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

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.