1) 16S rRNA sequencing is the gold standard for bacterial identification and can identify novel, rare, or aberrant bacterial strains that other phenotypic methods cannot. 2) The document presents data from sequencing 300 clinical isolates, identifying many new species and some new genera, and providing definitive identifications in 88% of cases. 3) While powerful, 16S rRNA sequencing has some limitations like requiring a pure culture and difficulty differentiating closely related species, and interpretation requires consideration of database issues.

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. 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. 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. 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. 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. Protocol:
Lasergene DNASTAR
EditSeq
MegAlign
SeqMan

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. Protocol:
• Identification
~99-100% confirm species
~97-99% confirm genus, new species
<97% new species, new genus

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. 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. 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. 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. 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. 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. 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. THANKS
Richard C. Huard, Ph.D.
rchuard@nyp.org