How do you trace the source of a serious sterility failure when the bacteria lack a paper trail? Microphyles Case No. 7  

Multilocus sequence typing (MLST) is the process of characterizing microbial isolates by sequencing multiple housekeeping genes. Whether isolates are related is determined based on their sequence variations. MLST is invaluable for the pharmaceutical industry in determining the similarity of contaminant strains during sterility failures. It also helps us determine the source of the contamination.

Tracing the source of a contamination helps to implement effective Corrective and Protective Actions required by the US Food and Drug Administration, and thereby minimize the chances of recontamination. Thus, when a customer submits a sample for MLST, it is either for a critical investigation or for verifying an important production strain. As such, we strive to provide the customer with accurate and timely strain comparisons.

Unfortunately, in the complex world of gene sequencing and taxonomy, it is not always that simple.

We recently received a batch of critical samples to test via MLST that were part of a serious sterility failure. The first step was to confirm the identification of the samples in order to know what gene targets to amplify as these targets for strain-level differentiation vary by species. In this particular case, based on the 16S rRNA gene sequence, we identified the 17 of the samples as Cutibacterium acnes (formerly Propionibacterium acnes)—a ubiquitous Gram-positive bacterium that is part of the normal skin flora. Nothing about the 16S rDNA sequences raised any red flags. Based on phylogenetic trees and sequence alignment, the samples were very closely related to the C. acnes type strain.

We proceeded with the MLST assay for C. acnes. Fifteen of the samples amplified as expected with the remaining two yielding no usable information. Of the fifteen successful samples, we saw six that were unequivocally related suggesting these samples were a source of the contaminant. However, the results we were getting for the two troublesome strains were truly perplexing.

Investigations into this inconsistency led to the discovery of an article published the previous year proposing a new Cutibacterium species.1 This new species, named C. namnetense (formerly Propionibacterium namnetense), was isolated from bone and tissue samples and is closely related to C. acnes. However, based on several of the protein-coding genes, it is clearly a novel species. Additionally, during our investigation we found that many members of the Propionibacterium genus were reclassified to Cutibacterium.2

With that dilemma solved, we provided the customer with an accurate MLST reflecting the relatedness between the C. acnes strains and informed them of the newly described species of which the two remaining samples were members.

The results of the present study signify the importance of having a curated and up-to-date library database, as well as the importance of having experienced microbial phylogeneticists interpreting the sequence data. Having current taxonomic names is crucial to providing accurate identifications and a necessity when performing MLST.

This scenario also highlights the importance of investigating unexpected results as they can be an indicator of new information. Comprehensive evaluation of data during investigations will lead to a high level of confidence in the resulting actions.

References:

  1. Propionibacterium namnetense sp. nov., isolated from a human bone infection. Guillaume Ghislain Aubin​,  Pascale Bémer​,  Stanimir Kambarev​,  Nisha B. Patel,  Olivier Lemenand​,  Jocelyne Caillon, Paul A. Lawson, Stéphane Corvec. 2016; International Journal of Systematic and Evolutionary Microbiology 66: 3393-3399.
  2. The natural history of cutaneous propionibacteria, and reclassification of selected species within the genus Propionibacterium to the proposed novel genera Acidipropionibacterium gen. nov., Cutibacterium gen. nov. and Pseudopropionibacterium gen. nov., Scholz, C. F. P. and Kilian, M., 2016; Int. J. Syst. Evol. Microbiol., 66: 4422-4432.