The dependence of the antibacterial effect of the polycationic peptide warnerin on the energy state of target cells

The bactericidal effect of the polycationic peptide warnerin, produced by Staphylococcus warneri IEGM KL-1, was found to depend on the energy state of susceptible Staphylococcus epidermidis cells. The pretreatment of these cells with compounds that diminish the proton-motive force of plasma membranes enhanced cell tolerance to warnerin. The components and pH of the membrane proton potential influenced the antibacterial activity of warnerin in different ways. In particular, the antibacterial activity of warnerin decreased when the electric component of the proton-motive force of target membranes declined.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 166–171.Original Russian Text Copyright © 2005 by Korobov, Titova, Lemkina, Polyudova, Pankova.     

Characterization of Staphylococcus aureus mutants expressing reduced susceptibility to common house-cleaners

AIMS: To characterize mutants of Staphylococcus aureus expressing reduced susceptibility to house cleaners (HC), assess the impact of the alternative sigma factor SigB on HC susceptibility, and determine the MIC of clinical methicillin-resistant S. aureus (MRSA) to a HC. METHODS AND RESULTS: Susceptibility to HC, HC components, H2O2, vancomycin and oxacillin and physiological parameters were determined for HC-reduced susceptibility (HCRS) mutants, parent strain COL and COLsigB::kan. HCRS mutants selected with three HC expressed reduced susceptibility to multiple HC, HC components, H2O2 and vancomycin. Two unique HCRS mutants also lost the methicillin resistance determinant. In addition, all HCRS mutants exhibited better growth at two temperatures, and one HCRS mutant expressed reduced carotenoid production. COLsigB::kan demonstrated increased susceptibility to all HC and many HC components. sigB operon mutations were not detected in one HCRS mutant background. Of 76 clinical MRSA, 20 exhibited reduced susceptibility to a HC. CONCLUSIONS: HCRS mutants demonstrate altered susceptibility to multiple antimicrobials. While sigB is required for full HC resistance, one HCRS mechanism does not involve sigB operon mutations. Clinical MRSA expressing reduced susceptibility to a common HC were detected. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that HCRS mutants are not protected against, nor selected by, practical HC concentrations.     

Dissimilatory Fe(III) reduction by an electrochemically active lactic acid bacterium phylogenetically related to Enterococcus gallinarum isolated from submerged soil

AIMS: The isolation and identification of a glucose-oxidizing Fe(III)-reducing bacteria (FRB) with electrochemical activity from an anoxic environment, and characterization of the role of Fe(III) in its metabolism. METHODS AND RESULTS: A Gram-positive (Firmicutes), nonmotile, coccoid and facultative anaerobic FRB was isolated based on its ability to reduce Fe(III). Using the Vitek Gram-positive identification card kit and 16S rRNA gene sequence analysis, the isolate was identified as Enterococcus gallinarum, designated strain MG25. On glucose this isolate produced lactate plus small amounts of acetate, formate and CO2 and its growth rates were similar in the presence and absence of Fe(O)OH. These results suggest that MG25 can couple glucose oxidation to Fe(III) reduction, but without conservation of energy to support growth. Cyclic voltammetry showed that strain MG25 was electrochemically active. CONCLUSIONS: An electrochemically active and FRB, E. gallinarum MG25, was isolated from submerged soil. Fe(III) is used in the bacterial metabolism as an electron sink. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report concerning the electrochemical activity of glucose-oxidizing FRB, E. gallinarum. This organism and others like it could be used as new biocatalysts to improve the performance of a mediator-less microbial fuel cell.     

An investigation of the thermal inactivation of Staphylococcus aureus and the potential for increased thermotolerance as a result of chilled storage

AIMS: The aims of this study were; (i) to provide thermal inactivation data for Staphylococcus aureus; (ii) to examine the kinetics, including decimal reduction times (D-value) and rate constants (k), that describe the thermal inactivation of Staph. aureus and to compare two different methods of calculating D-values and (iii) to determine whether or not chilled storage would toughen these microorganisms resulting in increased thermotolerance. METHODS AND RESULTS: Isolates of Staph. aureus recovered from domestic refrigerators were grown in shaken culture for 8 h at 37 degrees C, recovered and washed by centrifugation and combined to form a cocktail of five strains. Samples from this cocktail were (a) heat treated at 50, 55 and 60 degrees C or (b) held under simulated domestic refrigeration conditions for 72 h and then heat treated as above. The numbers of Staph. aureus in heat treated and chill held, heat treated samples were enumerated by direct selective plating onto Baird Parker Agar (BPA) and recovery plating on Tryptone Soya Agar (TSA) subsequently overlaid with BPA. D-values were obtained using two different methods both of which may be used when the thermal inactivation follows first order kinetics. In the first method D-values are obtained by plotting the Log(10) of the surviving cells against time and using the equation D = -1/slope. The second method uses the rate constant (k) which is obtained from the slope of a plot of ln N/N(0)vs time and D is obtained using the equation D = 2.303 k(-1). D(50), D(55) and D(60) values ranged from 94.3 to 127.9 min, 13 to 21.7 min and 4.8 to 6.5 min. Prechilling did not enhance thermal resistance. The method of calculation did not affect the D-values obtained because the thermal inactivation of Staph. aureus in this study followed first order kinetics with r(2) values of 0.91-0.99. CONCLUSIONS: The thermal inactivation of Staph. aureus in tryptone soya broth (TSB) follows first order kinetics and in general chilling of these bacteria does not increase the resistance to thermal destruction during subsequent thermal processes such as cooking. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides much needed data on the thermal resistance of Staph. aureus and validates chilling as a food storage activity which does not cause toughening of the microorganisms to subsequent cooking. However, the data generated strongly suggests that Staph. aureus is more thermotolerant than Listeria monocytogenes and should be used as the target microorganism in designing mild thermal treatments for food, in which case the current recommendations for pasteurization (70 degrees C for 2 min, minimum) should be revised.     

Dissemination and survival of non-indigenous bacterial genomes in pristine Antarctic environments

Continental Antarctic is perceived as a largely pristine environment, although certain localized regions (e.g., parts of the Ross Dependency Dry Valleys) are relatively heavy impacted by human activities. The procedures imposed on Antarctic field parties for the handling and disposal of both solid and liquid wastes are designed to minimise eutrofication and contamination (particularly by human enteric bacteria). However, little consideration has been given to the significance, if any, of less obvious forms of microbial contamination resulting from periodic human activities in Antarctica. The predominant commensal microorganism on human skin, Staphylococcus epidermidis, could be detected by PCR, in Dry Valley mineral soils collected from heavily impacted areas, but could not be detected in Dry Valley mineral soils collected from low impact and pristine areas. Cell viability of this non-enteric human commensal is rapidly lost in Dry Valley mineral soil. However, S. epidermidis can persist for long periods in Dry Valley mineral soil as non-viable cells and/or naked DNA.     

Identification and characterization of bacterial-binding property in the type III repeat domain of fibronectin

To characterize fibronectin binding with Granulicatella adiacens, a causative agent of infective endocarditis, monoclonal antibodies were generated against human fibronectin and selected for their capacity to inhibit the fibronectin binding of the organism. Thermolysin and lysyl-endopeptidase digests of fibronectin were characterized by Western blot. The epitope of inhibitory monoclonal antibody was found in the central portion of fibronectin known as the cell-binding domain, and not in the N-terminal portion known to be the binding region of most microbial species, e.g., Staphylococcus aureus and Streptococcus pyogenes. While these two species could bind to both the N-terminal and central portion, Escherichia coli and G. adiacens bind only to the latter. Excess amounts of free fibronectin in the solution inhibited the bacterial adherence to the N-terminal fibronectin fragment, but not to the central region, thereby suggesting the central region plays a significant role for in vivo bacterial colonization in the presence of high concentrations of soluble fibronectin.     

S. aureus-dependent microglial activation is selectively attenuated by the cyclopentenone prostaglandin 15-deoxy-Delta12,14- prostaglandin J2 (15d-PGJ2)

Microglial activation is a hallmark of brain abscess. The continual release of proinflammatory mediators by microglia following bacterial challenge may contribute, in part, to the destruction of surrounding normal tissue characteristic of brain abscess. Therefore, attenuating chronic microglial activation during the course of CNS bacterial infections may have therapeutic benefits. The purpose of this study was to evaluate the ability of the natural peroxisome proliferator-activated receptor (PPAR)-gamma agonist 15-deoxy-Delta12,14- prostaglandin J2 (15d-PGJ2) to modulate microglial activation in response to Staphylococcus aureus, one of the main etiologic agents of brain abscess in humans. 15d-PGJ2 was a potent inhibitor of proinflammatory cytokine (IL-1beta, TNF-alpha, IL-12 p40) and CC chemokine (MIP-1beta, MCP-1) production in primary microglia, but had no effect upon the expression of select CXC chemokines (MIP-2, KC). 15d-PGJ2 also selectively inhibited the S. aureus-dependent increase in microglial TLR2, CD14, MHC class II, and CD40 expression, whereas it had no effect on the co-stimulatory molecules CD80 and CD86. Microarray analysis revealed additional inflammatory mediators modulated by 15d-PGJ2 in primary microglia following S. aureus exposure, the majority of which were chemokines. These results suggest that suppressing microglial activation through the use of 15d-PGJ2 may lead to the sparing of damage to normal brain parenchyma that often results from brain abscess.     

我国奶牛乳房炎致病性金黄色葡萄球菌血清型分布

本试验采用玻片凝集法对从临床型乳房炎病乳中分离获得的56株金黄色葡萄球菌进行血清型分型。结果表明336型占67.9%(38/56),5型占7.1%(4/56),8型占3.6%(2/56)。     

Proteomic profiling of cell envelope-associated proteins from Staphylococcus aureus

The emergence of highly virulent community acquired Staphylococcus aureus and continued progression of resistance to multiple antimicrobials, including methicillin and vancomycin, marks the reemergence of S. aureus as a serious health care threat. Investigation of proteins localized to the cell surface could help to elucidate mechanisms of virulence and antibiotic resistance in S. aureus. In this study, proteomic profiling methods were developed to solubilize, display, and evaluate abundance levels of proteins present in the supernatants of the lysostaphin-digested cell envelope from cultured vancomycin-intermediate S. aureus (VISA) cells. Combining approaches of 2-DE or chromatographic separation of proteins with MS analyses resulted in the identification of 144 proteins of particular interest. Of these proteins, 48 contained predicted cell wall localization or export signal motifs, including 14 with distinct covalent peptidoglycan-anchor sites, four of which are uncharacterized to date. One of the two most abundant cell envelope proteins, which showed remarkably high variations in MW and pI in the 2-DE gel display, was the S. aureus surface protein G. The display of numerous secreted proteins that are not covalently cell wall-anchored, suggests that, in the exponential growth phase, secreted proteins can be retained physiologically in the cell envelope and may interact with cell wall-anchored proteins and carbohydrate structures in a manner yet to be determined. The remaining 96 proteins, devoid of recognizable motifs, were repeatedly profiled in the VISA cell envelope fractions. We describe a novel semiquantitative method to determine abundance factors of such proteins in 2-DE gels of cell envelope fractions relative to whole cell lysates and discuss these data in the context of true cell envelope localization versus experimentally caused cell lysis.     

Anti‐infectious activity of synbiotics in a novel mouse model of methicillin‐resistant Staphylococcus aureus infection

The anti‐infectious activity of synbiotics against methicillin‐resistant Staphylococcus aureus (MRSA) infection was evaluated using a novel lethal mouse model. Groups of 12 mice treated with multiple antibiotics were infected orally with a clinical isolate of MRSA at an inoculum of 10⁸ CFU on day 7 after starting the antibiotics. A dose of 400 mg/kg 5‐fluorouracil (5‐FU) was injected intraperitoneally on day 7 after the infection. A dose of 10⁸ CFU Bifidobacterium breve strain Yakult and 10 mg of galactooligosaccharides (GOS) were given orally to mice daily with the antibiotic treatment until day 28. The intestinal population levels of MRSA in the mice on multiple antibiotics were maintained stably at 10⁸ CFU/g of intestinal contents after oral MRSA infection and the subsequent 5‐FU treatment killed all the mice in the group within 14 days. B. breve administration saved most of the mice, but the synbiotic treatment saved all of the mice from lethal MRSA infection. The synbiotic treatment was effective for the treatment of intestinal infection caused by four MRSA strains with different toxin productions. There was a large difference among the six Bifidobacteria strains that were naturally resistant to the antibacterial drugs used. B. breve in combination with GOS is demonstrated to have valuable preventive and curative effects against even fatal MRSA infections.