CRISPRS Molecular Subtyping And Whole Genome Sequencing Analysis For Salmonella Derby Isolates

Salmonella Derby is one of the important pathogens,which can cause systemic diseases in humans.It can cause outbreak of diarrhea in infants and toddlers in domestic and overseas.The CDC data show that the positive rate of Salmonella Derby isolation in pork production chain tends to be the first.Salmonella Derby can maitain a long period of asymptomatic infection and can be planted in various tissues of sows and piglets.It is so easy to be ignored due to their inapparent presence over a long period of time on farms.Recently,it has been found that the isolation rate of the bacteria in layers and chicken is also increasing.Therefore,Salmonella Derby is a great threat to food safety.Hence it is necessary to carry out a systematic study of Salmofnella Derby,so as to provide data support for the prevention and control of Salmonella Derby.Based on the CRISPRS sequence,Salmonella Derby was subtyped firstly in this study.According to the results of genotyping,we selected 2 representative Salmonella Derby strains and sequenced their whole genome,then analyzed the genetic information.1.CRISPRS molecular subtyping for Salmonella DerbyA total of 84 strains of Salmonella were isolated from the 151 pork samples from 2015 to 2016,and the positive rate of Salmonella was 55.6%.Different batches(spring,summer,autumn,winter,)in the retail markets,the positive rate of Salmonella in pork fluctuated greatly,ranging from 45.6%-75.0%,indicating that the seasonal factor did effect on the prevalence of Salmonella and reached the peak in summer.Different retail market Salmonella isolation rate also has the difference,the prevalence rate of 65.7%in T retail market and 47.6%in S retail market.Statistical analysis showed that there was significant difference between the two retail markets(P<0.05).Identification of Salmonella serovars showed that 84 Salmonella strains were confirmed 14 serotypes and Salmeonella Derby was the most dominant serotype,accounting for 28.6%(24/84)of the total.Other serovars such as S Rissen(17.9%,15/84),S.Anatum(13.1%,11/84),S.Muenster(11.9%,10/84)and S.Typhimurium(10.7%,9/84)were also widely distributed in the retail markets.In the previous epidemiological investigation of our laboratory in pork,swine,chicken,human,dog and goose samples,from this study 376 strains of Salmonella Derby were isolated and stocked.Combined with the 24 strains of Salmonella Derby,a total 400 of Salmonella Derby strains were subtyped by CRISPRS technique.Through analysis of the CRISPRS gene locus,the results showed that the spacer of Salmonella Derby contained 2 CRISPRS loci,CRISPR1 and CRISPR2,with a total of 101 spacers.CRISPR1 includes 62 different spacer sequences,among them there are 18 novel spacers.CRISPR2 has 39 different spacer sequences,among them there are 17 novel spacers.The number of spacers and polymorphism of CRISPR1 locus is higher than those of the CRISPR2 locus spacers.According to the distribution of spacers in 400 Salmonella Derby strains,CRISPRs typing identified 51 different Derby CRISPR types(DCTs),among them DCT24(including 110 Salmonella Derby strains)and DCT31(including 109 Salmonella Derby strains)were the most dominant CRISPRS types.Through the phylogenetic tree analysis,51 DCTs are divided into two lineages,L1 and L2,and these two lineages are divided into several substrain.MLST result of all strains in L1 lineage was ST40 and the ST type of all strains in L2 lineage was ST71.Interestingly,we also found that the spacers arrangements were distinct between ST40 and ST71 strains,except for STMlvarl and DerBl spacer.This analysis revealed that CRISPR genes are highly polymorphic even in the same serotype,which could be tremendously useful for bacterial subtyping during molecular epidemiological investigations.As a result,the strains of the two lineages are distantly related,possibly from different ancestors.264 Salmonella Derby isolates from Yangzhou slaughter houses,its downstream retail markets and human source were identified 37 CRISPRS types,in which DCT24 and DCT31 as the dominant CRISPRS types.A total of 185 strains of slaughter houses were divided into 18 CRISPRS types,and 73 strains of retail markets were divided into 28 CRISPRS types.The results showed that the strains of retail markets were more diverse than those of slaughter house isolates.Salmonella Derby strains of human source were identified DCT14,DCT20 and DCT24,and the difference of spacers' number among the three CRISPRS types were 5 to 14 spacers.They shared same CRISPRS type with those of pork source isolates,it indicated that Salmonella Derby have been spread in the food chain.A comparative analysis of the Huaian slaughterhouse results showed that 30 strains of Salmonella Derby were identified 7 CRISPRS types,and the difference of spacers' number among the seven CRISPRS types were 1 to 9 spacers.Isolates of all these times samples shared DCT24 type,the result revealed that long-term existence of this type in the slaughterhouse of Huaian.The isolates from the same slaughter link belong to different CRISPRS types,which suggested that these Salmonella Derby isolates have diversity sources.The same batch of slaughterhouses in different links have the same CRISPRS type,indicating that the same Salmonella Derby strain are transmitted propagate in slaughter line.Therefore,each link should be performed the hygienic measures to prevent cross contaminant.Through cluster analysis of Salmonella Derby in 9 regions,the results showed that 60 strains of Salmonella Derby were identified 11 CRISPRS types,with DCT24 and DCT47 as the dominant CRISPRS types.The difference of spacers' number among the 11 CRISPRS types were 1 to 22 spacers.The result showed that CRISPRS typing method can well distinguish to Salmonella Derby isolates.According to the distribution of spacers,it is more intuitive to see the differences of the strains,this method has wide application value in bacteria subtyping in molecular epidemiology and to analyze their evolutional characteristic.2.Biological characteristics and whole genome sequencing analysis of Salmonella Derby strains R10 and T12According to the CRISRPS typing results the human isolate R10 from L1 lineage and the pork source strain T12 from L2 were studied on the biological characteristics.Biochemical identification results showed that ILATK was negative in R10 strain,but T12 strain was positive.R10 strain showed resistance to tetracycline(TET),trimethoprim-sulfamethoxazole(SXT),streptomycin(STR)and chloramphenicol(CHL),and the T12 strain was sensitive to all these antibiotics.The adhesion capacity of R10 strain to mouse macrophage RAW264.7 was significantly lower than that of T12 strain(P<0.05),and the invasion ability of R10 strain was extremely significant lower than that of T12 strain(P<0.01).The adhesion and invasion ability of R10 strain to porcine jejunum epithelial cells IPEC-J2 was extremely significant lower than that of T12 strain(P<0.01).BALB/c mice were used as experimental animals to determine the LD50 of the two strains.The LD50 of R10 strain was 1.17×107CFU,the T12 strain was 2.64×107 CFU,and there was no significant difference between them.Biological characteristics results showed that the two strains had great difference,it need to conduct whole genome sequencing and analyze genome characteristics.The genome of R10 strain consists of 4881181 bp,which was a circular structure and has one plasmid of 2834 bp.The content of GC was about 52%and the coding region GC content was 53.7%.Annotation result of the R10 genome showed that there are 4678 protein coding genes and the coding region accounted for 86.9%,107 structural RNA genes.The genome site of T12 strain is 4,920,869 bp,which the GC content was 52.1%and the coding region GC content was 53.2%.The results of genome annotation showed that there are 4755 protein coding genes and coding region accounted for 87.7%,106 structural RNA genes.There is a new virulence island 23(SPI-23),which has 36.4 Kb in the genome of Salmonella Derby R10 and 36 coding genes.However,only one relative conservative gene(docB)was found in the T12 strain.Based on comparison of resistance genes in their genomes,it was shown that R10 strains has 7 more resistance genes than T12 strains,including aadA2,floR,FosA2,dfrAl,tetG,2 sull genes.They are resistant genes againts streptomycin,chloramphenicol,fosfomycin,sulfa Antibiotic synergist,tetracycline and trimethoprim-sulfamethoxazole respectively.There results are coincided with resistant pattern of R10 and T12 strains.In the genome synteny analysis,there are four LCBs(locally colinear blocks,LCB),the inverted phenomenon existed between the genomes,and some insertions or deletions.It suggested that there are big difference between the two genome structures.The site of inversion area is 928 Kb.By comparing the nucleotide acid differences between the two genomes,it was found that there are more than forty thousand differences.They may come from different ancestors.The completion of the whole genome sequencing of Salmonella Derby laid the theoretical foundation for the further research and analysis of its biological function and genetic evolution.