Implementation of PCR-based replicon typing PBRT system and automatic amplicon detection

Background: Plasmids are the main vectors of antimicrobial resistance genes in Enterobacteriaceae and plasmid typing is basic for the study of evolution, epidemiology and spread of antibacterial resistance. The PCR-Based Replicon Typing (PBRT) is a method for plasmid identification and typing in Enterobacteriaceae, updated from 2005 to date (Carattoli et al. 2005, J. Microbiol. Methods 63:219-228; Carloni et al. 2017, Plasmid 90:10-14), detecting 28 different replicons in 8 multiplex PCRs. The aims of this work were: a) to implement and validate the PBRT scheme, based on the epidemiological relevance of new replicons; b) to improve the PBRT method, using a new high-throughput instrument for the amplicon detection. Materials/methods: Primers were designed to target new replicons (P1, N2, FIB-KN, FIB-KQ, X4) and multiplex PCR conditions optimized. The new PBRT scheme (PBRT kit 2.0, Diatheva) has been validated for specificity and sensitivity testing 39 Enterobacteriaceae strains (Escherichia coli, Klebsiella spp., Salmonella spp., Proteus vulgaris, Aeromonas punctata) from different origin and sources and 20 K. pneumoniae clinical strains obtained during routine hospital surveillance. The amplicons of the new PBRT scheme were analysed both by standard electrophoresis on agarose gel and by the AATI Fragment Analyzer instrument (Advanced Analytical). Results: The new PBRT scheme detected 30 replicons (HI1, HI2, I1-α, M, N, I2, B/O, FIB, FIA, P1, W, L, X3, I1-γ, T, A/C, FIIS, N2, U, X1, R, FIIK, FIB-KQ, X2, FIB-KN, K, HIB-M, FIB-M, and FII, X4) and identified them in 100% of the analysed strains, matching the data obtained by WGS and PlasmidFinder. All the 20 clinical isolated carried FIIK (pKPN3-like plasmid) and 10/20 carried FIB-KQ, detecting the KPC-pKpQIL-plasmid. The analysis of amplicons by Fragment Analyzer instrument showed a good resolution of the peaks. To reduce the number of loadings, T-tail primers were devised to adjust the amplicon sizes and 8 multiplex reactions could be assembled in 5 capillary electrophoresis runs. Conclusions: The updated PBRT scheme exhibited a good specificity and sensitivity, detecting new epidemiologically relevant replicons. The proposed method for the amplicon analysis by the Fragment Analyzer instrument reduces the detection time and simplifies the electrophoresis step with an automated workflow