Spectro-electrochemical and DFT studies of a planar Cu(II)–phenolate complex active in the aerobic oxidation of primary alcohols

A square-planar compound [Cu(pyrimol)Cl] (pyrimol = 4-methyl-2-N-(2-pyridylmethylene)aminophenolate) abbreviated as CuL–Cl) is described as a biomimetic model of the enzyme galactose oxidase (GOase). This copper(II) compound is capable of stoichiometric aerobic oxidation of activated primary alcohols in acetonitrile/water to the corresponding aldehydes. It can be obtained either from Hpyrimol (HL) or its reduced/hydrogenated form Hpyramol (4-methyl-2-N-(2-pyridylmethyl)aminophenol; H2L) readily converting to pyrimol (L-) on coordination to the copper(II) ion. Crystalline CuL–Cl and its bromide derivative exhibit a perfect square-planar geometry with Cu–O(phenolate) bond lengths of 1.944(2) and 1.938(2) Å. The cyclic voltammogram of CuL–Cl exhibits an irreversible anodic wave at +0.50 and +0.57 V versus ferrocene/ferrocenium (Fc/Fc+) in dry dichloromethane and acetonitrile, respectively, corresponding to oxidation of the phenolate ligand to the corresponding phenoxyl radical. In the strongly donating acetonitrile the oxidation path involves reversible solvent coordination at the Cu(II) centre. The presence of the dominant CuII–L. chromophore in the electrochemically and chemically oxidised species is evident from a new fairly intense electronic absorption at 400–480 nm ascribed to a several electronic transitions having a mixed pi-pi(L.) intraligand and Cu–Cl -> L. charge transfer character. The EPR signal of CuL–Cl disappears on oxidation due to strong intramolecular antiferromagnetic exchange coupling between the phenoxyl radical ligand (L.) and the copper(II) centre, giving rise to a singlet ground state (S = 0). The key step in the mechanism of the primary alcohol oxidation by CuL–Cl is probably the alpha-hydrogen abstraction from the equatorially bound alcoholate by the phenoxyl moiety in the oxidised pyrimol ligand, Cu–L., through a five-membered cyclic transition state.

Community patterns of soil bacteria and nematodes in relation to geographic distance

Ecosystems consist of aboveground and belowground subsystems and the structure of their communities is known to change with distance. However, most of this knowledge originates from visible, aboveground components, whereas relatively little is known about how soil community structure varies with distance and if this variability depends on the group of organisms considered. In the present study, we analyzed 30 grasslands from three neighboring chalk hill ridges in southern UK to determine the effect of geographic distance (1e198 km) on the similarity of bacterial communities and of nematode communities in the soil. We found that for both groups, community similarity decayed with distance and that this spatial pattern was not related to changes either in plant community composition or soil chemistry. Site history may have contributed to the observed pattern in the case of nematodes, since the distance effect depended on the presence of different nematode taxa at one of the hill ridges. On the other hand, site-related differences in bacterial community composition alone could not explain the spatial turnover, suggesting that other factors, such as biotic gradients and local dispersal processes that we did not include in our analysis, may be involved in the observed pattern. We conclude that, independently of the variety of causal factors that may be involved, the decay in similarity with geographic distance is a characteristic feature of both communities of soil bacteria and nematodes.

Detection and identification of species-specific bacteria associated with synanthropic mites

Internal bacterial communities of synanthropic mites Acarus siro, Dermatophagoides farinae, Lepidoglyphus destructor, and Tyrophagus putrescentiae (Acari: Astigmata) were analyzed by culturing and culture-independent approaches from specimens obtained from laboratory colonies. Homogenates of surface-sterilized mites were used for cultivation on non-selective agar and DNA extraction. Isolated bacteria were identified by sequencing of the 16S rRNA gene. PCR amplified 16S rRNA genes were analyzed by terminal restriction fragment length polymorphism analysis (T-RFLP) and cloning sequencing. Fluorescence in situ hybridization using universal bacterial probes was used for direct bacterial localization. T-RFLP analysis of 16S rRNA gene revealed distinct species-specific bacterial communities. The results were further confirmed by cloning and sequencing (284 clones). L. destructor and D. farinae showed more diverse communities then A. siro and T. putrescentiae. In the cultivated part of the community, the mean CFUs from four mite species ranged from 5.2 × 102 to 1.4 × 103 per mite. D. farinae had significantly higher CFUs than the other species. Bacteria were located in the digestive and reproductive tract, parenchymatical tissue, and in bacteriocytes. Among the clones, Bartonella-like bacteria occurring in A. siro and T. putresecentiae represented a distinct group related to Bartonellaceae and to Bartonella-like symbionts of ants. The clones of high similarity to Xenorhabdus cabanillasii were found in L. destructor and D. farinae, and one clone related to Photorhabdus temperata in A. siro. Members of Sphingobacteriales cloned from D. farinae and A. siro clustered with the sequences of “Candidatus Cardinium hertigii” and as a separate novel cluster.

In vitro fermentation by human gut bacteria of proteolytically digested caseinomacropeptide nonenzymatically glycosylated with prebiotic carbohydrates

The in vitro fermentation selectivity of hydrolyzed caseinomacropeptide (CMP) glycosylated, via Maillard reaction (MR), with lactulose, galacto-oligosaccharides from lactose (GOSLa), and galacto-oligosaccharides from lactulose (GOSLu) was evaluated, using pH-controlled small-scale batch cultures at 37 °C under anaerobic conditions with human feces. After 10 and 24 h of fermentation, neoglyconjugates exerted a bifidogenic activity, similar to those of the corresponding prebiotic carbohydrates. No significant differences were found in Bacteroides, Lactobacillus�Enterococcus, Clostridium histolyticum subgroup, Atopobium and Clostridium coccoides�Eubacterium rectale populations. Concentrations of lactic acid and short-chain fatty acids (SCFA) produced during the fermentation of prebiotic carbohydrates were similar to those produced for their respective neoglycoconjugates at both fermentation times. These findings, joined with the functional properties attributed to CMP, could open up new applications of MR products involving prebiotics as novel multiple-functional ingredients with potential beneficial effects on human health.

In vitro fermentation of alternansucrase raffinose-derived oligosaccharides by human gut bacteria

In this work, in vitro fermentation of alternansucrase raffinose-derived oligosaccharides, previously fractionated according to their degree of polymerization (DP; from DP4 to DP10), was carried out using small-scale pH-controlled batch cultures at 37 °C under anaerobic conditions with human feces. Bifidogenic activity of oligosaccharides with DP4�6 similar to that of lactulose was observed; however, in general, a significant growth of lactic acid bacteria Bacteroides, Atopobium cluster, and Clostridium histolyticum group was not shown during incubation. Acetic acid was the main short chain fatty acid (SCFA) produced during the fermentation process; the highest levels of this acid were shown by alternansucrase raffinose acceptor pentasaccharides at 10 h (63.11 mM) and heptasaccharides at 24 h (54.71 mM). No significant differences between the gas volume produced by the mixture of raffinose-based oligosaccharides (DP5�DP10) and inulin after 24 h of incubation were detected, whereas lower gas volume was generated by DP4 oligosaccharides. These findings indicate that novel raffinose-derived oligosaccharides (DP4�DP10) could be a new source of prebiotic carbohydrates.

Effect of dextransucrase cellobiose acceptor products on the growth of human gut bacteria

The selective fermentation by human gut bacteria of gluco-oligosaccharides obtained from the reaction between the glucosyl group of sucrose and cellobiose, catalyzed by dextransucrases (DSR) from Leuconostoc mesenteroides, has been evaluated. Oligosaccharides were fractionated according to their molecular weight, and their effect on the growth of different bacterial groups was studied. To determine the structure (position and configuration of glycosidic linkages)�function relationship, their properties were compared to those of DSR maltose acceptor products (DSRMal) and of recognized prebiotic carbohydrates (fructo-oligosaccharides, FOS). Cellobiose acceptor products (DSRCel) showed bifidogenic properties similar to those of FOS. However, no significant differences related to molecular weight or isomeric configurations were found for DSRCel and DSRMal products.

High prevalence of multidrug-tolerant bacteria and associated antimicrobial resistance genes isolated from ornamental fish and their carriage water

Background: Antimicrobials are used to directly control bacterial infections in pet (ornamental) fish and are routinely added to the water these fish are shipped in to suppress the growth of potential pathogens during transport. Methodology/Principal Findings: To assess the potential effects of this sustained selection pressure, 127 Aeromonas spp. isolated from warm and cold water ornamental fish species were screened for tolerance to 34 antimicrobials. Representative isolates were also examined for the presence of 54 resistance genes by a combination of miniaturized microarray and conventional PCR. Forty-seven of 94 Aeromonas spp. isolates recovered from tropical ornamental fish and their carriage water were tolerant to >= 15 antibiotics, representing seven or more different classes of antimicrobial. The quinolone and fluoroquinolone resistance gene, qnrS2, was detected at high frequency (37% tested recent isolates were positive by PCR). Class 1 integrons, IncA/C broad host range plasmids and a range of other antibiotic resistance genes, including floR, blaTEM21, tet(A), tet(D), tet(E), qacE2, sul1, and a number of different dihydrofolate reductase and aminoglycoside transferase coding genes were also detected in carriage water samples and bacterial isolates. Conclusions: These data suggest that ornamental fish and their carriage water act as a reservoir for both multi-resistant bacteria and resistance genes.

Development of a miniaturised microarray-based assay for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria

We describe the development of a miniaturised microarray for the detection of antimicrobial resistance genes in Gram-negative bacteria. Included on the array are genes encoding resistance to aminoglycosides, trimethoprim, sulphonamides, tetracyclines and beta-lactams, including extended-spectrum beta-lactamases. Validation of the array with control strains demonstrated a 99% correlation between polymerase chain reaction and array results. There was also good correlation between phenotypic and genotypic results for a large panel of Escherichia coli and Salmonella isolates. Some differences were also seen in the number and type of resistance genes harboured by E. coli and Salmonella strains. The array provides an effective, fast and simple method for detection of resistance genes in clinical isolates suitable for use in diagnostic laboratories, which in future will help to understand the epidemiology of isolates and to detect gene linkage in bacterial populations. (C) 2008 Published by Elsevier B.V. and the International Society of Chemotherapy.

Hcp2, a secreted protein of the phytopathogen Pseudomonas syringae pv. tomato DC3000, is required for competitive fitness against bacteria and yeasts

When analysing the secretome of the plant pathogen Pseudomonas syringae pv. tomato (Pst) DC3000, we identified hemolysin co-regulated protein (Hcp) as one of the secreted proteins. Hcp is assumed to be an extracellular component of the type VI secretion system (T6SS). Two copies of hcp genes are present in the Pst DC3000 genome, hcp1 (PSPTO_2539) and hcp2 (PSPTO_5435). We studied the expression patterns of hcp genes and tested the fitness of hcp knock-out mutants in host plant colonization and in inter-microbial competition. We found that the hcp2 gene is expressed, most actively at the stationary growth phase, and that the Hcp2 protein is secreted via T6SS and appears in the culture medium as covalently linked dimers. Expression of hcp2 is not induced in planta and it does not contribute to virulence or colonisation in tomato or Arabidopsis plants. Instead, hcp2 is required for survival in competition with enterobacteria and yeasts, and its function is associated with suppression of the growth of these competitors. This is the first report on bacterial T6SS-associated genes functioning in competition against yeast. Our results suggest that the T6SS of P. syringae may play an important role in bacterial fitness, allowing this plant pathogen to survive in conditions where it has to compete with other micro-organisms for resources.

Screening for Bacillus isolates in the broiler gastrointestinal tract

Spores from a number of different Bacillus species are currently being used as human and animal probiotics, although their mechanisms of action remain poorly understood. Here we describe the isolation of 237 presumptive gut-associated Bacillus spp. isolates that were obtained by heat and ethanol treatment of fecal material from organically reared broilers followed by aerobic plating. Thirty-one representative isolates were characterized according to their morphological, physiological, and biochemical properties as well as partial 16S rRNA gene sequences and screening for the presence of plasmid DNA. The Bacillus species identified included B. subtilis, B. pumilus, B. licheniformis, B. clausii, B. megaterium, B. firmus, and species of the B. cereus group, whereas a number of our isolates could not be classified. Intrinsic properties of potential importance for survival in the gut that could be advantageous for spore-forming probiotics were further investigated for seven isolates belonging to five different species. All isolates sporulated efficiently in the laboratory, and the resulting spores were tolerant to simulated gastrointestinal tract conditions. They also exhibited antimicrobial activity against a broad spectrum of bacteria, including food spoilage and pathogenic organisms such as Bacillus spp., Clostridium perfringens, Staphylococcus aureus, and Listeria monocytogenes. Importantly, the isolates were susceptible to most of the antibiotics tested, arguing that they would not act as donors for resistance determinants if introduced in the form of probiotic preparations. Together, our results suggest that some of the sporeformers isolated in this study have the potential to persist in or transiently associate with the complex gut ecosystem.

Effect of the growth promoter avilamycin on emergence and persistence of antimicrobial resistance in enteric bacteria in the pig

Aim: To assess the effect of the growth promoter avilamycin on emergence and persistence of resistance in enteric bacteria in the pig. Methods and Results: Pigs ( treated with avilamycin for 3 months and controls) were challenged with multiresistant Salmonella Typhimurium DT104 and faecal counts were performed for enterococci, Escherichia coli, S. Typhimurium and Campylobacter ( before, during and 5 weeks post-treatment). Representative isolates were tested for antibiotic resistance and for the presence of resistance genes. Avilamycin-resistant Enterococci faecalis (speciated by PCR) were isolated from the treated pigs and continued to be detected for the first week after treatment had ceased. The avilamycin- resistance gene was characterized by PCR as the emtA gene and speciation by PCR. MIC profiling confirmed that more than one strain of Ent. faecalis carried this gene. There was no evidence of increased antimicrobial resistance in the E. coli, Salmonella and Campylobacter populations, although there was a higher incidence of tetB positive E. coli in the treated pigs than the controls. Conclusion: Although avilamycin selects for resistance in the native enterococci population of the pig, no resistant isolates were detected beyond 1 week post-treatment. This suggests that resistant isolates were unable to persist once selective pressure was removed and were out-competed by the sensitive microflora. Significance and Impact of the Study: Our data suggest the risk of resistant isolates becoming carcass contaminants and infecting humans could be minimized by introducing a withdrawal period after using avilamycin and prior to slaughter.

Attaching-effacing lesions associated with Escherichia coli O157:H7 and other bacteria in experimentally infected conventional neonatal goats

Four conventionally reared goats aged 6 days were inoculated orally with approximately 10(10) colony-forming units (cfu) of a non-verotoxigenic strain of Escherichia coli O157:H7. All remained clinically normal. Tissues were sampled under terminal anaesthesia at 24 (two animals), 48 and 72 h post-inoculation (hpi). E. coli O157:H7 was cultured from the ileum, caecum, colon and rectum of all animals, but the number of bacteria recovered at these sites varied between animals. Attaching-effacing (AE) lesions associated with O157 organisms, as confirmed by immunolabelling, were observed in the ileum of one of the two animals examined at 24 hpi, and in the ileum, caecum and proximal colon of an animal examined at 72 hpi. E. coliO157 organisms were detected at > 105 cfu/g of tissue at these sites. In addition, A-E lesions associated with unidentified bacteria were observed at various sites in the large bowel of the same animals. Lesions containing both E. coliO157 and unidentified bacteria (non-O157) were not observed. Non-O157 AE lesions were also observed in the large bowel of one of two uninoculated control animals. This indicated that three (one control and two inoculated) animals were colonized with an unidentified AE organism before the commencement of the experiment. The O157-associated AE lesions were observed only in animals colonized by non-O157 AE organisms and this raises questions about individual host susceptibility to AE lesions and whether non-O157 AE organisms influence colonization by E. coli O157.

Attaching-effacing bacteria in animals

Enteric bacteria with a demonstrable or potential ability to form attaching-effacing lesions, so-called attaching-effacing (AE) bacteria, have been found in the intestinal tracts of a wide variety of warm-blooded animal species, including man. In some host species, for example cattle, pigs, rabbits and human beings, attaching-effacing Escherichia coli (AEEC) have an established role as enteropathogens. In other host species, AE bacteria are of less certain significance. With continuing advances in the detection and typing of AE strains, the importance of these bacteria for many hosts is likely to become clearer. The pathogenic effects of AE bacteria result from adhesion to the intestinal mucosa by a variety of mechanisms, culminating in the formation of the characteristic intimate adhesion of the AE lesion. The ability to induce AE lesions is mediated by the co-ordinated expression of some 40 bacterial genes organized within a so-called pathogenicity island, known as the "Locus for Enterocyte Effacement". It is also believed that the production of bacterial toxins, principally Vero toxins, is a significant virulence factor for some A-EEC strains. Recent areas of research into AE bacteria include: the use of Citrobacter rodentium to model human AEEC disease; quorum-sensing mechanisms used by AEEC to modulate virulence gene expression; and the potential role of adhesion in the persistent colonization of the intestine by AE bacteria. This review of AE bacteria covers their molecular biology, their occurrence in various animal species, and the diagnosis, pathology and clinical aspects of animal diseases with which they are associated. Reference is made to human pathogens where appropriate. The focus is mainly on natural colonization and disease, but complementary experimental data are also included. (C) 2004 Elsevier Ltd. All rights reserved.

Layer-by-layer coating of alginate matrices with chitosan–alginate for the improved survival and targeted delivery of probiotic bacteria after oral administration

The oral administration of probiotic bacteria has shown potential in clinical trials for the alleviation of specific disorders of the gastrointestinal tract. However, cells must be alive in order to exert these benefits. The low pH of the stomach can greatly reduce the number of viable microorganisms that reach the intestine, thereby reducing the efficacy of the administration. Herein, a model probiotic, Bifidobacterium breve, has been encapsulated into an alginate matrix before coating in multilayers of alternating alginate and chitosan. The intention of this formulation was to improve the survival of B. breve during exposure to low pH and to target the delivery of the cells to the intestine. The material properties were first characterized before in vitro testing. Biacore™ experiments allowed for the polymer interactions to be confirmed; additionally, the stability of these multilayers to buffers simulating the pH of the gastrointestinal tract was demonstrated. Texture analysis was used to monitor changes in the gel strength during preparation, showing a weakening of the matrices during coating as a result of calcium ion sequestration. The build-up of multilayers was confirmed by confocal laser-scanning microscopy, which also showed the increase in the thickness of coat over time. During exposure to in vitro gastric conditions, an increase in viability from <3 log(CFU) per mL, seen in free cells, up to a maximum of 8.84 ± 0.17 log(CFU) per mL was noted in a 3-layer coated matrix. Multilayer-coated alginate matrices also showed a targeting of delivery to the intestine, with a gradual release of their loads over 240 min.