C.W.Y. Ha1, S. Yilmaz2, S. Devkota1.
1F. Widjaja Foundation Inflammatory Bowel and Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA. 2 General Automation Lab Technologies, 953 Indiana St., San Francisco, CA 94107, USA.
A novel high-throughput system for isolation, cultivation and screening of microorganisms from complex ecosystems
BACKGROUND
Defined communityMixture obligate anaerobes and facultative anaerobes§ 42 organisms from 5 different phyla.§ Included slow- and fast-growing bacteria.§ One species was at ~0.01% relative abundance in the
starting community, representing the ”rare” taxa.
Complex communitySpecimen from human gut mucosa§ Starting composition was unclear at the time of
inoculation, but typically comprised of anaerobes.
42 species that made up the mock community Protocol
Cedars-Sinai Medical CenterEmail: [email protected]
Phone: 310-423-4165
Species recovery and distribution of taxa post-incubation
§ A key challenge in the microbiome field is to movebeyond correlative gene-based bacterial profiling and tobetter understand the ecological function of individualspecies within a community.
§ There is a renewed interest in microbial cultivation inbiomedical, agricultural and biotechnology research.
§ Limitations of existing cultivation methods:- Space and labor intensive and thus, difficult to scale.- Bias towards fast growing or swarming organisms
when non-selective media is used.- Difficult to isolate rare taxa from a complex community.- Agar plates: Only 50-100 discernible isolates per dish.- Culture tubes: Growth affected by niche competition.
§ The GALT array: a newly-developed microcultivationplatform, which consists of 50,000 miniaturizedgrowth chambers in a 3 x 2 x 0.1 inch chip.
1) Determine whether the microcultivation platformperforms better than commonly used approaches inisolating members of a heterogenous population.
2) Determine whether this cultivation approach can beused to optimize media composition for improveddiversity recovery.
§ Individually grown cultures were diluted with pre-reducedmedia to the same OD600. P. distasonis was further diluted.
§ Bacterial suspensions were combined in equal volumes anddiluted to ~1000 cells/uL.
§ Agar plate: Serial dilutions of the suspension were plated.Culture tube: Diluted suspension was used for inoculation.Microcultivation: Mixture was loaded (1 cell per well), chipwas sealed with adhesive film.
§ Cultures were incubated anaerobically. Cells were thencollected for DNA extraction and 16S sequencing.
The performance of three cultivation platforms (agarplates, culture tubes with liquid media and microcultivation)were assessed using consortia with distinct structures:
Number of taxa Simpson index
Media 1Media 2
2 types of growth media were tested in this study.
Array- based microcultivation promotes isolation and even representation of cultivable taxa from a complex microbiome
100 micron
Empty microwell
Inoculated microwell
§ The GALT array is compatible with aerobic andanaerobic cultivation.
§ Pure cultures on the array can be visualizedunder a light microscope.
§ Fast growing organisms were less likely todominate in the microcultivation systemcompared to the culture tube counterpart.
§ Low abundance taxa in the defined consortiumwas successfully captured by microcultivation.
§ Slow growing organisms were represented inboth culture tubes and the high densitymicrowells.
§ The array based platform yielded the highestnumber of distinct taxa.
§ Despite this, there are some taxa exclusive tothe culture tube method. May be due to cross-feeding between species.
§ Results from all three cultivation platformssupport that Media 2 is better suited forcultivation of mucus-associated bacteria thanMedia 1.
§ Microcultivation was as efficient as culturetubes in recovering distinct taxa fromheterogeneous microbial populations, whileagar based method was the least effective.
RECOVERY AND CULTIVATION OF ANAEROBES – HUMAN GUT MUCOSA
Agar plate
Culture tube
Micro-cultivation
Agar plate
Culture tube
Micro-cultivation
Alp
ha d
iver
sity
mea
sure
ActinobacteriaActinomyces odontolyticusCollinsella aerofaciensEggerthella lentaGardnerella vaginalisRothia mucilaginosa
FusobacteriaFusobacterium nucleatum
FirmicutesBlautia hanseniiBlautia productaClostridium innocuumClostridium perfringens
Clostridium ramosumCoprobacillus sp.Eisenbergiella tayiEnterococcus faecalisFlavonifractor plautiiGemella sanguinisLactobacillus rhamnosus GGLactobacillus gasseriOscillibacter ruminantiumParvimonas micraPhascolarctobacterium sp.Ruminococcus gnavusRuminococcus torquesStaphylococcus epidermidisStreptococcus australisStreptococcus anginosusStreptococcus lutetiensisTyzzerella nexilisVeillonella atypicaVeillonella parvula
ProteobacteriaBilophila wadsworthiaEscherichia coliHaemophilus parainfluenzaeKlebsiella sp.Proteus mirabilis
BacteroidetesBacteroides fragilisBacteroides ovatusBacteroides thetaiotaomicronBacteroides uniformisBacteroides vulgatusParabacteroides distasonisParabacteroides merdae
Fast grower; slow grower; low abundance species
STUDY DESIGN
Diversity of post-incubation cultures Relative abundance of taxa in each cultivation method
§ These results demonstrate that the GALT arrayis both a space-saving and effective way toprofile the cultivable microbiome.
§ This highly-parallelized array technologysupports multiple core workflows within amicrobiome lab, including cultivation and mediaoptimization.
§ The microcultivation array is agnostic to thesource of microbial community. Thus,organisms from various environments canbe sampled, suggesting broad applicationsin basic and applied research.
CONCLUSION
The number of species captured by each method
Relative abundance of the “rare” taxa
Microcultivation array at day 4
Relative abundance of the slow growing organism
Recovery rates MEDIA 1 MEDIA 2
Agar plate 34/42 = 81% 30/42 = 71.4%
Culture tube 37/42 = 88% 39/42 = 93%
Microcultivation 38/42 = 90.5% 38/42 = 90.5%
Relative abundance of the fast growing organism
Coprobacillus sp. MEDIA 1 MEDIA 2
Agar plate 0.02% 0.04%
Culture tube 0.02% 0.2%
Microcultivation 0.1% 0.08%
C. perfringens MEDIA 1 MEDIA 2
Agar plate 4.2% 5.6%
Culture tube 4.3% 15.6%
Microcultivation 3.6% 5.7%
P. distasonis MEDIA 1 MEDIA 2
Agar plate - 0.4%
Culture tube - 0.6%
Microcultivation 0.8% 1.5%
AIMS
RECOVERY AND CULTIVATION OF ANAEROBES - DEFINED COMMUNITY SUMMARY
U.S. quarter
coin
Observable cultivable taxa from human gut mucosa across three cultivation platforms. Left) Alpha diversity measures of primary cultures after four days of anaerobic growth. 16S data were analyzed using DADA2 and phyloseq pipelines. Diversity was assessed after normalization to 53,000 reads per sample. Right) Distribution of the top 24 genera in each cultivation method. The dividers within each bar reflect the number of taxa that make up the genus.
§ Test the compatibility of this array basedcultivation platform with fluorescent assaysand functional assays for high-throughputscreening of intestinal organisms withspecific traits.
§ Determine whether the microcultivationplatform is suitable for fungal growth.
Genus
Micro-
cultivationCulture
tubeAgar plate
FUTURE DIRECTION
