Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

The overall goal of this procedure is to provide a streamlined approach for detecting class one inter grounds and their associated gene cassettes from foodstuffs. This is accomplished by first making an enriched bacterial culture from your chosen food item. The second step is to screen the culture for the presence of class one inters using PCR and isolate single colonies from the inter positive mixed culture.

After screening the single colonies for the presence of integrons, prepare genomic DNA from the single colonies for diagnostic PCRs to differentiate bacterial species and detect the presence of gene cassettes. The final step is to purify PCR products and submit them for DNA sequence analysis. Ultimately, DNA sequence analysis is used to characterize gene cassettes within a class one inter and identify the species in which they reside.

Antibiotic resistance is one of the major problems facing medical treatment in the 21st century. One of the ways that we can manage antibiotic resistance is to detect and trace antibiotic resistance genes in the environment from humans to the environment and back again. This protocol is going to show you how to detect antibiotic resistance.

Genes in food stuffs that we might eat raw or with minimal processing. The procedure is going to be demonstrated by Leet Waldron, a postdoc working in my laboratory. Foods that are eaten raw or lightly cooked, such as fruits, vegetables, and shellfish are of the most concern for human health.

Only the preparation of enriched cultures from seafood will be demonstrated here. Please refer to the protocol text for the preparation of cultures from fruits and vegetables. To prepare an enriched culture from seafood, first use sterile forceps and tweezers to dissect out the digestive tract from prawns.

Place the dissected material into a sterile 1.5 milliliter tube containing 200 microliters of a sodium phosphate buffer. Macerate the sample using a pestle to create a homogenate and vortex. Briefly dispense five milliliters of LB broth into two sterile five milliliter tubes and inoculate each with the seafood homogenate.

Shake both tubes overnight at 200 OPM incubating one tube at 25 degrees Celsius and the other at 37 degrees Celsius on the following day. Store the cultures at four degrees Celsius until required to recover single colonies from A PCR positive mixed culture. First serially dilute the culture tenfold in sodium phosphate buffer by adding one milliliter of the mixed culture to nine milliliters of sodium phosphate buffer mixed by vortex.

Add one milliliter of the dilution to a further nine milliliters of sodium phosphate buffer and repeat until a serial dilution of 10 to the minus eight is reached plate. The diluted culture spread 100 microliters of the 10 to the minus four to 10 to the minus eight dilution onto LB agar. In duplicate, incubate the plates overnight at the same temperature used for the initial mixed culture on the following day.

Single colonies can be selected from the serial dilution plates. For DNA preparation, make sure all plates and sample tubes are clearly labeled prior to starting. Touch a sterile toothpick to a single colony and then transfer into a PCR tube containing 100 microliters of sterile water.

Spin the toothpick between the fingers to dislodge some of the cells into the water using the same toothpick, inoculate an LB plate with a streak of each picked colony. Large numbers of colonies can be stored on labeled plates. If each streak is about one centimeter long, close the lid immediately to reduce contamination between samples.

Select as many different colony types as possible using criteria such as colony size, shape, and color. Incubate the LB plates overnight at the same temperature as that used for the initial enriched culture. Cultures can then be stored at four degrees Celsius until required to prepare DNA from the bacterial suspensions.

Heat the samples to 99 degrees Celsius for 10 minutes using a thermocycler micro centrifuge at 14, 000 times, G for five minutes at room temperature to pellet cell debris return to ice. Subsequently screen the DNA by PCR with primer's HS 4 63 A and HS 4 64. Following the procedures described in the protocol text, it is important to avoid any potential sources of contaminating DNA when performing the PCR to assess the results of the amplification electro referee.

Seven microliters of each PCR product on a 2%aros gel poured and run in TBE buffer after post staining the gel with the DNA stain. Visualize the DNA using a UV transluminator Isolates that carry a class one integrin will generate a strong single band at 471 base pairs genomic DNA prepared from isolates that return a positive PCR are used for these analyses. Thaw the genomic DNA on ice and briefly vortex and pulse spin at 14, 000 times G for 20 seconds.

The primer sequences and PCR conditions used for diagnostic PCRs are shown in table one in the manuscript. First, repeat the PCR for the class oneone integrase gene to confirm that the isolate is positive for the 471 base pair amplicon. Next, identify the positive cultures to the species level by amplifying the small subunit ribosomal RNA gene 16 S-R-D-N-A.

All bacterial targets should generate a 16 S amplicon of about 1, 450 base pairs since many of these positive cultures will be the same. Bacterial species distinguish the different species by restriction digestion of the 16 SPCR product. The restriction enzyme HIF one is recommended since it generates good diversity from 16 s targets and is a reliable, inexpensive enzyme.

Set up a 30 microliter restriction digest master mix in a sterile tube and add 20 microliters of un purified 16 SPCR product. Incubate in a 37 degree Celsius water bath overnight on the following day, check the digest on a 2%AROS gel selected 16 SPCR products are then purified using a commercial kit and sent to a sequencing facility for DNA sequencing. Use the resulting DNA sequence to interrogate the N-C-B-I-D-N-A database with the Blast N function greater than 97%nucleotide identity is usually taken as species identity.

Mixed cultures antibacterial isolates are screened for the presence of the class one integrase gene by PCR. Using the primers HS 4 63 A and HS 4 64 positive colonies generate a strong single band at 471 base pairs. Genomic, DNA from pure isolates is used for characterization of inter cassette arrays, amplification of cassette arrays from TN 4 0 2 integrons using primers HS 4 58 and HS 4 59 generates variable product sizes dependent on the identity and size of the component cassettes in the array.

Identification of these cassettes requires DNA sequencing Environmental Class one integrons are often embedded in bacterial chromosomes. Their consider rays can be amplified by primer's MG 2 84 and MG 2 85 that target the proximal and most distal recombination sites respectively. This PCR also generates variable product sizes dependent on the identity and size of the component cassettes in the array.

However, cassettes in environmental arrays are unlikely to encode antibiotic resistance. Bacterial species are identified by amplifying 16 S-R-D-N-A and screening the PCR products by digestion. With HIF one individual species will generate distinctive patterns such the isolates of the same species can be easily identified and confirmed by DNA sequencing.

After watching this video, you should now have a good idea of how to isolate Class one ingrams and their antibiotic resistance cassettes from everyday foods.