Genomic characterization of a mobile-element-associated resistance island in trimethoprim-sulfamethoxazole-resistant Stenotrophomonas maltophilia isolates sharing the same PFGE genotype

Abstract

Background Stenotrophomonas maltophilia is an opportunistic nosocomial pathogen with intrinsic multidrug resistance and limited therapeutic options. This study integrated phenotypic susceptibility testing, targeted molecular screening, PFGE and short-read whole-genome sequencing (WGS) to characterize resistance determinants and their local genomic context in clinical-culture isolates. Methods Thirty-four non-duplicate clinical-culture isolates were analyzed. Susceptibility to trimethoprim-sulfamethoxazole (TMP-SMX), levofloxacin and minocycline was determined by disk diffusion. Minocycline testing was repeated with acceptable quality control, and the repeat-run zone records were reassessed using current CLSI breakpoints. A predefined, hypothesis-driven PCR panel was used to screen selected resistance-, efflux- and biofilm-associated targets, and gyrA, gyrB, parC and parE were sequenced. Genetic relatedness was assessed by XbaI-PFGE. Three selected isolates (S05, S08 and S25) underwent WGS as a targeted genomic-context analysis rather than a whole-collection population-genomic study. Results Resistance rates were 5.9% for TMP-SMX, 8.8% for levofloxacin and 11.8% for minocycline after reassessment using current CLSI minocycline breakpoints. Smqnr- and sul1-target PCR positivity was observed in 61.8% and 8.8% of isolates, respectively. A dfrA27-target PCR amplicon was observed in 82.4% of isolates but was not associated with TMP-SMX resistance and was not confirmed in the three sequenced genomes. No QRDR hotspot mutations were identified. The two TMP-SMX-resistant isolates, S05 and S08, shared the same PFGE genotype. Short-read WGS identified an identical 19,361-bp mobile-element-associated resistance region in these isolates, containing a putative Sul-family/folP-like dihydropteroate synthase (DHPS) annotation, two assembly-predicted hits each of blaOXA-2 and ant(3’’), multiple insertion sequences and a mercury-resistance module; the region was absent from S25. Conclusions The identical resistance region in two TMP-SMX-resistant isolates sharing the same PFGE genotype is compatible with shared ancestry, vertical inheritance or persistence of an island-carrying PFGE-defined genotype; horizontal transfer was not demonstrated. The discordance between dfrA27-target PCR positivity, phenotype and WGS highlights the need to interpret molecular screening together with phenotypic susceptibility and orthogonal confirmation. The WGS component provides a local genomic-context characterization rather than evidence of cross-genotype dissemination.