The Study On Genomic Diversity Of Burkholderia Pseudomallei On Hainan Island,China

Burkholderia pseudomallei,the environmental Gram-negative bacillus,is commonly found in soil and water in tropical and subtropical regions between 20°north and south latitude with the known hyperendemicity foci in Australasia and Southeast Asia,especially in northern Australia and Thailand.B.pseudomallei is the causative agent of melioidosis,which is difficult to treat clinically with a case fatality rate（CFR）of up to40%,and ineffective antimicrobial treatment may cause the CFR to exceed 70%.Due to the common clinical drug resistance,some melioidosis patients may even be in a state of chronic infection for decades,which poses a serious threat to human health and brings great pressure to public health in endemic areas.Hainan Island is the most endemic area of B.pseudomallei in mainland China.In1989,the first case of melioidosis in Hainan Island was reported in Sanya city.Since the turn of this century,the number of clinically reported cases has increased year by year due to the improvement of the understanding of this pathogen and the clinical diagnosis technology,which poses severe pressure to local prevention and control of B.pseudomallei in Hainan.However,current genetic diversity and molecular epidemiological researches on B.pseudomallei in our country are limited to traditional molecular biological methods with low resolution,such as MLST,MLVA,etc.,and the research based on the whole genome sequencing is still a blank.Objective:This study aims to reveal the genomic epidemiological characteristics of B.pseudomallei on Hainan Island,including population structure,genetic diversity,and the transmission relationship,as well as the distribution and variation of pathogenicity related genetic elements in B.pseudomallei genome,so as to provide a theoretical fundament for our surveillance,prevention,control and clinical treatment of this pathogen.Methods:1.Preparation of genome datasetA total of 1,654 B.pseudomallei genomes were included in this study,including 122newly-sequenced Hainan genomes and 1,532 publicly available genomes downloaded from the NCBI（up to March 2019）.The newly-sequenced Hainan strains which were composed of 119 clinical strains and 3 environmental strains were collected from 12different cities on Hainan Island,and the sampling time ranged from 2002 to 2018.The publicly available genomes were collected from 22 different countries,including Australia and Thailand,etc.,and the sampling time ranged from 1942 to 2018.2.Whole genome sequencingDNA of the 122 Hainan strains were extracted and sent to the Novogene company for whole genome sequencing.DNA libraries were prepared with the Illumina protocol and sequenced on the Illumina Nova Seq 6000 system,with paired-end runs and a read length of 150 bp.3.Quality control and assemblyLow-quality reads were filtered with Trimmomatic v0.38,and the genomes were assembled de novo with high-quality reads through softwear shovill,the draft assemblies have been uploaded to the NCBI under the Bio Project PRJNA669904.4.Variant callingThe assemblies were aligned against the reference genome（strain K96243,NC＿006350.1,NC＿006351.1）,using MUMmer v3.1,to generate a whole-genome alignment and identify the SNPs in the core genome.The SNPs in the repetitive regions were excluded and only high-quality SNPs were used in the subsequent analysis.5.Phylogenetic analysisThe identified SNPs were used to construct a maximum likelihood（ML）tree with Fast Tree v2,using the default settings.The phylogenetic tree was visualized with i To L（https://itol.embl.de/）.6.Population structure analysisThe software hier BAPS（Hierarchical Bayesian Analysis of Population Structure）was used to identify the population structure of B.pseudomallei based on the core genome.The key parameter of this software is the preset maximum number of populations（K）.In this study,the hier BAPS were run with the K of 100,200 and 300,respectively.7.Transmission relationshipFirst,we identified the core genome SNPs of the main lineages of Hainan genomes,and the recombinant fragments were removed through software Gubbins.Second,the ML trees of corresponding lineages were constructed through RAx ML-NG v.0.9.Finally,with the input files of ML trees and isolation location of each strain,the directions and numbers of transmission events were predicted through software phytools v0.5.To improve the credibility,the phytools v0.5 was run with 100 iterations,and the median was chosen as the transmission number.8.Analysis of virulence factors and antimicrobial-resistance genesWe compared the assembled genomes of all 1,654 strains with the core dataset of the Virulence Factors Database（VFDB）and the Comprehensive Antibiotic Resistance Database（CARD）to detect possible virulence factors and antimicrobial-resistance genes,using BLASTn and Resistance Gene Identifier（RGI）v5.1.1,respectively.A gene/segment was considered present if the overall hit coverage and identity were both≥70%.Then,we compared all the assembled genomes with the four regulators（Amr R[BPSL1805],Bpe R[BPSL0812],Bpe T[BPSS0290]and Bpe S[BPSL0731]）of three efflux pumps（Amr AB-Opr A,Bpe AB-Opr B and Bpe EF-Opr C）in B.pseudomallei.We also compared the assembled genomes with theβ-lactamase Pen A[BPSS0946]and the penicillin-binding protein 3（PBP3,[BPSS1219]）,to identify the mutations through BLASTn.Sequence alignment was performed using MEGA v7.0.14.Results:1.Population structure of B.pseudomallei in HainanA total of 16 phylogenic groups（PGs）were detected,and all the Hainan strains were distributed within nine PGs,however,80%of them were concentrated in five major PGs（>5%;group 1,12.3%;group 3,31.1%;group 9,13.1%;group 11,8.2%;group 15,15.6%）,other strains were scattered in other 4 minor groups.2.Transmissions across Hainan and surrounding regionsIn current study,a total of six transmission events of B.pseudomallei between Hainan Island and neighboring countries or regions were detected,including two from Singapore to Hainan,one from Thailand to Hainan,one from Hainan to Singapore,one from Hainan to Taiwan,and one from Hainan to Malaysia.The results proved the two original sources of Hainan strains in recent years:Thailand and Singapore,and also showed that there were not only import events from Southeast Asian countries to Hainan,but also export events from Hainan to neighboring countries or regions,including Singapore,Malaysia,and Taiwan-CHN.We also calculated the evolutionary time of these transmission events which ranged from 10 to 39 years.3.Local transmissions of B.pseudomallei in HainanA total of 21 across-city transmission events were detected,18 of which originated from the two southern hotspot cities,Sanya and Lingshui,and then spread to the northern,western,and eastern areas of the island.Besides,the evolution time of these transmission events is all within 20 years,except for the two transmission events from Sanya to Lingshui,which were 73 years and 41 years respectively.4.Identification of virulence factorsA total of 160 virulence factor genes were identified,which were annotated with the following functions:‘cell motility’,‘intracellular trafficking,secretion and vesicular transport’,‘cell wall,membrane,envelope biogenesis’,and‘signal transduction mechanism’.Various kinds of virulence factors with different functions indicate the strong pathogenic capacity of B.pseudomallei.Furthermore,a population-specific virulence gene boa B that was related to adhesion and invasion was identified in Hainan strains in group 1.5.Identification of antimicrobial-resistance genesVarious types of mutations were identified on clinically relevant antimicrobial-resistance genes,including point mutations and indels,such as Amr R_V222M,Amr R_A121G,Amr RInsertion45,Amr RInsertion68,Amr RInsertion73,Amr RDeletion159-end,Amr RDeletion199-205,Pen A_I139M,Pen A_P145L,Pen A_T147A,Pen A_T264A,and short fragments deletion on penicillin-binding protein 3（PBP3）.Conclusions:In summary,through population genomics analysis,this study revealed that B.pseudomallei on Hainan Island could be divided into five major populations and several minority populations,showing very high genetic diversity.Secondly,two origins of Hainan B.pseudomallei have been confirmed in current study,that were Thailand and Singapore.Besides,we also revealed the transmission relationship of Hainan B.pseudomallei.There were not only mutual transmissions between B.pseudomallei strains in Hainan and neighboring countries or regions,but also frequent mutual transmissions between B.pseudomallei strains in different local cities in Hainan.Finally,the identification of virulence factors,antimicrobial-resistance genes and relevant mutations confirmed the high pathogenicity of Hainan strains.These results have deepened our understanding of the genetic characteristics and pathogenic mechanism of B.pseudomallei on Hainan Island,and also provided basic genomic data and scientific support for the surveillance,prevention,control,vaccine design and clinical treatment of this pathogen.