Differential prevalence of pathobionts and host gene polymorphisms in chronic inflammatory intestinal diseases: Crohn’s disease and intestinal tuberculosis

Background and objectives Crohn’s disease (CD) and Intestinal tuberculosis (ITB) are chronic inflammatory ulcero-constrictive intestinal diseases with similar phenotype. Although both are disease models of chronic inflammation and their clinical presentations, imaging, histological and endoscopic findings are very similar, yet their etiologies are diverse. Hence, we aimed to look at differences in the prevalence of pathobionts like adherent-invasive Escherichia coli (AIEC), Listeria monocytogenes, Campylobacter jejuni and Yersinia enterocolitica in CD and ITB as well as their associations with host-associated genetic polymorphisms in genes majorly involved in pathways of microbial handling and immune responses. Methods The study cohort included 142 subjects (69 patients with CD, 32 with ITB and 41 controls). RT- PCR amplification was used to detect the presence of AIEC, L. monocytogenes, C. jejuni, and Y. enterocolitica DNA in colonic mucosal biopsies. Additionally, we tested three SNPs in IRGM (rs13361189, rs10065172, and rs4958847), one SNP in ATG16L1 (rs2241880) and one SNP in TNFRSF1A (rs4149570) by real-time PCR with SYBR green from peripheral blood samples in this cohort. Results In patients with CD, AIEC was most frequently present (16/ 69, 23.19%) followed by L. monocytogenes (14/69, 20.29%), C. jejuni (9/69, 13.04%), and Y. enterocolitica (7/69, 10.14%). Among them, L. monocytogenes and Y. enterocolitica were significantly associated with CD (p = 0.02). In addition, we identified all the three SNPs in IRGM (rs13361189, rs10065172, and rs4958847), one SNP in ATG16L1 (rs2241880) and TNFRSF1A (rs4149570) with a significant difference in frequency in patients with CD compared with ITB and controls (p<0.05). Conclusion Higher prevalence of host gene polymorphisms, as well as the presence of pathobionts, was seen in the colonic mucosa of patients with CD as compared to ITB, although both are disease models of chronic inflammation.

The research article entitled "Differential prevalence of pathobionts and host gene polymorphisms in chronic inflammatory intestinal diseases: Crohn's Disease and Intestinal Tuberculosis" (PONE-D-21-12204) by Khan et.al highlights the prevalence of pathobionts like adherent-invasive Escherichia coli (AIEC), Listeria monocytogenes, Campylobacter jejuni and Yersinia enterocolitica in Crohn's Disease (CD) and Intestinal Tuberculosis (ITB). Consistent with previous studies, this study reveal a significantly enhanced prevalence of the risk alleles of IRGM (rs13361189, rs4958847 and rs10065172) and ATG16L1 (rs2241880) TNFRSF1A polymorphism (rs4149570) genes with CD.
The manuscript is well written and suitable statistical tools and analysis have been applied to determine the significance of the data. Although, the study is limited with lack of statistically significant data, possibly due to the small sample size as also admitted by authors in the manuscript, the findings from this study are very important especially the prevalence of specific SNPs in genes implicated in IBD, in a North Indian cohort. This study takes the field one step closer to a non-invasive and affordable diagnostic procedure for IBD and may prove to be useful for the follow up studies with a bigger sample size in Indian population. Such investigative studies should be pursued with further experiments involving the colonization of wild type, germ-free, and genetically modified mice with an individual bacterial species or with a combination of bacteria, in order to identify the exact causal bacterial strain or core microbiome and clarify the fate of the gut microbiota in IBD.
The manuscript may be considered for publication after addressing the following comments:

Comments:
1. In the introduction, authors mentioned Mycobacterium avium subspecies paratuberculosis (MAP) as one of the pathogen associated with CD. Also there is moderately high seroprevalence ( 3. Since sample storage conditions can affect the quantification of the target microbial markers especially fast growing E.coli, the storage conditions of the clinical samples (Biopsies) before the isolation of the nucleic acid should be described in the methods.
4. Refer lines 183-184 (C. jejuni prevalence) & 186-188 (AIEC prevalence). Since the Prevalence data for these two strains is not statistically significant, affirmative statements should be avoided. Sentences can be rewritten for better clarity and avoiding any misunderstanding.
5. Refer to lines 258-260, Pls provide suitable references of the previously published studies from the literature, if any, where the comparative assessment of the prevalence of various pathobionts, between CD and ITB was done.
A comparison between their methods & findings with that in the present study will be useful.
6. Refer lines 269-270, The statement "The control and ITB groups were found to have low infection incidences" holds true only for L. monocytogenes and Y. enterocolitica as only these were significantly less prevalent in the control and ITB groups as compared to CD patients, whereas there was no significant difference in the incidence of the AIEC and C. jejuni between the groups. So sentence need to be modified accordingly.

7.
Recent case study of an Asian female patient (Korean) with Crohn's Disease reported her case to be initially misdiagnosed as Intestinal Tuberculosis due to active pulmonary tuberculosis (Park et.al., Korean J Gastroenterol, 2021). Given that India is TB endemic country, it may be worthy to include the active pulmonary tuberculosis in the clinical history of the patient to avoid such misdiagnosis specifically in studies involving comparison of CD Vs. ITB. Also, since the baseline features of gut microbiota after or during anti-TB treatment among ITB patients may differ, it may be worth to mention the ATT treatment in recent past of subjects included in this study. The inclusion and exclusion criteria (especially history of tuberculosis, previous/existing antitubercular drug therapy at the time of specimen collection) for various groups (CD, ITB and Controls) under this study have not been defined and should be included in the manuscript.
8. As this study doesn't demonstrate any association between the CD associated genetic polymorphisms and the prevalence of various pathobionts, its implications in pathogenesis of CD and ITB should have been discussed in correlation with clinical symptoms of patients under different groups in this study. This would have provided more insights to understand the role of these marker genes in etiology of CD and ITB.