bioRxiv preprint doi: https://doi.org/10.1101/2020.07.13.200808; this version posted July 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 1 Microbiome Dynamics of Bovine Mastitis Progression and Genomic Determinants 2 M. Nazmul Hoque1,2, Arif Istiaq1,3,4, M. Shaminur Rahman1, M. Rafiul Islam1, Azraf Anwar5, 3 AMAM Zonaed Siddiki6, Munawar Sultana1, Keith A. Crandall7, M. Anwar Hossain1,8,* 4 1Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh 5 2Department of Gynecology, Obstetrics and Reproductive Health, Bangabandhu Sheikh Mujibur 6 Rahman Agricultural University, Gazipur-1706, Bangladesh 7 3Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan 8 4Present address: Department of Stem Cell Biology, Faculty of Arts and Science, Kyushu 9 University, Fukuoka, Japan 10 5Independent Researcher, 47-07 41st Street, New York, USA, Email: [email protected] 11 6Department of Pathology and Parasitology, Chattogram Veterinary and Animal Sciences 12 University, Chattogram-4202, Bangladesh 13 7Computational Biology Institute and Department of Biostatistics & Bioinformatics, Milken 14 Institute School of Public Health, The George Washington University, Washington, DC, USA 15 8Present address: Vice-Chancellor, Jashore University of Science and Technology, Jashore-7408, 16 Bangladesh 17 18 *Correspondence to: [email protected] 19 20 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.13.200808; this version posted July 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 21 Abstract 22 The milk of lactating cows presents a complex ecosystem of interconnected microbial 23 communities which can impose a significant influence on the pathophysiology of mastitis. 24 Previously, we reported the alteration of microbiome (bacteria, archaea, virus) composition 25 between clinical mastitis (CM) and healthy (H) milk. We hypothesized possible dynamic shifts 26 of microbiome compositions with the progress of different pathological states of mastitis (CM, 27 Recurrent CM; RCM, Subclinical Mastitis; SCM) determined by its favoring genomic potentials. 28 To evaluate this hypothesis, we employed whole metagenome sequencing (WMS) in 20 milk 29 samples (CM = 5, RCM = 6, SCM = 4, H = 5) to unravel the microbiome dynamics, 30 interrelation, and relevant metabolic functions. PathoScope (PS) and MG-RAST (MR) analyses 31 mapped the WMS data to 442 bacterial, 58 archaeal and 48 viral genomes with distinct variation 32 in microbiome composition and abundances across these metagenomes (CM>H>RCM>SCM). 33 PS analysis identified 385, 65, 80 and 144 bacterial strains in CM, RCM, SCM, and H milk, 34 respectively, with an inclusion of 67.19% previously unreported opportunistic strains in mastitis 35 metagenomes. Moreover, MR detected 56, 13, 9 and 46 archaeal, and 40, 24, 11 and 37 viral 36 genera in CM, RCM, SCM and H-milk metagenomes, respectively. The CM-microbiomes had 37 closest association with RCM-microbiomes followed by SCM, and H-microbiomes. 38 Furthermore, we identified 333, 304, 183 and 50 virulence factors-associated genes (VFGs), and 39 48, 31, 11 and 6 antibiotic resistance genes (AGRs) in CM, RCM, SCM, and H-microbiomes, 40 respectively, showing a significant correlation between the relative abundances of VFGs (p = 41 0.001), ARGs (p = 0.0001), and associated bacterial taxa. We also detected correlated variations 42 in the presence and abundance of several metabolic functional genes related to bacterial 43 colonization, proliferation, chemotaxis, motility and invasion, oxidative stress, virulence and 44 pathogenicity, phage integration and excision, biofilm-formation, and quorum-sensing to be 45 associated with different episodes of mastitis. Therefore, profiling the dynamics of microbiome 46 in different states of mastitis, concurrent VFGs, ARGs, and genomic functional correlations will 47 contribute to developing microbiome-based diagnostics and therapeutics for bovine mastitis, and 48 carries significant implications on curtailing the economic fallout from this disease. 49 Key words: Whole metagenome sequencing, microbiome, shift, clinical, recurrent, subclinical 50 mastitis, virulence, and antibiotics resistant genes. bioRxiv preprint doi: https://doi.org/10.1101/2020.07.13.200808; this version posted July 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 51 Introduction 52 Mastitis is the inflammation of the mammary gland and/or quarters1 and represents the 53 foremost disease-driven challenge in production faced by the global dairy industry2. The milk 54 microbiota composition is an important determinant of mammalian health3,4, plays an important 55 role in udder health by interacting with the immune and metabolic functions of the cow5,6, the 56 spread of virulence factors7, and the spread of antimicrobial resistance genes (ARGs)8,9. 57 Previously, we reported that the microbiome signature in bovine clinical mastitis (CM) has 58 functional biasness from a genomic standpoint6, and observed distinct shifts and differences in 59 abundance between the microbiome of CM and H milk. Furthermore, bovine CM milk had a 60 68.04% inclusion of previously unreported and/or opportunistic strains6. In addition, a plethora 61 of archaeal and viral entities6 might also be present in association with the bacteria, suggesting 62 relevant physiological and pathological implications for their host. Although bovine mastitis 63 microbiome comprises of bacteria, archaea, viruses, and other microorganisms, until now, most 64 researchers have mainly focused on the bacterial component of the milk microbiota11-14. 65 We also reported that the resistomes (the total collection of antibiotic resistance genes) 66 act as a potential key factor in disease complication and recurrence, could be concurrent to 67 microbiome signature10. However, no association was found with cattle breed, despite significant 68 differences in microbiome diversity among different breeds10. Bovine mastitis microbiomes may 69 show a shift in taxonomic diversity and composition according to disease states (clinical, 70 subclinical and recurrent)6,11,14,15. Following pathogen invasion and subsequent establishment in 71 the mammary gland, either CM or subclinical mastitis (SCM) may present itself. Bovine CM is 72 one of the most frequent diseases (with visibly abnormal milk, and swollen, hot, painful, and 73 reddened udder) affecting the global dairy industry16,17, and diverse groups of microbial bioRxiv preprint doi: https://doi.org/10.1101/2020.07.13.200808; this version posted July 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 74 communities colonizing the mammary gland and/or quarters have evolved novel mechanisms 75 that facilitate their proliferation during the disease process6. One of the very frustrating aspects 76 of bovine CM is its recurrent nature, reinfection of a quarter or udder after bacteriological cure18. 77 Recurrent CM (RCM) is caused by persistent intra-mammary infections (IMI)17, and specific 78 differences in the milk microbiota may contribute to recurrence susceptibility18. IMI may persist 79 beyond the resolution of the clinical symptoms of CM, and sub-sequent RCM flare-ups may be 80 observed17. Almost every dairy herd has cows with SCM15, and a variety of pathogens can 81 establish chronic infections which may only occasionally manifest the clinical signs of mastitis19. 82 During the progression of mastitis, dysbiosis in the milk microbiome can occur, with an 83 increase in opportunistic pathogenic bacteria, and a reduction in healthy milk commensal 84 bacteria6, 20, 21. Researchers have now started to explore the complexities of the microbial 85 ecology within this niche through disease progression6,7,10,20. While understanding the role of all 86 interacting microbial players contributing to disease progression is becoming increasingly crucial 87 in the face of large-scale epidemics22, 23, such a study has yet to be completed for dairy animal 88 mastitis. The microbial community present within the mammary gland and/or quarters is being 89 increasingly recognized as a key regulator of metabolic and immune homeostasis5,6, and a 90 mediator of antimicrobial resistance6. The resistomes or ARGs, which exist in both 91 pathogenic9,10 and nonpathogenic commensal bacteria9 are frequently carried on mobile genetic 92 elements. Similarly, the virulome, the set of genes encoding virulence, can also be carried on the 93 mobilome9, and thus, many virulence factors associated genes (VFGs) easily spread in bacterial 94 populations by horizontal gene transfer, converting mutualistic or commensal bacteria into 95 potential opportunistic pathogens7,21,24. Though the exact functions of archaea and viruses in the 96 progression of mastitis are not fully explored like bacterial