Heterogeneity of SOS response expression in clinical isolates of Escherichia coli influences adaptation to antimicrobial stress.

In recent years, new evidence has shown that the SOS response plays an important role in the response to antimicrobials, with involvement in the generation of clinical resistance. Here we evaluate the impact of heterogeneous expression of the SOS response in clinical isolates of Escherichia coli on response to the fluoroquinolone, ciprofloxacin. In silico analysis of whole genome sequencing data showed remarkable sequence conservation of the SOS response regulators, RecA and LexA. Despite the genetic homogeneity, our results revealed a marked differential heterogeneity in SOS response activation, both at population and single-cell level, among clinical isolates of E. coli in the presence of subinhibitory concentrations of ciprofloxacin. Four main stages of SOS response activation were identified and correlated with cell filamentation. Interestingly, there was a correlation between clinical isolates with higher expression of the SOS response and further progression to resistance. This heterogeneity in response to DNA damage repair (mediated by the SOS response) and induced by antimicrobial agents could be a new factor with implications for bacterial evolution and survival contributing to the generation of antimicrobial resistance.

Impact of suppression of the SOS response on protein expression in clinical isolates of Escherichia coli under antimicrobial pressure of ciprofloxacin.

Introduction/objective: Suppression of the SOS response in combination with drugs damaging DNA has been proposed as a potential target to tackle antimicrobial resistance. The SOS response is the pathway used to repair bacterial DNA damage induced by antimicrobials such as quinolones. The extent of lexA-regulated protein expression and other associated systems under pressure of agents that damage bacterial DNA in clinical isolates remains unclear. The aim of this study was to assess the impact of this strategy consisting on suppression of the SOS response in combination with quinolones on the proteome profile of Escherichia coli clinical strains. Materials and methods: Five clinical isolates of E. coli carrying different chromosomally- and/or plasmid-mediated quinolone resistance mechanisms with different phenotypes were selected, with E. coli ATCC 25922 as control strain. In addition, from each clinical isolate and control, a second strain was created, in which the SOS response was suppressed by deletion of the recA gene. Bacterial inocula from all 12 strains were then exposed to 1xMIC ciprofloxacin treatment (relative to the wild-type phenotype for each isogenic pair) for 1 h. Cell pellets were collected, and proteins were digested into peptides using trypsin. Protein identification and label-free quantification were done by liquid chromatography-mass spectrometry (LC-MS) in order to identify proteins that were differentially expressed upon deletion of recA in each strain. Data analysis and statistical analysis were performed using the MaxQuant and Perseus software. Results: The proteins with the lowest expression levels were: RecA (as control), AphA, CysP, DinG, DinI, GarL, PriS, PsuG, PsuK, RpsQ, UgpB and YebG; those with the highest expression levels were: Hpf, IbpB, TufB and RpmH. Most of these expression alterations were strain-dependent and involved DNA repair processes and nucleotide, protein and carbohydrate metabolism, and transport. In isolates with suppressed SOS response, the number of underexpressed proteins was higher than overexpressed proteins. Conclusion: High genomic and proteomic variability was observed among clinical isolates and was not associated with a specific resistant phenotype. This study provides an interesting approach to identify new potential targets to combat antimicrobial resistance.

Selection, characterization, and biosensing applications of DNA aptamers targeting cyanotoxin BMAA.

Scientists have established a connection between environmental exposure to toxins like ß-N-methylamino-l-alanine (BMAA) and a heightened risk of neurodegenerative disorders. BMAA is a byproduct from certain strains of cyanobacteria that are present in ecosystems worldwide and is renowned for its bioaccumulation and biomagnification in seafood. The sensitivity, selectivity, and reproducibility of the current analytical techniques are insufficient to support efforts regarding food safety and environment monitoring adequately. This work outlines the in vitro selection of BMAA-specific DNA aptamers via the systematic evolution of ligands through exponential enrichment (SELEX). Screening and characterization of the full-length aptamers was achieved using the SYBR Green (SG) fluorescence displacement assay. Aptamers BMAA_159 and BMAA_165 showed the highest binding affinities, with dissociation constants (Kd) of 2.2 ± 0.1 µM and 0.32 ± 0.02 µM, respectively. After truncation, the binding affinity was confirmed using a BMAA-conjugated fluorescence assay. The Kd values for BMAA_159_min and BMAA_165_min were 6 ± 1 µM and 0.63 ± 0.02 µM, respectively. Alterations in the amino proton region studied using solution nuclear magnetic resonance (NMR) provided further evidence of aptamer-target binding. Additionally, circular dichroism (CD) spectroscopy revealed that BMAA_165_min forms hybrid G-quadruplex (G4) structures. Finally, BMAA_165_min was used in the development of an electrochemical aptamer-based (EAB) sensor that accomplished sensitive and selective detection of BMAA with a limit of detection (LOD) of 1.13 ± 0.02 pM.

Decoding Non-coding Variants: Recent Approaches to Studying Their Role in Gene Regulation and Human Diseases.

Genome-wide association studies (GWAS) have mapped over 90% of disease- and quantitative-trait-associated variants within the non-coding genome. Non-coding regulatory DNA (e.g., promoters and enhancers) and RNA (e.g., 5' and 3' UTRs and splice sites) are essential in regulating temporal and tissue-specific gene expressions. Non-coding variants can potentially impact the phenotype of an organism by altering the molecular recognition of the cis-regulatory elements, leading to gene dysregulation. However, determining causality between non-coding variants, gene regulation, and human disease has remained challenging. Experimental and computational methods have been developed to understand the molecular mechanism involved in non-coding variant interference at the transcriptional and post-transcriptional levels. This review discusses recent approaches to evaluating disease-associated single-nucleotide variants (SNVs) and determines their impact on transcription factor (TF) binding, gene expression, chromatin conformation, post-transcriptional regulation, and translation.

Implications of two-component systems EnvZ/OmpR and BaeS/BaeR in in vitro temocillin resistance in Escherichia coli.

BACKGROUND: BaeS/BaeR is a two-component system of Escherichia coli that controls the expression of porins and efflux pumps. Its role in beta-lactam resistance is limited. OBJECTIVES: To study the role of baeS/baeR two-component system in temocillin resistance in E. coli. METHODS: E. coli strain BW25113 and single-gene deletion mutants related to two-component systems were collected from the KEIO collection. Double-gen deletion mutants were generated. Temocillin-resistant mutant frequencies were determined at 32 mg/L. E. coli BW25113 mutants were selected by selective pressure from serial passages. Biological costs were analysed by growth curves. Genomes of the generated mutants were sequenced. The expression level of the mdtA, mdtB, mdtC, acrD and tolC in the ΔbaeS mutant was determined by RT-PCR (with/without temocillin exposure). RESULTS: The frequency of temocillin mutants ranged from 2.12 × 10-8 to 4.51 × 10-8 in single-porin mutants. No mutants were recovered from E. coli BW25113 (>10-9). Selection of temocillin-resistant variants by serial passage yielded mutants up to 128 mg/L. Mutations were found in the baeS gene. Temocillin MICs ranged from 4 to 32 mg/L (highest MICs for ΔbaeS and ΔompR). The efflux pumps mdtA, mdtB, mdtC and acrD pumps were overexpressed 3-10-fold in the presence of temocillin in ΔbaeS compared to control. CONCLUSIONS: Mutations in the sensor histidine kinase, baeS, may be involved in temocillin resistance through the expression of the efflux pumps mdtABC and acrD. In addition, the low mutation rate may be a good predictor of temocillin activity.

Evaluation of temocillin efficacy against KPC-2-producing Klebsiella pneumoniae isolates in a hollow-fibre infection model.

BACKGROUND: Temocillin is an old antimicrobial that is resistant to hydrolysis by ESBLs but has variable activity against carbapenemase-producing Enterobacteriaceae. The current EUCAST susceptibility breakpoints for Enterobacterales are set at ≤16 mg/L (susceptible with increased exposure) based on a dose of 2 g q8h, but there is limited information on the efficacy of this dose against temocillin-susceptible carbapenemase-producing Klebsiella pneumoniae isolates. OBJECTIVES: To evaluate the efficacy of this dose using a hollow-fibre infection model (HFIM) against six KPC-2-producing clinical isolates of K. pneumoniae. METHODS: The isolates were characterized by WGS and temocillin susceptibility was determined using standard and high inoculum temocillin. Mutant frequencies were estimated and temocillin activity was tested in time-kill assays and in the HFIM. At standard conditions, three of the isolates were classified as susceptible (MIC ≤ 16 mg/L) and three as resistant (MIC > 16 mg/L). The HFIM was performed over 3 days to mimic human-like pharmacokinetics of 2 g q8h. Bacterial counts were performed by plating on Mueller-Hinton agar (MHA) and MHA containing 64 mg/L temocillin to detect resistant subpopulations. RESULTS: All isolates showed a reduction in bacterial population of at least 3 log cfu/mL within the first 8 h of simulated treatment in the hollow-fibre assay. Regrowth was observed for the three resistant isolates and one of the susceptible ones. The MIC value for these isolates was higher by at least two dilutions compared with their initial values. CONCLUSIONS: These data suggest that an optimized pharmacokinetic regimen may be of clinical interest for the treatment of KPC-2-producing K. pneumoniae susceptible to temocillin. These data showed activity of temocillin against KPC-2-producing K. pneumoniae susceptible to temocillin; however, a dose of 2g q8h administered over 30 min may be inadequate to prevent the emergence of resistant variants.

Prioritizing cardiovascular disease-associated variants altering NKX2-5 and TBX5 binding through an integrative computational approach.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide and are heavily influenced by genetic factors. Genome-wide association studies have mapped >90% of CVD-associated variants within the noncoding genome, which can alter the function of regulatory proteins, such as transcription factors (TFs). However, due to the overwhelming number of single-nucleotide polymorphisms (SNPs) (>500,000) in genome-wide association studies, prioritizing variants for in vitro analysis remains challenging. In this work, we implemented a computational approach that considers support vector machine (SVM)-based TF binding site classification and cardiac expression quantitative trait loci (eQTL) analysis to identify and prioritize potential CVD-causing SNPs. We identified 1535 CVD-associated SNPs within TF footprints and putative cardiac enhancers plus 14,218 variants in linkage disequilibrium with genotype-dependent gene expression in cardiac tissues. Using ChIP-seq data from two cardiac TFs (NKX2-5 and TBX5) in human-induced pluripotent stem cell-derived cardiomyocytes, we trained a large-scale gapped k-mer SVM model to identify CVD-associated SNPs that altered NKX2-5 and TBX5 binding. The model was tested by scoring human heart TF genomic footprints within putative enhancers and measuring in vitro binding through electrophoretic mobility shift assay. Five variants predicted to alter NKX2-5 (rs59310144, rs6715570, and rs61872084) and TBX5 (rs7612445 and rs7790964) binding were prioritized for in vitro validation based on the magnitude of the predicted change in binding and are in cardiac tissue eQTLs. All five variants altered NKX2-5 and TBX5 DNA binding. We present a bioinformatic approach that considers tissue-specific eQTL analysis and SVM-based TF binding site classification to prioritize CVD-associated variants for in vitro analysis.

The mef(A)/msr(D)-carrying streptococcal prophage Φ1207.3 encodes an SOS-like system, induced by UV-C light, responsible for increased survival and increased mutation rate.

Bacterial SOS response is an inducible system of DNA repair and mutagenesis. Streptococci lack a canonical SOS response, but an SOS-like response was reported in some species. The mef(A)-msr(D)-carrying prophage Ф1207.3 of Streptococcus pyogenes contains a region, spanning orf6 to orf11, showing homology to characterized streptococcal SOS-like cassettes. Genome-wide homology search showed the presence of the whole Φ1207.3 SOS-like cassette in three S. pyogenes prophages, while parts of it were found in other bacterial species. To investigate whether this cassette confers an SOS-mutagenesis phenotype, we constructed Streptococcus pneumoniae R6 isogenic derivative strains: (i) FR172, streptomycin resistant, (ii) FR173, carrying Φ1207.3, and (iii) FR174, carrying a recombinant Φ1207.3, where the SOS-like cassette was deleted. These strains were used in survival and mutation rate assays using a UV-C LED instrument, for which we designed and 3D-printed a customized equipment, constituted of an instrument support and swappable-autoclavable mini-plates and lids. Upon exposure to UV fluences ranging from 0 to 6,400 J/m2 at four different wavelengths, 255, 265, 275, and 285 nm, we found that the presence of Φ1207.3 SOS-like cassette increases bacterial survival up to 34-fold. Mutation rate was determined by measuring rifampicin resistance acquisition upon exposure to UV fluence of 50 J/m2 at the four wavelengths by fluctuation test. The presence of Φ1207.3 SOS-like cassette resulted in a significant increase in the mutation rate (up to 18-fold) at every wavelength. In conclusion, we demonstrated that Φ1207.3 carries a functional SOS-like cassette responsible for an increased survival and increased mutation rate in S. pneumoniae. IMPORTANCE Bacterial mutation rate is generally low, but stress conditions and DNA damage can induce stress response systems, which allow for improved survival and continuous replication. The SOS response is a DNA repair mechanism activated by some bacteria in response to stressful conditions, which leads to a temporary hypermutable phenotype and is usually absent in streptococcal genomes. Here, using a reproducible and controlled UV irradiation system, we demonstrated that the SOS-like gene cassette of prophage Φ1207.3 is functional, responsible for a temporary hypermutable phenotype, and enhances bacterial survival to UV irradiation. Prophage Φ1207.3 also carries erythromycin resistance genes and can lysogenize different pathogenic bacteria, constituting an example of a mobile genetic element which can confer multiple phenotypes to its host.

Prioritizing Cardiovascular Disease-Associated Variants Altering NKX2-5 Binding through an Integrative Computational Approach.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide and are heavily influenced by genetic factors. Genome-wide association studies (GWAS) have mapped > 90% of CVD-associated variants within the non-coding genome, which can alter the function of regulatory proteins, like transcription factors (TFs). However, due to the overwhelming number of GWAS single nucleotide polymorphisms (SNPs) (>500,000), prioritizing variants for in vitro analysis remains challenging. In this work, we implemented a computational approach that considers support vector machine (SVM)-based TF binding site classification and cardiac expression quantitative trait loci (eQTL) analysis to identify and prioritize potential CVD-causing SNPs. We identified 1,535 CVD-associated SNPs that occur within human heart footprints/enhancers and 9,309 variants in linkage disequilibrium (LD) with differential gene expression profiles in cardiac tissue. Using hiPSC-CM ChIP-seq data from NKX2-5 and TBX5, two cardiac TFs essential for proper heart development, we trained a large-scale gapped k-mer SVM (LS-GKM-SVM) predictive model that can identify binding sites altered by CVD-associated SNPs. The computational predictive model was tested by scoring human heart footprints and enhancers in vitro through electrophoretic mobility shift assay (EMSA). Three variants (rs59310144, rs6715570, and rs61872084) were prioritized for in vitro validation based on their eQTL in cardiac tissue and LS-GKM-SVM prediction to alter NKX2-5 DNA binding. All three variants altered NKX2-5 DNA binding. In summary, we present a bioinformatic approach that considers tissue-specific eQTL analysis and SVM-based TF binding site classification to prioritize CVD-associated variants for in vitro experimental analysis.

Hidden modes of DNA binding by human nuclear receptors.

Human nuclear receptors (NRs) are a superfamily of ligand-responsive transcription factors that have central roles in cellular function. Their malfunction is linked to numerous diseases, and the ability to modulate their activity with synthetic ligands has yielded 16% of all FDA-approved drugs. NRs regulate distinct gene networks, however they often function from genomic sites that lack known binding motifs. Here, to annotate genomic binding sites of known and unexamined NRs more accurately, we use high-throughput SELEX to comprehensively map DNA binding site preferences of all full-length human NRs, in complex with their ligands. Furthermore, to identify non-obvious binding sites buried in DNA-protein interactomes, we develop MinSeq Find, a search algorithm based on the MinTerm concept from electrical engineering and digital systems design. The resulting MinTerm sequence set (MinSeqs) reveal a constellation of binding sites that more effectively annotate NR-binding profiles in cells. MinSeqs also unmask binding sites created or disrupted by 52,106 single-nucleotide polymorphisms associated with human diseases. By implicating druggable NRs as hidden drivers of multiple human diseases, our results not only reveal new biological roles of NRs, but they also provide a resource for drug-repurposing and precision medicine.

CREPE: a Shiny app for transcription factor cataloguing.

Summary: Transcription factors (TFs) are proteins that directly interpret the genome to regulate gene expression and determine cellular phenotypes. TF identification is a common first step in unraveling gene regulatory networks. We present CREPE, an R Shiny app to catalogue and annotate TFs. CREPE was benchmarked against curated human TF datasets. Next, we use CREPE to explore the TF repertoires of Heliconius erato and Heliconius melpomene butterflies. Availability and implementation: CREPE is available as a Shiny app package available at GitHub (github.com/dirostri/CREPE). Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Disease-associated non-coding variants alter NKX2-5 DNA-binding affinity.

Genome-wide association studies (GWAS) have mapped over 90 % of disease- or trait-associated variants within the non-coding genome, like cis-regulatory elements (CREs). Non-coding single nucleotide polymorphisms (SNPs) are genomic variants that can change how DNA-binding regulatory proteins, like transcription factors (TFs), interact with the genome and regulate gene expression. NKX2-5 is a TF essential for proper heart development, and mutations affecting its function have been associated with congenital heart diseases (CHDs). However, establishing a causal mechanism between non-coding genomic variants and human disease remains challenging. To address this challenge, we identified 8475 SNPs predicted to alter NKX2-5 DNA-binding using a position weight matrix (PWM)-based predictive model. Five variants were prioritized for in vitro validation; four of them are associated with traits and diseases that impact cardiovascular health. The impact of these variants on NKX2-5 binding was evaluated with electrophoretic mobility shift assay (EMSA) using purified recombinant NKX2-5 homeodomain. Binding curves were constructed to determine changes in binding between variant and reference alleles. Variants rs7350789, rs7719885, rs747334, and rs3892630 increased binding affinity, whereas rs61216514 decreased binding by NKX2-5 when compared to the reference genome. Our findings suggest that differential TF-DNA binding affinity can be key in establishing a causal mechanism of pathogenic variants.

Effect of Glycerol on Fosfomycin Activity against Escherichia coli.

Fosfomycin is an antimicrobial that inhibits the biosynthesis of peptidoglycan by entering the bacteria through two channels (UhpT and GlpT). Glycerol is clinically used as a treatment for elevated intracranial pressure and induces the expression of glpT in Escherichia coli. Glycerol might offer synergistic activity by increasing fosfomycin uptake. The present study evaluates the use of glycerol at physiological concentrations in combination with fosfomycin against a collection of isogenic mutants of fosfomycin-related genes in E. coli strains. Induction of fosfomycin transporters, susceptibility tests, interaction assays, and time-kill assays were performed. Our results support the notion that glycerol allows activation of the GlpT transporter, but this induction is delayed over time and is not homogeneous across the bacterial population, leading to contradictory results regarding the enhancement of fosfomycin activity. The susceptibility assays showed an increase in fosfomycin activity with glycerol in the disk diffusion assay but not in the agar dilution or broth microdilution assays. Similarly, in the time-kill assays, the effect of glycerol was absent by the emergence of fosfomycin-resistant subpopulations. In conclusion, glycerol may not be a good candidate for use as an adjuvant with fosfomycin.

A butterfly pan-genome reveals that a large amount of structural variation underlies the evolution of chromatin accessibility.

Despite insertions and deletions being the most common structural variants (SVs) found across genomes, not much is known about how much these SVs vary within populations and between closely related species, nor their significance in evolution. To address these questions, we characterized the evolution of indel SVs using genome assemblies of three closely related Heliconius butterfly species. Over the relatively short evolutionary timescales investigated, up to 18.0% of the genome was composed of indels between two haplotypes of an individual Heliconius charithonia butterfly and up to 62.7% included lineage-specific SVs between the genomes of the most distant species (11 Mya). Lineage-specific sequences were mostly characterized as transposable elements (TEs) inserted at random throughout the genome and their overall distribution was similarly affected by linked selection as single nucleotide substitutions. Using chromatin accessibility profiles (i.e., ATAC-seq) of head tissue in caterpillars to identify sequences with potential cis-regulatory function, we found that out of the 31,066 identified differences in chromatin accessibility between species, 30.4% were within lineage-specific SVs and 9.4% were characterized as TE insertions. These TE insertions were localized closer to gene transcription start sites than expected at random and were enriched for sites with significant resemblance to several transcription factor binding sites with known function in neuron development in Drosophila We also identified 24 TE insertions with head-specific chromatin accessibility. Our results show high rates of structural genome evolution that were previously overlooked in comparative genomic studies and suggest a high potential for structural variation to serve as raw material for adaptive evolution.

Prevalence, Incidence, and Risk Factors for Intestinal Colonization Due to Fluoroquinolone-Resistant ST131 Escherichia coli: a Longitudinal Study in Highly Dependent, Long-Term Care Facility Residents.

Escherichia coli ST131 clade C is an important driver for fluoroquinolone resistance (FQ-R). We conducted a prospective observational study in residents from two long-term care facilities (LTCFs) in Seville, Spain, in 2018. Fecal swabs and environmental samples were obtained. E. coli isolates were screened for clade C, FQ-R ST131 by PCR, and molecular typing by PFGE; representatives from pulsotypes were studied by whole-genome-sequencing (WGS) and assigned to lineages (cgSTs). Prevalence of colonization at each time point, incidence density, and risk factors for acquisition were studied. Seventy-six FQ-R ST131 E. coli isolates belonging to 34 cgSTs were obtained; 24 belonging to subclade C1 (116 isolates, 65.9%) and 10 to C2 (60, 34.1%). C1 lineages showed lower virulence scores than C2 (median [IQR], 19 [18 to 20] versus 21 [20 to 21.5], P = 0.001) and higher number of plasmids (4 [3 to 5] versus 2 [2 to 3], P = 0.01). aac(6')-Ib-cr and blaOXA-1 were less frequent in C1 than C2 (2 [8.3%] versus 6 [60%], P = 0.003 for both); ESBL genes were detected in eight (33.3%) C1 (5 blaCTX-M-27) and three (30%) C2 (all blaCTX-M-15). Of the 82 residents studied, 49 were colonized at some point (59.7%), with a pooled prevalence of 38.6%. Incidence density of new lineage acquisition was 2.22 per 100 resident weeks (1.28 and 0.93 C1 and C2 subclades, respectively). Independent risk factors for acquisitions were having a colonized roommate (HR = 4.21; 95% CI = 1.71 to 10.36; P = 0.002) and urinary or fecal incontinence (HR = 2.82; 95% CI = 1.21 to 6.56; P = 0.01). LTCFs are important reservoirs of clade C ST131 E. coli. The risk factors found suggest that cross-transmission is the most relevant transmission mechanisms. IMPORTANCE We aimed at investigating the microbiological and epidemiological features of clade C fluoroquinolone-resistant ST131 E. coli isolates colonizing highly dependent residents in long-term care facilities (LTCFs) during 40 weeks and the risk factors of acquisition. Isolates from C1 and C2 subclades were characterized in this environment. The clonality of the isolates was characterized and they were assigned to lineages (cgSTs), Resistance genes, virulence factors, and plasmids were also described. This study suggests that cross-transmission is the most relevant transmission mechanisms; however, environmental colonization might also play a role. We believe the data provide useful information to depict the epidemiology of these bacteria by merging detailed microbiological and epidemiological information.

Role of inorganic phosphate concentrations in in vitro activity of fosfomycin.

OBJECTIVES: The objective of this study was to evaluate the in vitro activity of fosfomycin under different physiological concentrations of inorganic phosphate (Pi). METHODS: The wild-type BW25113 strain, four isogenic mutants (ΔglpT, ΔuhpT, ΔglpT-uhpT, and ΔphoB) and six clinical isolates of Escherichia coli with different fosfomycin susceptibilities were used. EUCAST breakpoints were used. Susceptibility was evaluated by agar dilution using standard Mueller-Hinton agar (Pi concentration of 1 mM similar to human plasma concentration) and supplemented with Pi (13 and 42 mM, minimum and maximum urinary Pi concentrations) and/or glucose-6-phosphate (25 mg/L). Fosfomycin transporter promoter activity was assayed using PglpT::gfpmut2 or PuhpT::gfpmut2 promoter fusions in standard Mueller-Hinton Broth (MHB), supplemented with Pi (13 or 42 mM) ± glucose-6-phosphate. Fosfomycin activity was quantified, estimating fosfomycin EC50 under different Pi concentrations (1, 13 and 42 mM + glucose-6-phosphate) and in time-kill assays using fosfomycin concentrations of 307 (maximum plasma concentration (Cmax)), 1053 and 4415 mg/L (urine Cmax range), using MHB with 28 mM Pi (mean urine Pi concentration) + 25 mg/L glucose-6-phosphate. RESULTS: All the strains showed decreased susceptibility to fosfomycin linked to increased Pi concentrations: 1-4 log2 dilution differences from 1 to 13 mM, and 1-8 log2 dilution differences at 42 mM Pi. Changes in phosphate concentration did not affect the expression of fosfomycin transporters. By increasing Pi concentrations higher fosfomycin EC50 bacterial viability was observed, except against ΔglpT-uhpT. The increase in Pi reduced the bactericidal effect of fosfomycin. DISCUSSION: Pi variations in physiological fluids may reduce fosfomycin activity against E. coli. Elevated Pi concentrations in urine may explain oral fosfomycin failure in non-wild-type but fosfomycin-susceptible E. coli strains.

Higher prevalence of CTX-M-27-producing Escherichia coli belonging to ST131 clade C1 among residents of two long-term care facilities in Southern Spain.

Recently, the emergence of an international lineage of the CTX-M-27-producing clade C1 of Escherichia coli ST131 is being observed. The aim is to see if this strain has also been introduced in our area. Twenty-eight (33%) out of 86 individuals from two LTCFs in Seville were found to be colonized with fluoroquinolone-resistant E. coli ST131 and 46% isolates were ESBL/pAmpC producers. C1 isolates were more common than C2 and more frequently produced blaESBL/pAmpC genes (53% vs 33%). Strain sharing was observed in 6 groups of 2-5 cases (61%). A differentiated cluster of 5 C1-CTX-M-27 isolates was found which lacked the M27PP1 region.

Interplay between IncF plasmids and topoisomerase mutations conferring quinolone resistance in the Escherichia coli ST131 clone: stability and resistance evolution.

The Escherichia coli ST131 H30-Rx subclone vehicles CTX-M-15 plasmids and mutations in gyrA and parC conferring multidrug resistance successfully in the clinical setting. The aim of this study was (1) to investigate the relationship of specific topoisomerase mutations on the stability of IncF (CTX-M producing) plasmids using isogenic E. coli mutants and (2) to investigate the impact of the IncF-type plasmids present in the E. coli clone ST131 on the evolution of quinolone resistance. E. coli ATCC 25922 (background strain) and derived mutants encoding specific QRDR substitutions were used. Also, NGS-characterized IncFIA and IncFIB plasmids (encoding CTX-M genes) were included. Plasmid stability was evaluated by sequential dilutions into Luria broth medium without antibiotics for 7 days. Mutant frequency to ciprofloxacin was also evaluated. Moderate differences in the IncF plasmids stability were observed among E. coli ATCC 25922 and isogenic mutants. Under our experimental conditions, the fluctuation of bacteria harboring plasmids was less than 0.5-log(10) in all cases. In the mutant frequency tests, it was observed that the presence of these IncF plasmids increased this value significantly (10-1000-fold). Quinolone resistance substitutions in gyrA or parC genes, frequently found associated with E. coli clone ST131, do not modify the stability of ST131-associated IncFIA and IncFIB plasmids under in vitro conditions. IncF-type plasmids present in E. coli clone ST131 facilitate the selection of resistance to quinolones. These results are consistent with the clinical scenario in which the combination of resistance to quinolones and beta-lactams is highly frequent in the E. coli clone ST131.