Subinhibitory Levels of Oxytetracycline in Earthworm Meal Significantly Boost Resistance-Mutation Rates in Bacillus spp. within the Gut of Eisenia fetida

Aims: To estimate changes in the number of oxytetracycline -resistant strains, a dynamic population of Bacillus in the gut of Eisenia fetida cultured in processed-cow-dung (PrCD) supplemented with subinhibitory amounts of oxytetracycline, was studied.

Study Design: Sterile water containing oxytetracycline was sprayed over PrCD at concentration of 0.012 µg per mg dehydrated PrCD per spraying. The number of oxytetracycline-resistant Bacillus spp. microbiota of E. fetida’s gut was compared to the number of oxytetracycline-resistant Bacillus spp. in its feed (PrCD). For this purpose, Luria Agar (LA) and Bacillus Agar (BA) plates amended with or without oxytetracycline were used.

Place and Duration of Study: The study was carried out in the Omics Laboratory of Department of Biotechnology, University of North Bengal situated in Darjeeling district of West Bengal, India during 2017-18.

Methodology: E. fetida gut content and PrCD samples were collected on different days of the experiment for bacteriological analysis. Dilution plating on LA yielded the total number of cultivable heterotrophic bacteria. LA plates amended with oxytetracycline (15 µg/ml) were used to acquire the fraction of heterotrophic bacteria resistant to oxytetracycline. BA plates were also utilized to obtain the fraction of resistant Bacillus spp. population. The frequency of mutation was determined using a conventional formula.

Results: In the gut of E. fetida reared in PrCD supplemented with sub-inhibitory concentration of oxytetracycline, a rise in the oxytetracycline-resistant Bacillus population was observed. On day 1, the frequency of oxytetracycline-resistant Bacillus spp. (5×10-8) matched the spontaneous mutation frequency, however higher frequencies on days 2 and 7 (1.6×10-4 and 3.5 x 10-6 respectively) suggested significant dissemination of oxytetracycline resistance in the gut environment.

Conclusion: Subinhibitory oxytetracycline concentrations in earthworm diet had a significant effect on mutation rates, showing that evolutionary forces on the gut microbiota may be determining their responses to antibiotic stress.