Background Discontinuation of inappropriate antimicrobial therapy is an important target for stewardship intervention. The drug and duration-dependent effects of antibiotics on the developing neonatal gut microbiota needs to be precisely quantified. Methods In this retrospective, cross-sectional study, we performed 16S rRNA sequencing on stool swab samples collected from neonatal intensive care unit patients within 7 days of discontinuation of therapy who received ampicillin and tobramycin (AT), ampicillin and cefotaxime (AC), or ampicillin, tobramycin, and metronidazole (ATM). We compared taxonomic composition within term and preterm infant groups between treatment regimens. We calculated adjusted effect estimates for antibiotic type and duration of therapy on the richness of obligate anaerobes and known butyrate-producers in all infants. Results A total of 72 infants were included in the study. Term infants received AT (20/28; 71%) or AC (8/28; 29%) with median durations of 3 and 3.5 days, respectively. Preterm infants received AT (32/44; 73%) or ATM (12/44; 27%) with median durations of 4 and 7 days, respectively. Compositional analyses of 67 stool swab samples demonstrated low diversity and dominance by potential pathogens. Within 1 week of discontinuation of therapy, each additional day of antibiotics was associated with lower richness of obligate anaerobes (adjusted risk ratio [aRR], 0.84; 95% confidence interval [CI], .73–.95) and butyrate-producers (aRR, 0.82; 95% CI, .67–.97). Conclusions Each additional day of antibiotics was associated with lower richness of anaerobes and butyrate-producers within 1 week after therapy. A longitudinally sampled cohort with preexposure sampling is needed to validate our results.

Systematically determining Illumina sequencing performance characteristics for detection of ARGs in metagenomic samples is essential to inform study design and appraisal of human, animal, and environmental metagenomic antimicrobial resistance studies. In this study, we quantified the performance characteristics of ARG detection in E. coli genomes and metagenomes and established a benchmark of ~15× coverage for ARG detection for E. coli in metagenomes.