Project description:New and rapid antimicrobial susceptibility/resistance testing methods are required for bacteria from positive blood cultures. In the current study we developed and evaluated a targeted LC-MS/MS assay for the detection of beta-lactam, aminoglycoside and fluoroquinolone resistance mechanisms in blood cultures positive for E. coli or K. pneumoniae. Selected targets were the beta-lactamases SHV, TEM, OXA-1-like, CTX-M-1-like, CMY-2-like, chromosomal E. coli AmpC, OXA-48-like, NDM, VIM and KPC, the aminoglycoside modifying enzymes AAC(3)-Ia, AAC(3)-II, AAC(3)-IV, AAC(3)-VI, AAC(6’)-Ib, ANT(2”)-I and APH(3’)-VI, the 16S-RMTases ArmA, RmtB, RmtC and RmtF, the quinolone resistance mechanisms QnrA, QnrB, AAC(6’)-Ib-cr, the wildtype QRDR of GyrA, and for E. coli, the porins OmpC and OmpF. The developed assay was evaluated using 100 prospectively collected positive blood cultures, 100 negative blood cultures inoculated with isolates that were previously collected from blood cultures, and 48 isolates inoculated with isolates carrying genes of less prevalent resistance mechanisms.
Project description:To demonstrate plasmid transferability by conjugation, cultures of the donor S. Infantis, and recipient Escherichia coli (E. coli) K12 were mated. S. Infantis and transconjugant were screened for resistance genes.
Project description:Global response to ciprofloxacin in low level quinolone resistant Escherichia coli: a shorter path to survival. Background: Bactericidal activity of quinolones in bacteria has been related to a combination of DNA fragmentation, ROS production and programmed dead cell systems. Subjacent molecular systems responsible for reduction of bactericidal effect in low-level quinolone resistance (LLQR) phenotypes remain to be clarified. To answer this question and to define new possible antimicrobial targets, the transcriptomic profile in isogenic Escherichia coli harbouring quinolone resistance mechanisms in the presence of ciprofloxacin was evaluated. Materials and methods: E. coli 25922 was used as background strain. Four LLQR isogenic strains were tested for transcriptomic assays: ATCC 25922 (wild-type), EC14 (coding for QnrS1), EC19 (marR deletion and coding for QnrS1) and EC24 (Ser83Leu substitution in GyrA and coding for QnrS1). Cells in exponential phase (DO600=0.4) were exposed to 1 mg/L of ciprofloxacin (breakpoint for reduced susceptibility according to CLSI) during 1 hour and, further, RNA was purified. Gene expression analysis was performed using AGILENT technology. Data obtained for each strain were always normalized to the wild-type E. coli ATCC 25922. Specific ROS modulation targets were validated by genetic and biochemical approach. Results: A radical differential response to ciprofloxacin in LLQR strains, either up or downregulation, was observed (proportional to the MIC values). Multiple genes implicated in ROS production (related to TCA cycle, aerobic respiration or detoxification systems) were upregulated (sdhC up to 63.5-folds) in LLQR mutants. SOS system components were downregulated (recA up to 30.7-folds). yihE, coding for a protective kinase of programmed cell death, was also upregulated (up to 5.2-folds). SdhC inhibition sensitized LLQR phenotypes (up to Log=2.3 after 24 hours). Conclusions: At clinical relevant concentration of ciprofloxacin, the pattern of genes expression of critical systems for bacterial survival and mutant development were significantly modified in LLQR phenotypes. This approach allowed validating ROS modulation as an interesting target in terms of bacterial sensitization.
