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The use of germ line-mutated mice in understanding host-pathogen interactions.

Microbial pathogenesis reflects an imbalance between parasite and host factors that favour pathogen multiplication and tissue destruction over those required for microbial elimination and preservation of the integrity of host tissues. In vivo analysis of host-pathogen interactions has been revolutionized by the ability to engineer specific genetic alterations including loss of function mutations and transgenes into the mouse germline. This brief review recapitulates what we have learned about the host response to Toxoplasma gondii infection to illustrate the usefulness of gene-altered mice in microbial pathogenesis research. A consideration of the pitfalls and limitations of experiments in knockout mice and ways of addressing these concerns are discussed. Finally, advances in inducible and tissue-restricted alterations in gene function are presented and their possible applications to microbiology research are considered.

Animals↗

Interactions of microbial populations in cellulose fermentation.

Fermentation of cellulose in the rumen occurs through the interactions of many microbial species. The initial degradation of cellulose is caused by cellulase-producing organisms. The soluble hydrolysis products are used by both cellulolytic and noncellulolytic organisms to produce acetate, propionate, and butyrate and the important intermediates H2 and succinate. Interactions between species are necessary for the decarboxylation of succinate to propionate. H2 is used by methanogenic bacteria to reduce CO2 to CH4. The removal of H2 by methanogenesis increases the production of acetate from carbohydrates by several important cellulose- and carbohydrate-fermenting microbial species. Monensin and lasalocid appear to alter the overall fermentation by selecting for populations that produce relatively larger amounts of propionate and against populations that produce relatively larger amounts of acetate and H2. Cellulose fermentation in the human large intestine is compared with fermentation in the rumen.

Anaerobiosis↗

Specificity of marine microbial surface interactions.

The macromolecular surface components involved in intraspecific cell surface interactions of the green microalga Chlorella vulgaris and closely associated bacteria were investigated. The specific surface attachment between this alga and its associated bacteria is mediated by lectin-like macromolecules associated with the surfaces of these cells. The binding activity of these surface polymers was inhibited by specific simple sugars; this suggests the involvement of specific receptor-ligand binding sites on the interactive surfaces. Epifluorescent microscopic evaluation of bacteria-alga interactions in the presence and absence of the macromolecules that mediate these interactions showed that the glycoproteins active in these processes were specific to the microbial sources from which they were obtained. The demonstration and definition of the specificity of these interactions in mixed microbial populations may play an important role in our understanding of the dynamics of marine microbial populations in the sea.

Adhesiveness↗

Microbial surface interactions: reduction of the haemagglutination activity of the oral bacterium Fusobacterium nucleatum by absorption with Streptococcus and Bacteroides.

Oral strains of Fusobacterium nucleatum showed haemagglutination (HA) of sheep red blood cells and attachment of HA-active F. nucleatum fragments to other microorganisms allowed a means of studying microbial surface interactions. HA-active sonicated fragments (SF) prepared from F. nucleatum were mixed with whole cell suspensions of 48 bacterial strains and, after incubation, the whole cells were separated from the non-absorbed fragments by differential centrifugation. Attachment of F. nucleatum fragments to the cells was indicated by a reduction in the HA activity of the SF in the supernatant fluid remaining after absorption with whole cells. HA activity of the microbial cells used for absorption and the detection of F. nucleatum fragments on these cells by an indirect fluorescent antibody technique provided further evidence of attachment. Of the 48 strains tested, 10 absorbed F. nucleatum HA-active fragments. They included Bacteroides gingivalis, Bacteroides fragilis subsp. distasonis, Bacteroides corrodens, Streptococcus morbillorum, Streptococcus sanguis (Blackburn and JC 74) and Streptococcus mutans AHT, BHT, 10449 and 6715. Chelators revealed that F. nucleatum attached to the microorganisms via a Ca2+-dependent interaction. Sugar inhibition demonstrated that F. nucleatum attached to the microorganisms via a D-galactose-containing moiety on their surface. A reduction in the absorption of F. nucleatum HA-active fragments by Strep. mutans grown in a higher concentration of sucrose was observed.

Absorption↗

Pseudomonas aeruginosa phenazines dictate site-specific competitive interactions with Klebsiella pneumoniae.

Pseudomonas aeruginosa and Klebsiella pneumoniae are Gram-negative opportunistic pathogens that frequently colonize the human body and are major causes of infection. These bacteria are often co-isolated in polymicrobial urinary tract and lung infections, the latter of which is associated with increased disease severity and worse clinical outcomes. Despite their overlapping niches and clinical relevance, little is known about how these two pathogens interact and how those interactions influence human health. Given the growing recognition that microbial interactions are key drivers of disease, we investigated how P. aeruginosa and K. pneumoniae influence one another. We discovered an antagonistic interaction in which P. aeruginosa restricts the growth of K. pneumoniae. This inhibition is driven by phenazine production in P. aeruginosa, specifically the secondary metabolites pyocyanin and pyorubin, which are both necessary and sufficient to suppress K. pneumoniae growth. Using a diverse set of clinical isolates, we found that this antagonism is strain dependent. Both the susceptibility of K. pneumoniae to phenazines and the ability of P. aeruginosa to restrict K. pneumoniae growth varies between strains. Moreover, the necessity of phenazine production is specific to the site of infection. Together, these findings demonstrate that strain background and environmental context are critical determinants of pathogen interactions. Our work underscores the importance of considering these variables when investigating how microbial interactions influence infection and disease outcomes.

Journal Article↗

Effects of initial corncob particle size on the short-term composting for preparation of cultivation substrates for Pleurotus ostreatus.

The short-term composting based on corncob for preparing Pleurotus ostreatus cultivation medium originated from agricultural production practices and so lacked systematic investigation. In this study, the influences of a Dafen (15 mm, DFT) and Xiaofen (5 mm, XFT) initial particle size (IPS) of corncob on the microbial succession and compost quality were examined. Results demonstrated that XFT compost was better suited for mushroom cultivation due to its high biological efficiency of 70 % and the absence of contamination. The composting microbes differed significantly between the DFT and XFT composts. During composting, the genera of Bacillus, Acinetobacter, Lactobacillus, Streptomyces, and Paenibacillus were majorly found in the DFT compost, while Acinetobacter, Lactobacillus, Puccinia, Bacteroides, and Bacillus genera dominated the XFT compost. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that throughout the thermophilic phase, XFT compost had much greater relative abundances of sequences relevant to energy, carbohydrate, and amino acid metabolism than DFT compost. Analysis of network correlations and Mantel tests indicated that IPS reduction could increase microbial interactions. Overall, adjusting the IPS of corncob to 5 mm increased microbial interactions, improved compost quality, and thereby boosted the P. ostreatus yield. These findings will be pertinent in optimizing the composting process of cultivation medium for P. ostreatus.

Composting↗

Effects of microbial community interactions on transformation rates of xenobiotic chemicals.

The effects of culture filtrates, mixed populations, and common microbial exudates on bacterial transformations of three agricultural and industrial chemicals were investigated. Test chemicals included methyl parathion, diethyl phthalate, and 2,4-dichlorophenoxyacetic acid butoxyethyl ester. The presence of various cultures, filtrates, or exudates of algae, fungi, or other bacteria either stimulated or inhibited bacterial transformation rates. Inhibition resulted from treatments that lowered the pH, and stimulation resulted from an increase in cell biomass (based on plate counts) and from a different process whereby rates of transformation per bacterial cell rapidly increased as much as 10-fold.

2,4-Dichlorophenoxyacetic Acid↗

Mycorrhizosphere interactions to improve plant fitness and soil quality.

Arbuscular mycoruhizal fungi are key components of soil microbiota and obviously interact with other microorganisms in the rhizosphere, i.e. the zone of influence of plant roots on microbial populations and other soil constituents. Mycorrhiza formation changes several aspects of plant physiology and some nutritional and physical properties of the rhizospheric soil. These effects modify the colonization patterns of the root or mycovrhizas (mycorrhizosphere) by soil microorganisms. The rhizosphere of mycorrhizal plants, in practice a mycorrhizosphere, harbors a great array of microbial activities responsible for several key ecosystem processes. This paper summarizes the main conceptual principles and accepted statements on the microbial interactions between mycorrhizal fungi and other members of rhizosphere microbiota and discusses current developments and future trends concerning the following topics: (i) effect of soil microorganisms on mycorrhiza formation; (ii) mycorrhizosphere establishment; (iii) interactions involved in nutrient cycling and plant growth; (iv) interactions involved in the biological control of plant pathogens; and (v) interactions to improve soil quality. The main conclusion is that microbial interactions in the rhizosphere of mycorrhizal plants improve plant fitness and soil quality, critical issues for a sustainable agricultural development and ecosystem functioning.

Bacteria↗

Cooperative anaerobic catabolism of chlorinated organic compounds: implications for sustainable bioremediation.

Biodegradation research historically followed a reductionist approach focused on axenic (pure) cultures capable of catabolizing the specific contaminant(s) of interest. While this approach has substantially advanced our understanding of the microbiology, physiology, biochemistry, and genetics of contaminant degradation under laboratory conditions, it does not capture the complexity of natural and engineered environments. During in situ bioremediation, microbiomes are exposed to mixtures of contaminants, and microbial interactions profoundly influence contaminant transformation and fate. In anoxic environments, degradation of chlorinated compounds is often sustained by metabolic cooperation among taxonomically and physiologically distinct microorganisms. Through the exchange of metabolites such as hydrogen, formate, acetate, and other nutrients, microbial populations establish interdependent networks that overcome thermodynamic and physiological constraints, enabling self-sustaining systems of contaminant transformations that would be inefficient or impossible with individual organisms. We highlight examples of microbial interactions that underpin anaerobic catabolism of chlorinated contaminants, including systems resulting in self-sustained anaerobic bioremediation.

Biodegradation, Environmental↗

The virulence of mixed infection with Streptococcus constellatus and Fusobacterium nucleatum in a murine orofacial infection model.

Orofacial infections are usually polymicrobial, and it is the microbial interactions of pathogenic species that cause tissue destruction. In this study, the microbial interaction between Streptococcus constellatus and Fusobacterium nucleatum was characterized using a murine orofacial infection model. A mixture of viable S. constellatus and F. nucleatum cells (both 2 x 10(8) CFU/mouse) was injected into the submandible; as a result, all of the test mice died. In contrast, none of the experimental animals monoinjected with either S. constellatus or F. nucleatum died (P<0.001), indicating that the synergism between the two resulted in the virulence. When a mixture of viable S. constellatus cells and a culture filtrate of F. nucleatum was tested, lethality and the bacterial cell count per lesion were significantly enhanced as compared with monoinjections (P<0.02). However, the virulence of F. nucleatum was not enhanced by infection of a culture filtrate of S. constellatus. The enhancement of virulence was observed even when viable S. constellatus cells and the culture filtrate of F. nucleatum were injected at separate sites. Heat treatment of the culture filtrate of F. nucleatum did not affect the enhancement. These results indicate that a heat-stable substance(s) produced by F. nucleatum contributes to the microbial synergy of S. constellatus and F. nucleatum in orofacial infections.

Abscess↗

Postmortem alterations of bacterial localization.

Localization of bacteria in mammalian gastrointestinal tracts by electron microscopy has yielded much information on our understanding of intimate host-microbial interactions, but because of difficulties in obtaining human tissue, the human host-microbial interactions have not been well studied. In this report, an attempt was made to ascertain the feasibility of using gastrointestinal tissues obtained at autopsy. A rat model was used to produce samples that would simulate conditions that autopsy samples might undergo. Rats, left at room temperature for 1 hr and then refrigerated at 2C for time intervals up to 24 hours after death were autopsied and samples from the stomach, ileum and cecum were removed and processed for light and scanning microscopy. Tissues were fixed in buffered glutaraldehyde, postfixed in buffered osmium tetroxide, dehydrated in an ethanol series, critical point dried, coated with gold-palladium and examined. Keratinized murine stomachs showed little or no changes in the localization of the gram-positive layer of bacteria over 24 hours. However, major changes were noted in the ileum and colon with the ileum showing the most dislocation of the flora. Colonic tissues were altered by invading bacteria although some regions had a relatively normal appearing host-flora relationship. Slow cooling rates of the gastrointestinal tracts were implicated as a major cause for alterations in bacterial localization. The data predict that studies of normal microbial location with human autopsy samples will be best pursued on keratinized tissue.

Animals↗

The ES-242s, novel N-methyl-D-aspartate antagonists of microbial origin, interact with both the neurotransmitter recognition site and the ion channel domain.

ES-242-1 approximately 5 are novel microbial bioxanthracenes which do not contain nitrogen. The ES-242s inhibited the binding of [3H]TCP and [3H]CGS19755 to the N-methyl-D-aspartate (NMDA) receptor complex. They had no effect on the binding of the specific ligands for the non-NMDA receptor. The biochemical and pharmacological properties of ES-242-1 were fully examined since it is the most potent of the five compounds. ES-242-1 is highly specific for the NMDA receptor; it has no effect on other receptors. Kinetic analyses indicated that ES-242-1 inhibited the binding of [3H]TCP and [3H]CGS19755 in a competitive manner, respectively, suggesting that ES-242-1 interacts with both the transmitter recognition site and the channel domain. ES-242-1 selectively inhibited NMDA-induced Ca2+ influx in primary cultures of mouse hippocampal neurons. ES-242-1 also specifically blocked the increase in cyclic GMP accumulation induced by NMDA or L-glutamate in rat cerebellar slices. In a concentration range of 0.1-1.0 microM, ES-242-1 was as potent as MK-801 in preventing glutamate-induced neurotoxicity in primary cultures of mouse hippocampal neurons. These results show that ES-242-1 is a potent and specific antagonist for the NMDA receptor. The antagonistic properties of the ES-242s appear to be due to a novel mechanism of action at the NMDA receptor.

Animals↗

Modelling Yersinia enterocolitica inactivation in coculture experiments with Lactobacillus sakei as based on pH and lactic acid profiles.

In food processing and preservation technology, models describing microbial proliferation in food products are a helpful tool to predict the microbial food safety and shelf life. In general, the available models consider microorganisms in pure culture. Thus, microbial interactions are ignored, which may lead to a discrepancy between model predictions and the actual microbial evolution, particularly for fermented and minimally processed food products in which a background flora is often present. In this study, the lactic acid mediated negative microbial interaction between the lactic acid bacterium Lactobacillus sakei and the psychrotrophic food pathogen Yersinia enterocolitica was examined. A model describing the lactic acid induced inhibition (i.e., early induction of the stationary phase) of the pathogen [Vereecken, K.M., Devlieghere, F., Bockstaele, A., Debevere, J., Van Impe, J.F., 2003. A model for lactic acid induced inhibition of Yersinia enterocolitica in mono- and coculture with Lactobacillus sakei. Food Microbiology 20, 701-713.] was extended to describe the subsequent inactivation (i.e., decrease of the cell concentration to values below the detection limit). In the development of a suitable model structure to describe the inactivation process, critical points in the variation of the specific evolution rate mu [1/h] with the dynamic (time-varying) pH and undissociated lactic acid profiles were taken into account. Thus, biological knowledge, namely, both pH and undissociated lactic acid have an influence on the microbial evolution, was incorporated. The extended model was carefully validated on new data. As a result, the newly developed model is able to accurately predict the growth, inhibition and subsequent inactivation of Y. enterocolitica in coculture as based on the dynamic pH and lactic acid profiles of the medium.

Antibiosis↗

Microbial-host interactions in the airways in chronic respiratory infection.

The pathogenic events that take place in chronic respiratory infection highlight the successful microbial strategy of survival by persistence, or colonization. Microorganisms implement their strategy of persistence by two principal tactics: (1) sabotage of the host's bronchial defenses (ie, direct microbe-mediated damage to the host), and (2) subversion of the host's normally protective defenses into damaging host tissue itself (ie, indirect host-mediated damage provoked by the microbe). Among the various ways in which microorganisms directly damage host defenses and facilitate their own persistence in the respiratory tract are inhibition of ciliary function, inhibition of mucociliary transport, alteration of ion transport in respiratory epithelium, stimulation of mucus production, and damage to respiratory epithelium. Patients with chronic respiratory infection suffer a vicious circle of events leading to progressive lung damage and cardio-respiratory failure. Treatments to break this circle include antimicrobial therapy to reduce microbial colonization and anti-inflammatory/immunosuppressive therapy to modulate damaging host responses.

Bacterial Physiological Phenomena↗

Spatial Metabolomics Reveals the Role of Penicillic Acid in Cheese Rind Microbiome Disruption by a Spoilage Fungus.

Microbial interactions in cheese rinds influence community structure, food safety, and product quality. But the chemical mechanisms that mediate microbial interactions in cheeses and other fermented foods are generally not known. Here, we investigate how the spoilage mold Aspergillus westerdijkiae chemically inhibits beneficial cheese-rind bacteria using a combination of omics technologies. In cheese rind community and co-culture experiments, A. westerdijkiae strongly inhibited most cheese rind community members. In co-culture with Staphylococcus equorum, A. westerdijkiae strongly affected bacterial gene expression, including upregulation of a putative bceAB gene cluster that is associated with resistance to antimicrobial compounds in other bacteria. Mass spectrometry imaging (MSI) revealed spatially localized production of secondary metabolites, including penicillic acid and ochratoxin B at the fungal-bacterial interface. Integration of LC-MS/MS and genome annotations confirmed the presence of additional bioactive metabolites, such as notoamides and circumdatins. Fungal metabolic responses varied by bacterial partner, suggesting species-specific chemical strategies. Notably, penicillic acid levels increased 2.5-fold during interaction with Brachybacterium, and experiments with purified penicillic acid showed inhibition of a range of cheese rind bacteria. These findings show that A. westerdijkiae deploys a context-dependent arsenal of mycotoxins and other metabolites, disrupting microbial community assembly in cheese rinds.

Aspergillus westerdijkiae↗

Computational and experimental approaches to studying metal interactions with microbial biofilms.

The structural and compositional heterogeneity of biofilms poses unique problems in metal fate and transport. A starting point for quantitative understanding of biofilm-metal interactions is surface complexation theory, with roots in chemical equilibria and thermodynamics. This approach permits fitting of experimental data to a variety of mathematical models from which predictive parameters, such as K, may be extracted. Applications of more sophisticated fitting routines such as tableau (as in FITEQL) or spectra pK methods provide a better measure of the heterogeneity. There remain large theoretical and computational challenges, as there is ample evidence to suggest that the principle of additivity is problematic, owing to chemical interactions between individual sorbent phases within biofilms. And finally, the question of how bacterial metabolic activity is likely to influence metal uptake by biofilms adds yet another layer of complexity for future investigations.

Bacteria↗

Microbial-host interactions specifically control the glycosylation pattern in intestinal mouse mucosa.

The glycosylation of the intestinal cell layer is thought to control several key functions of the gut such as vectorial transports, defence against microbial agents or immunological processes. It has been assumed that the gut microflora may modulate the glycosylation pattern of the intestinal cell layer. However, there is no direct evidence for this regulatory process. The first goal of this work was to establish the germ-free mice intestinal glycosylation baseline using a histochemical approach and a panel of ten lectins with defined glycan specificities to tissue sections prepared from various cellular compartments of the small and large intestine. Using this baseline, we have studied the contribution of the gut microflora on the carbohydrate composition of glycoconjugates of intestinal cells by comparing the germ-free and conventional mice glycosylation patterns. Analysis of the germ-free mice intestinal glycosylation baseline revealed that the expression of glycans depends on the proximodistal gradient (small to large intestine) and on the cell lineage (absorptive, goblet, crypt, and Paneth cells), indicating that mice are able to create and maintain a strict topological and cell lineage-specific regulation of glycosyltransferase expression. By comparing germ-free and conventional mice, we find that the gut microflora specifically modulates the gut glycosylation pattern, quantitatively as well as qualitatively by changing the cellular and subcellular distribution of glycans. This is the first report in mice to directly demonstrate the critical contribution of microflora to intestinal glycosylation, a key characteristic of the gut.

Animals↗

Interaction of microbial agents with the immune system during infectious disease.

Research during the last years has revealed a considerable complexity of the immune system. It is clear that immunological reactions depend on extensive and only partly clarified interactions between a number of different cell types (e.g. B lymphocytes, plasma cells, T cell subpopulations, cytotoxic K and NK cells, monocytic cells, neutrophilic and eosinophilic granulocytes) and their molecular products (e.g. immunoglobulins, lymphokines and interleukins). These components further interact with the complement system, as well as with immunologically nonspecific components like acute phase proteins (e.g. C-reactive protein) and with other pathophysiological phenomena occurring during infections, e.g. the fever response. The application of these observations from basic and experimental immunology to the investigation of antimicrobial immune reactions is still only in its beginning, but has already resulted in new concepts of clinical value for the understanding of infectious diseases. The present paper briefly describes certain aspects of the immune response to infections with various microbial agents, with particular emphasis on reactions of clinical importance. In addition to B and T cell reactions, possible antimicrobial functions of K cells and NK cells are discussed, and the possible importance in infectious disease of various T cell subpopulations, particularly T suppressor cells, is discussed. Lastly, various escape mechanisms are mentioned whereby certain microbial agents may evade elimination by the immune response of the host.

Antibody Formation↗