鸡场饮水中肺炎克雷伯氏菌的分离鉴定及耐药性研究

【目的】研究养殖场鸡饮水中肺炎克雷伯氏菌（Klebsiella pneumoniae）的耐药表型和耐药基因的携带情况，并分析其相关性，为有效预防该病提供参考。【方法】从养殖场鸡饮水系统中分离、鉴定肺炎克雷伯氏菌，采用药敏纸片扩散法检测分离菌株对25种常用抗菌药的耐药性，通过PCR检测菌株携带的常见耐药基因。对其中4株肺炎克雷伯氏菌进行全基因组测序，利用CARD抗性基因数据库预测耐药基因的携带情况。【结果】从养殖场鸡饮水系统中共分离到9株肺炎克雷伯氏菌，药敏试验表明这些菌株呈现出多重耐药，仅对多粘菌素敏感。PCR检测发现，9株肺炎克雷伯氏菌共携带13种常见的耐药基因，其中，β-内酰胺酶类耐药基因CMY-1、BlaSHV和BlaCTX-M，碳青霉烯类耐药基因KPC，喹诺酮类耐药基因QnrB、QnrS和QnrA，氨基糖苷类耐药基因Aph(3’)-Ia，磺胺类耐药基因Sul2等12种耐药基因与其耐药表型表现一致，而Mcr-1基因与其表型相反。全基因组扫描分析结果显示，4株肺炎克雷伯氏菌共携带了59种耐药基因，涉及31类抗生素药物，其中四环素类抗生素的耐药基因最多，其次是大环内酯类抗生素和氟喹诺...

Isolation, Identification and Drug Resistance Research of Klebsiella pneumoniae in Drinking Water from a Chicken Farm

【Objective】It aims to investigate resistance phenotypes and resistance genes of Klebsiella pneumoniae in drinking water of chicken farm, and analyze the correlation between them, to provide references for effective prevention of the disease.【Method】Strains of K. pneumoniae were isolated and identified from the drinking water system of a chicken farm. The resistances of the isolated strains to 25 antibiotics were detected by the drug-sensitive disk diffusion method. The presence of common drug-resistance genes in all these strains was detected by PCR. The whole genomes of four strains of K. pneumoniae were sequenced to analyze and predict the carrying of drug resistance genes by the Comprehensive Antibiotic Research Database (CARD).【Result】Nine strains of K. pneumoniae were isolated from the drinking water system of the chicken farm. The results of drug sensitivity testing revealed that these strains were multiple drug resistant, and only susceptive to colistin sulfate. Thirteen common drug resistance genes carried by all nine strains were confirmed by PCR, and consistency between drug resistance phenotypes and genes was observed in 12 resistant genes including β-lactamase resistance genes CMY-1, BlaSHV and BlaCTX-M; carbapenem resistance gene KPC; quinolone resistance genes QnrB, QnrS and QnrA; aminoglycoside resistance gene Aph(3'')-Ia; sulfa resistance gene Sul2 and etc; while inconsistency between the presence of Mcr-1 and its corresponding phenotype was observed. A total of 59 resistance genes, involving 31 classes of antibiotics, were carried by four strains of K. pneumoniae through whole genome scanning analysis. Among these, the number of genes resistant to tetracycline antibiotics was maximum, followed by genes resistant to macrolide and fluoroquinolone antibiotics. Additionally, a total of 23 drug-resistant genes were predicted when the coverage and homology were both set at a rate of higher than 98%, including FosA6, CRP, KpnF, KpnFG, KpnE, OqxA, OqsB, SHV-11, Tet(D), CTX-M-27, QnrB65, DHA-1, Aph(4'')-Ia, MsrE, QnrB2, Aph(3'')-Ia, Sul1, CmlA6, FloR, AadA12, Ant(3'')-IIa, ArmA and Sul2.【Conclusion】Strains of K. pneumoniae isolated from the drinking water of the chicken farm show serious drug resistance and carried multiple drug resistance genes, among which Mcr-1 gene is detected on the isolated strains. However, the isolated strains are sensitive to polymyxin, and the D-type carbapenemase OXA-48 gene was not detected in the isolated strains.