Enteric pathogens engage in complex interactions with the host and the resident microbiota to establish gut colonization1-3. Although mechanistic interactions between enteric pathogens and bacterial commensals have been extensively studied, whether and how commensal fungi affect enteric infections remain largely unknown1. Here we show that colonization with the common human gut commensal fungus Candida albicans worsened infections with the enteric pathogen Salmonella enterica subsp. enterica serovar Typhimurium. The presence of C. albicans in the mouse gut increased Salmonella caecal colonization and systemic dissemination. We investigated the underlying mechanism and found that Salmonella binds to C. albicans via type 1 fimbriae and uses its type 3 secretion system to deliver effector proteins into C. albicans. A specific effector, SopB, was sufficient to manipulate C. albicans metabolism and trigger the release of millimolar amounts of arginine into the extracellular environment. The released arginine, in turn, induced expression of the type 3 secretion system in Salmonella, increasing its invasion of epithelial cells. C. albicans deficient in arginine production was unable to increase Salmonella virulence. Arginine-producing C. albicans also dampened the inflammatory response during Salmonella infection. Arginine supplementation in the absence of C. albicans increased the systemic spread of Salmonella and decreased the inflammatory response, phenocopying the presence of C. albicans. In summary, we identified C. albicans colonization as a susceptibility factor for disseminated Salmonella infection and arginine as a central metabolite in the cross-kingdom interaction between fungi, bacteria and host.

Asymptomatic colonization is often critical for persistence of antimicrobial-resistant pathogens, such as Acinetobacter baumannii, and can increase the risk of clinical infections. However, the ecological factors shaping A. baumannii gut colonization remain unclear. We show that A. baumannii and other pathogenic Acinetobacter evolved to utilize the amino acid ornithine, a non-preferred carbon source, to compete with resident microbiota and persist in the gut in mice. A. baumannii encodes ornithine succinyltransferase (AstO) necessary for catabolizing ornithine, especially in conditions of increased microbial diversity. Supplemental dietary ornithine promotes long-term fecal shedding of A. baumannii. By contrast, supplementation of preferred carbon sources-monosodium glutamate or histidine-abolishes the requirement for ornithine catabolism. Additionally, A. baumannii gut carriage is higher in formula-fed human infants, who generally consume higher levels of protein, revealing dietary impacts on Acinetobacter colonization. Together, these results reveal that ornithine catabolism facilitates A. baumannii colonization, providing a reservoir for pathogen spread.

SummaryAntimicrobial resistance is an urgent threat to human health. Asymptomatic colonization is often critical for persistence of antimicrobial-resistant pathogens. Gut colonization by the antimicrobial-resistant priority pathogenAcinetobacter baumanniiis associated with increased risk of clinical infection. Ecological factors shapingA. baumanniigut colonization remain unclear. Here we show thatA. baumanniiand other pathogenicAcinetobacterevolved to utilize the amino acid ornithine, a non- preferred carbon source.A. baumanniiutilizes ornithine to compete with the resident microbiota and persist in the gut in mice. Supplemental dietary ornithine promotes long-term fecal shedding ofA. baumannii. By contrast, supplementation of a preferred carbon source—monosodium glutamate (MSG)— abolishes the requirement forA. baumanniiornithine catabolism. Additionally, we report evidence for diet promotingA. baumanniigut carriage in humans. Together, these results highlight that evolution of ornithine catabolism allowsA. baumanniito compete with the microbiota in the gut, a reservoir for pathogen spread.

Candida albicans and Staphylococcus aureus are two commonly associated pathogens that cause nosocomial infections with high morbidity and mortality. Our prior and current work using a murine model of polymicrobial intra-abdominal infection (IAI) demonstrates that synergistic lethality is driven by Candida-induced upregulation of functional S. aureus α-toxin leading to polymicrobial sepsis and organ damage. In order to determine the candidal effector(s) mediating enhanced virulence, an unbiased screen of C. albicans transcription factor mutants was undertaken revealing that zcf13Δ/Δ fails to drive augmented α-toxin or lethal synergism during co-infection. A combination of transcriptional and phenotypic profiling approaches shows that ZCF13 regulates genes involved in pentose metabolism, including RBK1 and HGT7 that contribute to fungal ribose catabolism and uptake, respectively. Subsequent experiments reveal that ribose inhibits the staphylococcal agr quorum sensing system and concomitantly represses toxicity. Unlike wild-type C. albicans, zcf13Δ/Δ did not effectively utilize ribose during co-culture or co-infection leading to exogenous ribose accumulation and agr repression. Forced expression of RBK1 and HGT7 in the zcf13Δ/Δ mutant fully restores pathogenicity during co-infection. Collectively, our results detail the interwoven complexities of cross-kingdom interactions and highlight how intermicrobial metabolism impacts polymicrobial disease pathogenesis with devastating consequences for the host.

A diverse array of commensal microorganisms inhabits the human intestinal tract. The most abundant and most studied members of this microbial community are undoubtedly bacteria. Their important role in gut physiology, defense against pathogens, and immune system education has been well documented over the last decades. However, the gut microbiome is not restricted to bacteria. It encompasses the entire breadth of microbial life: viruses, archaea, fungi, protists, and parasitic worms can also be found in the gut. While less studied than bacteria, their divergent but important roles during health and disease have become increasingly more appreciated. This review focuses on these understudied members of the gut microbiome. We will detail the composition and development of these microbial communities and will specifically highlight their functional interactions with enteric pathogens, such as species of the family Enterobacteriaceae. The interactions can be direct through physical interactions, or indirect through secreted metabolites or modulation of the immune response. We will present general concepts and specific examples of how non-bacterial gut communities modulate bacterial pathogenesis and present an outlook for future gut microbiome research that includes these communities.

Candida albicans and Staphylococcus aureus are among the most prevalent nosocomial pathogens that are responsible for severe morbidity and mortality, even with appropriate treatment. Using a murine model of polymicrobial intra-abdominal infection (IAI), we have previously shown that coinfection with these pathogens results in synergistic lethality that is partially dependent on exacerbated prostaglandin signaling, while monomicrobial infection is nonlethal. Therefore, the objective of this study was to identify staphylococcal virulence determinants that drive lethal synergism during polymicrobial IAI. Using the toxigenic S. aureus strain JE2, we observed that coinfection with C. albicans led to a striking 80 to 100% mortality rate within 20 h postinoculation (p.i.) while monomicrobial infections were nonlethal. Use of a green fluorescent protein (GFP)-P3 promoter S. aureus reporter strain revealed enhanced activation of the staphylococcal agr quorum sensing system during in vitro polymicrobial versus monomicrobial growth. Analyses by quantitative real-time PCR (qPCR), Western blot, and toxin functional assays confirmed enhanced agr-associated gene transcription and increases in secreted alpha-and delta-toxins. C. albicans-mediated elevated toxin production and hemolytic activity were determined to be agrA dependent, and genetic knockout and complementation of hla identified alpha-toxin as the key staphylococcal virulence factor driving lethal synergism. Analysis of mono-and polymicrobial infections 8 h p.i. demonstrated equivalent bacterial burdens in the peritoneal cavity but significantly elevated levels of alpha-toxin (3-fold) and the eicosanoid prostaglandin E2 (PGE2) (4-fold) during coinfection. Importantly, prophylactic passive immunization using the monoclonal anti-alpha-toxin antibody MEDI4893* led to significantly improved survival rates compared to those following treatment with isotype control antibody. Collectively, these results define alpha-toxin as an essential virulence determinant during C. albicans-S. aureus IAI and describe a novel mechanism by which a human-pathogenic fungus can augment the virulence of a highly pathogenic bacterium in vivo. IMPORTANCE Relatively little is known about the complex interactions and signaling events that occur between microbes and even less so about how microbial "cross talk" shapes human health and disease. Candida albicans (a fungus) and Staphylococcus aureus (a bacterium) are formidable human nosocomial pathogens, causing severe morbidity and mortality. Moreover, they are frequently coisolated from central venous catheters and deep-seated infections, including intra-abdominal sepsis. In this work, we have shown that coinfection with C. albicans and S. aureus is highly lethal, leading to >80% mortality by day 1 postinfection, whereas monoinfection with C. albicans or S. aureus does not cause mortality. This infectious synergism is dependent on the expression of staphylococcal alpha-toxin, and secretion of this potent virulence factor is actually augmented by C. albicans via an agr-dependent mechanism. Moreover, prophylactic neutralization of alpha-toxin with a monoclonal antibody is sufficient to elicit protection during coinfection. Therefore, we have demonstrated that a pathogenic fungus can enhance virulence determinants of a bacterium in vivo with devastating consequences to the host. These results have important implications in the surveillance and treatment of polymicrobial disease and highlight the dynamic intersection of environment, pathogens, and host.

Candida albicans and Staphylococcus aureus are commonly coisolated from central venous catheters and deep-seated infections, including intra-abdominal sepsis. Thus, they represent a significant cause of nosocomial morbidity and mortality. Yet how these organisms behave in the context of polymicrobial growth remains poorly understood. In this work, we set out to determine the mechanism by which activation of the staphylococcal agr quorum sensing system and production of its major virulence effector alpha-toxin is enhanced during coculture with C. albicans . Surprisingly, we likely ruled out that a secreted candidal factor drives this process. Instead, we demonstrated that alkalinization of the extracellular milieu by C. albicans and other Candida species correlated with elevated agr activity. Thus, we propose a mechanism where modulation of the extracellular pH by fungal opportunists can indirectly alter virulence of a bacterial pathogen. Uncovering molecular events that drive interkingdom pathogenicity mechanisms may enhance surveillance and treatment for devastating polymicrobial infections.

While Koch’s Postulates have established rules for microbial pathogenesis that have been extremely beneficial for monomicrobial infections, new studies regarding polymicrobial pathogenesis defy these standards. The explosion of phylogenetic sequence data has revolutionized concepts of microbial interactions on and within the host. However, there remains a paucity of functional follow-up studies to delineate mechanisms driven by such interactions and how they shape health or disease. That said, one particular microbial pairing, the fungal opportunist Candida albicans and the bacterial pathogen Staphylococcus aureus, has received much attention over the last decade. Therefore, the objective of this review is to discuss the multi-faceted mechanisms employed by these two ubiquitous human pathogens during polymicrobial growth, including how they: establish and persist in inter-Kingdom biofilms, tolerate antimicrobial therapy, co-invade host tissue, exacerbate quorum sensing and staphylococcal toxin production, and elicit infectious synergism. Commentary regarding new challenges and remaining questions related to future discovery of this fascinating fungal–bacterial interaction is also provided.

Magnetic biochar (MBC) was produced by magnetite (Fe3O4) precipitation onto Douglas fir biochar (NBC) that had been generated by wet fast pyrolysis. Treating an aqueous Fe3+/Fe2+ solution with NaOH induced Fe3O4 to nucleate and deposit on NBC. The NBC and the resulting MBC were used to remove Pb2+ and Cd2+ from water. Both biochars were characterized by SEM, EDX, TEM, PZC, XRD, elemental analysis, and surface area measurements. Batch sorption studies were carried out from pH 2–7 with adsorbate concentrations from 10 to 250 mg/L at 25, 35 and 45 °C. MBC and NBC suspensions in the contaminated solutions were vortexed for two min. Then MBC was magnetically removed, while NBC required filtration. Remediated solutions were then analyzed using AAS. The amounts of lead and cadmium adsorbed onto both NBC and MBC were lower at low pH values and increased with increasing pH. The Langmuir and Freundlich adsorption isotherm models were applied to describe equilibrium data. The maximum Langmuir adsorption capacities at pH 5 and 45 °C for Pb2+ and Cd2+ uptake were ∼40 and ∼16 mg/g for NBC and ∼27 and ∼11 mg/g for MBC, respectively. NBC and MBC recycling was carried out after metal ion extraction with 0.1 M HCl and water. Adsorption kinetics of NBC and MBC were compared with four other biochars (mixed feed, magnetized mixed feed, pinewood and magnetized switchgrass). Adsorption equilibria of Pb2+ and Cd2+ onto both NBC and MBC were reached within 2 min while the other biochars required from 2 to 20 h. NBC and MBC have potential as low cost, green adsorbents for rapid lead and cadmium remediation as replacements for more expensive commercial activated carbon.

Biochar was produced from the fast pyrolysis of Douglas fir (DFBC). Magnetic biochar (MDFBC) was prepared by magnetite (Fe3O4) precipitation onto the biochar’s surface from an aqueous Fe3+/Fe2+ solution upon NaOH treatment. The resulting MDFBC was used to remove 4-nitroaniline (4NA), salicylic acid (SA), benzoic acid (BA) and phthalic acid (PA) from water. The surface chemistry and composition of the MDFBC were examined by SEM, SEM-EDX, TEM, PZC, XPS, XRD, elemental analysis, and surface area measurements. Batch sorption studies were carried out from pH 2 to 10 and adsorbate concentrations from 25 to 500mg/L at 15, 25, 35 and 45°C. MDFBC suspensions in the contaminated solutions were vortexed for two min and then magnetically removed. Remediated solutions were then analyzed using UV–Visible spectroscopy. The amounts of 4NA, SA, BA and PA adsorbed onto MDFBC was higher at low pH values and decreased with increasing pH. MDFBC sorption at 15, 25, 35 and 45°C was evaluated using the Langmuir, Freundlich, Sips, Redlich–Peterson, and Toth adsorption isotherm models. Langmuir adsorption capacities at pH 5 and 45°C for 4NA, SA, BA and PA were 114, 109, 90 and 86mg/g, respectively. Thrice recycled MDFBC using extraction with methanol (5×10mL) and water (10mL) had 90% of the original adsorption capacities for 4NA, SA, BA and PA, respectively. The adsorption kinetics of MDFBC and DFBC were far faster than four other biochars (mixed feed, magnetized mixed feed, pinewood and magnetized switchgrass) and also faster than a commercial activated carbon. Equilibrium for MDFBC and its DFBC precursor was reached within 2min, while the other biochars took between 8 and 20h. The fast adsorption kinetics and high adsorption capacities of MDFBC could be advantageously employed in filtration devices, columns, or as shown here, in batch operations with stirring to speed adsorption, followed by magnetic separation of the adsorbent for regeneration.

Adsorption studies of salicylic acid (SA) and 4-nitroaniline (4NA) from aqueous solutions were performed with magnetic biochar (MBC) in order to train students in analytical techniques such as standard calibration curves, UV-vis spectrophotometry, and chemical separations within the context of wastewater purification. Analysis of samples purified by MBC enhances student understanding of water quality and the importance of potable water production, a growing worldwide concern. MBC was prepared by iron oxide precipitation onto the biochar surface using an aqueous Fe3+/Fe2+ solution followed by NaOH treatment. MBC effectiveness in removing organic contaminants from water was evaluated using UV-vis spectroscopy, Beer's law, and a comparison to standard calibration curves while determining the concentrations of two contaminant compounds. The use of MBC to remove organic contaminants in water highlights the effectiveness of this technique for wastewater remediation and prompt discussion of water quality concerns with undergraduate students.