| Salmonella not only causes huge economic losses to the livestock and poultry industry,but also poses a serious threat to human health.The drug resistance was analyzed from the phenotype and genotype level within different Salmonella strains that were obtained from 2012 to 2017 in Jiangsu Province.The molecular characteristics(drug resistance genes,virulence factors,metal resistance genes,and mobile genetic elements,etc.)of S.Indiana were analyzed based on genome sequencing and the biological information analysis platform.The relationship between the class 1 integrons and the multi-drug resistance was analyzed to explore the mechanism of multi-drug.resistance(MDR)formation in S.Indiana.In addition,through comparing with other serotypes ofSalmonella,the pathogenicity of S.Indiana to mice was systematically studied in vivo and in vitro.1.Typing and drug resistance analysis of Salmonella from different sourcesIn this study,254 Salmonella strains isolated from different sources was analyzed,including animal sources(pig and chicken),healthy human beings and-patients with foodborne diseases from 2012 to 2017 in Jiangsu Province.Their differences in serotype,drug resistance genes,multilocus sequence typing(MLST),cluster analysis,and class 1 integrons were investigated to find the epidemic strains and spreading rules.After serotyping the 254 Salmonella strains,they were mainly divided into 9 serotypes.The isolation rate of S.Enteritidis(37.01%,94/254)was highest,followed by S.Indiana(18.90%,48/254)and S.Derby(14.17%,36/254).S.Enteritidis(38.38%71/185)and S.Indiana(25.41%,47/185)were the most isolated strains of Salmonella from animal sources.Salmonella from chickens were mainly S.Enteritidis(53.79%,71/132)and S.Indiana(35.61%,47/132),and S.Derby(52.83%,28/53)was mainly isolated from pigs.Salmonella of human origin were mainly S.Enteritidis(33.33%,23/69)and S.Typhimurium(27.54%,19/69).Only one S.Indiana strain was isolated from a foodborne disease patient.The 254 strains of Salmonella were divided into 19 STs using MLST method,among which ST 11 account for the highest proportion,followed by ST17,ST40,and ST19.In addition,all S.Indiana belonged to ST17.The cluster analysis of STs and Neighbor-Joining(NJ)tree showed that 254 Salmonella strains were divided into 5 main clonal complexes,CC11,CC17,CC19,CC40,and CC68.The results of antimicrobial susceptibility test showed that the most severely resistant serotype was S.Indiana,followed by S.Enteritidis.Most S.Indiana strains were MDR strains.Most of drug resistance genes could be detected in S.Indiana,especially aminoglycoside resistance genes,followed by chloramphenicol,β-lactam and trimethoprim amine resistance genes.Most S.Indiana strains contained a gene cluster comprising blaCTXM(and/or blaOXA-1-like together with blaTEM-1-like),sull,aacC4,aac(6’)-Ib,floR,and dfA17.The results of class 1 integrons analysis showed that 22.83%(58/254)of Salmonella strains were positive.The highest detection rate of class 1 integrons was 64.58%(31/48)in S.Indiana,followed by S.Thompson,44.44%(4/9).Taken together,S.Indiana has become the second highest isolation rate Salmonella from chickens,most of which were MDR strains,and most of S.Indiana carried class 1 integrons and a large number of resistance genes.2.Mechanism of multi-drug resistance formation of S.IndianaTo explore and verify the mechanism of its multi-drug resistance,the complete nucleotide sequences of six isolates of S.Indiana were determined to analyze their drug resistance genes,metal resistance genes,virulence factors,and mobile genetic elements(MGEs),etc..The synteny analysis and evolutionary analysis based on chromosomes and plasmids were performed.The results showed that most S.Indiana carried 11-22 drug resistance genes,and its large plasmid carried a huge number of MGEs and drug resistance genes.The large plasmid in S.Indiana 222 strain had a size of 308.6 kb and carried 22 drug resistance genes in.8 classes,which is the plasmid with the most drug resistance genes known so far.Most S.Indiana carried about 10 different transposons,and some strains even carried a large number of insertion sequences.Most Salmonella carried 8-10 genes encoding tellurium resistance proteins.In addition,some S.Indiana also carried mercury,copper,and silver resistance genes,and these metal resistance genes were mostly distributed in two sides of integrons in the plasmid.We speculated that these metal resistance genes may be related to the MDR of S.Indiana.Synteny analysis showed that the synteny of the plasmids was weak,and there were a large number of gene rearrangements,inversions,and deletions of fragments,which indicated that the plasmids of Salmonella strains had undergone frequent recombination to obtain the resistance genes.A novel integrase gene which was only 699 bp was detected for the first time based on wholegenome sequencing in S.Indiana.Compared with intI1,it has 67.06%nucleotide identity,and we named it intI1-15.In vitro integration system was constructed to compare the integration functions of these two integrases.It was found that the integration frequency of intI1-15 integrase was significantly lower than that of intI1.However,carrying two integrases broadened the way to obtain resistance genes for S.Indiana,which promoted the formation of MDR in S.Indiana.The correlation between the resistance genes and class 1 integrons of showed that the resistance genes in S.Indiana were strongly correlated with class 1 integrons,while other serotypes were weakly correlated.Plasmid conjugative transfer experiments showed that the frequency of recipient strains with integrons was significantly higher than that of without integrons.In summary,S.Indiana had a plasmid that carried the most drug resistance genes so far.At the same time,there were two integrases in S.Indiana,which were the main reasons for the formation of MDR.3.Systematic evaluation of the pathogenicity of S.IndianaAbove data showed that S.Indiana carried a large number of drug-resistant gene s,and there was a potential risk of spread to humans.In this study,we evaluated the pathogenicity of S.Indiana compared with other serotypes of Salmonella using in vivo and animal and-cellular experimental model systems.The clinical symptoms,pathological changes,and median lethal dose(LD50)in mice were investagted.The results showed that S.Indiana,S.Enteritidis,S.Typhimurium,and S.Thompson caused histopathological changes in most organs to varying degrees,primarily in the liver and intestine of mice.The gross lesions included white necrotic foci on the liver surface with different levels.The histopathological changes of monocyte/macrophage infiltration and coagulative necrosis were observed in the liver.Intestinal villi became short and was sloughed off,and lymphocyte infiltration was found in the submucosa.Compared with the other serotypes,S.Indiana induced a slighter pathological changes.The LD50 of S.Indianawas above 105 CFU/mL,which was several times or even-dozens of times the LD50 of S.Enteritidis and S.Typhimurium to mice.The adhesion of Salmonella in vitro was measured with the primary mouse intestinal epithelial cells(IECs)by laser confocal microscope.The adhesion,invasiveness and intracellular proliferative capacity of Salmonella in vitro were measured with the primary mouse intestinal epithelial cells(IECs),and murine macrophagelike cell line-RAW264.7 cells and human colon adenocarcinoma cell line Caco-2 cells by CFU counting.Compared with the other three serotypes,most S.Indiana strains exhibited significantly lower adhesion-and invasiveness.The results of adhesion and invasion tests showed that the intracellular growth trend of most Salmonella strains was positively correlated with the number of pathogens adhering to and invading cells.In particular,the number of S.Indiana-222 isolates adhered and invaded into the IECs and Caco-2 cells were the least among the 11 Salmonella isolates,but its intracellular proliferation rate was the fastest,which is worthy of our further in-depth study.In conclusion,S.Indiana could cause pathological ch anges in the liver and intestines,but its pathogenicity was lower than that of S.Typhimurium and S.Enteritidis.In summary,the isolation rate of S.Indiana was second only to that of S.Enteritidis,and it was a MDR strain mainly from chickens,but it can also be occasional isolated in thehuman being.S.Indiana had the plasmid that carried the most drug-resistance genes at present.Moreover,S.Indiana carried a-newly discovered integrase,which accelerated the formation of its MDR.Compared with other serotypes of Salmonella,S.Indiana was less pathogenic,but still had the potential risk to humans.Therefore,it was necessary to strengthen the surveillance and control of S.Indiana. |
