Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

1. Collection of flies

1. Collect individual flies using sterile entomological sweep nets. Put the nets in a cooler and transfer them to the lab.

2. Dissection of Flies

1. Immobilize aseptically collected flies by placing them at -20 °C for 5 – 7 min.
2. Using sterile forceps place one fly in a sterile 2 ml tube containing 1 ml of pre-warmed (37 °C) buffered peptone water (BPW). Mix the tube gently by inversion for 2 min. It is essential that the whole body of the fly be in contact with the media so that the microbiota present on the body surface (S) of the fly will be transferred to the BPW (BPW-S). Label the tube with a number and body part of the fly (i.e., 1S).
   NOTE: Please see Table of Specific Reagents/Equipment for a detailed description of materials and reagents mentioned in this protocol.
3. Using sterile forceps remove the fly from the BPW-S media and transfer it to an empty and clean 2 ml tube to surface disinfect the fly. Incubate the tube containing the BPW-S media at 37 °C while performing the disinfection and dissection protocol.
   1. Surface-disinfect the fly by immersing it in 1 ml of 70% ethanol for 1 min, followed by a rinse with sterile distilled water before immersing it in 1 ml of freshly prepared 0.05% (v/v) bleach solution. Rinse 3 times with sterile distilled water. Transfer water from the last rinse to an autoclaved 2 ml tube.
      NOTE: Discard the liquid each time by using a 1,000 µl micropipette or by inverting the tube, making sure the fly remains inside the tube. Mix gently by inversion at each step of the surface-disinfection process.
   2. To evaluate the effectiveness of the disinfection process, transfer 100 µl of the water from the last rinse to a trypticase soy agar (TSA) plate and spread using a sterile L-shaped disposable spreader. Incubate the plate at 37 °C for 24 hr. After incubation, register the presence of any bacterial colonies.
      NOTE: The presence of bacterial colonies on the TSA plates indicates an inefficient surface-disinfection process. If this occurs, the presence of foodborne pathogens should only be reported on the body surface of the fly because cross-contamination between the body surface and the alimentary canal cannot be ruled out.
4. After surface-disinfecting the fly, transfer it to a piece of autoclaved paper towel to remove excess water and then to a sterile 60 mm disposable Petri dish.
5. Place the Petri dish under a dissecting scope and identify the fly to the species level using dichotomous keys for dipteran families^20,21.
6. Using autoclaved fine tip forceps gently pull the anus and the whole alimentary canal (A) out of the fly and aseptically transfer it to another sterile 2 ml tube containing 1 ml of pre-warmed (37 °C) BPW with 0.5 mm zirconia/silica beads (BPW-A). Label the tube with the same number selected for the individual fly and the body part of the fly (i.e., 1A).
7. Mix the tube containing the BPW-A thoroughly for 5 – 10 min using a cell disruptor. Incubate at 36 ± 1 °C while performing the rest of the protocol.
8. To voucher and/or store the specimen for long-term, place the remainder of the fly in a clean 2 ml tube and add 1 – 2 ml of 95% ethanol.

3. Primary and Secondary Enrichment

1. Label all primary and secondary enrichment tubes containing media according to the sample number and the body part of the fly.
2. Under a sterile hood, transfer 300 µl of BPW-S (surface) to sterile 2 ml tubes containing the following media:
   1. For Salmonella, use 1 ml of pre-warmed (42 °C) BPW. Incubate in a recirculating water bath at 42.5 °C for 22 – 24 hr. For secondary enrichment, transfer 100 µl of enriched BPW to 400 µl of pre-warmed (37 °C) brain heart infusion (BHI) broth previously placed in sterile cluster tubes. Incubate at 37 °C for 3 hr.
   2. For Cronobacter, use 1 ml of pre-warmed (37 °C) BPW with novobiocin (10 mg/L; Wallace, M., personal communication). Alternatively, use 1 ml of the R & F Enterobacter sakazakii enrichment broth with supplement (vancomycin and cefsulodin) as primary enrichment. Incubate at 37 °C for 22 – 26 hr. For secondary enrichment, transfer 100 µl of enriched BPW with novobiocin to 400 µl of pre-warmed (37 °C) BHI broth previously placed in sterile cluster tubes. Incubate at 37 °C for 3 hr.
   3. For L. monocytogenes, use 1 ml of freshly prepared RT 24 Listeria enrichment broth (24 LEB) with selective supplement. Incubate at 37 °C for 44 ± 5 hr. No secondary enrichment is required for the detection of L. monocytogenes.
   4. Repeat steps 3.2.1 – 3.2.3 above using the tube labeled as BPW-A.

4. Preparation of the PCR-Based System for Amplification and Detection of the Target Foodborne Pathogen

Steps 4-8 use a commercial PCR cycler/detector system, a computer workstation, and ready-to-use kits to screen for Salmonella (Salmonella 2 standard assay kit), Cronobacter species (E. sakazakii standard assay kit), and Listeria monocytogenes (L. monocytogenes 24E assay kit). Standard assays use PCR end-point detection. Each kit contains PCR-ready tablets with an intercalating dye that emits a fluorescence signal when binding to double-stranded DNA. The signal is captured during the detection phase of the PCR system program, generating a melting curve that is interpreted by the software as positive or negative.

1. Prepare reagents and equipment as specified by the manufacturer’s protocol per each target foodborne pathogen.
   NOTE: The protocols for detecting Salmonella and Cronobacter require a one-step lysis procedure whereas the protocol for detecting L. monocytogenes requires a two-step lysis procedure (see sections 5 and 6, respectively).
2. Turn on the automated heating block selecting the specific program for the target pathogen. Alternatively, if the heating blocks are manual, set temperatures to 37 °C (for Salmonella, Cronobacter spp., and L. monocytogenes) or to 55 ± 2 °C (for L. monocytogenes part 2 of lysis – see step 6.2) and 95 ± 3 °C.
3. Make sure that the cooling blocks have been refrigerated O/N, otherwise chill them at 2 – 8 °C for at least 2 hr.
4. Using the computer software of the PCR-based detection system, create a rack file following manufacturer’s instructions.
5. Label and arrange cluster tubes containing the lysis reagent in the rack, according to the rack file.
6. Initialize the PCR-based detection system instrument.

5. Perform Lysis for the Detection of Salmonella and Cronobacter

1. Prepare the lysis reagent by adding 150 µl of protease to one 12 ml bottle of lysis buffer.
2. Transfer 200 µl of lysis reagent to each of the previously labeled cluster tubes.
   NOTE: Cluster tubes containing the lysis reagent can be stored at 2 – 8 °C for up to 2 weeks.
3. Using long pipette tips, transfer 20 µl of secondary enriched samples (see steps 3.2.1 and 3.2.2) to corresponding cluster tubes containing 200 µl of lysis reagent. Use new pipette tips for each sample.
   NOTE: Keep tubes from primary and secondary enrichment in the refrigerator (Salmonella) or at RT (Cronobacter) for further confirmation analysis of PCR-positive/ negative samples.
4. Prepare negative controls by adding 20 µl of sterile BHI media to cluster tubes containing 200 µl of lysis reagent.
5. Prepare positive controls by adding 20 µl of O/N bacterial cultures (grown in BHI) of any known Salmonella or Cronobacter strain to cluster tubes containing 200 µl of lysis reagent.
6. Cap cluster tubes and secure tightly using the capping tool.
7. Place the rack of cluster tubes in the automated heating block after selecting the specific program for the target pathogen. Alternatively, incubate cluster tubes at 37 ± 2 °C for 20 min, followed by incubation at 95 ± 3 °C for 10 min. Finally, transfer the cluster tubes to cooling blocks (2 – 8 °C) for 5 min.
   NOTE: Cluster tubes containing the lysate can be stored at -20 °C for up to 2 weeks.

6. Perform Lysis for the Detection of L. monocytogenes

1. Perform part one of lysis as follows:
   1. Add 1.8 ml of sterile deionized water to the bottle of fully thawed lysing agent 1.
      NOTE: Store lysing agent 1 at 2 – 8 °C until ready to use. After opening and diluting, store at RT (20 – 30 °C) for up to 6 months.
   2. Combine lysing agents 1 and 2 in a 4:1 ratio (40 µl of diluted lysing agent 1 and 10 µl of lysing agent 2 per each sample). Transfer 50 µl of the combined lysing agents to cluster tubes. Use the mixture within 4 hr.
   3. Add 500 µl of primary enriched sample (see step 3.2.3) to the cluster tube containing the 50 µl of the combined lysing agents.
   4. Prepare a negative control by adding 500 µl of sterile 24 LEB to 50 µl of the combined lysing agents.
   5. Prepare a positive control by adding 500 µl of O/N L. monocytogenes culture grown in 24 LEB to 50 µl of the combined lysing agents.
   6. Cap the cluster tubes, mix gently and place in heating block at 37 ± 1 °C for 30 min.
      NOTE: Keep tubes from primary enrichment in the refrigerator for further confirmation analysis of PCR-positive/negative samples.
2. Perform part 2 of lysis as follows:
   1. Prepare the lysis reagent as instructed in steps 5.1 and 5.2.
   2. Using long pipette tips transfer 20 µl of part one lysate to cluster tubes containing 200 µl of lysis reagent. Use new pipette tips for each sample.
   3. Cap cluster tubes and secure tightly using the capping tool.
   4. Place cluster tubes in automated heating block selecting the specific program for L. monocytogenes. Alternatively, incubate cluster tubes at 55 ± 2 °C for 30 min, followed by incubation at 95 ± 3 °C for 10 min. Finally, transfer the cluster tubes to cooling blocks (2 – 8 °C) for 5 min.
      NOTE: Cluster tubes containing the lysate can be stored at -20 °C for up to 2 weeks.

7. Hydrate PCR-Ready Tablets

1. Select a chilled (4 °C) PCR cooling block and place a PCR tube rack over the insert.
2. Place corresponding PCR tubes containing the PCR-ready tablets (included with each kit) for the target foodborne pathogen in the holder, according to the rack file.
3. Using the decapping tool, carefully remove the caps from PCR-tubes. Discard the caps and verify that each tube contains a tablet.
4. Transfer 50 µl (for Salmonella and Cronobacter) or 30 µl (for L. monocytogenes) of lysate to specific PCR tubes. Use new optical caps and secure tightly onto the PCR tubes using the capping tool.
   NOTE: After adding the lysate to PCR-ready tablets, samples must remain chilled at 2 – 8 °C until loaded into the PCR-based detection system. The PCR tubes can be centrifuged at 2,500 x g for a few seconds to assure that the full volume is in the bottom the tube.
5. Load the PCR tubes into the PCR cycler/detection system instrument by opening the instrument drawer.
6. Place the rack of PCR tubes into the wells in the drawer and check that the tubes are seated correctly.
7. Close the drawer and initiate the program as described by the manufacturer’s protocol.
   NOTE: The PCR-based instrument has preset cycling parameters for each foodborne pathogen.
8. Verify that the PCR cycling status bar displays a blue bar indicating that the amplification portion of the program is running.
   NOTE: For standard PCR assays, the processing time of the full program (amplification and detection) takes approximately 3 – 3.5 hr to complete.

8. Review Results

1. After processing is complete, follow the screen prompts from the PCR-based system instrument to remove samples and review results.
2. If the target foodborne pathogen is present in the sample (either the surface or the alimentary canal of the fly) the well is red with a ‘plus’ sign (positive). If the pathogen is absent, the well is green with a ‘minus’ sign (negative).
3. If the well is yellow with a red bar across the center, it indicates a signal error.

9. Isolation of Bacterial Pathogens from PCR-Positive Results

1. Select tubes from the primary (for L. monocytogenes) or secondary (for Salmonella and Cronobacter) enrichment of those samples that were PCR-positive. Also, randomly select 3 – 5% of samples that were PCR-negative and proceed as follows:
2. For Salmonella:
   1. Add 100 µl of the secondary enrichment media to 10 ml of Rappaport-Vassiliadis (RV) medium and to 1 ml of tetrathionate (TT) broth. Incubate tubes at 42.5 °C in a recirculating water bath for 22 – 24 hr.
   2. After incubation, streak a 3 mm loopful (10 µl) of each, RV and TT media on bismuth sulfite (BS) agar, xylose lysine desoxycholate (XLD) agar, and Hektoen enteric (HE) agar. Incubate plates at 35 ± 1 °C for 22 – 24 hr.
   3. After incubation, examine plates for the presence of typical Salmonella colonies on each media. If no isolated colonies can be obtained after several sub-culturing steps, consider the sample as negative and report as a false positive for the PCR-based system.
      NOTE: For typical Salmonella colonies on specific media see ²². Select five presumptive typical Salmonella colonies and subculture them on BS, XLD, or HE until pure cultures of isolated/single colonies are obtained.
   4. Select one pure colony and identify presumptive Salmonella by using biochemical commercial tests such as the VITEK 2 identification card or API biochemical identification system, following the manufacturer’s instructions.
3. For Cronobacter:
   1. Streak a 3 mm loopful (10 µl) of the secondary enrichment media on two plates of chromogenic culture media such as R&F Enterobacter sakazakii (Cronobacter) chromogenic plating medium, and/or ChromID Sakazakii Agar. Incubate plates at 35 °C for 22 – 24 hr.
   2. After incubation, examine plates for the presence of typical Cronobacter colonies (blue-black to blue-gray). Select 5 presumptive Cronobacter colonies and subculture them onto R&F Enterobacter sakazakii (Cronobacter) chromogenic plating medium, ChromID Sakazakii Agar, or TSA until pure cultures of isolated/single colonies are obtained.
      NOTE: If no isolated colonies can be obtained after several sub-culturing steps, consider the sample as negative and report as a false positive for the PCR-based detection system.
   3. Select one pure colony and identify presumptive Cronobacter by using biochemical commercial tests such as the VITEK 2 identification card or API 20E biochemical identification system, following the manufacturer’s instructions.
4. For L. monocytogenes:
   1. Streak a 3 mm loopful (10 µl) of the primary enrichment media on two plates of Brilliance Listeria agar (BLA). Incubate plates at 36 ± 1 °C for 22 – 26 hr.
   2. After incubation, examine plates for the presence of presumptive L. monocytogenes (blue-green) colonies. Select 5 presumptive L. monocytogenes colonies and subculture them on BLA until obtaining pure cultures of isolated/single colonies. Re-incubate negative plates at 36 ± 1 °C for an additional 22 – 26 hr.
   3. Select one pure colony and identify presumptive L. monocytogenes by using commercial biochemical tests such as the VITEK 2 identification card or API Listeria biochemical identification system, following the manufacturer’s instructions.
5. Presumptive foodborne pathogens isolated from insects should be further confirmed and serotyped in a reference laboratory.