The effect of quercetin on genetic expression of the commensal gut microbes Bifidobacterium catenulatum, Enterococcus caccae and Ruminococcus gauvreauii.

Quercetin is one of the most abundant polyphenols found in fruits and vegetables. The ability of the gut microbiota to metabolize quercetin has been previously documented; however, the effect that quercetin may have on commensal gut microbes remains unclear. In the present study, the effects of quercetin on the commensal gut microbes Ruminococcus gauvreauii, Bifidobacterium catenulatum and Enterococcus caccae were determined through evaluation of growth patterns and cell morphology, and analysis of genetic expression profiles between quercetin treated and non-treated groups using Single Molecule RNA sequencing via Helicos technology. Results of this study revealed that phenotypically, quercetin did not prevent growth of Ruminococcus gauvreauii, mildly suppressed growth of Bifidobacterium catenulatum, and moderately inhibited growth of Enterococcus caccae. Genetic analysis revealed that in response to quercetin, Ruminococcus gauvreauii down regulated genes responsible for protein folding, purine synthesis and metabolism. Bifidobacterium catenulatum increased expression of the ABC transport pathway and decreased metabolic pathways and cell wall synthesis. Enterococcus caccae upregulated genes responsible for energy production and metabolism, and downregulated pathways of stress response, translation and sugar transport. For the first time, the effect of quercetin on the growth and genetic expression of three different commensal gut bacteria was documented. The data provides insight into the interactions between genetic regulation and growth. This is also a unique demonstration of how RNA single molecule sequencing can be used to study the gut microbiota.

Photo-catalytic inactivation of an Enterococcus biofilm: the anti-microbial effect of sulphated and europium-doped titanium dioxide nanopowders.

The control and prevention of biofilm-related infections is an important public healthcare issue. Given the increasing antibiotic resistance among bacteria and fungi that cause serious infections in humans, promotion of new strategies combating microorganisms has been essential. One attractive approach to inactivate microorganisms is the use of semiconductor photo-catalysis, which has become the subject of extensive research. In this study, the bactericidal properties of four photo-catalysts, TiO2, TiO2-S, TiO2-Eu and TiO2-Eu-S, were investigated against established 24, 48, 72 and 96 h biofilms of Enterococcus The exposure of biofilms to the catalysts induced the production of superoxide radical anions. The best photo-catalytic inactivation was achieved with the TiO2-Eu-S and TiO2-S nanopowders and 24 h biofilms. Transmission electron microscopy images showed significant changes in the structure of the biofilm cells following photo-inactivation. The results suggest that doping with europium and modifying the surface with sulphate groups enhanced the bactericidal activity of the TiO2 nanoparticles against enterococcal biofilms.

The coffee-machine bacteriome: biodiversity and colonisation of the wasted coffee tray leach.

Microbial communities are ubiquitous in both natural and artificial environments. However, microbial diversity is usually reduced under strong selection pressures, such as those present in habitats rich in recalcitrant or toxic compounds displaying antimicrobial properties. Caffeine is a natural alkaloid present in coffee, tea and soft drinks with well-known antibacterial properties. Here we present the first systematic analysis of coffee machine-associated bacteria. We sampled the coffee waste reservoir of ten different Nespresso machines and conducted a dynamic monitoring of the colonization process in a new machine. Our results reveal the existence of a varied bacterial community in all the machines sampled, and a rapid colonisation process of the coffee leach. The community developed from a pioneering pool of enterobacteria and other opportunistic taxa to a mature but still highly variable microbiome rich in coffee-adapted bacteria. The bacterial communities described here, for the first time, are potential drivers of biotechnologically relevant processes including decaffeination and bioremediation.

[Sonosensitizing Action of Teraphtal in Bacterial Media].

Employing bacterial model systems it was shown that the introduction of the soluble compound--sodium salt of cobalt octacarboxyphthalocyanine (teraphtal)--into the medium led in an ultrasonic field to a decrease in the proportion of survived bacteria. It is suggested that in the bacterial environment teraphtal forms a solid phase, which in the ultrasonic field causes destruction of the structures adjacent to the nanocrystals due to localized cavitation processes.

Mechanisms of linezolid resistance in staphylococci and enterococci isolated from two teaching hospitals in Shanghai, China.

BACKGROUND: Linezolid is one of the most effective treatments against Gram-positive pathogens. However, linezolid-resistant/intermediate strains have recently emerged in worldwide. The purpose of this study was to analyse the prevalence and resistance mechanisms of linezolid-resistant/intermediate staphylococci and enterococci in Shanghai, China. RESULTS: Thirty-two linezolid-resistant/intermediate strains, including 14 Staphylococcus capitis, three Staphylococcus aureus, 14 Enterococcus faecalis and one Enterococcus faecium clinical isolates, were collected in this study which displayed linezolid MICs of 8 to 512 µg/ml, 8-32 µg/ml, 4-8 µg/ml and 4 µg/ml, respectively. All linezolid-resistant S. capitis isolates had a novel C2131T mutation and a G2603T mutation in the 23S rRNA region, and some had a C316T (Arg106Cys) substitution in protein L4 and/or harboured cfr. Linezolid-resistant S. aureus isolates carried a C389G (Ala130Gly) substitution in protein L3, and/or harboured cfr. The cfr gene was flanked by two copies of the IS256-like element, with a downstream orf1 gene. Linezolid-resistant/intermediate enterococci lacked major resistance mechanisms. The semi-quantitative biofilm assay showed that 14 linezolid-resistant E. faecalis isolates produced a larger biofilm than linezolid-susceptible E. faecalis strains. Transmission electron microscopy showed the cell walls of linezolid-resistant/intermediate strains were thicker than linezolid-susceptible strains. CONCLUSION: Our data indicated that major resistance mechanisms, such as mutations in 23S rRNA and ribosomal proteins L3 and L4, along with cfr acquisition, played an important role in linezolid resistance. Secondary resistance mechanisms, such as biofilm formation and cell wall thickness, should also be taken into account.

The elongation of ovococci.

The morphogenesis of ovococci has been reviewed extensively. Recent results have provided new insights concerning the mechanisms of elongation in ovoid bacteria. We present here the proteins involved in the elongation (firmly established and more or less hypothetical) and discuss the relationship between elongation and division of ovococci.

Integration of microfiltration and anion-exchange nanoparticles-based magnetic separation with MALDI mass spectrometry for bacterial analysis.

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is powerful in characterizing and identifying bacterial isolates. However, sufficient quantities of bacterial cells are required for generating MALDI mass spectra and a procedure to isolate and enrich target bacteria from sample matrix prior to MALDI-MS analysis is often necessary. In this paper, anion-exchange superparamagnetic nanoparticles (NPs), i.e., fluidMAG-DEAE and fluidMAG-Q, were employed to capture Aeromonas, Salmonella, Pseudomonas, Enterococcus, Bacillus, Staphylococcus and Escherichia coli from aqueous solutions and fresh water. The magnetically isolated bacteria were then characterized by whole cell MALDI-MS. The capture efficiency was found to be dependent on bacterial species, medium pH, the functional group and concentration of the NPs. The experimental results demonstrated that fluidMAG-DEAE and fluidMAG-Q were broad spectrum probes for bacteria. Furthermore, both dead and live bacteria could be captured by the NPs, and the live bacteria captured remained viable. Membrane filtration prior to the magnetic isolation could increase enrichment factor and eliminate potential matrix interference. A detection limit of 1 x 10(3)cfu/ml was achieved for the bacteria spiked in tap water and reservoir water, and the analytical time was around 2h.

Ultrastructural effects of oritavancin on methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus.

The ultrastructural effects of the lipoglycopeptide oritavancin on methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus (VRE) were examined by transmission electron microscopy. Oritavancin but not vancomycin induced aberrant septum formation and loss of staining of nascent septal cross walls in MRSA. Septal distortions were also observed in VRE exposed to oritavancin.

Factors affecting the adsorption of bacteriocins to Lactobacillus sakei and Enterococcus sp.

Bacteriocins bacJW3BZ and bacJW6BZ produced by Lactobacillus plantarum, and bacJW11BZ and bacJW15BZ produced by Lactobacillus fermentum, inhibit Gram-positive and Gram-negative bacteria. Treatment of Enterococcus sp. HKLHS and Lactobacillus sakei DSM 20017 with these bacteriocins deformed the cells and resulted in DNA and beta-galactosidase leakage. The bacteriocins adsorbed to sensitive and resistant strains. Optimal adsorption of bacJW3BZ and bacJW6BZ to Enterococcus sp. HKLHS was recorded at pH 10.0, whereas adsorption of bacJW11BZ and bacJW15BZ was favored at pH 4.0-8.0 and 2.0-4.0, respectively. Adsorption to L. sakei DSM 20017 was less influenced by pH. Incubation temperature had a major influence on the adsorption of bacJW6BZ and bacJW11BZ to sensitive cells, with better results recorded below 30 degrees C. Although variable results were recorded for bacJW3BZ and bacJW15BZ, optimal adsorption occurred between 37 and 60 degrees C. Variable levels of adsorption were recorded in the presence of inorganic salts and solvents, and this seems to be species-specific. Maximal adsorption (100%) was recorded for bacJW3BZ and bacJW15BZ to L. sakei DSM 20017 in the presence of most inorganic salts and solvents tested. Maximal adsorption of bacJW6BZ to Enterococcus sp. HKLHS (50%) was recorded in the presence of Triton X-114 and little (17%) or no adsorption in the presence of other reagents.

Granular layer in the periplasmic space of gram-positive bacteria and fine structures of Enterococcus gallinarum and Streptococcus gordonii septa revealed by cryo-electron microscopy of vitreous sections.

High-resolution structural information on optimally preserved bacterial cells can be obtained with cryo-electron microscopy of vitreous sections. With the help of this technique, the existence of a periplasmic space between the plasma membrane and the thick peptidoglycan layer of the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus was recently shown. This raises questions about the mode of polymerization of peptidoglycan. In the present study, we report the structure of the cell envelope of three gram-positive bacteria (B. subtilis, Streptococcus gordonii, and Enterococcus gallinarum). In the three cases, a previously undescribed granular layer adjacent to the plasma membrane is found in the periplasmic space. In order to better understand how nascent peptidoglycan is incorporated into the mature peptidoglycan, we investigated cellular regions known to represent the sites of cell wall production. Each of these sites possesses a specific structure. We propose a hypothetic model of peptidoglycan polymerization that accommodates these differences: peptidoglycan precursors could be exported from the cytoplasm to the periplasmic space, where they could diffuse until they would interact with the interface between the granular layer and the thick peptidoglycan layer. They could then polymerize with mature peptidoglycan. We report cytoplasmic structures at the E. gallinarum septum that could be interpreted as cytoskeletal elements driving cell division (FtsZ ring). Although immunoelectron microscopy and fluorescence microscopy studies have demonstrated the septal and cytoplasmic localization of FtsZ, direct visualization of in situ FtsZ filaments has not been obtained in any electron microscopy study of fixed and dehydrated bacteria.

The increase in the number of accessible SH-groups in the enterococcal membrane vesicles by ATP and nicotinamide adenine dinucleotides.

The number of accessible SH groups was determined in membrane vesicles prepared from Enterococcus hirae grown under anaerobic conditions at alkaline pH (pH 8.0). Addition of ATP or nicotinamide adenine dinucleotides (NAD(+) +NADH) to the vesicles caused a approximately 4-fold or approximately 1.9-fold increase in the number of SH-groups, respectively. This was inhibited by treatment with N-ethylmaleimide. The increase was significant when ATP and NAD(+) +NADH both were added. The change was lacking in the presence of the F0F1-ATPase inhibitors N,N'-diclohexylcarbodiimide or sodium azide. This was also absent in atp mutant with defect in the F0F1-ATPase and, in addition, it was less in potassium ion-free medium. These results are correlated with data about K+-dependent F0F1-ATPase activity, suggesting a relationship between the F0F1-ATPase and K+ uptake Trk-like system. The latter may be regulated by NAD or NADH mediating conformational changes.

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.

Isolation and identification of biofilm microorganisms from silicone gastrostomy devices.

BACKGROUND/PURPOSE: Silicone gastrostomy devices (tubes and "buttons") are used extensively for long-term feeding and administration of special diets and medications. However, their potential for harboring microorganisms and possibly compromising the host largely is unknown. This study was conducted to isolate and identify the microbial species in viable biofilms attached to these devices in a pediatric cohort. METHODS: A total of 78 domains on 18 silicone gastrostomy devices (12 "buttons" and 6 tubes converted to skin level devices), previously used for feeding (3 to 47 months) in children ranging in age from 6 months to 17 years were analyzed for microbial content. Biofilms were removed from the silicone tube surfaces and inoculated into enriched nutrient media using standard procedures. Intact biofilms also were observed using scanning electron microscopy (SEM) and confocal scanning laser microscopy. RESULTS: All devices analyzed in this investigation were found to exhibit biofilm growth. Of the 24 identified bacterial species, the predominant genera included Bacillus, Enterococcus, and Staphylococcus. Control studies of the tubes under SEM showed a multitude of crevices serving as niches for microbial colonization. Observation of the attached biofilm by SEM showed various biomasses with numerous morphologies. CONCLUSIONS: Biofilm composition and attachment to silicone enteral access devices has not received appropriate attention previously. This study shows that devices are colonized with various bacteria and fungi posing a potential threat to patients, particularly those who are immunocompromised. These microorganisms also may play a significant role in the formation of granulation tissue and contribute to device failure.

Anaerobic transformation of quercetin-3-glucoside by bacteria from the human intestinal tract.

From human feces two phenotypically different types of bacteria were isolated on quercetin-3-glucoside as carbon and energy source. Isolates of one type were identified as strains of Enterococcus casseliflavus. They utilized the sugar moiety of the glycoside, but did not degrade the aglycon further. The sugar moiety (4 mM) was fermented to 5.5 +/- 2.1 mM formate, 2.1 +/- 0.7 mM acetate, 1.6 +/- 0.3 mM l-lactate, and 1.3 +/- 0.4 mM ethanol. The second type of isolate was identified as Eubacterium ramulus. This organism was capable of degrading the aromatic ring system. Growing cultures of Eubacterium ramulus converted 5 mM quercetin-3-glucoside to 1.7 +/- 0.6 mM 3,4-dihydroxyphenylacetic acid, 7.6 +/- 1.0 mM acetate, and 4.0 +/- 0.4 mM butyrate. Molecular hydrogen, 3,4-dihydroxybenzaldehyde, and ethanol were detected in small amounts. Phloroglucinol was a transient intermediate in the breakdown of quercetin-3-glucoside. Eubacterium ramulus did not grow on the aglycon quercetin or the ring-fission intermediate phloroglucinol, but cleaved the flavonoid ring system when glucose was present as a cosubstrate. The most probable number of quercetin-3-glucoside-degrading bacteria determined in nine human fecal samples was 10(7)-10(9)/g dry mass. Isolates from these experiments were all identified as Eubacterium ramulus.

Isolation and characterization of an Enterococcus-like bacterium causing muscle necrosis and mortality in Macrobrachium rosenbergii in Taiwan.

A Gram-positive, ovoid, diplococoid bacterium tentatively identified as Enterococcus-like was isolated from diseased Macrobrachium rosenbergii in Taiwanese aquaculture ponds. The diseased prawns displayed poor growth, anorexia, inactivity, opaque and whitish musculature, and mortality. In histological preparations, melanized hemocytic granulomas were seen in the connective tissue around hemal sinuses together with hemocytic aggregation in necrotic musculature. Five isolates of diplococci were collected from diseased prawns at 4 farms and these were evaluated for 93 characteristics including morphology, physiology, biochemistry and sensitivity to antibiotics. The results indicated that the isolates belonged to a single species. They grew in 0.5 to 6.0% NaCl, at 10 to 40 degrees C, at pH 9.6 and on bile esculin medium, gave positive pyrrolidonylarylamidase, arginine dehydrolase and Voges-Proskauer tests, were resistant to bacitracin and SXT, and were CAMP-negative and non-hemolytic on sheep blood agar. These findings indicated an Enterococcus-like bacterium closely related to Enterococcus seriolicida (recently reduced to synonymy with Lactococcus garvieae). Experimental injection of 3 x 10(5) cells of strain KM002 of this Enterococcus-like bacterium into the ventral sinus of the prawn cephalothorax caused 100% mortality in 11 d, and induced muscular necrosis and hepatopancreatitis, gross signs and histopathology similar to those observed in the naturally infected prawns. It was concluded that this Enterococcus-like bacterium was the etiological agent associated with mortality of the farmed, diseased prawns.

Is there a synergistic effect between mixed bacterial infection in biofilm formation on biliary stents?

BACKGROUND: Biliary sludge which forms as a result of bacterial adherence and biofilm formation in the biliary system is a recognized cause of blockage of plastic stents. Bacteriological cultures of sludge have revealed a mixed infection with gram-positive and gram-negative bacteria. Animal studies have shown that prophylactic ciprofloxacin, which selectively suppress gram-negative bacteria, results in prolonged stent patency despite colonization of the stents by gram-positive bacteria. METHODS: We tested a possible synergistic effect between gram-negative and gram-positive bacteria in adherence and biofilm formation on plastic stents. Clinical isolates of Escherichia coli and Enterococcus were cultured in separate chemostats to achieve a steady growth. Adherence of the two bacteria on plastic stent surface were tested separately by perfusing infected bile with the respective bacteria through different modified Robbins devices containing 10F polyethylene stent pieces up to 4 days. In a second experiment, Enterococcus was perfused through stent pieces precolonized with E. coli for 24 hours. The stent pieces were then removed daily and analyzed by bacteriologic culture and scanning electron microscopy for bacterial adherence and biofilm formation. RESULTS: Gram-negative E. coli were more adherent than gram-positive Enterococcus. Precolonization with E. coli facilitates subsequent attachment of Enterococcus. CONCLUSIONS: We concluded that there is a synergistic effect between gram-positive and gram-negative bacteria in adherence and biofilm formation.

Encapsulation of streptococci isolated from bovine milk.

Capsule expression was assessed by India ink staining in 95 streptococcal and enterococcal isolates representing 8 species. Organisms were evaluated after primary culture on blood agar and after storage in 10% skim milk at -80 degrees C. Seventeen of 95 primary culture samples were positive for an unstained halo (capsule) by India ink. Only 3 isolates were positive for capsule after being maintained for an extended period of time. To confirm results of India ink tests, an unencapsulated and encapsulated strain of each species was observed by electron microscopy. Cells of encapsulated strains were surrounded by thread-like fibers indicative of capsule, whereas cells of unencapsulated strains had a smooth surface. The importance of capsule in the pathogenesis of streptococcal mastitis is unknown. Studies on capsule expression by streptococci may facilitate investigations on the pathogenesis of streptococcal mastitis.

Differences in the formation of poles of Enterococcus and Bacillus.

The pole of Enterococcus hirae (Streptococcus faecium) is more pointed than that of Bacillus subtilis; i.e. the pole of the former is prolate and the latter is oblate. Both species form their poles by constructing annular additions on the inside surface. In both cases, the thick septum starts to split from the outside before the septum is complete. Physiochemical considerations dictate that the peptidoglycan must be unstretched as laid down. However, it later becomes stressed and may stretch to increase its surface area or to change its shape. Our earlier analysis for B. subtilis demonstrated that, without the addition of new peptidoglycan, the nascent wall is stretched after it is externalized to 1.51 times the original area. The wall of partially formed poles that is already exteriorized continues to deform with further development. For E. hirae, Higgins & Shockman's measurements showed that the completed pole has a surface area 2.18 times larger than a completed septal disk and the wall changes shape very little after exteriorization. A model is presented here for the streptococcus in which the septal wall does not increase its surface area on exteriorization either by expansion or by murein insertion. Instead, the septal wall as it is split and exteriorized twists to become oblique, increasing the inner radius of the incomplete septum. In consequence of this rotation, extra layers of peptidoglycan are added to the inside face of the developing septum. This additional murein forms the more pointed pole shape for E. hirae. This "split-and-splay" model thus refines and extends the surface stress theory of E. hirae developed a decade ago by proposing a source of the extra wall needed for the formation of its prolate, more pointed, pole.