生物膜形成对铜绿假单胞菌产不同种β-内酰胺酶的影响

The effect of biofilms on the production of β-lactamases in Pseudomonas aeruginosa

目的探讨生物膜形成对铜绿假单胞菌产β-内酰胺酶的影响。方法观察4株临床分离产TEM型超广谱β-内酰胺酶（简称TEM）和产CARB型超广谱β-内酰胺酶（简称CARB）的铜绿假单胞菌，在生物膜形成前后分别用低剂量抗菌药物诱导，利用相对定量PCR的方法检测各菌株所产β-内酰胺酶mRNA的相对表达量。结果生物膜形成前，铜绿假单胞菌TEM mRNA的相对表达量为1．000±0．495，经环丙沙星、头孢他啶、亚胺培南诱导后TEM mRNA的相对表达量分别为2．038±0．036、3．308±0．010、8．654±0．026；生物膜形成后，铜绿假单胞菌TEM mRNA的相对表达量为1．766±0．026，而经环丙沙星、头孢他啶、亚胺培南诱导后所产TEM mRNA的相对表达量分别为13．849±0．004、16．372±0．044和34．581±0．143。生物膜形成前，铜绿假单胞菌CARB mRNA的相对表达量为1．000±0．069，经环丙沙星、头孢他啶、亚胺培南诱导后CARB mRNA的相对表达量分别为3．506±0．036、5．878±0．190、8．771±0．066；生物膜形成后，铜绿假单胞菌CARB mRNA的相对表达量为2．937±0．164，经环丙沙星、头孢他啶、亚胺培南诱导后CARB mRNA的相对表达量分别为9．528±0．050、10．844±0．062和21．900±0．087。结论生物膜形成可影响铜绿假单胞菌产酶的能力；生物膜形成后，抗菌药物诱导后的铜绿假单胞菌TEM mRNA相对表达量的增幅高于CARB mRNA，证实生物膜形成对铜绿假单胞菌分泌β-内酰胺酶可产生不同的影响。

Objective To investigate the effect of formation of biofilms on the expression of β-lactamases in Pseudomonas aeruginosa. Method Four strains of Pseudomonas aeruginosa producing TEM and CARB extended spectrum β-lactamases were induced by 3 antimicrobials （Ciprofloxacin, Ceftazidime and Imipenem） to generate β-lactamases before and after the formation of biofilms. Different expression of β-lactamases-mRNA was determined by relatively quantitative polymerase chain reaction. Results Before biofilm formation, the relative level of mRNA expression of TEM β-lactamase was 1. 000 ± 0. 495 before and 2. 038 ±0. 036, 3. 308±0. 010, and 8. 654±0. 026 after induction with the 3 antimicrobials. After biofilm formation, the relative level of mRNA expression of TEM β-lactamase was 1. 766 ± 0. 026 before and 13. 849±0. 004, 16. 372 ± 0. 044 and 34. 581 ±0. 143 respectively after antimicrobial induction with the above 3 antimicrobials （ t = 6. 94 - 582. 14, P = 0. 000）. Before biofilm formation, the relative level of mRNA expression of CARB β-lactamase was 1. 000 ± 0. 069 before and 3. 506 ± 0. 036, 5. 878 ± 0. 190, and 8. 771± 0. 066 after induction with the 3 antimicrobials;while for biofilm strains, the relative level of mRNA expression of CARB β-lactamase was 2. 937±0. 164 before and 9. 528± 0. 050, 10. 844± 0. 062, and 21. 900 ± 0. 087 resoectivelv after induction with the 3 antimicrobials （t = 5. 77 -20. 03. P = 0. 000 ）. Before biofilm formation, the increase of relative level of TEM mRNA expression after induction by antimirobials was lower than that of CARB mRNA （5.49 ±4. 32 vs 6. 15 ±2. 26; t = 3.31, P = 0.05）, while after biofilm formation, the increase of the relative level of TEM mRNA after antimicrobial induction was higher than that of CARB mRNA （ 12.40 ±6.95 vs 4. 92 ± 2. 33 ; t = 5. 94, P = 0. 01 ）. Conclusions The formation of biofilms could influence the ability of Pseudomonas aeruginosa in expressing different β-1actamases ; and the amplitude of TEM β-lactamase was higher than that of CARB β-1actamase after biofilm formation, suggesting that the formation of biofilms may have different effects on Pseudomonas aeruginosa strains with different genotypes.