Gram-Positive Bacteria Responses to Antibiotics When Incubated in Fecal in Vitro Microcosms from Swine Supplemented with Chlorophyllin

Standardized antimicrobial susceptibility assays use defined culture media and may therefore not fully account for gastrointestinal constituents that influence bacterial responses to antibiotics. This study evaluated whether swine fecal extracts, including extracts associated with dietary chlorophyllin supplementation, altered the antimicrobial susceptibility of multidrug-resistant Gram-positive bacteria. Fecal material collected from pigs fed either a non-chlorophyllin or chlorophyllin-supplemented diet was pooled by dietary group, processed to produce clarified fecal extracts, and incorporated at 20% (vol/vol) into Mueller–Hinton (MH) broth. Minimum inhibitory concentrations (MICs) of chlortetracycline, penicillin, carbadox, tylosin, ceftazidime, and norfloxacin were determined for selected Staphylococcus , Streptococcus , and Enterococcus strains using broth microdilution and compared with MICs obtained in standard MH broth. Fecal extracts altered MICs in a strain- and antibiotic-dependent manner. Extracts from chlorophyllin-fed pigs produced particularly pronounced reductions in susceptibility thresholds for several combinations, including >128- to >256-fold decreases in chlortetracycline MICs for Streptococcus agalactiae , Staphylococcus hyicus , and Enterococcus faecium H 1019 V; >253-fold decreases in penicillin MICs for several strains; and >32- to >256-fold decreases in ceftazidime MICs for four strains. Carbadox MICs were also reduced in several organisms. However, these effects were not universal: tylosin MICs were generally unchanged or increased, and norfloxacin MICs were unchanged or increased in all strains tested. Fecal extracts from non-chlorophyllin-fed pigs also altered MICs, demonstrating that the fecal matrix itself influenced phenotypic antimicrobial susceptibility. These findings indicate that antimicrobial susceptibility measured under standard laboratory conditions may differ substantially in the presence of fecal-derived constituents and support further characterization of the metabolites responsible for these effects.