The genus Bacillus includes species with diverse natural histories, including free-living nonpathogenic heterotrophs such as B. subtilis and host-dependent pathogens such as B. anthracis (the etiological agent of the disease anthrax) and B. cereus, a cause of food poisoning. Although highly similar genotypically, the ecological niches of these three species are mutually exclusive, which raises the untested hypothesis that their metabolism has speciated along a nutritional tract. Here, we developed a pipeline for quantitative total assessment of the use of diverse sources of carbon for general metabolism to better appreciate the “culinary preferences” of three distinct Bacillus species, as well as related Staphylococcus aureus. We show that each species has widely varying metabolic ability to utilize diverse sources of carbon that correlated to their ecological niches. This approach was applied to the growth and survival of B. anthracis in a blood-like environment and find metabolism shifts from sugar to amino acids as the preferred source of energy. Finally, various nutrients in broth and host-like environments are identified that may promote or interfere with bacterial metabolism during infection.

Pathogenic bacteria take host nutrients to support their growth, division, survival, and pathogenesis. The genus Bacillus includes species with diverse natural histories, including free-living nonpathogenic heterotrophs such as B. subtilis and host-dependent pathogens such as B. anthracis (the etiological agent of the disease anthrax) and B. cereus, a cause of food poisoning. Although highly similar genotypically, the ecological niches of these three species are mutually exclusive, which raises the untested hypothesis that their metabolism has speciated along a nutritional tract. Here, we employed a quantitative measurement of the number of reducing equivalents as a function of growth on hundreds of different sources of carbon to gauge the “culinary preferences” of three distinct Bacillus species, and related Staphylococcus aureus. We show that each species had widely varying metabolic ability to utilize diverse sources of carbon that correlated to their ecological niches. In addition, carbohydrates are shown to be the preferred sources of carbon when grown under ideal in vitro conditions. Rather unexpectedly, these metabolic utilizations did not correspond one-to-one with an increase in biomass, which brings to question what cellular activity should be considered productive when it comes to virulence. Finally, we applied this system to the growth and survival of B. anthracis in a blood-based environment and find that amino acids become the preferred source of energy while demonstrating the possibility of applying this approach to identifying xenobiotics or host compounds that can promote or interfere with bacterial metabolism during infection.Author summarySuccessful organisms must make nutritional adaptations to thrive in their environment. Bacterial pathogens are no exception, having evolved for survival inside their hosts. The host combats these pathogens by depriving them of potential biochemical resources, termed nutritional immunity. This places pathogens under pressure to utilize their resources efficiently and strategically, and their metabolism must in turn be tailored for this situation. In this study, we examined the carbon metabolism of three human pathogens of varying virulence (Bacillus anthracis, Bacillus cereus, and Staphylococcus aureus) and one nonpathogenic Bacillus (Bacillus subtilis) via a phenotype microarray that senses reducing equivalents produced during metabolism. Our analysis shows the existence of distinct preferences by these pathogens towards only a select few carbohydrates and implies reliance on specific metabolic pathways. These metabolic signatures obtained could be distinguished from one bacterial species to another, and we conclude that nutrient preferences offer a new perspective into investigating how pathogens can thrive during infection despite host-induced starvation.

We describe the epidemiology and clinical characteristics of 29 patients with cancer and diarrhea in whom Enteroaggregative Escherichia coli (EAEC) was initially identified by GI BioFire panel multiplex. E. coli strains were successfully isolated from fecal cultures in 14 of 29 patients. Six of the 14 strains were identified as EAEC and 8 belonged to other diverse E. coli groups of unknown pathogenesis. We investigated these strains by their adherence to human intestinal organoids, cytotoxic responses, antibiotic resistance profile, full sequencing of their genomes, and annotation of their functional virulome. Interestingly, we discovered novel and enhanced adherence and aggregative patterns for several diarrheagenic pathotypes that were not previously seen when co-cultured with immortalized cell lines. EAEC isolates displayed exceptional adherence and aggregation to human colonoids compared not only to diverse GI E. coli, but also compared to prototype strains of other diarrheagenic E. coli. Some of the diverse E. coli strains that could not be classified as a conventional pathotype also showed an enhanced aggregative and cytotoxic response. Notably, we found a high carriage rate of antibiotic resistance genes in both EAEC strains and diverse GI E. coli isolates and observed a positive correlation between adherence to colonoids and the number of metal acquisition genes carried in both EAEC and the diverse E. coli strains. This work indicates that E. coli from cancer patients constitute strains of remarkable pathotypic and genomic divergence, including strains of unknown disease etiology with unique virulomes. Future studies will allow for the opportunity to re-define E. coli pathotypes with greater diagnostic accuracy and into more clinically relevant groupings.