Evaluation of four methods of assigning species and genus to medically important bacteria using 16S rRNA gene sequence analysis

Abstract

The four methods for assigning bacterial species are the Clinical and Laboratory Standards Institute (CLSI), modified CLSI (mCLSI), phylogenetic analysis (PA) and closest match (CM) methods, these are used to identify the genus and species using 16S rRNA gene sequence results. In this study, the results of identification by these four methods of 37 aerobic reference strains, 30 anaerobic reference strains, 15 Acinetobacter reference strains and 167 Acinetobacter clinical strains were compared. The rates of accurate identification to the species level using the CLSI, mCLSI, PA and CM methods were as follows: 24.3, 86.5, 86.5 and 89.2%, respectively, for the 37 aerobic reference strains; 73.3%, 96.7%, 90.0% and 93.3%, respectively, for the 30 anaerobic reference strains; 40.0%, 93.3%, 100% and 93.3%, respectively, for the 15 Acinetobacter reference strains; and 53.9%, 90.4%, 95.8% and 90.4%, respectively, for the 167 Acinetobacter clinical strains. The rates of accurate identification to the genus level using the CLSI, mCLSI, PA, and CM methods were as follows: 91.9%, 91.9%, 94.6% and 91.9%, respectively, for the 37 aerobic reference strains; 100%, 100%, 100% and 100%, respectively, for all of the 30 anaerobic reference strains, 15 Acinetobacter reference strains and the 167 Acinetobacter clinical strains. The mCLSI is the most practical and pragmatic method for identification of species based on 16S rRNA sequences for hospital, research or industry laboratories because it performs well and involves a simple procedure.

Test Agreement among Biochemical Methods, Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry, and 16S rRNA Sequencing for Identification of Microorganisms Isolated from Bovine Milk

“…Sequences were compared to the GenBank database using the publicly available nucleotide-BLAST algorithm (www.ncbi.nlm.nih.gov). The modified CLSI method was used for diagnosis, assigning a species with Ն99% identity, regardless of the similarity score differences (20). The diagnostic results of the other test methods were not known to those performing the 16S rRNA testing.…”

Next-generation systematics: An innovative approach to resolve the structure of complex prokaryotic taxa

“…>98.7% identity between their 16S rRNA gene sequences and a high mutual phenetic similarity456. However, the value of phenotyping and DDH is limited, not least by a lack of reproducibility and compatibility of results between different laboratories78 while 16S rRNA gene sequences tend to provide insufficient resolution to distinguish between closely related species910. Moreover, it is not possible to apply the ‘polyphasic’ approach to unculturable bacteria311, the so called ‘microbial dark matter’.…”

Targeted next-generation sequencing of the 16S-23S rRNA region for culture-independent bacterial identification - increased discrimination of closely related species

“…Therefore, Park et al . 12 proposed a modified CLSI (mCLSI) method which was more practical and pragmatic for identification of species based on 16S rRNA sequences than the CLSI method. The mCLSI method assigns bacterial species when the similarity score is 99% or higher but irrespective of the similarity score differences.…”