蛙源米尔伊丽莎白菌的生物膜形成能力及β内酰胺类和喹诺酮类耐药性分析

BIOFILM FORMATION ABILITY AND RESISTANCE TO Β-LACTAMS AND FLUOROQUINOLONES OF ELIZABETHKINGIA MIRICOLA ISOLATED FROM FROG

  • 摘要: 研究旨在探讨蛙源米尔伊丽莎白菌(Elizabethingia miricola)的药敏特性, 生物膜形成能力及对β-内酰胺类和喹诺酮类药物的耐药机制。通过16S rRNA和rpoB基因序列系统发育分析对分离菌株进行分子鉴定。采用肉汤稀释法、结晶紫微孔板法和卡尔加里生物膜装置分别测定其最小抑菌浓度(MIC)、生物膜形成能力及最低生物膜清除浓度(MBEC)。利用PCR方法分析β-内酰胺耐药基因及喹诺酮类耐药决定区(QRDR)突变, 并通过双纸片法检测其超广谱β-内酰胺酶(ESBL)和金属β-内酰胺酶(MBL)的产生。结果显示, 从患病蛙类样本中分离到的9株优势菌株均为E. miricola; 各菌株对β-内酰胺类、氨基糖苷类及喹诺酮类药物耐药, 但对多西环素和氟苯尼考敏感; 所有菌株均可形成生物膜, 生物膜形成菌的MBEC比对应浮游菌的MIC提高8—>256倍; 所有菌株均携带β-内酰胺类耐药相关基因(blaCMEblaGOBblaB), 并产生ESBL及MBL; QRDR的gyrA基因均检测到了可介导喹诺酮类耐药的非同义突变S83R, gyrBparCparE未发现突变。结果表明, 蛙源E. miricola呈现多重耐药性, 普遍具有生物膜形成能力, β-内酰胺酶的产生及喹酮类药物作用靶点突变与其β-内酰胺及喹诺酮类耐药表型相符。多西环素和氟苯尼考的体外敏感性较高, 具有治疗该菌感染的潜力。研究可为E. miricola感染的治疗及耐药机制的深入研究提供参考。

     

    Abstract: Meningitis-like epidemic diseases is the largest threat to the frog farming industry. The causing pathogen often exhibits extensive antibiotic resistance, but the resistance mechanisms remain poorly understood. Our aim was to examine the antimicrobial susceptibility and biofilm forming ability of the Elizabethkingia miricola strains isolated from frogs and to determine their mechanisms of β-lactams and fluoroquinolones resistance. Isolates were re-identified to precise species using 16S rRNA and rpoB gene phylogenetic tree. Antimicrobial susceptibility, biofilm formation, and the minimum biofilm eradication concentration (MBEC) were examined by using broth dilution method, crystal microtiter plate assay, and the calgary biofilm device (CBD), respectively. β-lactamase genes and mutations in the quinolone-resistance determining regions (QRDR) were identified using PCR. ESBLs and MBLs production were screened using a combined disc test. All dominant strains in diseased frogs collected from different farms were identified as Elizabethingia miricola. The isolates were resistant to β-lactams, aminoglycosides, and fluoroquinolones tested, whereas all isolates were susceptible only to doxycycline and florfenicol among 17 antibiotics. The positive rate of biofilm formation was 100% among the 9 E. miricola isolates, with the majority being low to moderate biofilm formers. The MBEC of antibiotics tested on biofilm bacteria were 8—256 times higher than the MIC on planktonic bacteria. All strains carried β-lactam resistance genes (blaCME, blaGOB, and blaB), ESBL and MBL phenotypes were observed for all isolates. A single mutation was found in the gyrA gene (S83R), which confer fluoroquinolone resistance, while no mutations were found in the gyrB, parC, or parE gene of these isolates. The results indicate that frog-derived E. miricola exhibits multidrug resistance and generally has the ability to form biofilms. The production of β-lactamase and mutation at QRDR correspond with its β-lactam and fluoroquinolone resistance phenotypes. Doxycycline and florfenicol exhibited good sensitivity in vitro and have the potential for treating severe E. miricola infections.

     

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