Cellular Response to Ciprofloxacin in Low-Level Quinolone-Resistant Escherichia coli.

Bactericidal activity of quinolones has been related to a combination of DNA fragmentation, reactive oxygen species (ROS) production and programmed cell death (PCD) systems. The underlying molecular systems responsible for reducing bactericidal effect during antimicrobial therapy in low-level quinolone resistance (LLQR) phenotypes need to be clarified. To do this and also define possible new antimicrobial targets, the transcriptome profile of isogenic Escherichia coli harboring quinolone resistance mechanisms in the presence of a clinical relevant concentration of ciprofloxacin was evaluated. A marked differential response to ciprofloxacin of either up- or downregulation was observed in LLQR strains. Multiple genes implicated in ROS modulation (related to the TCA cycle, aerobic respiration and detoxification systems) were upregulated (sdhC up to 63.5-fold) in mutants with LLQR. SOS system components were downregulated (recA up to 30.7-fold). yihE, a protective kinase coding for PCD, was also upregulated (up to 5.2-fold). SdhC inhibition sensitized LLQR phenotypes (up to ΔLog = 2.3 after 24 h). At clinically relevant concentrations of ciprofloxacin, gene expression patterns in critical systems to bacterial survival and mutant development were significantly modified in LLQR phenotypes. Chemical inhibition of SdhC (succinate dehydrogenase) validated modulation of ROS as an interesting target for bacterial sensitization.

Comparison of clinical categories for Escherichia coli harboring specific qnrand chromosomal-mediated fluoroquinolone resistance determinants according to CLSI and EUCAST / Comparación de las categorías clínicas de cepas de Escherichia coli que contienen definidos mecanismos cromosómicos y plasmídicos de resistencia a quinolonas según CLSI y EUCAST

EUCAST breakpoints are more restrictive than those defined by CLSI. This study highlights the discrepancies between CLSI and EUCAST in a well characterized isogenic Escherichia coli collection and their correlations with specific quinolone resistance mechanisms. The greatest number of discrepancies was observed in strains containing 2-4 resistance mechanisms (MIC values on the borderline of clinical resistance). Bearing in mind that quinolones are concentration dependent antimicrobial agents, small changes in MIC may have relevant consequences for treatment outcomes

Los puntos de corte de EUCAST son más restrictivos que los definidos por CLSI. Este estudio analiza las discrepancias entre CLSI y EUCAST en una colección isogénica de Escherichia coli y su correlación con mecanismos específicos de resistencia a quinolonas. El mayor número de discrepancias se observó en cepas que contienen 2-4 mecanismos de resistencia (con CMI en el límite de la resistencia clínica). Teniendo en cuenta que las quinolonas son agentes antimicrobianos concentración-dependientes, pequeños cambios en el valor de la CMI pueden tener consecuencias relevantes para el resultado del tratamiento

Comparison of clinical categories for Escherichia coli harboring specific qnr and chromosomal-mediated fluoroquinolone resistance determinants according to CLSI and EUCAST.

EUCAST breakpoints are more restrictive than those defined by CLSI. This study highlights the discrepancies between CLSI and EUCAST in a well characterized isogenic Escherichia coli collection and their correlations with specific quinolone resistance mechanisms. The greatest number of discrepancies was observed in strains containing 2-4 resistance mechanisms (MIC values on the borderline of clinical resistance). Bearing in mind that quinolones are concentration dependent antimicrobial agents, small changes in MIC may have relevant consequences for treatment outcomes.

Effect of the efflux pump QepA2 combined with chromosomally mediated mechanisms on quinolone resistance and bacterial fitness in Escherichia coli.

OBJECTIVES: The aim of the study was to determine the interplay between the plasmid-mediated qepA2 gene and multiple chromosomally mediated fluoroquinolone resistance determinants in the development of fluoroquinolone resistance in Escherichia coli and its influence on bacterial fitness. METHODS: E. coli ATCC 25922 and derived isogenic strains harbouring different chromosomally mediated fluoroquinolone resistance determinants were electroporated with pBK-CMV vector encoding QepA2. The MICs of fluoroquinolones were determined by standardized microdilution. The mutant prevention concentration (MPC) was evaluated. Bacterial fitness was analysed using ΔlacZ system competition assays. RESULTS: The ciprofloxacin MIC for strains harbouring the qepA2 gene was 4- to 8-fold higher compared with strains without the qepA2 gene. The qepA2 gene also increased the MPC of ciprofloxacin 4- to 16-fold. Combination of the qepA2 gene plus two to three additional mechanisms conferred a clinically relevant resistance level. The presence of the qepA2 gene was associated with fitness costs in strains with mutations in the gyrA and/or parC genes, although the presence of an additional deletion of the marR gene compensated for this fitness cost by increasing bacterial fitness by 5%-23%. CONCLUSIONS: The additive effect of chromosomally mediated fluoroquinolone resistance mechanisms and the qepA2 gene led to clinical levels of fluoroquinolone resistance. Under competitive conditions, the qepA2 gene had a biological cost in E. coli that was compensated for by the presence of an additional deletion in the marR gene.

Interplay between plasmid-mediated and chromosomal-mediated fluoroquinolone resistance and bacterial fitness in Escherichia coli.

OBJECTIVES: The aim of this study was to analyse the interplay among plasmid-mediated qnr genes, alone or in combination with multiple chromosomal-mediated fluoroquinolone (FQ) resistance determinants, susceptibility to FQs and bacterial fitness in an isogenic Escherichia coli collection. METHODS: E. coli ATCC 25922 was used to modify or delete chromosomal genes. qnr genes were cloned into the pBK-CMV vector. The MICs of FQs were determined by microdilution. Mutant prevention concentration and frequency of mutants were evaluated. Bacterial fitness was analysed using ΔlacZ system competition assays using in vitro and in vivo models. RESULTS: The relationships between the number of resistance mutations and bacterial fitness were complex. With specific combinations of resistance mechanisms the addition of a new resistance mutation was shown to improve bacterial fitness. qnrA1 caused a decrease in fitness (7%-21%) while qnrS1 caused an increase in fitness (9%-21%) when combined with chromosomal mutations. We identified susceptible triple mutants in which the acquisition of a fourth resistance mutation significantly increased fitness and at the same time reached the clinical resistance level (the acquisition of qnrS1 in a S83L + D87N + ΔmarR genetic background). A strong correlation with the production of reactive oxygen species, as well as changes in susceptibility, was observed following treatment with ciprofloxacin. CONCLUSIONS: Our data indicate that there may be critical stages (depending on the genotype) in resistance development, including chromosomal- and plasmid-mediated mechanisms, at which some low-fitness mutants below the resistance breakpoint are able to evolve clinical resistance with just one or two mutations, and show increased fitness.

Bactericidal effect of colistin on planktonic Pseudomonas aeruginosa is independent of hydroxyl radical formation.

The bactericidal effect of several major types of antibiotics has recently been demonstrated to be dependent on the formation of toxic amounts of hydroxyl radicals (OH·) resulting from oxidative stress in metabolically active cells. Since killing by the antimicrobial peptide colistin does not require bacterial metabolic activity, we tested whether the bactericidal effect of colistin depends on the formation of OH·. In Pseudomonas aeruginosa cultures, OH-mediated killing by ciprofloxacin was demonstrated by decreased bacterial survival and induction of 3'-(p-hydroxyphenyl) fluorescein (HPF) fluorescence. OH·-mediated killing by ciprofloxacin was further confirmed by rescue of cells and reduction of HPF fluorescence due to prevention of OH· accumulation by scavenging with thiourea, by chelating with dipyridyl, by decreasing metabolism as well as by anoxic growth. In contrast, no formation of OH· was seen in P. aeruginosa during killing by colistin, and prevention of OH· accumulation could not rescue P. aeruginosa from killing by colistin. These results therefore demonstrate that the bactericidal activity of colistin on P. aeruginosa is not dependent on oxidative stress. In conclusion, antimicrobial peptides that do not rely on OH· formation should be considered for treatment of Gram-negative bacteria growing at low oxygen tension such as in endobronchial mucus and paranasal sinuses in cystic fibrosis patients, in abscesses and in infectious biofilm.

Formation of hydroxyl radicals contributes to the bactericidal activity of ciprofloxacin against Pseudomonas aeruginosa biofilms.

Antibiotic-tolerant, biofilm-forming Pseudomonas aeruginosa has long been recognized as a major cause of chronic lung infections of cystic fibrosis patients. The mechanisms involved in the activity of antibiotics on biofilm are not completely clear. We have investigated whether the proposed induction of cytotoxic hydroxyl radicals (OH˙) during antibiotic treatment of planktonically grown cells may contribute to action of the commonly used antibiotic ciprofloxacin on P. aeruginosa biofilms. For this purpose, WT PAO1, a catalase deficient ΔkatA and a ciprofloxacin resistant mutant of PAO1 (gyrA), were grown as biofilms in microtiter plates and treated with ciprofloxacin. Formation of OH˙ and total amount of reactive oxygen species (ROS) was measured and viability was estimated. Formation of OH˙ and total ROS in PAO1 biofilms treated with ciprofloxacin was shown but higher levels were measured in ΔkatA biofilms, and no ROS production was seen in the gyrA biofilms. Treatment with ciprofloxacin decreased the viability of PAO1 and ΔkatA biofilms but not of gyrA biofilms. Addition of thiourea, a OH˙ scavenger, decreased the OH˙ levels and killing of PAO1 biofilm. Our study shows that OH˙ is produced by P. aeruginosa biofilms treated with ciprofloxacin, which may contribute to the killing of biofilm subpopulations.

Genomewide overexpression screen for fosfomycin resistance in Escherichia coli: MurA confers clinical resistance at low fitness cost.

To determine whether the overexpression of chromosomal genes can confer fosfomycin resistance, genomewide screening of a complete set of 5,272 plasmid-expressed open reading frames of Escherichia coli (ASKA collection) was performed. Major results are that (i) no clinical level of resistance is achieved by overexpressing chromosomal genes, except murA; (ii) this level is reached at a low fitness cost; and (iii) this cost is much lower than that imposed by other mutations conferring fosfomycin resistance.

Genetic characterization of an extended-spectrum AmpC cephalosporinase with hydrolysing activity against fourth-generation cephalosporins in a clinical isolate of Enterobacter aerogenes selected in vivo.

BACKGROUND: Extended-spectrum AmpC cephalosporinases (ESACs) have been reported in Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii. Here, we characterize a new AmpC variant presenting a broadened substrate activity towards fourth-generation cephalosporins, selected in vivo following cefepime treatment for Enterobacter aerogenes. METHODS: Two consecutive clonally related isolates of E. aerogenes were evaluated. Screening for ESAC production was performed using plates containing 200 mg/L cloxacillin. MICs were determined by microdilution (CLSI guidelines). bla(AmpC) genes were cloned into a pCR-Blunt II-TOPO vector and expressed in Escherichia coli. The ampC genes were cloned into vector pGEX-6P-1 for protein purification. RESULTS: Isolate Ea595 was resistant to two fourth-generation cephalosporins, cefepime and cefpirome; using plates containing cloxacillin, susceptibility to ceftazidime and cefepime was restored, suggesting overproduction of the ESAC ß-lactamase. Sequencing identified a new AmpC ß-lactamase variant presenting one amino acid substitution, Val291Gly, inside the H-10 helix. Recombinant plasmids harbouring this ESAC ß-lactamase conferred a broadened resistance profile to cefepime and cefpirome, with resistance levels increasing from 16- to 32-fold in E. coli. AmpC-Ea595 hydrolysed ceftazidime, cefepime and cefpirome at high levels, presenting a lower K(m) and enabling us to classify the enzyme as an ESAC. Homology modelling suggested that the size of the active site could have increased. CONCLUSIONS: We characterized an ESAC ß-lactamase selected in vivo and conferring a high level of resistance to fourth-generation cephalosporins in E. aerogenes. The broadened spectrum was caused by a new modification to the H-10 helix, which modified the active site.