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LLQR E. coli ATCC 25922 vs wild-type E. coli ATCC 25922

ABSTRACT: 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.

ORGANISM(S): Escherichia coli O157:H7 str. Sakai Escherichia coli ATCC 25922 Escherichia coli str. K-12 substr. MG1655 Escherichia coli CFT073 Escherichia coli O157:H7 str. EDL933

PROVIDER: GSE86341 | GEO | 2017/01/01

REPOSITORIES: GEO

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Project description:Background: Antibiotic resistance is an urgent threat to public health. Prior to the evolution of antibiotic resistance, bacteria frequently undergo response and tend to develop a state of adaption to the antibiotic. Ciprofloxacin is a broad-spectrum antibiotic by damaging DNA. With the widespread clinical application, the resistance of bacteria to ciprofloxacin continues to increase. This study aimed to investigate the transcriptome changes under the action of high concentration of ciprofloxacin in Escherichia coli. Results: We identified 773 up-regulated differentially expressed genes (DEGs) and 645 down-regulated DEGs in ciprofloxacin treated cells. Enriched biological pathways reflected the up-regulation of biological process such as DNA damage and repair system, toxin/antitoxin systems, formaldehyde detoxification system, peptide biosynthetic process and cellular protein metabolic process. With KEGG pathway analysis, up-regulated DEGs of kdsA and waa operon were associated with “LPS biosynthesis”. rfbABC operon was related to “streptomycin biosynthesis” and “polyketide sugar unit biosynthesis ”. Down-regulated DEGs of thrABC and fliL operons were associated with “flagellum-dependent cell motility” and “bacterial-type flagellum” in GO terms, and enriched into “biosynthesis of amino acids” and “flagellar assembly” in KEGG pathways. After treatment of ciprofloxacin, bacterial lipopolysacchride (LPS) release was increased by two times, and the mRNA expression level of LPS synthesis genes, waaB, waaP and waaG were elevated (P < 0.05). Conclusions: Characterization of the gene clusters by RNA-seq showed high dose of ciprofloxacin not only lead to damage of bacterial macromolecules and components, but also induce protective response against antibiotic action by up-regulating the SOS system, toxin/antitoxin system and formaldehyde detoxification system. Moreover, genes related to biosynthesis of LPS were also upregulated by the treatment indicating that ciprofloxacin can enhance the production of endotoxin on the level of transcription. These results demonstrated that transient exposure of high dose ciprofloxacin is double edged. Cautions should be taken when administering the high dose antibiotic treatment for infectious diseases.

Project description:Infections of the udder by Escherichia coli very often elicit acute inflammation, while Staphylococcus aureus infections tend to cause mild, subclinical inflammation and persistent infections. The molecular causes undercovering the different disease patterns are poorly understood. We therefore profiled kinetics and extent of global changes in the transcriptome of primary bovine mammary epithelia cells (MEC) subsequent to challenging them with heat inactivated preparations of E. coli or S. aureus pathogens. E. coli swiftly and strongly induced expression of cytokines and bactericidal factors. S. aureus elicited a retarded response and failed to quickly induce expression of bactericidal factors. Both pathogens induced a similar pattern of chemokines for cell recruitment into the udder, but E. coli stimulated their synthesis much faster and stronger. The genes which are exclusively and most strongly up-regulated by E. coli may be clustered into a regulatory network with Tumor necrosis factor alpha (TNF-a) and Interleukin 1 (IL-1) in a central position. In contrast, the expression of these master cytokines is barely regulated by S. aureus. Both pathogens quickly trigger enhanced expression of IL-6. This is still possible after completely abrogating MyD88 dependent TLR-signalling in MEC. The E. coli specific strong induction of TNF-a and IL-1 expression may be causative for the severe inflammatory symptoms of animals suffering from E. coli mastitis while avoidance to quickly induce synthesis of bactericidal factors may support persistent survival of S. aureus within the udder. We suggest that S. aureus subverts MyD88-dependent activation of immune gene expression in MEC. We challenged pbMEC cultures with 1E+07 particles per ml of heat inactivated E. coli strain 1303 for 1, 3, 6, and 24 h and compared their transcriptomes to that of untreated control cells. The experiment included three biological replicas (rep1, rep2, rep3), each from a different cow We challenged pbMEC cultures with 1E+07 particles per ml of heat inactivated S. aureus strain 1027 for 1, 3, 6, and 24 h and compared their transcriptomes to that of untreated control cells. The experiment included three biological replicas (rep1, rep2, rep3), each from a different cow

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LLQR E. coli ATCC 25922 vs wild-type E. coli ATCC 25922

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