Metabolic responses of indigenous bacteria in chicken faeces and maggots to multiple antibiotics via heavy water labeled single-cell Raman spectroscopy

The use of maggots derived from chicken faeces as fish diets might serve as a vehicle for the widespread of multiple antibiotic resistant bacteria (ARB) in the environment. Heavy water labeled single-cell Raman spectroscopy (D₂O-Raman) was applied to detect the metabolic responses of indigenous bacteria in chicken faeces and maggots to different concentrations of combined colistin, kanamycin, and vancomycin. By incubating the samples with D₂O and antibiotics, metabolically active bacterial cells to antibiotics were distinguished from those inactive by the exhibition of C-D Raman band. Using the C-D band as a universal metabolic biomarker, 96% and 100% of cells in chicken faeces and maggots were revealed to be metabolically active to 1 × minimum inhibition concentration (MIC) of the afore-mentioned antibiotics. A noticeable decrease in the percentage of active cells from 96% to 76% in faeces and 100% to 93% in maggots was observed at 5 × MIC of antibiotics. However, these ratios were still far above that obtained from the same faeces (1.84%) and maggots (0.51%) samples using a cultivation method, indicating the wide presence of nongrowing but metabolically active bacterial cells under antibiotic treatment. Conclusively, the culture-independent D₂O-Raman approach detected and quantified a large portion of metabolically active indigenous bacteria to multiple antibiotics in their native environments, illustrating the great potential risks of these active cells to spread antibiotic resistance via food chain.