Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

Summary

This protocol describes the detection of class 1 integrons and their associated gene cassettes in foodstuffs.

Abstract

Antibiotic resistance is one of the greatest threats to health in the 21^st century. Acquisition of resistance genes via lateral gene transfer is a major factor in the spread of diverse resistance mechanisms. Amongst the DNA elements facilitating lateral transfer, the class 1 integrons have largely been responsible for spreading antibiotic resistance determinants amongst Gram negative pathogens. In total, these integrons have acquired and disseminated over 130 different antibiotic resistance genes. With continued antibiotic use, class 1 integrons have become ubiquitous in commensals and pathogens of humans and their domesticated animals. As a consequence, they can now be found in all human waste streams, where they continue to acquire new genes, and have the potential to cycle back into humans via the food chain. This protocol details a streamlined approach for detecting class 1 integrons and their associated resistance gene cassettes in foodstuffs, using culturing and PCR. Using this protocol, researchers should be able to: collect and prepare samples to make enriched cultures and screen for class 1 integrons; isolate single bacterial colonies to identify integron-positive isolates; identify bacterial species that contain class 1 integrons; and characterize these integrons and their associated gene cassettes.

Introduction

The discovery of antibiotics was one of the greatest scientific achievements of the 20^th century. However, the use and abuse of antibiotics has led to the rapid evolution of antibiotic resistant bacteria, and these now pose a serious threat to public health in the 21^st century. The rise of bacterial strains resistant to most treatment options raises the possibility we are entering an era where antimicrobial drugs are no longer effective^1,2.

The genetic machinery that confers antibiotic resistance is an ancient system, predating humans and antibiotic selection pressures by millions of years³. Mobile genetic elements, such as plasmids, transposons, genomic islands, integrative conjugative elements and integrons can disseminate antibiotic resistance genes (ARG) both within and between bacterial species⁴. Of these, integrons have played a central role in the spread of ARG, despite the fact that they rely upon plasmids and transposons for mobilization and insertion into bacterial genomes⁵. Integrons capture gene cassettes using an integron-integrase, and then express cassettes using an integron encoded promoter^6,7 (Figure 1). Integron gene cassettes are small mobile elements consisting of single open reading frames (ORF) whose products can confer resistance to antibiotics or disinfectants⁸. Class 1 integrons are the integrons most commonly recovered from clinical isolates⁵, where they have collectively acquired over 130 different antibiotic resistance gene cassettes⁹.

The spread of class 1 integrons into human-associated commensal and pathogenic bacteria generates human waste streams that contain large numbers of these genetic elements¹⁰. An estimated 10¹⁹ bacteria that contain class 1 integrons are released via sewage sludge each year in the United Kingdom¹¹. It is therefore not surprising that class 1 integrons conferring antibiotic resistances are now being detected in microbiota of wild birds, fish, and other native wildlife^12-14. Releasing integrons back into the environment poses a significant public health threat, since acquisition of new gene cassettes and complex rearrangements with other mobile elements continues to occur, particularly in sewage treatment plants and other water bodies^15-18. The natural environment then becomes a fertile recruiting ground for new resistance determinants and opportunistic pathogens^19,20. Novel integron-containing bacteria and new ARGs can circle back into the human community through contaminated water and food^21,22. Surveillance of environmental ARGs is a key strategy for understanding and managing antibiotic resistance in the future²³. In particular, attention should be paid to foodstuffs that are eaten raw or lightly cooked, since these present the greatest threat for transmission of new mobile elements and pathogens.

In this protocol, a streamlined approach for detecting, identifying and characterizing class 1 integrons and their associated gene cassettes in foodstuffs are outlined (Figure 2). Using a combination of culturing and polymerase chain reaction (PCR), integrons can be rapidly detected in complex bacterial communities and individual isolates. Methods for identifying the species of bacteria and the conformation and identity of the integron-associated gene cassettes are given. The method is suitable for a broad range of plant and animal foods, and examples of typical workflows are given for each of these food types.

Protocol

Foodstuffs that are eaten raw or lightly cooked are of most concern for human health. Examples include salad vegetables, fruit, shellfish and crustaceans.

1. Sample Collection

1. Collect samples under conditions that minimize contamination, and stored in separate, clean bags during transport. Once collected, samples should be stored at 4 °C and processed within 24 hr.

2. Enriched Culture Preparation

1. Fruits and vegetables:
   1. Place approximately 10 g of material in a durable plastic bag. If processing larger material, concentrate on the skin of the fruit or vegetable. Add 90 ml 0.1 M sodium phosphate buffer, pH 7.0 and process in a paddle blender for 30 sec. Alternatively, the material can be manually agitated if a paddle blender is not available.
   2. Collect the liquid into two 50 ml Falcon tubes. Centrifuge tubes at 4,000 x g for 7 min to pellet bacteria.
   3. Carefully remove the supernatant and suspend the pelleted bacteria in 30 ml of sterile Luria Bertani (LB) broth.
   4. Incubate on a shaker O/N, holding one tube at 25 °C and the other at 37 °C. Cultures should be shaken at 200 oscillations per minute (opm). Store cultures at 4 °C until required.
2. Seafood:
   1. Dissect out the stomach (oysters) or digestive tract (prawns) using sterile forceps and tweezers and place into a sterile 1.5 ml tube containing 200 µl of 0.1 M sodium phosphate buffer, pH 7.0. Macerate the sample to create a homogenate.
   2. Dispense 5 ml of LB broth into two sterile 5 ml tubes, and inoculate each with the seafood homogenate.
   3. Incubate on a shaker O/N, holding one tube at 25 °C and the other 37 °C. Cultures should be shaken at 200 opm. Store cultures at 4 °C until required.

3. Screening Cultures for Integrons

NOTE: Standard PCR protocols are used throughout this methodology, using buffers supplied with the enzyme, and a final MgCl₂ concentration of 2.5 mM. If required, Lorenz (2011)²⁴ has outlined PCR optimization and troubleshooting methods in an earlier issue of this journal.

1. Boiled DNA preparation:
   1. Dispense 100 µl of enriched culture into a sterile 0.5 ml PCR tube. Heat the sample to 99 °C for 10 min, using a water bath or heat block. Snap chill on ice for 2 min.
   2. Microcentrifuge at 14,000 x g for 5 min to pellet cell debris. Return to ice. DNA samples can be frozen at -20 °C until required, but must be thawed on ice.
2. Screening for class 1 integrons using PCR:
   NOTE: Mixed cultures from the O/N incubation are screened for class 1 integrons using the HS463a/HS464 PCR protocol (Table 1).
   1. Thaw all PCR reagents on ice, away from any potential sources of contaminating DNA. Make up 48 µl PCR mastermix per sample, including negative controls (Table 2). Dispense 48 µl of PCR master mix to each tube, using barrier tips.
   2. Add 2 µl of thawed DNA to individual PCR tubes using barrier tips. Run appropriate program in the PCR thermocycler.
   3. To assess the results of the amplification, electrophorese 7 µl of PCR product on a 2% agarose gel poured and run in TBE buffer (90 mM TRIS-borate, 2 mM EDTA). Include a molecular weight marker, such as a 100 bp ladder.
   4. Post stain the gel with a DNA stain, and visualize using a UV transilluminator. Check that there has been no amplification in the negative control. A strong band at around 470 bp for any particular sample indicates that the mixed culture contains bacteria that carry a class 1 integron. Pure colonies will now be isolated from any mixed culture that returned a positive PCR.

4. Screening of Single Colonies for Class 1 Integrons

1. Serial dilutions and spread plates:
   NOTE: To isolate single colonies from the PCR-positive mixed cultures, the mixed culture is serially diluted 10-fold in sodium phosphate buffer, and then plated to recover single colonies.
   1. Add 1 ml of mixed culture to 9 ml of 0.1 M sodium phosphate buffer, pH 7.0 and mix by inversion. Add 1 ml of the dilution to a further 9 ml of phosphate buffer, and repeat until a serial dilution of 10^-8 is reached.
   2. Spread 100 µl of the 10^-4 to 10^-8 dilutions onto LB agar, in duplicate. Incubate the plates O/N at the same temperature used for the initial mixed culture. Plates can then be stored at 4 °C until required, however it is best to process the single colonies fairly promptly.
2. Single colony selection and boiled DNA preparation:
   1. Pick single colonies from the serial dilution plates, selecting as many different colony types as possible, using criteria such as colony size, shape and color. Use sterile toothpicks to select single colonies from the spread plate.
   2. Touch the toothpick to the colony, and then transfer into a PCR tube containing 100 µl of sterile water. If large numbers of colonies are to be screened, then colonies can be prepared in a microtiter tray. Spin the toothpick between the fingers to dislodge some of the cells into the water.
   3. Using the same toothpick, inoculate an LB plate with a streak of the colony. Large numbers of colonies can be stored on labelled plates if each streak is about 1 cm long. Repeat steps 1 to 3 until you have selected an appropriate number of isolates.
   4. Incubate the LB plates O/N, at the same temperature as that used for the initial enriched culture. Cultures can then be stored at 4 °C until required.
   5. For preparing DNA from the bacterial suspensions, follow the steps as outlined in section 3.1. For screening of pure culture lysates, perform the HS463a/HS464 PCR as outlined in section 3.2. Isolates that return a positive PCR will be used for preparation of genomic DNA (section 5), and for further analyses.

5. Genomic DNA Extraction of Integron-positive Single Colonies Using Bead Beating²⁵

1. Inoculate 5 ml of LB broth with a sample of the pure culture isolated in section 4.2. Incubate O/N, shaking at the same temperature that was used for the initial enriched culture.
2. Pellet cells at 4,000 x g for 7 min, then remove the supernatant. Resuspend the pelleted cells in 1 ml CLS-TC solution in a Lysing Matrix E fast prep tube. Using a bead beating machine, process the sample at 5.5 m/sec for 30 sec.
3. Centrifuge tube at 14,000 x g for 5 min and recover 700 µl of supernatant into a sterile 1.5 ml tube.
4. Add 700 µl of binding matrix diluted 1:5 with 6 M guanidinium thiocyanate and mix at RT for 5 min. Centrifuge at 14,000 x g for 1 min, discard supernatant.
5. Add 800 µl salt ethanol wash (70% ethanol, 0.1 M sodium acetate) and vortex until the pellet is fully resuspended. Wash for 5 min at RT, centrifuge at 14,000 x g for 1 min and discard supernatant. Ensure all supernatant has been removed, using a micropipette if necessary, then allow to air dry for 5 min.
6. Resuspend the pellet matrix in 200 µl TE (10 mM Tris-HCl, 1mM EDTA, pH 7.6) by pipetting up and down. Incubate at RT for 3 min. Centrifuge at 14,000 x g for 3 min and transfer 160 µl of the eluted DNA into a sterile tube. Run an aliquot of the genomic DNA extraction on a 2% w/v agarose gel to check the yield and integrity of the DNA extraction. The purified genomic DNA is stored at -20 °C and defrosted on ice when required for PCR testing.

6. Diagnostic PCRs and DNA Sequencing

NOTE: The genomic DNA prepared in section 5 will be used for all diagnostic PCRs, and to confirm the positive test for class 1 integrons.

1. Thaw genomic DNA on ice, briefly vortex the DNA sample and pulse spin at 14,000 x g for 20 sec. Repeat the PCR for the class 1 integron-integrase gene using HS463a/HS464 (Table 1), confirming that the isolate is positive for the 471 bp amplicon (Section 3.2).
2. Identification of positive cultures to species level is achieved by analysis of the small subunit rRNA gene (16S rDNA). Amplify the 16S gene using primers f27/r1492 (Table 1). Check the PCR products using electrophoresis. All bacterial targets should generate a 16S amplicon of about 1450 bp.
3. Sequence the 16S rRNA gene amplicon to determine species identity, but since there are likely to be multiple positive PCRs from step 6.2, and many of these positives will be the same bacterial species, we will first distinguish different species using restriction digestion of the 16S PCR product.
   1. Digest an aliquot of the 16S PCR with the restriction enzyme Hinf1. Any enzyme that recognizes 4 bp cleavage sites will do, but Hinf1 generates good diversity from 16S targets, and is a reliable, inexpensive enzyme.
4. Set up a 30 µl restriction digest mastermix (Table 2) in a sterile tube and add 20 µl of un-purfied 16S PCR product. Incubate in a 37 °C water bath O/N. Check the digest on a 2% agarose gel.
5. Score the digestion patterns produced by Hinf1. Isolates with the same 16S restriction profile are likely to be the same species. Rather than sequencing every 16S PCR product, sequence at most three representatives of each Hinf1 restriction pattern.
6. Purify the 16S PCR products using a commercial kit. A single stranded exonuclease kit can be used, but column based or precipitation methods also work well to remove unincorporated primers and single stranded DNAs. Sequence the 16S amplicons using primer r910 (Table 1), setting up the reactions as specified by the sequencing facility.
7. Use the resulting DNA sequence to interrogate the NCBI DNA database with the blastn function (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Greater than 97% nucleotide identity is usually taken as species identity.

7. Mapping and Characterization of Integrons and Cassette Arrays

NOTE: Class 1 integrons emanating from human-dominated ecosystems are likely to be the most common integrons in all your samples. These integrons all have a recent single origin, and therefore have a highly conserved DNA sequence⁵.

1. Differentiate between a class 1 integrons emanating from human sources and those that occur naturally in environmental samples by performing a PCR with primer set intI1F165/intI1R476¹⁰ (Table 1).
   NOTE: Class 1 integrons from clinical or commensal bacteria will generate a ~300 bp product, while environmental class 1 integrons will not generate any product.
2. Characterize the clinical integrons by amplifying the cassette array with primer set HS458/HS459. This PCR will amplify the region between intI1 and the 3’ conserved segment at the terminus of most cassette arrays. Because the number and identity of cassettes is variable, the size of the PCR product will also vary.
3. Purify amplicons and sequence the DNA using each of the amplification primers. Use sequences to interrogate DNA databases using the blastn function (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
4. Ensure naming of cassettes follow standardized nomenclature⁹, since many data depositions for integron gene cassettes use inconsistent or ambiguous names. Give particular attention to novel gene cassettes, since these may encode new phenotypes, including genes that confer increased transmissibility, pathogenicity or virulence^8,21.
   NOTE: Preclinical forms of the class 1 integron still circulate in natural environments. In particular, those that are linked to active Tn402 transposons are of most interest²⁶. The cassette arrays of these integrons will not amplify with primer set HS458/HS459, but can be amplified with the MRG284/MRG285 primer set (Table 1), generating amplicons of variable size dependent on cassette content. PCR products should be purified and DNA sequenced as outlined in 7.2.

Results

Screening of mixed cultures and bacterial isolates for intI1

Primer set HS463a/HS464 PCR can be used to detect the presence of the class 1 integron-integrase gene, intI1 (Figure 1). This primer set works well for detecting intI1 in mixed cultures, and is also used to screen bacterial colonies harvested from spread plates (Figure 2). Positive isolates should generate a single strong band at 471 bp using this primer set (Figure 3A...

Discussion

The identification of integrons and their associated gene cassettes is potentially a key step in predicting the emergence of new opportunistic pathogens, tracking pathways for pathogens into the human food chain, and identifying new resistance and virulence determinants^8,21,26. The aim of this paper was to describe a streamlined approach for screening samples for class 1 integrons, characterizing their cassette arrays and identifying the bacterial species in which they reside. Critical steps in the protocol in...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
GoTaq Colourless Mastermix	Promega	M7132	Used in all PCRs
RNAse (Ribonuclease A from bovine pancreas)	Sigma	R6513-10MG	Used in all PCRs
HinFI restriction enzyme	Promega	R6201	Used to digest 16S rDNA PCR poducts. Enzyme comes with optimal buffer and BSA
100 bp ladder	GE Healthcare	27400701	Used as a size standard on all agarose gels
GelRed DNA stain	Biotium	41003	CAUTION: Personal protection must be worn when handling this material
Guanidinium thiocyanate	Life Technologies	AM9422	CAUTION: Personal protection must be worn when handling this material
CLS-TC Solution	MP Biomedicals	6540409	Resuspension solution used at the begining of the genomic DNA extraction
Lysing Matrix E FastPrep tubes	MP Biomedicals	116914500	Tube required for mechanical disruption of bacterial cell walls. This code is used for packs of 500 tubes, smaller quantities are available.
Binding matrix	MP Biomedicals	116540408	Diluted 1:5 with 6 M guanidinium thiocyanate and used in the genomic DNA extraction method.
Fast Prep machine	MP Biomedicals	Number of options available	MP Biomedicals has a number of FastPrep machines available to purchase. Visit http://www.mpbio.com for more information