Bordin Amanda, Pandey Sushil, Coulter Christopher, Syrmis Melanie, Pardo Carolyn, Hackett Hazel, Bell Scott C, Wainwright Claire E, Nimmo Graeme R, Jennison Amy V, Clark Julia E, Whiley David M
The University of Queensland Centre for Clinical Research, University of Queensland, Brisbane, Queensland, Australia.
Queensland Mycobacterium Reference Laboratory, Pathology Queensland, Brisbane, Queensland, Australia.
J Med Microbiol. 2021 Apr;70(4). doi: 10.1099/jmm.0.001349.
complex (MABSC) is an environmental organism and opportunistic pathogen. MABSC pulmonary infections in people with cystic fibrosis are of growing clinical concern. Resistance data guide the use of macrolides and amikacin in MABSC pulmonary disease treatment. MABSC can acquire resistance against macrolides or amikacin via 23S or 16S rRNA gene mutations, respectively. Current culture-based methods for MABSC detection and antibiotic resistance characterization are typically prolonged, limiting their utility to directly inform treatment or clinical trials. Culture-independent molecular methods may help address this limitation. To develop real-time PCR assays for characterization of key 23S or 16S rRNA gene mutations associated with constitutive resistance in MABSC. We designed two real-time PCR assays to detect the key 23S and 16S rRNA gene mutations. The highly conserved nature of rRNA genes was a major design challenge. To reduce potential cross-reactivity, primers included non-template bases and targeted single-nucleotide polymorphisms unique to MABSC. We applied these assays, as well as a previously developed real-time PCR assay for MABSC detection, to 968 respiratory samples from people with cystic fibrosis. The results from the molecular methods were compared to those for gold standard culture methods and 23S and 16S rRNA gene sequencing.The real-time PCR MABSC detection assay provided a sensitivity of 83.8 % and a specificity of 97.8 % compared to culture. The results from the real-time PCR resistance detection assays were mostly concordant (>77.4 %) with cultured isolate sequencing. The real-time PCR resistance detection assays identified several samples harbouring both resistant and susceptible MABSC, while culture-dependent methods only identified susceptible MABSC in these samples. Using the molecular methods described here, results for health care providers or researchers could be available days or weeks earlier than is currently possible via culture-based antibiotic susceptibility testing.
非结核分枝杆菌复合群（MABSC）是一种环境微生物和机会致病菌。囊性纤维化患者的MABSC肺部感染在临床上越来越受到关注。耐药数据指导大环内酯类药物和阿米卡星在MABSC肺部疾病治疗中的使用。MABSC可分别通过23S或16S rRNA基因突变获得对大环内酯类药物或阿米卡星的耐药性。目前基于培养的MABSC检测和抗生素耐药性鉴定方法通常耗时较长，限制了它们直接指导治疗或临床试验的效用。非培养分子方法可能有助于解决这一局限性。为了开发实时PCR检测方法，以鉴定与MABSC固有耐药性相关的关键23S或16S rRNA基因突变。我们设计了两种实时PCR检测方法来检测关键的23S和16S rRNA基因突变。rRNA基因的高度保守性是一个主要的设计挑战。为了减少潜在的交叉反应，引物包含非模板碱基，并靶向MABSC特有的单核苷酸多态性。我们将这些检测方法以及之前开发的用于MABSC检测的实时PCR检测方法应用于968份囊性纤维化患者的呼吸道样本。将分子方法的结果与金标准培养方法以及23S和16S rRNA基因测序的结果进行比较。与培养法相比，实时PCR MABSC检测方法的灵敏度为83.8%，特异性为97.8%。实时PCR耐药性检测方法的结果与培养分离株测序结果大多一致（>77.4%）。实时PCR耐药性检测方法鉴定出几个同时含有耐药和敏感MABSC的样本，而基于培养的方法仅在这些样本中鉴定出敏感的MABSC。使用本文所述的分子方法，医疗保健提供者或研究人员获得结果的时间可能比目前通过基于培养的抗生素敏感性检测提前数天或数周。
