Data_Sheet_1_Detection of blaCTX-M and blaDHA genes in stool samples of healthy people: comparison of culture- and shotgun metagenomic-based approaches.pdf

We implemented culture- and shotgun metagenomic sequencing (SMS)-based methods to assess the gut colonization with extended-spectrum cephalosporin-resistant Enterobacterales (ESC-R-Ent) 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-Ent strains (24 Escherichia coli) 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 blaCTX-M/blaDHA genes (native SMS reads mapping to blaCTX-M/blaDHAs 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 blaCTX-M/blaDHA genes were detected than in the native assay, including in stools that were pre-enriched culture-negative (pre-enriched SMS reads mapping to blaCTX-M/blaDHAs 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-Ent (average: ~105 vs. ~107 CFU/g) and E. coli classified reads (average: 193,959 vs. 1.45 million) than those of native SMS positive samples. Finally, the detection of blaCTX-M/blaDHA genes was compared with two well-established bioinformatic tools. In conclusion, only the pre-enriched SMS assured detection of most carriers of ESC-R-Ent. However, its performance was not comparable to the pre-enriched culture-based approach.

本研究通过文化和宏基因组测序（SMS）相结合的方法，对42名志愿者的肠道中广谱β-内酰胺酶耐药肠杆菌科（ESC-R-Ent）的定植情况进行评估。两种方法均采用原样和预先富集（加入头孢呋辛的肉汤）粪便样本进行。在CHROMID® ESBL板上进行原样培养筛选，得到17份阳性样本，而预先富集培养（金标准）则鉴定出23名携带者。总计鉴定出26株ESC-R-Ent菌株（24株大肠杆菌）：25株为CTX-M产生者，3株为DHA-1产生者（其中2株同时产生CTX-M）。在原样粪便上进行SMS（称为“原样SMS”），设定身份和覆盖率的阈值均≥60%，仅在23份预先富集培养阳性样本中检测到7份blaCTX-M/blaDHA基因（原样SMS读段映射到金标准中发现的blaCTX-M/blaDHAs：灵敏度，59.0%；特异性，100%）。此外，在23份预先富集培养阳性样本和19份阴性样本中，分别检测到平均31.5和24.6个抗菌药物耐药基因（ARGs）。当采用预先富集SMS时，检测到的blaCTX-M/blaDHA基因比原样检测方法更多，包括在预先富集培养阴性粪便中（预先富集SMS读段映射到金标准中发现的blaCTX-M/blaDHAs：灵敏度，78.3%；特异性，75.0%）。此外，预先富集SMS平均每样本鉴定出38.6个ARGs，而相应的原样SMS为每样本29.4个ARGs。值得注意的是，使用原样SMS检测出的假阴性粪便中ESC-R-Ent（平均浓度：~105 vs. ~107 CFU/g）和大肠杆菌分类读段（平均：193,959 vs. 1.45百万）的浓度低于原样SMS阳性样本。最后，blaCTX-M/blaDHA基因的检测与两种成熟的生物信息学工具进行了比较。总之，只有预先富集SMS能够确保大多数ESC-R-Ent携带者的检测。然而，其性能并不等同于基于预先富集培养的方法。