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How 16S rRNA Can Be Used For Identification of Bacteria

28 May 2024by Manoj Dadlani

In the realm of microbiology, accurately identifying bacterial species is crucial for understanding microbial communities, diagnosing infections, and guiding treatment strategies. Traditional methods of bacterial identification, such as culture-based techniques, have limitations in providing rapid and comprehensive identification, especially for clinically unidentifiable bacterial isolates.

However, advancements in molecular biology have revolutionized bacterial identification, with 16S rRNA gene sequencing emerging as a powerful tool. In this article, we delve into the intricacies of 16S rRNA identification of bacteria, exploring its principles, applications, and advantages over conventional methods.

What is the 16S rRNA Gene?

16S rRNA sequencing is a highly effective method for identifying bacteria, particularly those belonging to the same species or those considered to be clinical unidentifiable bacterial isolates. It is based on the comparison of the 16S ribosomal RNA (rRNA) gene sequences, which are highly conserved among different bacteria but vary enough to allow distinction at the species level.

By extracting the DNA from a sample and performing 16S rRNA gene sequencing, researchers and clinicians can obtain a detailed view of the novel, rare and abundant species present within it.

This technique has proven invaluable in microbiology, with improved capabilities to rapidly and accurately identify bacteria. The 16S data analysis process further enables the differentiation of closely related bacterial strains, offering insights that are critical for the diagnosis and treatment of various microbial diseases.

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16S rRNA & Identification of Bacteria

Bacterial identification through 16S rDNA sequencing involves analyzing the genetic material of bacteria to determine their species. The 16S rRNA gene, present in the prokaryotic ribosome, serves as a molecular marker for bacterial identification.

This gene exhibits conserved regions, essential for ribosomal function, interspersed with variable regions that are unique to different bacterial taxa. The variability in these regions allows for discrimination between bacterial species, making 16S rRNA sequence data an ideal target for taxonomic classification and species identification.

Moreover, the 16S rRNA gene is present in all bacteria, making it a universal tool for bacterial identification. This means that regardless of the type of sample or bacterial species being tested, the same primers can be used for PCR amplification and sequencing.

Recommended guidelines for use of 16S rRNA gene sequencing for microbial identification

Why is 16S rRNA Used for Bacterial Identification? 5 Benefits

The 16S rRNA gene forms the basis of a leading method of DNA sequence analysis. Ribosomal DNA sequencing is a powerful tool for identifying bacteria, and the 16S rRNA gene offers several advantages over traditional methods.

16S rRNA gene sequencing has revolutionized bacterial identification in microbiology, offering a rapid, accurate and comprehensive approach to studying microbial communities. Its universal applicability and numerous benefits make it a valuable tool for both research and clinical purposes, with the potential to further advance our understanding of bacterial diversity and pathogenicity.

Key benefits of 16s rRNA gene sequencing include:

  1. Conserved and Variable Regions: The 16S rRNA gene contains conserved regions that are crucial for ribosomal function across bacterial species. These conserved regions provide a stable backbone for primer binding during PCR (Polymerase Chain Reaction), facilitating amplification of the target gene. Additionally, the variable regions interspersed between conserved regions contain sequence differences unique to each bacterial species, enabling differentiation between closely related organisms.
  2. Evolutionary Conservation: Despite variations in bacterial genomes, the 16S rRNA gene exhibits a relatively slow rate of evolution, making it suitable for phylogenetic classification and evolutionary studies. The evolutionary conservation of this gene ensures its presence in diverse bacterial taxa, allowing for the identification of both common and rare species within microbial communities.
  3. High-throughput Sequencing Technology: Recent advancements in DNA sequencing technology have significantly enhanced the efficiency and throughput of 16S rRNA sequencing. Next-generation sequencing platforms enable the simultaneous comprehensive data analysis of multiple samples, facilitating large-scale studies of microbial diversity and community composition.
  4. Rapid Identification: Compared to traditional methods of bacterial identification, 16S rRNA gene sequencing offers a rapid turnaround time. This is particularly crucial for clinical applications, where timely and accurate diagnosis is paramount for effective treatment.
  5. Non-Culture Based Identification: Unlike traditional culture-based techniques that rely on isolating and growing bacteria in the laboratory, 16S rRNA gene sequencing can identify bacteria directly from clinical samples, without the need for culturing. This allows for the detection of fastidious and slow-growing bacterial species that may be missed using conventional methods.

Universal phylogenetic tree based on the 16S rRNA gene sequence comparisons.

Differences Between 16S rRNA Sequencing & Alternative Types of Bacterial Identification

There are a number of other methods used for bacterial identification, each with its own advantages and limitations. Below, we’ll dive into some of the key differences between 16S rRNA sequencing and its traditional alternatives.

Culture-based Techniques

A 16S rRNA gene sequence does not depend on bacterial viability and can identify a broader range of species, including those that are difficult to culture.

In contrast, traditional methods of bacterial identification rely on culturing microorganisms on selective media followed by biochemical assays or phenotypic characterization. While culture-based techniques are cost-effective and widely available, they are limited by the requirement for viable organisms and the inability to identify unculturable or fastidious bacteria.

One study published in the National Library of Medicine states:

“Although being inexpensive and allowing both quantitative and qualitative information about the diversity of microorganisms present in a sample, however, these methods are laborious and time consuming (media preparation, dilution, plating, incubation, counting, isolation, and characterization), and results are only observed after several days, and frequently false positives are obtained especially when considering similar microbial species. Another problem associated with culture-based methods is the fact that they cannot identify non-culturable cells.”

DNA-DNA Hybridization

16S rRNA sequencing offers a rapid and cost-effective alternative for bacterial identification, bypassing the need for pure cultures and providing phylogenetic information beyond species boundaries.

In comparison, DNA-DNA hybridization involves measuring the extent of nucleotide sequence similarity between the DNA of an unknown bacterium and that of known reference strains. While this method provides accurate species-level identification, it is labor-intensive and time-consuming. Moreover, DNA-DNA hybridization requires pure cultures of bacteria, which may not always be available.

MALDI-TOF Mass Spectrometry

16S rRNA sequencing offers superior resolution at the species level, particularly for taxonomically complex or clinically relevant bacterial isolates.

In contrast, a routine clinical microbiology laboratory may instead utilize Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), which is a rapid and high-throughput technique for bacterial identification based on the mass spectra of microbial proteins.

While MALDI-TOF MS is valuable for identifying common bacterial species in routine clinical microbiology laboratories, its accuracy may be compromised when distinguishing closely related taxa or identifying novel species, as the limited sensitivity of whole-cell MALDI-TOF MS makes the direct analysis of clinical samples difficult. 

Applications of 16S rRNA Bacterial Identification

16s sequencing has become a popular method for studying microbial diversity and identifying bacteria in various settings. Some of the key applications of this technique include:

  • Species-Level Identification: One of the primary advantages of 16S rRNA sequencing is its ability to provide species-level identification of bacteria. By comparing the 16S rRNA gene sequences of unknown isolates to reference databases, researchers can accurately classify and identify bacteria and different bacterial species to uncover phylogenetic relationships within microbial communities.
  • Clinical Diagnostics: In clinical microbiology, rapid and accurate identification of bacterial pathogens is essential for diagnosing infectious diseases and guiding antimicrobial therapy. 16S rRNA sequencing enables the identification of clinically relevant species, including those associated with antibiotic resistance, allowing for tailored treatment strategies and improved patient outcomes.
  • Environmental Microbiology: The application of 16S rRNA sequencing extends beyond clinical settings to environmental microbiology, where it is used to characterize microbial communities in diverse habitats. By profiling the 16S rRNA gene diversity in soil, water, air, and other environments, researchers can elucidate ecological interactions, monitor ecosystem health, and identify potential bioindicators of environmental disturbance.
  • Microbiome Studies: The human microbiome, consisting of trillions of microorganisms inhabiting various body sites, plays a critical role in health and disease. 16S rRNA sequencing enables the comprehensive analysis of microbial communities associated with the human microbiome, offering insights into microbial diversity, community structure, and functional potential. Such studies have implications for understanding host-microbiome interactions, identifying disease biomarkers, and developing targeted interventions for microbiome-related disorders.


In summary, 16S rRNA sequencing and 16S data analysis represents a powerful tool for the identification of bacterial species, offering numerous advantages over traditional methods of bacterial identification. By leveraging the conserved and variable regions of the 16S rRNA gene, researchers can accurately classify bacteria at the species level, even in the absence of pure cultures.

The widespread adoption of next-generation sequencing technologies has further accelerated the application of 16S rRNA sequencing in diverse fields, including clinical microbiology, environmental science, and microbiome research. 

As our understanding of microbial diversity continues to evolve, 16S rRNA sequencing will remain indispensable for unraveling the complex microbial world and its impact on human health and the environment.

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Manoj Dadlani

Mr. Manoj Dadlani serves as Chief Executive Officer at CosmosID, Inc., the Maryland based provider of industry-leading solutions for unlocking the microbiome. Previously, Mr. Dadlani served as a partner at Applied Value Group, a management consulting and investment firm, and was co-founder and CEO at Rasa Industries, Ltd., a leading beverage manufacturing company. Mr. Dadlani has substantial experience in strategy, M&A, supply chain management, product development, marketing and business development. Mr. Dadlani received his bachelor’s and master’s degrees in Biological Engineering from Cornell University. Services offered by CosmosID’s CLIA certified and GLP laboratory cover the entire workflow from study design to sample collection, extraction, library preparation, sequencing, data analysis and publication support. CosmosID’s cloud-based metagenomics application offers user-friendly access to the largest curated databases for microbial genomics, antimicrobial resistance and virulence data and has been independently validated to return metagenomic analyses at strain level resolution with industry-leading sensitivity and precision.