Franconi Iacopo, Poma Noemi, Rizzato Cosmeri, Maltinti Lorenzo, Falcone Marco, Tavanti Arianna, Lupetti Antonella
Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa, Italy.
SD Mycology Unit, Azienda Ospedaliero-Universitaria Pisana, Pisa, Italy.
JAC Antimicrob Resist. 2025 Apr 30;7(3):dlaf051. doi: 10.1093/jacamr/dlaf051. eCollection 2025 Jun.
is an opportunistic pathogen with increasing rates of resistance to fluconazole and voriconazole. Recently, in an outbreak at the Azienda Ospedaliero-Universitaria Pisana, a new amino acid substitution, S862C in the CpMrr1 protein, was found only in azole-resistant strains. The contribution of this mutation to the acquisition of an azole-resistant phenotype was investigated in this study.
Antifungal resistance in clinical strains isolated from the outbreak ( = 16) was tested by the broth microdilution method and Etest strip. WGS and Sanger sequencing analyses were used for the detection of SNPs. A CRISPR-Cas9-based genome editing strategy was used to induce the C2585G substitution in the gene of susceptible isolates to investigate its role in the acquisition of azole resistance.
The A395T and the newly found C2585G substitution in the gene were present in all resistant isolates, but not in the susceptible ones. Such mutations were later induced in the reference strain ATCC 22019 and in two azole-susceptible clinical isolates in homozygosis, and in heterozygosis only for ATCC 22019 and one azole-susceptible clinical isolate. Both heterozygous and homozygous mutants carrying the C2585G mutation were fluconazole resistant, with some clones also presenting intermediate susceptibility or resistance to voriconazole.
To the best of our knowledge, this is the first study to report the effect on azole resistance of a novel C2585G nucleotide substitution in the gene found in clinical isolates recovered during an outbreak of azole-resistant in a healthcare setting.
是一种机会致病菌，对氟康唑和伏立康唑的耐药率不断上升。最近，在比萨大学医院爆发的疫情中，仅在耐唑类菌株中发现了CpMrr1蛋白中的一种新的氨基酸取代，即S862C。本研究调查了这种突变对获得唑类耐药表型的贡献。
通过肉汤微量稀释法和Etest试纸条检测从疫情中分离出的临床菌株（n = 16）的抗真菌耐药性。全基因组测序（WGS）和桑格测序分析用于检测单核苷酸多态性（SNP）。采用基于CRISPR-Cas9的基因组编辑策略，在敏感菌株的基因中诱导C2585G取代，以研究其在获得唑类耐药性中的作用。
该基因中的A395T和新发现的C2585G取代存在于所有耐药菌株中，而在敏感菌株中不存在。随后，在参考菌株ATCC 22019和两个唑类敏感的临床分离株中纯合诱导了此类突变，而在ATCC 22019和一个唑类敏感的临床分离株中杂合诱导。携带C2585G突变的杂合和纯合突变体均对氟康唑耐药，一些克隆对伏立康唑也呈现中介敏感性或耐药性。
据我们所知，这是第一项报道在医疗机构耐唑类疫情期间回收的临床分离株中发现的基因中一种新的C2585G核苷酸取代对唑类耐药性影响的研究。
