Biocompatibility analysis and chemical characterization of Mn-doped hydroxyapatite.

The present work studies the effect of Mn doping on the crystalline structure of the Hap synthesized by the hydrothermal method at 200 °C for 24 h, from Ca(OH)2 and (NH4)2HPO4, incorporating MnCl2 to 0.1, 0.5, 1.0, 1.5 and 2.0 %wt of Mn concentrations. Samples were characterized by the X-Ray Diffraction technique, which revealed the diffraction peaks that corresponded to the hexagonal and monoclinic phase of the Hap; it was observed that the average size of crystallite decreased from 23.67 to 22.69 nm as the concentration of Mn increased. TEM shows that in all samples, there are two distributions of particle sizes; one corresponds to nanorods with several tens of nanometers in length, and the other in which the diameter and length are very close. FTIR analysis revealed absorption bands corresponding to the PO4-3 and OH- groups characteristic of the Hap. It was possible to establish a substitution mechanism between the Mn and the ions of Ca+2 of the Hap. From the Alamar blue test, a cell viability of 86.88% ± 5 corresponding to the sample of Hap at 1.5 %wt Mn was obtained, considered non-cytotoxic according to ISO 10993-5. It also evaluated and demonstrated the good osteoinductive properties of the materials, which were verified by histology and immunofluorescence expression of osteogenic markers. Adhesion, viability, biocompatibility and osteoinductive properties, make these materials candidates for future applications in bone tissue engineering with likely uses in regenerative medicine.

Synergistic Effect of SOS Response and GATC Methylome Suppression on Antibiotic Stress Survival in Escherichia coli.

The suppression of the SOS response has been shown to enhance the in vitro activity of quinolones. Furthermore, Dam-dependent base methylation has an impact on susceptibility to other antimicrobials affecting DNA synthesis. Here, we investigated the interplay between these two processes, alone and in combination, in terms of antimicrobial activity. A genetic strategy was used employing single- and double-gene mutants for the SOS response (recA gene) and the Dam methylation system (dam gene) in isogenic models of Escherichia coli both susceptible and resistant to quinolones. Regarding the bacteriostatic activity of quinolones, a synergistic sensitization effect was observed when the Dam methylation system and the recA gene were suppressed. In terms of growth, after 24 h in the presence of quinolones, the Δdam ΔrecA double mutant showed no growth or delayed growth compared to the control strain. In bactericidal terms, spot tests showed that the Δdam ΔrecA double mutant was more sensitive than the ΔrecA single mutant (about 10- to 102-fold) and the wild type (about 103- to 104-fold) in both susceptible and resistant genetic backgrounds. Differences between the wild type and the Δdam ΔrecA double mutant were confirmed by time-kill assays. The suppression of both systems, in a strain with chromosomal mechanisms of quinolone resistance, prevents the evolution of resistance. This genetic and microbiological approach demonstrated the enhanced sensitization of E. coli to quinolones by dual targeting of the recA (SOS response) and Dam methylation system genes, even in a resistant strain model.

RecA inactivation as a strategy to reverse the heteroresistance phenomenon in clinical isolates of Escherichia coli.

RecA inhibition could be an important strategy to combat antimicrobial resistance because of its key role in the SOS response, DNA repair and homologous recombination contributing to bacterial survival. This study evaluated the impact of RecA inactivation on heteroresistance in clinical isolates of Escherichia coli and their corresponding recA-deficient isogenic strains to multiple classes of antimicrobial agents. A high frequency (>30%) of heteroresistance was observed in this collection of clinical isolates. Deletion of the recA gene led to a marked reduction in heteroresistant subpopulations, especially against quinolones or ß-lactams. The molecular basis of heteroresistance was associated with an increase in copy number of plasmid-borne resistance genes (blaTEM-1B) or tandem gene amplifications (qnrA1). Of note, in the absence of the recA gene, the increase in copy number of resistance genes was suppressed. This makes the recA gene a promising target for combating heteroresistance.

Activity of fosfomycin and amikacin against fosfomycin-heteroresistant Escherichia coli strains in a hollow-fiber infection model.

Objectives:To evaluate human-like intravenous doses of fosfomycin (8g/Q8h) and amikacin (15mg/kg/Q24h) efficacy in monotherapy and in combination against six fosfomycin-heteroresistant Escherichia coli isolates using a hollow-fiber infection model (HFIM).Materials and methods:Six fosfomycin-heteroresistant E. coli isolates (4 with strong mutator phenotype) and the control strain E. coli ATCC 25922 were used. Mutant frequencies for rifampin (100mg/L), fosfomycin (50 and 200mg/L) and amikacin (32mg/L) were determined. Fosfomycin and amikacin MICs were assessed by agar dilution (AD), gradient strip (GSA) and broth microdilution (BMD) assays. Fosfomycin and amikacin synergies were studied by checkerboard and time-kill assays at different concentrations. Fosfomycin (8g/Q8h) and amikacin (15mg/kg/Q24h) efficacy alone and in combination were assessed using a HFIM.Results:Five isolates were resistant to fosfomycin by AD and BMD, but all susceptible by GSA. All isolates were considered susceptible to amikacin. Antibiotic combinations were synergistic in two isolates and no antagonism was detected. In time-kill assays, all isolates survived under fosfomycin at 64mg/L, although, at 307mg/L, only the normomutators and two hypermutators survived. Four isolates survived under 16mg/L amikacin and none at 45mg/L. No growth was detected under combination conditions. In HFIM, fosfomycin and amikacin monotherapies failed to sterilise bacterial cultures, however, fosfomycin and amikacin combination showed a rapid eradication.Conclusions.There may be a risk of treatment failure of fosfomycin-heteroresistant E. coli isolates using either amikacin or fosfomycin in monotherapy. These results support that the combination amikacin-fosfomycin can rapidly decrease bacterial burden and prevent the emergence of resistant subpopulations against fosfomycin-heteroresistant strains.

Effect of RecA inactivation and detoxification systems on the evolution of ciprofloxacin resistance in Escherichia coli.

BACKGROUND: Suppression of SOS response and overproduction of reactive oxygen species (ROS) through detoxification system suppression enhance the activity of fluoroquinolones. OBJECTIVES: To evaluate the role of both systems in the evolution of resistance to ciprofloxacin in an isogenic model of Escherichia coli. METHODS: Single-gene deletion mutants of E. coli BW25113 (wild-type) (ΔrecA, ΔkatG, ΔkatE, ΔsodA, ΔsodB), double-gene (ΔrecA-ΔkatG, ΔrecA-ΔkatE, ΔrecA-ΔsodA, ΔrecA-ΔsodB, ΔkatG-ΔkatE, ΔsodB-ΔsodA) and triple-gene (ΔrecA-ΔkatG-ΔkatE) mutants were included. The response to sudden high ciprofloxacin pressure was evaluated by mutant prevention concentration (MPC). The gradual antimicrobial pressure response was evaluated through experimental evolution and antibiotic resistance assays. RESULTS: For E. coli BW25113 strain, ΔkatE, ΔsodB and ΔsodB/ΔsodA mutants, MPC values were 0.25 mg/L. The ΔkatG, ΔsodA, ΔkatG/katE and ΔrecA mutants showed 2-fold reductions (0.125 mg/L). The ΔkatG/ΔrecA, ΔkatE/ΔrecA, ΔsodA/ΔrecA, ΔsodB/ΔrecA and ΔkatG/ΔkatE/ΔrecA strains showed 4-8-fold reductions (0.03-0.06 mg/L) relative to the wild-type. Gradual antimicrobial pressure increased growth capacity for ΔsodA and ΔsodB and ΔsodB/ΔsodA mutants (no growth in 4 mg/L) compared with the wild-type (no growth in the range of 0.5-2 mg/L). Accordingly, increased growth was observed with the mutants ΔrecA/ΔkatG (no growth in 2 mg/L), ΔrecA/ΔkatE (no growth in 2 mg/L), ΔrecA/ΔsodA (no growth in 0.06 mg/L), ΔrecA/ΔsodB (no growth in 0.25 mg/L) and ΔrecA/ΔkatG/ΔkatE (no growth in 0.5 mg/L) compared with ΔrecA (no growth in the range of 0.002-0.015 mg/L). CONCLUSIONS: After RecA inactivation, gradual exposure to ciprofloxacin reduces the evolution of resistance. After suppression of RecA and detoxification systems, sudden high exposure to ciprofloxacin reduces the evolution of resistance in E. coli.

Synergistic Quinolone Sensitization by Targeting the recA SOS Response Gene and Oxidative Stress.

Suppression of the recA SOS response gene and reactive oxygen species (ROS) overproduction have been shown, separately, to enhance fluoroquinolone activity and lethality. Their putative synergistic impact as a strategy to potentiate the efficacy of bactericidal antimicrobial agents such as fluoroquinolones is unknown. We generated Escherichia coli mutants that exhibited a suppressed ΔrecA gene in combination with inactivated ROS detoxification system genes (ΔsodA, ΔsodB, ΔkatG, ΔkatE, and ΔahpC) or inactivated oxidative stress regulator genes (ΔoxyR and ΔrpoS) to evaluate the interplay of both DNA repair and detoxification systems in drug response. Synergistic sensitization effects, ranging from 7.5- to 30-fold relative to the wild type, were observed with ciprofloxacin in double knockouts of recA and inactivated detoxification system genes. Compared to recA knockout, inactivation of an additional detoxification system gene reduced MIC values up to 8-fold. In growth curves, no growth was evident in mutants doubly deficient for recA gene and oxidative detoxification systems at subinhibitory concentrations of ciprofloxacin, in contrast to the recA-deficient strain. There was a marked reduction of viable bacteria in a short period of time when the recA gene and other detoxification system genes (katG, sodA, or ahpC) were inactivated (using absolute ciprofloxacin concentrations). At 4 h, a bactericidal effect of ciprofloxacin was observed for ΔkatG ΔrecA and ΔahpC ΔrecA double mutants compared to the single ΔrecA mutant (Δ3.4 log10 CFU/ml). Synergistic quinolone sensitization, by targeting the recA gene and oxidative detoxification stress systems, reinforces the role of both DNA repair systems and ROS in antibiotic-induced bacterial cell death, opening up a new pathway for antimicrobial sensitization.

Effect of RecA inactivation on quinolone susceptibility and the evolution of resistance in clinical isolates of Escherichia coli.

BACKGROUND: SOS response suppression (by RecA inactivation) has been postulated as a therapeutic strategy for potentiating antimicrobials against Enterobacterales. OBJECTIVES: To evaluate the impact of RecA inactivation on the reversion and evolution of quinolone resistance using a collection of Escherichia coli clinical isolates. METHODS: Twenty-three E. coli clinical isolates, including isolates belonging to the high-risk clone ST131, were included. SOS response was suppressed by recA inactivation. Susceptibility to fluoroquinolones was determined by broth microdilution, growth curves and killing curves. Evolution of quinolone resistance was evaluated by mutant frequency and mutant prevention concentration (MPC). RESULTS: RecA inactivation resulted in 2-16-fold reductions in fluoroquinolone MICs and modified EUCAST clinical category for several isolates, including ST131 clone isolates. Growth curves and time-kill curves showed a clear disadvantage (up to 10 log10 cfu/mL after 24 h) for survival in strains with an inactivated SOS system. For recA-deficient mutants, MPC values decreased 4-8-fold, with values below the maximum serum concentration of ciprofloxacin. RecA inactivation led to a decrease in mutant frequency (≥103-fold) compared with isolates with unmodified SOS responses at ciprofloxacin concentrations of 4×MIC and 1 mg/L. These effects were also observed in ST131 clone isolates. CONCLUSIONS: While RecA inactivation does not reverse existing resistance, it is a promising strategy for increasing the effectiveness of fluoroquinolones against susceptible clinical isolates, including high-risk clone isolates.

Interplay among Different Fosfomycin Resistance Mechanisms in Klebsiella pneumoniae.

The objectives of this study were to characterize the role of the uhpT, glpT, and fosA genes in fosfomycin resistance in Klebsiella pneumoniae and evaluate the use of sodium phosphonoformate (PPF) in combination with fosfomycin. Seven clinical isolates of K. pneumoniae and the reference strain (ATCC 700721) were used, and their genomes were sequenced. ΔuhpT, ΔglpT, and ΔfosA mutants were constructed from two isolates and K. pneumoniae ATCC 700721. Fosfomycin susceptibility testing was done by the gradient strip method. Synergy between fosfomycin and PPF was studied by checkerboard assay and analyzed using SynergyFinder. Spontaneous fosfomycin mutant frequencies at 64 and 512 mg/liter, in vitro activity using growth curves with fosfomycin gradient concentrations (0 to 256mg/liter), and time-kill assays at 64 and 307 mg/liter were evaluated with and without PPF (0.623 mM). The MICs of fosfomycin against the clinical isolates ranged from 16 to ≥1,024 mg/liter. The addition of 0.623 mM PPF reduced fosfomycin MIC between 2- and 8-fold. Deletion of fosA led to a 32-fold decrease. Synergistic activities were observed with the combination of fosfomycin and PPF (most synergistic area at 0.623 mM). The lowest fosfomycin-resistant mutant frequencies were found in ΔfosA mutants, with decreases in frequency from 1.69 × 10-1 to 1.60 × 10-5 for 64 mg/liter of fosfomycin. In the final growth monitoring and time-kill assays, fosfomycin showed a bactericidal effect only with the deletion of fosA and not with the addition of PPF. We conclude that fosA gene inactivation leads to a decrease in fosfomycin resistance in K. pneumoniae The pharmacological approach using PPF did not achieve enough activity, and the effect decreased with the presence of fosfomycin-resistant mutations.

Role of low-level quinolone resistance in generating tolerance in Escherichia coli under therapeutic concentrations of ciprofloxacin.

BACKGROUND: Tolerance (including persistence) and resistance result in increased survival under antibiotic pressure. OBJECTIVES: We evaluated the interplay between resistance and tolerance to ciprofloxacin under therapeutic and killing conditions to determine the contribution of low-level quinolone resistance (LLQR) mechanisms to tolerance. We also determined how the interaction between resistance (LLQR phenotypes) and tolerance was modified under SOS response suppression. METHODS: Twelve isogenic Escherichia coli strains harbouring quinolone resistance mechanisms combined with SOS response deficiency and six clinical E. coli isolates (LLQR or non-LLQR) were evaluated. Survival (tolerance or persistence) assays were used to measure surviving bacteria after a short period (up to 4 h) of bactericidal antibiotic treatment under therapeutic and killing concentrations of ciprofloxacin [1 mg/L, EUCAST/CLSI breakpoint for resistance; and 2.5 mg/L, peak serum concentration (Cmax) of this drug]. RESULTS: QRDR substitutions (S83L in GyrA alone or combined with S80R in ParC) significantly increased the fraction of tolerant bacteria (2-4 log10 cfu/mL) after exposure to ciprofloxacin at clinically relevant concentrations. The impact on tolerant bacteria due to SOS response suppression (including persistence mediated by the tisB gene) was reversed by LLQR mechanisms at therapeutic concentrations. Furthermore, no reduction in the fraction of tolerant bacteria due to SOS response suppression was observed when S83L in GyrA plus S80R in ParC were combined. CONCLUSIONS: Tolerance and quinolone resistance mutations interact synergistically, giving LLQR mechanisms an additional role in allowing bacterial survival and evasion of therapeutic antimicrobial conditions by a combination of the two strategies. At clinically relevant concentrations, LLQR mechanisms reverse further impact of SOS response suppression in reducing bacterial tolerance.

Suppression of the SOS response modifies spatiotemporal evolution, post-antibiotic effect, bacterial fitness and biofilm formation in quinolone-resistant Escherichia coli.

Background: Suppression of the SOS response has been proposed as a therapeutic strategy for potentiating quinolones against susceptible, low-level quinolone-resistant (LLQR) and resistant Enterobacteriaceae. Objectives: To monitor the functionality of the SOS response in the evolution towards clinical quinolone resistance and study its impact on the evolution of spatiotemporal resistance. Methods: An isogenic collection of Escherichia coli (derived from the strain ATCC 25922) carrying combinations of chromosomally and plasmid-mediated quinolone resistance mechanisms (including susceptible, LLQR and resistant phenotypes) and exhibiting a spectrum of SOS activity was used. Relevant clinical parameters such as mutation rate, mutant prevention concentration (MPC), bacterial fitness, biofilm formation and post-antibiotic effect (PAE) were evaluated. Results: Inactivating the SOS response (recA deletion) led to a decrease in mutation rate (∼103 fold) in LLQR compared with WT strains at ciprofloxacin concentrations of 1 mg/L (the EUCAST breakpoint for resistance) and 2.5 mg/L (Cmax), as well as a remarkable delay in the spatiotemporal evolution of quinolone resistance. For all strains, there was an 8-fold decrease in MPC in RecA-deficient strains, with values for LLQR strains decreasing below the Cmax of ciprofloxacin. Inactivation of the SOS response reduced competitive fitness by 33%-50%, biofilm production by 22%-80% and increased the PAE by ∼3-4 h at sub-MIC concentrations of ciprofloxacin. Conclusions: Our data indicate that suppression of the SOS response affects key bacterial traits and is a promising strategy for reversing and tackling the evolution of antibiotic resistance in E. coli, including low-level and resistant phenotypes at therapeutic quinolone concentrations.

Random distributions of initial porosity trigger regular necking patterns at high strain rates.

At high strain rates, the fragmentation of expanding structures of ductile materials, in general, starts by the localization of plastic deformation in multiple necks. Two distinct mechanisms have been proposed to explain multiple necking and fragmentation process in ductile materials. One view is that the necking pattern is related to the distribution of material properties and defects. The second view is that it is due to the activation of specific instability modes of the structure. Following this, we investigate the emergence of necking patterns in porous ductile bars subjected to dynamic stretching at strain rates varying from 103 s-1 to 0.5×105 s-1 using finite-element calculations and linear stability analysis. In the calculations, the initial porosity (representative of the material defects) varies randomly along the bar. The computations revealed that, while the random distribution of initial porosity triggers the necking pattern, it barely affects the average neck spacing, especially, at higher strain rates. The average neck spacings obtained from the calculations are in close agreement with the predictions of the linear stability analysis. Our results also reveal that the necking pattern does not begin when the Considère condition is reached but is significantly delayed due to the stabilizing effect of inertia.

Urinary Tract Conditions Affect Fosfomycin Activity against Escherichia coli Strains Harboring Chromosomal Mutations Involved in Fosfomycin Uptake.

The steps by which Escherichia coli strains harboring mutations related to fosfomycin (FOS) resistance arise and spread during urinary tract infections (UTIs) are far from being understood. The aim of this study was to evaluate the effects of urine, pH, and anaerobiosis on FOS activity against a set of isogenic strains carrying the most prevalent chromosomal mutations conferring FOS resistance (ΔuhpT, ΔglpT, ΔcyaA, and ΔptsI), either singly or in combination. We also studied fosfomycin-resistant E. coli clinical isolates from patients with UTI. Our results demonstrate that urinary tract physiological conditions might have a profound impact on FOS activity against strains with chromosomal FOS resistance mutations. Specifically, acidic pH values and anaerobiosis convert most of the strains categorized as resistant to fosfomycin according to the international guidelines to a susceptible status. Therefore, urinary pH values may have practical interest in the management of UTIs. Finally, our results, together with the high fitness cost associated with FOS resistance mutations, might explain the low prevalence of fosfomycin-resistant E. coli variants in UTIs.

Quinolone Resistance Reversion by Targeting the SOS Response.

Suppression of the SOS response has been postulated as a therapeutic strategy for potentiating antimicrobial agents. We aimed to evaluate the impact of its suppression on reversing resistance using a model of isogenic strains of Escherichia coli representing multiple levels of quinolone resistance. E. coli mutants exhibiting a spectrum of SOS activity were constructed from isogenic strains carrying quinolone resistance mechanisms with susceptible and resistant phenotypes. Changes in susceptibility were evaluated by static (MICs) and dynamic (killing curves or flow cytometry) methodologies. A peritoneal sepsis murine model was used to evaluate in vivo impact. Suppression of the SOS response was capable of resensitizing mutant strains with genes encoding three or four different resistance mechanisms (up to 15-fold reductions in MICs). Killing curve assays showed a clear disadvantage for survival (Δlog10 CFU per milliliter [CFU/ml] of 8 log units after 24 h), and the in vivo efficacy of ciprofloxacin was significantly enhanced (Δlog10 CFU/g of 1.76 log units) in resistant strains with a suppressed SOS response. This effect was evident even after short periods (60 min) of exposure. Suppression of the SOS response reverses antimicrobial resistance across a range of E. coli phenotypes from reduced susceptibility to highly resistant, playing a significant role in increasing the in vivo efficacy.IMPORTANCE The rapid rise of antibiotic resistance in bacterial pathogens is now considered a major global health crisis. New strategies are needed to block the development of resistance and to extend the life of antibiotics. The SOS response is a promising target for developing therapeutics to reduce the acquisition of antibiotic resistance and enhance the bactericidal activity of antimicrobial agents such as quinolones. Significant questions remain regarding its impact as a strategy for the reversion or resensitization of antibiotic-resistant bacteria. To address this question, we have generated E. coli mutants that exhibited a spectrum of SOS activity, ranging from a natural SOS response to a hypoinducible or constitutively suppressed response. We tested the effects of these mutations on quinolone resistance reversion under therapeutic concentrations in a set of isogenic strains carrying different combinations of chromosome- and plasmid-mediated quinolone resistance mechanisms with susceptible, low-level quinolone resistant, resistant, and highly resistant phenotypes. Our comprehensive analysis opens up a new strategy for reversing drug resistance by targeting the SOS response.

Molecular insights into fosfomycin resistance in Escherichia coli.

Objectives: Fosfomycin activity in Escherichia coli depends on several genes of unknown importance for fosfomycin resistance. The objective was to characterize the role of uhpT , glpT , cyaA and ptsI genes in fosfomycin resistance in E. coli. Methods: WT E. coli BW25113 and null mutants, Δ uhpT , Δ glpT , Δ cyaA , Δ ptsI , Δ glpT-uhpT , Δ glpT-cyaA , Δ glpT-ptsI , Δ uhpT-cyaA , Δ uhpT-ptsI and Δ ptsI-cyaA , were studied. Susceptibility to fosfomycin was tested using CLSI guidelines. Fosfomycin mutant frequencies were determined at concentrations of 64 and 256 mg/L. Fosfomycin in vitro activity was tested using time-kill assays at concentrations of 64 and 307 mg/L (human C max ). Results: Fosfomycin MICs were: WT E. coli BW25113 (2 mg/L), Δ glpT (2 mg/L), Δ uhpT (64 mg/L), Δ cyaA (8 mg/L), Δ ptsI (2 mg/L), Δ glpT-uhpT (256 mg/L), Δ glpT-cyaA (8 mg/L), Δ glpT-ptsI (2 mg/L), Δ uhpT-cyaA (512 mg/L), Δ uhpT-ptsI (64 mg/L) and Δ ptsI-cyaA (32 mg/L). In the mutant frequency assays, no mutants were recovered from BW25113. Mutants appeared in Δ glpT , Δ uhpT , Δ cyaA and Δ ptsI at 64 mg/L and in Δ uhpT and Δ cyaA at 256 mg/L. Δ glpT-ptsI , but not Δ glpT-cyaA , Δ uhpT-cyaA or Δ uhpT-ptsI , increased the mutant frequency compared with the highest frequency found in each single mutant. In time-kill assays, all mutants regrew at 64 mg/L. Initial bacterial reductions of 2-4 log 10 cfu/mL were observed for all strains, except for Δ uhpT-ptsI , Δ glpT-uhpT and Δ uhpT-cyaA . Only Δ glpT and Δ ptsI mutants were cleared using 307 mg/L. Conclusions: Fosfomycin MIC may not be a good efficacy predictor, as highly resistant mutants may appear, depending on other pre-existing mutations with no impact on MIC.

Role of inoculum and mutant frequency on fosfomycin MIC discrepancies by agar dilution and broth microdilution methods in Enterobacteriaceae.

OBJECTIVES: Fosfomycin is re-evaluated as a treatment of multidrug-resistant Enterobacteriaceae infections. However, MIC differences have been described among the different susceptibility testing. The aim was to study the role of the different inoculum size used in agar dilution with respect to broth microdilution, according to CLSI, in the fosfomycin MIC discrepancies. METHODS: Fosfomycin MICs were determined using agar dilution (reference) and broth microdilution in 220 Escherichia coli (n=81) and Klebsiella pneumoniae (n=139) clinical isolates. Fosfomycin mutant frequencies were determined in 21 E. coli (MIC=1mg/L) and 21 K. pneumoniae (MIC=16mg/L). The emergence of resistant subpopulations of five E. coli strains (MIC=1mg/L) was monitored over the time by microdilution assay using 0, 4 and 8 mg/L of fosfomycin, and eight different inocula (5×105-3.91×103 CFU/well, 1 : 2 dilutions). RESULTS: For E. coli, 86.4% of categorical agreement (CA), 9.1% very major errors (VME), 3.3% major errors (ME) and 9.9% minor errors (mE) were found. For K. pneumoniae, CA was 51.1%, VME 15.7%, ME 28.4% and mE 25.2%. Essential agreement (±1-log2) was observed in 55.45%. By microdilution, 35.9% of the MICs showed discrepancies of ≥2 dilutions. Initial inoculum used was 5.63 times higher in the microdilution method, in range with CLSI methodology for both techniques. Fosfomycin mutant frequencies were 6.05×10-5 (4×MIC) to 5.59×10-7 (256×MIC) for E. coli, and 1.49×10-4 (4×MIC) to 1.58×10-5 (16×MIC) for K. pneumoniae. Resistant subpopulations arose mainly after 8 h of incubation with inocula >3.13×104 CFU/well. CONCLUSIONS: The higher inoculum used in the microdilution method enriched the initial inoculum with resistant subpopulations and could partially explain the fosfomycin MIC discrepancies with respect to the agar dilution method.

Plasmid-mediated quinolone resistance in Enterobacteriaceae: a systematic review with a focus on Mediterranean countries.

Quinolones are a family of synthetic broad-spectrum antimicrobial drugs. These molecules have been widely prescribed to treat various infectious diseases and have been classified into several generations based on their spectrum of activity. Quinolones inhibit bacterial DNA synthesis by interfering with the action of DNA gyrase and topoisomerase IV. Mutations in the genes encoding these targets are the most common mechanisms of high-level fluoroquinolone resistance. Moreover, three mechanisms for plasmid-mediated quinolone resistance (PMQR) have been discovered since 1998 and include Qnr proteins, the aminoglycoside acetyltransferase AAC(6')-Ib-cr, and plasmid-mediated efflux pumps QepA and OqxAB. Plasmids with these mechanisms often encode additional antimicrobial resistance (extended spectrum beta-lactamases [ESBLs] and plasmidic AmpC [pAmpC] ß-lactamases) and can transfer multidrug resistance. The PMQR determinants are disseminated in Mediterranean countries with prevalence relatively high depending on the sources and the regions, highlighting the necessity of long-term surveillance for the future monitoring of trends in the occurrence of PMQR genes.

Pharmacodynamics of fosfomycin: insights into clinical use for antimicrobial resistance.

The aim of this study was to improve the understanding of the pharmacokinetic-pharmacodynamic relationships of fosfomycin against extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strains that have different fosfomycin MICs. Our methods included the use of a hollow fiber infection model with three clinical ESBL-producing E. coli strains. Human fosfomycin pharmacokinetic profiles were simulated over 4 days. Preliminary studies conducted to determine the dose ranges, including the dose ranges that suppressed the development of drug-resistant mutants, were conducted with regimens from 12 g/day to 36 g/day. The combination of fosfomycin at 4 g every 8 h (q8h) and meropenem at 1 g/q8h was selected for further assessment. The total bacterial population and the resistant subpopulations were determined. No efficacy was observed against the Ec42444 strain (fosfomycin MIC, 64 mg/liter) at doses of 12, 24, or 36 g/day. All dosages induced at least initial bacterial killing against Ec46 (fosfomycin MIC, 1 mg/liter). High-level drug-resistant mutants appeared in this strain in response to 12, 15, and 18 g/day. In the study arms that included 24 g/day, once or in a divided dose, a complete extinction of the bacterial inoculum was observed. The combination of meropenem with fosfomycin was synergistic for bacterial killing and also suppressed all fosfomycin-resistant clones of Ec2974 (fosfomycin MIC, 1 mg/liter). We conclude that fosfomycin susceptibility breakpoints (≤64 mg/liter according to CLSI [for E. coli urinary tract infections only]) should be revised for the treatment of serious systemic infections. Fosfomycin can be used to treat infections caused by organisms that demonstrate lower MICs and lower bacterial densities, although relatively high daily dosages (i.e., 24 g/day) are required to prevent the emergence of bacterial resistance. The ratio of the area under the concentration-time curve for the free, unbound fraction of fosfomycin versus the MIC (fAUC/MIC) appears to be the dynamically linked index of suppression of bacterial resistance. Fosfomycin with meropenem can act synergistically against E. coli strains in preventing the emergence of fosfomycin resistance.

Presence of quinolone resistance to qnrB1 genes and blaOXA-48 carbapenemase in clinical isolates of Klebsiella pneumoniae in Spain.

A study is presented on the presence of quinolone resistance qnrB1 genes in clinical isolates belonging to the largest series of infections caused by OXA-48-producing Klebsiella pneumoniae in a single-centre outbreak in Spain. Evidence is also provided, according to in vitro results, that there is a possibility of co-transfer of plasmid harbouring blaOXA-48 with an other plasmid harbouring qnrB1 in presence of low antibiotic concentrations of fluoroquinolones, showing the risk of multi-resistance screening.

Impact of qnrA1, qnrB1 and qnrS1 on the efficacy of ciprofloxacin and levofloxacin in an experimental pneumonia model caused by Escherichia coli with or without the GyrA mutation Ser83Leu.

OBJECTIVES: The aim of this study was to evaluate the impact of qnrA1, qnrB1 and qnrS1 on the in vivo efficacies of ciprofloxacin and levofloxacin in an experimental model of pneumonia caused by Escherichia coli. METHODS: Two isogenic groups of E. coli transformants, based on two ATCC 25922 strains, with or without the GyrA mutation Ser83Leu, and carrying qnrA1, qnrB1 or qnrS1, were used in an experimental pneumonia model. The efficacies of ciprofloxacin (40 mg/kg/day) and levofloxacin (50 and 150 mg/kg/day) were evaluated. RESULTS: For the pneumonia caused by the parental strains lacking qnr genes, both fluoroquinolones significantly (P<0.05) reduced the bacterial lung concentration by >7 log10 cfu/g against E. coli ATCC/pBK and between 5.09 and 6.34 log10 cfu/g against E. coli ATCC-S83L/pBK. The presence of any qnr genes in the strains of both isogenic groups diminished the reduction of bacterial lung concentration with any therapy (P<0.05). Furthermore, all therapeutic schemes reduced the percentage of positive blood cultures in both isogenic groups (P<0.05). Finally, the survival results suggest a higher mortality with the strains expressing qnr genes. CONCLUSIONS: The presence of qnrA1, qnrB1 and qnrS1 in E. coli reduced the efficacy of ciprofloxacin and levofloxacin in a murine pneumonia model.