Analysis Of S.aureus AIPs Using LC-MS/MS And The Effect Of AIPs On SEA And Bioflim Formation

Staphylococcus aureus(S.aureus)is a major source for bacteria-caused food poisoning,in which 80% of food poisoning was caused by S.aureus enterotoxin A(SEA).ST6、ST5 and ST1 are common bacterial strains in food poisoning and food contamination.At the same time,S.aureus is also one of the main pathogens to produce biofilm,which can protect strains escaping the obliterate effects from host as well as enhance the antibiotic resistance.Recently,scientists have discovered that quorum sensing(QS)plays a key role in physiological activities such as generation of SEA and biofilm.The signal molecules for S.aureus are oligopeptides(AIPs).For the reasons above,it is of great importance to understand the relationship between AIPs,SEA and biofilm.In this study,we first established a chromatography-mass spectrometry(LC-MS/MS)method for qualitative and quantitative detection of AIPs.And then we used vitro plate assay and ELISA to test the formation of biofilm and SEA for ST6、ST5 and ST1 strains,respectively.This study aims to confirm the effects of environment on AIPs and to test the correlation between SEA and biofilm generation.The key discoveries are as follows:(1)An effective detection method was developed using LC-MS/MS.We have found the optimal condition by conducting ion detection in MRM mode within 0.1% formic acid water solution and 0.1% formic acid acetonitrile solution of mobile phase.Under such condition,we were able to quantify the AIPs from the S.aureus cultures directly,with a limit of detection(LOD)range of 0.001~0.006 mg/L and a linear dynamic range of 0.1~4.0 mg/L.The recycle rate is between 65.7% and 122.3%.(2)We have discovered that there is a statistically significant correlation between ST-type of S.aureus and the generation of AIPs.However,most strains only generate one type of AIPs by one ST-type S.aureus in our study.We have discovered that ST6,ST7,ST239,ST398 and ST59 strains will generate AIP-I,ST5 strains will generate AIP-II and ST1 strains will generate AIP-III.The type of AIPs is only determined by the strain and independent of the culture time,the yield of AIPs decreases with increase of culture time.(3)We have discovered that NaCl concentration had a highly significant effect on AIPs produced by ST6 strains(P<0.01),had a significant effect on AIPs produced by ST5 strains(P<0.05)but had no obvious effect on AIPs produced by ST1 strains;glucose and sucrose concentrations had no obvious effect on AIPs produced by either of the three types of S.aureus strains.Moreover,AIPs produced by all three types of strains have shown a highly significant correlation(P<0.01)on pH value and temperature.(4)ST6 strains were the high-yielding clones of AIPs in NaCl,glucose and sucrose conditions.Under the different concentration of NaCl,glucose and sucrose,three types of strains were showed the same change trend,namely as with the concentration of NaCl,glucose and sucrose increased,the output of AIPs reduced.When the pH value of nutrient solution from acidic to neutral,AIPs was increasing,but with the continue increasing,the amount of AIPs reduce.The quantity of three ST-types increased with the increase of temperature.(5)The same change trend between AIPs and SEA generation has been discovered for all three ST-types of S.aureus under different culture conditions.Except that AIPs yield from ST1 strains has a negative correlations with biofilm formation under NaCl environment,we have found no other obvious correlation under other culture conditions.