Monday, 24 April 2017 09:19

16S Alphabet Soup: A Quick Guide

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As the concepts of gene sequencing and the microbiome are becoming more common in our everyday vocabulary in healthcare, the demand for more sophisticated clinical testing is rising. It is likely that the number of bacterial sequencing requests received by clinical microbiology laboratories for direct testing of clinical specimens is also rising. The terms used by clinicians to order such testing are diverse and sometimes confusing. Below is one example of an informal consult between a clinical team and a microbiologist; this conversation may not be too foreign to your own lab:

  • Medical student #1: We’d like to order “16S” for this patient.
  • Medical student #2: I thought Infectious Diseases told us to order “broad-range bacterial sequencing.”
  • Clinical Microbiology Fellow: How do 16S and broad range sequencing differ from whole genome sequencing?
  • Infectious Diseases Fellow: I’m not sure. Do you think that this test will also detect mycobacteria? And what about fungi?

Broad-range Bacterial PCR, Bacterial Sequencing, 16S rDNA Sequencing: What Do All These Names Mean?

The names listed above are usually aliases for the same test – broad-range bacterial amplification followed by sequencing. Basically, a portion of the bacterial 16S rRNA gene is amplified by PCR and the amplified DNA is then sequenced. Analysis of the sequence using a genetic sequence database such as GenBank of the NIH typically allows species-level identification of the organism. In this section, we’ll review the concept of broad-range bacterial PCR and sequencing, the testing process, and various aliases for this method. We’ll also broadly cover clinical applications and limitations that may be used as talking points with clinicians asking for advice on this test.

Basic Molecular Biology Refresher. Ribosomal RNA (rRNA) molecules are components of the ribosome of the cell. Ribosomes the organelles responsible for protein synthesis. Prokaryotes have ribosomes composed of 23S, 16S, and 5S subunits. The “S” stands for Svedberg unit, a measurement of how quickly the particles settle (sedimentation rate), a property related to mass, density, and shape. Prokaryotic ribosomes differ from those of eukaryotes. Eukaryotic ribosome components are 28S, 18S, 5.8S, and 5S rRNAs. Thus, targeting the genes for these 16S rRNA molecules prevents cross-reaction with human cells.

16S as a Near-Perfect Target. The 16S rRNA gene (rDNA) is a near-perfect target for a molecular test that is meant to cast a wide net to detect and identify all bacterial genus or species level. The 16S rRNA gene has highly conserved sequences - sometimes nearly identical - that are shared among many different genera and species of bacteria and mycobacteria. However, the 16S rRNA  gene also has particular regions that are highly conserved within a species or genus, but differ between species or genera. Thus, the conserved regions bracketing variable regions can be used to design PCR primers that anneal to these targets and amplify the variable regions for sequencing and analysis. Depending on the region of the 16S rDNA targeted by the assay, bacteria, including mycobacteria, may be identifiable to the family, genus or species level. At this time all 16S rDNA sequencing assays are laboratory-developed and, therefore, the performance characteristics may vary significantly between assays. However, the general process is very similar.

Alphabet soup 1An amplicon sequence may solve the mystery of the microbial source of infection, especially when the microbe can't be cultured. source

The Process: Broad-range bacterial PCR and sequencing undergoes the 4-step process outlined below.

  1. Bacterial DNA is extracted.
  2. A portion of 16S rDNA is amplified.
  3. The amplicon (amplified area) is sequenced.
  4. The amplicon sequence is compared to a library of organisms with known 16S rDNA sequences in order to identify the organism.

Sequencing Approaches. Typically, Sanger, or traditional, sequencing is performed on the amplicon. However, other sequencing approaches such as next-generation sequencing have more recently been applied to the amplicon for identification as well. Which sequencing approach to employ depends upon many factors, such as the length of amplicon, the source of the clinical specimen, and the degree of specimen sterility. For example, Sanger sequencing cannot be utilized when more than one amplicon is present, such as would be expected in a polymicrobial specimen. Broad-range bacterial PCR and sequencing is also typically limited to testing from sterile sources, since contaminating or colonizing bacteria will also result in a signal and may confuse the clinical picture. However, different sequencing methods such as next-generation sequencing have been used in concert with 16S rDNA amplification to allow for detection and identification of multiple organisms in mixed populations. Further details on the choice of sequencing approaches are nicely detailed by Salipante SJ et al.

Aliases. Broad-range bacterial PCR and sequencing is referred to by many names: 16S, broad range, rRNA, and bacterial sequencing, among others. Laboratories typically use the term broad-range or 16S rRNA sequencing. “Broad range” essentially refers to the broad target of the 16S in amplifying regions that are common to most bacteria, including mycobacteria.

Applications of Broad-Range Bacterial PCR and Sequencing. This approach can be helpful when traditional cultures from patients with suspected bacterial or mycobacterial infections fail to grow microorganisms. It can be more sensitive than traditional culture techniques. The diagnostic utility of 16S bacterial sequencing has been particularly evident through the many publications on its application in cases of endocarditis when applied to resected or excised heart valves . This approach has been also used with success in bone and joint infections when run on tissue or fluid from sterile sites. Of course, this approach can be used for bacterial isolates either from solid media or in broth, but the focus of this discussion is on direct clinical specimen testing. Finally, this method has been used for identifying bacteria from formalin-fixed, paraffin-embedded surgical pathology tissues, but the sensitivity of the assay drops when microorganisms are not visible using special stains on the tissue.

Limitations of Broad-Range Bacterial PCR and Sequencing. The utilization of 16S rRNA gene in these applications is limited to amplification of bacteria and mycobacteria as fungi, viruses, and parasites do not possess the 16S rRNA gene. A similar approach to identifying fungi may be used, but a different target gene (not 16S) common to fungi is utilized and this is not further discussed here. Additionally, this method should not be used as a test of cure, because nucleic acids – from alive or dead organisms – may persist for long periods of time after successful treatment. 16S sequences are highly conserved in some groups of bacteria, including some streptococci, so additional testing  might be needed for identification of these organisms. Finally, broad-range bacterial PCR and sequencing is less sensitive as a diagnostic method compared to a targeted PCR assay for a particular bacterium. For instance, if a clinician or pathologist is highly suspicious of a particular bacterium or mycobacterium in a specimen (i.e., cat scratch disease is suspected clinically, or Whipple’s disease is suspected based on histopathologic picture), then a targeted PCR assay for the organism causing that particular disease will usually be more sensitive than running the more general broad-range bacterial amplification and sequencing assay.

Summary. Broad-range bacterial PCR and sequencing (or 16S rDNA PCR and sequencing) is a sensitive method for detecting bacterial nucleic acids in direct clinical specimens. The assay covers bacteria and mycobacteria but not fungi, viruses, or parasites, although similar methods may be applied to fungi using different genetic targets (a separate assay). If Sanger sequencing is employed after amplification, specimens from sterile sources (without contaminating or colonizing bacteria) should be used. The literature supports various uses of broad-range bacterial sequencing, most notably for excised heart valves in cases of suspected endocarditis, and also in bone and joint infections. If a particular bacterial or mycobacterial agent is suspected and a targeted PCR is available for testing for that organism, the more sensitive diagnostic step would be to run the targeted PCR rather than the broad-range bacterial PCR.


The above post reflects the thoughts of its author, Dr. Audrey Schuetz, and not the American Society for Microbiology.


Alphabet Soup 2Print and share this flyer that discusses the vital role of clinical microbiologists in fighting the spread of antimicrobial resistance! Click here for a high-resolution version!

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Want to hear more about what clinical microbiologists do, possible career trajectories for clinical microbiologists, and how a medical technician changed the standard of care for lung transplant patients? Click play (below) to listen to an interview with Dr. Robin Patel on This Week in Microbiology to hear all this and more.


ASM knows many of of its scientific members are also amazing artists. Medical Laboratory Scientist Caitlin Cahek spoke with ASM about how she uses her agar art as an online teaching tool

For an in-depth look at applications of next-generation sequencing in the clinical microbiology lab, please see our report: Applications of Clinical Microbial Next-Generation Sequencing.

Last modified on Monday, 24 April 2017 14:07
Audrey Schuetz

Audrey Schuetz is Associate Professor of Laboratory Medicine and Pathology at Mayo Clinic College of Medicine and Science in Rochester, Minnesota. She is a pathologist and microbiologist. She enjoys studying antimicrobial resistance, anaerobes, and Infectious Diseases pathology. You can follow her on Twitter at @schuetz_audrey.