Media supplements and methods to culture human gastrointestinal anaerobic microorganisms

a technology of anaerobic microorganisms and media supplements, which is applied in the field of media supplements and methods to culture human gastrointestinal anaerobic microorganisms, can solve the problems of difficult culture of gut microorganisms, hampered studies of gut microorganisms, and novel culture techniques to grow these microorganisms. to achieve the effect of enhancing the growth of axenic bacteria in vitro

Inactive Publication Date: 2014-11-20
UNIVERSITY OF GUELPH
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  • Abstract
  • Description
  • Claims
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Benefits of technology

[0011]In an aspect, there are provided methods for culturing microorganisms from the human distal colon using a media supplement termed “Liquid Gold”. Liquid Gold refers to a 0.2 μm filtrate of spent culture media or effluent from a chemostat vessel in which a target ecosystem is cultured. Liquid Gold is used to supplement culture media, e.g., standard laboratory media. Supplementation with Liquid Gold allows culturing of “unculturable” microorganisms, i.e., microorganisms which are refractory to culture axenically using traditional methods. Without wishing to be limited by theory, it is believed that Li

Problems solved by technology

This is because the individual species of the gut microbiota are difficult to culture axenically in vitro (Hart et al., 2002).
It is recognized that novel culture techniques are required to grow these “unculturable” microorganisms.
Studies of gut microbiota have been hampered by a lack of model systems.
For example, different study participants can each have unique, host-specific community profiles representing their gut microbiota, making comparison of the gut microbiota between subjects difficult, especially when attempting to correlate the effects of a treatment to changes in the gut microbiota.
In vivo models also often limit the dynamic monitoring of the gut microbiota by deriving th

Method used

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  • Media supplements and methods to culture human gastrointestinal anaerobic microorganisms
  • Media supplements and methods to culture human gastrointestinal anaerobic microorganisms
  • Media supplements and methods to culture human gastrointestinal anaerobic microorganisms

Examples

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example 1

Threshold for DGGE Analysis

[0125]A study by Possemiers et al. (Possemiers, S. et al., FEMS Microbiol. Ecol., 49(3):495-507, 2004) established a threshold stability criterion of 80% similarity (or 20% variability) for DGGE studies based on within-gel variability seen between identical marker lanes. According to this study, the threshold can be used in conjunction with moving window correlation analysis to monitor the dynamics of the community. This approach allows us to examine the similarity of a vessel to itself over time. The 80% similarity threshold can then be used to determine how long it takes a vessel to rise above this cut-off and reach steady state.

[0126]In order to apply this threshold to our studies, a similar analysis was carried out on the marker lanes from our DGGE gels. We found an average of 80.4±8.9% similarity, or 19.6±8.9% variability, between these marker lanes. Since these values correspond well with the values used in the Possemiers et al. study, a threshold of...

example 2

Comparison of Same Donor Over Time

[0128]The DGGE pattern of 10% inocula from Donor 2 (a 38-year old healthy female) over an 8 month period is shown in FIG. 2. As seen by visual inspection, the variation in the profiles seems to be due to differences in band brightness, not the appearance or disappearance of bands. The inocula isolated from this donor had an average correlation coefficient of 76.9±8.7%. Slight differences between profiles were shown in FIG. 2 where samples showed slightly higher similarity depending on the time of sample collection. Overall, the gut microbiota of this healthy donor remained stable over time.

[0129]The gut microbiota of this donor maintained a high diversity over time, with an average Shannon-Weaver index value of 3.39±0.08. The donor's microbiota also maintained a very high average range-weighted richness at 776.5±27.7. Finally, the community evenness was stable over time, with an average Shannon equitability value of 0.82±0.02.

example 3

Comparison of Different Donors

[0130]We used DGGE to compare fecal inocula isolated from four different healthy donors (from 38 to 43 years of age with no recent history of antibiotic treatment) (FIG. 3). We found that the fecal community of each donor was different from the communities of other individuals (as expected, Tannock, G. W., Eur. J. Clin. Nutr., 56 Suppl. 4:S44-9, 2002). The average correlation coefficient between the inocula from different donors was 74.4±8.4%.

[0131]The gut microbiota of all four donors had high diversity, with an average Shannon-Weaver index value of 3.42±0.04. The donor microbiota also maintained a very high average range-weighted richness at 585.3±26.2. The community evenness between the different donors was quite similar, with an average Shannon equitability value of 0.86±0.01.

[0132]We also used DGGE to compare two different communities, seeded by fecal samples from two different healthy donors, each of whom donated on at least two separate occasions...

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Abstract

A media supplement for culturing anaerobic bacteria is provided which comprises a filtrate of effluent from a chemostat vessel in which a target bacterial ecosystem has been cultured. Methods of using the supplement for culturing or isolating anaerobic microbial strains or communities, particularly anaerobic bacteria from the human gut, are also provided.

Description

RELATED APPLICATIONS[0001]This application claims priority to U.S. provisional application No. 61 / 534,456, filed on Sep. 14, 2011, the entire contents of which are hereby incorporated by reference. This application is related to copending international PCT application titled “Method for Treatment of Disorders of the Gastrointestinal System,” filed Sep. 14, 2012, the entire contents of which are hereby incorporated by reference.FIELD OF THE INVENTION[0002]This invention relates to methods for culturing microbial communities from the human distal colon and methods for isolating anaerobic microbes from such communities, as well as media supplements for use in such methods.BACKGROUND OF THE INVENTION[0003]The human gut is the most densely inhabited ecosystem on Earth (Marchesi and Shanahan, 2007). Like other complex microbial ecosystems, the human microbiota has not been sampled to completion (Eckburg et al., 2005). This is because the individual species of the gut microbiota are diffic...

Claims

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Application Information

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IPC IPC(8): C12N1/20
CPCC12N1/20A61P1/12A61P29/00A61P31/04C12R2001/145C12N1/205C12R2001/01A61K35/74A61K35/745A61K35/747
Inventor ALLEN-VERCOE, EMMAMCDONALD, JULIE
Owner UNIVERSITY OF GUELPH
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