Phylogeny and Comparative Genomics Unveil Independent Diversification Trajectories ofqnrBand Genetic Platforms within Particular Citrobacter Species

Abstract

ABSTRACTTo gain insights into the diversification trajectories ofqnrBgenes, a phylogenetic and comparative genomics analysis of these genes and their surrounding genetic sequences was performed. For this purpose,Citrobactersp. isolates (n= 21) and genome or plasmid sequences (n= 56) available in public databases harboring complete or truncatedqnrBgenes were analyzed.Citrobacterspecies identification was performed by phylogenetic analysis of different genotypic markers. The clonal relatedness among isolates, the location ofqnrBgenes, and the genetic surroundings ofqnrBgenes were investigated by pulsed-field gel electrophoresis (PFGE), S1-/I-CeuI-PFGE and hybridization, and PCR mapping and sequencing, respectively. Identification ofCitrobacterisolates was achieved usingleuSandrecNgene sequences, and isolates characterized in this study were diverse and harbored chromosomalqnrBgenes. Phylogenetic analysis of all knownqnrBgenes revealed seven main clusters and two branches, with most of them included in two clusters. Specific platforms (comprisingpspFandsapAand varying in synteny and/or identity of other genes and intergenic regions) were associated with each one of theseqnrBclusters, and the reliable identification of allCitrobacterisolates revealed that each platform evolved in different recognizable (Citrobacter freundii,C. braakii,C. werkmanii, andC. pasteurii) and putatively new species. A high identity was observed between some of the platforms identified in the chromosome ofCitrobacterspp. and in different plasmids ofEnterobacteriaceae. Our data corroborateCitrobacteras the origin ofqnrBand further suggest divergent evolution of closely relatedqnrBgenes/platforms in particularCitrobacterspp., which were delineated using particular genotypic markers.