16S Metagenomics Reveals Unique Diversity and Novel Gut Microbiota Associated with Reactive Arthritis

Abstract

Introduction: Reactive arthritis (ReA) provides a unique opportunity to comprehend how a mucosal infection leads to inflammatory arthritis at a distant site without the apparent invasion of the pathogen. Unfortunately, conventional stool cultures after ReA provide limited information, and there is a dearth of metagenomic studies in ReA.

Methods: Patients who meeting the criteria for ReA or undifferentiated peripheral spondyloarthritis (upSpA: similar ReA phenotype  with exclusion of psoriatic arthritis and inflammatory bowel disease) were included if they presented within 4 weeks of the onset of the current episode of arthritis. Metagenomic DNA was extracted from the stools of these patients and of 36 age- and sex-similar controls. Sequencing and analysis were done using a standard 16S ribosomal pipeline.

Results: Of 55 patients, 20 had postdiarrheal ReA, and 35 were classified as upSpA. A total of 7421 unique OTUs were identified. First, the alpha and beta diversities were similar between ReA and upSpA. Additionally, the microbial abundance at various taxonomical levels was similar, baring one exception.Comparing the gut microbiota of patients versus healthy controls, the patients had significantly higher alpha (Observed, Chao1, Shannon, Simpson, Fisher) and beta diversity measures (nonmetric dimension reduction and principal component analysis using unweighted UniFrac distances).After stringency filters, Proteobacteria were found to have high abundance in cases, while Firmicutes were more abundant in the controls at the phylum level. Six families were overexpressed in patients, while another five were overexpressed in controls. Sixteen genera and 18 species were significantly different between the groups in the DEseq2 analysis. The abundant species in patients were pathobionts such as Staphylococcus aureus (FDR = 5.76E-09), Escherichia coli, (FDR = 2.52E-08), Klebsiella pneumoniae (FDR = 6.37E-08) and Clostridium septicum (FDR = 7.34E-05). Novel species overexpressed in the patient sample included Empedobacter brevis (FDR = 4.15E-04), Roseburia hominis, (FDR = 2.96E-03), Bacillus velezensis (FDR = 4.87E-03), and Crassaminicella, while some Prevotella and related species were underrepresented. Linear discriminant analysis confirmed the strong association of Staphylococcus aureus, Klebsiella pneumoniae and Escherichia coli with ReA.

Conclusion: The microbiota of classical gut-associated ReA and upSpA is similar. These patients have higher alpha and beta diversities in their gut microbiota compared to healthy controls. We identified known and previously unreported species associated with ReA/upSpA that need further targeted studies to explore their role in the pathogenesis of inflammatory arthritis.

Funding: This project was funded by an APLAR Research Grant to SA (2019).

Declaration of Interest: SA has received honorarium as speaker from Pfizer, Dr Reddy’s, Cipla and Novartis (unrelated to the current work) and travel grants from the Indian Rheumatology Association. The other authors do not have any interests to declare.

Ethical Approval: All patients gave their informed consent, and the study was approved by the Institutional Ethics Committee of the Kalinga Institute of Medical Sciences.

Keywords: reactive arthritis, post-infectious arthritis, spondyloarthritis, microbiome