Genetic and Biochemical Characterization of Six Lactobacillus Isolates from American Quarter Horses

Main Article Content

Racheal L Baartmans Mary Mendoza Allison Dickey Hosni M. Hassan


The internal cavity of equine contains symbiotic microorganisms that are collectively referred to as the gut microbiota, which interact with the host immune system from birth. The microorganisms in the gut microbiota are shaped by their interactions with the gut environment throughout the life of the host, i.e., exposure to antibiotics and diet.  Lactobacilli are one of the major groups found in the gastrointestinal tracts of humans and animals. Lactobacilli are members of the lactic acid bacteria, and they help to maintain a balanced gut microbiome and stimulate the host’s immune system. In this study six equine Lactobacillus spp. were isolated from three American Quarter horses of different ages (i.e., 1-day post-weaning, 1.5-month post-weaning, and 10-year-old mature gelding). The metabolic properties that allowed the isolates to survive in the harsh environment of the gut were characterized. Thus, we evaluated their abilities to metabolize different carbohydrates and to withstand acidic pH, bile salts, antibiotics, and to inhibit pathogenic bacteria which may be encountered during their passage to the small/large intestine. We also identified the genetic elements that allow the isolates to survive and persist in the host’s gut environment by using data generated from whole genome sequencing. The data indicated that the isolates were metabolically adapted to the age of the host and the type of feed consumed. The characterized isolates are potential probiotic candidates for enhancing the gut health of equines.

Keywords: Horse Lactobacilli, Gut microbiome, Probiotics, Bile tolerance, Acid tolerance, Salmonella, Genomic

Article Details

How to Cite
BAARTMANS, Racheal L et al. Genetic and Biochemical Characterization of Six Lactobacillus Isolates from American Quarter Horses. Medical Research Archives, [S.l.], v. 11, n. 1, jan. 2023. ISSN 2375-1924. Available at: <>. Date accessed: 23 july 2024. doi:
Research Articles


1. Dobrogosz WJ, Peacock TJ, Hassan HM. Evolution of the probiotic concept from conception to validation and acceptance in medical science. Adv Appl Microbiol. 2010;72:1-41. doi:10.1016/S0065-2164(10)72001-3
2. Reid G, Sanders ME, Gaskins HR, et al. New scientific paradigms for probiotics and prebiotics. J Clin Gastroenterol. Aug 2003;37(2):105-18. doi:10.1097/00004836-200308000-00004
3. Food, Agriculture Organization WHO-z. Report of a Joint FAO/WHO working group on drafting guidelines for the evaluation of probiotics in food. London Ontario, Canada; 2002.
4. Gibson G, Fuller R. Aspects of In Vitro and In Vivo Research Approaches Directed Toward Identifying Probiotics and Prebiotics for Human Use Journal of Nutrition 2000:391S-395S.
5. Nicholson JK, Holmes E, Kinross J, et al. Host-gut microbiota metabolic interactions. Science. Jun 8 2012;336(6086):1262-7. doi:10.1126/science.1223813
6. Backhed F, Ley RE, Sonnenburg JL, Peterson DA, Gordon JI. Host-bacterial mutualism in the human intestine. Science. Mar 25 2005;307(5717):1915-20. doi:10.1126/science.1104816
7. Harper A, Naghibi MM, Garcha D. The Role of Bacteria, Probiotics and Diet in Irritable Bowel Syndrome. Foods. Jan 26 2018;7(2)doi:10.3390/foods7020013
8. Ward RE, Ninonuevo M, Mills DA, Lebrilla CB, German JB. In vitro fermentation of breast milk oligosaccharides by Bifidobacterium infantis and Lactobacillus gasseri. Appl Environ Microbiol. Jun 2006;72(6):4497-9. doi:10.1128/AEM.02515-05
9. Dagher SF, Azcarate-Peril MA, Bruno-Barcena JM. Heterologous expression of a bioactive beta-hexosyltransferase, an enzyme producer of prebiotics, from Sporobolomyces singularis. Appl Environ Microbiol. Feb 2013;79(4):1241-9. doi:10.1128/AEM.03491-12
10. Paradis DMV MR. Normal Foal Nutrition 2012;58:399-401.
11. Schoster A, Arroyo LG, Staempfli HR, Weese JS. Comparison of microbial populations in the small intestine, large intestine and feces of healthy horses using terminal restriction fragment length polymorphism. BMC Res Notes. Mar 12 2013;6:91. doi:10.1186/1756-0500-6-91
12. Schoster A, Weese JS, Guardabassi L. Probiotic use in horses - what is the evidence for their clinical efficacy? J Vet Intern Med. Nov-Dec 2014;28(6):1640-52. doi:10.1111/jvim.12451
13. Kido Y, Maeno S, Tanno H, Kichise Y, Shiwa Y, Endo A. Niche-specific adaptation of Lactobacillus helveticus strains isolated from malt whisky and dairy fermentations. Microb Genom. Apr 2021;7(4)doi:10.1099/mgen.0.000560
14. Moeller AH, Gomes-Neto JC, Mantz S, et al. Experimental Evidence for Adaptation to Species-Specific Gut Microbiota in House Mice. mSphere. Jul 10 2019;4(4)doi:10.1128/mSphere.00387-19
15. De Man JC, Rogosa M, Sharpe ME. A medium for the cultivation of Lactobacilli. Journal of Applied Bacteriology. 1960;23(1):130-135. doi:10.1111/j.1365-2672.1960.tb00188.x
16. Pierce BEL, M.J. Exercises for the Microbiology Laboratory. Morton publishing company; 2005.
17. Crabtree KT, Hinsdill, R.D. Litmus milk and other general Microbiology techniques. Fundamental Experiments in Microbiology. 1974:350.
18. Zeng Z, Zuo F, Marcotte H. Putative Adhesion Factors in Vaginal Lactobacillus gasseri DSM 14869: Functional Characterization. Appl Environ Microbiol. Oct 1 2019;85(19)doi:10.1128/AEM.00800-19
19. Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol. Apr 1966;45(4):493-6.
20. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. Oct 5 1990;215(3):403-10. doi:10.1016/S0022-2836(05)80360-2
21. Meinders RL, Mendoza, M., Dickey, A. N., Scholl, E. H., Hassan, H. M. Complete Genome Sequences of Six Lactobacilli Isolated from American Quarter Horses. American Society for Microbiology. 2020;9(47)(Microbiology Resource Announcements)
22. Tatusova T, DiCuccio M, Badretdin A, et al. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res. Aug 19 2016;44(14):6614-24. doi:10.1093/nar/gkw569
23. Haft DH, DiCuccio M, Badretdin A, et al. RefSeq: an update on prokaryotic genome annotation and curation. Nucleic Acids Res. Jan 4 2018;46(D1):D851-D860. doi:10.1093/nar/gkx1068
24. Alikhan NF, Petty NK, Ben Zakour NL, Beatson SA. BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics. Aug 8 2011;12:402. doi:10.1186/1471-2164-12-402
25. Aziz RK, Bartels D, Best AA, et al. The RAST Server: rapid annotations using subsystems technology. BMC Genomics. Feb 8 2008;9:75. doi:10.1186/1471-2164-9-75
26. Overbeek R, Olson R, Pusch GD, et al. The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST). Nucleic Acids Res. Jan 2014;42(Database issue):D206-14. doi:10.1093/nar/gkt1226
27. Brettin T, Davis JJ, Disz T, et al. RASTtk: a modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes. Sci Rep. Feb 10 2015;5:8365. doi:10.1038/srep08365
28. Costa MC, Silva G, Ramos RV, et al. Characterization and comparison of the bacterial microbiota in different gastrointestinal tract compartments in horses. Vet J. Jul 2015;205(1):74-80. doi:10.1016/j.tvjl.2015.03.018
29. Schierl EA, Blazevic DJ. Rapid identification of enterococci by reduction of litmus milk. J Clin Microbiol. Aug 1981;14(2):227-8. doi:10.1128/jcm.14.2.227-228.1981
30. Willis AT. Anaerobic bacteriology : clinical and laboratory practice. 3 ed. Butterworths; 1977.
31. Beebe JA, Frey PA. Galactose mutarotase: purification, characterization, and investigations of two important histidine residues. Biochemistry. Oct 20 1998;37(42):14989-97. doi:10.1021/bi9816047
32. Bouffard GG, Rudd KE, Adhya SL. Dependence of lactose metabolism upon mutarotase encoded in the gal operon in Escherichia coli. J Mol Biol. Dec 2 1994;244(3):269-78. doi:10.1006/jmbi.1994.1728
33. Douillard FP, Ribbera A, Kant R, et al. Comparative genomic and functional analysis of 100 Lactobacillus rhamnosus strains and their comparison with strain GG. PLoS Genet. 2013;9(8):e1003683. doi:10.1371/journal.pgen.1003683
34. Wang S, Yang B, Ross RP, et al. Comparative Genomics Analysis of Lactobacillus ruminis from Different Niches. Genes (Basel). Jan 8 2020;11(1)doi:10.3390/genes11010070
35. Ventura M, O'Flaherty S, Claesson MJ, et al. Genome-scale analyses of health-promoting bacteria: probiogenomics. Nat Rev Microbiol. Jan 2009;7(1):61-71. doi:10.1038/nrmicro2047
36. Kandler O. Carbohydrate metabolism in lactic acid bacteria. Antonie van Leeuwenhoek. 1983;49(3):209-224. doi:10.1007/bf00399499
37. Arskold E, Lohmeier-Vogel E, Cao R, Roos S, Radstrom P, van Niel EW. Phosphoketolase pathway dominates in Lactobacillus reuteri ATCC 55730 containing dual pathways for glycolysis. J Bacteriol. Jan 2008;190(1):206-12. doi:10.1128/JB.01227-07
38. Zaunmuller T, Eichert M, Richter H, Unden G. Variations in the energy metabolism of biotechnologically relevant heterofermentative lactic acid bacteria during growth on sugars and organic acids. Appl Microbiol Biotechnol. Sep 2006;72(3):421-9. doi:10.1007/s00253-006-0514-3
39. Talarico TL, Casas IA, Chung TC, Dobrogosz WJ. Production and isolation of reuterin, a growth inhibitor produced by Lactobacillus reuteri. Antimicrob Agents Chemother. Dec 1988;32(12):1854-8. doi:10.1128/AAC.32.12.1854
40. Engels C, Schwab C, Zhang J, et al. Acrolein contributes strongly to antimicrobial and heterocyclic amine transformation activities of reuterin. Sci Rep. Nov 7 2016;6:36246. doi:10.1038/srep36246
41. Research KE. Understanding pH in the Equine Digestive Tract. 2022.
42. Cubitt DT. Basic Equine Digestive Function. Standlee Premium Western Forage. 2022.
43. Cotter PD, Hill C. Surviving the acid test: responses of gram-positive bacteria to low pH. Microbiol Mol Biol Rev. Sep 2003;67(3):429-53, table of contents. doi:10.1128/MMBR.67.3.429-453.2003
44. Azcarate-Peril MA, Altermann E, Hoover-Fitzula RL, Cano RJ, Klaenhammer TR. Identification and inactivation of genetic loci involved with Lactobacillus acidophilus acid tolerance. Appl Environ Microbiol. Sep 2004;70(9):5315-22. doi:10.1128/AEM.70.9.5315-5322.2004
45. Castro-Bravo N, Wells JM, Margolles A, Ruas-Madiedo P. Interactions of Surface Exopolysaccharides From Bifidobacterium and Lactobacillus Within the Intestinal Environment. Front Microbiol. 2018;9:2426. doi:10.3389/fmicb.2018.02426
46. Fukao M, Zendo T, Inoue T, et al. Plasmid-encoded glycosyltransferase operon is responsible for exopolysaccharide production, cell aggregation, and bile resistance in a probiotic strain, Lactobacillus brevis KB290. J Biosci Bioeng. Oct 2019;128(4):391-397. doi:10.1016/j.jbiosc.2019.04.008
47. How Often Should Horses Eat? Constantly. Succeed Digestive Conditioning Program.
48. Rodriguez-Pozo ML, Armengou L, Viu J, Rios J, Jose-Cunilleras E. Peritoneal bile acids concentration in adult horses with hepatic and gastrointestinal disorders. Equine Vet J. Sep 2022;54(5):914-921. doi:10.1111/evj.13538
49. Ruiz L, Margolles A, Sanchez B. Bile resistance mechanisms in Lactobacillus and Bifidobacterium. Front Microbiol. Dec 24 2013;4:396. doi:10.3389/fmicb.2013.00396
50. Lv LX, Yan R, Shi HY, et al. Integrated transcriptomic and proteomic analysis of the bile stress response in probiotic Lactobacillus salivarius LI01. J Proteomics. Jan 6 2017;150:216-229. doi:10.1016/j.jprot.2016.08.021
51. Gueimonde M, Sanchez B, C GdLR-G, Margolles A. Antibiotic resistance in probiotic bacteria. Front Microbiol. 2013;4:202. doi:10.3389/fmicb.2013.00202
52. Czaran TL, Hoekstra RF, Pagie L. Chemical warfare between microbes promotes biodiversity. Proc Natl Acad Sci U S A. Jan 22 2002;99(2):786-90. doi:10.1073/pnas.012399899
53. Walsh MC, Gardiner GE, Hart OM, et al. Predominance of a bacteriocin-producing Lactobacillus salivarius component of a five-strain probiotic in the porcine ileum and effects on host immune phenotype. FEMS Microbiol Ecol. May 2008;64(2):317-27. doi:10.1111/j.1574-6941.2008.00454.x
54. Dobson A, Cotter PD, Ross RP, Hill C. Bacteriocin production: a probiotic trait? Appl Environ Microbiol. Jan 2012;78(1):1-6. doi:10.1128/AEM.05576-11
55. Palmer DJ, Metcalfe J, Prescott SL. Preventing disease in the 21st century: the importance of maternal and early infant diet and nutrition. J Allergy Clin Immunol. Sep 2012;130(3):733-4. doi:10.1016/j.jaci.2012.06.038
56. Belizario JE, Faintuch J. Microbiome and Gut Dysbiosis. Exp Suppl. 2018;109:459-476. doi:10.1007/978-3-319-74932-7_13
57. La Reau AJ, Meier-Kolthoff JP, Suen G. Sequence-based analysis of the genus Ruminococcus resolves its phylogeny and reveals strong host association. Microb Genom. Dec 2016;2(12):e000099. doi:10.1099/mgen.0.000099