Study On The Bacterial Biofilm Resistance And The Gene Regulation Mechanism Related To Biofilm Formation

Objective:To study the pathogenic distribution of bacterial biofilm on endotracbeal tube in mechanically ventilated patients, and to discuss the antibiotic resistance of bacterial biofilm by comparing the antimicrobial resistance between the biofilm-forming bacteria and the corresponding planktonic bacteria cultured in vitro. Further to study the staphylococcus epidermidis biofilm isolated from clinic and the corresponding planktonic bacteria to discuss the relationship between the gene distribution concerning to the staphylococcus epidermidis biofilm formation, such as the operon-icaA gene, the transcriptional regulator-sigB gene, operon regulation gene-icaR gene and the virulence factor regulation system-agrA gene, and the phenotype formation of biofilm.methods:(1)The endotracheal tubes removed from 110 patients who were implemented artificial airway (endotracheal intubation and tracheostomy) and also ventilated mechanically were studied. The biofilm-forming bacteria strains and corresponding planktonic bacteria strains were isolated by Congo red medium, and the biofilm bacteria strains were detected in semi-quantitative method. Antibiotic sensitivity testing was used to analyze the difference of drug resistance between the biofilm-forming bacteria strains and the corresponding planktonic bacteria on general MH medium, and the difference of antimicrobial resistance between the biofilm-positive bacteria on Poloxamer medium(Poloxamer, F-127) and that on general MH medium.(2)Altogether isolates of staphylococcus epidermidis were collected from the endotracheal tubes, and were divided into biofilm group and the corresponding planktonic bacteria group. The luminance ratios of icaA,sigB,icaR and agrA of staphylococcus epidermidis in biofilm group and planktonic bacteria group were detected by RT-PCR. And the expression of icaA,sigB,icaR,agrA genes in different groups were detected by Real-time PCR and were relatively quantitated through 2-△△Ct method. Moreover, the relevancies between that four genes and biofilm formation in Staphylococcus epidermidis were analyzed respectively.Results:(1)In 110 mechanically ventilated patient cases,61 biofilm-forming isolates cultured from the endotracheal tubes (55.45%) were selected. The most common isolates included Staphylococcus, Enterococcus faecalis, and Pseudomonas aeruginosa. The antibiotic sensitivity testing results of the biofilm-forming bacteria and that of the corresponding planktonic bacteria on general MH medium were similar. The difference had no statistical significance (P> 0.05). The antibiotic sensitivity testing results of the biofilm-forming bacteria on MH medium and that on Poloxamer medium were different. The statistical difference was significant (P<0.05). Moreover, the biofilm-forming bacteria were more antimicrobial resistant on Poloxamer medium.(2)The luminance ratios of icaA,sigB,icaR and agrA were not significant between the biofilm-forming bacteria and the corresponding planktonic bacteria (P> 0.05),but the mean rank and sum ranks of icaA and sigB in biofilm group were significantly higher than that in planktonic bacteria group, the mean rank and sum ranks of icaR and agrA were significantly smaller than planktonic bacteria group.(3)The expression of icaA and sigB in biofilm group were 418.8 times and 81 times more than the planktonic bacteria group. The expression level of icaR and agrA in biofilm group are 1/145 and 1/270 times less than the planktonic bacteria group.Conclusions:(1)The pathogenic distribution of bacterial biofilm on endotracheal tube in mechanically ventilated patients from our hospital was based on Staphylococcus, Enterococcus faecalis, and Pseudomonas aeruginosa. The difference of antibiotic resistance was not significant with the biofilm-forming bacteria and planktonic bacteria on MH medium, while the difference was significant with the biofilm-forming bacteria on MH medium and on Poloxamer medium. The study indicates that Poloxamer medium might show the real state of the drug resistance of biofilm-forming bacteria, and it has greater resistance.(2) icaA,SigB,icaR,agrA genes are closely related to biofilm formation of staphylococcus epidermidis. icaA,sigB can promote biofilm-forming, and icaR,agrA can inhibit biofilm-forming. Agr system can regulate the expression of icaA,SigB and icaR, that reducing icaA,SigB and increasing icaR.