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. Mobil....

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 ....

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