Abstract
The gut microbiome of preterm infants is highly vulnerable to perturbations. Members of the class Clostridia are among the first anaerobes colonizing the preterm gut, yet their ecological roles and antimicrobial resistance (AMR) properties remain poorly understood. We characterized 98 Clostridia isolates from fecal samples of preterm infants, spanning 17 species and 11 genera. Isolates were identified by MALDI-TOF and 16S rRNA sequencing, colonization levels were quantified, and antimicrobial susceptibility was assessed by disk diffusion and E-test. Resistance determinants were screened by PCR and sequenced. We focused on Clostridia that were present at low colonization levels (mean 5.3 log10 CFU g-1 of feces). While most isolates were susceptible to amoxicillin-clavulanic acid, imipenem, and metronidazole, resistance to tetracycline (12%), clindamycin (35%), and cefotaxime (35%) was observed. Distinct species-specific resistance included linezolid (Clostridium argentinense), chloramphenicol (Clostridium innocuum), and tigecycline (Paeniclostridium sordellii), and one Robinsonella peoriensis isolate displayed vancomycin resistance. The detection of tet and erm genes corresponded with phenotypic resistance, while β-lactamase activity was uncommon. Although colonizing at low levels, these findings highlight the ecological significance of rarely studied commensal Clostridia and their contribution to the neonatal resistome, acting as underappreciated reservoirs of AMR genes during a critical window of microbiome assembly.