Microbial library curation is interdependent between ID technologies. Micro-Phyles Case No. 3.

It has been nearly 60 years since molecular biology’s central dogma was first proposed by Nobel laureate Frances Crick1. Over the years, the development of various technologies for microbial diagnostics have been linked to these same stages of a microbial cell’s inner workings. Genotypic technologies, the gold standard for identifying microbes, are primarily based on DNA sequence while proteotypic t20160328-MicroPhylesLogo-294x152-EurekaHomeechnologies are based on the analysis of abundant cellular proteins using Matrix-assisted laser desorption/ionization–time of flight mass spectrometry, otherwise known as MALDI-TOF MS or just MALDI.

Multiple identification technologies applied under the right circumstances can find and fix errors that may not be obvious when only a single technology is used. We received an isolate from a customer for identification with our AccuPRO-ID service, which is based on MALDI. The top library match was Odoribacter splanchnicus, usually an inhabitant of the human intestine. At CRL, every identification undergoes a quality check before the report is finalized and shared with the customer. In this particular case, a red flag was immediately raised because colony morphology– characteristics of a colony (size, color, shape, odor etc.) on solid growth media—did not resemble Odoribacter splanchnicus. In such cases, we rely on DNA sequencing for resolution and when we tested the isolate the ID by sequencing was Bacillus pumilus/safensis—found in diverse environments including spacecraft assembly facility2,3 which matched the colony morphology.

For a scenario when two technologies give discrepant results from presumably the same isolate, an internal inquiry is triggered to resolve it. There are typically three main reasons that may lead to different IDs by different technologies. The first occurs when the two discrepant species in question are closely related. But different IDs may also occur if the culture plate contains mixed colonies or if the library entries of one of the two technologies is erroneous. The first reason was ruled out because the two IDs in question represent species that are phylogenetically distant. Odoribacter species are Gram-negative, anaerobic, non-spore forming and fusiform, whereas Bacillus species are Gram-positive, aerobic, spore-forming and rod-shaped. When the original culture plate was subcultured and re-tested by both DNA sequencing and MALDI, the same discrepancy was observed, therefore the second reason was also ruled out. This left us with the third reason, which was the possibility of a problematic library entry.

The accuracy of DNA sequence in our library was confirmed by the correct phylogenetic placement of the type strain of Bacillus pumilus/safensis. The MALDI entry for O. splanchnicus was not created by Charles River, but was made from the type strain (DSM 20712T, ATCC 29572). Since this entry was not created by CRL, we could not analyze the exact same isolate. As an alternative path forward, we purchased the type strain from the American Type Culture Collection (ATCC), verified its identity with 16S rDNA sequencing and created a new O. splanchnicus MALDI entry. None of the previous MALDI spectra from this isolate matched the new O. splanchnicus entry that we created. When the previous O. splanchnicus MALDI entry was excluded, then the MALDI best match was now Bacillus species. Based on the results of this investigation, we inactivated the original MALDI entry in 2015, and the manufacturer also deleted this entry during their 2016 library release.

This investigation shows how CRL can test the same isolate using multiple Accugenix services to resolve discrepancies quickly for an accurate ID for our customers. Furthermore, it enables MALDI library curation that is continually verified with genotypic and phylogenetic approaches.

References:

  1. Central Dogma of Molecular Biology, Frances Crick, Nature, Vol 227, August 1970.
  2. Bacillus safensis nov., isolated from spacecraft and assembly-facility surfaces. Satomi et al., IJSEM, 2006 Aug;56(Pt 8):1735-40. 3.
  3. Recurrent isolation of hydrogen peroxide-resistant spores of Bacillus pumilus from a spacecraft assembly facility. Kempf et al., Astrobiology. 2005 Jun;5(3):391-405.

How to cite:

Khot, Prasanna. You say Odoribacter, I say… Bacillus?. Eureka blog. Oct. 19, 2016. Available: http://eureka.criver.com/you-say-odoribacter-I-say-bacillus/