禽致病性大肠杆菌生物被膜形成能力及相关特性分析

Analysis of biofilm formation ability and related characteristics of avian pathogenic Escherichia coli

[目的]本研究旨在分析能形成生物被膜的临床分离禽致病性大肠杆菌(APEC)菌株的生物学特征。[方法]用刚果红平板法对70株APEC进行筛选,并用结晶紫染色法鉴定菌株生物被膜的形成能力;通过测定不同时间生物被膜生长情况,推断菌株的膜成熟时间;依据玻璃试管法判断菌株是否有卷曲菌毛以及形成生物被膜基质中是否含有胞外纤维素,同时用苯酚-硫酸法测定膜基质中胞外多糖的浓度。对与生物被膜形成相关群体感应系统、菌毛、鞭毛、蛋白、多糖的基因进行PCR检测,最后通过最小抑菌浓度法检测形成生物被膜的菌株对10种抗菌药的耐药性并分析它们的耐药特性。[结果]在鉴定出能形成生物被膜的31株菌中,强、中等和弱成膜能力的分别有10、7和14株。强成膜菌株膜成熟时间比弱成膜菌株的短且差异显著(P<0.05),但与中等成膜菌株膜成熟时间差异不显著(P>0.05)。强成膜能力的菌株中含有菌毛和产生胞外纤维素的菌株所占的比例最高,且胞外多糖含量极显著高于中等和弱成膜菌株(P<0.01)。生物被膜相关基因的检测结果显示,群体感应系统相关基因检出率最高,达到100.00%,其他从高到低依次为菌毛、蛋白、鞭毛、多糖相关基因。耐药性测定结果表明,生物被膜阳性菌株对10种抗菌药有不同程度的耐药性,且成膜能力越强,对10种抗菌药的耐药程度越严重。[结论]强成膜能力菌株的膜成熟时间短,产生膜基质的量多、组成更复杂,且对10种抗菌药的耐药程度更严重。

[Objectives]The aim of this study was to analyze the biological characteristics of avian pathogenic Escherichia coli(APEC)strains isolated in clinic which could form biofilm.[Methods]Seventy strains of APEC were screened by Congo red plate method,and the ability to form biofilm was identified by crystal violet staining.Then,the maturation time of biofilm was inferred by measuring the growth of the biofilm at different times.According to the glass tube test method,whether the strains had curli and whether there was extracellular cellulose in the matrix of biofilm were determined.Meanwhile,the concentration of extracellular polysaccharide in the biofilm matrix was determined by phenol-sulfuric acid method.And then,the genes of quorum sensing system,curli,flagella,protein and polysaccharide which were to biofilm formation were detected by PCR.Finally,the resistance of the biofilm-forming strains to 10 kinds of antimicrobial drugs was determined by the minimum inhibitory concentration method,and the characteristics of their resistance were analyzed.[Results]Among the 31 strains that could form biofilm,the numbers of strong,medium and weak biofilm-forming strains were 10,7 and 14,respectively.Although,the biofilm maturation time of strong biofilm-forming ability strains was significant short to weak strains(P<0.05),there was no significant difference between strong and medium strains(P>0.05).The strains that carried curli and produced extracellular cellulose in biofilm matrix had the highest proportion in strong film-forming ability strains,and the content of extracellular polysaccharide was extremely significantly higher than that of medium and weak film-forming strains(P<0.01).The results of biofilm related genes showed that the detection rate of quorum sensing system was the highest,up to 100.00%,and the other average detection rates were curli,protein,flagella and polysaccharide related genes in order.Meanwhile,the results of drug resistance determination showed that the biofilm positive strains had different degrees of resistance to 10 kinds of antimicrobial drugs,and the stronger the film forming ability,the more serious the resistance to 10 kinds of antimicrobial drugs.[Conclusions]It was found that the strains with strong biofilm-forming ability had shorter biofilm maturity time,more biofilm matrix,more complex composition,and more severe resistance to 10 kinds of antimicrobial agents.This study provides an important reference for the prevention and control of APEC and the rational use of antimicrobial drugs.