DOI: 10.5061/dryad.6wwpzgn13
title: Stenotrophomonas isolates from patients with cystic fibrosis and other chronic respiratory infections

description: 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.
description: <strong>The study/bacterial
 isolates</strong> 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 <em>S.
 maltophilia</em>. 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.  
 <strong>Whole genome sequencing</strong>
 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 <em>S.
 maltophilia</em> [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)
 <br><strong>Analysis of Average Nucleotide identity
 by BLAST (ANIb)</strong> 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].
   <strong>Phylogenetic
 trees</strong> Neighbor-joining trees of the
 concatenated alleles of the MLST scheme for<em> S.
 malthophilia</em>, 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
 <em>S. tumilicola</em> 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  
 <strong>Antimicrobial susceptibility
 testing</strong> 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.   <strong>Protease and esterase activity</strong> 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 KH<sub>2</sub>PO<sub>4</sub>, 540 mL glycerol and 400 mL water to 500 mL molten nutrient broth agar medium.  Bacteria were grown to 1 OD<sub>600</sub> 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 <em>Pseudomonas aeruginosa</em> 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 <em>Stenotrophomonas maltophilia</em>. Lett Appl Microbiol. 2006;43:443-9. doi: 10.1111/j.1472-765X.2006.01965.x