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

Abstract

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.

A new in vivo model of intestinal colonization using Zophobas morio larvae: testing hyperepidemic ESBL- and carbapenemase-producing Escherichia coli clones

“…Notably, direct culture-based analyses on MacConkey II and Columbia sheep blood agar plates (Becton-Dickinson) revealed that disinfected pears were contaminated with Bacillus cereus [~3 × 10 1 colony forming units (CFU)/mL], whereas dry food contained ,afaD,chuA,fimH,fimC,gad,hra,iha,iss,kpsE,kpsMII_K5,ompT,papA_ F43,sat,senB,traT fimH,gad,terC,yehA,yehB,yehC,yehD csgA,gad,hra,irp2,terC Moraxella osloensis and Micrococcus luteus (~2-5 × 10 2 CFU/mL); the oat substrate was constantly negative. Moreover, the three types of food were regularly screened for the presence of extended-spectrum cephalosporin-resistant (ESC-R)-Ent using an overnight broth enrichment and selective agar plates as previously done (Endimiani et al, 2020;Campos-Madueno et al, 2023a). As a result, no food used throughout experiments resulted contaminated with ESC-R-Ent (data not shown).…”