Genotypic and phenotypic insights into virulence factors of nosocomial Stenotrophomonas maltophilia isolates collected in Bulgaria (2011-2022).

The present study aimed to explore the virulence characteristics in 221 Bulgarian nosocomial Stenotrophomonas maltophilia isolates (2011-2022) via screening for the presence of virulence genes, their mutational variability, and the corresponding enzyme activity. PCR amplification, enzymatic assays, whole-genome sequencing (WGS), and biofilm quantification on a polystyrene plate were performed. The incidence of virulence determinants was as follows: stmPr1 (encoding for the major extracellular protease StmPr1) 87.3%, stmPr2 (minor extracellular protease StmPr2) 99.1%, Smlt3773 locus (outer membrane esterase) 98.2%, plcN1 (non-hemolytic phospholipase C) 99.1%, and smf-1 (type-1 fimbriae, biofilm-related gene) 96.4%. The 1621-bp allele of stmPr1 was most frequently found (61.1%), followed by the combined allelic variant (17.6%), stmPr1-negative genotype (12.7%), and 868-bp allele (8.6%). Protease, esterase, and lecithinase activity was observed in 95%, 98.2%, and 17.2% of the isolates, respectively. The WGS-subjected isolates (n = 9) formed two groups. Five isolates possessed only the 1621-bp variant of stmPr1, higher biofilm formation ability (Optical Density at λ = 550 nm (OD550): 1.253-1.789), as well as a low number of mutations in the protease genes and smf-1. Three other isolates had only the 868-bp variant, weaker biofilm production (OD550: 0.788-1.108), and higher number of mutations within these genes. The only weak biofilm producer (OD550 = 0.177) had no stmPr1 alleles. In conclusion, the similar PCR detection rates did not allow differentiation of the isolates. In contrast, WGS permitted stmPr1 alleles-based differentiation. To the best of our knowledge, this is the first Bulgarian study presenting genotypic and phenotypic insights into virulence factors of S. maltophilia isolates.