Campos-Madueno Edgar I, Aldeia Claudia, Perreten Vincent, Sendi Parham, Moser Aline I, Endimiani Andrea
Institute for Infectious Diseases (IFIK), University of Bern, Bern, Switzerland.
Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland.
Front Microbiol. 2023 Aug 31;14:1236208. doi: 10.3389/fmicb.2023.1236208. eCollection 2023.
We implemented culture- and shotgun metagenomic sequencing (SMS)-based methods to assess the gut colonization with extended-spectrum cephalosporin-resistant (ESC-R-) in 42 volunteers. Both methods were performed using native and pre-enriched (broth supplemented with cefuroxime) stools. Native culture screening on CHROMID ESBL plates resulted in 17 positive samples, whereas the pre-enriched culture (gold-standard) identified 23 carriers. Overall, 26 ESC-R- strains (24 ) were identified: 25 CTX-M and 3 DHA-1 producers (2 co-producing CTX-Ms). Using the SMS on native stool ("native SMS") with thresholds ≥60% for both identity and coverage, only 7 of the 23 pre-enriched culture-positive samples resulted positive for / genes (native SMS reads mapping to / identified in gold-standard: sensitivity, 59.0%; specificity 100%). Moreover, an average of 31.5 and 24.6 antimicrobial resistance genes (ARGs) were detected in the 23 pre-enriched culture-positive and the 19 negative samples, respectively. When the pre-enriched SMS was implemented, more / genes were detected than in the native assay, including in stools that were pre-enriched culture-negative (pre-enriched SMS reads mapping to / identified in gold-standard: sensitivity, 78.3%; specificity 75.0%). In addition, the pre-enriched SMS identified on average 38.6 ARGs/sample, whereas for the corresponding native SMS it was 29.4 ARGs/sample. Notably, stools resulting false-negative by using the native SMS had lower concentrations of ESC-R- (average: ~10 vs. ~10 CFU/g) and classified reads (average: 193,959 vs. 1.45 million) than those of native SMS positive samples. Finally, the detection of / genes was compared with two well-established bioinformatic tools. In conclusion, only the pre-enriched SMS assured detection of most carriers of ESC-R-. However, its performance was not comparable to the pre-enriched culture-based approach.
我们采用基于培养和鸟枪法宏基因组测序（SMS）的方法，对42名志愿者肠道中耐超广谱头孢菌素（ESC-R-）的定植情况进行评估。两种方法均使用原始粪便和预富集（添加头孢呋辛的肉汤）粪便进行。在CHROMID ESBL平板上进行原始培养筛选，得到17个阳性样本，而预富集培养（金标准）鉴定出23名携带者。总体而言，共鉴定出26株ESC-R-菌株（24株 ）：25株CTX-M生产者和3株DHA-1生产者（2株同时产生CTX-M）。对原始粪便使用SMS（“原始SMS”），同一性和覆盖率阈值均≥60%，在23个预富集培养阳性样本中，只有7个样本的 / 基因检测呈阳性（原始SMS读数映射到金标准中鉴定的 / ：敏感性，59.0%；特异性，100%）。此外，在23个预富集培养阳性样本和19个阴性样本中，分别平均检测到31.5个和24.6个抗菌抗性基因（ARG）。采用预富集SMS时，检测到的 / 基因比原始检测更多，包括在预富集培养阴性的粪便中（预富集SMS读数映射到金标准中鉴定的 / ：敏感性，78.3%；特异性，75.0%）。此外，预富集SMS平均每个样本鉴定出38.6个ARG，而相应的原始SMS为每个样本29.4个ARG。值得注意的是，使用原始SMS检测为假阴性的粪便中，ESC-R-的浓度（平均：~10 vs. ~10 CFU/g）和分类读数（平均：193,959 vs. 145万）低于原始SMS阳性样本。最后，将 / 基因的检测结果与两种成熟的生物信息学工具进行了比较。总之，只有预富集SMS能确保检测到大多数ESC-R-携带者。然而，其性能与基于预富集培养的方法不可比。
