Molecular Biology Reports

, Volume 41, Issue 10, pp 6405–6417 | Cite as

Insights into resistome and stress responses genes in Bubalus bubalis rumen through metagenomic analysis

  • Bhaskar Reddy
  • Krishna M. Singh
  • Amrutlal K. Patel
  • Ancy Antony
  • Harshad J. Panchasara
  • Chaitanya G. Joshi


Buffalo rumen microbiota experience variety of diets and represents a huge reservoir of mobilome, resistome and stress responses. However, knowledge of metagenomic responses to such conditions is still rudimentary. We analyzed the metagenomes of buffalo rumen in the liquid and solid phase of the rumen biomaterial from river buffalo adapted to varying proportion of concentrate to green or dry roughages, using high-throughput sequencing to know the occurrence of antibiotics resistance genes, genetic exchange between bacterial population and environmental reservoirs. A total of 3914.94 MB data were generated from all three treatments group. The data were analysed with Metagenome rapid annotation system tools. At phyla level, Bacteroidetes were dominant in all the treatments followed by Firmicutes. Genes coding for functional responses to stress (oxidative stress and heat shock proteins) and resistome genes (resistance to antibiotics and toxic compounds, phages, transposable elements and pathogenicity islands) were prevalent in similar proportion in liquid and solid fraction of rumen metagenomes. The fluoroquinolone resistance, MDR efflux pumps and Methicillin resistance genes were broadly distributed across 11, 9, and 14 bacterial classes, respectively. Bacteria responsible for phages replication and prophages and phage packaging and rlt-like streptococcal phage genes were mostly assigned to phyla Bacteroides, Firmicutes and proteaobacteria. Also, more reads matching the sigma B genes were identified in the buffalo rumen. This study underscores the presence of diverse mechanisms of adaptation to different diet, antibiotics and other stresses in buffalo rumen, reflecting the proportional representation of major bacterial groups.


Resistome Personal genome machine MG-RAST Virulence genes Stress gene 



This research work was supported by Niche area of excellence project funded by Indian Council of Agricultural Research, New Delhi, India.

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  1. 1.
    Hespell RB, Mackie RI, White BA, Isaacson R (1997) Gastrointestinal microbiology, vol 2. Chapman and Hall, LondonGoogle Scholar
  2. 2.
    Klieve AV, Bauchop T (1988) Morphological diversity of ruminal bacteriophages from sheep and cattle. Appl Environ Microbiol 54:1637–1641PubMedCentralPubMedGoogle Scholar
  3. 3.
    Handelsman J, Rondon MR, Brady SF, Clardy J, Goodman RM (1998) Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products. Chem Biol 5:R245–R249CrossRefPubMedGoogle Scholar
  4. 4.
    Donato JJ, Moe LA, Converse BJ, Smart KD, Berklein FC et al (2010) Metagenomic analysis of apple orchard soil reveals antibiotic resistance genes encoding predicted bifunctional proteins. Appl Environ Microbiol 76:4396–4401PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Diaz-Torres ML, McNab R, Spratt DA, Villedieu A, Hunt N et al (2003) Novel tetracycline resistance determinant from the oral metagenome. Antimicrob Agents Chemother 47:1430–1432PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Mori T, Mizuta S, Suenaga H, Miyazaki K (2008) Metagenomic screening for bleomycin resistance genes. Appl Environ Microbiol 74:6803–6805PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Bockelmann U, Dorries HH, Ayuso-Gabella MN, Salgot de Marcay M, Tandoi V et al (2009) Quantitative PCR monitoring of antibiotic resistance genes and bacterial pathogens in three European artificial groundwater recharge systems. Appl Environ Microbiol 75:154–163PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Edwards RA, Rodriguez-Brito B, Wegley L, Haynes M, Breitbart M et al (2006) Using pyrosequencing to shed light on deep mine microbial ecology. BMC Genomics 7:57PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Graham NA, Chabanet P, Evans RD, Jennings S, Letourneur Y et al (2011) Extinction vulnerability of coral reef fishes. Ecol Lett 14:341–348PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Storteboom H, Arabi M, Davis JG, Crimi B, Pruden A (2010) Identification of antibiotic-resistance-gene molecular signatures suitable as tracers of pristine river, urban, and agricultural sources. Environ Sci Technol 44:1947–1953CrossRefPubMedGoogle Scholar
  11. 11.
    Soni DK, Singh RK, Singh DV, Dubey SK (2013) Characterization of Listeria monocytogenes isolated from Ganges water, human clinical and milk samples at Varanasi, India. Infect Genet Evol 14:83–91CrossRefPubMedGoogle Scholar
  12. 12.
    Romero-Perez GA, Ominski KH, McAllister TA, Krause DO (2011) Effect of environmental factors and influence of rumen and hindgut biogeography on bacterial communities in steers. Appl Environ Microbiol 77:258–268PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Singh KM, Jakhesara SJ, Koringa PG, Rank DN, Joshi CG (2012) Metagenomic analysis of virulence-associated and antibiotic resistance genes of microbes in rumen of Indian buffalo (Bubalus bubalis). Gene 507:146–151CrossRefPubMedGoogle Scholar
  14. 14.
    Swanson KS, Dowd SE, Suchodolski JS, Middelbos IS, Vester BM et al (2011) Phylogenetic and gene-centric metagenomics of the canine intestinal microbiome reveals similarities with humans and mice. ISME J 5:639–649Google Scholar
  15. 15.
    Durso LM, Harhay GP, Smith TP, Bono JL, DeSantis TZ et al (2011) Bacterial community analysis of beef cattle feedlots reveals that pen surface is distinct from feces. Foodborne Pathog Dis 8:647–649Google Scholar
  16. 16.
    Brulc JM, Antonopoulos D, Berg-Miller ME, Wilson KM, Yannarell CA, Dinsdale AE, Edwards ER, Frank ED, Emerson BJ, Wacklin P, Coutinho MP, Henrissat B, Nelson EK, White AB (2009) Gene-centric metagenomics of the fiber-adherent bovine rumen microbiome reveals forage specific glycoside hydrolases. Proc Natl Acad Sci USA 106:1948–1953Google Scholar
  17. 17.
    Singh KM, Ahir V, Tripathi AK et al (2012) Metagenomic analysis of Surti buffalo 400 366 (Bubalus bubalis) rumen: a preliminary study. Mol Biol Rep 39(4):4841–4848Google Scholar
  18. 18.
    Overbeek R, Overbeek R, Begley T, Butler RM, Choudhuri JV, Chuang HY, Cohoon M, Crécy-Lagard DV, Diaz N, Disz T, Edwards R et al (2005) The subsystems approach to genome annotation and its use in the project to annotate 1000 genomes. Nucleic Acids Res 33:5691–5702Google Scholar
  19. 19.
    Durso LM, Miller DN, Wienhold B (2012) Distribution and Quantification of Antibiotic Resistant Genes and Bacteria across Agricultural and Non Agricultural Metagenomes. PLoS ONE 7(11):e48325Google Scholar
  20. 20.
    Cardoso AM, Cavalcante JJ, Cantao ME, Thompson CE, Flatschart RB et al (2012) Metagenomic analysis of the microbiota from the crop of an invasive snail reveals a rich reservoir of novel genes. PLoS One 7:e48505PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Dinsdale EA, Edwards RA, Hall D, Angly F, Breitbart M et al (2008) Functional metagenomic profiling of nine biomes. Nature 452:629–632CrossRefPubMedGoogle Scholar
  22. 22.
    Allen HK, Cloud-Hansen KA, Wolinski JM, Guan C, Greene S, Lu S, Boeyink M, Broderick NA, Raffa KF, Handelsman J (2009) Resident microbiota of the gypsy moth midgut harbors antibiotic resistance determinants. DNA Cell Biol 28(3):109–117Google Scholar
  23. 23.
    Allen HK, Donato J, Wang HH, Cloud-Hansen KA, Davies J, Handelsman J (2010) Call of the wild: antibiotic resistance genes in natural environments. Nat Rev Microbiol 8(4):251–259CrossRefPubMedGoogle Scholar
  24. 24.
    Allen HK, Looft T, Bayles DO, Humphrey S, Levine UY, Alt D, Stanton TB (2011) Antibiotics in feed induce prophages in swine fecal microbiomes. mBio 2(6):00260-11. doi: 10.1128/mBio.00260-11 CrossRefGoogle Scholar
  25. 25.
    Reyes A, Haynes M, Hanson N, Angly FE, Heath AC et al (2010) Viruses in the faecal microbiota of monozygotic twins and their mothers. Nature 466:334–338PubMedCentralCrossRefPubMedGoogle Scholar
  26. 26.
    Canton R (2009) Antibiotic resistance genes from the environment: a perspective through newly identified antibiotic resistance mechanisms in the clinical setting. Clin Microbiol Infect 15(Suppl 1):20–25CrossRefPubMedGoogle Scholar
  27. 27.
    Kim KH, Bae JW (2011) Amplification methods bias metagenomic libraries of uncultured single-stranded and double-stranded DNA viruses. Appl Environ Microbiol 77:7663–7668PubMedCentralCrossRefPubMedGoogle Scholar
  28. 28.
    Dantas G, Sommer MO, Oluwasegun RD, Church GM (2008) Bacteria subsisting on antibiotics. Science 320:100–103CrossRefPubMedGoogle Scholar
  29. 29.
    Hacker J, Kaper JB (2000) Pathogenicity islands and the evolution of microbes. Annu Rev Microbiol 54:641–679CrossRefPubMedGoogle Scholar
  30. 30.
    Minot S, Sinha R, Chen J, Li H, Keilbaugh SA et al (2011) The human gut virome: inter-individual variation and dynamic response to diet. Genome Res 21:1616–1625PubMedCentralCrossRefPubMedGoogle Scholar
  31. 31.
    Rohwer F, Prangishvili D, Lindell D (2009) Roles of viruses in the environment. Environ Microbiol 11:2771–2774CrossRefPubMedGoogle Scholar
  32. 32.
    Rodrigues DF, Tiedje JM (2008) Coping with our cold planet. Appl Environ Microbiol 74:1677–1686PubMedCentralCrossRefPubMedGoogle Scholar
  33. 33.
    Hoper D, Volker U, Hecker M (2005) Comprehensive characterization of the contribution of individual SigB-dependent general stress genes to stress resistance of Bacillus subtilis. J Bacteriol 187:2810–2826PubMedCentralCrossRefPubMedGoogle Scholar
  34. 34.
    Cavicchioli R (2006) Cold-adapted archaea. Nat Rev Microbiol 4:331–343CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Bhaskar Reddy
    • 1
  • Krishna M. Singh
    • 1
    • 3
  • Amrutlal K. Patel
    • 1
  • Ancy Antony
    • 1
  • Harshad J. Panchasara
    • 2
  • Chaitanya G. Joshi
    • 1
  1. 1.Department of Animal Biotechnology, College of Veterinary Science and Animal Husbandry (CVSAH)Anand Agricultural University (AAU)AnandIndia
  2. 2.Department of Animal NutritionSardarkrushinagar Dantiwada Agricultural UniversitySardarkrushinagarIndia
  3. 3.Xcelris GenomicsXcelris LabsAhmedabadIndia

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