Bacteria and pathogen identification and methods

Microbes or pathogens identification is common for both research (microbial community studies) and clinics (pathogen identification for infectious diseases). In clinical microbiology labs, fast and correct identification of bacteria or pathogens provides guidance for getting accurate treatment in-time. Traditionally, this is based on phenotypic characteristics such as culture and biochemical testing (CBtest). Although the test is not as accurate as molecular genotypic method such as 16S rRNA gene sequencing, CBtest is the gold standard in hospitals in middle-income countries.

The conserved and variable regions in the 1500 bp 16S rRNA gene provide the foundation for 16S rRNA gene sequencing. The technique is used to accurately determine phylogenetic relationship between bacteria. With over 100,000 sequences available, 16S rRNA sequencing becomes gold-standard for microbial identification and classification.

Compared to the traditional CBtest, the 16S rRNA gene sequencing method is more reliable. CBtest may not provide accurate identification because the patient profile can change based on physical conditions. In addition, some bacteria grow in traditional cultures, others either grow very slowly or do not grow at all, leading to wrong conclusions. The traditional method can cause delay in treatment, wrong treatment, or even loss of patients’ lives.

In contrast, sequencing methods can reveal all pathogens present in the samples, including the unculturable. 16S rRNA sequencing is often performed by Sanger sequencing and more recently next generation sequencing (NGS).

16S rRNA gene sequencing by Sanger sequencing

Since 1980s, 16S rRNA gene sequencing by PCR and direct Sanger sequencing has gained popularity for microbial identification and taxonomy studies.  Sanger sequencing can reveal the sequence of all the 1500 bp rRNA gene in 6 reactions and provide accurate data for taxonomy resolution down to genus and species. This is critical when species determination is necessary to rule out species that are drug resistant or pathogenic. Lately, to improve the Sanger sequencing method, some of the labs have made modifications to make it less time consuming or less expensive.

16S rRNA gene sequencing by NGS

With the advent of the NGS in mid-2000s, the high-throughput technique has been widely used for 16S rRNA sequencing. NGS can sequence multiple 16S rRNA genes from a complex microbial environment simultaneously, which Sanger sequencing can’t do. It is a common practice for NGS to only sequence a few variable regions, for instance, from V1 to V3, or V3 and V4. However, the short-read NGS cannot provide the taxonomic resolution that is achieved by sequencing of the entire (~1500 bp) gene by Sanger sequencing. And the bacteria diversity is often either under- or over-estimated.

Sanger sequencing or NGS for pathogen identification in clinical microbiology lab?

NGS as a new method for pathogen identification in clinical microbiology lab has been evaluated in different labs. NGS still has some limitations, especially in mid-income countries. The NGS method needs costly instrument set-up, maintenance, training, and complex bioinformatic analysis. Most of all, the NGS method lacks standardization and validation between labs. This makes it hard to be the standard method for all the clinical labs across the board. Before NGS overcomes this critical problem, it is not likely becoming the routine method of choice in these clinical labs.

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