Use of Axenic Culture Tools to Study Coxiella burnetii.

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.

Physicochemical and Nutritional Requirements for Axenic Replication Suggest Physiological Basis for Coxiella burnetii Niche Restriction.

Bacterial obligate intracellular parasites are clinically significant animal and human pathogens. Central to the biology of these organisms is their level of adaptation to intracellular replication niches associated with physicochemical and nutritional constraints. While most bacterial pathogens can adapt to a wide range of environments, severe niche restriction-an inability to thrive in diverse environments-is a hallmark of bacterial obligate intracellular parasites. Herein the physicochemical and nutritional factors underlying the physiological basis for niche restriction in the zoonotic bacterial obligate intracellular parasite and Q fever agent Coxiella burnetii are characterized. Additionally, these factors are reviewed in the context of C. burnetii evolution and continued (patho) adaptation. C. burnetii replication was strictly dependent on a combination of moderately acidic pH, reduced oxygen tension, and presence of carbon dioxide. Of macronutrients, amino acids alone support replication under physicochemically favorable conditions. In addition to utilizing gluconeogenic substrates for replication, C. burnetii can also utilize glucose to generate biomass. A mutant with a disruption in the gene pckA, encoding phosphoenolpyruvate carboxykinase (PEPCK), the first committed step in gluconeogenesis, could be complemented chemically by the addition of glucose. Disruption of pckA resulted in a moderate glucose-dependent growth defect during infection of cultured host cells. Although, C. burnetii has the theoretical capacity to synthesize essential core metabolites via glycolysis and gluconeogenesis, amino acid auxotrophy essentially restricts C. burnetii replication to a niche providing ample access to amino acids. Overall, the described combination of physiochemical and nutritional growth requirements are strong indicators for why C. burnetii favors an acidified phagolysosome-derived vacuole in respiring tissue for replication.

Anaplasma marginale superinfection attributable to pathogen strains with distinct genomic backgrounds.

Strain superinfection occurs when a second pathogen strain infects a host already infected with a primary strain. The selective pressures that drive strain divergence, which underlies superinfection, and allow penetration of a new strain into a host population are critical knowledge gaps relevant to shifts in infectious disease epidemiology. In regions of endemicity with a high prevalence of infection, broad population immunity develops against Anaplasma marginale, a highly antigenically variant rickettsial pathogen, and creates strong selective pressure for emergence of and superinfection with strains that differ in their Msp2 variant repertoires. The strains may emerge either by msp2 locus duplication and allelic divergence on an existing genomic background or by introduction of a strain with a different msp2 allelic repertoire on a distinct genomic background. To answer this question, we developed a multilocus typing assay based on high-throughput sequencing of non-msp2 target loci to distinguish among strains with different genomic backgrounds. The technical error level was statistically defined based on the percentage of perfect sequence matches of clones of each target locus and validated using experimental single strains and strain pairs. Testing of A. marginale-positive samples from tropical regions where A. marginale infection is endemic identified individual infections that contained unique alleles for all five targeted loci. The data revealed a highly significant difference in the number of strains per animal in the tropical regions compared to infections in temperate regions and strongly supported the hypothesis that transmission of genomically distinct A. marginale strains predominates in high-prevalence areas of endemicity.