Activation of class 1 integron integrase is promoted in the intestinal environment

Abstract

Class 1 integrons are widespread genetic elements playing a major role in the dissemination of antibiotic resistance. They allow bacteria to capture, express and exchange antibiotic resistance genes embedded within gene cassettes. Acquisition of gene cassettes is catalysed by the class 1 integron integrase, a site-specific recombinase playing a key role in the integron system. In in vitro planktonic culture, expression of intI1 is controlled by the SOS response, a regulatory network which mediates the repair of DNA damage caused by a wide range of bacterial stress, including antibiotics. However, in vitro experimental conditions are far from the real lifestyle of bacteria in natural environments such as the intestinal tract which is known to be a reservoir of integrons. In this study, we developed an in vivo model of intestinal colonization in gnotobiotic mice and used a recombination assay and quantitative real-time PCR, to investigate the induction of the SOS response and expression and activity of the class 1 integron integrase, IntI1. We found that the basal activity of IntI1 was higher in vivo than in vitro. In addition, we demonstrated that administration of a subinhibitory concentration of ciprofloxacin rapidly induced both the SOS response and intI1 expression that was correlated with an increase of the activity of IntI1. Our findings show that the gut is an environment in which the class 1 integron integrase is induced and active, and they highlight the potential role of integrons in the acquisition and/or expression of resistance genes in the gut, particularly during antibiotic therapy.

Fig 1. Bacterial colonization and plasmid stability in the mouse gut.

On day 0, three groups of germ-free mice were inoculated with 10⁸ CFU of MG/pZE1 control strain (carrying p6851 and pZE1 that did not carry the intI1 gene, n = 4) (A), MG/intI1 strain (carrying p6851 and pZE1intI1 allowing the expression of intI1 SOS-regulated, n = 4) (B) or MG/intI1* strain (carrying p6851 and pZE1intI1* allowing the constitutive expression of intI1, n = 3) (C). Bacterial colonization in the mouse gut was monitored by counting the CFU/g of faeces on non-selective medium (MG1656). Plasmid stability was evaluated by counting the CFU/g of faeces on selective media supplemented with kanamycin (p6851) or ampicillin (pZE1, pZE1intI1 and pZE1intI1*). Symbols represent the average of the CFU/g of faeces per day and error bars indicate the SD.

Fig 2. Recombination activity of IntI1 integrase in the mouse gut.

On day 0, two groups of germ-free mice were inoculated with 10⁸ CFU of MG/intI1 (carrying p6851 and pZE1intI1 allowing the expression of intI1 SOS-regulated, n = 4) (A) or MG/intI1* (carrying p6851 and pZE1intI1* allowing the constitutive expression of intI1, n = 3) (B). Recombination activity of IntI1, reflected by the frequency of recombinants (FR), was estimated by determining the frequency of emergence of tobramycin-resistant recombinants, as a result of specific recombination between attC sites located on a synthetic array of two cassettes (attC_aadA7-cat(T4)-attC_VCR2-aac(6’)-Ib*) carried on plasmid p6851. Each symbol represents the FR in the gut calculated from a single mouse exhibiting recombinants. For each sampling day, the average FR is shown as a black horizontal line.

Fig 3. Comparisons of recombination activity of IntI1 and expression of intI1 and SOS regulon genes, in vivo versus in vitro.

(A) Recombination activity of IntI1, reflected by the frequency of recombinants (FR), was estimated by determining the frequency of emergence of tobramycin-resistant recombinants, as a result of specific recombination between attC sites located on a synthetic array of two cassettes (attC_aadA7-cat(T4)-attC_VCR2-aac(6’)-Ib*) carried on plasmid p6851. The FR was calculated in MG/intI1 (carrying p6851 and pZE1intI1 allowing the expression of intI1 SOS-regulated) and MG/intI1* (carrying p6851 and pZE1intI1* allowing the constitutive expression of intI1), in the mouse gut and in 24-h-old-planktonic cultures. Each symbol represents the FR calculated from a single mouse exhibiting recombinants on days 2 and 3 post inoculation (n = 3 for MG/intI1 and n = 4 for MG/intI1*) and from planktonic cultures (n = 28 for MG/intI1 and n = 15 for MG/intI1*). For each condition, the average FR is shown as a black horizontal line. Transcript levels of (B) the intI1 gene in MG/intI1 and MG/intI1* and (C) the SOS regulon genes dinD, recN and sfiA, in MG/intI1 in the mouse gut and in 24-h-old-planktonic cultures are represented. Transcript levels were normalized to the housekeeping gene dxs. Error bars indicate the SD. Differences were determined using the Mann-Whitney U test. *p < 0.05, **p < 0.01 and ****p < 0.0001.

Fig 4. Effect of ciprofloxacin on the induction of the SOS response and the IntI1 integrase expression and activity in the mouse gut.

On day 0, three germ-free mice were inoculated with 10⁸ CFU of MG/intI1 (carrying p6851 and pZE1intI1 allowing the expression of intI1 SOS-regulated). Ciprofloxacin was added to the drinking water of mice on day 11 post inoculation, just after the fecal sampling on that day. (A) Recombination activity of IntI1, reflected by the frequency of recombinants (FR), was estimated by determining the frequency of emergence of tobramycin-resistant recombinants, as a result of specific recombination between attC sites located on a synthetic array of two cassettes (attC_aadA7-cat(T4)-attC_VCR2-aac(6’)-Ib*) carried on plasmid p6851. Each symbol represents the FR in the gut calculated from a single mouse exhibiting recombinants. For each sampling day, the average FR is shown as a black horizontal line. (B) Transcript levels of sfiA and intI1 genes in the mouse gut are represented. Transcript levels were normalized to the housekeeping gene dxs. Error bars indicate the SD. Differences were determined using the Mann-Whitney U test. *p < 0.05.