Properties And Biofilm Control Of Cold Induced Staphylococcus Aureus

Staphylococcus aureus is a common foodborne pathogenic bacteria,which has been seriously polluted in frozen food in recent years.The pathogenic Staphylococcus aureus can produce enterotoxin.Once the frozen food is polluted,it will be able to multiply and produce toxin at proper temperature after thawing,thus causing food poisoning.The detection of Staphylococcus aureus in many frozen foods in China these years has caused serious economic losses and food safety accidents.Staphylococcus aureus has strong tolerance to low temperature.Thus,study the change of staphylococcus aureus after induced at low temperature can provides a theoretical basis for the control of Staphylococcus aureus in frozen foods.In this paper,several strains of Staphylococcus aureus were isolated from frozen food,and their ability of regrowth and freezing tolerance were evaluated.Then we selected a strain with the strongest low temperature tolerance and studied its adaptation to other environment after cold induction.Meanwhile,prolonged cold induction could induce Staphylococcus aureus to form small colony variants(SCVs).Compared with the wild type,the growth morphology,biofilm formation and resistance to antibiotics of SC Vs were changed.4 kinds of common fungicides were selected to remove the biofilm produced by SCVs,providing theoretical methods and guidance for controlling this kind of SCVs.The main conclusions were drawn as follows:1.Isolation of Staphylococcus aureus from frozen food and determination of their freezing tolerance.From the supermarket and the farm product market,36 frozen foods were collected to detect Staphylococcus aureus,of which the frozen meat products had a high pollution rate and the total physical examination rate was 16.7%.Isolation of Staphylococcus aureus from frozen food samples by traditional culture and PCR detection,a total of 7 strains of Staphylococcus aureus were obtained,named W1,W2,W3,J1,J3,J4,J5.Prolonged cold stress was carried out for the isolated Staphylococcus aureus,and its survival rate was measured.The results showed that W3 had the strongest tolerance to-20?,and the bacteria number was hardly changed after 8w cold stress.J3 had the strongest tolerance to 4?.After 8 weeks of cold storage,the number of mycelium decreased only 0.761g(cfu/ml).However,the tolerance of standard strains to-20? was worse than that of all isolated strains.After 8 weeks,the number of mycelium decreased 2.271g(cfu/ml).In addition,the tolerance of the isolated strains to freeze thawing cycle was evaluated.The results showed that after 9 freezing and thawing cycles,the number of isolated strains showed an upward trend,and the J1 increased most significantly.With the increase of the number of freeze-thaw cycles,the survival rate of bacteria in each group decreased gradually.Meanwhile the standard strains generally showed a downward trend,showing a stronger sensitivity to the freeze thawing cycle than the isolated strains.2.Cold-induced Staphylococcus aureus tolerance to various environment and control.The cold tolerance strain W3 and the cold sensitive strain CICC10201 were selected,and their tolerance to different temperatures,different concentrations of pH,osmotic pressure,hydrogen peroxide and alcohol were measured at after cold stress at 4?for 5h.The results show that the cold resistant strains was more tolerant to high temperature than cold sensitive strains.After cold induction,the survival rates of the two bacteria were improved at 45-60?.Cold induction increased the sensitivity of Staphylococcus aureus to the acid pH environment but had cross resistance to the alkaline pH environment.The survival rate of the cold induced bacteria in the hypertonic environment decreased,and the lowest decreased by 24.09%.In addition,after cold induction,the sensitivity of the two strains to different disinfectants also changed.After 30%alcohol stress,the number of two strains was increased 1.92 and 2.071g(cfu/ml)compared with that before the cold induction.This showed that compared with its wild type,cold-induced Staphylococcus aureus is more tolerant to alcohol.W3 was no significant change in the tolerance of 35 to 50 mM hydrogen peroxide before and after cold induction,but the sensitivity of the standard strain CICC10201 was increased and the survival rate was decreased.However,standard strain CICC 10201 has increased sensitivity to hydrogen peroxide and decreased survival rate after cold stress.W3 has stronger biofilm-forming ability than the standard strain CICC 10201,and its biofilm can be effectively removed with 1%84 disinfectant.3.Propertiesandcontrol of cold-induced small colony variants ofS.aureus.Staphylococcus aureus was incubated to form small colony variants(SCVs)at-20? for 3 weeks.We measured the growth,auxotrophy,morphology,drug resistance and biofilm formation capacity of this strain.SC Vs growth slower which the delay period was postponed by 2 h.While its cell was smaller than wild-type(WT)cells(p<0.05),the feature of Gram staining and hemolysis were decreased.Cold-induced SCVs were identified as menadione-deficient strains that hardly grew in MHB medium,but grew normally after addition of 0.1?g/ml menadione.Subculturing could lead to SCVs reversion.SCVs had increased tolerance to various antibiotics,particularly aminoglycosides(gentamicin,kanamycin),and had increased sensitivity to tetracycline,chloramphenicol,and ciprofloxacin.Additionally,SCVs showed stronger biofilm-forming ability and expression of biofilm-related genes(icaADBC)was higher than WT.1%'84' disinfection and 3%hydrogen peroxide could effectively eliminated biofilms.