2011-2013年河北地区医院烧伤患者铜绿假单胞菌耐药株oprD基因突变分析

Analysis of mutations of the oprD gene of drug-resistant strains of Pseudomonas aeruginosainfecting patients with burns in a hospital

目的分析铜绿假单胞菌oprD基因突变情况及其与耐药性的关系,以指导烧伤患者感染的临床治疗。方法采用体外药敏试验分析铜绿假单胞菌的耐药情况,然后进行oprD基因的PCR扩增,采用双脱氧链末端终止法对其进行测序分析。结果药敏试验中头孢噻肟、氨苄西林、头孢唑林对铜绿假单胞菌的抑菌圈直径均为6mm,即该菌对上述抗生素均具有耐药性。头孢哌酮、哌拉西林、头孢吡肟、美罗培南、阿米卡星、环丙沙星对铜绿假单胞菌的抑菌环直径范围分别为6~22mm、6~20mm、6~20mm,6~28mm,6~22mm和6~32mm,即该菌对这6种抗生素产生了不同程度的耐药。美罗培南最大抑菌直径为28mm,环丙沙星为32mm,可优先用于治疗烧伤患者铜绿假单胞菌感染。通过PCR扩增oprD基因,其大小为1 340bp;采用双脱氧链终止法对铜绿假单胞菌菌株oprD基因测序,在279位发生C→G的碱基替换,302位发生T→C的碱基替换,334位发生A→G的碱基替换,350位发生T→C的碱基替换,380位发生A→G的突变。结论 oprD基因缺失或其基因片段上的碱基替换引起的基因突变与铜绿假单胞菌的耐药性密切相关,可供烧伤患者感染的临床治疗及抗生素使用提供指导。

Objective To analyze mutations of the oprD gene of Pseudomonas aeruginosa and their relationship to drug resistance in order to guide the clinical treatment of infections and antibiotic use in patients with burns. Methods The drug resistance of P.aeruginosa was analyzed using an in vitro drug susceptibility test.The oprD gene was amplified using PCR,and dideoxy chain termination was used for sequencing. Results Drug susceptibility testing revealed that cefotaxime,ampicillin,and cefazolin resulted in an inhibition zone of 6mm for P.aeruginosa,indicating that P.aeruginosa has resistance to these drugs and should not be used to clinically treat P.aeruginosainfecting patients with burns.Cefoperazone resulted in an inhibition zone of 6-22 mm,piperacillin resulted in a zone of 6-20 mm,cefepime resulted in a zone of 6-20 mm,meropenem resulted in a zone of 6-28 mm,amikacin resulted in a zone of 6-22 mm,and norfloxacin resulted in a zone of 6-32 mm,indicating that P.aeruginosa had different levels of resistance to these 6antibiotics.Meropenem had a maximum inhibition zone of up to 28 mm while norfloxacin had a zone of up to 32 mm.These two antibiotics should be chosen to clinically treat P.aeruginosainfecting patients with burns.The oprD gene was amplified using PCR and was found to be 1 340 bp in size.Sequencing of the oprD gene using dideoxy chain termination revealed that the gene had a base substitution of G for C at exon 279,substitution of C for T at exon 302,substitution of G for A at exon 334,substitution of C for T at exon 350,and substitution of G for A at exon 380.Mutations resulting from gene deletion or base substitutions were directly related to the drug resistance of P.aeruginosa. Conclusion Gene mutations caused by oprD gene deletion or substitution of its bases were closely associated with the drug resistance of P.aeruginosa.This finding can help to guide clinical treatment of infections and antibiotic use in patients with burns.