Effect of HIV-infection & highly-active antiretroviral therapy on the human
gut microbiome investigated by phylogenetic-targeted pyrosequencing:
A meta-genomic profile of duodenal biopsies,
aspirates, and stool over time
C.L. Ellis1,2, C.-S. Li2, S. Mann3, Z.-M. Ma4, H. Overman2, A. Maniar1,
T. Yotter1, E. Tsuchida5, T.H. Knight1, J.C. Rutledge1,
C.J. Miller1,4, R.B. Pollard1, D.M. Asmuth1
1University
of California Davis Medical School , Sacramento, CA, USA
2University of California Davis, Davis, CA, USA
3Mather Veteran's Administration Hospital, Internal Medicine, Mather, CA, USA
4California National Primate Research Center, Davis, CA, USA
5CARES Clinic, Sacramento, CA, USA
IAS 2011, July 18, Rome, Italy
Abs. MOAA0104
Conflict of Interest Disclosures
• None to declare
We house >trillion gut microbes (~99%
Bacteria) outnumbering our own cells & genes
Majority of the microbial ‘communities’ composing this
gut microbial meta-genome, called the gut microbiome
(or microbiota), cannot be studied by classical culturing
Characterize based on evolutionary- or phylo- genetic signatures
within specific 16S ribosomal RNA (rRNA) gene sequences (rDNA)
16S rDNA Quantitative PCR
16S rDNA Sequencing
environmental or meta- genomics
Hamaday M & Knight R. Gen Res (2009)
HIV Disease & The Gut Microbiome:
Overall Research Questions
 Is the gut microbiome altered during acute/chronic HIV
disease (i.e. any microbial communities depleted or that
actually thrive in an infected/inflammed gut)?
 What about after therapy and immune reconstitution?
Any relationships to gut inflammation, barrier
destruction,
microbial
translocation,
and
systemic immunopathy/inflammation
Marchettia G et al.
Ellis CL et al.
Merlini E. et al.
Gori A et al.
Estes et al.
McKenna P et al.
Hunt P et al.
Wolf BW et al.
Anukam KC et al.
Brenchley JM
Jiang W et al.
Gautraux MD et al.
Higher-Taxonomic qPCR Assessment
of Stool Microbiota in HIV Patients
We observed an unexpected increase in the
proportion
of
‘yet-unidentified’
microbiota
not
covered by common primers which accounted for
majority of seronegative microbiota
Ellis CL et al. CROI, 317 (2011)
HIV Seronegative
Control (n=5)
HIV Positive
pre-ART (n=18)
Bacteroidales
Clostridiales
Lactobacilliales
Other/Unidentified
HIV Positive 9 mo
post-ART (n=13)
HIV Seronegative
Control (n=5)
HIV Positive 9 mo
post-ART (n=13)
HIV Positive
pre-ART (n=18)
Bacteroidales
Lactobacilliales
% Enterobacteriales
Clostridiales
0.06
% Enterobacteriales
(Gamma-Proteobacteria)
0.04
Control
Baseline
9 month
0.02
m
on
th
9
B
as
el
in
e
Other/Unidentified
C
on
tro
l
0.00
What Taxa Compose
That ‘Yet-Unidentified’ Proportion?
• Next-generation sequencing superior to traditional
sanger sequencing for this ecological question
Example: 454 pyro-sequencing
Pros:
Better coverage (orders of
magnitude), accuracy, sensitivity
(parallel sample analysis)
Cons:
Computationally challenging
$$$$$$$$
Benson A et al. PNAS (2010)
Hypothesis-Generating Study Objectives
• Our objective with HIV/AIDS patients was to apply
pyrosequencing, during a HAART trial, in order to:
– Develop a panel of potential taxa which may
compose the yet-unidentified stool microbiota and
thereby guide qPCR-based characterization
– Explore, using different intestinal specimen-types,
the creation of a spatial meta-genomic 16S pilot
profile before and after HAART
at the stable higher-taxonomic community level
Pilot Clinical Trial - Cohort Design
14 chronically-infected HIV patients with
race/ethnic/sex variation
Distal duodenal biopsy and aspirate specimen,
and a stool specimen, at 0 mo and 9 mo > HAART
2 paired biopsies, 2 aspirates, and
16 stools (1/2 are paired/longitudinal)
3 seronegative control subjects volunteered
(family/friends of patients)
1 biopsy, 2 stools
Total: 1/4 of a 96-well plate split for submission to UN-L
CAGE (Core for Applied Genomics & Ecology), USA
Wet Lab:

Microbial gDNA extracted by beadbeating* and
phenol/chloroform-based methods

The chosen primer set for pyro-seq covered V1,
V2 and V3 16S rRNA gene regions
 long read lengths, spikes *actinobacterial taxa
Dry Lab (Bioinformatics):
CLASSIFIER algorithm:
Ribosomal Database Project Website (Free)
Full method incl. taxa binning/classification, scoring,
sequence matching/alignment described:
Benson A et al. PNAS (2010)
DATA
Higher-Taxonomic Microbial Community Snapshot
Stool Summary
G(+) Clostridia and G(-) Bacteroidetes dominated most samples
Also a great abundance of G(+) Erysipelotrichi and Actinobacteria (owing
to beadbeating) in all samples (inconsistent pre/post therapy changes)
Actino appear to be increased in some HIV+ specimens vs controls
Anti-HIV Activity of Biochemicals from
Strains within the Actinobacteria class
Tanaka et al. Jpn J Cancer Res (1986)
Higher-Taxonomic Microbial Community Snapshot
Distal Duodenal Biopsies Summary
Facultative anaerobic taxa, e.g. G(-) Gammaproteobacteria, were
present in greater numbers in biopsies (site-specific) vs the stool (mixture)
Though obligate anaerobes were also detected in high abundance….
Interestingly, Cyanobacteria (primitive BGA) was also detected
Unexpected
Cyanobacteria/blue-green algae observed in a pre-HAART
biopsy of a non-vegetarian patient at >26% vs. control
Obesity and The Gut Microbiota
Cyanobacterial sequences
detected in obese mouse cecum:
May represent descendants of nonphotosynthetic ancestral cyanobacteria that
have adapted to life in lightless animal guts
Ley R et al. PNAS (2005)
Extracts & Compounds from Algae &
Cyanobacteria Possess Anti-HIV Activity
Scheffer et al. Ecotox Enviro Safety (2000)
Higher-Taxonomic Microbial Community Snapshot
Distal Duodenal Luminal Aspirates Summary
A liquid-phase sample-type new to this sequencing approach: a
spread of facultative anaerobes but also some obligate anaerobes
also Fusobacteria (a potent LPS-producing G[-] taxa) and, again,
Cyanobacteria
Summary/Conclusion
1. Pyrosequencing for patient gut microbiome profiling: orders of
magnitude > information/insight than traditional methods!
much more sensitive to inter-subject differences
2. Our prelim pyro dataset revealed taxa which likely accounted for the
previously-unidentified seropositive microbiota (Erysipelotrichi and
Fusobacteria as well as Actinobacteria and Cyanobacteria)
Does increased anti-HIV active microbes
(Actinoand
Cyano- taxa) indicate a mutualistic symbiosis-based
response to protect the infected host? Cause or effect?
A link to gut inflammation/translocation, immunopathy, CVD?
3. Post-therapy immune reconstitution likely affects host-microbiota
interactions, however, few consistent 0mo9mo changes observed
 HAART may not be sufficient to restore any alterations in overall gut
microbiome composition during HIV disease
Much opportunity for more research
LIMITATIONS
• Low n, especially controls
• Only 2 time points for each patient
>Currently expanding our n and surveying
multiple timepoints over the 9mo of HAART
However, objectives were met, we now have a:
1- Panel of potential taxa to guide qPCR design
2- Spatial pilot profile of the HIV+ gut microbiome
Take Home Message
Attention to the ‘other’ druggable genome(s):
Sequencing of a patient's personal microbial
(meta)genome or microbiome could help design
future ancillary therapeutic-interventions
Targeted pro-biotics, pre-biotics, or both
(syn-biotics), and selective anti-biotics (or all?)
*Some success in IBD, Atherosclerosis, Obesity
Acknowledgements
•Project PI and Mentor: David M. Asmuth MD
•Major Prof/Project Co-PI: John C. Rutledge
•Thesis Committee:
Jonathan A. Eisen PhD
Charles L. Bevins MD, PhD
•Other Dissertation Mentors
Mark A. Underwood MD, MAS
Britt M. Burton-Freeman PhD
Amber L. Hartman PhD
Eugene S. Lee MD, PhD
THANK YOU FOR YOUR ATTENTION
AND ANY FEEDBACK!
Correspondence:
david.asmuth@ucdmc.ucdavis.edu
Project Team:
Andrew Tritt, BS
Zhong-Min Ma PhD
MD
Surinder K. Mann MD
Chin-Shang Li PhD
Jian Wu MD, PhD
Thomas H. Knight MS
Tammy Yotter BS, RN, CCRP
Jian Wu MD, PhD
Timothy L. Hayes PhD
Archana H. Maniar MD
Paolo V. Troia-Cancio MD
Heather A. Overman BS
Natalie J. Torok MD
Anthony Albanese MD
Christopher J. Miller DVM, PhD
Richard B. Pollard MD
*Our Subjects/Patients
and Their Families
NIH - NCRR