Impacts of anthropogenic activity on the ecology of class 1 integrons and integron-associated genes in the environment

Abstract

The impact of human activity on the selection for antibiotic resistance in the environment is largely unknown, although considerable amounts of antibiotics are introduced through domestic wastewater and farm animal waste. Selection for resistance may occur by exposure to antibiotic residues or by co-selection for mobile genetic elements (MGEs) which carry genes of varying activity. Class 1 integrons are genetic elements that carry antibiotic and quaternary ammonium compound (QAC) resistance genes that confer resistance to detergents and biocides. This study aimed to investigate the prevalence and diversity of class 1 integron and integron-associated QAC resistance genes in bacteria associated with industrial waste, sewage sludge and pig slurry. We show that prevalence of class 1 integrons is higher in bacteria exposed to detergents and/or antibiotic residues, specifically in sewage sludge and pig slurry compared with agricultural soils to which these waste products are amended. We also show that QAC resistance genes are more prevalent in the presence of detergents. Studies of class 1 integron prevalence in sewage sludge amended soil showed measurable differences compared with controls. Insertion sequence elements were discovered in integrons from QAC contaminated sediment, acting as powerful promoters likely to upregulate cassette gene expression. On the basis of this data, >1 × 10(19) bacteria carrying class 1 integrons enter the United Kingdom environment by disposal of sewage sludge each year.

Figure 1

Gene prevalence in total bacterial population from QAC contaminated reed bed (RB), sewage sludge (SS), pig slurry (PS) and Cotswold soil (CW). Diagonal lines, intI1; stippled bars, qacEΔ1; grey bars, qacE; white bars, qacG and vertical lines qacH. Error bars are s.d. of four biological replicates, each composed of three technical replicates. Prevalence is statistically greater for all genes in bacteria from RB, SS and PS than from CW (χ²-test for comparisons of two proportions; from independent samples).

Figure 2

Gene prevalence in total bacterial population from limed, dewatered sewage sludge (cake), agricultural soil 1, 12 and 24 months after cake application. Control samples were taken from an area adjacent to the agricultural amended plots, which had not received sludge. Diagonal lines, intI1; stippled bars, qacEΔ1; grey bars qacE. Error bars are s.d. of three biological replicates for cake and 10 for amended soils, each composed of three technical replicates. All intI1 prevalences are statistically different from one another.

Figure 3

Location of ISPpu17 (a) and ISUnCu13 (b) relative to intI1 and qacE/qacEΔ1. Large arrows show orientation of ORFs, inverted repeats (IR) in red and underlined in sequences; LH IR, left hand IR, RH IR, right hand IR; part of attI supplied by integron, vertical lined areas; part of attI supplied by cassette, cross hatched areas; attIΔ, attI interrupted by IS; Pc, promoter driving cassette gene expression; RBS, ribosomal-binding site, underlined by dashed line; attI, recombination site, underlined by dashed and dotted line in sequence.

Figure 4

Promoter activity measured in lacZ reporter constructs; Pc, main promoter driving gene cassette expression in class 1 integrons, amplified with Pc promoter forward and Pc promoter reverse primers (Supplementary Table S1; template DNA accession no. FJ663012); qacE, promoter from qacE gene cassette, amplified with qacE promoter forward and reverse (accession no. FJ663012); ISPpu17/qacE, fusion promoter, amplified with ISUnCu13 forward and qacE promoter reverse (accession no. FJ663011); ISUnCu13+qacE, 3′ region of ISUnCu13 and qacE promoter, amplified with ISUnCu13 forward primer and qacE reverse (accession no. FJ663010); pbad, promoter amplified with pBad forward and pBad reverse; neg, negative control with coding region of araC with no promoter amplified by araC forward and reverse primers. Miller Units=1000 × absorbance₄₂₀/(absorbance₅₉₅ × reaction time × volume).