Identification of Macrolide Resistance Alleles in Environmental Metagenomes Robert Schmieder1,2, Anca Segall3, Molly Schmid4 and Robert Edwards2 1 Computational Science Research Center, SDSU; 2 Department of Computer Science, SDSU; 3 Department of Biology, SDSU; 4 Keck Graduate Institute of Applied Life Sciences E D W A R D S L A B A T S A N D I E G O S T A T E U N I V E R S I T Y Why do we care about antibiotic resistance ? How do we identify resistance alleles ? What did we find ? (cont.)

Anbiocs are substances Anbioc misuse 1 Idenfy 23S rRNA‐like sequences in metagenomes Base frequencies that kill or inhibit growth contributes to emergence of 296 publicly available metagenomes (over 42M sequence reads) DB of 23S rRNA sequences Base frequencies for the 13 of bacteria from fully sequenced bacterial posions of interest listed by resistant bacteria genomes in NCBI their posion in the E. coli K‐12 MG1655 23S rRNA gene. The Bacteria can bars are in order of (S)ILVA, (D)B, and (M)etagenomes. develop resistance Growing problem of BLAST 480 Species 23S DB SILVA counts are based on anbioc resistance 755 Strains 6,685 23S rRNA sequences in health care and with length between 2,200 bp and 3,200 bp (as of version Modified image from: www.answersingenesis.org 2211 Sequences commercial farming 100). DB counts are based on Aim: Examine 23S rRNA drug resistance determinants in metagenomes from 2,211 unique 23S rRNA 2 Idenfy base substuons in candidate sequences sequences of all completely different environments. The results may contribute to characterizing the sequenced bacterial genomes reservoir of anbioc resistance determinants in an environment. Map mutaon sites in reference organism E. coli K‐12 MG1655 to other species in the NCBI database and with using global mulple alignments length between 2,200 bp and 3,200 bp (as of 11/23/09). The number of metagenomic What is macrolide resistance ? A2058C/G/T A2059C/T Mutaon sites sequences are shown above G2057A A2062C the bars. Macrolides are anbiocs that inhibit bacterial protein synthesis G2032A/C/T Sequence logo of region composed of 2 amino or neutral sugars aached to lactone ring of variable size in domain V showing high conservaon over Several key posions were invariant in all tested metagenomes. Posion 754 shows the 480 different species highest frequency of base substuons, followed by posion 2057, 2611, and 2032. Overall Macrolide groups infrequent occurrence of resistance alleles. 14‐membered: Clarithromycin, Dirithromycin, 2% G2032A 7% G2057A Erythromycin and Roxithromycin G2032A: Helicobacter pylori J99 ‐ source paent was exposed to macrolides Mutaon frequencies # Metagenomes 15‐membered: Azithromycin G2057A: Chlamydia trachomas D/UW‐3/CX ‐ clinical isolates obtained aer ineffecve therapy Frequency of reference and altered # Sequences of urogenital chlamydiasis with fluoroquinolones and macrolides 16‐membered: Josamycin, Midecamycin, Miocamycin, sequences in the metagenomes. The Rokitamyin, Spiramycin and Tylosin majority of sequences similar to the Figure taken from Poehlsgaard and Douthwaite (2005) Analysis workflow resistance regions were found in the Macrolide resistance mechanisms microbial metagenomes. The human Preprocess metagenomic dataset Summary of the computaonal gut samples contained the highest • Ribosomal modificaon (methylaon or mutaon) process that was integrated into a number of altered and/or resistance sequences. • Efflux of the macrolide out of the cell Point mutaons in 23S rRNA Run BLASTn against 23S rRNA database pipeline for automac analysis An addional web‐service allows the • Macrolide inacvaon Parse BLAST results data processing through a user‐ friendly interface. Modificaon by point mutaon(s) in 23S rRNA Realign candidate sequences The bases at the alleles known to Domain II Domain V (pepdyl‐transferase region) determine resistance to macrolides Idenfy and count base substuons were used to calculate the frequency of resistance for each Calculate mutaon frequencies single metagenome.

Modified image from rna.ucsc.edu/rnacenter/ribosome_images.html Mutaon site What did we find ? What comes next ? Single base substuon Streptococcus pneumoniae at posion 2059 will cause a >100‐fold increase in resistance against the macrolide Erythromycin Healthy Japanese individuals The study will be extended to analyze addional sites in the 23S rRNA gene that allow No anbiocs for at least 4 weeks bacteria to gain resistance against other anbiocs such as Oxazolidinones. Furthermore, a Why use metagenomes ? web‐service is under development to idenfy resistance alleles in metagenomic data sets. Metagenomes are community genome sequences with minimal sampling bias. Global ocean survey Further information In combinaon with next‐generaon sequencing techniques, analysis of metagenomic sequences can efficiently assess the reservoir of resistance Please contact [email protected]. This poster is available online at determinants in an environment. hp://edwards.sdsu.edu/posters/macrolide_resistance2.pdf