and S. typhimurium, respectively. Membrane disruption was detected by measuring the proton flow across the membrane. CONCLUSIONS: Disturbance in membrane potential and structural changes was caused by LAE, although cells were not disrupted. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time the cellular effects of LAE on bacterial cells were studied.     

Fabrication of silver nanoparticles by Phoma glomerata and its combined effect against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus

Aims:  We report extracellular synthesis of silver nanoparticles (Ag-NPs) from Phoma glomerata and its efficacy against Escherichia coli , Staphylococcus aureus and Pseudomonas aeruginosa . The bacteria exhibiting resistance to various antibiotics showed remarkable sensitivity, when used in combination of antibiotics and Ag-NPs.
Methods and Results:  Biosynthesis of Ag-NPs was carried out by challenging the fungal cell filtrate with 1 mmol l⁻¹ silver nitrate. The Ag-NPs were characterized with the help of UV–Visible spectrophotometer and Fourier transform infrared spectroscopy. Scanning electron microscopy was carried out to detect the size of Ag-NPs. Evaluation of the combined effect(s) was studied by disc diffusion method against E. coli , Staph. aureus and Ps. aeruginosa .
Conclusions:  The biosynthesis route seems to be eco-friendly and easy to scale up the process. Thus, these Ag-NPs may prove as a better candidate for drugs and can potentially eliminate the problem of chemical agents because of their biogenic nature.
Significance and Impact of the Study:  The bacterial resistance against antibiotics has been increasing with alarming rate. To overcome this problem, there is a pressing need to develop bactericidal agents. Ag-NPs may prove to be an answer to drug-resistant bacteria.     

How much ozone bactericidal activity is compromised by plasma components?

Aims:  Evaluation of bactericidal effect of different concentrations of ozone when used (a) as a gas, or (b) dissolved in saline. The addition of hydrogen peroxide or 4-hydroxynonenal dissolved in saline was also tested, as well as the effect of human plasma.
Methods and Results:  Staphylococcus aureus , methicillin-resistant Staph. aureus (MRSA), and Pseudomonas aeruginosa , suspended in their culture media were tested. While all bacteria suspended in protein-free saline were killed at high ozone concentrations, they survived when as little as 5% human plasma was present. Hydrogen peroxide was 100-fold less active than ozone and needed to remain in contact with bacteria for at least 60 min. 4-hydroxynonenal (2 μmol l⁻¹) was inhibitory for proliferation of both Staph. aureus and MRSA, but not for Ps. aeruginosa .
Conclusions:  Ozone and the cascade of its derivative products are potent bactericidal agents, but even small amounts of human plasma, hence of hydro- and liposoluble antioxidants, in bacterial suspensions inhibit oxidation and protect bacteria.
Significance and Impact of the Study:  Any substantial in vivo cytocidal effect of ozone and its derivatives can be excluded. On the other hand, topical and continuous action of various ozone preparations remains valuable in a variety of skin and mucosal infections.     

Comparison of bacteriostatic and bactericidal activity of 13 essential oils against strains with varying sensitivity to antibiotics

Aims:  To compare the bacteriostatic and bactericidal activity of 13 chemotyped essential oils (EO) on 65 bacteria with varying sensitivity to antibiotics.
Methods and Results:  Fifty-five bacterial strains were tested with two methods used for evaluation of antimicrobial activity (CLSI recommendations): the agar dilution method and the time-killing curve method. EO containing aldehydes ( Cinnamomum verum bark and Cymbopogon citratus ), phenols ( Origanum compactum , Trachyspermum ammi , Thymus satureioides , Eugenia caryophyllus and Cinnamomum verum leaf) showed the highest antimicrobial activity with minimum inhibitory concentration (MIC) <2% (v/v) against all strains except Pseudomonas aeruginosa . Alcohol-based EO ( Melaleuca alternifolia , Cymbopogon martinii and Lavandula angustifolia ) exhibited varying degrees of activity depending on Gram status. EO containing 1·8-cineole and hydrocarbons ( Eucalyptus globulus , Melaleuca cajeputii and Citrus sinensis ) had MIC_90% ≥ 10% (v/v). Against P. aeruginosa , only C. verum bark and O. compactum presented MIC ≤2% (v/v). Cinnamomum verum bark, O. compactum , T. satureioides , C. verum leaf and M. alternifolia were bactericidal against Staphylococcus aureus and Escherichia coli at concentrations ranging from to 0·31% to 10% (v/v) after 1 h of contact. Cinnamomum verum bark and O. compactum were bactericidal against P. aeruginosa within 5 min at concentrations <2% (v/v).
Conclusions:  Cinnamomum verum bark had the highest antimicrobial activity, particularly against resistant strains.
Significance and Impact of the Study:  Bacteriostatic and bactericidal activity of EO on nosocomial antibiotic-resistant strains.     

Comparative analysis of antibiotic and antimicrobial biocide susceptibility data in clinical isolates of methicillin-sensitive Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa between 1989 and 2000

AIMS: To analyse population minimum inhibitory concentrations (MICs) data from clinical strains of Staphylococcus aureus and Pseudomonas aeruginosa for changes over a 10-year period and to look for correlations between the antimicrobials tested. METHODS AND RESULTS: Data from the MIC study of 256 clinical isolates of Staph. aureus [169 methicillin-sensitive Staph. aureus (MSSA), 87 methicillin-resistant Staph. aureus (MRSA)] and 111 clinical isolates of Ps. aeruginosa against eight antimicrobial biocides and several clinically relevant antibiotics was analysed using anova, Spearman-Rho correlation and principal component analysis. Comparisons suggest that alterations in the mean susceptibility of Staph. aureus to antimicrobial biocides have occurred between 1989 and 2000, but that these changes were mirrored in MSSA and MRSA suggests that methicillin resistance has little to do with these changes. Between 1989 and 2000 a sub-population of MRSA has acquired a higher resistance to biocides, but this has not altered the antibiotic susceptibility of that group. In both Staph. aureus and Ps. aeruginosa several correlations (both positive and negative) between antibiotics and antimicrobial biocides were found. CONCLUSIONS: From the analyses of these clinical isolates it is very difficult to support a hypothesis that increased biocide resistance is a cause of increased antibiotic resistance either in Staph. aureus or in Ps. aeruginosa. SIGNIFICANCE AND IMPACT OF THE STUDY: The observation of negative correlations between antibiotics and biocides may be a useful reason for the continued use of biocides promoting hygiene in the hospital environment.     

Activity and mechanisms of action of selected biocidal agents on Gram-positive and -negative bacteria

AIMS: This study investigates the antimicrobial activity and mode of action of two natural products, eugenol and thymol, a commonly utilized biostatic agent, triclocarban (TCC), and two surfactants, didecyldimethylammonium chloride (DDDMAC) and C10-C16 alkyldimethyl amine N-oxides (ADMAO). METHODS AND RESULTS: Methods used included: determination of minimum inhibitory concentrations (MICs), lethal effect studies with suspension tests and the investigation of sub-MIC concentrations on growth of E. coli, Staph. aureus and Ps. aeruginosa using a Bioscreen microbiological analyser. Leakage of intracellular constituents and the effects of potentiating agents were also investigated. Only DDDMAC was bactericidal against all of the organisms tested. Eugenol, thymol and ADMAO showed bacteriostatic and bactericidal activity, but not against Ps. aeruginosa. TCC was only bacteristatic against Staph. aureus, but like the other agents, it did affect the growth of the other organisms in the Bioscreen experiments. All of the antimicrobial agents tested were potentiated by the permeabilizers to some extent and leakage of potassium was seen with all of the agents except TCC. CONCLUSIONS: DDDMAC was bactericidal against all organisms tested and all compounds had some bacteriostatic action. Low level static effects on bacterial growth were seen with sub-MIC concentrations. Membrane damage may account for at least part of the mode of action of thymol, eugenol, DDDMAC and ADMAO. SIGNIFICANCE AND IMPACT OF THE STUDY: The ingredients evaluated demonstrated a range of bactericidal and bacteriostatic properties against the Gram-negative and -positive organisms evaluated and the membrane (leakage of intracellular components) was implicated in the mode of action for most (except TCC). Sub-MIC levels of all ingredients did induce subtle effects on the organisms which impacted bacterial growth, even for those which had no true inhibitory effects.     

Inhibition of quorum sensing regulated bacterial functions by plant essential oils with special reference to clove oil

Aims:  To evaluate quorum sensing (QS) inhibitory activity of plant essential oils using strains of Chromobacterium violaceum (CV12472 and CVO26) and Pseudomonas aeruginosa (PAO1).
Methods and Results:  Inhibition of QS-controlled violacein production in C. violaceum was assayed using disc diffusion and agar well diffusion method. Of the 21 essential oils, four oils showed varying levels of anti-QS activity. Syzygium aromaticum (Clove) oil showed promising anti-QS activity on both wild and mutant strains with zones of pigment inhibition 19 and 17 mm, respectively, followed by activity in cinnamon, lavender and peppermint oils. The effect of clove oil on the extent of violacein production was estimated photometrically and found to be concentration dependent. At sub-MICs of clove oil, 78·4% reduction in violacein production over control and up to 78% reduction in swarming motility in PAO1 over control were recorded. Gas chromatography–mass spectrometry analysis of clove oil indicated presence of many phytocompounds. Eugenol, the major constituent of clove oil could not exhibit anti-QS activity.
Conclusions:  Presence of anti-QS activity in clove oil and other essential oils has indicated new anti-infective activity. The identification of anti-QS phytoconstituents is needed to assess the mechanism of action against both C. violaceum and Ps. aeruginosa .
Significance and Impact of the study:  Essential oils having new antipathogenic drugs principle because of its anti-QS activity might be important in reducing virulence and pathogenicity of drug-resistant bacteria in vivo .     

Effectiveness of cleaning techniques used in the food industry in terms of the removal of bacterial biofilms

The effectiveness of cleaning was investigated through food factory trials and laboratory experiments using a naturally occurring biofilm from a food factory environment and generated biofilms. The efficacy of factory cleaning and disinfection programmes was assessed by swabbing and total viable count (TVC) analysis of surfaces before cleaning, after cleaning and after disinfection. Cleaning produced a 0.91 log reduction in the attached population. Investigation of the effectiveness of a variety of cleaning methods in the removal of a naturally occurring food factory biofilm showed that the high pressure spray and the mechanical floor scrubber, which use a high degree of mechanical action, were most effective. Cleaning trials with biofilms of Pseudomonas aeruginosa or Staphylococcus aureus showed that spraying with water at pressures of 34.5, 51.7 and 68.9 bar did not significantly increase the removal, as assessed by direct epifluorescent microscopy (DEM) and swabbing and TVC analysis, beyond the three log reduction observed at 17.2 bar. The effect of spray time at 17.2 bar showed that increasing spray time from 1 to 10 s did not significantly increase removal of Ps. aeruginosa biofilm. Investigation of the optimum distance of the spray lance from the surface at 17.2 bar was found to be between 125 and 250 mm. The use of an alkaline, acidic or neutral detergent prior to spraying with water at 17.2 bar did not significantly increase the removal of Ps. aeruginosa or Staph. aureus. However, the acidic and alkaline products significantly (P = 0.05) affected the viability of Staph. aureus and Ps. aeruginosa, respectively, thereby minimizing the potential for the spread of contamination.     

Decontamination of dental unit water systems with hydrogen peroxide

AIMS: Transmission of microbial pathogens to patients from water in dental units is a concern. To reduce this risk, the decontaminating efficiency of hydrogen peroxide was evaluated. METHODS AND RESULTS: Three percent hydrogen peroxide diluted 1 : 4 in distilled water (contact time 15 min) was used daily to disinfect the waterlines of a pilot unit previously contaminated with Pseudomonas aeruginosa or Staphylococcus aureus. The behaviour of the test bacteria was seen to differ over time. Staph. aureus numbers slowly decreased until only low numbers were recovered, after which the levels remained stable. Ps. aeruginosa abatement was more rapid and the density of the bacteria reached a peak when the circuit was empty. CONCLUSIONS: Staph. aureus and Ps. aeruginosa treated with hydrogen peroxide fell from 6 to 4 log. SIGNIFICANCE AND IMPACT OF THE STUDY: Treatment of dental unit waterlines with hydrogen peroxide was seen to be able to keep the number of the bacteria under control, as long as the treatment was repeated daily.     

Effects of ozone on membrane permeability and ultrastructure in Pseudomonas aeruginosa

Aims: To examine the mechanism of ozone‐induced damage to cytoplasmic membrane and cell ultrastructure of Pseudomonas aeruginosa ATCC27853. Methods and Results: Cell suspensions of Ps. aeruginosa ATCC27853 were treated with ozonated water. The leakages of cellular potassium (K⁺), magnesium (Mg²⁺) and adenosine triphosphate (ATP), determined by inductively coupled plasma/mass spectrometry (ICP/MS) and a commercial bioluminescence assay kit, were to assess ozone‐induced damage to the cytoplasmic membrane. Maximum leakages of K⁺ and Mg²⁺ were attained, respectively, at 0·53 mg l^?1 ozone after 0·5 and 2 min with >99% inactivation of culturable bacteria, while that of ATP was achieved at 0·67 mg l^?1 ozone after 1 min. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) revealed that treated cells retained intact shapes and cytoplasm agglutinations and vacuoles occurred. Conclusions: Ozone inactivates Ps. aeruginosa ATCC27853 by the combined results of increased cytoplasmic membrane permeability and cytoplasm coagulation, rather than by severe membrane disruption and cell lysis. Significance and Impact of the Study: Pseudomonas aeruginosa is a common water‐related pathogen. These insights into the leakage of cytoplasmic components and ultrastructural changes provide evidence for the mechanisms of ozone‐mediated inactivation.     

The mode of antimicrobial action of the essential oil of Melaleuca alternifolia (tea tree oil)

The essential oil of Melaleuca alternifolia (tea tree) exhibits broad-spectrum antimicrobial activity. Its mode of action against the Gram-negative bacterium Escherichia coli AG100, the Gram-positive bacterium Staphylococcus aureus NCTC 8325, and the yeast Candida albicans has been investigated using a range of methods. We report that exposing these organisms to minimum inhibitory and minimum bactericidal/fungicidal concentrations of tea tree oil inhibited respiration and increased the permeability of bacterial cytoplasmic and yeast plasma membranes as indicated by uptake of propidium iodide. In the case of E. coli and Staph. aureus, tea tree oil also caused potassium ion leakage. Differences in the susceptibility of the test organisms to tea tree oil were also observed and these are interpreted in terms of variations in the rate of monoterpene penetration through cell wall and cell membrane structures. The ability of tea tree oil to disrupt the permeability barrier of cell membrane structures and the accompanying loss of chemiosmotic control is the most likely source of its lethal action at minimum inhibitory levels.     

Bactericidal and sporicidal performance of a polymer-encapsulated chlorine dioxide-coated surface

Aims:  To investigate the physical characteristics and the bactericidal and sporicidal potential of a polymer-encapsulated ClO₂ coating.
Methods and Results:  An antimicrobial coating based on polymer-encapsulated ClO₂ was developed. A low viscosity, water/oil/water double emulsion coating was formulated for easy on-site application. Escherichia coli , Pseudomonas aeruginosa , Bacillus subtilis and Staphylococcus aureus were applied onto the coating to study the bactericidal capabilities of the coating. The bactericidal performance of the coating increased when the contact time with the tested bacteria increased. Over 99% of the E. coli , Ps. aeruginosa , B. subtilis were killed with a contact time of 30 min. Although endospores of B. subtilis are more resistant, about 75% of the spores were killed after 72 h on the coating. Moreover, a sustained release of gaseous ClO₂ was achieved to maintain about 90% removal of B. subtilis with a 10-min contact time during a 28-day study period. The coating also exhibits antiadhesive properties against bacteria.
Conclusions:  A polymer-encapsulated ClO₂ coating with sustained release of ClO₂ and promising bactericidal and sporicidal features was tested for 28 days.
Significance and Impact of the Study:  This study provides a new direction for developing polymer-encapsulated ClO₂ coatings that possess persistent bactericidal and sporicidal properties.     

In vitro antibacterial activity and antifungal mode of action of flocculosin, a membrane-active cellobiose lipid

Aims:  To investigate the in vitro antibacterial activity and antifungal mode of action of flocculosin, a cellobiose lipid produced by Pseudozyma flocculosa .
Methods and Results:  When tested against clinical bacterial isolates, the compound was particularly active against Gram-positive bacteria and its effect was not mitigated against isolates known as resistant to other antibiotics. The antifungal activity of flocculosin was found to be rapid and concentration-dependent. At lethal concentrations against Candida albicans , flocculosin caused a rapid leakage of intracellular potassium and inhibited acidification of the medium by plasma membrane ATPases suggesting a physical rather than a biochemical effect. TEM observations of cells exposed 6 h to flocculosin revealed disrupted membranes and disorganized mitochondria.
Conclusions:  Data obtained in this study confirm that flocculosin acts by disrupting the membrane surface of sensitive micro-organisms.
Significance and Impact of the Study:  The elucidation of an antifungal mode of action of flocculosin can be exploited in furthering its antimicrobial potential against fungi and bacteria whose cell membranes are particularly sensitive to the action of the molecule.     

Inhibition spectrum studies of microthecin and other anhydrofructose derivatives using selected strains of Gram-positive and -negative bacteria, yeasts and moulds, and investigation of the cytotoxicity of microthecin to malignant blood cell lines

Aims:  To prepare 1,5-anhydro- d -fructose (AF) derivatives, test their microbial inhibition spectrum, and to further examine the most effective AF derivative against Pseudomonas aeruginosa and malignant blood cell lines.
Methods and Results:  Microthecin and nine other AF derivatives were synthesized from AF. The 10 compounds were tested in vitro against Gram-positive (GP) and Gram-negative (GN) bacteria, yeasts and moulds using a well diffusion method and in a Bioscreen growth analyser. Of the test compounds, microthecin exhibited the most significant antibacterial activity at 100–2000 ppm against both GP and GN bacteria, including Ps. aeruginosa. Further tests with three malignant blood cell lines ( Mutu, Ramos, Raji ) and one normal cell line indicated that microthecin was a cell toxin, with a cell mortality >85% at 50 ppm. The other nine AF derivatives demonstrated low or no antimicrobial activity.
Conclusions:  Microthecin was active 100–2000 ppm against GP and GN bacteria including Ps. aeruginosa , but was inactive against yeasts and moulds. Microthecin was also a cytotoxin to some mammalian cell lines.
Significance and Impact of the Study:  Microthecin might have potential for development as a novel drug against Ps. aeruginosa and to target cancer cells. It might also be developed as a food processing aid to control bacterial growth.     

Bacterial contamination on touch surfaces in the public transport system and in public areas of a hospital in London

Aims:  To investigate bacterial contamination on hand-touch surfaces in the public transport system and in public areas of a hospital in central London.
Methods and Results:  Dipslides were used to sample 118 hand-touch surfaces in buses, trains, stations, hotels and public areas of a hospital in central London. Total aerobic counts were determined, and Staphylococcus aureus isolates were identified and characterized. Bacteria were cultured from 112 (95%) of sites at a median concentration of 12 CFU cm⁻². Methicillin-susceptible Staph. aureus (MSSA) was cultured from nine (8%) of sites; no sites grew methicillin-resistant Staph. aureus (MRSA).
Conclusions:  Hand-touch sites in London are frequently contaminated with bacteria and can harbour MSSA, but none of the sites tested were contaminated with MRSA.
Significance and Impact of the Study:  Hand-touch sites can become contaminated with staphylococci and may be fomites for the transmission of bacteria between humans. Such sites could provide a reservoir for community-associated MRSA (CA-MRSA) in high prevalence areas but were not present in London, a geographical area with a low incidence of CA-MRSA.     

Destruction of Bacillus licheniformis spores by microwave irradiation

Aims:  To investigate the sporicidal mechanisms of microwave irradiation on Bacillus licheniformis spores.
Methods and Results:  We measured spore viability and the release of DNA and proteins, and performed transmission electron microscopy (TEM). A microwave oven (0·5 kW) was modified to output power at 2·0 kW, which allowed a shorter sterilization cycle. A 2·0 kW microwave treatment at the boiling temperature for 1 min did not kill all spores, but killed most spores. The spore inactivation rate was faster than that of boiling and 0·5 kW microwave oven. In contrast to boiling and 0·5 kW microwave treatments, the 2·0 kW microwave resulted in significant leakage of proteins and DNA from spores due to injury to the spore structure. TEM revealed that 2·0 kW microwave irradiation affected spore cortex hydrolysis and swelling, and ruptured the spore coat and inner membrane.
Conclusions:  These results suggest that 2·0 kW microwave irradiation ruptures the spore coat and inner membrane, and is significantly different from boiling.
Significance and Impact of the Study:  This study provides information on the sporicidal mechanisms of microwave irradiation on B. licheniformis spores.     

Antibiotic activity of isoxazolylnaphthoquinone imines on mice infected with Staphylococcus aureus

I. ALBESA, P. BOGDANOV, A. ERASO, N.R. SPERANDEO AND M.M. DE BERTORELLO. 1995. The antibiotic activity of new synthetic isoxazolylnaphthoquinone imines was studied. Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 were resistant to the four compounds studied (MIC > 128 µg ml⁻¹), but Staphylococcus aureus ATCC 25923, ATCC 29213 and 30 clinical isolates of Staph. aureus were inhibited by 2-hydroxy- N -(3,4-dimethyl-5-isoxazolyl)-1,4-naphthoquinone-4-imine (I). This compound diminished bloodstream infection of mice injected i.m. with Staph. aureus; septicaemia decayed significantly when I was applied at the beginning of the infection while when I was given 3 d after bacterial challenge, a significant protection was afforded. Bactericidal activity in serum increased during the 5 h after I was administered i.p.
The acetyl derivative of I had a high MIC but when inoculated orally in mice decreased the Staph. aureus counts in circulation. This protection occurred only when the schedule of administration started close to the bacterial challenge. Antibiotic activity in vivo may be associated with in vitro effects.     

Plasma membrane damage to Candida albicans caused by chlorine dioxide (ClO2)

Aim:  To investigate the plasma membrane damage of chlorine dioxide (ClO₂) to Candida albicans ATCC10231 at or below the minimal fungicidal concentration (MFC).
Methods and Results:  ClO₂ at MFC or below was adopted to treat the cell suspensions of C. albicans ATCC10231. Using transmission electron microscopy, no visible physiological alteration of cell shape and plasma membrane occurred. Potassium (K⁺) leakages were significant; likewise, it showed time- and dose-dependent increases. However, adenosine triphosphate (ATP) leakages were very slight. Research shows that when 99% of the cells were inactivated, the leakage was measured at 0·04% of total ATP. Compared with the mortality-specific fluorescent dye of DiBAC₄(3), majority of the inactivated cells were poorly stained by propidium iodide, another mortality-specific fluorescent dye which can be traced by flow cytometry.
Conclusion:  At or below MFC, ClO₂ damages the plasma membranes of C. albicans mainly by permeabilization, rather than by the disruption of their integrity. K⁺ leakage and the concomitant depolarization of the cell membrane are some of the critical events.
Significance and Impact of the Study:  These insights into membrane damages are helpful in understanding the action mode of ClO₂.