Development A Multilocus Sequence Analysis For Identification Of The Proteus Species And Discovery Of Novel Proteus Species

We developed a multilocus sequence analysis(MLSA)approach based on five housekeeping genes(HKGs)to delineate genetic similarities and differences among Proteus species.Of all 219 Proteus strains collected in current study,phylogenetic tree of concatenated five HKGs(dnaJ,mdh,pyrC,recA and rpoD)divided into seven clusters,representative of seven genospecies.Meanwhile,phylogenetic trees of the four individual HKGs can also be divided into seven clusters,representative for seven genospecies,all of which corresponded to that of the concatenated tree.An exception is phylogenetic tree of recA,which clustered four strains at different genospecies in comparison with other four individual HKGs and concatenated HKGs phylogenetic trees.The evaluation of inter-and intragenospecies distance of HKGs concatenation,all intergenospecies indicated more significant different distances than those of intragenospecies,which revealed these HKGs concatenation can be used as gene marker to distinguish different Proteus genospecies.Further web-based genomic relationship(is DDH and ANI)estimated by draft genome indicated that,among the seven genospecies defined in this study,the is DDH and ANI values of the type/representative strains were less than the proposed cutoff level for species delineation,i.e.,70%and 95%,respectively,which were congruent with the MLSA method.Notably,among the three subclusters within genospecies 5,either among the three type strains or representative strain(CA142267)among the three subclusters,their is DDH and ANI values were more than the proposed cutoff level for species delineation.The results indicated strains within the genospecies 5 actually belonged to same species.In conclusion,our data suggested the MLSA was a powerful method for the discrimination and classification of the Proteus at species level.As a result,two species(P.cibarius and P.terrae)should be emended to same species of genospecies 5.Base on this MLSA,of all elven Proteus strains were divided into four clusters which were clearly distinct from all known seven genospecies of genus Proteus.The results suggested the four clusters represent four novel species of genus Proteus.The strains of four clusters were identified by biochemical reactions,sugar fermentation experiment,enzyme activities,the utilization of sole carbon sources,16S rRNA and rpoB gene sequences phylogenetic analysis,genomic relationship(is DDH and ANI),the strains of four clusters represent novel species in the genus Proteus and named Proteus columbae,Proteus alimentorum,Proteus faecis and Proteus cibi.The cellular fatty acid and antimicrobial susceptibilities were also described of the four novel species.