Abstract: Objective　To perform single⁃molecule, real⁃time sequencing of ceftazidime⁃avibactam (CAZ⁃AVI)⁃resistant Pseudomonas aeruginosa and to investigate the mechanism underlying ceftazidime⁃avibactam resistance in P. aeruginosa.  Methods    The susceptibility of 89 P. aeruginosa isolates randomly sampled from clinical specimens in Sanming First Hospital Affiliated to Fujian Medical University from November 2021 through July 2023 to common antimicrobial agents was tested, and the minimum inhibitory concentration (MIC) of CAZ⁃AVI was determined against P. aeruginosa with a broth microdilution assay, with CAZ⁃AVI MICs of 8 mg/L and lower defined as susceptible and 16 mg/L and higher as resistant. The expression of drug⁃resistant genes ampC, oxa⁃488, oprD, mexA, oxa⁃10, oxa⁃14, vim and tem was quantified in P. aeruginosa using a real⁃time quantitative reverse transcription PCR (qPCR) assay. CAZ⁃AVI⁃susceptible and ⁃resistant P. aeruginosa isolates from the same case were selected for PacBio single⁃molecule, real⁃time sequencing, and sequencing results were subjected to genome structure and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotations.  Results　The 89 P. aeruginosa isolates showed a relatively high level of resistance to meropenem (75.28%) and imipenem (74.16%) and the highest susceptibility to amikacin (91.01%). There were 49 CAZ⁃AVI⁃resistant P. aeruginosa isolates and 40 susceptible isolates. qPCR assay detected lower oprD gene expression in CAZ⁃AVI⁃resistant P. aeruginosa isolates [0.104 (2.385)] than in susceptible isolates [0.551 (17.885)] (Z = -2.958, P < 0.01), and there were no significant differences between CAZ⁃AVI⁃susceptible and ⁃resistant P. aeruginosa isolates in terms of ampC, oxa⁃488, mexA or tem gene expression (all P values > 0.05), while oxa⁃10, oxa⁃14 and vim gene was expressed in few P. aeruginosa isolates. There were 1 729, 3 936, 3 737 and 3 955 genes in CAZ⁃AVI⁃resistant P. aeruginosa isolates PA⁃762 and PA⁃M174 and susceptible isolates PA⁃885 and PA⁃808 that were annotated to GO terms, with the highest numbers of genes enriched in the molecular function of catalytic activity, high numbers of genes enriched in biological processes of metabolic process, single⁃organism process and cellular process, and high numbers of genes enriched in cellular components of cell and cell membranes. There were 1 803, 4 084, 3 915 and 4 066 genes in the PA⁃762, PA⁃M174, PA⁃885 and PA⁃808 isolates enriched in the KEGG signaling pathway, and the majority of genes were enriched in four primary signaling pathways of metabolism, genetic information processing, environmental information processing and cellular process, with the highest number of genes associated with metabolic pathways. Both CAZ⁃AVI⁃resistant P. aeruginosa isolates PA⁃762 and PA⁃M174 carried multiple efflux pumps systems, including MexAB⁃OprM, MexCD⁃OprJ, MexEF⁃OprN and MexXY⁃OprM. Single nucleotide substitution was found at position 169 in the DNA sequence of the PA⁃762 isolate, leading to substitution of serine for glycine at position 57 in the protein sequence, and there are deletions of two bases at positions 307 and 308 in the DNA sequence of the PA⁃M174 isolate, leading to substitution of threonine for arginine at position 103 in the protein sequence.  Conclusion　Mutation or downregulation of oprD gene may lead to CAZ⁃AVI resistance in P. aeruginosa.

Key words: Pseudomonas aeruginosa, Ceftazidime?avibactam, Drug resistance, Single?molecule, real?time sequencing

摘要： 目的　对耐头孢他啶⁃阿维巴坦（ceftazidime⁃avibactam，CAZ⁃AVI）铜绿假单胞菌进行三代测序，探索铜绿假单胞菌CAZ⁃AVI耐药机制。方法　自2021年11月至2023年7月于福建医科大学附属三明第一医院采集的临床标本分离的铜绿假单胞菌中，随机选择89株铜绿假单胞菌，测定其对常见抗菌药物的敏感性，采用微量肉汤稀释法测定铜绿假单胞菌对CAZ⁃AVI的最低抑制浓度（minimal inhibitory concentration，MIC），分别以CAZ⁃AVI MICs ≤ 8 mg/L和≥ 16 mg/L定义为敏感和耐药。采用实时荧光定量PCR（real⁃time quantitative reverse transcription PCR，qPCR）法检测铜绿假单胞菌耐药基因ampC、oxa⁃488、oprD、mexA、oxa⁃10、oxa⁃14、vim和tem表达水平，分别挑选两组源自同一病例的CAZ⁃AVI敏感及耐药铜绿假单胞菌进行细菌PacBio三代测序，对测序结果进行基因组结构及基因本体论（Gene Ontology，GO）和京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）功能注释。结果　89株铜绿假单胞菌对美罗培南（75.28%）和亚胺培南耐药率（74.16%） 较高，对阿米卡星（91.01%）敏感率最高；CAZ⁃AVI耐药和敏感铜绿假单胞菌分别有49株和40株。qPCR法检测结果显示，CAZ⁃AVI耐药和敏感铜绿假单胞菌菌株中oprD基因表达水平中位数（四分位间距）分别为0.104（2.385）和0.551（17.885），差异有统计学意义（Z = -2.958，P < 0.01），ampC、oxa⁃488、mexA、tem基因表达水平差异均无统计学意义（P均> 0.05），而oxa⁃10、oxa⁃14、vim基因仅在少量铜绿假单胞菌菌株中表达。CAZ⁃AVI耐药铜绿假单胞菌PA⁃762株与PA⁃M174株、CAZ⁃AVI敏感PA⁃885株与PA⁃808株分别有1 729、3 936、3 737、3 955个基因注释到GO功能条目，其中催化活性分子功能富集到的基因数最多，代谢过程、单一生物体过程和细胞过程等生物学过程富集到的基因数较多，与细胞、细胞膜等细胞组分相关的基因数较多。PA⁃762、PA⁃M174、PA⁃885、PA⁃808株分别有1 803、4 084、3 915、4 066个基因富集到 KEGG信号通路，大部分基因被富集到代谢、遗传信息加工、环境信息加工和细胞过程等4个一级信号通路，其中与代谢通路有关的基因数最多。PA⁃762、PA⁃M174株均携带MexAB⁃OprM、MexCD⁃OprJ、MexEF⁃OprN、MexXY⁃OprM等多种外排泵系统。PA⁃762株DNA序列的第169位发生单核苷酸替换，导致蛋白质序列第57位的丝氨酸被替换为甘氨酸；PA⁃M174株DNA序列的第307、308位发生两个碱基缺失，导致蛋白质序列第103位的苏氨酸被替换为精氨酸。结论　oprD基因突变或其表达水平下调可能导致铜绿假单胞菌对CAZ⁃AVI产生耐药性。

关键词: 铜绿假单胞菌, 头孢他啶?阿维巴坦, 耐药性, 三代测序