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  1. 1. 16S rRNA SEQUENCING IN THE CLINICAL MICROBIOLOGY LABORATORY Richard C. Huard, Ph.D. Department of Pathology Clinical Microbiology Service New York-Presbyterian Hospital Columbia University Medical Center rchuard@nyp.org
  2. 2. 16S rRNA SEQUENCING • Gold standard for bacterial identification • 16S rRNA gene ~ 1500 bp Small subunit of ribosome Common to all bacteria Present in 1 or more copies Critical to cell function
  3. 3. 16S rRNA • Analogous to 18S - eukaryotes + fungi • Taxonomy Evolutionary distance Relatedness of microorganisms • RNA gene product – base-pairing forms a complex tertiary architecture • Few genes are as relatively unchanged in evolution • Nearly all bacteria share highly sequence conserved regions bracketing regions that are variable species-specific manner
  4. 4. 16S rRNA Gene • most sequencing efforts focus on the 5’ end of the gene • target the entire gene for amplification - universal primers • use a minimal set of universal internal primers to sequence • C. Petti, 2007; CID 44:1108-14.
  5. 5. Protocol: 1. Bacteria in pure culture 2. DNA isolation 3. PCR amplification (standard program, universal primers) 4. Agarose gel electrophoresis 5. Purify PCR products 6. Sequence
  6. 6. Protocol: Lasergene DNASTAR EditSeq MegAlign SeqMan
  7. 7. Protocol: • NCBI GenBank webpage: http://www.ncbi.nlm.nih.gov/BLAST/ • - an annotated collection of nucleotide sequences • - short sequences to whole genomes • - open access • Nucleotide-nucleotide BLAST • - paste in the linear sequence data, submit - search is performed • - list of matches is provided
  8. 8. Protocol: • Identification ~99-100% confirm species ~97-99% confirm genus, new species <97% new species, new genus
  9. 9. Our Data: 16S rRNA Sequencing • Built a database of 16S rRNA sequences 175 isolates of known identity QC strains, outside culture collections, internally validated Clinically relevant species Cross-referenced to GenBank • Evaluated 300 clinical isolates 200 BacT Section 100 NTM / Nocardia Inclusion criteria: • Failed to give a definitive and/or rapid identification by routine methods • Potentially clinically relevant • Unique antibiogram
  10. 10. Our Data: 16S rRNA Sequencing • Definitive identification: 88% overall 16S PCR worked 100% of the time • 34 isolates were new species: Mycobacterium sp. (3) Nocardia sp. (1) Moraxella sp. (4) Acinetobacter sp. (5) Streptococcus sp. (6) • 3 isolates were of new genera: Enterobacteriaceae Family (2) Rhizobiales Family (1)
  11. 11. Our Data: 16S rRNA Sequencing • Difficult to Identify • Rarely Reported Clinically NTM, Nocardia Francisella philomiragia • Difficult to Differentiate Tsukamurella tyrosinosolvens Burkholderia cepacia Weissella confusa complex • Not Previously Reported USA • Phenotypic Variants Nocardia cyriacigeorgica GNR in CF patients Shineria larvae • Long Germination Time • Not Previously Reported Clinically HACEK Rothia aeria
  12. 12. CAVEATS TO 16S SEQUENCING • 16S rRNA Limitations Requires pure culture Different spp. can have an identical 16S • B. bronchoseptica + B. parapertussis, M. gastri + M. kansasii Different spp. can have minimally variable 16S • S. pneumoniae + S. mitis, M. abscessus + M. chelonae Genomovars of a single “species” may have relatively different 16S sequences (P. vulgaris, E. cloacae, B. fragilis) Multiple 16S alleles within a strain
  13. 13. Caveats to Using GenBank • Issues of which to be aware Not a quality-controlled database • Many sequencing errors (N, misreads, gaps) • Many incorrect IDs Lots of junk sequences (anaerobes) Paucity of entries (Coaggulase-negative Staphylococci) Dated entries (Legionella micdadei vs. Tatlockia micdadei) Submitters assign names to new species that are not validly published Highest score is not necessarily the correct species Alternatives (RIDOM) lack the same breadth
  14. 14. SUMMARY: 16S SEQUENCING • Can better discriminate bacterial isolates than many phenotypic methods • ID novel, poorly described, rarely isolated, or phenotypically aberrant strains • Clarify clinical importance, guide choice of treatment • With slow-growers – can speed TAT and be cost-effective • Unlikely to ever completely do away with culture
  15. 15. THANKS • Clinical Microbiology Service Dr. Phyllis Della-Latta, Director Dr. Susan Whittier, Asst. Director • IDSA Committee Richard C. Huard, Ph.D. rchuard@nyp.org
  16. 16. THANKS Richard C. Huard, Ph.D. rchuard@nyp.org