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

Christine E. Farrance, Anne Buboltz, Bindhu Verghese, Sunhee Hong and Douglas H. Smith

Ralstonia pickettii

Poster 9 Accugenix, Inc. 223 Lake Drive Newark, DE 19702

Abstract
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.

Methods
Confirmation of species identity for nine isolates was determined by standard 16S rDNA sequencing by the proprietary methods and library database at Accugenix, Inc. The genetic distance of each isolate from the type strain was determined and a Neighbor Joining tree (NJ tree) showing the phylogenic relationships was created. Strain characterization on six of the isolates was also performed on the RiboPrinter using the recommended methods from DuPont, Qualicon, and RiboGroups assigned. In general, SLST methods involve sequencing one gene that is known to harbor variable DNA sequences, and with MLST multiple genes of moderate variability are sequenced. The gene sequences from each isolate are then aligned and compared in a phylogenetic tree to show the amount of conservation and divergence in the DNA of that gene region. In situations where a single gene fails to resolve to the strain level, the sequences from the multiple gene targets are concatenated, aligned and compared. This comparison enables the level of divergence between microorganisms to be calculated and displayed in a phylogenetic tree. The goal is to determine genes or gene combinations that can give a high level of variability to differentiate to the strain or subspecies level while being rapid and low cost, compared to other typing methods. In the present study, an initial screen of potential strain typing genes for R. pickettii revealed a complex genome organization consisting of multiple circular chromosomes and plasmids in each cell. The development of sequence typing loci proceeded by examining the sequence data for the chromosomes to identify potential targets. Although no protein coding genes were previously described in the literature as having been used to differentiate this bacterium at the strain level, there were genes, such as rpoB, that have been used for species level differentiation or used for sequence typing of closely related organisms. Five genes were identified that were of interest for strain typing R. pickettii. The target genes were amplified using specific primers and subjected to comparative DNA sequencing analysis. Two targets produced multiple PCR products, indicating possible gene duplication or multiple primer annealing sites and were excluded from this study. The remaining sequences were aligned and compared to the other organisms sequence data. The percent divergence between organisms was calculated, assembled into a phylogenetic tree and used to determine strain differentiation power. At this point another target was eliminated from consideration due to indications of horizontal gene transfer. The data from the remaining two targets were used for strain-level identification.

Introduction
Subspecies level identification of microorganisms, as well as discrimination between closelyrelated species, is a challenging goal that is in increasing demand. 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. As a solution to these drawbacks, single- and multi-locus sequence typing (S/MLST) methods can be developed to accurately, reproducibly and cost-effectively differentiate closely-related microorganisms. MLST and SLST are well-established, highly accurate sequence-based methods that can be used to distinguish closely-related microorganisms to the subspecies or strain level by analyzing essential outer membrane protein coding genes which contain more variability or housekeeping genes that encode for proteins necessary for the normal cellular functions of the bacterium. 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 in a population of organisms. The analysis of multiple loci provides additional variation to further differentiate between closely related strains. This genotypic technique is designed to deliver the most informative data and, as a result, is highly discriminatory. 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. Additionally, databases can be created for historical trending and tracking. 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. As an example of sequence typing for a commonly found environmental contaminant, we undertook the development and evaluation of single or multi-locus targets for differentiation of Ralstonia pickettii. Ralstonia pickettii is a gram-negative, rod shaped beta proteobacterium found in moist environments such as soils, rivers and lakes. It has also been identified in biofilms in plastic water pipes and, as it is an oligotrophic organism, it is capable of surviving in areas with a very low concentration of nutrients. The ability to persist in these harsh conditions makes R. pickettii a unique candidate for bioremediation; however, these characteristics also make R. pickettii an organism of great concern in the pharmaceutical manufacturing environment. Indeed, it is one of the top frequently occurring organisms identified from environmental monitoring programs by Accugenix. Because of this, we initiated a study to identify protein coding gene targets that have the potential to repeatedly differentiate between environmental strains.
PDA's 6th Annual Global Conference on Pharmaceutical Microbiology Challenges Facing Pharmaceutical Microbiology in the 21st Century October 17-19, 2011 | Bethesda North Marriott Hotel | Bethesda, Maryland

Figure 2. RiboPrinter analysis of Ralstonia pickettii indicates four to five RiboGroups depending on the restriction enzyme used for the analysis. Six of the strains originally sequenced were subjected to ribotyping analysis with DuPont Qualicons RiboPrinter an automated system that generates genetic banding patterns of each isolate and using both EcoRI and PvuII restriction enzymes. Two samples were not analyzed since they were no longer in-house, and one strain did not yield sufficient growth for digestion. Results from the PvuII digest of the six strains showed four distinct RiboGroups as assigned by the commercial software (left panel). Isolates 5 and 6 were assigned to Group 7-S-6, strains 8 and 9 to Group 7S-3, while strains 4 and 7 were assigned to different groups as they have distinctly different banding patterns. Although, inspection of the raw data indicates that the DNA for strain 4 was uncut (data not shown). Results from the EcoRI digest of the six strains showed five RiboGroups as assigned by the commercial software (right panel). However, two sets of the strains assigned to different RiboGroups, 5/6 and 4/7, have banding patterns with a high apparent similarity after digestions with EcoRI, and furthermore, seem to have a similar degree of differences in the banding patterns as PvuII-digested strains 8/9 and 5/6 which were assigned to the same RiboGroup. Because of the differences seen with the two restriction enzymes, EcoRI and PvuII, ribotyping was unable to adequately, repeatedly discriminate between the R. pickettii isolates.

Figure 4. Single locus sequence typing using the housekeeping gene recombinase A (recA) can differentiate five distinct sequence types for R. pickettii. The ability to differentiate these strains was also tested using the recA SLST target. The recA gene from each isolate was sequenced, the sequences aligned and a NJ tree assembled. The phylogenetic analysis revealed that these seven strains fell into five separate sequence types, indicating that this single locus was also successfully able to discriminate between these closely related R. pickettii strains, and was able to identify an additional strain type than with the rpoB sequence as rpoB is a more conserved gene than recA (Figure 4a). Of significant note is that the phylogenetic relationships identified with recA (Figure 4a) are the same as those determined with rpoB (Figure 3a), with the additional strain type included in the same species cluster which includes strains 5, 6 and 7. Both SLST data complement each other and thus have the same consistent differences with the RiboPrinter RiboGroup designations as described in Figure 3. A comparative concise alignment of the sequences showed 76 single nucleotide polymorphisms in the 1093 bp sequence (Figure 4b).

Conclusions
The data described here indicate that 16S rDNA sequence is sufficient for species-level identification, but does not have enough discrimination for the strain or subspecies-level. The single locus sequence typing targets described here, recA and rpoB, are able to discriminate between closely related Ralstonia pickettii strains. The two individual gene targets are consistent with each other for the identification of the sequence clusters, with a small increase in variation detected with recA. This preliminary study indicates the necessity of further development in the sequence typing of Ralstonia pickettii with the analysis of an additional single locus or multi-loci combination. More testing, with more isolates, is needed. If there had not been significant differentiation with either of these two single locus targets, additional genes would have been identified and sequenced and their sequences used in the phylogenic analysis to achieve additional discrimination between closely related strains of R. pickettii. The resulting phylogenetic relationships determined by the recA and rpoB SLST targets were similar to the identified RiboGroups, but had notable differences. The RiboPrinter produced different Group designations for the same isolates with the two restriction enzymes. Ribotyping had variabilty in the quality of the data (uncut DNA samples, smears in the lanes and high background) resulting in subjective interpretation of the banding patterns. Problematic methodologies seem to lead to inconsistent data and difficulties in interpretation. Sequence typing is a reliable method, as it is not dependent on the reagents, equipment or supply chain of a single source provider. Trending and tracking is simplified when a historical database is maintained with the sequence information, as it is not subjective - no interpretation of the sequence is needed. SLST is more cost effective: the RiboPrinter costs $450 while SLST is $270 for one day turnaround time on the analysis. The simpler methodology of the sequence-based technologies allows for faster turnaround times once species-specific primers are developed and the process validated. Accugenix has always been committed to providing the most accurate and reliable specieslevel 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. The development of MLST and SLST methods is just another example of how Accugenix continues to be the first contract laboratory service provider to offer leading-edge technologies as solutions for our clients.

Results and Discussion

Figure 1. DNA sequencing of the 16S rRNA region of Ralstonia pickettii results in species level identification, but limited subspecies or strain level differentiation. The approximately first 500 base pairs (bp) of the 16S rRNA gene of nine isolates of Ralstonia pickettii were sequenced and compared in a pairwise fashion to the DSM 6297 type strain and the sequence distances calculated. A NJ tree was constructed that indicated three clusters for R. pickettii strains (Figure 1a). The NJ tree shows that seven of the isolates had a 0.00% sequence distance from the type strain, while strain 6 and strain 7 showed greater sequence diversity (0.19% and 0.38% respectively). A comparative concise alignment of the sequences showed that isolate 6 has two polymorphic base positions at sites 254 and 264, while isolate 7 has two definitive changes at the same positions (Figure 1b). While Accugenixs standard genotypic identification method 16S rDNA sequencing was useful for species-level identification, it could not discriminate all the isolates at the strain level.

Figure 3. Single locus sequence typing using the housekeeping gene RNA polymerase B (rpoB) can differentiate four distinct sequence types for R. pickettii. The ability to differentiate these strains was tested using SLST. Initially, the rpoB gene from seven environmental isolates and the DSM 6297 type strain was sequenced. Phylogenetic analysis revealed that these seven strains fell into four separate sequence types, indicating that this single locus was successfully able to discriminate between these closely related R. pickettii strains (Figure 3a). Interestingly, the phylogenetic relationship determined by the pairwise comparisons of the sequence distances are consistent with the ribotyping data with strains 8 and 9, which had the same RiboGroup assignment with both restriction enzymes (Figure 2) and are the same sequence type. Additionally, strain 7 is a unique sequence type based on the rpoB sequence, and was determined to be in a different RiboGroup with both enzymes. Strains 5 and 6 also cluster together in the NJ tree and have the same RiboGroup designation when cut with PvuII, but not EcoRI. A comparative concise alignment of the sequences showed 48 single nucleotide polymorphisms in the 1160 bp sequence (Figure 3b).