Basic Life Science Research Associate, Sarafan ChEM-H
Design, construction, and invivo augmentation of a complex gut microbiome.
Efforts to model the human gut microbiome in mice have led to important insights into the mechanisms of host-microbe interactions. However, the model communities studied to date have been defined or complex, but not both, limiting their utility. Here, we construct and characterize invitro a defined community of 104 bacterial species composed of the most common taxa from the human gut microbiota (hCom1). We then used an iterative experimental process to fill open niches: germ-free mice were colonized with hCom1 and then challenged with a human fecal sample. We identified new species that engrafted following fecal challenge and added them to hCom1, yielding hCom2. In gnotobiotic mice, hCom2 exhibited increased stability to fecal challenge and robust colonization resistance against pathogenic Escherichia coli. Mice colonized by either hCom2 or a human fecal community are phenotypically similar, suggesting that this consortium will enable a mechanistic interrogation of species and genes on microbiome-associated phenotypes.
View details for DOI 10.1016/j.cell.2022.08.003
View details for PubMedID 36070752
Establishing Stable Binary Cultures of Symbiotic Saccharibacteria from the Oral Cavity
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Many bacterial species cannot be cultured in the laboratory using standard methods, posing a significant barrier to studying the majority of microbial diversity on earth. Novel approaches are required to culture these uncultured bacteria so that investigators can effectively study their physiology and lifestyle using the powerful tools available in the laboratory. The Candidate Phyla Radiation (CPR) is one of the largest groups of uncultivated bacteria, comprising ~15% of the living diversity on earth. The first isolate of this group was a member of the Saccharibacteria phylum, 'Nanosynbacter lyticus' strain TM7x. TM7x is an unusually small bacterium that lives as a symbiont in direct contact with a bacterial host, Schaalia odontolytica, strain XH001. Taking advantage of the unusually small cell size and its lifestyle as a symbiotic organism, we developed a protocol to rapidly culture Saccharibacteria from dental plaque. This protocol will show how to filter a suspension of dental plaque through a 0.2 µm filter, then concentrate the collected Saccharibacteria cells and infect a culture of host organisms. The resulting coculture can be passaged as any normal bacterial culture and infection is confirmed by PCR. The resulting binary culture can be maintained in the laboratory and used for future experiments. While contamination is a possibility, the binary culture can be purified by either further filtering and reinfection of host, or by plating the binary culture and screening for infected colonies. We hope this protocol can be expanded to other sample types and environments, leading to the cultivation of many more species in the CPR.
View details for DOI 10.3791/62484
View details for Web of Science ID 000646217000090
View details for PubMedID 33938896
A method for detection of SARS-CoV-2 RNA in healthy human stool: a validation study.
The Lancet. Microbe
Background: Faecal shedding of SARS-CoV-2 has raised concerns about transmission through faecal microbiota transplantation procedures. Validation parameters of authorised tests for SARS-CoV-2 RNA detection in respiratory samples are described in product labelling, whereas the published methods for SARS-CoV-2 detection from faecal samples have not permitted a robust description of the assay parameters. We aimed to develop and validate a test specifically for detection of SARS-CoV-2 in human stool.Methods: In this validation study, we evaluated performance characteristics of a reverse transcriptase real-time PCR (RT-rtPCR) test for detection of SARS-CoV-2 in human stool specimens by spiking stool with inactivated SARS-CoV-2 material. A modified version of the US Centers for Disease Control and Prevention RT-rtPCR SARS-CoV-2 test was used for detection of viral RNA. Analytical sensitivity was evaluated in freshly spiked stool by testing two-fold dilutions in replicates of 20. Masked samples were tested by a second laboratory to evaluate interlaboratory reproducibility. Short-term (7-day) stability of viral RNA in stool samples was assessed with four different stool storage buffers (phosphate-buffered saline, Cary-Blair medium, Stool Transport and Recovery [STAR] buffer, and DNA/RNA Shield) kept at -80°C, 4°C, and ambient temperature (approximately 21°C). We also tested clinical stool and anal swab specimens from patients who were SARS-CoV-2 positive by nasopharyngeal testing.Findings: The lower limit of detection of the assay was found to be 3000 viral RNA copies per g of original stool sample, with 100% detection across 20 replicates assessed at this concentration. Analytical sensitivity was diminished by approximately two times after a single freeze-thaw cycle at -80°C. At 100 times the limit of detection, spiked samples were generally stable in all four stool storage buffers tested for up to 7 days, with maximum changes in mean threshold cycle values observed at -80°C storage in Cary-Blair medium (from 29·4 [SD 0·27] at baseline to 30·8 [0·17] at day 7; p<0·0001), at 4°C storage in DNA/RNA Shield (from 28·5 [0·15] to 29·8 [0·09]; p=0·0019), and at ambient temperature in STAR buffer (from 30·4 [0·24] to 32·4 [0·62]; p=0·0083). 30 contrived SARS-CoV-2 samples were tested by a second laboratory and were correctly identified as positive or negative in at least one of two rounds of testing. Additionally, SARS-CoV-2 RNA was detected using this assay in the stool and anal swab specimens of 11 of 23 individuals known to be positive for SARS-CoV-2.Interpretation: This is a sensitive and reproducible assay for detection of SARS-CoV-2 RNA in human stool, with potential uses in faecal microbiota transplantation donor screening, sewage monitoring, and further research into the effects of faecal shedding on the epidemiology of the COVID-19 pandemic.Funding: National Institute of Allergy and Infectious Diseases, US National Institutes of Health; Center for Biologics Evaluation and Research, US Food and Drug Administration.
View details for DOI 10.1016/S2666-5247(21)00059-8
View details for PubMedID 33821247
Insights Obtained by Culturing Saccharibacteria With Their Bacterial Hosts
JOURNAL OF DENTAL RESEARCH
2020; 99 (6): 685-694
Oral microbiome research has moved from asking "Who's there?" to "What are they doing?" Understanding what microbes "do" involves multiple approaches, including obtaining genomic information and examining the interspecies interactions. Recently we isolated a human oral Saccharibacteria (TM7) bacterium, HMT-952, strain TM7x, which is an ultrasmall parasite of the oral bacterium Actinomyces odontolyticus. The host-parasite interactions, such as phage-bacterium or Saccharibacteria-host bacterium, are understudied areas with large potential for insight. The Saccharibacteria phylum is a member of Candidate Phyla Radiation, a large lineage previously devoid of cultivated members. However, expanding our understanding of Saccharibacteria-host interactions requires examining multiple phylogenetically distinct Saccharibacteria-host pairs. Here we report the isolation of 3 additional Saccharibacteria species from the human oral cavity in binary coculture with their bacterial hosts. They were obtained by filtering ultrasmall Saccharibacteria cells free of other larger bacteria and inoculating them into cultures of potential host bacteria. The binary cocultures obtained could be stably passaged and studied. Complete closed genomes were obtained and allowed full genome analyses. All have small genomes (<1 Mb) characteristic of parasitic species and dramatically limited de novo synthetic pathway capabilities but include either restriction modification or CRISPR-Cas systems as part of an innate defense against foreign DNA. High levels of gene synteny exist among Saccharibacteria species. Having isolates growing in coculture with their hosts allowed time course studies of growth and parasite-host interactions by phase contrast, fluorescence in situ hybridization, and scanning electron microscopy. The cells of the 4 oral Saccharibacteria species are ultrasmall and could be seen attached to their larger Actinobacteria hosts. Parasite attachment appears to lead to host cell death and lysis. The successful cultivation of Saccharibacteria species has significantly expanded our understanding of these ultrasmall Candidate Phyla Radiation bacteria.
View details for DOI 10.1177/0022034520905792
View details for Web of Science ID 000514540000001
View details for PubMedID 32075512
View details for PubMedCentralID PMC7243422
Isolation and cultivation of candidate phyla radiationSaccharibacteria(TM7) bacteria in coculture with bacterial hosts
JOURNAL OF ORAL MICROBIOLOGY
2020; 12 (1): 1814666
The vast majority of bacteria on earth have not yet been cultivated. There are many bacterial phyla with no cultivated examples including most members of the Candidate Phylum Radiation with the exception of human oral isolates from the phylum Saccharibacteria.The aims of this research were to develop reproducible methods and validate approaches for the cultivation of human oral Saccharibacteria and to identify the conceptual pitfalls that delayed isolation of these bacteria for 20 years after their discovery.Oral samples were dispersed and passed through 0.2 µm membrane filters. The ultrasmall saccharibacterial cells in the filtrate were pelleted, inoculated into broth cultures of potential bacterial host cells and passaged into fresh medium every 2-3 days.Thirty-two isolates representing four species of Saccharibacteria were isolated in stable coculture with three species of host bacteria from the phylum Actinobacteria. Complete genome sequences were obtained for 16 isolates.Human oral Saccharibacteria are obligate bacterial parasites that can be stably passaged in coculture with specific species of host bacteria. Isolating these important members of the human oral microbiome, and many natural environments, requires abandoning many of Koch's concepts and methods and embracing novel microbiological approaches.
View details for DOI 10.1080/20002297.2020.1814666
View details for Web of Science ID 000566651300001
View details for PubMedID 33209205
View details for PubMedCentralID PMC7651992
Complete Genome Sequence of Strain AC001, a Novel Cultured Member of the Human Oral Microbiome from the Candidate Phylum Saccharibacteria (TM7)
MICROBIOLOGY RESOURCE ANNOUNCEMENTS
2019; 8 (42)
Strain AC001 is a cultured representative of human microbial taxon 488, a bacterium from the candidate phylum Saccharibacteria It is an obligate parasite with a genome of <0.9 Mb and grows in coculture with its host, Pseudopropionibacterium propionicum The complete genome sequence is presented here.
View details for DOI 10.1128/MRA.01158
View details for Web of Science ID 000490937700015
View details for PubMedID 31624156
View details for PubMedCentralID PMC6797545
Complete Genome Sequence of Strain PM004, a Novel Cultured Member of the Human Oral Microbiome from the Candidate Phylum Saccharibacteria (TM7)
MICROBIOLOGY RESOURCE ANNOUNCEMENTS
2019; 8 (42)
Strain PM004 is a cultured representative of human microbial taxon 955, a bacterium from the phylum Saccharibacteria It is an obligate parasite with a genome of <0.9 Mb and can be grown in coculture with its host, Pseudopropionibacterium propionicum The complete genome sequence is presented here.
View details for DOI 10.1128/MRA.01159-19
View details for Web of Science ID 000490937700016
View details for PubMedID 31624157
View details for PubMedCentralID PMC6797546