High-Throughput Short Sequence Typing Schemes forPseudomonas aeruginosaandStenotrophomonas maltophiliapure culture and environmental DNA

Molecular typing techniques are employed to determine the genetic similarities between bacterial isolates. These methods primarily utilize specific genetic markers or analyze the complete genome sequence of pure bacterial cultures. However, the use of environmental DNA profiling to assess epidemiologic links between patients and their environment has not been explored in depth. This work reports on the development and validation of two High-Throughput Short Sequence Typing (HiSST) schemes targeting the opportunistic pathogens Pseudomonas aeruginosa and Stenotrophomonas maltophilia , along with a modified SM2I medium for specific isolation of S. maltophilia . Our HiSST schemes are based on four discriminative loci for each species and demonstrate high discrimination power, comparable to pairwise whole genomes comparison. Moreover, each scheme includes species-specific PCR primers, enabling precise differentiation from closely related taxa without the need for upstream culture-dependent methods. For example, the primers designed to target the bvgS locus allow to distinguish P. aeruginosa from the very closely related Pseudomonas paraeruginosa sp. nov. The selected loci included in the schemes for P. aeruginosa ( pheT , btuB , sdaA , bvgS ) and for S. maltophilia ( yvoA , glnG , ribA , tycC) , are within the range of 271 to 330 base pairs adapted to massive parallel amplicon sequencing technology. A R-based script implemented in the DADA2 pipeline was assembled to facilitate HiSST analysis for efficient and accurate genotyping of P. aeruginosa and S. maltophilia . The performance of both schemes was demonstrated through in-silico validations, assessments against reference culture collections, and a case study involving environmental samples.