Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

The overall goal of this procedure is to screen for the presence of bacterial foodborne pathogens from the body surface and the elementary canal of individual flies using a commercially available PCR based detection system. This is achieved by first collecting wild flies from the trash area of restaurants. Individual flies are immersed in nutrient broth to collect microbiota from the body surface.

Then the elementary canal is extracted, placed in another tube containing nutrient broth and disrupted to liberate the bacteria from within the tissue. The next step is to transfer the samples to pathogen specific and enrichment broth for bacterial growth, which will increase the concentration of the bacteria above the detection limits of the assay. Next, the enriched samples are lysed and added to PCR tubes containing ready to use tablets for PCR based detection of the target pathogens.

The final step is to isolate pure bacterial colonies from the samples that were PCR positive by plating the enrichment broth on selective and differential media. Ultimately, the combination of PCR based detection and pure colony isolation is used to assess the prevalence of kob backer, salmonella, and listeria monocytogenes from the elementary canals and the body surfaces of individual wild caught flies eyes. The main advantage of this technique or existing methods like traditional plating, is that it is rapid and reliable, as well as sensitive enough to screen the body surface and the elementary canal of a single fly for the presence of bacterial footwear pathogens.

This method can help answer key questions in the investigation of foodborne illness outbreaks, such as the mechanisms of transmission by insects and the sources of the pathogens. The implications of this technique extend beyond diagnoses of foodborne pathogens from food and environmental samples, and now include the ability to detect those bacterial pathogens from individual insects like flies. To begin place aseptically collected flies in a minus 20 degrees Celsius freezer for five to seven minutes to immobilize them.

Then place one, fly into a sterile two milliliter tube containing one milliliter of pre-warned buffered peptol water known as BPW. Using sterile forceps, gently mix the contents of the tube by inversion for two minutes to ensure that the whole body of the fly contacts the medium. This transfers the microbiota on the surface of the fly to the BPW.

Next, transfer the fly to a new two milliliter tube using sterile forceps surface. Disinfect the fly by immersing it in 70%ethanol and mix gently for one minute, and then remove the ethanol. Rinse the fly with sterile distilled water following the rinse, add freshly prepared 0.05%bleach solution to the tube.

Mix gently for one minute and then remove the bleach. Rinse the fly three times with sterile distilled water to confirm sterilization. Transfer the water from the last rinse to a sterile two milliliter tube and test for contaminating bacteria as described in the text protocol.

Next, place the fly on an autoclave paper towel to remove any excess water. Once dry, transfer the fly to a sterile 60 millimeter disposable Petri dish and place the dish under a dissecting microscope. Carefully pull the anus and the entire elementary canal out of the fly using sterile fine tip forceps.

Next aseptically. Transfer the elementary canal to another two milliliter tube containing one milliliter of prewarm BPW and 0.5 millimeter zirconia silica beads. Use a cell disruptor to mix the tube containing the elementary canal for five to 10 minutes to release bacteria from within the tissue.

Place the remainder of the fly in a clean two milliliter tube containing 95%ethanol for long-term storage at minus 20 degrees Celsius. For primary bacterial enrichment, distribute the BPW containing microbiota from the surface or the elementary canal into three, two milliliter tubes With the appropriate prewarm medium, incubate the tubes at the indicated temperature and time that is required for each pathogen for secondary enrichment of salmonella and kob backer. Transfer 100 microliters of the enriched culture to sterile cluster tubes containing 400 microliters of prewarm brain heart infusion broth.

Incubate the tubes in a heating block at 37 degrees Celsius for three hours. To prepare for cell lysis, add 150 microliters of the kit supplied protease to one 12 milliliter bottle of lysis buffer and mix Transfer 200 microliters of the lysis reagent to each of the labeled cluster tubes. Next, transfer 20 microliters of the secondary enriched samples to the corresponding cluster tubes using long pipette tips for negative controls.

Add 20 microliters of sterile medium for positive controls. Add 20 microliters from overnight bacterial cultures of known salmonella or kob backer strains. Once all of the samples have been added, secure caps onto the cluster tubes with the capping tool.

Next, lice the cells by placing the rack of cluster tubes in the automated heating block and run the specific program for the target pathogen to begin PCR based detection. First, chill a PCR tube rack on a cooling block. Then place the corresponding PCR tubes containing PCR ready tablets for the target foodborne pathogen into the holder.

Carefully remove the caps from the PCR tubes using the decapping tool and discard them. Verify that each tube contains a tablet. Then transfer 50 microliters of lysate to the appropriate PCR tubes.

Secure new optical caps onto the PCR tubes using the capping tool. After assembling the reactions, load the PCR tubes into the PCR cycler detection system. Then run the program and view the results as described in the manufacturer's protocol.

To isolate pure bacterial colonies, select the primary enrichment tubes of samples that were PCR positive. For salmonella, add 100 microliters of the enrichment culture to 10 milliliters of Rappaport vasilis medium, and to one milliliter of tetra anate broth. Incubate the tubes at 42.5 degrees Celsius for 22 to 24 hours after incubation streak, a three millimeter loop full of each medium on bismuth sulfite auger XLD, auger and hector and enteric auger.

Then set the plates in a 35 degree Celsius incubator. After 22 to 24 hours of growth, examine the plates for typical salmonella colonies on each medium streak a positive colony on selective medium, and isolate single colonies. After incubation.

Use one pure colony to confirm the identity of presumptive salmonella using a commercial biochemical test like the API 20 E test for KOB backer streak. A loop full from the primary enrichment tube on two plates of chromogenic culture medium, such as RNF enterobacter Sakaki eye chromogenic plating medium. Incubate the plates at 35 degrees Celsius for 22 to 24 hours, and then examine them for blue, black, or blue gray colonies for listeria monocytogenes streak.

A loop full from the enrichment tube on two plates of brilliance. Listeria agar incubate plates at 35 degrees Celsius for 22 to 26 hours, and then examine them for blue-green colonies. This standardized protocol was tested on 100 wild flies caught from the dumpster area of 10 urban restaurants.

Shown here is the percentage of flies that were confirmed positive for the target pathogens. 22%of the elementary canals and 8%of the body surfaces were positive. For at least one of the three foodborne pathogens evaluated.

The overall presence of foodborne bacteria in the elementary canal of flies was significantly higher than on the body surface. The prevalence of kob backer species on the body surfaces and elementary canals of flies was statistically higher than the prevalence of salmonella and tarica and listeria monocytogenes. However, no statistical differences were observed when performing pairwise comparisons between the body surfaces and elementary canals of flies for each bacterial pathogen After its development.

This technique paved the way for other professionals in the field of foodborne disease outbreak investigations to screen individual flies for the presence of bacterial pathogens and to gather more information about the mechanisms by which foodborne microorganisms are spread. While attempting this Procedure, it's important to remember that confirmation of PCR positive results is part of the standard operating procedure of many laboratories, and for regulatory purposes, it's recommended to isolate the culture for Confirmation following this procedure. Other methods like yp, DNA fingerprinting or whole genome sequencing can be performed in order to answer additional questions such as the strain of the foodborne bacterial pathogen carried by the fly.