Antibiotic Resistance And Biofilm Formation Of Staphylococcus Aureus After Cold Stress

Staphylococcus aureus has become one of the most common bacteria causing food poisoning and has a high possibility of contaminating food.In addition,S.aureus is highly resistant to cold and is often found in frozen foods such as meat products,ice cream,dumplings and dumplings.Under external stress(such as antibiotic stress),in order to increase the survival opportunity,bacteria may have colony morphological variation and cross resistance.Biofilm formation plays an important role in bacterial survival and tolerance to environmental stress.The formation of biofilm,the increase of antibiotic resistance and virulence will aggravate the risk of food pollution and food safety hazards.However,there are few studies on the drug resistance and biofilm formation of S.aureus after cold stress.Therefore we comprehensively tested the properties of S.aureus after cold stress on the antibiotic resistance,biofilm formation and toxicity.DNA sequencing,quantitative PCR,transmission electron microscopy and confocal laser scanning microscope were used to detect and analyze the research.Meanwhile,enzymes,drug loaded liposomes and natural essential oil liposomes were developed to remove the biofilm,so as to provide theoretical basis and application solutions for food safety.1.The properties of S.aureus after cold stress formed small colony morphological variation.First of all,the small colony morphology variant(SCV)of S.aureus CICC10201under cold stress-20℃was identified by gene sequencing as menadione auxotrophic types,with thicker cell well,reduced membrane potential,increased resistance to aminoglycosides,ampicillin,cefotetan,doxycycline and chloromycetin.Secondly,the effects of food environmental conditions(temperature,p H,glucose or salt)on the growth of SCV were studied.When the temperature was lower than 30℃,p H<6.0 or p H>9,and glucose concentration was higher than 2%or lower than 0.1%,the growth of SCV cells was significantly inhibited.And when the Na Cl concentration is in the 0%-2%range,and the effect on SCV growth is not obvious.The absence of menadione interrupts the current in the electron transport chain and reduces the intake of aminoglycosides.Furthermore,SCV could revert to a normal phenotype through subculture.And the reversion mutation(R)cells increased expression of virulence factors.The findings in this paper reminded us to strictly monitor S.aureus in low temperature environment to avoid the formation of SCV and cause cross-contamination during food processing and production.2.Properties of drug resistance of S.aureus after cold stress.Firstly,the minimal inhibitory concentration(MIC)of quinolones and aminoglycosides to 8 strains of S.aureus(CICC10201,C4,CICC10788,CICC21600,CICC22942,W1,W3 and C1)was detected after cold stress for 4-24 weeks at 4℃or-20℃.It was found that,the resistance of S.aureus CICC10201 cells to quinolones and aminoglycosides increased significantly after cold stress for 4 weeks at 4℃or-20℃.Simlarly,the resistance of S.aureus CICC22942 strain after cold stress to quinolones increased.And the resistance of S.aureus C1 and W1 cells to aminoglycosides increased after cold stress for 8 weeks at 4℃.The drug resistance of 8strains of bacteria in other treatment groups decreased or the difference was not obvious.Secondly,the changes of antibiotic resistance were analyzed by detecting the target-site of antibiotic,gene expression level of efflux pump or modifying enzyme and cell membrane potential.After cold stress for 4 weeks,the efflux pump gene nor A of S.aureus CICC10201was over-expressed>50-fold.And the target-site Y410F mutation of topoisomerase IV subunit-Glr A reduced the binding efficiency of quinolones.In addition,reasons for the MIC increase in aminoglycoside were the over-expression of the lmr S and aph A3 genes,and an increase in cell membrane permeability.Moreover,the virulence genes(fnbp A,hla,fuh D or sst D)of S.aureus CICC10201 and W3 were overexpressed after cold stress.3.Properties of biofilm formation in S.aureus after cold stress.Firstly,the biofilm formation of 8 strains(S.aureus CICC21600,CICC22942,W1,W3,C1,C4,CICC10210and CICC10788)after cold stress for 4-24 weeks was detected by crystal violet staining.It was found that the biofilm formation of S.aureus CICC10210 and C4 cells decreased with the prolongation of cold stress,and that of S.aureus CICC21600,W1 or W3 increased after cold stress for 8-20 weeks at-20℃.Similarly,the biofilm formation of S.aureus CICC22942 and C1 enhanced after cold stress for 12-20 weeks at-20℃.And the difference of S.aureus CICC10788 was not obvious.Then the growth of biofilm bacteria,the m RNA expression levels of related genes and the components of biofilm matrix were detected.The biofilm formation of S.aureus CICC21600,CICC22942,W1,W3 and C1were significantly increased after cold stress of 20w at-20℃.Corresponding to the result that the cell accumulation of biofilm bacteria of S.aureus CICC22942,W1,W3 or C1 was higher than that of strains without cold stress.The RT-PCR detection showed that the m RNA levels of ica A,ica B,ica C or ica D gene in these S.aureus CICC21600,CICC22942,W1,W3 or C1 cells were overexpressed,which was consistent with the increase of extracellular polysaccharide content measured by sulfuric acid phenol method.What’s more,the BCA kit was used to detect the content of protein in the biofilm matrix.The content of protein in S.aureus W1 or CICC22942 increased significantly,that of S.aureus CICC10201 or C4 cells was significantly reduced.It was consistent with the change of biofilm formation of the four strains,further indicating that adhesion protein promoted biofilm formation.In addition,the hydrophobicity of cells in S.aureus CICC21600,W1and W3 biofilm increased significantly after cold stress,which promoted the formation of their biofilms.The biofilm matrix is complex,and the increase of polysaccharide content is one of the main reasons for the improvement of biofilm formation.4.Remove of S.aureus biofilm.First of all,the biofilm was treated with 0.125mg/ml snelase for 1h,and the removal efficiency was higher than 50%.The biofilm of S.aureus strains were treated with snailase and NOR,snailase and proteinase K,or proteinase K and NOR respectively,and the clearance rate was higher than 80%.In addition,lipid nanoparticle was prepared by encapsulation of erythromycin with phospholipids,0.8%Surfactin and 0.2%sodium tripolyphosphate(TPP).The size of the lipid nanoparticle was about 52 nm and the potential was-67 m V which measured by Marvin laser particle size analyzer.The encapsulation rate of erythromycin was about 65%and the actual content of erythromycin was 31.56μg/m L.What’s more,the lipid nanoparticle could significantly remove the biofilms of S.aureus W1,W3,CICC22942 and CICC10788 on polyethylene,and significantly remove the biofilms of S.aureus C1,W3,CICC10788 and C4 on stainless steel,with redcuing the total viable count of bacteria in biofilms by 10~3 CFU/m L.Moreover,lipid nanoparticle integrated the effects of Surfactin,erythromycin and TPP,which could obviously remove polysaccharides and protein components in the biofilm matrix.In addition,results of laser confocal microscopy showed that lipid nanoparticle could effectively kill residual bacteria in the biofilm and avoid repeated contamination.Last but not least,the origanum vulgare essential oil nanoparticle was prepared.The nanoparticle size was 192 nm and potential was about-21.8 m V.And the S.aureus biofilm bacteria on cabbage could be effectively removed by the origanum vulgare essential oil nanoparticle.These methods of removal of S.aureus biofilm could be applied to food industry,medicine or other fields.