Taxonomic position, antibiotic resistance and virulence factor production by Stenotrophomonas isolates from patients with cystic fibrosis and other chronic respiratory infections

Abstract

Background: The potential pathogenic role of Stenotrophomonas maltophilia in lung disease and in particular in cystic fibrosis is unclear. To develop further understanding of the biology of this taxa, the taxonomic position, antibiotic resistance and virulence factors of S. maltophilia isolates from patients with chronic lung disease were studied.

Results: A total of 111 isolates recovered between 2003 and 2016 from respiratory samples from patients in five different countries were included. Based on a cut-off of 95%, analysis of average nucleotide identity by BLAST (ANIb) showed that the 111 isolates identified as S. maltophilia by Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS) belonged to S. maltophilia (n = 65), S. pavanii (n = 6) and 13 putative novel species (n = 40), which each included 1-5 isolates; these groupings coincided with the results of the 16S rDNA analysis, and the L1 and L2 ß-lactamase Neighbor-Joining phylogeny. Chromosomally encoded aminoglycoside resistance was identified in all S. maltophilia and S. pavani isolates, while acquired antibiotic resistance genes were present in only a few isolates. Nevertheless, phenotypic resistance levels against commonly used antibiotics, determined by standard broth microbroth dilution, were high. Although putative virulence genes were present in all isolates, the percentage of positive isolates varied. The Xps II secretion system responsible for the secretion of the StmPr1-3 proteases was mainly limited to isolates identified as S. maltophilia based on ANIb, but no correlation with phenotypic expression of protease activity was found. The RPF two-component quorum sensing system involved in virulence and antibiotic resistance expression has two main variants with one variant lacking 190 amino acids in the sensing region.

Conclusions: The putative novel Stenotrophomonas species recovered from patient samples and identified by MALDI-TOF/MS as S. maltophilia, differed from S. maltophilia in resistance and virulence genes, and therefore possibly in pathogenicity. Revision of the Stenotrophomonas taxonomy is needed in order to reliably identify strains within the genus and elucidate the role of the different species in disease.

Keywords: Antibiotic resistance; Cystic fibrosis; Respiratory infection; Stenotrophomonas; Taxonomy; Virulence.

Fig. 1

Heatmap based on percentage of ANIb for strains belonging to the different STs that were analyzed in this study (one isolate per ST was included) as well as available type strain sequences. The color bar shows the percentage of ANIb between any two strains starting from blue (0.8 or 80%) to red (1 or 100%); the blue/red cutoff is 0.94/0.95 (see Methods section for more details). Type strains are indicated by (T) behind the strain identification. The putative species (see text) are indicated at the right. For details about strains and the similarity between strains see Supplementary Fig. 1

Fig. 2

Evolutionary history of Stenotrophomonas strains based on concatenated alleles from the S. maltophilia MLST scheme (pubmlst.org). The figure combines data from Ochoa-Sánchez and Vinuesa and our study (Ochoa-Sánchez and Vinuesa, 2017). Lineages #1-#10 were defined by Hauben et al. (Hauben et al., 1999). Lineages (A) to (F) were defined by Kaiser et al. (Kaiser et al., 2009) and Ochoa-Sanchez et al. (Ochoa-Sanchez 2017) expanded this further. The putative species A-N from the ANIb analysis are indicated by ps and the letter. The evolutionary history was inferred using the Neighbor-Joining method (Saitou, et al., 1987). The optimal tree with the sum of branch length = 1.82770971 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The analysis involved 190 amino acid sequences. All ambiguous positions were removed for each sequence pair. There were a total of 3710 positions in the final dataset. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018). The left column denotes the isolates and strain identification. The right column the grouping. Type strains are indicated by (T) behind the name.

Fig. 3

Evolutionary history of Stenotrophomonas strains belonging to unknown species based on concatenated alleles of the MLST scheme using 16S rRNA sequences was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 0.18050158 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1050 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The analysis involved 57 nucleotide sequences. There were a total of 1392 positions in the final dataset

Fig. 4

The evolutionary history of L1 ß-lactamases was inferred using the Neighbor-Joining method. One amino acid sequence per ST was included. L1a-L1d were used as reference sequences from literature (Walsh et al., 1994; Crowder et al., 1998). The optimal tree with the sum of branch length = 1.25626229 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1050 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The analysis involved 57 amino acid sequences. There were a total of 295 positions in the final dataset. No L1 sequences were found for the ST34, STH, and STI isolates. The letter behind the strain number indicate the presumptive species (Sm = Stenotrophomonas maltophilia, Sp = S. pavani) or putative novel species (A-M) as based on the analysis of the ANIb

Fig. 5

The evolutionary history of L2 ß-lactamases was inferred using the Neighbor-Joining method. One amino acid sequence per ST was included. L2a-L2d were used as reference sequences from literature (Walsh et al., 1997). Strains M15 and W540 are the type strains for S. lactitubi and S. indicatrix, respectively. The optimal tree with the sum of branch length = 1.04210294 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1050 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The analysis involved 59 amino acid sequences. There were a total of 305 positions in the final dataset. There were a total of 304 positions in the final dataset. No L2 ß-lactamase sequences for ST89 and ST215 isolates were found