Purpose: The development of an in situ crop vessel assay to investigate bacterial growth within fly crops up to 48 h post-ingestion.
Methods: Flies were fasted for 12 h prior to feeding E. coli ATCC 43895 with pEGFP. Flies were fed bacteria suspended in sterile M9 media with 1% glucose, ampicillin and IPTG. Red food coloring was added to confirm that flies had consumed the bacteria. After feeding flies E. coli O157:H7, the flies with red abdomens were chilled at 0°C; 70% ethanol was used to externally disinfect the fly. Crops were removed and maintained in sterile phosphate buffered saline in microtiter plates held at 32°C. For each time point, five crops were homogenized individually using a tissue grinder and bacterial levels (CFU/crop) were monitored using plate counts. Confocal microscopy of intact crops was used to monitor biofilm development.
Results: There was no statistical increase in cell numbers (CFU/crop) over the 48 h incubation period. Microscopy shows that upon prolonged incubation, GFP-expressing E. coli within the crop produced biofilms. This method showed greater reproducibility in studying bacterial interactions with the crop, than using a live fly feeding study.
Significance: This assay is a reproducible in situ assay that can be used to study the biofilm formation of E. coli O157:H7 within the crop of M. domestica. This system will allow further studies to better study the relationship and dissemination of E. coli O157:H7 from the environment to food by house fly.