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 1. Metagenomic analysis 2. Culturable isolate analysis

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 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 ??