| Objective:Bacterial biofilm is a growth mode relative to planktonic bacteria,and most of the bacteria in the clinic can form biofilms.This study detects the biofilm forming ability of common clinical strains,and analyzes the correlation between the drug resistance of clinical strains and the biofilm forming ability;This study explores the anti-biofilm activity of the fermentation product of Klebsiella pneumoniae and analyzes its chemical properties,which is a clinical anti-biofilm activity.The research and development of biofilm drugs provides experimental basis.Methods:1.We collected the Staphylococcus aureus,Staphylococcus epidermidis,Enterococcus faecalis,Escherichia coli,Pseudomonas aeruginosa,and Acinetobacter baumannii clinical strains each of 60isolated from Changsha Central Hospital of South China University from October 2019 to August 2020.2.We collected 20 strains of Klebsiella pneumoniae and culture with shaking for 24 hours,filtered and sterilized the supernatant to extract the supernatant,and detected the anti-biofilm activity of the Klebsiella pneumoniae fermentation product by 96-well plate method.The chemical properties of anti-biofilm active substances were analyzed by boiling,enzymatic method,and sodium periodate oxidation method.The extracellular polysaccharide was extracted by ethanol method,and the content of Klebsiella pneumoniae extracellular polysaccharide was determined by concentrated sulfuric acid-phenol method.3.The concentration-dilution method explored the concentration dependence of the anti-biofilm activity of Klebsiella pneumoniae polysaccharides,and the crystal violet staining method was used to detect the broad-spectrum biofilm activity of Klebsiella pneumoniae extracellular polysaccharides.Results:1.Investigation on the biofilm-forming ability of common clinical strains:Among the clinical strains of Escherichia coli,8 strains(13.33%)have strong biofilm-forming ability,21 strains have medium biofilm-forming ability(35.00%),22 strains have weak biofilm-forming ability(36.67%),and 9 strains(15.00%)have no biofilm-forming ability.Among the clinical strains of Pseudomonas aeruginosa,15 strains(25.00%)have strong biofilm-forming ability,29 strains(48.33%)have medium biofilm-forming ability(35.00%),14 strains(23.33%)have weak biofilm-forming ability,and 2 strains(3.34%)without biofilm-forming ability.Among the clinical strains of Acinetobacter baumannii,strains with strong biofilm-forming ability account for 24(40.00%),strains with medium biofilm-forming ability accounted for 15(25.00%),and strains with weak biofilm-forming ability accounted for 21(35.00%).There is no Acinetobacter baumannii without biofilm-forming ability.Among the clinical strains of Staphylococcus aureus,7 strains(11.67%)have strong biofilm-forming ability,19 strains(31.67%)have medium biofilm-forming ability,and 23 strains have weak biofilm-forming ability(38.33%),11 strains(18.33%)have no biofilm-forming ability.Strains with strong film-forming ability account for 4(6.67%)of clinical strains of Staphylococcus epidermidis,and 10 strains have medium biofilm-forming ability(16.67%),14 strains(23.33%)have weak biofilm-forming ability,32 strains(53.33%)have no biofilm-forming ability.Among clinical strains of Enterococcus faecalis,15 strains(25.00%)have strong biofilm-forming ability,17 strains(28.33%)have medium biofilm-forming ability,17 strains(28.33%)have weak ability,and 11 strains(18.34%)have no biofilm-forming ability.2.The relationship between the drug resistance of clinical strains and the ability of biofilm formation:The resistance rate of Escherichia coli to ceftazidime,ceftriaxone,cefepime,and levofloxacin is related to the formation of biofilm(P<0.05).The resistance rate of Escherichia coli with strong biofilm-forming ability to ceftazidime was significantly higher than weak biofilm-forming ability(c~2=17.995,P<0.005).The resistance rate of Pseudomonas aeruginosa to imipenem,meropenem,tobramycin,ceftazidime,cefepime,cefoperazone/sulbactam and levofloxacin was correlated with biofilm formation(P<0.05).For imipenem,meropenem and levofloxacin,the resistance rate of Pseudomonas aeruginosa in the strong film-forming ability group was significantly higher than that in the weak film-forming ability group(c~2=21.277,P<0.005;c~2=17.253,P<0.005;c~2=10.934,P<0.005).The resistance rate of Pseudomonas aeruginosa to tobramycin in the strong film-forming ability group was significantly higher than that in the medium film-forming ability group and weak film-forming ability group(c~2=25.881,P<0.005;c~2=25.262,P<0.005).The resistance rate of Pseudomonas aeruginosa to ceftazidime in strong film-forming ability group was significantly higher than that in medium and weak film-forming ability group(c~2=12.310,P<0.005;c~2=16.541,P<0.005).The resistance rate of Pseudomonas aeruginosa to cefoperazone/sulbactam in strong film forming ability group was significantly higher than that in medium film forming ability group and weak film forming ability group(c~2=11.630,P<0.005;c~2=19.106,P<0.005).There is no correlation between the drug resistance of Acinetobacter baumannii and the ability of biofilm-formation(P>0.05).The resistance rate of Staphylococcus aureus to gentamicin,clindamycin,erythromycin and oxacillin is related to the formation of biofilm(P<0.05).The resistance rate of Staphylococcus epidermidis to oxacillin is related to the biofilm formation(P<0.05).The resistance rate of Enterococcus faecalis to erythromycin and tetracycline is related to the ability of biofilm formation(P<0.05),and the resistance rate of Enterococcus faecalis to erythromycin with strong biofilm ability was significantly higher than the Enterococcus faecalis with weak biofilm ability(c~2=28.235,P<0.005).3.c Chemical properties of anti-biofilm active substances of Klebsiella pneumoniae:The clinically isolated non-repetitive Klebsiella pneumoniae fermentation products can inhibit ATCC35984(strong biofilm strain of Staphylococcus epidermidis)biofilm formation to varying degrees,and the inhibition rate is between 16.1%and 73.5%.The chemical substance in the supernatant of Klebsiella pneumoniae that inhibits Staphylococcus epidermidis is polysaccharide,with a concentration of 200.13 mg/L.4.Anti-Staphylococcus epidermidis ATCC35984 biofilm ability and broad-spectrum anti-biofilm activity of Klebsiella pneumoniae extracellular polysaccharide:5%,10%,20%,30%,40%,50%concentration of polysaccharide can make the amount of Staphylococcus epidermidis biofilm decreased from 2.7781±0.0400 to 2.7307±0.0312(P>0.05),2.6897±0.0285(P>0.05),2.5119±0.0430(P<0.05),1.1310±0.0535(P<0.01),0.6097±0.0453(P<0.01),0.6093±0.0262(P<0.01).The inhibitiom rate of Klebsiella pneumoniae extracellular polysaccharides on Pseudomonas aeruginosa PAO1,Pseudomona aeruginosa 15,Pseudomonas aeruginosa 16,Escherichia coli 06,Escherichia coli 39,Acinetobacter baumannii 02,Acinetobacter baumannii 10,Enterococcus faecalis 05,Enterococcus faecalis 18,Staphylococcus epidermidis 06,Staphylococcus epidermidis 13,Staphylococcus epidermidis ATCC35984,Staphylococcus aureus 11,and Staphylococcus aureus 23 are 40.3%,15.1%,13.0%,28.2%,33.3%,62.5%,64.7%,77.1%,88.9%,69.4%,72.9%,75.5%,85.9%,87.4%,and Klebsiella pneumoniae extracellular polysaccharide has anti-biofilm activity of Staphylococcus aureus,Escherichia coli,Staphylococcus epidermidis,Pseudomonas aeruginosa,Enterococcus faecalis,Acinetobacter baumannii.Conclusions:1.Most of the clinically common strains collected in this research can form biofilms.2.The drug resistance of clinical strains other than Acinetobacter baumannii has a certain correlation with the ability of biofilm formation.3.Klebsiella pneumoniae extracellular polysaccharide has broad-spectrum anti-biofilm activity and is concentration-dependent.The polysaccharide have no effect of inhibiting bacterial growth,and its anti-biofilm mechanism needs further study. |
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