鸭源鸡杆菌的药物敏感性及其对氟喹诺酮类药物的耐药机制

Drug sensitivity and fluoroquinolones resistance gene of Gallibacterium anatis

为了解近年来鸭源鸡杆菌(Gallibacterium anatis)的药物敏感性及其对氟喹诺酮类药物的耐药机制,以204株G.anatis为对象,选取11种抗菌药,用纸片法检测其药物敏感性,随后从中随机选取30株进行gyrA、parC基因的PCR扩增和测序,并分析喹诺酮耐药决定区编码氨基酸的突变情况。结果显示,试验菌株对复方新诺明、四环素、氟喹诺酮类药物的耐药率达86.76%以上,对头孢曲松与头孢噻肟的耐药率较低,分别为11.27%和7.35%;99.02%的菌株耐3种以上药物,92.16%的菌株耐6种以上药物,其中有6株对11种药物全部耐药;30株G.anatis全部扩增出parC基因,其中22株扩增出gyrA基因,GyrA亚基存在Ser83→Phe、Asp87→Ala/Tyr/Asn和Asp139→Tyr 3个位点氨基酸置换,ParC亚基存在Thr84→Ile、Glu88→Gly、Ala94→Val和Gly179→Val 4个位点氨基酸置换,敏感菌株不存在氨基酸位点突变,中度敏感菌株发生了3处氨基酸的替换,94.7%的耐药菌株发生了4处及以上氨基酸位点的置换。结果表明,G.anatis分离株产生了严重的多重耐药性;该菌对氟喹诺酮类药物的耐药性与喹诺酮耐药决定区编码氨基酸突变的关系密切,突变位点数量与耐药程度呈正相关;推测GyrA83、GyrA87和ParC84位氨基酸的突变对耐药起着决定性作用,其余4个位点对突变起协同增强作用。

To understand the drug susceptibility and resistance mechanisms of Gallibacterium anatis(G.anatis),which has been isolated in recent years,to fluoroquinolones,we performed an experiment on 204 strains.The strains were tested with 11 antimicrobial drugs,and the disk diffusion was used to conduct the tests.Thirty strains were randomly selected for gyrA and parC gene PCR amplification and sequencing.Mutations in the amino acids encoding the quinolone resistance-determining regions were compared.The results showed that the tested strains were>86.76%resistant to cotrimoxazole,tetracycline,and fluoroquinolones and less resistant to ceftriaxone and cefotaxime(11.27%and 7.35%,respectively).Of the strains tested,99.02%were resistant to more than three drugs and 92.16%were resistant to more than six drugs;six strains were resistant to all 11 drugs selected.The parC gene was amplified from thirty strains of G.anatis,of which the gyrA gene was amplified from 22 strains.The gyrA subunit had three loci of amino acid substitution(Ser83→Phe,Asp87→Ala/Tyr/Asn,and Asp139→Tyr).The parC subunit had four such loci(Thr84→Ile,Glu88→Gly,Ala94→Val,and Gly179→Val).There were no amino acid site mutations in the sensitive strains.Three amino acid substitutions occurred in the moderately sensitive strains.Four or more amino acid site substitutions occurred in 94.7%of the resistant strains.Our results indicated that the G.anatis isolate developed severe multi-drug resistance.The resistance of the bacterium to fluoroquinolones was closely related to the amino acid mutations encoded in its quinolone resistance-determining region,and the number of mutated loci was positively correlated with the degree of resistance.We hypothesized that the mutation of amino acids at GyrA83,GyrA87,and ParC84play a fundamental role in the drug resistance and that the mutation of the remaining four loci play a role in synergistic enhancement.