Abstract
Whole genome sequencing (WGS) is increasingly used for epidemiological investigations of pathogens. While SNP variant calling is currently considered as the most suitable method, the choice of a representative reference genome and the isolate dependency of results limit standardization and affect resolution in an unknown manner. Whole or core genome Multi Locus Sequence Typing (wg-, cg-MLST) represents an attractive alternative. Here, we assess the accuracy of wg- and cg-MLST by comparing results of four Pseudomonas aeruginosa datasets for which epidemiological and genomic data were previously described. Three datasets included 155 isolates from three different sequence types (ST) of P. aeruginosa collected in our ICUs over a 5-year period. The fourth dataset consisted of 10 isolates from an investigation of P. aeruginosa contaminated hand soap. All isolates were previously analyzed by a core SNP approach. In this study, wg- and cg-MLST were performed in BioNumericsTM using a scheme developed by Applied-Maths. Correlation between SNP calling and wg- or cg-MLST results were evaluated by calculating linear regressions and their coefficient of correlations (R2) between the number of SNPs and the number of allele differences in pairwise comparison of isolates. The number of SNPs and allele difference between isolates with close epidemiological linkage varies between 0–26 and 0–13, respectively. When compared to core-SNP calling, a higher coefficient of correlation was obtained with cgMLST (R2 of 0.92–0.99) than with wgMLST (0.78–0.99). In one dataset, a putative homologous recombination of a large DNA fragment (202 loci) was identified among these isolates, affecting its phylogeny, but with no impact on the epidemiological analysis of outbreak isolates. In conclusion, we showed that the P. aeruginosa wgMLST scheme in BioNumericsTM is as discriminatory as the core-SNP calling approach and apparently useful for outbreak investigations. We also showed that epidemiological linked isolates showed less than 26 SNPs or 13 allele differences. These are important figures for the distinction between outbreak and non-outbreak isolates when interpreting WGS results. However, as P. aeruginosa is highly recombinant, a cgMLST approach is preferable and caution should be addressed to possible recombination of large DNA fragments.
Highlights
Pseudomonas aeruginosa is considered one of the main Gram-negative bacterial species causing health-care associated infections (Gellatly and Hancock, 2013)
The identification of genetically highly related isolates was concordant with previously acquired epidemiological information: differences of 0–16 Single Nucleotide Polymorphism (SNP) and 0–12 alleles were observed among outbreak’ isolates, whereas differences of 108– 120 SNPs and 74–101 alleles were observed between these isolates and the two non-outbreak isolates of patient #1
The identification of genetically closely related isolates using wgMLST was highly concordant with epidemiological data and SNP calling results for all outbreaks in this study
Summary
Pseudomonas aeruginosa is considered one of the main Gram-negative bacterial species causing health-care associated infections (Gellatly and Hancock, 2013). P. aeruginosa is widely present in the environment and can be retrieved from different sources, such as respiratory therapy equipment, antiseptics, soap, sinks, and hydrotherapy pools (Morrison and Wenzel, 1984). This opportunistic pathogen was found to be part of the endogenous microbiota of up to 24% of hospitalized patients (Morrison and Wenzel, 1984). P. aeruginosa has been previously described as the second most common organism responsible for infections acquired in intensive care units (ICUs) (Vincent et al, 1995). As P. aeruginosa possesses a very complex ecology, only powerful typing methods can give insight on the relatedness of strains and, on the routes of colonization and/or infection (Maiden et al, 2013)
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