水产动物源嗜水气单胞菌药物敏感性及QRDR基因突变分析

Antimicrobial susceptibility and mutations of QRDR in Aeromonas hydrophila isolated from aquatic animals

为了解临床分离的59株嗜水气单胞菌对喹诺酮类药物的耐药性及其靶基因gyrA和parC编码的喹诺酮类耐药决定区(QRDR)的突变情况。采用纸片琼脂扩散法测定15种抗菌药物对水产动物源嗜水气单胞菌的耐药情况,采用PCR法扩增gyrA和parC,并通过测序分析其靶基因突变情况。结果表明:24株(40.7%)嗜水气单胞菌对喹诺酮类药物耐药,其中对萘啶酸、诺氟沙星、氧氟沙星、环丙沙星的耐药率依次为40.7%、16.9%、20.3%、8.5%。敏感菌株QRDR靶位点未发生突变,24株喹诺酮类耐药菌株中gyrA基因编码的第83位氨基酸均存在Ser→Ile突变;19株菌parC基因编码的第87位氨基酸也发生突变,其中18株为Ser→Ile突变,1株为Ser→Arg突变,有5株未检测到突变;ParC亚基氨基酸突变的菌株其GyrA亚基均同时发生氨基酸突变。表明临床分离的嗜水气单胞菌对喹诺酮类药物耐药程度不一,其中萘啶酸的耐药最为严重,诺氟沙星、氧氟沙星和环丙沙星敏感性较高。喹诺酮类耐药菌株GyrA亚基QRDR氨基酸突变,可能是引起萘啶酸耐药的主要原因。临床分离嗜水气单胞菌QRDR区域的突变具有随机性,但靶位点GyrA的Ser83→Ile以及ParC的Ser87→Ile是最主要的突变方式,另外喹诺酮类药物耐药性的产生可能还与其他耐药机制存在关联。

The aim of this study was to investigate antimicrobial susceptibility of 59 Aeromonas hydrophila isolated to quinolones and the GyrA and ParC mutation of the quinolone resistance determining regions （QRDR） in quinolones resistant isolates. In this study, disc diffusion method was used to determine the susceptibility of A. hydrophila isolated from aquatic animals against 15 antimicrobial agents. The fragments of the gyrA gene and parC gene were amplified by PCR. Twenty-four （40.7%） isolated were resistant to quinolones, and the resistance rates of nalidixic acid, norfloxacin, ofloxacin, and ciprofloxacin were 40.7%,16.9%,20.3% and 8.5%, respectively. Sequence analysis results indicated that 24 resistant strains had a mutation point Ser→Ile at codon 83 in GyrA and 19 resistant strains had a mutation point at condon 87 in ParC, generating a mutation of Ser→Ile （18 strains）, and a mutation of Ser→Arg was found in one strain. The strains with ParC mutations were also associated with GyrA mutation, 5 resistant strains were not detected any mutation in ParC but with mutation in GyrA. No mutations were detected in quinolone-susceptible isolated. Although there were different quinolone resistance level of A. hydrophila isolated from aquatic animal, nalidixic acid seems more serious than norfloxacin, ofloxacin, and ciprofloxacin. All the quinolone resistant isolated had mutations in GyrA, indicating that mutation of GyrA contributed to the resistance of nalidxic acid. The resuhs also showed that random mutation sites of QRDR in A. hydrophila from clinical isolated, where The Ser83→Ile in GyrA and Ser87→Ile in ParC were the dominant substitutions. In addition, the results indicated that quinolone resistance mechanisms were not only relate to mutations but also including other resistance mechanisms.