Providing our clients custom subspecies-level identification and differentiation through protein-targeted gene sequencing
Subspecies level identification of microorganisms, as well as discrimination between closely-related species, is a challenging goal that an increasing number of our clients are requesting as a service. Some of the common approaches used to differentiate closely-related strains compare organisms by considering their genotypic, phenotypic, serological, spatial or their temporal characteristics. While the combination of these traits can result in subspecies level identifications, the analysis of multiple characteristics increases the time, labor and expense needed to differentiate isolates, as well as increases the errors that can arise from qualitative and subjective analyses. To remedy these drawbacks, Accugenix is endorsing the use of single- and multi-locus sequence typing (S/MLST) methods to accurately, reproducibly and cost-effectively differentiate closely-related microorganisms.
SLST and MLST are well-established methods that can be used to distinguish closely-related microorganisms. Since the foundation of S/MLST is built upon DNA sequencing results, which can be easily cataloged and referenced, these techniques are highly reproducible, unambiguous and scalable. In fact, web-based databases containing MLST data serve as primary resources for tracking and documenting the genetic relationship of bacteria at a global-scale. Given the reproducibility of S/MLST between experiments, and over time, these methods can be used to determine if isolates recovered from one area are the same or different as another isolate – a trait that allows for high-resolution trending and tracking projects.
S/MLST methods involve sequencing one to ten genes that are known to harbor moderately, to highly, variable DNA sequences, such as protein-coding or housekeeping genes. Housekeeping genes are genes which encode for proteins necessary for the normal cellular functions of the bacteria. By using the gene sequence, as opposed to the gene product as in enzyme electrophoresis, more variation can be detected resulting in more alleles per locus. The addition of multiple loci provides additional variation to further differentiate between closely related strains. After sequencing, the gene sequences from each isolate are aligned and compared in an evolutionary tree to show the amount of conservation and divergence in that gene region, to standardize variability and to strengthen the comparative tree. All the sequences from the multiple gene targets are also concatenated (placed end-to-end), aligned and compared to sequences from other organisms. This comparison enables the level of divergence/conservation between microorganisms to be calculated and displayed with a phylogenetic tree. The goal is to determine gene combinations that can give a high level of variability to differentiate to the strain or subspecies level.
A Case in Point
Most recently, Accugenix evaluated subspecies typing methods using 31 isolates of Propionibacterium acnes, which is a highly clonal species that exhibits very little genetic variability between strains. While Accugenix’s standard genotypic identification method – 16S rDNA target sequencing – was useful for species-level identification, it could not 
discriminate strains at the subspecies level (Table 1, Figure 1).
Next, the strains were compared using DuPont Qualicon’s RiboPrinter® – an automated system that generates genetic snapshots of each isolate. Although this assay was run at its highest level of discrimination by using two restriction enzymes, EcoRI and PvuII, ribotyping was ineffective in discriminating between P. acnes strains (Figure 2).
Following ribotyping, the discriminatory power of repetitive element PCR (rep-PCR) was tested. While one minor shift was observed with this method, indicating strain variation, it was not large enough to pass the cutoff standards for a subspecies level differentiation of strains (Figure 3).
Subsequently, the ability to differentiate these strains was tested using MLST. Eight housekeeping genes were sequenced from all P. acnes strains (Table 1). After concatenation and alignment of these sequences, phylogenetic analysis revealed that these 31 P. acnes strains fell into eight separate outgroups, indicating that MLST was successfully able to discriminate between these closely related P. acnes strains (Figure 4). Finally, these P. acnes strains were subjected to SLST analysis, which consisted of sequencing one, highly-variable, gene that is under increased selective pressure – dermatan sulfate-binding protein (Table 1). Phylogenetic analysis completed on these sequences also revealed eight outgroups (Figure 5), suggesting that SLST can successfully discriminate between closely related strains of P. acnes.
The data described here show that SLST and MLST perform equally when making subspecies level identifications between P. acnes strains and indicate that these sequences typing methods are able to discriminate between closely-related strains better than other subspecies identification systems. However with P. acnes, SLST is the favored method as it only requires sequencing of a single gene target.
SLST and MLST subspecies typing methods are not limited to P. acnes. Many other sequence typing targets have been identified for a variety of other species that are commonly encountered by our clients – of which several have been performed at Accugenix.
Accugenix has always been committed to providing the most accurate and reliable species-level identifications to our clients. Now, we are proud to include sub-species level identifications to our service list. We are the leader in innovation in the field of microbial identification, strain typing and genotypic methods. MLST and SLST is just another example of Accugenix being the first contract laboratory service provider to offer this technology as a solution for our clients.
