Going for the Gut
Research Models
Axel Kornerup Hansen

Going for the Gut

The microbiome is far more likely to influence research than any bacteria routinely monitored in rodents today.

Bacteriology has changed dramatically since 1890, when German physician Robert Koch introduced four postulates that, in theory at least, were designed to establish a causative relationship between a microbe and disease.

The criteria specified that a pathogen: (1) should not be found in healthy organisms; (2) should be cultivable; (3) should cause disease when inoculated in a healthy, susceptible lab animal and; (4) must be re-isolated from the new host and shown to be identical to the disease-causing pathogen.

Nos. 1 and 2 were soon dropped due to the concept of latent infections—which covers most of what is monitored in rodents today—and the recognition that certain pathogens, such as Clostridium piliforme, were uncultivable. And if No. 1 was questionable, then so was No. 4, leaving No. 3 as the only postulate that still carried weight in modern bacteriology. Yet while this postulate fit most of what was included when rodent health monitoring was evolving in the 1950s, the only bacterium found in rodent bacteriology screenings today is Pasteurella pneumotropica. Funny thing is, 10 years after Charles River’s founder Henry Foster produced the world’s first health-monitored rodents in 1959, Patricia Brennan from the Argonne National Laboratory showed it was impossible to fulfill postulate No. 3 for P. pneumotropica without adding Mycoplasma pulmonis.[1]

So, today enormous resources are spent on an agent that, strictly speaking, can’t really be regarded a pathogen. Call it my “gut instinct” but maybe time has come to spend these resources differently and more broadly.

Microbial hot spots
The gut microbiota contains a hundred trillion (1014) organisms dispersed on 500 to 1000 different species,  only 10-20 % of which can be cultivated. However, molecular methods such as qPCR, gel electrophoresis (GGE), terminal restriction fragment length polymorphism analysis (T-RFLP) and, least but not least, high-throughput sequencing have evolved over the last decade, allowing a full characterization of the entire microbiome. This has shown  the microbiota to have an essential impact on a wide range of animal models that apply to  inflammatory bowel disease (IBD),[2-8] diabetes type 1 [9-11] and type 2[12, 13], obesity,[14-17] dermatitis[18] and psychiatric disease.[19, 20].

Different bacterial species work in conjunction with one another, making it unclear just who is the pathogen, the co-pathogen or the symbiont.[13, 17] The level of the entire phylum Firmicutes correlates to leptin-deficiency[15], stress responses[20, 21] and vitamin D receptor function,[22] and for some animal models the microbiota is responsible for between 35%[13] to more than 80 %[18]of the variation in some parameters.

Bacterial species with a strong impact on animal models, have been revealed, but can they be regarded as pathogens or symbionts, and should they be absent or present in rodents purchased to model disease? For instance, in mice a high abundance of Akkermansia muciniphila may protect against diabetes type 1,[23] while a low abundance may protect against colorectal cancer.[24] Segmented filamentous bacteria (SFB) drive arthritis development[25] but also seems to protect mice against diabetes type 1.[26] Stress,[20] dietary fluctuations,[9, 27] and other factors may change the gut microbiota, and with this the animal model.

Different strategies may be pursued to control the variation caused by the gut microbiota. Standardization may be achieved by inoculation of tailor-made microbiotas[28] or feeding animals certain prebiotic diets[29], but we would probably have to use different standards for different types of research.

Alternatively, animals for microbiota-sensitive studies could be screened to incorporate this information in data evaluation, thereby turning this uncontrolled variation into controlled variation. With rapidly declining prices for full sequencing we are reaching a state where, without additional cost, it is now possible to replace bacteriological screenings of little customer relevance with screenings of high value for the animal user.


  1. Brennan PC, Fritz TE, Flynn RJ. Role of Pasteurella pneumotropica and Mycoplasma pulmonis in murine pneumonia. JBacteriol 1969; 97(1): 337-349
  2. Kang CS, Ban M, Choi EJ, Moon HG, Jeon JS, Kim DK, Park SK, Jeon SG, Roh TY, Myung SJ, Gho YS, Kim JG, Kim YK. Extracellular Vesicles Derived from Gut Microbiota, Especially Akkermansia muciniphila, Protect the Progression of Dextran Sulfate Sodium-Induced Colitis.Plos One 2013; 8(10) [PMID: WOS:000326152300006  DOI: 10.1371/journal.pone.0076520]
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  8. Nell S, Suerbaum S, Josenhans C. The impact of the microbiota on the pathogenesis of IBD: lessons from mouse infection models. Nature Reviews Microbiology 2010; 8(8): 564-577
  9. Hansen AK, Ling F, Kaas A, Funda DP, Farlov H, Buschard K. Diabetes preventive gluten-free diet decreases the number of caecal bacteria in non-obese diabetic mice. Diabetes-Metabolism Research & Reviews 2006; 22(2): 220-225
  10. Hansen CH, Krych L, Buschard K, Metzdorff SB, Nellemann C, Hansen LH, Nielsen DS, Frokiaer H, Skov S, Hansen AK. A maternal gluten-free diet reduces inflammation and diabetes incidence in the offspring of NOD mice. Diabetes 2014; In press [PMID: 24696449  DOI: 10.2337/db13-1612]
  11. Sofi MH, Gudi R, Karumuthil-Melethil S, Perez N, Johnson BM, Vasu C. pH of Drinking Water Influences the Composition of Gut Microbiome and Type 1 Diabetes Incidence. Diabetes 2014; 63(2): 632-644 [PMID: WOS:000331110000030  DOI: 10.2337/db13-0981]
  12. Bech-Nielsen GV, Hansen CH, Hufeldt MR, Nielsen DS, AASTED B, Vogensen FK, Midtvedt T, Hansen AK. Manipulation of the gut microbiota in C57BL/6 mice changes glucose tolerance without affecting weight development and gut mucosal immunity. ResVetSci 2012; 92: 501-508
  13. Ellekilde M, Krych L, Hansen CH, Hufeldt MR, Dahl K, Hansen LH, Sorensen SJ, Vogensen FK, Nielsen DS, Hansen AK. Characterization of the gut microbiota in leptin deficient obese mice - Correlation to inflammatory and diabetic parameters. Res Vet Sci 2014; 96(2): 241-250 [PMID: 24556473  DOI: 10.1016/j.rvsc.2014.01.007]
  14. Everard A, Belzer C, Geurts L, Ouwerkerk JP, Druart C, Bindels LB, Guiot Y, Derrien M, Muccioli GG, Delzenne NM, De Vos WM, Cani PD. Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity. Proceedings of the National Academy of Sciences of the United States of America 2013; 110(22): 9066-9071 [DOI: 10.1073/pnas.1219451110]
  15. Ley RE, Backhed F, Turnbaugh P, Lozupone CA, Knight RD, Gordon JI. Obesity alters gut microbial ecology. Proc Natl Acad Sci U S A 2005; 102(31): 11070-11075 [PMID: 16033867 PMCID: 1176910 DOI: 10.1073/pnas.0504978102]
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  17. Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 2006;444(7122): 1027-1031 [PMID: 17183312  DOI: 10.1038/nature05414]
  18. Lundberg R, Clausen SK, Pang W, Nielsen DS, Möller K, Josefsen K, Hansen AK. Gastrointestinal Microbiota and Local Inflammation during Oxazolone-induced Dermatitis in BALB/cA Mice. Comparative Medicine 2012; 62(5): 371-380
  19. Hsiao EY, McBride SW, Hsien S, Sharon G, Hyde ER, McCue T, Codelli JA, Chow J, Reisman SE, Petrosino JF, Patterson PH, Mazmanian SK. Microbiota Modulate Behavioral and Physiological Abnormalities Associated with Neurodevelopmental Disorders. Cell  2013;155(7): 1451-1463 [PMID: WOS:000328693300004  DOI: 10.1016/j.cell.2013.11.024]
  20. Bangsgaard Bendtsen KM, Krych L, Sørensen DB, Pang W, Nielsen DS, Josefsen K, Hansen LH, Sørensen SJ, Hansen AK. Gut Microbiota Composition Is Correlated to Grid Floor Induced Stress and Behavior in the BALB/c Mouse. Plos One 2012; 7(10): e46231
  21. Bravo JA, Forsythe P, Chew MV, Escaravage E, Savignac HM, Dinan TG, Bienenstock J, Cryan JF. Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve. Proc Natl Acad Sci U S A 2011; 108(38): 16050-16055 [PMID: 21876150 PMCID: 3179073 DOI: 10.1073/pnas.1102999108]
  22. Ooi JH, Li YF, Rogers CJ, Cantorna MT. Vitamin D Regulates the Gut Microbiome and Protects Mice from Dextran Sodium Sulfate-Induced Colitis. Journal of Nutrition 2013;143(10): 1679-1686 [PMID: WOS:000330331700021  DOI: 10.3945/jn.113.180794]
  23. Hansen CHF, Krych L, Nielsen DS, Vogensen FK, Hansen LH, Sørensen SJ, Hansen AK. Early life treatment with vancomycin propagates Akkermansia muciniphila and reduces diabetes incidence in non-obese diabetic (NOD) mice. Diabetologia 2012; 55(8): 2285-2294
  24. Zackular JP, Baxter NT, Iverson KD, Sadler WD, Petrosino JF, Chen GY, Schloss PD. The gut microbiome modulates colon tumorigenesis. mBio 2013; 4(6): e00692-00613 [PMID: 24194538  DOI: 10.1128/mBio.00692-13]
  25. Wu HJ, Ivanov II, Darce J, Hattori K, Shima T, Umesaki Y, Littman DR, Benoist C, Mathis D. Gut-Residing Segmented Filamentous Bacteria Drive Autoimmune Arthritis via T Helper 17 Cells. Immunity 2010; 32(6): 815-827 [PMID: WOS:000279365800012  DOI: DOI 10.1016/j.immuni.2010.06.001]
  26. Kriegel MA, Sefik E, Hill JA, Wu HJ, Benoist C, Mathis D. Naturally transmitted segmented filamentous bacteria segregate with diabetes protection in nonobese diabetic mice.ProcNatlAcadSciUSA 2011; 108(28): 11548-11553 [DOI: 1108924108 [pii];10.1073/pnas.1108924108 [doi]]
  27. Pyndt BM, Hansen JT, Krych L, Larsen C, Klein AB, Nielsen DS, Josefsen K, Hansen AK, Sørensen DB. A possible Link between Food and Mood: Dietary Impact on Gut Microbiota and Behavior in BALB/c Mice. Plos One 2014; In Press
  28. Hansen CHF, Nielsen DS, Kverka M, Zakostelska Z, Klimesova K, Hudcovic T, Tlaskalova-Hogenova H, Hansen AK. Patterns of early gut colonization shape future immune responses of the host. Plos One 2012; 7(3): e34043 [DOI: 10.1371/journal.pone.0034043 [doi];PONE-D-11-21227 [pii]]
  29. Hansen CHF, Frokiaer H, Christensen AG, Bergstrom A, Licht TR, Hansen AK, Metzdorff SB. Dietary Xylooligosaccharide Downregulates IFN-gamma and the Low-Grade Inflammatory Cytokine IL-1 beta Systemically in Mice. Journal of Nutrition 2013; 143(4): 533-540 [PMID: WOS:000316437300020  DOI: 10.3945/jn.112.172361]