Tracking the Untrackable Microbe: The Use of SLST and MLST in Environmental Monitoring Programs

Tracking and trending of microorganisms in industrial settings, especially in sterile pharmaceutical manufacturing, is an important part of a comprehensive environmental monitoring program. Environmental monitoring programs that use phenotypic identification systems, and track and trend microorganisms by name, do not always incorporate enough information to develop contamination response protocols or provide definitive conclusions. While standard genotypic sequence-based identification systems, such as 16S, D2 and ITS ribosomal DNA (rDNA) sequencing, not only negate the need for phenotypic identification systems, but they also and increase the ability to accurately track and trend microorganisms at the species-level by using the sequence rather than the name. However, some common pharmaceutical contaminants cannot be resolved by this approach alone. By combining standard genotypic identification methods with multi-locus sequence typing (MLST) or single-locus sequence typing (SLST), it is possible to resolve some of the most difficult organisms to trend and track in the pharmaceutical industry. As an illustration of this point, we present a case-study involving Ralstonia pickettii, an organism found in moist environments, which commonly contaminates pharmaceutical manufacturing facilities. While isolates of this species are difficult to resolve or characterize with consistency by standard phenotypic or genotypic methods, the combination of 16S rDNA sequencing and SLST, or MLST, differentiates isolates at the strain level, enabling reliable tracking and trending of this organism. Given the power of microbial differentiation provided by this method, we expect it will be highly complementary to forthcoming changes to USP <1113>, where environmental monitoring programs will likely need to track and trend microorganisms with increased resolution. The case study presented here shows the preliminary development of single locus sequence typing for strain-level differentiation of Ralstonia pickettii that is inexpensive and rapid.