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.

The study/bacterial isolates

The isolates were collected for the iABC-project, in which novel antibiotics for CF and bronchiectasis are being developed. A total of 111 isolates were included in the study. These isolates were recovered from respiratory samples of CF (n=103) and diverse pulmonary infections (n=8) between 2003 and 2016 from five different countries: Australia (n=1), United Kingdom (n=41), Spain (n=35), the Netherlands (n=33), and USA (n=1) (Table 1).  Matrix-assisted laser desorption/ionization time of flight mass spectrometry  (MALDI-TOF/MS, Bruker) identified all isolates as S. maltophilia.

The Spanish and UK isolates were described in prior studies [de Dios Caballero et al, 2016; Muhlebach et al, 2018].

-de Dios Caballero J, Del Campo R, Royuela A, Solé A, Máiz L, Olveira C, Quintana-Gallego E, de Gracia J, Cobo M, de la Pedrosa EG, Oliver A, Cantón R. 2016. Bronchopulmonary infection-colonization patterns in Spanish cystic fibrosis patients: Results from a national multicenter study. J Cyst Fibros 15:357-365.

-Muhlebach MS, Hatch JE, Einarsson GG, McGrath SJ, Gilipin DF, Lavelle G, Mirkovic B, Murray MA, McNally P, Gotman N, Davis Thomas S, Wolfgang MC, Gilligan PH, McElvaney NG, Elborn JS, Boucher RC, Tunney MM. 2018. Anaerobic bacteria cultured from cystic fibrosis airways correlate to milder disease: a multisite study. Eur Respir J 52: pii=1800242.

Whole genome sequencing

Bacterial DNA was purified using the Qiacube with the DNeasy Blood & Tissue kit with the enzymatic lysis protocol (Qiagen, Carlsbad, CA) and used to prepare a library for sequencing with the MiSeq or Nextseq (Illumina, San Diego, CA) platforms, using the Nextera XT library prep kit (Illumina).

These steps followed the protocol of the manufacturer. After DNA isolation and a DNA concentration measurement was made using Qubit (Thermo Fisher) with the ds DNA HS Reagent kit. Final clean up used Agencourt AMPure XP magnetic beads according to the protocol of the manufacturer (Beckman Coulter).

Contigs were assembled with SPAdes genome assembler v.3.6.2.

The assembled contigs were analyzed for the presence of resistance genes by ResFinder [Resfinder] from the Center for Genomic Epidemiology (DTU, Copenhagen, Denmark) [Zankari et al, 2012].

Multi-locus Sequence Typing (MLST) was performed using ResFinder and Pubmlst using the scheme for S. maltophilia [PubMLST]. Novel alleles and sequence types (ST) were submitted to the database.

-ResFinder. https://cge.cbs.dtu.dk/services/ResFinder/ (accessed October 28, 2019)

-Zankari E, Hasman H, Cosentino S Vestergaard M., Rasmussen S, Lund O, et al. J Antimicrob Chemother. 2012;67:2640-4. doi: 10.1093/jac/dks261

-PubMLST. https://pubmlst.org/general.shtml (accessed November 10, 2020)

Analysis of Average Nucleotide identity by BLAST (ANIb)

Average nucleotide identity (ANI) among the genomes was calculated using ANIb algorithm of pyani tool version 0.2.3 [41], which uses nucleotide BLAST (version 2.2.28+) alignment for whole genome alignment. Genomes were fragmented into genomic fragments of 1020 bases long. After pairwise alignments of all fragments of each genome, ANI was calculated as the percentage of nucleotide identity for matching regions of all genomes. In biclustering analysis of ANI scores, complete linkage was used as a hierarchical clustering method with the Euclidean distance metric. A heatmap of all genomes was generated using biclustering, where a color scale bar shows the pairwise ANI scores above 75% are shown in a color range starting from blue (ANI 75%) through white to red (ANI 100%). In the heatmap, each species is shown in a different color next to a leaf node of a tree.  A heatmap of all genomes was generated using biclustering, with a color scale bar showing the pairwise ANI score for values above the threshold value of 0.75. A cut-off of 0.95 was used to define groups, which is also the conservative cut-off used to define species [21, 42].

Phylogenetic trees

Neighbor-joining trees of the concatenated alleles of the MLST scheme for S. malthophilia, the 16S rRNA gene, and the L1 and L2 ß-lactamase sequences were generated with WebPrank and Clustal Omega for the alignments, and with MEGA X for the generation of the tree, using 500 bootstraps [40, 43]. The trees were drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method, in the units of the number of base substitutions per site for the 16S rDNA sequences. For the other sequences, the evolutionary distances were computed using the Poisson correction method, in the units of the number of amino acid substitutions per site [Zuckerkandl and Pauling, 1965; Felsenstein, 1985; Saitou and Nei, 1987; Tamura and Nei, 2004; Kumar et al, 2018]. For the ß-lactamase sequences, only one sequence per ST was included. KODON (Applied Maths, Belgium) was used for the whole genome alignments.

The whole genome sequences of the type strains of all species except S. tumilicola were available for analysis.

-Zuckerkandl E, Pauling L. (1965). “Evolutionary divergence and convergence in proteins”. In evolving genes and proteins. Eds. V. Bryson and H.J. Vogel (New York, NY: Academic Press), 97-166.

-Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap. Evol. 1985;39;783-91. doi: 10.1111/j.1558-5646.1985.tb00420.x

-Saitou N, Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Bio. Evol. 1987:4:406-25. 10.1093/oxfordjournals.molbev.a040454

-Tamura K, Nei M, Kumar S. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Nat Acad Sc. USA. 2004;101:11030-5. doi: 10.1073/pnas.0404206101

-Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35:1547-9. doi: 10.1093/molbev/msy096

Antimicrobial susceptibility testing

Minimum inhibitory concentrations (MICs) were determined by the standard ISO broth microdilution method with frozen panels (Trek Diagnostic Systems, Westlake, OH) using EUCAST methodology [European Committee on Antimicrobial Susceptibility Testing, 2019].

MICs were determined in cation-adjusted Mueller-Hinton broth. The antimicrobial agents and the concentration ranges tested were as follows: aztreonam (0.25–256 mg/L); ceftazidime (0.25–256 mg/L); ciprofloxacin (0.03–32 mg/L); colistin (0.25–16 mg/L); tobramycin (0.125–128 mg/L); imipenem (0.125–128 mg/L); meropenem (0.06–64 mg/L); and trimethoprim/sulfamethoxazole (co-trimoxazole) (0.06–32 mg/L).

-European Committee on Antimicrobial Susceptibility Testing. (2019). Breakpoint tables for interpretation of MICs and zone diameters, version 9.0. http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/v_9.0_Breakpoint_Tables.pdf.

Protease and esterase activity

Esterase indicator plates were prepared by addition of  15 mL of an emulsion of  3 mL tributyrin (glyceryl tributyrate), 47 mL water, and 10 mL emulsion reagent composed of 6 g arabic gum, 17.9 g NaCl, 0,41 g KH₂PO₄, 540 mL glycerol and 400 mL water to 500 mL molten nutrient broth agar medium.  Bacteria were grown to 1 OD₆₀₀ in LB broth and 2 µl was spotted onto the plates and incubated overnight at 37°C to respectively assess protease activity by halo formation [Wilhelm et al, 2007].

Protease indicator plates were prepared by adding 2% skim milk to nutrient broth agar. Plates were incubated overnight at 37°C to respectively assess protease activity by halo formation [Figueirêdo et al, 2006]

-Wilhelm S, Gdynia A, Tielen P, Rosenau, F, Jaeger KE. The autotransporter esterase EstA of Pseudomonas aeruginosa is required for rhamnolipid production, cell motility, and biofilm formation. J Bacteriol. 2007;189:6695-703. doi: 10.1128/JB.00023-07

-Figueirêdo PMS, Furumura MT, Santos A M, Sousa ACT, Kota DJ, Levy CE, et al. Cytotoxic activity of clinical Stenotrophomonas maltophilia. Lett Appl Microbiol. 2006;43:443-9. doi: 10.1111/j.1472-765X.2006.01965.x