Genome Sequencing And Comparative Genomics Analysis Of The Cronobacter Spp.

Cronobacter spp.is one of the most dangerous pathogens in powdered infant formula(PIF),which can causes severe infections among neonates with a high fatality rate.This species is also widely distributed in nature owing to its strong resistance to the environment,so it becomes the main reason for PIF contaminated.Cronobacter spp.has rich genetic diversity,and its population characteristics is not only universal but also special.In other words,the population characteristics and genetic material of Cronobacter are diverse in different environments.In previous work,our team analyzed the population structure and tolerance of Cronobacter isolated from PIF and production environments in China.The results showed that the isolated strains had unique population characteristics in comparison to other countries isolates,and the tolerance of different ST strains was different significantly.In order to further investigate the functional genes related to environmental adaptation and the variation genes group of interspecies and intraspecific of Cronobacter,the genetic evolution of the genus was explored.In this study,15 different ST strains isolated from PIF and production environments were selected for de novo sequencing in combination with 12 completed genome sequences from the Genbank database.In total,27 genomes of Cronobacter were employed to analyze their gene and genome compositions and evaluation from the comparative genomic sight.The main content and results of this study were as follows:(1)The genomes of 15 Cronobacter isolates were sequenced using the Illumina HiSeq 2500 platform.The genome sequences of 15 Cronobacter strains were assembled using SOAP denovo(v2.04)range in size from 4.34 to 4.57 Mb,with an average GC content of 56.88%.The strains contained an average of 4042 CDSs.The number of predicted tRNA of sequenced genomes was between 70 and 87.The reference strain had 22 rRNAs,but the number of sequenced strains was less than 5.Using the Mauve 2.3.1 software for the analysis of the inter-specific and intra-specific genomes of the seven standard strains of Cronobacter spp.and C.sakazakii,respectively.There were 4411 locally collinear blocks(LCBs),which were basically homologous regions identified by the MAUVE alignment of all 27 genomes.The results showed that there were no large-scale insertions,deletions,inversions,etc.among the Cronobacter spp.However,C.sakazakii had poor collinearity and large-scale genome rearrangements.(2)ANI values among 27 study genomes had been calculated by Jspeceis with BLASTn analysis.The ANI value of 14 newly sequenced strains including CE63,CE28,CE55,CE16,CE52,CE32,CE75,CE62,CE29,CE64,CE15,CE56,CE65,CE13 and 5 reference strains of C.sakazakii was all larger than 97%.Similarly,the ANI value of CMa3 and C.malonaticus were also greater than 97%.While,the ANI value of other five species was all less than 95%.The sequenced strains can be classified into the species of C.malonaticus CMCC 45402 and C.malonaticus LMG23826 respectively.It can be seen that the calculation of the average nucleotide identity based on the whole genome sequence could be an accurate and effective method for species definition and taxonomic of the Cronobacter spp.(3)According to PGAP clustering results,the dilution curves of 27 Cronobacter genome were drawn using PanGP.The results showed that the genome belonged to the open pan-genome,indicating frequent gene exchange among strains under selective pressure.It suggested that the gene pool of Cronobacter had high diversity.As the number of genomes increased,a large number of new genes had been discovered continuously,which further demonstrated that Cronobacter had a high degree of plasticity.The pan genome and core genome of 27 Cronobacter were defined using the Roary software and comparative genomics methods,and functional annotation was performed using the COG database.The results showed that 27 Cronobacter were divided into11262 orthologous clusters,of which the unique genes accounted for 43%.Core genes,accessory genes and specific genes were all enriched in 26 functional categories,indicating that the functions of Cronobacter were regulated by pan genome.(4)C.sakazakii unique genes were identified by comparing Cronobacter interspecies genomes.The specific gene included 44 virulence and bio-related genes and 20 hypothetical proteins.As below,absorption and utilization of exogenous sialic acid gene nanKCATE,icsA autotransporter precursor,toxin-antitoxin biofilm protein tabA,protease htpX,diguanylate cyclase dosC,murein DD-endopeptidase mepS,S-fimbrial adhesin protein sfaS precursor,DNA ligase B,leucine-specific-binding protein precursor livK,HTH-type transcriptional regulator lurR and leuO.The ST1 strain included the unique virulence gene regions such as the tellurite resistance genes terBDZWXY,von Willebrand factor type A domain protein,and 13 hypothetical proteins.The ST4 strain included the putative virulence factor ypjF,putative prophage CPS-53 integrase and 19 putative proteins.No specific virulence factor was found in strain ST8.(5)The antibiotic resistance gene was obtained by using the BLASTP program to compare the protein sequences of 27 Cronobacter to the homologous protein model sequence in the CARD database.The results showed that 27 Cronobacter contained 134 ARO,which contained antibiotic resistance genes,multiple drug resistance genes,multiple efflux pump and their regulatory factor.The cluster analysis showed that there was no significant difference in drug resistance genes carried by the strains(6)The PhyMLpackage and Mega software were used to complete the construction of the phylogenetic trees based on the sequence of whole genome,16 S rDNA and housekeeping genes.The results showed that Cronobacter had been differentiated into two branches,C.muytjensii-C.dublinensis and C.muytjensii-C.turicensis-C.sakazakii-C.malonaticus.The former had evolved to be adapted for environmental and plant association niche,and the latter had evolved andacquired accessory genes that have enhanced its virulence capacity.(7)The ClonalFrameML and CODEML programs were used to determine the recombination regions and evolution selection directions of the 27 Cronobacter core genomes.As a result,it was found that there were recombination events in the core genomes of 27 strains,and were all underwent to purification selection.The IS and GI of 27 Cronobacter genomes were predicted by using ISFinder and IslandViewer software,respectively.The result showed that the distribution of a large number of IS may be formed through transposition under selective pressure.The homologous regions of T6 SS between Citrobacter and Cronobacter confirmed the occurrence of gene transfer events,which should increase the genetic diversity and strong environmental adaptability of the species.Analyze the genetic evolutionary characteristics of Cronobacter from the perspective of genomics,thereby applying for actual production.This project can provide new ideas and theoretical basis for monitoring,prevention and treatment the Cronobater spp.in PIF.