An in vivo method for assessing the antimicrobial properties of topical antiseptics using artifically contaminated skin

A method is described for assessing the antimicrobial activity of topical antiseptics on human skin. The test, carried out on the forearms of volunteers, uses an inoculum of Stuphylococcus aureus . Up to four preparations may be tested at one time plus a placebo and untreated control site. The method has been used to compare four proprietary antiseptics and to evaluate their activity for up to one hour and has demonstrated a chloroxylenol, triclosan, EDTA combination product to be the most effective. The method is highly adaptable and may be used to evaluate activity versus other organisms and to examine other factors affecting antiseptic efficacy     

In vivo standardization of cutaneous bactericidal activity of antiseptics by using monoxenic hairless mice.

This study was designed to investigate the bactericidal activities of antiseptics on the cutaneous flora of hairless mice monoxenic to Staphylococcus epidermidis, Staphylococcus aureus, or Pseudomonas aeruginosa in vivo. A standardized method for testing such antiseptics to compare their bactericidal effectiveness in humans in described. Seven antiseptics belonging to seven different chemical groups (iodine derivatives, alcohols, mercury compounds, quaternary ammonium salts, biguanides, phenols, and carbanilides) were used as recommended by the manufacturers (conditions of contact or prolonged contact time followed by washing with distilled water). Germfree hairless mice were infected with various bacterial strains by gastric intubation, producing levels of about 10(3) CFU/cm2 of skin. The antiseptic under test was placed on the right or left side of the ventral region. The contralateral side served as a control. In order to standardize the method, a number of crucial parameters were carefully controlled: the amount of antiseptic applied, the area of skin treated, the duration of treatment, and the washing procedure. Skin samples were obtained by cutaneous biopsy, which effectively removed all the bacteria along with the sample. The bacterial populations were counted before and after application of the antiseptic. Reductions of between 0.5 and 1.9 log units were obtained; these are comparable to those observed in humans. The standardization of our procedure and the use of animals with a strictly controlled flora eliminated much of the variability and sources of error inherent in human studies. This model could be of value for the study of resistant bacterial strains responsible for nosocomial infections and for investigations of damaged skin.     

Comparison of tolerance of four bacterial nanocellulose-producing strains to lignocellulose-derived inhibitors

Background

Through pretreatment and enzymatic saccharification lignocellulosic biomass has great potential as a low-cost feedstock for production of bacterial nanocellulose (BNC), a high value-added microbial product, but inhibitors formed during pretreatment remain challenging. In this study, the tolerance to lignocellulose-derived inhibitors of three new BNC-producing strains were compared to that of Komagataeibacter xylinus ATCC 23770. Inhibitors studied included furan aldehydes (furfural and 5-hydroxymethylfurfural) and phenolic compounds (coniferyl aldehyde and vanillin). The performance of the four strains in the presence and absence of the inhibitors was assessed using static cultures, and their capability to convert inhibitors by oxidation and reduction was analyzed.

Results

Although two of the new strains were more sensitive than ATCC 23770 to furan aldehydes, one of the new strains showed superior resistance to both furan aldehydes and phenols, and also displayed high volumetric BNC yield (up to 14.78 ± 0.43 g/L) and high BNC yield on consumed sugar (0.59 ± 0.02 g/g). The inhibitors were oxidized and/or reduced by the strains to be less toxic. The four strains exhibited strong similarities with regard to predominant bioconversion products from the inhibitors, but displayed different capacity to convert the inhibitors, which may be related to the differences in inhibitor tolerance.

Conclusions

This investigation provides information on different performance of four BNC-producing strains in the presence of lignocellulose-derived inhibitors. The results will be of benefit to the selection of more suitable strains for utilization of lignocellulosics in the process of BNC-production.

     

Comparison of mechanical properties of four large, wave-exposed seaweeds

Seaweeds have a simple structural design compared to most terrestrial plants. Nonetheless, some species have adapted to the severe mechanical conditions of the surf zone. The material properties of either tissue sections or the whole stipe of four wave-exposed seaweeds, Durvillaea antarctica, D. willana, Laminaria digitata, and L. hyperborea, were tested in tension, bending, and torsion. Durvillaea has a very low modulus of elasticity in tension (E(tension) = 3-7 MN·m(-2)) and in bending (E(bending) = 9-12 MN · m(-2)), torsion modulus (G = 0.3 MN · m(-2)) and strength (σ(b)rk = 1-2 MN · m(-2)), combining a compliable and twistable stipe "material" with a comparatively high breaking strain (ε(brk) = 0.4-0.6). In comparison, the smaller stipes of Laminaria have a higher modulus of elasticity in tension (E(tension) = 6-28 MN·m(-2)) and in bending (E(bending) = 84-109 MN·m(-2)), similar strength (σ(brk) = 1-3 MN·m(-2)), and a higher torsion modulus (G = 0.7-10 MN·m(-2)), combined with a lower breaking strain (ε(brk) = 0.2-0.3) than Durvillaea. Time-dependent, viscoelastic reactions were investigated with cycling tests. The tested species dissipated 42-52% of the loading energy in tension through plastic-viscoelastic processes, a finding that bears important ecological implications. Overall, there seems to be no correlation between single material properties and the size or habitat position of the tested seaweed species.     

Comparison of the bactericidal activity of different vertebrate sera

Schwab, G. E. (University of Adelaide, Adelaide, South Australia), and P. R. Reeves. Comparison of the bactericidal activity of different vertebrate sera. J. Bacteriol. 91:106-112. 1966.-The bactericidal activity for gram-negative bacteria of normal sera from eight species of vertebrates was investigated to compare complement-mediated killing in sera from animals representing various classes of vertebrates. Although all the sera were bactericidal, there was considerable variation in the range of bacterial species killed and in the bactericidal titers. The temperature dependence of the bactericidal and hemolytic complement activities was also studied. The curves relating activity to temperature were similar in shape for sera from a homeotherm and poikilotherms, but those for the homeotherm reached their maximum at temperatures of 5 to 10 C higher than the poikilotherms. The role of lysozyme and complement in killing rough gram-negative bacteria was examined, and the results suggest that, as for smooth organisms, killing is due to antibody plus complement. These natural antibodies, like those for smooth strains, were shown to be specific.     

Effect of suramin on pinocytosis by resident rat peritoneal macrophages: an analysis using four different substrates

The effect of suramin on pinocytosis and intralysosomal proteolysis by resident rat peritoneal macrophages cultured in vitro has been studied. Suramin had little effect on the rate of pinocytic uptake of two non-adsorptive substrates [14C]sucrose and [3H]dextran, but unexpectedly enhanced uptake of a third, 125I-labelled polyvinylpyrrolidone (PVP). Since this enhanced uptake was completely abolished by NaF at a concentration known to inhibit pinocytosis, it clearly represented an increased internalization of substrate and not merely a superficial binding to the cell surface. It was concluded that suramin (i) does not affect the rate of formation of pinocytic vesicles but (ii) acts as a bivalent ligand, binding to both the macrophage surface and the 125I-labelled polyvinylpyrrolidone, thus converting a non-adsorptive into an adsorptive substrate. Suramin (500 micrograms/ml) decreased both the rate of uptake of formaldehyde-denatured 125I-labelled bovine serum albumin (BSA) (an adsorptive substrate) and the rate of its subsequent intracellular degradation. Thus, depending on the substrate chosen to measure pinocytosis, the same modifier may stimulate or inhibit uptake or be without effect.     

Redox properties of several bacterial ferredoxins using square wave voltammetry

The equilibrium reduction potential of the 2[4Fe-4S] ferredoxin (Fd) isolated from four different bacterial strains was determined at a methyl viologen-modified gold electrode using square wave voltammetry. The observed reduction potential at pH 8 for Clostridium thermoaceticum Fd was -385 mV; Clostridium pasteurianum, -393 mV; Clostridium thermosaccharolyticum, -408 mV; and Chromatium vinosum, -460 mV versus normal hydrogen electrode at 25 degrees C. The reduction potential of the C. pasteurianum Fd was found to be pH independent from pH 6.4 to 8.7, indicating that the electron transfer mechanism does not involve proton exchange. In contrast, the reduction potential of the C. thermosaccharolyticum Fd was found to be pH dependent from pH 6.4 to 8.7, with pKox approximately 7 and pKred approximately 7.5. The +30 mV change in reduction potential from pH 8.7 to 6.4 was attributed to an electrostatic interaction between the iron-sulfur cluster II and the protonated histidine 2 residue located about 6 A away. The Ch. vinosum Fd interacted reversibly at the methyl viologen-modified gold electrode, and its reduction potential was verified using visible spectroelectrochemistry. The reduction potential of Ch. vinosum Fd was found to be 30 mV more positive than previously reported. The similarities of the bacterial Fd reduction potentials are discussed in terms of the homology of their primary structure as reflected by the similarities in the visible and circular dichroic spectra.     

Measuring the rate of conjugal plasmid transfer in a bacterial population using quantitative PCR

Horizontal transfer of genes between species is an important mechanism for bacterial genome evolution. In Escherichia coli, conjugation is the transfer from a donor (F⁺) to a recipient (F⁻) cell through cell-to-cell contact. We demonstrate what we believe to be a novel qPCR method for quantifying the transfer kinetics of the F plasmid in a population by enumerating the relative abundance of genetic loci unique to the plasmid and the chromosome. This approach allows us to query the plasmid transfer rate without the need for selective culturing with unprecedented single locus resolution. We fit the results to a mass action model where the rate of plasmid growth includes the lag time of newly formed F⁺ transconjugants and the recovery time between successive conjugation events of the F⁺ donors. By assaying defined mixtures of genotypically identical donor and recipient cells at constant inoculation densities, we extract an F plasmid transfer rate of 5 × 10⁻¹⁰ (cells/mL · min)⁻¹. We confirm a plasmid/chromosome ratio of 1:1 in homogenous F⁺ populations throughout batch growth. Surprisingly, in some mixture experiments we observe an excess of F plasmid in the early saturation phase that equilibrates to a final ratio of one plasmid per chromosome.     

A quantitative test of population genetics using spatiogenetic patterns in bacterial colonies

It is widely accepted that population-genetics theory is the cornerstone of evolutionary analyses. Empirical tests of the theory, however, are challenging because of the complex relationships between space, dispersal, and evolution. Critically, we lack quantitative validation of the spatial models of population genetics. Here we combine analytics, on- and off-lattice simulations, and experiments with bacteria to perform quantitative tests of the theory. We study two bacterial species, the gut microbe Escherichia coli and the opportunistic pathogen Pseudomonas aeruginosa, and show that spatiogenetic patterns in colony biofilms of both species are accurately described by an extension of the one-dimensional stepping-stone model. We use one empirical measure, genetic diversity at the colony periphery, to parameterize our models and show that we can then accurately predict another key variable: the degree of short-range cell migration along an edge. Moreover, the model allows us to estimate other key parameters, including effective population size (density) at the expansion frontier. While our experimental system is a simplification of natural microbial community, we argue that it constitutes proof of principle that the spatial models of population genetics can quantitatively capture organismal evolution.     

Role of charge properties of bacterial envelope in bactericidal action of human group IIA phospholipase A2 against Staphylococcus aureus

Mammalian Group IIA phospholipases A(2) (PLA(2)) potently kill Staphylococcus aureus. Highly cationic properties of these PLA(2) are important for Ca(2+)-independent binding and cell wall penetration, prerequisites for Ca(2+)-dependent degradation of membrane phospholipids and bacterial killing. To further delineate charge properties of the bacterial envelope important in Group IIA PLA(2) action against S. aureus, we examined the effects of mutations that prevent specific modifications of cell wall (dltA) and cell membrane (mprF) polyanions. In comparison to the parent strain, isogenic dltA(-) bacteria are approximately 30-100x more sensitive to PLA(2), whereas mprF(-) bacteria are <3-fold more sensitive. Differences in PLA(2) sensitivity of intact bacteria reflect differences in cell wall, not cell membrane, properties since protoplasts from all three strains are equally sensitive to PLA(2). A diminished positive charge in PLA(2) reduces PLA(2) binding and antibacterial activity. In contrast, diminished cell wall negative charge by substitution of (lipo)teichoic acids with d-alanine reduces antibacterial activity of bound PLA(2), but not initial PLA(2) binding. Therefore, the potent antistaphylococcal activity of Group IIA PLA(2) depends on cationic properties of the enzyme that promote binding to the cell wall, and polyanionic properties of cell wall (lipo)teichoic acids that promote attack of membrane phospholipids by bound PLA(2).     

Comparison of the structural-functional properties of DNA-dependent RNA polymerase of the tularemia microbe and intestinal bacterial

The subunit compositions of RNA-polymerases from Escherichia coli and Francisella tularensis were compared. The activities of the enzymes on the corresponding chromosomal DNAs and their mixtures were defined.     

Comparison of two skin antiseptics in blood transfusion. Comparison with the results of the bacteriologic control of labile blood products

The comparison of two skin antiseptics (70 degrees alcohol and 0,5% alcoholic chlorhexidine solution) was carried out by the method of in vivo impressions. The study used 45 healthy volonteers from whom a bacteriological sample was taken from both bends of the elbow, without use of an antiseptic, and after the application of one according to usual sample taking methods. The subjects were divided into two sets according to the antiseptic being tested. The results of the cultures after 24 and 48 hours are expressed in the number of germs per cm2, which thus permits us to calculate a reduction in percentage or log 10 form, and to appreciate the efficiency of the antiseptic studied. Routine bacteriological inspection of labile blood products was carried out on products picked at random every week during 18 months of exclusive use of 70 degrees alcohol by the sample taking services. The results show a comparable in vivo effectiveness of the two antiseptics on the superficial aerobic flora of the bend of the elbow, and the ease of use of 70 degrees alcohol has led to its choice. This choice is confronted with the results of the inspection of blood products: out of 1 293 inspections, only one slight contamination (cocci gram+) was found on a unit of whole blood. These data as a whole can be compared with the work of various authors.     

Comparison of Agrobacterium-mediated transformation of four barley cultivars using the GFP and GUS reporter genes

Experiments were conducted to produce transgenic barley plants following infection of immature embryos with Agrobacterium tumefaciens. Transformed callus was obtained using hygromycin resistance as a selectable marker and either green fluorescent protein (GFP) or -glucuronidase (GUS) as a reporter. Significantly reduced plant transformation frequencies were obtained with the GFP gene compared to GUS. However, GFP proved to be an excellent reporter of early transformation events and was used to compare four barley cultivars for efficiency in two phases of transformation: the generation of stably transformed barley callus and the regeneration of plantlets from transformed callus. Transformed callus was generated at a high frequency (47–76%) in all four cultivars. Regeneration of transformed plantlets was also achieved for all four cultivars although the frequency was much higher for Golden Promise than for the other three genotypes, reiterating that genotype is an important determinant in the regenerative ability of barley. This study has demonstrated for the first time that Agrobacterium-mediated transformation can be used to transform the Australian cultivars Sloop and Chebec.Communicated by W. Harwood     

The effects of the bacterial interaction with visible-light responsive titania photocatalyst on the bactericidal performance

Bactericidal activity of traditional titanium dioxide (TiO₂) photocatalyst is effective only upon irradiation by ultraviolet light, which restricts the potential applications of TiO₂ for use in our living environments. Recently carbon-containing TiO₂ was found to be photoactive at visible-light illumination that affords the potential to overcome this problem; although, the bactericidal activity of these photocatalysts is relatively lower than conventional disinfectants. Evidenced from scanning electron microscopy and confocal Raman spectral mapping analysis, we found the interaction with bacteria was significantly enhanced in these anatase/rutile mixed-phase carbon-containing TiO₂. Bacteria-killing experiments indicate that a significantly higher proportion of all tested pathogens including Staphylococcus aureus, Shigella flexneri and Acinetobacter baumannii, were eliminated by the new nanoparticle with higher bacterial interaction property. These findings suggest the created materials with high bacterial interaction ability might be a useful strategy to improve the antimicrobial activity of visible-light-activated TiO₂.