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Human bacteria isolation Study

Purpose

The purpose of this study is to collect fecal samples from 200 healthy human donors. We will use the stool samples to:

  • Estimate presence and abundance of Clostridia Cluster III, IV and XIVa species via DNA bacterial sequencing

  • Estimate presence and abundance of siderophore-antimicrobial peptide producing Enterobacteriaceae species

  • Anaerobically isolate and bank fecal bacteria in order to build a frozen bacterial biorepository

Background

The gut microbiome is the community of microbes that inhabit the gastrointestinal tract. A plethora of studies from the last 10 years has shown how microbiome reconstitution with defined combination of bacteria that evolved and adapted to the human gastrointestinal tract is a powerful tool for treatment and prevention of multiple inflammatory and infectious diseases. For example, work from Dr. Bucci and collaborators have shown that the administration of short-chain fatty acids producing Clostridia is a potent inducer of regulatory T-cells (Tregs) and can cure colitis in a mouse models1,2. Similarly we have previously shown that combinations of gastrointestinal members of the Bacteroides and Parabacteroides genera induce potent CD8+ response in animal models of melanomoma3. Finally in collaborations with groups at Weill Cornell and the NIH we have established the role of gut bacteria in affecting immunity in patients undergoing anti-microbial treatment for Tb4,5.

In addition to the immune-modulatory properties of gut bacteria, our group has provided extensive evidence that these human-adapted microbes can provide potent colonization resistance against enteric pathogens and antimicrobial-resistant bacteria6. For example, we have demonstrated the microbiota supplementation with the commensal bacteria C. scindens protects mice from C. difficile-induced colitis via C. scindens ability in transforming primary into secondary bile acids which prevent C. difficile germination7. We have demonstrated that secondary metabolites that are produced by gastrointestinal Enterobacteriaceae such as Microcin MccH47 and microcin MccI47 are potent inhibitor of drug-resistant E. coli, Salmonella and Klebsiella species8,9.

Based on this background, we aim to leverage our expertise in anaerobic microbiology, biochemistry, systems and synthetic biology to construct a biorepository of bacteria isolated from healthy humans. These bacteria will be stored in glycerol stocks and will be used for in vitro and in vivo experiments.

The fecal matter collected from human individuals will be used solely as source of bacteria. We will not collect any human data or individual information. Samples received will be completely deidentified upon reception.

References:

1. Atarashi K, Tanoue T, Oshima K, Suda W, Nagano Y, Nishikawa H, Fukuda S, Saito T, Narushima S, Hase K, Kim S, Fritz JV, Wilmes P, Ueha S, Matsushima K, Ohno H, Olle B, Sakaguchi S, Taniguchi T, Morita H, Hattori M, Honda K. Treg induction by a rationally selected mixture of Clostridia strains from the human microbiota. Nature 500(7461), 232–6, 2013. 

2. Stein RR, Tanoue T, Szabady RL, Bhattarai SK, Olle B, Norman JM, Suda W, Oshima K, Hattori M, Gerber GK, Sander C, Honda K, Bucci V. Computer-guided design of optimal microbial consortia for immune system modulation. eLife 7, e30916, 2018.

3. Tanoue T, Morita S, Plichta DR, Skelly AN, Suda W, Sugiura Y, Narushima S, Vlamakis H, Motoo I, Sugita K, Shiota A, Takeshita K, Yasuma-Mitobe K, Riethmacher D, Kaisho T, Norman JM, Mucida D, Suematsu M, Yaguchi T, Bucci V, Inoue T, Kawakami Y, Olle B, Roberts B, Hattori M, Xavier RJ, Atarashi K, Honda K. A defined commensal consortium elicits CD8 T cells and anti-cancer immunity. Nature 565(7741), 600–605, 2019.

4. Wipperman MF, Fitzgerald DW, Juste MAJ, Taur Y, Namasivayam S, Sher A, Bean JM, Bucci V, Glickman MS. Antibiotic treatment for Tuberculosis induces a profound dysbiosis of the microbiome that persists long after therapy is completed. Sci Rep 7(1), 10767, 2017.

5. Vorkas CK, Wipperman MF, Li K, Bean J, Bhattarai SK, Adamow M, Wong P, Aube J, Juste MAJ, Bucci V, Fitzgerald DW, Glickman MS. Mucosal-associated invariant and gammadelta T cell subsets respond to initial Mycobacterium tuberculosis infection. JCI Insight 3(19), 2018.

6. Kim S, Covington A, Pamer EG. The intestinal microbiota: Antibiotics, colonization resistance, and enteric pathogens. Immunol Rev 279(1), 90–105, 2017.

7. Buffie CG, Bucci V, Stein RR, McKenney PT, Ling L, Gobourne A, No D, Liu H, Kinnebrew M, Viale A, Littmann E, van den Brink MRM, Jenq RR, Taur Y, Sander C, Cross JR, Toussaint NC, Xavier JB, Pamer EG. Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile. Nature 517(7533), 205–208, 2015.

8. Palmer JD, Piattelli E, McCormick BA, Silby MW, Brigham CJ, Bucci V. Engineered Probiotic for the Inhibition of Salmonella via Tetrathionate-Induced Production of Microcin H47. ACS Infect Dis (Journal Article), 2017.

9. Sassone-Corsi M, Nuccio SP, Liu H, Hernandez D, Vu CT, Takahashi AA, Edwards RA, Raffatellu M. Microcins mediate competition among Enterobacteriaceae in the inflamed gut. Nature 540(7632), 280–283, 2016.

Inclusion and Exclusion Criteria

Inclusion

  • Male or female subjects 18 years of age, but not more than 60 years of age at the time of enrollment.
  • Must be able to provide signed and dated informed consent. 
  • Healthy subjects willing to provide stool specimens under no financial expectation or interest

Exclusion

  • Any subject who meets any of the following criteria will be excluded from participation in this study:
  • Body Mass Index greater than or equal to 35 or less than or equal to 18.
  • Use of any of the following drugs within the last 6 months:
    •  systemic antibiotics, antifungals, antivirals or antiparasitics (intravenous, intramuscular, or oral);
    •  oral, intravenous, intramuscular, nasal or inhaled corticosteroids;
    •  cytokines;
  • methotrexate or immunosuppressive cytotoxic agents;
  • large doses of commercial probiotics consumed (greater than or equal to 108 cfu or organisms per day) - includes tablets, capsules, lozenges, chewing gum or powders in which probiotic is a primary component. (Ordinary dietary components such as fermented beverages/milks, yogurts, foods do not apply);
  • Acute disease at the time of collection (defer sampling until subject recovers). Acute disease is defined as the presence of a moderate or severe illness with or without fever.
  • Chronic, clinically significant (unresolved, requiring on-going medical management or medication) pulmonary, cardiovascular, gastrointestinal, hepatic or renal functional abnormality, as determined by medical history or physical examination.
  • Unstable dietary history as defined by major changes in diet during the previous month, where the subject has eliminated or significantly increased a major food group in the diet.
  • Recent history of chronic alcohol consumption defined as more than five 1.5-ounce servings of 80 proof distilled spirits, five 12-ounce servings of beer or five 5-ounce servings of wine per day.
  • Positive test for HIV, HBV or HCV.
  • Any confirmed or suspected condition/state of immunosuppression or immunodeficiency (primary or acquired) including HIV infection.
  • Major surgery of the GI tract, with the exception of cholecystectomy and appendectomy, in the past five years. Any major bowel resection at any time.
  • History of active uncontrolled gastrointestinal disorders or diseases including:
    • inflammatory bowel disease (IBD) including ulcerative colitis (mild-moderate-severe), Crohn's disease (mild-moderate-severe), or indeterminate colitis;
    •  irritable bowel syndrome (IBS) (moderate-severe);
    •  persistent, infectious gastroenteritis, colitis or gastritis, persistent or chronic diarrhea of unknown etiology, Clostridium difficile infection (recurrent) or Helicobacter pylori infection (untreated);
    •  chronic constipation

Fecal Samples Collection

  • Upon consent and enrollment, the subject will receive a Fisherbrand commode specimen collection system. The subject will be provided a Sigma anaerobic sachet to be added to the specimen collection bucket upon closure in order to maintain anaerobiosis. The subject will also be provided with dry ice pouches and a Styrofoam box in where to place the closed bucket and the ice.

  • After sample collection at home, one of the researchers will pick up the sample from the subject. The subject will notify researcher/PI about location pickup. This researcher will transport the sample to campus and will place it into a dedicated -80 oC freezer in the Bucci laboratory in AS8. The sample will need to be retrieved within 24 hours. Also, alternatively to sample pick up by staff personnel, the subject will be able to drop-off the sample at UMMS AS8-2057 or AS8-1057.

  • Another researcher with no knowledge of pick up location will open the sample, assign a random barcode and perform microbial DNA extraction and bacterial isolation in anaerobic chamber.

  • No information on address or pickup location will be retained. At the end of the process there will be a barcode for the sample and a secondary barcoding set to link bacterial isolates to the sample of origin. There will be no possible way to link stool sample to

Fecal DNA Sequencing and Informatics

DNA will be extracted in the lab from the stool sample using Qiagen PowerSoil DNA extraction kit. Indexed DNA libraries will be prepared with the Illumina Nextera XT kit. DNA  will be sequenced and resulting sequences will be informatlically processed as in several publications  from the group4,5.

Bacterial Isolation

Bacterial isolation will be performed by streaking fecal matter on agar plates of different media composition optimized to obtain selective growth of specific bacterial members (e.g. Reinforced Clostridial Agar).

  • Agar plates will be prepared outside the chamber according to lab protocol instructions.

  • After the chamber is turned on, all plates, media, and equipment to be used will be placed in the anaerobic chamber ahead of time to allow to off-gas oxygen (minimum 24 hours)

  • Plates will be then put into the chamber at 35°C for at least 30 minutes before plating to allow media to come to temperature.

  • Fecal samples will be taken from the -80 °C freezer, where they are banked, one sample at a time

  • The sample will be introduced in the anaerobic chamber and let to partially thaw

  • A small piece of feces (less than pea sized) will be removed and transferred to a 1.5mL microfuge tube containing 500 uL of anaerobic saline buffer to resuspend

  • The mixture will be vigorously vortexed and resuspended into four serial dilutions

  • After ensuring that all plates are appropriately labeled with media type, antibiotic concentration if any, and the dilution and sample to be plated on them, add ~10 sterile glass beads to each plate.

  • 100uL from each dilution will be spotted in the center of each plate. This will be done in duplicate (2 plates per dilution)

  • Plates will be shaken in a side to side motion, avoiding swirling of the beads. Beads are decanted into the waste container with 70% ethanol, limiting the time that the plate is exposed.

  • Plates are incubated for 24 - 48 hours at 35°C inside the anaerobic chamber (incubator inside).

  • After incubation, single colonies are picked based on morphology to be grown in liquid culture (1.5 ml) in appropriate medium.

  • After 24-48 hours of growth in liquid, each culture is transferred into a cryotube containing 50% glycerol (1:1) ratio and appropriately labelled (glycerol stock).

  • Part of the liquid culture will be introduced into a single well within a 96-well plate and used to amplify the 16S ribosomal RNA (rRNA) gene via PCR.

  • The amplified 16S rRNA gene will be sent out for Sanger sequencing to determine the taxonomic assignment of the isolated bacterium (e.g. Escherichia coli).

  • The stock is removed from the chamber and stored in a dedicated rack in a -80°C freezer (biorepository).

For each sample we will store in a database (1) the results from microbial genome sequencing as fasta files, (2) the list of strain IDs in the bacterial biobank that were derived from that sample, (3) the location in the biorepository freezer of the raw stool sample and of the related bacterial isolates.

Data and Specimen Banking

SUBJECT IDENTIFIERS: There is no identification of study participants.

SPECIMEN IDENTIFIERS: All specimens collected will be assigned anonymous specimen identifiers. There will be no possibility to link specimen to human subject that provided the specimen

MANAGEMENTOF PATIENTDATA:No subject data will be collected. The subjects are considered as just fecal matter donors.

DATE INFORMATION: The date of specimen collection will be recorded in order to keep record on when the sample arrived in the laboratory. No effort will be made to associate dates of collection with any subject/donor. Publication or other public release of data or results will not include collection dates but will refer to time of collection as the number of days from initiation of study participation. The year of collection may be included in publication or public release of data.

SPECIMEN BANKING: All stool specimens and all derived materials (such as, but not limited to: RNA, DNA, protein) will be stored in research labs for future use unless consumed in research activities. All specimens will be stored de-identified