猪源肠外致病性大肠埃希氏菌的生物膜形成能力及耐药性

Biofilm-forming capability and drug resistance of extraintestinal pathogenic Escherichia coli isolated from diseased swine

摘要:

【背景】猪源肠外致病性大肠埃希氏菌(extraintestinal pathogenic Escherichia coli，ExPEc)是一种严重危害养猪业的病原菌，有关其生物膜形成能力与耐药性的研究报道很少。【目的】探讨从病猪肺脏中分离鉴定的3株ExPEc的生物膜形成能力及耐药性，为从抗生物膜形成角度防治猪肠外大肠埃希氏菌病提供参考。【方法】采用96孔板结晶紫染色法结合正交实验优化猪源ExPEc分离株的生物膜形成最佳条件与成膜能力；通过扫描电镜观察各菌株生物膜的形态结构；利用PCR方法检测其携带的生物膜形成相关基因；采用微量肉汤稀释法测定抗生素对生物膜态与浮游态下猪源ExPEc分离株的最小抑菌浓度(minimum inhibitory concentration，MIC)。【结果】3株猪源ExPEc的最佳成膜条件并不一致，但在各自最佳条件下均能形成很强的生物被膜且同时携带10个生物膜形成相关基因(pgaA，pgaB，pgaC，pgaD，luxS，fimA，hipA，iha，flhC，flhD)。扫描电镜观察显示，菌株SE-1聚集后可形成片状生物膜，菌株SE-2和SE-3聚集后可形成多层结构的生物膜，且聚集体间有明显空隙。部分受试抗菌药物对生物膜态SE-1、SE-2和SE-3菌株的MIC值较对应浮游菌提高了2?32倍，药物敏感性由原来的敏感转为中敏或耐药。【结论】实验结果为从抗生物膜形成角度防治猪肠外大肠埃希氏菌病奠定了基础。

关键词:猪，肠外致病性大肠埃希氏菌，生物膜形成能力，成膜相关基因，最小抑菌浓度

Abstract:

[Background] Porcine extraintestinal pathogenic Escherichia coli (ExPEc), a pathogen seriously endangers the swine industry. However, few studies have reported the biofilm-forming capability and drug resistance of porcine ExPEc. [Objective] To study the relationship between the biofilm-forming capability and drug resistance of 3 ExPEc strains isolated from the diseased swine lung, and provide the reference to prevent and treat the porcine ExPEc disease in the perspective of anti-biofilm formation. [Methods] The optimal conditions and capability for biofilm formation of porcine ExPEc strains were examined using 96-well plate crystal violet staining combined with orthogonal experiments. The morphologic features of the biofilms produced by each strain were observed using a scanning electron microscopy. Biofilm-forming related genes carried by each strain were detected by PCR, as well as the minimum inhibitory concentration (MIC) of antibiotic to porcine ExPEc strains both in floating form and biofilm form were determined by micro-broth dilution method, respectively. [Results] The optimal film-forming conditions of 3 ExPEc strains were not consistent, but showed strong biofilm formation under their optimum conditions, and carried synchronously 10 kinds of biofilm-forming related genes (pgaA, pgaB, pgaC, pgaD, luxS, fimA, hipA, iha, flhC, flhD). Scanning electron microscopic observation showed that the strain SE-1 aggregated to form a flaky biofilm, while both SE-2 and SE-3 strains aggregated to form multi-layered biofilms, and there was obvious space between aggregates. The MIC values of some tested antibiotics to 3 strains growing in a biofilm were 2?32 times higher than those of corresponding planktonic bacteria, and the drug sensitivity changed from sensitivity to medium or resistance. [Conclusion] The result in the study provides a basis for the prevention and treatment of porcine ExPEc infection from the perspective of anti-biofilm formation.

Key words:Swine, Extraintestinal pathogenic Escherichia coli, Biofilm-forming capability, Film-forming related genes, Minimum inhibitory concentration