Analysis of Phylogenetic Variation of Stenotrophomonas maltophilia Reveals Human-Specific Branches

Abstract

Stenotrophomonas maltophilia is a non-fermenting Gram-negative bacterium that is ubiquitous in the environment. In humans, this opportunistic multi-drug-resistant pathogen is responsible for a plethora of healthcare-associated infections. Here, we utilized a whole genome sequencing (WGS)-based phylogenomic core single nucleotide polymorphism (SNP) approach to characterize S. maltophilia subgroups, their potential association with human infection, and to detect any possible transmission events. In total, 89 isolates (67 clinical and 22 environmental) from Germany were sequenced. Fully finished genomes of five strains were included in the dataset for the core SNP phylogenomic analysis. WGS data were compared with conventional genotyping results as well as with underlying disease, biofilm formation, protease activity, lipopolysaccharide (LPS) SDS-PAGE profiles, and serological specificity of an antibody raised against the surface-exposed O-antigen of strain S. maltophilia K279a. The WGS-based phylogenies grouped the strains into 12 clades, out of which 6 contained exclusively human and 3 exclusively environmental isolates. Biofilm formation and proteolytic activity did correlate neither with the phylogenetic tree, nor with the origin of isolates. In contrast, the genomic classification correlated well with the reactivity of the strains against the K279a O-specific antibody, as well as in part with the LPS profiles. Three clusters of clinical strains had a maximum distance of 25 distinct SNP positions, pointing to possible transmission events or acquisition from the same source. In conclusion, these findings indicate the presence of specific subgroups of S. maltophilia strains adapted to the human host.

Keywords: Stenotrophomonas maltophilia; biofilm; cystic fibrosis; lipopolysaccharide; next generation sequencing; proteolytic activity; whole genome sequencing.

FIGURE 1

Maximum-likelihood tree calculated from the 408,860 aligned SNP positions of the 94 datasets of S. maltophilia and S. rhizophila. All but five strains can be classified into 12 major phylogenetic groups, designated groups 01–12. Groups of LPS profiles, presence of a K279a-like O-antigen, genotypes (GyrB, AFLP, rep-PCR, MLST) and origin of the isolate are indicated colored squares according to the legend on the left. On the right, the results of the biofilm (green bars) and protease (orange bars) assays are given as relative activities. Proteolytic activity is depicted as relative fluorescence/OD600 unit (110-min values) and values below 0 were set to 1. For the biofilm assay, the absorbance at 595 nm is depicted. Node resampling support of more than 90% is indicated by black dots. K279a-like: 75% of the K279a genome sequence could be recovered from WGS data. Black dots on nodes indicate a resampling support greater 90%.

FIGURE 2

S. maltophilia and S. rhizophila maximum-likelihood tree in radial tree layout. The 12 phylogenetic groups are indicated by numbers, and isolate origin mapped on the tree in green (environmental) and red (human). K279a-like strains for which 75% of the K279a genome sequence could be recovered from WGS data are indicated by a gray background.

FIGURE 3

Maximum-likelihood tree built from 649,312 SNP positions of the set of 73 S. maltophilia strains marked as K279a-like in Figure 1. Phylogenetic groups are indicated by numbers, and isolate origin shown as rectangles in the outer ring in green (environmental) and red (human). Excluding datasets from full genomes, 4 groups of strains were detected with a threshold of 25 distinct SNP positions [threshold used for M. abscessus by Bryant et al. (2013)] and are indicated by red stars next to isolate names and labeled G1 (ICU02, ICU06, ICU98, ICU119), G2 (U7, U8), G3 (SM527, SM530), and G4 (SM297, SM454). Black dots on nodes indicate a resampling support greater 90%.