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In a surprising twist, gut pathogens like Salmonella are not repelled by fecal indole; instead, they exploit it to locate nutrient-rich environments, turning a microbial defense into a colonization advantage.

Study: Navigating contradictions: Salmonella Typhimurium chemotaxis amidst conflicting stimuli of the intestinal environment. Image credit: Alpha Tauri 3D Graphics/Shutterstock.com

*Important notice: eLife publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Despite being a known bacterial repellent, indole in faeces does not prevent gut infections. A recent eLife study investigated how enteric bacteria navigate the conflicting chemotactic signals in the gut and how these interactions facilitate colonization.

Gut bacterial colonization

Motile gut bacteria colonize the gastrointestinal tracts of humans and other animals via chemotaxis. They determine chemical effectors in the lumen and navigate the internal environment to proliferate. This navigating process is regulated by chemoreceptor proteins, which identify chemical effectors and transfer signals through a phosphorylation cascade. The chemical signalling effectively controls bacterial flagellar rotation and swimming direction, ultimately forming bacterial colonization’s spatial and temporal patterns.

Most effectors have been analyzed as chemoattractants or chemorepellents in their pure form and under controlled conditions; however, natural environments, such as the gut, contain a complex mixture of contradictory signals. Therefore, it is essential to investigate how bacteria navigate conflicting chemical gradients and determine which signals to prioritize over others for their movement and colonization.

Indole is a crucial chemical effector for enteric bacterial communities that plays a significant role in interbacterial signaling. Gut bacteria excrete indole as a byproduct of tryptophan metabolism and accumulate to millimolar levels in human feces. This microbial metabolite is amphipathic and travels through bacterial membranes.

Indole has multiple functions, including suppressing virulence programs, regulating biofilm formation and motility, and promoting bacteriostatic and bactericidal effects at high concentrations. Most studies have analyzed the response of Escherichia coli (E. coli) chemotaxis in the presence of indole (chemorepellents) as a singular effector. However, whether these responses are preserved when indole is encountered alongside other gut-derived attractants remains unclear.

Enteric pathogens have demonstrated the ability to tolerate indole or evade its chemorepulsive effects under specific conditions. Therefore, it is essential to comprehend how bacteria navigate the conflicting chemotaxis signals of the intestinal environment and how they counter these stimuli to promote growth and colonization. It is also important to identify other bacteria, besides E. coli, which can chemotactically sense or respond to indole.

About the study

The current study hypothesized that indole plays a protective role against intestinal infection. It also investigated the chemotactic mechanism by which enteric pathogens navigate the complex mixture of opposing chemical cues present in fecal material, which is a key source of both indole and nutrients within the gut.

To test the hypothesis, Salmonella enterica serovar Typhimurium was used as a model pathogen because it requires chemotaxis and the chemoreceptor taxis to serine and repellents (Tsr) for cellular infection and invasion of intestinal tissue. Researchers had been particularly interested in Tsr because it involves chemorepellent and chemoattractant L-Serine (L-Ser) responses. S. Typhimurium and other Enterobacteriaceae that possess Tsr orthologues that help them navigate a complex and opposing chemical landscape.

Two types of quantification were performed to explain the explant infection: “invaded” (total Salmonella entering non-phagocytic host cells) and “total” bacteria.

Study findings

A swine colonic explant model that simulates the architecture and size of adult human colonic tissue demonstrated that fecal indole is insufficient to protect against pathogen invasion. To assess the role of chemotaxis in infection, a co-infection strategy was employed using S. Typhimurium strain IR715 wildtype (WT) and a cheY mutant (motile but non-responsive to chemoeffector stimuli) or a tsr deletion mutant.

Experimental findings indicated that under baseline conditions, the intestinal mucosa was accessible to the pathogen. In contrast to the study hypothesis, fecal treatment involving indole was found to provide a similar infection advantage as buffer treatment (without indole), and this effect was mediated by chemotaxis and Tsr.

Compared to the buffer treatment, the fecal treatments provided a higher competitive advantage for the WT-invaded population over the total population at three hours. WT was found to lose its competitive advantage over the chemotactic mutants when incubated with colonic tissue treated with 862 µM pure indole.

WT demonstrated a similar advantage to buffer when treated with 338 µM L-Ser alone, though fecal treatment provided a higher advantage at specific time points. WT also exhibited a colonization advantage at this concentration when L-Ser was co-administered with indole. The WT can infect the colon tissue at a higher level than the chemotactic mutant under all treatment conditions. The current study indicated that chemotaxis and Tsr increase the transit of pathogens to the chemical gradient and enhance their access to intestinal tissue in all conditions except when indole is the sole effector. This finding indicates a differential bacterial perception of indole when present as the sole effector or amidst other fecal effectors.

The CIRA analysis revealed that WT has a stronger chemoattraction response than cheY. Experimental findings demonstrated that S. Typhimurium is attracted to human feces through chemotaxis, involving Tsr, even in high indole concentration. Similar chemotactic navigation was also found in a diverse group of Enterobacteriaceae species that possess Tsr and are associated with human infections.

None of the tested bacterial strains exhibited chemorepulsion from feces despite its high indole content, indicating that the repellent effect is overridden in the native mixture of fecal signals.

The current study noted that the chemorepulsion response occurs much faster than chemoattraction. This finding was indicated through a zone of avoidance significantly visible within the first 10 seconds of indole exposure.

Isothermal titration calorimetry (ITC) analysis indicated that no binding occurs between the Tsr ligand-binding domain (LBD) and indole. This suggests that indole is sensed through a non-canonical mechanism, possibly via proton motive force perturbation or another region of Tsr. An increased amount of the nutrient L-Ser promoted the growth of all Salmonella strains analyzed; however, the growth benefits persisted as long as the concentration of indole was under one mM.

Chemohalation: A newly described bacterial behaviour

One of the novel contributions of this study is the identification of a distinct chemotactic response termed “chemohalation.” This behavior occurs when bacteria are exposed to mixtures of equal or near-equal concentrations of attractants and repellents. Rather than moving directly towards or away from the stimuli, the bacteria form a halo-like distribution around the effector source. This pattern represents a behavioural compromise between opposing cues and may reflect how bacteria fine-tune colonization in the gut’s complex landscape.

The researchers propose “chemohalation” as a new term in the chemotaxis lexicon, analogous to chemoattraction and chemorepulsion.

Conclusions

The results of this study alter the understanding of indole as merely a deterrent to pathogens. Instead, indole may help pathogens to detect niches that are low in microbial competitors and rich in nutrients.

The current study highlighted the limitation of using indole as a single effector when investigating bacterial behavior in a natural environment. This study emphasized that multiple opposing factors shape chemotaxis. It is also suggested that chemotaxis may enhance the probability of successful infection, not just bacterial access, by guiding pathogens to favorable intestinal zones.

Future studies should use other experimental models that fully replicate the complexity of in vivo infection dynamics in the human gut. Human-based or ileal models could provide further insight into bacterial behavior in distinct gut compartments. Targeted genetic analysis for different species is required to confirm whether Enterobacteriaceae use Tsr for fecal attraction.

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*Important notice: eLife publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

New research is shedding light on potential strategies for improving the effectiveness of fecal microbiota transplantation (FMT) for the treatment of Clostridioides difficile infection (CDI).¹

The study enrolled > 1000 adult patients with CDI treated with FMT at the Centre for Faecal Microbiota Transplantation (CEFTA) at Aarhus University Hospital and found the most effective FMT treatment strategy involved administering FMT as multiple-dose capsules or colonoscopy after an extended period of antibiotic pretreatment, regardless of CDI episode, and providing repeated FMT in patients with CDI recurrence. Of note, antibiotic pretreatment choice and prophylactic vancomycin in patients needing antibiotics following FMT did not impact outcomes.¹

Current treatment guidelines from the American Gastroenterological Association recommend the use of FMT-based therapy in patients with recurrent CDI at high risk of recurrence following standard-of-care antibiotics and in hospitalized patients with severe CDI after standard-of-care antibiotics if there is no improvement.²

“Real-world data indicate that one-third of patients do not respond adequately to their first FMT treatment, highlighting the need to optimize FMT treatment strategies,” Sara Ellegaard Paaske, a PhD student in the department of clinical medicine at Aahrus University, and colleagues wrote, calling attention to the current lack of large-scale systematic evidence to guide the decision on when to switch from repeated FMT treatments to antibiotic treatments or to assess the effectiveness of antibiotics in patients recently treated with FMT.¹

To address this gap in research and inform a rational clinical approach to FMT treatment for CDI, investigators conducted a multisite cohort study in the Central and North Denmark Regions. Eligible patients were ≥ 18 years of age with CDI treated with FMT applied through capsules, nasojejunal tube, or colonoscopy. CDI was defined as C. difficile-associated diarrhea (CDAD), characterised as diarrhea (≥ 3 daily Bristol Stool Form Scale 6–7) coupled with a positive C. difficile test.¹

The primary outcome was cure of CDAD 8 weeks after treatment, with cure failure defined as CDAD recurrence, suspected CDAD recurrence, death, or colectomy. Patients were followed until achieving CDAD cure 8 weeks after treatment or until they underwent colectomy, were lost to follow-up, or died.¹

Variables related to the FMT treatment strategy were categorised into pretreatment, FMT, and follow-up and included variation in choice and length of antibiotic pretreatment, FMT administration, FMT dosing, and use of prophylactic vancomycin in patients needing non-CDI antibiotics during follow-up.¹

From May 2016 to December 2023, investigators treated 1200 patients with CDI with FMT applied through capsules, nasojejunal tube, or colonoscopy at 10 hospital departments. Of these patients, 30 were lost to follow-up, leaving 1170 patients who received 1643 FMT treatments for inclusion.¹

Investigators noted vancomycin was the most common antibiotic pretreatment (91%), and capsule FMT was the most frequently used FMT administration (80%).¹

Among the included patients, 699 (60%; 95% CI, 57–63%) achieved cure at week 8 following their first FMT treatment, 342 (29%; 95% CI, 27–32%) had verified recurrence within 8 weeks, 49 (4%; 95% CI, 3–6%) had suspected recurrence in the 8-week follow-up, 73 (6%; 95% CI, 5–8%) died during follow-up, and 7 (1%; 95% CI, 0–1%) received colectomy in the follow-up.¹

Of the 393 patients with verified or suspected recurrence of CDAD, 326 (83%) were treated with up to 5 FMT treatments. Following repeated FMT treatments, 944 patients (81%; 95% CI, 78–83%) achieved CDAD cure at the 8-week follow-up, and 113 had CDAD recurrence or suspected CDAD recurrence (10%; 95% CI, 8–11%).¹

Upon analysis, prolonged antibiotic pretreatment was associated with greater cure rates (65%; odds ratio [OR], 1.22; 95% CI, 1.10–1.36; P <.001). Additionally, investigators noted FMT administration through oral, multi-dose capsules (69%; OR, 1.19; 95% CI, 1.11–1.27; P <.001) or colonoscopy (69%; OR, 1.14; 95% CI, 1.04–1.24; P = .01) resulted in the greatest cure rates.¹

Of note, neither antibiotic pretreatment type nor prophylactic vancomycin during non-CDI antibiotics affected cure rates, and in patients for whom FMT was initially unsuccessful, repeated FMT was more effective than antibiotic treatment alone.¹

“In conclusion, the most effective FMT treatment strategy involved administering FMT as multiple-dose capsules or colonoscopy after an extended period of antibiotic pretreatment, regardless of CDI episode, and providing repeated FMT in patients with CDI recurrence,” investigators wrote.¹ “Future guidelines should address FMT dosing, administration methods, and pretreatment duration to assist clinicians in selecting strategies with the highest likelihood of success.”

References

1. Paaske SE, Baunwall SMD, Rubak T, et al. Clinical management of Clostridioides difficile infection with faecal microbiota transplantation: a real-world cohort study. EClinicalMedicine. doi:10.1016/j.eclinm.2025.103302

2. Brooks A. AGA Supports Fecal Microbiota-Based Therapies for C Diff in New Guideline. HCPLive. February 21, 2024. Accessed July 16, 2025. https://www.hcplive.com/view/aga-supports-fecal-microbiota-based-therapies-for-c-diff-in-new-guideline