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.