Complex nutrient utilization strategies highlight the diverse functional capacity of the microbiota. For example, host consumption of a high-fat diet alters host colonocyte and microbial metabolism, stimulating the growth of IBD-associated intestinal E. coli (Turnbaugh et al., 2009; Litvak et al., 2018). Complex host:microbe interactions drive this phenomenon and have been well-reviewed (Winter and Bäumler, 2014; Litvak et al., 2017; Litvak et al., 2018; Shelton and Byndloss, 2020). Briefly, loss of butyrate-producing microbes in the inflamed gut reduces activation of butyrate sensor PPARγ in colonocytes, which prevents beta-oxidation and increases bioavailable oxygen (Byndloss et al., 2017). Epithelial oxygenation drives aerobic respiration of pathogenic and non-pathogenic Enterobacteriaceae and fuels their expansion at the mucosa (Litvak et al., 2017; Cevallos et al., 2019; Chanin et al., 2020). Currently, the impact of gut oxygenation and/or inflammation-associated loss of anaerobiosis on E. coli metabolite production has not been explored.

The flexibility of Enterobacteriaceae metabolism and carbon utilization are implicated in its ability to persist in the inflamed gut and promote inflammatory disease (Zhang et al., 2022). Undigested carbohydrates and complex sugars in the intestinal mucus layer serve as an important energy source for gut microbes, including AIEC (Kim et al., 2017). In fact, comparative genomics and functional assays have revealed that the metabolism of fucose, 1,2-propanediol, ethanolamine, and downstream metabolites present in the gut are important for AIEC persistence in the inflamed GI tract (Dogan et al., 2014; Delmas et al., 2019; Elhenawy et al., 2019; Ormsby et al., 2019; Viladomiu et al., 2021). E. coli from the stool of IBD patients often express genes for metabolism of ethanolamine (eutB, eutS) and 1,2-propanediol (pduC), which are thought to be a metabolic adaptation that is advantageous for AIEC to overcome nutritional limitation and colonize the gut (Ormsby et al., 2019; Viladomiu et al., 2021). However, there is still a lot to uncover and understand on how the metabolism of these substrates impact Enterobacteriaceae metabolite production and pro-inflammatory capacity.

During inflammation, the host limits nutrient availability to restrict growth of invading microbes (Cassat and Skaar, 2013; Becker and Skaar, 2014). Bioavailability of trace metals (e.g., iron, zinc, and copper) are tightly controlled and can change based on host diet and inflammation status (Pajarillo et al., 2021). Metals are vital nutrients for all forms of life and act as cofactors for essential proteins (Andreini et al., 2008; Hood and Skaar, 2012; Diaz-Ochoa et al., 2014; Palmer and Skaar, 2015). Thus, throughout evolution, the battle for metal acquisition has persisted between bacteria and host (Hood and Skaar, 2012; Skaar and Raffatellu, 2015). Hosts developed “Nutritional Immunity” to overcome sequestration of essential metals by invading pathogens, especially by controlling metal concentration through metal-binding proteins such as iron-binding lactoferrin, transferrin, lipocalin, and zinc-binding calprotectin (Oram and Reiter, 1968; Zackular et al., 2015; Palmer and Skaar, 2016). However, microbes also developed methods for effectively sequestering host metals, even during nutritional immunity. One means of evading nutritional immunity is via “stealth siderophores” which cannot be neutralized by the host.

In metal-limited environments, like the inflamed gut, bacteria produce siderophores to outcompete the host for essential metals (Ellermann and Arthur, 2017). Siderophores are low molecular weight compounds that bind various metals with high affinity (Hider and Kong, 2010; Holden and Bachman, 2015). The structural variety of siderophores makes it easy for bacteria to acquire essential metals and overcome growth limitations within the host (Hider and Kong, 2010; Holden and Bachman, 2015).

Enterobacteriaceae produce a siderophore called Enterobactin (Ent), which has an extremely high binding affinity to ferric iron  (Pollack and Neilands, 1970; Young and Gibson, 1979; Raymond et al., 2003). Ent is commonly produced in low iron environments through de-repression of the Ent biosynthetic gene cluster (BGC) by the DNA-binding Ferric Uptake Regulation protein, Fur (Crosa and Walsh, 2002). Host neutrophils and epithelial cells secrete lipocalin-2 (Lcn2) into the mucosa, which binds the Ent-iron complex and inhibits the growth of pathogenic bacteria (Flo et al., 2004; Bachman et al., 2012). Interestingly, Ent produced by commensal bacteria plays an important role in mitochondria iron uptake and homeostasis, which has a beneficial effect on host development (Qi and Han, 2018). Therefore, although siderophores have been thought to play a negative role in iron homeostasis and induce pathogenesis, Ent could provide a host advantage during colonization with certain bacterial strains.

Yersiniabactin (Ybt) is a stealth siderophore, harbored by some host-associated E. coli strains, that evades host nutritional immunity. The Ybt BGC, like Ent, is repressed by Fur in the presence of ample iron (Haag et al., 1993; Gehring et al., 1998; Pelludat et al., 1998; Crosa and Walsh, 2002). Ybt was originally described as an essential fitness factor for Yersinia pestis and is common in Klebsiella spp. and gut E. coli (Bach et al., 2000; Lawlor et al., 2007; Fetherston et al., 2010; Dogan et al., 2014; Brumbaugh et al., 2015; Ellermann et al., 2019). Ybt-mediated iron acquisition is important for virulence, niche formation by pathogens, and colonization resistance by commensal microbes (Koh et al., 2015; Ellermann and Arthur, 2017). Ybt uniquely binds biologically important metals other than iron to aid in infection, including zinc, copper, and nickel (Chaturvedi et al., 2012; Bobrov et al., 2014; Koh et al., 2015; Robinson et al., 2018) A recent paper demonstrated that Ybt-zinc acquisition allows the probiotic E. coli Nissle to tolerate host-mediated zinc sequestration, allowing beneficial Nissle to bloom in the gut (Behnsen et al., 2021). However, it is reasonable to suspect that Ybt also permits AIEC expansion and promotes further inflammation in the gut of IBD patients. In addition, Ybt can steal metal from host cells, disrupting host metal homeostasis and promoting disease. We demonstrated that AIEC production of Ybt, but not bacterial utilization of this siderophore, enhanced inflammation and induced intestinal fibrosis in the Il10^-/- mouse model (Ellermann et al., 2019). This study suggests that Ybt has direct effects on the host. Indeed, Ybt from Klebsiella spp. can deplete iron from airway epithelial cells and activate the hypoxia-inducible factor 1-alpha (HIF-1α) pathway (Holden et al., 2014). But how Ybt modulates host HIF-1α activation, and the biological consequences of this regulation remain unclear.

Intestinal E. coli compete for nutrients and niches in the intestine in a variety of ways, including Type VI secretion systems (T6SSs), siderophores, and microcins (Sorbara and Pamer, 2019). Microcins are small, secreted proteins with antimicrobial activities that can kill related bacteria by binding to siderophores and entering the cell like a “Trojan Horse” (Duquesne et al., 2007; Massip and Oswald, 2020). Microcins are produced by some uropathogenic, intestinal, and probiotic Enterobacteriaceae and may be exploited as therapeutics to prevent the bloom and persistent colonization of pathogens and inflammation-associated E. coli (Sassone-Corsi et al., 2016). While this area of research is understudied in the IBD field, the importance of microcins in infectious disease colonization resistance and their functional relationship with siderophores makes them an interesting target to pursue for microbiome modulation.

Colibactin is a microbially-derived genotoxin produced by a BGC known as the pks island. It is well appreciated that colibactin elicits host DNA damage and is implicated in the initiation of CRC (Nougayrède et al., 2006; Arthur et al., 2012; Arthur et al., 2014; Arthur, 2020; Dziubańska-Kusibab et al., 2020; Lopez et al., 2020; Pleguezuelos-Manzano et al., 2020). However, colibactin is a complex natural product with a high biosynthetic cost and no clear role in bacterial fitness, beyond the newly discovered role for the ClbS immunity protein that protects bacteria from colibactin-triggered prophage induction (Silpe et al., 2022). Although poorly understood, colibactin regulation is linked to E. coli virulence factors, including the Ent and Ybt siderophores, low iron, and antibiotic stress (Martin et al., 2017; Sadecki et al., 2021). Although mechanisms remain unclear, it has been postulated that colibactin protects against membrane stress and may have antibiotic activity against competing microbes. Sugimoto et al. developed a module to discover BGCs in metagenomic sequencing data of the human microbiome and revealed BGCs that encode 13 type II polyketides; among them, two polyketides have strong antibacterial activities (Sugimoto et al., 2019). This study suggests that uncharacterized BGC products may be important contributors to microbe:microbe competition in the gut.

Opposite of bacterial antagonism, neighboring microbes can promote functional synergy within polymicrobial communities. This can be detrimental in the context of host disease, as the microbiota can inadvertently heighten the virulence activity of pathogens. For example, Bacteroides thetaiotaomicron can alter gut mucins, enhancing E. coli virulence (Becattini et al., 2016). In addition, Enterococcus faecalis stimulates E. coli siderophore production that promotes E. coli growth and biofilm formation in metal-limited environments (Keogh et al., 2016). Propionate, an abundant SCFA commonly derived from Bacteroides spp., can mediate colonization resistance against Salmonella enterica serovar Typhimurium (S. Tm) (Shelton et al., 2022). However, in the inflamed gut, both S. Tm and E. coli use nitrate and nitrate-dependent propionate metabolism for intestinal expansion (Winter et al., 2013; Shelton et al., 2022) Furthermore, S. Tm and pathogenic E. coli utilize aspartate ammonia-lyase (aspA)-dependent fumarate respiration for growth in the murine gut during colitis (Yoo et al., 2022). Numerous instances of microbial functional synergy have been described in the inflamed gut (Hughes et al., 2017; Spiga et al., 2017; Gillis et al., 2018; Zhu et al., 2020), and it will be important to understand their contribution to disease in human IBD patients.

IBD anti-inflammatories, such as anti-tumor necrosis factor-alpha (TNFα), improve IBD disease activity and can modulate the microbiome in a way that reduces the development of IBD-associated CRC (Yang et al., 2020). This reduction in carcinogenicity of the microbiome is attributed to decreased abundance of colibactin-producing E. coli (Yang et al., 2020). Yet, it is poorly understood how colibactin production is modulated on a per-cell basis and whether xenobiotics can impact colibactin production and pro-carcinogenic effects.

Pharmaceuticals can be used as building blocks for bacterial metabolite production by bacteria. E. coli use an enzyme termed YbtS, a salicylate synthase, in the early steps of building Ybt. It is possible that exogenous salicylate (i.e., Asprin) can compensate for the loss of this enzyme and, even in its absence, promote Ybt production (Pelludat et al., 2003). Salicylates also alter bacterial susceptibility to antibiotics by activating E. coli multiple antibiotic resistance (Mar), leading to increased expression of the multidrug efflux pump AcrAB-TolC (Cohen et al., 1993; Sharma et al., 2017). TolC is also important in Ent efflux, so it will be valuable to assess how salicylates can impact bacterial fitness and pro-inflammatory potential in the gut.

Another salicylate-derived drug, mesalamine (5-aminosalicylic acid, 5-ASA), is an NSAID used in the treatment of IBD and specifically ulcerative colitis (Campieri et al., 1981; Dew et al., 1983). Mesalamine reduces microbial burden in the feces and at the mucosal niche, impacting AIEC virulence gene expression, motility, epithelial adherence/invasion, and intra-macrophage survival – all believed to help control active disease symptoms (Zhang et al., 2018; Dai et al., 2021). These direct effects of mesalamine on bacteria may be driven by the inhibition of polyphosphate kinase and rapid loss of bacterial polyphosphate levels, which can sensitize bacteria to oxidative stress and reduce colonization and biofilm formation in inflamed environments (Dahl et al., 2017). Mesalamine treatment of colibactin-producing E. coli can decrease transcription of the colibactin pks island, but this appears to be independent of the polyphosphate kinase, indicating a novel means of regulation (Zhang et al., 2018; Tang-Fichaux et al., 2020). Direct effects on gene expression also occur in S. Tm, where mesalamine impacts genes associated with bacterial invasion, cellular metabolism, and stress response (Kaufman et al., 2009). Furthermore, subinhibitory levels of the antibiotic sulfamethoxazole (SMX) induce E. coli metabolite production of antioxidants termed colipterins, which induce host anti-inflammatory IL-10 and reduce colitis in mouse models (Park et al., 2020). It will be important to determine how these clinically relevant IBD drugs impact E. coli behavior and metabolite production among the rich consortium of other microbes and both host and microbe-derived metabolites within the inflamed gut.

Modern-day antibiotics are invaluable in controlling pathogenic infections but can have long-lasting effects on the microbiota, including antibiotic resistance among resident gut microbes. Resistance mechanisms develop in natural environments and can easily diffuse through microbial populations (Becattini et al., 2016). In addition, large-scale antibiotic pressure through medical and industrial practices has led to a surge of antibiotic resistance. Based on a study of healthy subjects, it was estimated that 20% of bacteria isolated from feces harbored antibiotic resistance elements (Sommer et al., 2009). Another study found a greater incidence of antibiotic resistance among E. coli cultured from the ileum of IBD and non-IBD patients, most notably to antibiotics commonly used to treat GI infection (Dogan et al., 2013). Antibiotic resistance mechanisms often cause a shift in bacterial metabolism; thus, it is common for the downregulation of energy-intensive microbial activities to be associated with antibiotic resistance. We recently observed that evolved resistance of E. coli to polymyxin B surprisingly upregulates colibactin production, including elevated transcription and protein levels, which induces increased DNA damage in mammalian cells (Sadecki et al., 2021). Mechanisms driving this effect remain unknown, yet the impact of IBD-associated antibiotic resistance on bacterial metabolite production is an active area of research, due to its likely importance in intestinal disease.