Advantages and Limitations of 16S rRNA Next-Generation Sequencing for Pathogen Identification in the Diagnostic Microbiology Laboratory: Perspectives from a Middle-Income Country

Abstract

Bacterial culture and biochemical testing (CBtest) have been the cornerstone of pathogen identification in the diagnostic microbiology laboratory. With the advent of Sanger sequencing and later, next-generation sequencing, 16S rRNA next-generation sequencing (16SNGS) has been proposed to be a plausible platform for this purpose. Nevertheless, usage of the 16SNGS platform has both advantages and limitations. In addition, transition from the traditional methods of CBtest to 16SNGS requires procurement of costly equipment, timely and sustainable maintenance of these platforms, specific facility infrastructure and technical expertise. All these factors pose a challenge for middle-income countries, more so for countries in the lower middle-income range. In this review, we describe the basis for CBtest and 16SNGS, and discuss the limitations, challenges, advantages and future potential of using 16SNGS for bacterial pathogen identification in diagnostic microbiology laboratories of middle-income countries.

Combining Imaging Flow Cytometry and Molecular Biological Methods to Reveal Presence of Potentially Toxic Algae at the Ural River in Kazakhstan

“…Moreover, the traditional cyanobacterial taxonomy has been based on morphological features, but this classification is revised with an accumulation of molecular sequence data (Komárek et al, 2014;Hugenholtz et al, 2021). The taxonomic classification was done with a 16S NCBI database, however, the choice of database may have an effect on the results of taxonomic identification (Park and Won, 2018;Rizal et al, 2020;Winand et al, 2020). Metabarcoding based on 16S rRNA gene revealed a moderately diverse cyanobacterial community at S12 and S1 sampling points, and only a few cyanobacterial species were found at the S4 point (Figure 6) where the heated water stream of the artificial Peretaska channel (30 • C, October 2019) was represented mostly by Proteobacteria and Firmicutes.…”

Gardnerella vaginalis in Recurrent Urinary Tract Infection Is Associated with Dysbiosis of the Bladder Microbiome

“…Recently, through the new bacterial technique (16s RNA sequencing, EQUC), the classical perception of the bladder microbiome is being reconsidered, and the role of various microorganisms in urinary tract infection needs to be re-established [7,14]. According to a previous classical urine culture-based study, the most common causative bacteria of uncomplicated UTI were E. coli (58%), mixed flora (13.4%), and K. pneumoniae (6.5%) [29]. However, the pattern was different in our group's previous pilot study [14].…”

Protocol for correlation analysis of the murine gut microbiome and meta-metabolome using 16S rDNA sequencing and UPLC-MS

“…Most studies have characterized microbiome diversity based on a single gene, the small subunit ribosomal RNA (rRNA) gene, which is encoded by highly conserved 16S ribosomal DNA (rDNA). Characterized by high expression, stability and conservatism, 16S rDNA is the most common method for estimation among different species of bacteria and archaea ( Konstantinidis et al., 2006 ; Muhamad Rizal et al., 2020 ). Using Illumina high-throughput sequencing of 16S rDNA, we can fully describe bacterial diversity and community composition.…”

“…Using Illumina high-throughput sequencing of 16S rDNA, we can fully describe bacterial diversity and community composition. As delays or failures in the identification of pathogens could occur via bacterial culture and biochemical testing in clinical diagnostic microbiology ( Muhamad Rizal et al., 2020 ), NGS-based bacterial detection methods are emerging and becoming a mainstream technology in oncology. 16S rDNA sequencing provides an ideal method for the identification of unculturable and fastidious bacteria achieving more rapid and predictable turn-around time with a streamlined identification protocol.…”