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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

[email protected]

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

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

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.

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

Protocol:

� Lasergene DNASTAR

�EditSeq

�MegAlign

�SeqMan

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

• Identification

�~99-100% confirm species

�~97-99% confirm genus, new species

�<97% new species, new genus

Protocol:

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


• 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)


• Difficult to Identify

� NTM, Nocardia

• Difficult to Differentiate

� Burkholderia cepacia complex

• Phenotypic Variants

� GNR in CF patients

• Long Germination Time

� HACEK

• Rarely Reported Clinically

� Francisella philomiragia

� Tsukamurella tyrosinosolvens

� Weissella confusa

• Not Previously Reported USA

� Nocardia cyriacigeorgica

� Shineria larvae

• Not Previously Reported Clinically

� Rothia aeria

• 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

CAVEATS TO 16S SEQUENCING

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

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

THANKS


[email protected]

• Clinical Microbiology Service

� Dr. Phyllis Della-Latta, Director

� Dr. Susan Whittier, Asst. Director

• IDSA Committee

THANKS


[email protected]

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