The complicated world of fungi provides a valuable lesson about messy curating. Micro-Phyles Case No. 2
It all started with a question from a puzzled customer. Their production strain, which they may have obtained to synthesize the antibiotic penicillin, was identified as Penicillium rubens, different than the Penicillium notatum (ATCC 9478) and Penicillium chrysogenum (ATCC 11709) they thought they had purchased. How could this happen?
It’s not just an academic question. Mixing up strains can lead to QC failures and result in issues with maintaining compliance under Regulatory Guidance documents.
At the time we received the query, the samples were indeed catalogued by American Type Culture Collection (ATCC) as Penicillium chrysogenum. This triggered an investigation from our end, as we knew that these are strains commonly used in manufacturing. Penicillium chrysogenum, a commonly occurring mold in indoor environments, has gained much attention in the pharmaceutical industry for production of the antibiotic penicillin. Yet, the taxonomy of fungi is a complicated affair. For instance, Alexander Fleming’s strain, the original penicillin-producing strain, was initially identified as Penicillium rubrum, but was later re-identified as Penicillium notatum and finally as Penicillium chrysogenum. All these identifications were based on morphology, the branch of biology that deals with the form of living organisms, and with relationships between their structures.
This naming issue explains why different culture collections have catalogued these strains with different names. Nonetheless, recent studies using DNA sequencing analysis have shown that penicillin-producing strains, originally described by Fleming, are not P. rubrum, notatum or chrysogenum, but Penicillium rubens (Houbraken et al. 2011). Fleming’s strain is the strain that our customer sent us— ATCC 9478 (CBS 205.57 = NRRL 824 = IMI 015378). So even though ATCC and many other culture collections have changed their species identification for this strain, the customer is unfortunately unaware of this, or obtained the stock cultures prior to this change. The issue is further complicated by customers who use certain commercial identification systems that continue to misidentify this species as P. chrysogenum. Upon further investigation, we discovered that the issue is not just the name change but the lack of relevant species entries in the reference library, and the gene utilized for identification. One commercial sequencing system’s library, for instance, is missing the Penicillium rubens species entry entirely. And even if they were to add this species to their library, the D2 region which is sequenced to provide identification will not be able to differentiate between P. rubens and P. chrysogenum because the D2 sequence is too conserved.
This tale from the microphyles is another example of why it is critical to curate the names of the species in your reference library in order to ensure you have the most recent classification, enough species diversity to generate an ID, and an appropriate gene target to generate an accurate ID.
Figure 1: The phylogenetic Neighbor Joining trees constructed after searching two different reference libraries with the P. rubens sequence. Left: the Accugenix ITS2 fungal library provides a clear ID with a well-resolved tree. Right: the D2 database provides inaccurate species identification.
How to Cite:
Verghese, Bindhu. Straining to Get it Right. Eureka blog. July 11, 2016. Available: http://eureka.criver.com/straining-to-get-it-right/