*2.2. Investigators*

This multicenter study in the Valencian Community (Spain) includes eight academic medical centers. The patients will be recruited by the respective participating centers of the Public Health System of the Valencian Community, in which there are approximately 15,000 patients with IBD [24]. All the researchers will be gastroenterologists with experience in the follow-up and treatment of patients with CD. The centers participating in the study will have a total load of 3600 patients with CD, 10–15% of whom will be candidates for biological treatment according to estimated data from each center.

## *2.3. Study Objectives*

The main objective of this study is to evaluate the modification of the gu<sup>t</sup> microbiota in CD patients prior to and six months after anti-TNF therapy (infliximab or adalimumab). The secondary objectives are to evaluate the association between the changes in microbiota and the clinical, biological and endoscopic response of the patients; to correlate the normalization of the gu<sup>t</sup> microbiota with the response to anti-TNF treatment; to determine the level of biodiversity of the fecal microbiota at the inclusion and completion of the study in each participant; to evaluate the potential role of the *Faecalibacterium prausnitzii*/*Escherichia coli* and *Faecalibacterium prausnitzii*/*Clostridium coccoides* ratios as an indicator of therapeutic response; and finally, to describe the clinical, biological and epidemiological characteristics of the patients included in the study prior to and six months after anti-TNF treatment.

#### *2.4. Primary Study Variable*

Normalization of gu<sup>t</sup> microbiota after treatment: percentage of patients with dysbiosis of the gu<sup>t</sup> microbiota before the introduction of anti-TNF therapy whose microbiota is normalized after six months of treatment. Dysbiosis is defined as a gu<sup>t</sup> microbiota pattern di fferent from the established patterns of normality according to Arumugam et al. [5]: enterotype 1 (ET B) predominantly contains *Bacteroides*, enterotype 2 (ET P) is characterized by the high abundance of *Prevotella* inversely correlated with *Bacteroides*, and enterotype 3 (ET F) can be distinguished by the presence of *Firmicutes*, highlighting the genus *Ruminococcus*.

#### *2.5. Secondary Study Variables*

Percentage of patients with dysbiosis at inclusion (after initiation of anti-TNF treatment) and at study completion, measured as dichotomous qualitative variables.

Definitions:

Levels of biodiversity of the fecal microbiota for the microbiome analyzed are defined as follows: low biodiversity is a total of five or fewer species, medium biodiversity is a total of between 6 and 10 species, and high biodiversity is a total of 11 or more species present.

Determine the increase in biodiversity pre- and post-anti-TNF treatment, defining an improvement as an increase in the number of species greater than or equal to five with respect to baseline.

Determine the relationship between *Faecalibacterium prausnitzii*/*Escherichia coli* and *Faecalibacterium prausnitzii*/*Clostridium coccoides* pre- and post-anti-TNF treatment.

Associate the presence and type of modifications in the gu<sup>t</sup> microbiota after anti-TNF treatment with the clinical and biological response of the patient during the study.




Clinical, demographic and complementary test variables included in the study protocol (Table 2):

	- - Laboratory tests: complete blood count, erythrocyte sedimentation rate, CRP, fibrinogen, ferritin, transferrin saturation index, total proteins, albumin, urea, creatinine, GOT/AST (Aspartate Aminotransferase), GPT/ALT (Alanine Aminotransferase), GGT (Gamma-glutamyl transpeptidase), ALP (Alkaline Phosphatase), cholesterol and triglycerides
	- - FC
	- - Stool culture, parasites and *Clostridium di*ffi*cile* toxin in feces
	- - Stool collection for microbiota analysis
	- - Radiological (ultrasound/computerized tomography/magnetic resonance imaging) and/or endoscopic testing if available prior to treatment (at least 12 weeks prior to inclusion) or 6 months after treatment.
	- - 72-h dietary record prior to stool sample collection for microbiota analysis. Patients will be provided with a daily survey in which they must record food and beverage intake, specifying characteristics, quantity and brands of packaged products.
	- - Record of adherence to the Mediterranean diet. Together with the dietary record, the patients will also complete a validated survey of adherence to the Mediterranean diet [25] classifying this adherence as low (0–6 points), moderate (7–10 points) or high (11–14 points).

#### **Table 2.** Summary of variables to be collected during the study.


