Class 1 integrons are low-cost structures in Escherichia coli.

Resistance integrons are bacterial genetic platforms that can capture and express antibiotic resistance genes embedded within gene cassettes. The capture and shuffling of gene cassettes are mediated by the integrase IntI, the expression of which is regulated by the SOS response in Escherichia coli. Gene cassettes are expressed from a common Pc promoter. Despite the clinical and environmental relevance of integrons, the selective forces responsible for their evolution and maintenance are poorly understood. Here, we conducted pairwise competition experiments in order to assess the fitness cost of class 1 integrons in E. coli. We found that integrons are low-cost structures and that their cost is further reduced by their tight regulation. We show that the SOS response prevents the expression of costly integrases whose cost is activity dependent. Thus, when an integron is repressed, its cost depends mostly on the expression of its gene cassettes array and increases with Pc strength and the number of cassettes in the array. Furthermore, different cassettes have different costs. Lastly, we showed that subinhibitory antibiotic concentrations promoted the selection of integron-carrying bacteria, especially those with a strong Pc promoter. These results provide new insights into the evolutionary dynamics of integron-carrying bacterial populations.

Expression of the aac(6')-Ib-cr Gene in Class 1 Integrons.

aac(6')-Ib-cr is a plasmid-mediated quinolone resistance gene embedded within a gene cassette, most often within an integron. It confers resistance to quinolones and aminoglycosides. We investigated the role of a 101-bp fragment frequently present upstream of the aac(6')-Ib-cr gene cassette and found that it contributes to the expression of aac(6')-Ib-cr and provides an alternative start codon, confirming the length of the AAC(6')-Ib-cr protein to 199 amino acids.

Effects of antibiotics on Chlamydia trachomatis viability as determined by real-time quantitative PCR.

The objective of this study was to determine the effect of antibiotics on Chlamydia trachomatis viability by using a quantitative real-time PCR assay that measured DNA replication and mRNA transcription of the structural omp1 and omp2 genes, 16S rRNA and the groEL1 gene with and without antibiotics. Ofloxacin, moxifloxacin, azithromycin and doxycycline were tested against the serovar D and L2 reference strains and a derivative mutant resistant to fluoroquinolones, L2-OFXR, obtained by in vitro selection. Using DNA quantification, the antibiotic MIC was calculated when the number of DNA copies was equal to that of the chlamydial inoculum at time zero. This method allowed the easy determination of MICs by DNA quantification of the four selected genes and gave similar results to those obtained by immunofluorescence staining without biased interpretation. By using cDNA quantification, the lowest antibiotic concentration for which no RNA was transcribed corresponded to the minimum bactericidal concentration. C. trachomatis still transcribed the16S rRNA and groEL1 genes, even at concentrations well above the MIC, showing a bacteriostatic effect for all antibiotics tested. This method allows the study of antibiotic activity on growth and viability of C. trachomatis by DNA and RNA quantification at the same time without additional cell-culture passaging.

Semimechanistic pharmacokinetic-pharmacodynamic model with adaptation development for time-kill experiments of ciprofloxacin against Pseudomonas aeruginosa.

The objective of this study was to implement a semimechanistic pharmacokinetic-pharmacodynamic (PK-PD) model to describe the effects of ciprofloxacin against Pseudomonas aeruginosa in vitro. Time-kill curves were generated with an initial inoculum close to 5 x 10(6)CFU/ml of P. aeruginosa PAO1 and constant ciprofloxacin concentrations between 0.12 and 4.0 microg/ml (corresponding to 0.5x and 16x MIC). To support the model, phenotypic experiments were conducted with the PAO7H mutant strain, which overexpresses the MexEF OprN efflux pump and phenyl arginine beta-naphthylamide (PAbetaN), a known efflux inhibitor of main Mex multidrug efflux systems. A population approach was used for parameter estimation. At subinhibitory ciprofloxacin concentrations (0.12 and 0.25 microg/ml), an initial CFU decay followed by regrowth was observed, attesting to rapid emergence of bacteria with increased but moderate resistance (8-fold increase of MIC). This phenomenon was mainly due to an overexpression of the Mex protein efflux pumps, as shown by a 16-fold diminution of the MIC in the presence of PAbetaN in these strains with low-level resistance. A PK-PD model with adaptation development was successfully used to describe these data. However, additional experiments are required to validate the robustness of this model after longer exposure periods and multiple dosing regimens, as well as in vivo.

Real-time high resolution melting PCR for identification of the Swedish variant of Chlamydia trachomatis.

We developed a real-time High Resolution Melting PCR to identify the new Swedish variant of Chlamydia trachomatis ncCT. Of 1191 urogenital specimens C. trachomatis-positive by an omp1 real-time PCR, collected in France in 2007-2008, 1128 gave an interpretable profile corresponding to the wild-type strain; no nvCT was found. This test can be used on selected C. trachomatis-positive samples to monitor the nvCT spread.