678 - Polymorphic Locus Sequence Typing for Pseudomonas aeruginosa Outbreak Detection and Investigation

Background: Pseudomonas aeruginosa is a common agent of healthcare-associated infection (HAI), which can be challenging to treat due to intrinsic or acquired antibiotic resistance. In contrast to most HAI, the source of P. aeruginosa infection is typically environmental; e.g., from the faucets, drains, and showers in patient rooms. To unambiguously identify the source, strain typing of patient and environmental isolates is required. Typing methods include sequence-based MLST and WGS which resolve strains based on relatively rare SNPs, and length-based MLVA which resolves strains based on relatively frequent insertions/deletions within tandem repeats. These and other methods previously applied to P. aeruginosa have one or more limitations relating to cost, technical complexity, reproducibility, and turnaround time that preclude their routine use. Polymorphic locus sequence typing (PLST) is a hybrid approach that employs sequence analysis of the one or two most phylogenetically informative tandem repeat-containing loci within the genome of a microbial species. Strain resolution equals or exceeds MLST and MLVA and approaches that of WGS. Here, the identification and evaluation of candidate PLST loci for P. aeruginosa epidemiology are described.

Methods: Tandem repeats were bioinformatically identified in representative genome sequences, and screened by BLASTN queries of GenBank genome databases followed by clustal alignments and phylogenetic analysis. Candidate loci were amplified from heat-treated colony lysates and Sanger sequenced.

Results: PLST locus PaMT1 (Mbp 2.568-2.569 in the PA01 genome) resolved 99 alleles from 144 epidemiologically unrelated isolates with GenBank genome sequences, yielding diversity index = 0.991. Nineteen sets of isolates shared healthcare facility and PaMT1 allele, consistent with nosocomial transmission. Examples, confirmed by WGS-SNP analysis, include isolates from 8 VAP patients, 2 sets of cancer patient isolates, and a pair of isolates from patient and hospital sewage. In the laboratory, locus PaMT1 was readily amplified and sequenced from colony lysates, yielding typing data with 1-2 day turnaround.

Conclusion: PaMT1 PLST provides an affordable, user-friendly, and timely tool for P. aeruginosa outbreak detection and investigaton.