Quantification of class 1 integron abundance in natural environments using real-time quantitative PCR

Abstract

Integrons are bacterial genetic elements capable of capturing and expressing potentially adaptive genetic material. Class 1 integrons constitute the most intensely studied group of these elements to date, mainly due to their well-established role in the acquisition and dissemination of antibiotic resistance genes in clinical environments. However, virtually nothing is known about the distribution or abundance of class 1 integrons outside of the clinical context. Here we develop a SYBR Green-based real-time quantitative PCR assay capable of quantifying the abundance of class 1 integrons in environmental samples. It was shown that the abundance of the intI1 gene in creek sediment correlates with ecological condition, implying that class 1 integrons provide selective advantages relevant to environmental pressures other than the use of antibiotics. By comparing the quantities of intI1 and 16S rRNA gene in each sample, it was demonstrated that approximately 2.7% of cells potentially harbour a class 1 integron. These findings suggest that class 1 integrons are widespread in natural environments removed from clinical settings and occur in a broader range of host organisms than had previously been assumed on the basis of culture-dependent estimates.

Temporal changes of antibiotic-resistance genes and bacterial communities in two contrasting soils treated with cattle manure

“…Abundance of the intI1 gene (an integrase gene of class 1 integrons), tnpA (a transposase gene of the IS6 family transposons) was quantified on a Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad) using the primer sets HS463a/HS464 (Hardwick et al 2008) and tnpA-04F/tnpA-04R (Zhu et al 2013;Wang et al 2014), respectively. The total bacterial 16S rRNA gene was also quantified using the BACT1369F/PROK1492R with the probe TM1389F (Suzuki, Taylor and DeLong 2000).…”

“…Integrons possess a site specific recombination system that could capture and express mobile gene cassettes (Heuer, Schmitt and Smalla 2011a), and they were reported to often localize in broad-host-range IncP-1ε plasmids with a wide distribution in agricultural systems that further facilitates their mobility potential (Heuer et al 2012;Jechalke et al 2014c;Wolters et al 2015). The critical roles of integrons in the dissemination of ARGs in environments have been highlighted by cultivation methods and metagenomics approaches (Hardwick et al 2008;Stalder et al 2012;Gillings et al 2015). Class 1 integrons have been extensively studied due to their widespread distribution in Gram-negative bacteria of clinical importance (Stalder et al 2012), and the intI1 genes were reported to be very common in soils and related ecosystems (Gillings et al 2008;Jechalke et al 2014c).…”

Occurrence and composition of class 1 and class 2 integrons in clinical and environmental O1 and non-O1/non-O139 Vibrio cholerae strains from the Brazilian Amazon

“…In this context, class 1 integrons retain the gene cassettes that confer adaptive advantages to environmental pressures (Hardwick et al 2008).…”

Real-time PCR methods for quantitative monitoring of streptomycin and tetracycline resistance genes in agricultural ecosystems

“…When the antibiotic resistance gene was not detected in the sample no RQ value was calculated. The function of the endogenous control was to normalise the quantity of DNA in each of the samples (Hardwick et al, 2008), thus ensuring that the RQs of the target antibiotic resistance genes were not biased by differences in the quantity of bacteria or DNA present in the samples. Standard curve generation and delta-delta Ct values were calculated using Applied Biosystems software package.…”

“…In order to normalise the data the 16S rRNA gene was chosen. This gene has been previously used as the endogenous control gene in qPCR experiments (Hardwick et al, 2008;Zhang et al, 2000). As the genomes of all bacteria have not as yet been sequenced the exact number of copies of 16S rRNA genes per cell is currently unknown.…”