Download functional characterization of the antibiotic resistance reservoir in the

FUNCTIONAL CHARACTERIZATION OF THE ANTIBIOTIC
RESISTANCE RESERVOIR IN THE HUMAN MICROFLORA
Morten O.A Sommer, Gautam Dantas and George M.
Church
Presented by,
Sushmitha Paulraj
CONTENTS
 BACKGROUND
 PUBLISHED FINDINGS
 GOAL
 EXPERIMENT
 RESULTS
 CONCLUSION
Q&A
BACKGROUND
 Human Microflora
 Assemblage of microorganisms that reside on the
surface and in deep layers of skin, in the saliva and oral
mucosa, and in the gastrointestinal tracts
 Includes bacteria, fungi and archaea
 Some of these perform tasks that are useful for the
human host, while the majority have no known
beneficial or harmful effect
BACKGROUND
 Gut Microflora
 Consists of microorganisms that live in the digestive tracts of
animals, and is the largest reservoir of human flora
 Bacteria make up most of the flora in the colon and 60% of
the dry mass of feces
 500 different species live in the gut
 Bacteria in the gut fulfills a host of useful functions for
humans, including digestion of unutilized energy substrates;
stimulating cell growth; repressing the growth of harmful
microorganisms; training the immune system to respond only
to pathogens; and defending against some diseases
PUBLISHED FINDINGS I
 Methicillin resistant Staphylococcus aureus caused 18,964
mortalities in USA
 Genes harbored by the bacterial strains are acquired by
lateral (horizontal) gene transfer
 Increase in the Proteobacteria at the expense of Firmicutes
and Bacteroidetes during antibiotic treatment
 Resistance to ciprofloxacin in the gut microbiome and
clarithromycin resistance in commensal enterococci
PUBLISHED FINDINGS II
 Resistance to amoxicillin observed in oral bacterial isolates
in children not exposed to antibiotic treatment
 Abundance in ermB and tetQ, encoding resistance to
erythromycin and tetracycline has increased in cultured
human Bacteriodes
“These findings suggest that the human microflora could
constitute a substantial reservoir of antibiotic resistance
genes accessible to pathogens”
GOAL
“Perform functional
selection coupled
with
metagenomic analysis to characterize the antibiotic
resistance genes in the oral and gut microflora of
healthy individuals”
APPROACHES
Metagenomic analysis
2. Culturable isolate analysis
1.
1. METAGENOMIC ANALYSIS
 DNA was isolated directly from saliva and fecal samples
from 2 unrelated healthy humans not treated with
antibiotics for a year
 1-3 kb fragments of metagenomic DNA were cloned into an
expression vector and transformed into an E. coli strain
creating a library
 Antibiotic resistance clones from each library were selected
by plating Luria broth agar containing one of 13 antibiotics
belonging to 4 different amino acid classes, at conc. where
the strain is susceptible
 95 unique Inserts conferring resistance to all of the 13
antibiotics were sequenced and annotated
SIMILARITY TO GENBANK
 On average, the sequence similarity of the resistance genes
and the closest related gene in Genbank is 69.5% at the
nucleotide level and 65.3% at the amino acid level
 22 % had high homology to previously known genes,
tet(Q)-3 and CTX-M-15 enzyme
 Most of the related homolog's are derived from
commensals which are non pathogenic and commensals
with the capacity to become opportunistic pathogens
 Found genes 100% identical to hypothetical proteins of
unverified functions in Genbank, thus highlighting the
utility of a functional selection approach to improve
annotation of genomic and metagenomic sequencing data
from the human microbiome project
.. CONTINUED
 Antibiotic resistance genes harbored by the human microflora
were distantly related (60.7% nuc and 54.9% aa level) to
antibiotic resistance genes detected in pathogenic isolates
 78 inserts had low homology to genes to Genbank, encoding
resistance to the 13 antibiotics, implying the resistance genes of
the human microbiome are inaccessible or infrequently
exchanged with human pathogens
“However, all the genes identified in this study were functional in
E. coli suggesting that if a barrier to gene transfer exists between
the constituents of the human microbiome and pathogens, it
must stem from processes other than functional compatibility”
PHYLOGENETIC ANALYSIS
 Phylogenetic analysis of the inserts using Phylopythia
indiacated that they predominantly originate from the
phyla Bacteroidetes and Firmicutes, which dominate the
gut flora
 Low sequence identity to resistance genes were identified
in pathogens from these phyla and in pathogens from
Proteobacteria, the reason being Proteobacteria’s
underrepresentation in the metagenomic libraries as they
constitute 1% of the human gut flora
2. CULTURED ISOLATE ANALYSIS
 572 bacterial strains were isolated from fecal samples from
two healthy individuals
 Phylogenetic profiling revealed that they belonged
primarily to Proteobacteria, with a few Firmicutes and
Actinobacteria
 Isolates from individual 1 and 2 were resistant to 9/13 and
5/13 antibiotics
 Chloramphenicol and minocyclin were able to prevent the
growth of 99% of the isolates
ANTIBIOTIC RESISTANCE PROFILE
SIMILARITY TO PATHOGENS
 115
unique inserts encoding transferrable antibiotic
resistance genes from gut microbiome isolates were
identified
 95 % of the genes were over 90% identical at the nucleotide
level to the resistance genes in pathogenic isolates, and
almost half were 100% identical
 Resistance genes identical to those in pathogens belonged
to one class of TetA, two classes of AAC(3)-II and AAC(6)lb, and three classes of TEM, AmpC and CTX-M
 TEM1 gene variant were reported in pathogenic strains of
E.coli, Salmonella enterica etc. and most of these variants
were submitted to Genbank between 2007 and 2008
SIMILARITY TO BETA-LACTAMASE
 AmpC and CTX-M family of enzymes are extended-
spectrum beta-lactamases that hydrolyze a wider variety of
beta-lactums
 The CTX-M-15 beta lactamase was identified in libraries
from cultured gut microbiome isolates and in
metagenomic libraries from the same microbiome
 Global alignment of the 27 unique beta-lactamase
sequences from Morten’s study identified 15 distinct
sequence groups
 5/15 were characterized (CblA, CfxA, CTX-M, TEM and
AmpC) whereas 10/15 constitute unknown beta-lactamase
sequence families because of their amino-acid identity
level between 35%- 61% in Genbank
.. CONTINUED
 The known 5 were identified from cultured microbiome
isolated and 10 were identified by Morten’s culture
independent characterization
“Metagenomically derived resistance genes in this study
were more distantly related to previously identified genes
than those derived from aerobic gut isolates”
COMBINED ANALYSIS
 29 out of 210 (95+115) microbiome derived inserts encoding
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
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antibiotic resistance contained genes similar to previously
characterized transposases
14 were identical to those previously identified on
resistance and conjugative plasmids
90% of the identical transposases derived from culturable
aerobic isolates was significantly higher than those from
metagenomic sampling
Nearly half of the resistance genes identified in the
cultured gut isolates were identical at the nucleotide level
to resistance genes from human pathogenic isolates.
Although the identity does not provide any info on the
direction or mechanism of transfer
SPECULATIONS
Some speculations regarding the implication of the findings:
 The human microbiome may constitute a mobilizable
reservoir of antibiotic resistance genes that are accessed by
the pathogenic bacterium
 The aerobic cultured samples may be dormant pathogens
inhabiting the human microbiome
 Barrier to lateral gene transfer in vivo between the
dominant commensals in healthy humans and disease
causing isolates
 Under-sampling of antibiotic resistance genes in the
human microbiome
CONCLUSION
 Complete sequencing of these libraries would yield more
resistant genes
 65% of the resistance genes derived from cultured aerobes
were similar between the 2 individuals whereas less than
10% for metagenomically derived resistance genes
 Absence of in-depth characterization of the resistance
reservoir of the human microbiome might be elusive of the
process by which antibiotic resistance emerges in human
pathogens
QUESTIONS ??