Genomic Epidemiology Of Salmonella Pullorum

Salmonella enterica serovar Gallinarum biovar Pullorum(hereinafter referred to as S.Pullorum)is the causative agent of pullorum disease(PD).It's a poultry-specific pathogen which can be transmitted vertically through eggs.Chicks and poults are more susceptible to PD,which seriously threatened the poultry industry due to high mortality.PD was prevalent worldwide in early 1900s.However,it has been eradicated from commercial poultry in most developed countries,primarily due to those animal health programs like National Poultry Improvement Plan(NPIP).PD is still common in other parts of the world.In china,the epidemic of PD affect a wide range with high positive rates and increasing antimicrobial resistance.Quick and accurate identification and typing of pathogens is critical for disease surveillance and outbreak investigation.S.Pullorum is aflagellate and non-mobile,which is indistinguishable from Salmonella enterica serovar Gallinarum biovar Gallinarum(hereinafter S.Gallinarum)by normal serotyping,for both possess the common antigenic formula(1,9,12:-:-).Although differences of biochemical phenotypes exist between two biovars,there's no single reliable biochemical property that can completely distinguish the two.The isolates of S.Pullorum are genetically homogenous,which are difficult to distinguish by current molecular typing method with limited resolution.This challenges the application of conventional surveillance methods in PD.In recent years,the rapid development of next-generation sequencing(NGS)technology has led to a continuous increase in sequencing throughput and a straight decline in costs.With this opportunity,whole-genome sequencing(WGS)has become a viable application for epidemiological research.WGS achieves quick and accurate species identification,inference of antimicrobial resistance and virulence and high-resolution subtyping.In this study,97 isolates of S.pullorum sampled from 1962 to 2014 from 4 countries were preformed WGS.The phylogeny of S.pullorum was reconstructed based on core-genome singlenucleotide polymorphisms(SNPs).Bayesian analysis was used to estimate the mutation rate,divergence time and transmssion history of S.pullorum.By identifying pseudogenes and gene deletions,the extent and trends of genome degradation were assessed,and the inactivated metabolic pathways and virulence profiles were estimated.Identification of the mobile genetic elements(MGEs)present in the genome revealed the genetic mechanism of the antimicrobial resistance.1.Establishment of Bacterial Isolate Genome Sequence Analysis Pipeline(BIGSAP)and its application in surveillance of pullorum diseaseWith the advance of NGS,the bottlenecks in the application of WGS to epidemiological surveillance have shifted to data processing and analysis.To this end,we establish the Bacteria Isolate Genome Sequence Analysis Pipeline(BIGSAP),which integrates multiple databases and bioinformatics tools to enable "one-stop" analysis of bacterial genome data,including data quality control,genome assembly,species identification,serotype identification,molecular typing and drug sensitivity prediction.It compresses the tasks that cost one week or even weeks to archieve in a traditional microbiology lab to less than one hour.The use of BIGSAP does not require the background in bioinformatics,and the results are easy to read and meet the general needs of surveillance.Through BIGSAP analysis,we verified the species and serovars of 97 sequenced isolated of S.Pullorum.Eight acquired drug resistance genes were detected:aadA5,blaTEM-1B dfrA17,strA,strB,sull,sul2,and tet(A),as well as one gene mutation:gyrA(Ser83Phe).These genes and mutation mediate bacterial resistance to six classes of antibiotics:sulfonamides,?-lactams,aminoglycosides,tetracyclines,diaminopyrimidines,and fluoroquinolones.Nine sequence types(STs)were identified.In addition to the reported ST92,ST2151,and ST2453,six new STs were found:ST3717,ST3720,ST3721,ST3722,ST3723,and ST3727.All STs differ by only one or two loci,so they belong to the same eBurstGroup,eBG4.2.Phylogenetic analysis of Salmonella Pullorum revealed a transmission history of 1100 years of pullorum diseaseThe conventional molecular typing method has limited resolution,so that it is difficult to distinguish Salmonella pullorum isolates effectively.In this study,we used the genome of Salmonella Pullorum RKS5078 as a reference.After eliminating repeat,deletion,and recombination regions,a 4,160,759 bp core genome was obtained,from which 6,795 SNP sites were identified by mapping reads to reference genome.The typing method based on core-genome SNPs provides exceptional resolution,which can distinguish almost all isolates from each other.Subsequently,we performed a phylogenetic analysis on the population of Salmonella Pullorum by using maximum-likelihood and Bayesian method.We infer that the most recent common ancestor(MRCA)of S.Pullorum emerged in 914 CE(95%CI:565-1273),approximately 1,100 years ago,with an estimated mutation rate of 0.8 SNPs/genome/year,or 1.92×10-7 substitutions/site/year.The extant S.pullorum can be divided into four defined lineages,which we named L? to L?.L?,L?,and L? are Chinese native lineages.L? was introduced to Europe in 1837(CI95%1782-1889);L? may has originated in the Americas and was transmitted to Europe in 1842(CI95%1778-1902).The transmission event coincided with the "hen fever" that engulfed Europe and the United States in the middle of the 19th century.Chinese native chickens were transported to the United Kingdom and the United States as ornamental birds.We concluded from this that colonial trade promoted intercontinental spread of S.Pullorum.3.Continuing genome degradation of Salmonella Pullorum:pseudogene formation and deletionGenome degradation may be a common mechanism of host adaptation.Our study showed that the genome of S.Pullorum has undergone extensive degradation which is still continuing.A total of 552 pseudogenes and 55 deletion variations were identified which may result in the inactivation of 660 genes.Each isolate possesses an average of 167 inactive genes,among which 122 are shared by all isolates.It suggests that about 73%inactive mutations are already present in MRCA of S.Pullorum and are inherited to the offspring by vertical transfer.Gene inactivation didn't stop after diverging from MRCA,result in an average of 45 inactivated genes per strain and an average of 0.041 inactivated genes per year.85%of inactive genes are caused by pseudogene formation,and only a small proportion of mutations are caused by deletion.Flagella and fimbriae can mediate adhesion and invasion of host cells and activate pattern recognition receptors.The loss of flagellar and fimbrial genes in S.pullorum is the potential mechanism for avoiding stimulation of non-specific inflammation.The flagellar genes flhB and flgK were inactive before divergence of S.Pullorum and S.Gallinarum,which may be the direct cause of nonmotility.In L?,L? and L? the srfABC operon shows various types of degradation.It encode class 2 flagellar genes that are regulated by FlhDC.The MRCA of S.Pullorum inactivated seven fimbrial genes independently,including bcfE,stfC,pegCD,steB,lpfE and sefC.stfA and fimAI were truncated by deletion in L?.Inactive genes may affect the ability of pathogens to survive in the external environment and in the host tissues,which may be related to the change of lifestyle from gut to systemic of S.Pullorum.In the MRCA of S.Pullorum,eight metabolic pathways were inactivated,including heme synthesis(hemF),gluconeogenesis(ppsA),trehalose synthesis(treZ),L-leucine synthesis(ilvI),L-isoleucine synthesis(leuD),4-aminobutanoate degradation(gabT),pyruvate synthesis(gpmB)and allantoin degradation(allD).In addition,several transport systems associated with the uptake and secretion of substances are affected,such as sulfate/thiosulfate import(cysU),glutamate aspartate uptake(gltL),histidine translocation(hisJ),and betaine uptake(proW and osm V)and so on.Betaine is an osmoprotectant.The inability to uptake betaine results in bacteria that are difficult to maintain intracellular osmotic pressure.This may be one of the reasons why S.pullorum are significantly less resistant to chemical and mechanical stress.Besides,torAC and torR are inactivated in the MRCA of S.Pullorum,which may make it impossible to utilize trimethylamine N-oxidation for anaerobic respiration.4.Mobile genetic elements mediate virulence and antimicrobial resistance of Salmonella PullorumThe plasticity of Salmonella genomes benefits from lateral gene transfer(LGT),which confers the unique ecological and pathogenic characteristics of Salmonella.We identified a number of mobile genetic elements(MGEs)in the genome of S.Pullorum,which are vectors for LGT,including 4 prophages,3 small plasmids,and 5 resistance plasmids.The prophages show lineage-specificity.Among them,cpSPU1 is a P22-like prophage,in which we identified a possible secreted effector gene,named pipB3.Its putative protein product PipB3 contains multiple tandem pentapeptide repeat motifs resembled PipB/PipB2.PipB/PipB2 are effector proteins of Salmonella type 3 secretion system that can alter the physiology of host cells and promote bacteria survival in host tissues.?SPU1 is specifically present in isolates from China,especially from eastern and central China.?SPUl is highly similar to ST104,which is a prophage shared by isolates of S.Typhimurium DT104.The small plasmids are relatively conserved and present in most isolates.pSPUS1 encodes the virulence gene ipaJ,probably from the invasive plasmid of Shigella flexneri.The C-terminus of IpaJsF has a domain homologous to the C39-like protease that is able to cleave N-myristoylated proteins,thereby affecting cell growth,signaling,autophagosome maturation,and organelle function.The C-terminus of IpaJSPU has a conserved motif structure,including three catalytic amino acid residues Cys64,His206,and Asp218 consistent with IpaJSF,which imply that IpaJSPU may play a similar role as IpaJSF.Acquired antimicrobial resistance mediated by large plasmid is a major factor of MDR in S.Pullorum.The five resistance plasmids involved three incompatibility groups IncX,IncN,and IncQ.All the resistance clades exist in China's dominant lineage LI,which reflects the prevalence of overuse of antibiotics in China.The rapid growth of multi-drug resistance occurred after about 1990 years which is later than the emergence of MRCA in each resistant branch.This suggests that MDR strains already exist,and that their population size has increased significantly in the last two decades,which may be due to the excessive use of antibiotics in poultry farming.IncXl plasmid pSPUR1 and IncN plasmid pSPUR3 are potentially most harmful because they encode VirB/D4 and Trw T4SS,respectively.T4SS is a versatile secretion system that can transfer resistance plasmid horizontally to other recipient cell through conjugation.In addition,T4SS was also found to be associated with virulence,possibly conferring greater pathogenicity to S.Pullorum,and even altering its host adaptation.