Aztreonam-avibactam is a promising antimicrobial combination against multidrug-resistant organisms, such as carbapenemase-producing Enterobacterales. Resistance to aztreonam-avibactam has been found, but the resistance mechanism remains poorly studied. We recovered three Escherichia coli isolates of an almost identical genome but exhibiting varied aztreonam-avibactam resistance. The isolates carried a cephalosporinase gene, bla_CMY-42, on IncIγ plasmids with a single-nucleotide variation in an antisense RNA-encoding gene, inc, of the replicon. The isolates also had four extra amino acids (YRIK) in penicillin-binding protein 3 (PBP3) due to a duplication of a 12-nucleotide (TATCGAATTAAC) stretch in pbp3. By cloning and plasmid-curing experiments, we found that elevated CMY-42 cephalosporinase production or amino acid insertions in PBP3 alone mediated slightly reduced susceptibility to aztreonam-avibactam, but their combination conferred aztreonam-avibactam resistance. We show that the elevated CMY-42 production results from increased plasmid copy numbers due to mutations in inc. We also verified the findings using in vitro mutation assays, in which aztreonam-avibactam-resistant mutants also had mutations in inc and elevated CMY-42 production compared with the parental strain. This choir of target modification, hydrolyzing enzyme, and plasmid expression represents a novel, coordinated, complex antimicrobial resistance mechanism and also reflects the struggle of bacteria to survive under selection pressure imposed by antimicrobial agents.

中文翻译：

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努力生存：目标改变，水解酶和质粒表达的合唱团作为一种新型的氨曲南-阿维巴坦耐药机制

Aztreonam-avibactam是一种有前途的抗微生物药物组合，可抵抗多重耐药生物，例如产生碳青霉烯酶的肠杆菌。已经发现对氨曲南-阿维巴坦具有抗药性，但是抗药性机理的研究仍然很少。我们回收了三个几乎相同的基因组但表现出不同的氨曲南-avibactam耐药性的大肠杆菌分离株。分离株在复制子的反义RNA编码基因inc的单核苷酸变异的IncIγ质粒上带有头孢菌素酶基因bla _CMY-42。菌株也有在青霉素结合蛋白3（PBP3）四个额外的氨基酸（YRIK）由于12个核苷酸（TATCGAATTAAC）的复制在拉伸PBP3。通过克隆和质粒固化实验，我们发现仅在PBP3中CMY-42头孢菌素酶的产量增加或氨基酸插入介导的氨曲南-avibactam敏感性降低，但它们的组合赋予了氨曲南-avibactam耐药性。我们显示出升高的CMY-42生产是由于inc中的突变导致质粒拷贝数增加所致。我们也验证使用的调查结果体外突变试验，其中氨曲南，avibactam耐药突变体也有突变INC与亲本菌株相比，CMY-42产量增加。目标修饰，水解酶和质粒表达的这种合唱代表了一种新颖的，协调的，复杂的抗微生物耐药性机制，并且还反映了细菌在抗微生物剂施加的选择压力下生存的斗争。