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Coxiella burnetiiis a zoonotic Gram-negative obligate intracellular bacterial pathogen and the causative agent of Query (Q) fever in humans. Contamination of milk byC. burnetiias a consequence of livestock infection is a significant public health concern. Effective methods to inactivateC. burnetiiin milk is a critical aspect of food safety. In this study, we measured optical light attenuation factors; absorption, scattering, and reflection of skim milk (SM) and considered for evaluation of delivered UV dose under stirred conditions. The accuracy of the method followed for the estimation of delivered UV dose in SM was verified by comparative studies ofEscherichia coliATCC 25922 inactivation in phosphate buffer (transparent fluid), and humic acid (opaque fluid). Absorption, scattering coefficient, and the reflectance of SM at 254 nm was measured as 19 ± 0.3/cm. 26 ± 0.5/cm and 10.6 %, respectively. SM inoculated withC. burnetiiwas irradiated using a collimated beam device equipped with a low-pressure UV-C254 nmlamp at doses from 0 – 12 mJ·cm-2. Results showed a log-linear inactivation ofC. burnetiiin SM with UV-C sensitivity (D10) value of 4.1 ± 0.04 mJ·cm-2. Similar inactivation kinetics was observed withSalmonella enterica serovarMuenchen ATCC BAA 1674 in SM and thereby suggested as a suitable surrogate toC. burnetiifor pilot scale UV-C processing studies of SM.

The approximately 1011 viruses and microbial cells per gram of fecal matter (dry weight) in the large intestine are important to human health. The responses of three common gut bacteria species, and one opportunistic pathogen, to 117 commonly consumed foods, chemical additives, and plant extracts were tested. Many compounds, including Stevia rebaudiana and bee propolis extracts, exhibited species-specific growth inhibition by prophage induction. Overall, these results show that various foods may change the abundances of gut bacteria by modulating temperate phage and suggests a novel path for landscaping the human gut microbiome.

Coxiella burnetii is a bacterial obligate intracellular parasite and the etiological agent of query (Q) fever. While the C. burnetii genome has been reduced to ∼2 Mb as a likely consequence of genome streamlining in response to parasitism, enzymes for a nearly complete central metabolic machinery are encoded by the genome. However, lack of a canonical hexokinase for phosphorylation of glucose and an apparent absence of the oxidative branch of the pentose phosphate pathway, a major mechanism for regeneration of the reducing equivalent nicotinamide adenine dinucleotide phosphate (NADPH), have been noted as potential metabolic limitations of C. burnetii. By complementing C. burnetii with the gene zwf encoding the glucose-6-phosphate-consuming and NADPH-producing enzyme glucose-6-phosphate dehydrogenase (G6PD), we demonstrate a severe metabolic fitness defect for C. burnetii under conditions of glucose limitation. Supplementation of the medium with the gluconeogenic carbon source glutamate did not rescue the growth defect of bacteria complemented with zwf. Absence of G6PD in C. burnetii therefore likely relates to the negative effect of its activity under conditions of glucose limitation. Coxiella burnetii central metabolism with emphasis on glucose, NAD+, NADP+ and NADPH is discussed in a broader perspective, including comparisons with other bacterial obligate intracellular parasites., Expression of the gene zwf, a major mechanism for regenerating NADPH, in Coxiella burnetii leads to conditional impairment of growth.

Host organisms restrict the availability of iron to invading pathogens in order to reduce pathogen replication. To counteract the host’s response to infection, bacteria can rely on redundant mechanisms to obtain biologically diverse forms of iron during infection. C. burnetii appears specifically dependent on molecular iron for replication and viability and exhibits a response to iron akin to bacteria that colonize iron-rich environments. Physiological adaptation of C. burnetii to the unique acidic and degradative environment of the CCV is consistent with access of this pathogen to molecular iron., Coxiella burnetii, the causative agent of Query (Q) fever in humans, is a highly infectious obligate intracellular bacterium. Following uptake into a host cell, C. burnetii replicates within a phagolysosome-derived compartment referred to as the Coxiella-containing vacuole (CCV). During infection, C. burnetii exhibits tropism for tissues related to iron storage and recycling (e.g., the liver and splenic red pulp), suggesting that pathogen physiology is linked to host iron metabolism. Iron has been described to have a limited role in C. burnetii virulence regulation, despite evidence that C. burnetii-infected host cells increase expression of transferrin receptors, thereby suggesting that active iron acquisition by the bacterium occurs upon infection. Through the use of host cell-free culture, C. burnetii was separated from the host cell in order to directly assess the role of different forms of iron in C. burnetii replication and viability, and therefore virulence. Results indicate that C. burnetii tolerates molecular iron over a broad concentration range (i.e., ∼0.001 to 1 mM) and undergoes gross loss of viability upon iron starvation. C. burnetii protein synthesis and energy metabolism, however, occur nearly uninhibited under iron concentrations not permissive to replication. Despite the apparent absence of genes related to acquisition of host-associated iron-containing proteins, C. burnetii replication is supported by hemoglobin, transferrin, and ferritin, likely due to release of iron from such proteins under acidic conditions. Moreover, chelation of host iron pools inhibited pathogen replication during infection of cultured cells. IMPORTANCE Host organisms restrict the availability of iron to invading pathogens in order to reduce pathogen replication. To counteract the host’s response to infection, bacteria can rely on redundant mechanisms to obtain biologically diverse forms of iron during infection. C. burnetii appears specifically dependent on molecular iron for replication and viability and exhibits a response to iron akin to bacteria that colonize iron-rich environments. Physiological adaptation of C. burnetii to the unique acidic and degradative environment of the CCV is consistent with access of this pathogen to molecular iron.

Microbial viruses can control host abundances via density-dependent lytic predator-prey dynamics. Less clear is how temperate viruses, which coexist and replicate with their host, influence microbial communities. Here we show that virus-like particles are relatively less abundant at high host densities. This suggests suppressed lysis where established models predict lytic dynamics are favoured. Meta-analysis of published viral and microbial densities showed that this trend was widespread in diverse ecosystems ranging from soil to freshwater to human lungs. Experimental manipulations showed viral densities more consistent with temperate than lytic life cycles at increasing microbial abundance. An analysis of 24 coral reef viromes showed a relative increase in the abundance of hallmark genes encoded by temperate viruses with increased microbial abundance. Based on these four lines of evidence, we propose the Piggyback-the-Winner model wherein temperate dynamics become increasingly important in ecosystems with high microbial densities; thus 'more microbes, fewer viruses'.

There is a poor understanding of how the physiology of polymicrobial communities in cystic fibrosis (CF) lungs contributes to pulmonary exacerbations and lung function decline. In this study, a microbial culture system based on the principles of the Winogradsky column (WinCF system) was developed to study the physiology of CF microbes. The system used glass capillary tubes filled with artificial sputum medium to mimic a clogged airway bronchiole. Chemical indicators were added to observe microbial physiology within the tubes. Characterization of sputum samples from seven patients showed variation in pH, respiration, biofilm formation and gas production, indicating that the physiology of CF microbial communities varied among patients. Incubation of homogenized tissues from an explant CF lung mirrored responses of a Pseudomonas aeruginosa pure culture, supporting evidence that end-stage lungs are dominated by this pathogen. Longitudinal sputum samples taken through two exacerbation events in a single patient showed that a two-unit drop in pH and a 30% increase in gas production occurred in the tubes prior to exacerbation, which was reversed with antibiotic treatment. Microbial community profiles obtained through amplification and sequencing of the 16S rRNA gene showed that fermentative anaerobes became more abundant during exacerbation and were then reduced during treatment where P. aeruginosa became the dominant bacterium. Results from the WinCF experiments support the model where two functionally different CF microbial communities exist, the persistent Climax Community and the acute Attack Community. Fermentative anaerobes are hypothesized to be the core members of the Attack Community and production of acidic and gaseous products from fermentation may drive developing exacerbations. Treatment targeting the Attack Community may better resolve exacerbations and resulting lung damage.

Coxiella burnetii is a highly infectious obligate intracellular bacterium and the etiological agent of the zoonosis Query (Q) fever. This Gram-negative gamma-proteobacterium has adapted to replicate within a specialized compartment in mammalian phagocytic cells, known as the Coxiella-containing vacuole (CCV). Knowledge of critical characteristics of the CCV microenvironment (e.g., luminal pH), analysis of the C. burnetii genome sequence, and strategic metabolic profiling have provided the basis for determining the physicochemical and nutritional conditions necessary to support axenic replication of C. burnetii. In this unit, the media currently utilized for axenic culture of C. burnetii are described, with emphasis on application. To aid in experimental reproducibility and interpretation of results, considerations and limitations are discussed. Lastly, expected results for C. burnetii axenic growth under control conditions are provided as a reference. © 2018 by John Wiley & Sons, Inc.

Microbialization refers to the observed shift in ecosystem trophic structure towards higher microbial biomass and energy use. On coral reefs, the proximal causes of microbialization are overfishing and eutrophication, both of which facilitate enhanced growth of fleshy algae, conferring a competitive advantage over calcifying corals and coralline algae. The proposed mechanism for this competitive advantage is the DDAM positive feedback loop (dissolved organic carbon (DOC), disease, algae, microorganism), where DOC released by ungrazed fleshy algae supports copiotrophic, potentially pathogenic bacterial communities, ultimately harming corals and maintaining algal competitive dominance. Using an unprecedented data set of >400 samples from 60 coral reef sites, we show that the central DDAM predictions are consistent across three ocean basins. Reef algal cover is positively correlated with lower concentrations of DOC and higher microbial abundances. On turf and fleshy macroalgal-rich reefs, higher relative abundances of copiotrophic microbial taxa were identified. These microbial communities shift their metabolic potential for carbohydrate degradation from the more energy efficient Embden-Meyerhof-Parnas pathway on coral-dominated reefs to the less efficient Entner-Doudoroff and pentose phosphate pathways on algal-dominated reefs. This 'yield-to-power' switch by microorganism directly threatens reefs via increased hypoxia and greater CO2 release from the microbial respiration of DOC.

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Nature 531, 466–470 (2016); doi:10.1038/nature17193 In this Article, the ‘Predator–prey modelling’ section of the Methods shows Lotka–Volterra equations. Although these equations are meant to present a basic Lotka–Volterra model, the term ‘N/K’ in the second equation was inadvertently introduced during proofing, which makes the equations reflect the Piggyback-the-Winner model rather than basic Lotka–Volterra.