Citation: Gohil, P.; Patel, K.; Patel, S.; Pandit, R.; Suthar, V.; Duggirala, S.; Joshi, M.; Patil, D.; Joshi, C. In-Depth Analysis of an Obligate Anaerobe Paraclostridium bifermentans Isolated from Uterus of Bubalus bubalis. Animals 2022, 12, 1765. https:// doi.org/10.3390/ani12141765 Academic Editors: Luca Grispoldi and Beniamino T. Cenci-Goga Received: 23 March 2022 Accepted: 18 May 2022 Published: 9 July 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). animals Article In-Depth Analysis of an Obligate Anaerobe Paraclostridium bifermentans Isolated from Uterus of Bubalus bubalis Purva Gohil 1 , Kajal Patel 1 , Srushti Patel 2 , Ramesh Pandit 1 , Vishal Suthar 3, * , Srinivas Duggirala 2 , Madhvi Joshi 1 , Deepak Patil 3 and Chaitanya Joshi 1 1 Gujarat Biotechnology Research Centre, Gandhinagar 382011, India; purvagohil5@gmail.com (P.G.); kajalpatel302@gmail.com (K.P.); panditramesh.gbrc@gmail.com (R.P.); madhvimicrobio@gmail.com (M.J.); director.gbrc@gmail.com (C.J.) 2 Department of Microbiology, Gujarat Vidyapith, Gandhinagar 382320, India; srushtipatel.micro@gmail.com (S.P.); dsrinivasmurty@gmail.com (S.D.) 3 Directorate of Research, Kamdhenu University, Gandhinagar 382010, India; db1608@gmail.com * Correspondence: vsuthar28@gmail.com; Tel.: +91-982-449-1049 Simple Summary: Non-specific uterine infections in bovine (uterine line inflammation) are a sig- nificant issue for the dairy industry. These infections are responsible for significant financial losses all over the world. Paraclostridium bifermentans is an obligate anaerobe, gram-positive rod-shaped bacteria belonging to the Clostridia class and the Peptostreptococcaceae family produces endospores. This bacterium has the ability to infiltrate bovine uterine endometrial epithelial cells and cause infection in the endometrium epithelial cells. Our study found that an examination of a buffalo uterus with yellowish purulent discharge reported the presence of pathogenic bacteria Paraclostridium bifermentans, where its genomic characterization, substrate utilization, and antibiotic susceptibility po- tentiality was studied. This discovery indicates the presence of virulence genes as well as pathogenic features. This is the first report of P. bifermentans from the bovine uterus environment. Abstract: Chronic non-specific contamination of the reproductive tract in animals is a major issue during early postpartum, natural coitus, or artificial insemination. Uterine infection is one of the major concerns reducing fertility, production loss, and early culling of the animals. Therefore, the aim of this study was to identify any novel bacterium if present in the uterine environment of Bubalus bubalis causing infections. A strictly anaerobic bacterial strain designated as Paraclostridium bifermentans GBRC was isolated and characterized. Bacterium was found to be Gram positive moderate rod with motility. The optimum growth was observed at 40 ± 2 ◦ C. The pathogenic characteristics of the GBRC strain, such as hemolysis, gelatin hydrolysis, and the production of volatile sulfur compounds, were similar to those seen in the epithelial layer invading pathogenic strains. Assembled genome size was 3.6 MB, with 78 contigs, and a G + C content of 28.10%. Furthermore, the whole genome sequence analysis confirmed the presence of genes encoding virulence factors and provided genomic insights on adaptation of the strain in the uterine environment. Based on the phenotypic and genetic differences with phylogenetic relatives, strain GBRC is proposed to represent a first reported species of the genus Paraclostridium with potential pathogenic character, from the buffalo uterine environment. This study analysis of the GBRC strain serves as a key reference point for the investigation of potential pathogenic strains that may cause endometritis and metritis in bovine. Keywords: Bubalus bubalis; endometritis; Paraclostridium bifermentans; genomic research; toxin; virulence 1. Introduction The Bubalus bubalis species is a significant participant in the Indian dairy sector. Op- timum fertility of dairy buffaloes is inevitably vital for economically sustainable dairy production. Therefore, maintaining the reproduction performance at an optimal level is a Animals 2022, 12, 1765. https://doi.org/10.3390/ani12141765 https://www.mdpi.com/journal/animals