| Cronobacter（formerly Enterobacter sakazakii）is an important food-borne pathogen that can contaminate infant foods,which pose a serious threat to the health of infants and young children.Food contamination by Cronobacter may be related to the formation of biofilm on the food contact surface.The bacteria in the biofilm are not easily removed and widely spread.Therefore,studying of the biofilm-forming mechanism of Cronobacter is of great significance for controlling cross contamination in food production caused by biofilm.In addition,the drug-resistant strains of Cronobacter are repeatedly detected in foods,environmental and clinical infection cases.The spread of drug-resistant strains and the development of drug resistance spectrum have brought a great challenge to clinical anti-infective treatment.So it is particularly important to strengthen the surveillance of drug resistance and drug resistance mechanisms of Cronobacter.In this study,81 strains of Cronobacter isolated from food were molecularly classified by pulsed field gel electrophoresis（PFGE）,and 24 strains of different PFGE bands were selected for the study of biofilm-forming ability.The strains were treated with4℃cold stress and 4℃cold-adapted,and then compared their biofilm-forming ability,surface characteristics and the microstructure of the biofilm.And reversed transcript-quantitative PCR（qRT-PCR）was used to investigate the expression levels of five biofilm-related genes of gsiB,luxS,glpQ,sdiA and deoB,and whole-genome sequencing and analysis using high-throughput sequencing to mine genes associated with biofilm formation.The results showed that 81 strains of Cronobacter formed 71 PFGE type,and the type was more dispersed.24 strains of Cronobacter could form biofilm at 37℃,and the strains ES183,ES303,ES295,ES305 and ES304 had strong biofilm-forming ability.4℃cold stress could promote the formation of biofilm of Cronobacter,and the strains ES055,ES114,ES215 and ES300 were increased significantly.4℃cold-adaptation had little effect on biofilm-forming ability.Cronobacter were electron donors,and the electron donating ability of the strain cultured at 4℃was reduced.The surface hydrophobicity of each strain was significantly different at 37℃,and the strains ES017,ES298 and ES299 were more hydrophobic,the surface hydrophobicity of the strain cultured at 4℃reduced.4℃cold stress and 4℃cold-adapted strains had certain cell adhesion on the surface of stainless steel slides,strains ES017,ES055 and ES114 form micro-colony,ES113 had only a small amount of scattered bacteria.Compared with 37℃positive control strains,the expression levels of gsiB,luxS,glpQ,sdiA and deoB gene of 4℃cold stress strains reduced,except for a few strains,the expression levels of gsiB,luxS,glpQ and sdiA gene of 4℃cold-adapted strains reduced,deoB gene expression raised.33 genes regulating biological adhesion were found in the whole genome of Cronobacter,such as chiA,chiA-2,chiA-2,csgA-1,csgB,eae-1,eae-2,elfA,elfA-1,elfA-2,fimA,fimA-1,fimA-2,hifA,mrpA,papH,papH-1,papH-2,sfaH,smfA,mrkD,sfaS-1,sfaS-2,sfaS-3,sfaS-4,sfaS-5,fliD,prn,elfG,ycbV,nlpE,znuA,ydeH.In addition,this study also studied the drug resistance of Cronobacter.The PhoenixTM-100 automatic bacterial identification and drug susceptibility system was used to test the drug sensitivity of 305 strains of Cronobacter,19 antibiotics included amikacin,gentamicin,amoxicillin-clavulanic,piperacillin-tazobactam,ampicillin-sulbactam,ampicillin,piperacillin,cefazolin,cefepime,cefotaxime,ceftazidime,chloramphenicol,ciprofloxacin,levofloxacin,imipenem,meropenem,aztreonam,tetracycline,and trimethoprim-sulfamethoxazole.The results showed that the strains resistant to cefazolin and tetracycline were 8 strains and 2 strains,respectively;the strains intermediate to cefazolin and chloramphenicol were 79 strains and 8 strains,respectively;and strain ES008 was resistant to tetracycline and intermediate to chloramphenicol,strains ES024,ES029 and ES059 were simultaneously intermediate to cefazolin and chloramphenicol;all strains were sensitive to 16 other antibiotics.On the basis of drug sensitivity test,6cefazolin-resistant strains,2 tetracycline-resistant strains and 2 sensitive strains were subjected by high-throughput sequencing to obtain drug resistance-related factors.The results showed that there were 6 genes associated with cefazolin resistance,ampC,ompC,ompN,ompF,phoE,tolC,and the gene sequence alignment analysis of cefazolin-resistant and sensitive strains revealed that the gene ampC,ompC,ompF,phoE and tolC bases have large differences;and 2 tetracycline resistance genes,tetR and tetA,they code 14 proteins,TetR,TetG,TetY,Tet41,TetE,Tet39,TetJ,TetH,TetA,TetB,Tet30,Tet31,Tet33,TetZ. |
