The Biological Characterization Of Enterotoxin A-producing Staphylococcus Aureus From Different Origins And Whole Gemone Analysis

Staphylococcus aureus is an important foodborne pathogen due to its ability to produce staphylococcal enterotoxins(SEs),and therefore cause staphylococcal food poisoning(SFP).Staphylococcal enterotoxin A(SEA)is considered to be the most critical enterotoxin involved in SFP,and about 80% of SFP outbreaks are associated with SEA.After treated with high temperature,pepsin and trypsin digestion,the ability to cause emesis and vomiting by SEA would still exist.Therefore,the usual cooking and processing methods cannot completely inactivated SEA activity and bring possibility to cause SFP.In recent years,SFP related to ST6 S.aureus has been widely found in many Asian countries.Meanwhile,expression of SEA and Staphylococal Protein A gene spa-t701 were two of the most notable features of ST6 S.aureus isolated from SFP outbreaks in China.In addition,ST6-t701 strains were also detected in healthy people and food.Therefore,in this study,we characterization S.aureus isolated from hospital-related human and environment,food and processing chain,animal and food poisoning outbreaks from different geographical areas in China and pick out SEA-producing S.aureus.We analyzed the phenotypic and genotypic characteristics,and revealed the influence factors of enterotoxin expression and biofilm formation of SEA-producing S.aureus.The genomic information of food poisoning ST6-t701 strain were explored by whole genome sequencing(WGS),in order to reveal the genetic secret of food poisoning causing-S.aureus in China.The main results were shown as following:(1)The antimicrobial resistance of different sources S.aureus was not the same,the antimicrobial resistance of SFP strains was relatively low and human source S.aureus of antimicrobial resistance was significantly higher than that of SFP and foodborne strains.Hemolysis toxin genes hla,hld and luk ED were predominant in S.aureus.Compared with human and foodborne strains,SFP strain harbored the highest average number of toxin genes,and the rate of carrying more than 8 kinds of toxin genes(59.3%)was significantly higher;Moreover,the proportion of carrying sea and see genes was significantly higher than human and foodborne strains.There were 9 predominant spa types of 435 S.aureus,and t091 and t701 were the most frequently types.A total of 89.7% S.aureus were detected expression hemolysis,of which ?-toxin and ??-toxin were present 38.2% and 27.6% respectively.Compared with foodborne S.aureus,SFP and human isolates had more spa type overlap which indicated more contact between them.(2)Through screening the sea gene positive strains from more than 10913 samples and then validating by ELISA,a total of 108 SEA-producing S.aureus were picked out from food,SFP and human and/or related environment samples.No SEA-producing S.aureus were found in animal samples.The ST1(t127,t321),ST5(t002,t311,t570)and ST6(t304,t701)were the main sequence types and spa types of SEA-producing S.aureus.ST6 was the major sequence type of SFP and food sources isolates.ST239 and ST6 were the main type of human source isolates.SEA-producing S.aureus usually harbored many kinds of toxin genes,and the most frequent enterotoxin genes was see(98.1%),hemolysin genes were hld(95.1%)and hla(94.4%),leukotoxin gene was luk PV(80.6%).The predominant toxin genes profile in SEA-producing S.aureus was sea-see-hla-hlb-hld-hlg-hlgv-luk ED-luk PV(18/108,16.7%),which was also the main profile of SFP source SEA-producing S.aureus(33.3%).A total of16 clusters were identified by PFGE.One of the major clusters contains 97.4% of all ST6 strains,indicating that ST6 type strain had similar genetic background and high conserved.A total of 85.2% of SEA-producing S.aureus were resist at least one kinds of antimicrobial,and15.8%,21.5% and 71.5% of SFP,food and human were multi-drug resistance(?3)respectively.The ratio of human source strain resisted ?4 kinds of antimicrobials resistance was significantly higher than that of food and SFP SEA-producing S.aureus.A total of 15.7%MRSA-SEA-producing S.aureus strains were defined,all of which were isolated from human samples,and the main molecular type was ST239-t037.(3)SEA-producing S.aureus often expression more than one kinds of enterotoxins and SEA-SED-SEE(83.3%)was the main classical enterotoxins profile.Compared by ST types,the SEA expression of ST6 was significantly higher than ST1 and ST5.The expression of enterotoxin could be effected by nutrients.Na Cl concentration > or <1%,and low concentration of glucose(?0.25%)could inhibition enterotoxin expression.Acidic and alkaline(especially acidic)conditions and low temperature(?10?)could inhibition enterotoxin expression.(4)The autoinducing peptides(AIPs)were detected by HPLC-MS / MS and 12 h were chosen as the optimal detection time for the four AIPs.Results showed that closely connection among agr-type,AIP type and ST-types.Strain confirmed agr? also detected AIP-? and sequence type icluding ST6,ST7,ST239,ST398 and ST59;Strain confirmed agr? also detected AIP-? and main sequence type was ST5.Strain confirmed agr? also detected AIP-? and sequence type was ST1.However,no AIP-? was detected in SEA-producing S.aureus.We found AIP-? strains in non-SEA-producing S.aureus and spa type were all t2091.Results also showed that the high AIP-? detection also had high SEA expression and low AIP-? and AIP-? detection in ST5 and ST1 also confirmed had low SEA expression.(5)The biofilm formation ability of SEA-producing S.aureus was affected by various nutrient components.The 6%-9% concentration of Na Cl significantly increased the biofilm formation of SEA-producing S.aureus.Moreover,filamentous extracellular secretion was observed around the strain under field emission scanning electron microscope.Glucose and sucrose inhibited the biofilm formation;the concentration below 5.6% of maltose and lactose promoted the biofilm formation,while significantly inhibited by concentration increasing.Temperature and pH were the major factors of effecting the biofilm formation of SEA-producing S.aureus.When temperature was set below 10 ?,the biofilm formation ability of was weak.There was no significant difference in biofilm formation at 28 ?,37 ?and 42 ?.(6)One SFP outbreak,ST6-t701 type strain,named Wang1 was analyzed by WGS.Compared to reference strains,Wang1 carried a relatively less kinds of virulence genes.Through the alignment of the main virulence gene sequences,the conservatism of Wang1strain's toxin gene sequence is well and sea gene belongs to sea1,which showed high expression ability.With the nucleotides of luk M and hlb vary widely and the amino acid sequence of luk M changes,which might causing virulence changes.