| Among the numerous causes for foodborne diseases,Salmonella is composited by a group of gram-negative bacteria belonging to Enterobacteriaceae family.According to previous report,there are more than 30 million cases of foodborne infections in China every year,of which about 75%are caused by Salmonella(up to 20 million cases).There are,in general,two types of Salmonella(Typhoidal and Nontyphoidal Salmonella).Nontyphoidal Salmonella is one of leading four causes of diarrhoeal diseases globally,and most cases are mild with particular isolates or genotypes being life-threatening.Historically,Salmonella Goldcoast(SG),belonging to serogroup C2-C3,was a rarely detected serovar.During past two decades,SG infections have dramatically increased around the world,and resulted in several nation-wide and international outbreaks in Europe,US and Taiwan.However,there is still no complete monitoring data in mainland China.According to Hungarian reports in 2009,twelve antibiotics,including chloramphenicol,gentamicin and ciprofloxacin,had certain inhibitory effect on SG isolates,indicating that antibiotics could effectively kill or inhibit SG-induced infection.However,the sensitivity of SG strain to antibiotics in mainland China is unclear.And in recent years,SG strain resistance has been increasing due to over-reliance on antibiotic treatment and unreasonable drug use.Italy,Spain and other places have reported the detection of multidrug-resistant SG.In the face of SG strain with continuous expansion and multi-drug resistance trend,it is very important to explore the genetic evolution relationship and related drug resistance mechanism of SG strain,but there is no relevant systematic research on this.In addition,the biological characteristics of SG isolate,such as the capacilities for biofilm form,swarming and virulence,remain unclear.The main purpose of this study is as follows:(1)Based on the global SG epidemic data,in further explore the prevalence,distribution and drug resistance of SG in livestocks,food and human in China.(2)Based on whole genome sequencing,to determine the global SG sequence type(STs),antibiotics resistance gene(ARGs),virulence factor(VFs),plasmid type,and to understand the global genetic diversity,evolutionary origins and biological characteristics of SG.(3)Further explore the genomic structure characteristics and related drug resistance mechanism of SG isolates from pork in Chinese mainland.1.Epidemiological and antimicrobial resistance analysis of SG isolates in ChinaTo project the epidemiological trend for Salmonella in mainland China,all the Salmonella isolates between 2010 and 2019 were obtained from the Chinese Local Surveillance System for Salmonella(CLSSS).All these isolates were subjected to serological agglutination analysis according to the established Kauffmann-and-White classification scheme.Additional 133 SG isolates were recruited from public domains(Gen Bank,Enterobase,Sequence Read Archive)with the meta-data,of which 65 SG strains were from China.By comparison with the most recent two-time scale(2010-2014 vs 2015-2019),we found only 0.6-fold increase in total Salmonella samplings in the most recent period.In contrast with serovar Goldcoast,a 3.5-fold increase was observed in the most recent period(2015-2019),and mainly concentrated in the southeast coastal areas such as Shanghai,Guangdong and Taiwan.According to the information collected from the original samples of the strain,SG was determined to be from(1)human(blood,urine,saliva,feces).(2)Livestock,poultry and their metabolites and animal products(pigs,poultry,eggs,meat,etc.)(3)Environment(mainly water).For the 169 Chinese isolates,the minimum inhibitory concentration(MIC)of 14clinically relevant antimicrobials was conducted by the broth micro dilution assay according to the criteria recommended by the Clinical and Laboratory Standards Institute 2016.On the whole,SG strain was generally resistant to ciprofloxacin,ampicillin and streptomycin under 37?aerobic Environment,while naproxic acid,azithromycin,imipenem had certain inhibitory effect on SG strain.Analysis of antibiotic resistance of heterologous strains showed that the most effective antibacterial agents of human and environmental SG strains were azithromycin and imipenem,and cefotifurme had better antibacterial effect against animal strains.With the use of antibiotics,the multi-drug resistance trend of SG strain was further deterioration from2015 to 2019.Finally,most of the strains showed a drug-resistant phenotype.2.Genetic diversity,evolutionary relationship and biological characteristics of SGGenomic DNA of 169 SG isolates was extracted using the DNA Kit and quantified.Whole genome sequencing was performed for 146 strains at the Beijing Genomics institution.The remaining 23 strains were sequenced at Beijing Novogene.The whole genomic sequencing data of additional 133 SG isolates were recruited from the public domains.All the newly produced raw reads(n=302)were quality checked and assembled by using the in-house Galaxy Platform.In silico serovar prediction was analyzed with two different methods,including SISTR and Seq Sero2,which confirmed all the examined isolates as serovar Goldcoast.Sequence types were extracted from the assembled genomes using the seven housekeeping genes by MLST.The assembled contigs were submitted to the Galaxy server to scan the acquired AR determinants,coding virulence factors and the plasmid types by using the CGE Res Finder vs 3.1,VFDB,and CGE Plasmid Finder vs 2.0.To track of the host origin of SG,a maximum likelihood phylogenetic tree was built based on 70,574 SNPs,the complete genome sequence of NCTC13175 was used as a reference and SAL?GA9399AA strain as the outgroup.All the results showed that the population of SG strains was divided into two branches as a whole,which could be further subdivided into four Lineages according to the host geographical information,and Europe was the origin of each lineage.The main genotypes of SG strains are ST358 and ST2529,and there are obvious differences in ARGs,virulence factors and plasmid types among different lineages.In addition,the representative SG strains from each lineage were further screened out to explore the related biological functions.The results showed that all lineages of SG had the ability to form biofilm,cause movement and migration,and kill zebrafish.Specifically,we observed that SG has the ability to form biofilm and swarming motility,and most strains(27/29,93%)had a higher biofilm formation capacity in the bacterial solution at 37?than 28?,and the swarming motility capacity in the anaerobic Environment was stronger than that in the aerobic Environment.SG with a concentration of 105 CFU/ml was microinjected into zebrafish embryos,after 14 days of continuous culture and observed.The results showed that SG had strong killing ability to zebrafish.3.Genome structure characteristics and comparative genomics analysis of SG strain from pork in Chinese mainlandIn this study,the complete genome of SG strain Sal02792 from pork in Chinese mainland was determinated based on the third-generation sequencing technology Pac Bio.The result showed that the chromosome size of SG strain Sal02792 was4,677,190 bp,and the GC content was 52.23%,which was highly similar to SG strains NCTC13175 and SSDFZ69,but different from Sal5364.In addition,Sal02792 strain has a Inc HI2/Inc HI2A plasmid with a size of 237,330 bp and GC content of 46.96%.13 resistance genes,including oqx A,oqx B,qnr S1 and bla TEM-1B,are located on this plasmid.At last,conjugation transfer assay showed that most resistance genes in Sal02792 strain could be transferred to recipient bacteria with plasmid,which can lead to the horizenal gene transfer.This study confirmed that the acquisition of MDR phenotype of SG strain is dued to conjugated plasmid.To sum up,this study suggested a significant expansion and multi-drug resistance accumulation of SG in humans in Chinese coastal regions during 2015-2019,imipenem is an effective antimicrobial drug.Secondly,biological experiments show that SG strain has the ability to form biofilm on 96-well plate,swarming motility on semi-solid LB medium and strong lethal ability to zebrafish embryos.Third,based on the genetic evolution analysis of whole genome sequencing,this study defines SG strains as four lineages.The BEAST analysis suggested SG isolates circulation in animal(L-I)and human(L-II,L-III,and L-IV)were separated by an independent evolution history,but with the same possible origin from European countries.The sequence types,antibiotic resistance genes,virulence factors and plasmid types of SG strains are obviously different among lineages.Finally,the analysis of complete genome of SG strain from pork in Chinese mainland confirmed that the chromosome size of Sal02792 is4,677,190 bp,and the Inc HI2/Inc HI2A plasmid carries 13 drug-resistant genes,which is considered to be one of the major reasons for the development of MDR. |
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