AUTHOR=Campos-Madueno Edgar I. , Aldeia Claudia , Perreten Vincent , Sendi Parham , Moser Aline I. , Endimiani Andrea 

TITLE=Detection of blaCTX-M and blaDHA genes in stool samples of healthy people: comparison of culture- and shotgun metagenomic-based approaches

JOURNAL=Frontiers in Microbiology

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2023.1236208

DOI=10.3389/fmicb.2023.1236208

ISSN=1664-302X

ABSTRACT=<p>We implemented culture- and shotgun metagenomic sequencing (SMS)-based methods to assess the gut colonization with extended-spectrum cephalosporin-resistant <italic>Enterobacterales</italic> (ESC-R-<italic>Ent</italic>) in 42 volunteers. Both methods were performed using native and pre-enriched (broth supplemented with cefuroxime) stools. Native culture screening on CHROMID<sup>®</sup> ESBL plates resulted in 17 positive samples, whereas the pre-enriched culture (gold-standard) identified 23 carriers. Overall, 26 ESC-R-<italic>Ent</italic> strains (24 <italic>Escherichia coli</italic>) 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 <italic>bla</italic><sub>CTX-M</sub>/<italic>bla</italic><sub>DHA</sub> genes (native SMS reads mapping to <italic>bla</italic><sub>CTX-M</sub>/<italic>bla</italic><sub>DHAs</sub> 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 <italic>bla</italic><sub>CTX-M</sub>/<italic>bla</italic><sub>DHA</sub> genes were detected than in the native assay, including in stools that were pre-enriched culture-negative (pre-enriched SMS reads mapping to <italic>bla</italic><sub>CTX-M</sub>/<italic>bla</italic><sub>DHAs</sub> 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-<italic>Ent</italic> (average: ~10<sup>5</sup> vs. ~10<sup>7</sup> CFU/g) and <italic>E. coli</italic> classified reads (average: 193,959 vs. 1.45 million) than those of native SMS positive samples. Finally, the detection of <italic>bla</italic><sub>CTX-M</sub>/<italic>bla</italic><sub>DHA</sub> genes was compared with two well-established bioinformatic tools. In conclusion, only the pre-enriched SMS assured detection of most carriers of ESC-R-<italic>Ent</italic>. However, its performance was not comparable to the pre-enriched culture-based approach.</p>