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Segmented filamentous bacteria (SFB) are long-recognized prokaryotes that selec- tively colonize the ileal epithelium of a wide range of host species. SFB have gained considerable attention due to their ability to promote differentiation and proliferation of T

17 cells and IgA-producing plasma cells. Thus, while SFB are not pathogenic per se, their presence alters the consti- tutive immunological milieu and signifi- cantly affects mouse models of inflam- matory bowel disease (IBD) (1), rheumatoid arthritis (2), and multiple sclerosis (3), among other diseases.

SFB are common constituents of the gut microbiota (GM) of humans under the age of three years but are either cleared or recede to limits below detection in older individuals (4). A recent report suggested a link between persistent SFB colonization and

ulcerative colitis, one form of IBD, although that relationship is only correlative (5). Histor- ically, SFB have been refractory to culturing in the lab; however, a recent report from Schnupf et al. demonstrated successful in vitro cultivation of SFB from fecal material obtained from a colony of mono-associated mice (6). Here, we report a relatively simple and cost-effective method for isolating SFB from complex microbiota, thus enabling comparative evaluation of autochthonous SFB isolates (7) and providing a means of preparing pure inocula for use in prospective studies, with potential applications in novel host species.

SFB exclusively colonize the ileal mucosa of mice and are typically undetectable in the feces of pre-weanling mice. This is presumably due to the presence of maternal IgA since SFB flourish soon after weaning

(8). Thus, to optimize selection of SFB from an endogenous microbial population, mucosal scrapes were collected from thoroughly rinsed ileal tissue from 2- to 3-day post-weaning BALB/cAnNHsd mice (Harlan Laboratories, Indianapolis, IN) that were confirmed to be colonized with SFB via PCR (9). Briefly, immediately following humane euthanasia, approximately 4 cm of distal ileum was excised using aseptic technique and flushed thoroughly with sterile PBS to remove all visible luminal contents.

Ilea were placed on a sterile Petri dish and incised longitudinally to expose the mucosal surface. The mucosa was scraped with a sterile scalpel blade to collect epithelial cells and mucosa-associated bacteria.

The pooled material recovered from 4 mice was placed in 50 mL of sterile DMEM/

high glucose containing 4 mM L-glutamine, 4500 mg/L glucose, and sodium pyruvate (catalog #SH30243.02; Thermo Scien- tific, Waltham, MA). Pooled ileal mucosal scrape (IMS) material was plated at 2 mL per well in 24-well polystyrene plates (catalog #08–772–4G, Corning Primaria culture plates; Corning, Durham, NC) and incubated in a non-humidified incubator at 37°C under anaerobic (≥13% CO

,

<1% O

) (catalog #260678, BD GasPak EZ Anaerobe container system; Becton Dickinson, Franklin Lakes, NJ) or micro- aerophilic (2%–10% CO

, 6%–16% O

) (catalog #260680, BD GasPak EZ Campy container system; Becton Dickinson) condi- tions in sealed 4-L bell jars (Difco Labora- tories, Detroit MI), or under standard aerobic conditions. Media were supplemented with ciprofloxacin HCl (catalog #61–277-RG, Corning cellgro; Thermo Scientific) at 5 and 10 µg/mL, neomycin sulfate (catalog

#61–241-RG, Corning cellgro; Thermo Scientific) at 5 and 10 mg/mL, or a combi- nation of penicillin (10,000 IU/mL), strepto- mycin (10,000 µg/mL), and amphotericin (25 µg/mL) (catalog #1674049; MP Biomedicals, LLC, Santa Ana, CA), at full strength and at dilutions of 1:2 and 1:4. Media were supple- mented with antibiotics prior to inoculation with material collected during ileal muc osal scrapes; media were then replaced with antibiotic-free media at days 7 and 14 to account for consumption of factors in media and evaporation.

Isolation of segmented filamentous bacteria from complex gut microbiota

Aaron C. Ericsson

, Giedre Turner

, Lisa Montoya

, Annie Wolfe

, Stacey Meeker

, Charlie Hsu

, Lillian Maggio-Price

, and Craig L. Franklin

1

University of Missouri (MU) Mutant Mouse Resource and Research Center,

2

MU Metagenomics Center,

MU Rat Resource and Research Center,

4

Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, MO, and

Department of Comparative Medicine, School of Medicine, University of Washington, Seattle, WA

BioTechniques 59:94-98 (August 2015) doi 10.2144/000114319

Keywords: segmented filamentous bacteria; SFB; Candidatus Arthromitus; culture; microbiota;

commensal

Supplementary material for this article is available at www.BioTechniques.com/article/114319.

Segmented filamentous bacteria (SFB) modulate the ontogeny of the im- mune system, and their presence can significantly affect mouse mod- els of disease. Until recently, the inability to successfully culture SFB has made controlled studies on the mechanisms by which these bac- teria exert their influence problematic. Here, we report a new method for selecting SFB from complex microbial mixtures, providing research- ers a simple and cost-effective means to prepare pure infective inocu- la for prospective studies and also to compare individual SFB isolates.

Benchmarks

METHOD SUMMARY

We report a simple and cost-effective method for selecting segmented filamentous bacteria (SFB) from mice and rats that are colonized with a complex microbiota and confirm that both fresh and frozen isolates prepared using this method are infective.

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Y

Figure 1. Collection of segmented filamentous bacteria (SFB) filaments during ileal mucosal scrape, and enrichment in vitro. (A) Photomicrographs of ileal tissue collected via mucosal scrape showing multiple adherent SFB filaments (arrowheads); scale bars = 100 µm (left) and 200 µm (right). (B) Results of 16S rRNA amplicon sequencing of material recovered from 2 representative ileal mucosal scrapes (IMS) collected from specific pathogen- free mice or 4 wells of culture media inoculated 21 days earlier with similar IMS material. (C) Results of 16S rRNA amplicon sequencing of two IMS performed on rats or four similarly prepared cultures. Legends at right; bright red indicates sequences annotated to SFB (Candidatus Arthromitus).

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Figure 2. Gut microbial profiles of germ-free mice inoculated with culture or sham gavage. Results of 16S rRNA amplicon sequencing of feces of 3 germ-free male (A) or 3 germ-free female (B) mice inoculated with culture material or sham gavaged (C). Fecal samples were obtained prior to inoculation (pre; pooled from n = 3) or 1 week post-inoculation (1w) and analyzed. Ileal mucosal scrape (IMS) material obtained 2 weeks post-inoculation (end of study) and food provided to mice at 1 or 2 weeks post-inoculation were similarly analyzed. Numbers above bars indicate coverage. Samples marked NA (no amplification) yielded <10 sequences. Bright red indicates sequences annotated to Candidatus Arthromitus.

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Under microaerophilic conditions with starting concentrations of 5000 IU/mL, 5000 µg/mL, and 12.5 µg/mL of penicillin/

streptomycin/amphotericin respectively (1:2 PSA dilution), plates of cells inocu- lated with IMS material and colonized with SFB were routinely maintained with no detectable contaminants, for greater than 3 weeks (Figure 1A). All other combinations of gas mixtures and antibiotic concentra- tions resulted in either contamination with microbes other than SFB (aerobic condi- tions, 5 µg/mL ciprofloxacin, 5 mg/mL neomycin, 1:4 dilution PSA) or the elimination of SFB (10 µg/mL ciprofloxacin, 10 mg/mL neomycin, undiluted PSA). Under micro- aerophilic conditions and at a 1:2 dilution of PSA, culture media showed no evidence of acidification and no contaminating bacteria were detected upon repeated examination via inverted light microscopy. SFB filaments were seen throughout all inoculated wells, most commonly adherent to clumps of host epithelial cells.

These procedures have been repeated with highly consistent results. In one repre- sentative replicate, each well contained, on average, 70 pieces of tissue (range 20–120), each containing 12–24 adherent filaments, and approximately 100 free-floating filaments. Thus, each well contained an average of 1360 filaments of varying lengths.

Gram staining of culture material revealed Gram-positive characteristics with variable stain uptake by intrasegmental bodies (Supplementary Figure S1A). Staining with the LIVE/DEAD BacLight Bacterial viability kit (catalog #L-7007; Life Technologies, Eugene, OR) according to manufacturer’s instructions revealed substantial uptake of propidium iodide by the filaments.

Occasional gaps in stain uptake were noted, however, suggesting that although the majority of intrasegmental bodies were not viable, some remained viable (Supple- mentary Figure S2). DNA sequencing of 16S rRNA amplicons extracted from the IMS material according to published methods (10) detected a broad range of purity, with 7.4%–68% of sequences specific for Candi-

Between 10 and 21 additional sequences were also detected, indicating the presence of other contaminating bacteria or DNA despite thorough rinsing of ileal mucosa prior to the collection of scrape material.

Following 3 weeks of incubation under microaerophilic conditions in the presence of 1:2 PSA, sequencing revealed increased

relative abundance of SFB, although there was again contaminating microbial DNA.

Identical procedures performed using recently weaned Crl:SD rat pups (Charles River, Newton, MA) yielded similar results (Figure 1C).

Operational taxonomic units (OTUs) detected during sequencing were specu- lated to represent dead, non-viable bacteria.

To confirm this hypothesis, blood agar, MacConkey agar, and Hektoen enteric agar plates were inoculated with filament- containing culture media and incubated under aerobic, anaerobic, and microaero- philic conditions. No growth was observed on any plate under any culture condition, even when incubated 168 h at 37°C. Of note, the culture conditions used to screen for growth of contaminating bacteria included the conditions under which the original mucosal scrapes were cultured.

Thus, any live bacteria in the original plates would have presumably been cultured in the subcultures as well.

Although seemingly viable SFB filaments were consistently maintained using the described culture conditions, repeated attempts to passage and truly culture (i.e., propagate) SFB were unsuccessful. As SFB and the ileal cells to which they adhere were subcultured, their numbers consistently diminished. One benefit of a pure prepa- ration of SFB is as a controlled inoculum in prospective studies. With this in mind, SFB-negative weanling BALB/cJ mice (The Jackson Laboratory, Bar Harbor, ME) were inoculated via gastric gavage with culture media containing SFB filaments. Briefly, pooled material from 4 wells of a 24-well plate was centrifuged at 300 × g for 5 min, supernatant was discarded, and the pellet was resuspended in 0.3 mL sterile water.

This inoculum, containing between 5000 and 6000 SFB filaments, was then used to inoculate a single mouse. Fourteen days post- inoculation, fecal PCR performed according to published methods (9) confirmed coloni- zation of SFB, demonstrating that the SFB preparation was infective when taken directly from the plate (Supplementary Figure S3).

This same experiment was also performed using culture media frozen with or without the addition of glycerol. At 14 days post-gavage, all mice that received frozen culture media lacking glycerol were found to be colonized by SFB, suggesting infective SFB monocul- tures prepared as described can be stored as frozen aliquots. Of the mice that received

culture media frozen with 10% glycerol, 3 of

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4 tested positive for SFB (Supplementary Figure S4).

To determine if our method produced a pure inoculum, germ-free (GF) Swiss Webster mice, bred and housed at the University of Washington Gnotobiotic Animal Core facility in germ-free isolators (Class Biologically Clean, Ltd., Madison, WI), were inoculated with culture media initiated 21 days prior. Fecal material from the putatively mono-associated mice was collected 1 week post-inoculation (PI), and IMS material was collected 2 weeks PI. As a control, the same samples were collected from a cohort of sham-gavaged mice, along with food samples from all cages (at 1 week PI and at end of study). At necropsy, all mice possessed dilated ceca consistent with GF status, and no fecal odor (an indicator of contamination) was detected. DNA extracted from feces, IMS material, and food was interrogated using microbial community profiling as before. Unusually low DNA yields were noted for all fecal pellets, regardless of group, relative to yields from normally colonized mice. Additionally, ileal scrapes from sham-inoculated mice yielded low amounts of DNA whereas ileal scrapes from culture-inoculated mice yielded substantial DNA. Despite loading equivalent amounts of starting template, sequencing provided much greater coverage for samples from mice inoculated with culture material; 3 samples failed to amplify and returned

≤10 sequences. Profiles of the control mice showed consistency and harbored primarily OTUs that were also detected in food (Figure 2). However, there were rare species detected in controls not detected in the food. Pooled fecal material from the three control mice prior to inocu- lation returned only four sequences and thus proved inconclusive. The GM of the 2 groups of mice inoculated with culture material was clearly dominated by SFB, with 98.4–98.9% of sequences aligning to Candidatus Arthromitus. Among other OTUs detected, the majority were also found in either food or pre-inoculation profiles, suggesting residual DNA from non-viable bacteria might be present in the food. Of those OTUs detected as background in the culture-inoculated mice but not found in any food or pre-inoculation samples, all were notably low in abundance, often on the order of a single copy (Supplementary Table S1). Histopathological examination revealed SFB adherent to the mucosa of all culture-inoculated mice and no other forms

of bacteria (Supplementary Figure S5); ileal tissues from sham-inoculated mice were devoid of any bacterial organisms. Gram staining once again demonstrated Gram positive characteristics of the filaments and no contaminating organisms (Supple- mentary Figure S1B). Attempts to culture fecal material from SFB-inoculated GF mice on blood, MacConkey, and Hektoen enteric agars, in aerobic, anaerobic, and micro- aerophilic conditions, were unsuccessful.

Collectively, these data confirm that our culture material was indeed pure SFB and free of contaminating bacteria capable of colonizing the mouse GM.

Our technique described here will allow researchers to prepare and store infective doses of pure SFB, with minimal required resources or technical expertise, as well as to characterize, compare, and manipulate distinct SFB isolates. There is evidence that multiple genetically distinct isolates of SFB exist in mice (11), and techniques such as those described here, in conjunction with the findings of Schnupf et al. (6), will greatly facilitate research on the biological impact and therapeutic potential of SFB. Finally, considering the recent implication of SFB in ulcerative colitis, these techniques may also be valuable for the study of SFB in humans, as well as in other host species.

Author contributions

A.E. and C.F. conceived and designed the study, analyzed the data, and wrote and edited the manuscript. G.T., A.E., and L.M. collected ileal mucosal scrapes and performed the culture experiments. A.E.

and A.W. extracted and purified DNA. C.H., S.M., and L.M.P. performed inoculation of, and sample collection from, axenic mice.

Acknowledgments

The authors would like to acknowledge IDEXX BioResearch for their donation of culture media and use of Gram stain reagents, Greg Purdy for assistance with cell cultures, Karen Clifford for assis- tance formatting images, Kate Anderson for assistance formatting references, and funding from the NIH to the MU Mutant Mouse Resource and Research Center (2U42OD010918-16). Studies in germ-free mice were supported in part by funds from the Department of Comparative Medicine, University of Washington. This paper is subject to the NIH Public Access Policy.

Competing interests

The authors declare no competing interests.

References

1. Denning, T.L., B.A. Norris, O. Medina-Contreras, S. Manicassamy, D. Geem, R. Madan, C.L. Karp, and B. Pulendran. 2011. Functional specializations of intestinal dendritic cell and macrophage subsets that control Th17 and regulatory T cell responses are dependent on the T cell/APC ratio, source of mouse strain, and regional localization. J. Immunol.

187:733-747.

2. Wu, H.J. and J. Ivanov II. 2010. Darce, K. Hattori, T. Shima, Y. Umesaki, D.R. Littman, C. Benoist, and D. Mathis. Gut-residing segmented filamentous bacteria drive autoimmune arthritis via T helper 17 cells. Immunity 32:815-827.

3. Lee, Y.K., J.S. Menezes, Y. Umesaki, and S.K. Mazmanian. 2011. Proinflammatory T-cell responses to gut microbiota promote experimental autoimmune encephalomyelitis. Proc. Natl. Acad.

Sci. USA 108(Suppl 1):4615-4622.

4. Yin, Y., Y. Wang, L. Zhu, W. Liu, N. Liao, M. Jiang, B. Zhu, H.D. Yu, et al. 2013. Comparative analysis of the distribution of segmented filamentous bacteria in humans, mice and chickens. ISME J. 7:615-621.

5. Caselli, M., D. Tosini, R. Gafa, A. Gasbarrini, and G. Lanza. 2013. Segmented filamentous bacteria-like organisms in histological slides of ileo- cecal valves in patients with ulcerative colitis. Am. J.

Gastroenterol. 108:860-861.

6. Schnupf, P., V. Gaboriau-Routhiau, M. Gros, R. Friedman, M. Moya-Nilges, G. Nigro, N.

Cerf-Bensussan, and P.J. Sansonetti. 2015.

Growth and host interaction of mouse segmented filamentous bacteria in vitro. Nature. 520:99-103.

7. Pamp, S.J., E.D. Harrington, S.R. Quake, D.A.

Relman, and P.C. Blainey. 2012. Single-cell sequencing provides clues about the host inter- actions of segmented filamentous bacteria (SFB).

Genome Res. 22:1107-1119.

8. Ohashi, Y., M. Hiraguchi, C. Sunaba, C. Tanaka, T. Fujisawa, and K. Ushida. 2010. Colonization of segmented filamentous bacteria and its interaction with the luminal IgA level in conventional mice.

Anaerobe 16:543-546.

9. Snel, J., P.P. Heinen, H.J. Blok, R.J. Carman, A.J. Duncan, P.C. Allen, and M.D. Collins. 1995.

Comparison of 16S rRNA sequences of segmented filamentous bacteria isolated from mice, rats, and chickens and proposal of “Candidatus Arthromitus”.

Int. J. Syst. Bacteriol. 45:780-782.

10. Ericsson, A.C., J.W. Davis, W. Spollen, N. Bivens, S. Givan, C.E. Hagan, M. McIntosh, and C.L.

Franklin. 2015. Effects of vendor and genetic background on the composition of the fecal micro- biota of inbred mice. PLoS ONE 10:e0116704.

11. Chung, H., S.J. Pamp, J.A. Hill, N.K. Surana, S.M. Edelman, E.B. Troy, N.C. Reading, E.J.

Villablanca, et al. 2012. Gut immune maturation depends on colonization with a host-specific micro- biota. Cell 149:1578-1593.

Received 04 March 2015; accepted 19 June 2015.

Address correspondence to Craig L. Franklin, De- partment of Veterinary Pathobiology, University of Missouri, 4011 Discovery Dr., N128, Columbia, MO 65201. E-mail: [email protected]

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