DEVELOPMENT AND PILOT VALIDATION OF A NOVEL PCR-BASED REPLICON TYPING SCHEME FOR PLASMID FAMILIES ASSOCIATED WITH ANTIBIOTIC RESISTANCE IN PSEUDOMONAS SPP.

Background. Pseudomonas species are ubiquitous environmental Gram-negative bacteria increasingly associated with difficult to treat healthcare-associated infections. Along with their substantial intrinsic antimicrobial resistance, the ability to acquire additional resistance and pathogenicity determinants contributes to increased morbidity and mortality. Plasmids represent the major vehicles of gene transfer among hospital strains. Accumulation and dissemination of resistance genes through horizontal gene transfer is exceptionally problematic since it leads to the emergence of multi-resistant and stable phenotypes highlighting the importance of novel tools for studying plasmid epidemiology. Materials and Methods. In this study we introduce a novel PCR-based replicon typing (PBRT) scheme for differentiation of various Pseudomonas spp. plasmid families requiring only two multiplex PCR (mPCR) assays. mPCR 1 is composed of previously published primer sets for IncP-1, IncP-7, IncP-9, IncQ, A/C, N, W, IncU. Primers for multiplex PCR 2 were designed after an in-depth in-silico bioinformatic analysis of the repA gene of more than 50 reference IncP-2, IncP-6, IncP-10, pKLC102-like and pMOS94-like plasmids some of which studied for the first time as a group. Results. The scheme was tested on a set of 90 previously genotyped multi-resistant clinical Pseudomonas spp. isolates. The detection rate of the target plasmid families was low in our strain collection. Replicons were registered in only 3/90 isolates from the IncP-7 (n=1), IncP-10 (n=1), and pMOS94-like (n=1) families. This pilot study demonstrates a novel PBRT scheme applicable to Pseudomonas spp. targeting plasmids of incompatibility groups known to harbour genes associated with antibiotic resistance.