Central role of the ramAR locus in the multidrug resistance in ESBL-Enterobacterales.

The aim of this study was to evaluate the proportion of resistance to a temocillin, tigecycline, ciprofloxacin, and chloramphenicol phenotype called t2c2 that resulted from mutations within the ramAR locus among extended-spectrum ß-lactamases-Enterobacterales (ESBL-E) isolated in three intensive care units for 3 years in a French university hospital. Two parallel approaches were performed on all 443 ESBL-E included: (i) the minimal inhibitory concentrations of temocillin, tigecycline, ciprofloxacin, and chloramphenicol were determined and (ii) the genomes obtained from the Illumina sequencing platform were analyzed to determine multilocus sequence types, resistomes, and diversity of several tetR-associated genes including ramAR operon. Among the 443 ESBL-E strains included, isolates of Escherichia coli (n = 194), Klebsiella pneumoniae (n = 122), and Enterobacter cloacae complex (Ecc) (n = 127) were found. Thirty-one ESBL-E strains (7%), 16 K. pneumoniae (13.1%), and 15 Ecc (11.8%) presented the t2c2 phenotype in addition to their ESBL profile, whereas no E. coli presented these resistances. The t2c2 phenotype was invariably reversible by the addition of Phe-Arg-ß-naphthylamide, indicating a role of resistance-nodulation-division pumps in these observations. Mutations associated with the t2c2 phenotype were restricted to RamR, the ramAR intergenic region (IR), and AcrR. Mutations in RamR consisted of C- or N-terminal deletions and amino acid substitutions inside its DNA-binding domain or within key sites of protein-substrate interactions. The ramAR IR showed nucleotide substitutions involved in the RamR DNA-binding domain. This diversity of sequences suggested that RamR and the ramAR IR represent major genetic events for bacterial antimicrobial resistance.IMPORTANCEMorbimortality caused by infectious diseases is very high among patients hospitalized in intensive care units (ICUs). A part of these outcomes can be explained by antibiotic resistance, which delays the appropriate therapy. The transferable antibiotic resistance gene is a well-known mechanism to explain the high rate of multidrug resistance (MDR) bacteria in ICUs. This study describes the prevalence of chromosomal mutations, which led to additional antibiotic resistance among MDR bacteria. More than 12% of Klebsiella pneumoniae and Enterobacter cloacae complex strains presented mutations within the ramAR locus associated with a dysregulation of an efflux pump called AcrAB-TolC and a porin: OmpF. These dysregulations led to an increase in antibiotic output notably tigecycline, ciprofloxacin, and chloramphenicol associated with a decrease of input for beta-lactam, especially temocillin. Mutations within transcriptional regulators such as ramAR locus played a major role in antibiotic resistance dissemination and need to be further explored.

Impact of the phenotypic expression of temocillin resistance in Escherichia coli on temocillin efficacy in a murine peritonitis model.

BACKGROUND: Temocillin is a narrow spectrum ß-lactam active against MDR Enterobacterales. Mechanisms of acquired resistance to temocillin are poorly understood. We analysed resistance mechanisms in clinical isolates of Escherichia coli and evaluated their impact on temocillin efficacy in vitro and in a murine peritonitis model. METHODS: Two sets of isogenic clinical E. coli strains were studied: a susceptible isolate (MLTEM16S) and its resistant derivative, MLTEM16R (mutation in nmpC porin gene); and temocillin-resistant derivatives of E. coli CFT073: CFT-ΔnmpC (nmpC deletion), CFTbaeS-TP and CFTbaeS-AP (two different mutations in the baeS efflux-pump gene).Fitness cost, time-kill curves and phenotypic expression of resistance were determined. Temocillin efficacy was assessed in a murine peritonitis model. RESULTS: MICs of temocillin were 16 and 64 mg/L for MLTEM16S and MLTEM16R, respectively, and 8, 128, 256 and 256 mg/L for E. coli-CFT073, CFT-ΔnmpC, CFTbaeS-TP and CFTbaeS-AP, respectively. No fitness cost of resistance was evidenced. All resistant strains showed heteroresistant profiles, except for CFTbaeS-AP, which displayed a homogeneous pattern. In vitro, temocillin was bactericidal against MLTEM16R, CFT-ΔnmpC, CFTbaeS-TP and CFTbaeS-AP at 128, 256, 512 and 512 mg/L, respectively. In vivo, temocillin was as effective as cefotaxime against MLTEM16R, CFT-ΔnmpC and CFTbaeS-TP, but inefficient against CFTbaeS-AP (100% mortality). CONCLUSIONS: Heteroresistant NmpC porin alteration and active efflux modification do not influence temocillin efficacy despite high MIC values, unfavourable pharmacokinetic/pharmacodynamic conditions and the absence of fitness cost, whereas homogeneously expressed BaeS efflux pump alteration yielding similar MICs leads to temocillin inefficacy. MIC as sole predictor of temocillin efficacy should be used with caution.

Decentralized Coordination of a Multi-UAV System for Spatial Planar Shape Formations.

Motivated by feedback from firefighters in Normandy, this work aims to provide a simple technique for a set of identical drones to collectively describe an arbitrary planar virtual shape in a 3D space in a decentralized manner. The original problem involved surrounding a toxic cloud to monitor its composition and short-term evolution. In the present work, the pattern is described using Fourier descriptors, a convenient mathematical formulation for that purpose. Starting from a reference point, which can be the center of a fire, Fourier descriptors allow for more precise description of a shape as the number of harmonics increases. This pattern needs to be evenly occupied by the fleet of drones under consideration. To optimize the overall view, the drones must be evenly distributed angularly along the shape. The proposed method enables virtual planar shape description, decentralized bearing angle assignment, drone movement from takeoff positions to locations along the shape, and collision avoidance. Furthermore, the method allows for the number of drones to change during the mission. The method has been tested both in simulation, through emulation, and in outdoor experiments with real drones. The obtained results demonstrate that the method is applicable in real-world contexts.

The Role of Next-Generation Sequencing (NGS) in the Management of Tuberculosis: Practical Review for Implementation in Routine.

Next-generation sequencing (NGS) has modernized the field of tuberculosis (TB) research by enabling high-throughput sequencing of the entire genome of Mycobacterium tuberculosis (MTB), which is the causative agent of TB. NGS has provided insights into the genetic diversity of MTB, which are crucial for understanding the evolution and transmission of the disease, and it has facilitated the identification of drug-resistant strains, enabling rapid and accurate tailoring of treatment. However, the high cost and the technical complexities of NGS currently limit its widespread use in clinical settings. International recommendations are thus necessary to facilitate the interpretation of polymorphisms, and an experimental approach is still necessary to correlate them to phenotypic data. This review aims to present a comparative, step-by-step, and up-to-date review of the techniques available for the implementation of this approach in routine laboratory workflow. Ongoing research on NGS for TB holds promise for improving our understanding of the disease and for developing more efficacious treatments.

Updated Review on Clinically-Relevant Properties of Delafloxacin.

The extensive use of fluoroquinolones has been consequently accompanied by the emergence of bacterial resistance, which triggers the necessity to discover new compounds. Delafloxacin is a brand-new anionic non-zwitterionic fluoroquinolone with some structural particularities that give it attractive proprieties: high activity under acidic conditions, greater in vitro activity against Gram-positive bacteria-even those showing resistance to currently-used fluoroquinolones-and nearly equivalent affinity for both type-II topoisomerases (i.e., DNA gyrase and topoisomerase IV). During phases II and III clinical trials, delafloxacin showed non-inferiority compared to standard-of-care therapy in the treatment of acute bacterial skin and skin structure infections and community-acquired bacterial pneumonia, which resulted in its approval in 2017 by the Food and Drug Administration for indications. Thanks to its overall good tolerance, its broad-spectrum in vitro activity, and its ease of use, it could represent a promising molecule for the treatment of bacterial infections.

Recent Trends in Antimicrobial Resistance among Anaerobic Clinical Isolates.

Anaerobic bacteria are normal inhabitants of the human commensal microbiota and play an important role in various human infections. Tedious and time-consuming, antibiotic susceptibility testing is not routinely performed in all clinical microbiology laboratories, despite the increase in antibiotic resistance among clinically relevant anaerobes since the 1990s. ß-lactam and metronidazole are the key molecules in the management of anaerobic infections, to the detriment of clindamycin. ß-lactam resistance is usually mediated by the production of ß-lactamases. Metronidazole resistance remains uncommon, complex, and not fully elucidated, while metronidazole inactivation appears to be a key mechanism. The use of clindamycin, a broad-spectrum anti-anaerobic agent, is becoming problematic due to the increase in resistance rate in all anaerobic bacteria, mainly mediated by Erm-type rRNA methylases. Second-line anti-anaerobes are fluoroquinolones, tetracyclines, chloramphenicol, and linezolid. This review aims to describe the up-to-date evolution of antibiotic resistance, give an overview, and understand the main mechanisms of resistance in a wide range of anaerobes.

Differential response to antibiotic therapy in staphylococcal infective endocarditis: contribution of an ex vivo model.

OBJECTIVES: Staphylococcal infective endocarditis (IE) remains a hard-to-treat infection with high mortality. Both the evaluation of new innovative therapies and research on alternative models mimicking human IE are therefore urgently needed to improve the prognosis of patients with diagnosed IE. Dalbavancin is a novel anti-staphylococcal lipoglycopeptide but there are limited data supporting its efficacy on biofilm infections. This antibiotic could be an alternative to current therapies for the medical treatment of IE but it needs to be further evaluated. METHODS: Here we developed an original ex vivo model of Staphylococcus aureus IE on human heart valves and assessed biofilm formation on them. After validating the model, the efficacy of two antistaphylococcal antibiotics, vancomycin and dalbavancin, was compared by measuring and visualizing their respective ability to inhibit and eradicate late-formed biofilm. RESULTS: Determination of the minimum biofilm inhibitory (MbIC) and eradicating (MbEC) concentrations in our ex vivo model identified dalbavancin as a promising drug with much lower MbIC and MBEC than vancomycin (respectively <0.01 versus 28 mg/L and 0.03 versus 32 mg/L). CONCLUSIONS: These data highlight a strong bactericidal effect of dalbavancin, particularly on an infected heart valve compared with vancomycin. Dalbavancin could be a realistic alternative treatment for the management of staphylococcal IE.

Temocillin Resistance in the Enterobacter cloacae Complex Is Conferred by a Single Point Mutation in BaeS, Leading to Overexpression of the AcrD Efflux Pump.

The Enterobacter cloacae complex (ECC) has become a major opportunistic pathogen with antimicrobial resistance issues. Temocillin, an "old" carboxypenicillin that is remarkably stable toward ß-lactamases, has been used as an alternative for the treatment of multidrug-resistant ECC infections. Here, we aimed at deciphering the never-investigated mechanisms of temocillin resistance acquisition in Enterobacterales. By comparative genomic analysis of two clonally related ECC clinical isolates, one susceptible (Temo_S [MIC of 4 mg/L]) and the other resistant (Temo_R [MIC of 32 mg/L]), we found that they differed by only 14 single-nucleotide polymorphisms, including one nonsynonymous mutation (Thr175Pro) in the two-component system (TCS) sensor histidine kinase BaeS. By site-directed mutagenesis in Escherichia coli CFT073, we demonstrated that this unique change in BaeS was responsible for a significant (16-fold) increase in temocillin MIC. Since the BaeSR TCS regulates the expression of two resistance-nodulation-cell division (RND)-type efflux pumps (namely, AcrD and MdtABCD) in E. coli and Salmonella, we demonstrated by quantitative reverse transcription-PCR that mdtB, baeS, and acrD genes were significantly overexpressed (15-, 11-, and 3-fold, respectively) in Temo_R. To confirm the role of each efflux pump in this mechanism, multicopy plasmids harboring mdtABCD or acrD were introduced into either Temo_S or the reference strain E. cloacae subsp. cloacae ATCC 13047. Interestingly, only the overexpression of acrD conferred a significant increase (from 8- to 16-fold) of the temocillin MIC. Altogether, we have shown that temocillin resistance in the ECC can result from a single BaeS alteration, likely resulting in the permanent phosphorylation of BaeR and leading to AcrD overexpression and temocillin resistance through enhanced active efflux.

A 10-year microbiological study of Pseudomonas aeruginosa strains revealed the circulation of populations resistant to both carbapenems and quaternary ammonium compounds.

Pseudomonas aeruginosa is one of the leading causes of healthcare-associated infections. For this study, the susceptibility profiles to antipseudomonal antibiotics and a quaternary ammonium compound, didecyldimethylammonium chloride (DDAC), widely used as a disinfectant, were established for 180 selected human and environmental hospital strains isolated between 2011 and 2020. Furthermore, a genomic study determined resistome and clonal putative relatedness for 77 of them. During the ten-year study period, it was estimated that 9.5% of patients' strains were resistant to carbapenems, 11.9% were multidrug-resistant (MDR), and 0.7% were extensively drug-resistant (XDR). Decreased susceptibility (DS) to DDAC was observed for 28.0% of strains, a phenotype significantly associated with MDR/XDR profiles and from hospital environmental samples (p < 0.0001). According to genomic analyses, the P. aeruginosa population unsusceptible to carbapenems and/or to DDAC was diverse but mainly belonged to top ten high-risk clones described worldwide by del Barrio-Tofiño et al. The carbapenem resistance appeared mainly due to the production of the VIM-2 carbapenemase (39.3%) and DS to DDAC mediated by MexAB-OprM pump efflux overexpression. This study highlights the diversity of MDR/XDR populations of P. aeruginosa which are unsusceptible to compounds that are widely used in medicine and hospital disinfection and are probably distributed in hospitals worldwide.

Synergistic Activity of Pep16, a Promising New Antibacterial Pseudopeptide against Multidrug-Resistant Organisms, in Combination with Colistin against Multidrug-Resistant Escherichia coli, In Vitro and in a Murine Peritonitis Model.

Colistin is a drug of last resort to treat extreme drug-resistant Enterobacterales, but is limited by dose-dependent toxicity and the emergence of resistance. A recently developed antimicrobial pseudopeptide, Pep16, which acts on the cell membrane, may be synergistic with colistin and limit the emergence of resistance. We investigated Pep16 activity against Escherichia coli with varying susceptibility to colistin, in vitro and in a murine peritonitis model. Two isogenic derivatives of E. coli CFT073 (susceptible and resistant to colistin) and 2 clinical isolates (susceptible (B119) and resistant to colistin (Af31)) were used. Pep16 activity, alone and in combination with colistin, was determined in vitro (checkerboard experiments, time-kill curves, and flow cytometry to investigate membrane permeability). Toxicity and pharmacokinetic analyses of subcutaneous Pep16 were performed in mice, followed by the investigation of 10 mg/kg Pep16 + 10 mg/kg colistin (mimicking human concentrations) in a murine peritonitis model. Pep16 alone was inactive (MICs = 32-64 mg/L; no bactericidal effect). A concentration-dependent bactericidal synergy of Pep16 with colistin was evidenced on all strains, confirmed by flow cytometry. In vivo, Pep16 alone was ineffective. When Pep16 and colistin were combined, a significant decrease in bacterial counts in the spleen was evidenced, and the combination prevented the emergence of colistin-resistant mutants, compared to colistin alone. Pep16 synergizes with colistin in vitro, and the combination is more effective than colistin alone in a murine peritonitis by reducing bacterial counts and the emergence of resistance. Pep16 may optimize colistin use, by decreasing the doses needed, while limiting the emergence of colistin-resistant mutants.

Large-scale controlled coupling of single-photon emitters to high-index dielectric nanoantennas by AFM nanoxerography.

Improving the brightness of single-photon sources by means of optically resonant nanoantennas is a major stake for the development of efficient nanodevices for quantum communications. We demonstrate that nanoxerography by atomic force microscopy makes possible the fast, robust and repeatable positioning of model quantum nanoemitters (nitrogen-vacancy NV centers in nanodiamonds) on a large-scale in the gap of silicon nanoantennas with a dimer geometry. By tuning the parameters of the nanoxerography process, we can statistically control the number of deposited nanodiamonds, yielding configurations down to a unique single photon emitter coupled to these high index dielectric nanoantennas, with high selectivity and enhanced brightness induced by a near-field Purcell effect. Numerical simulations are in very good quantitative agreement with time-resolved photoluminescence experiments. A multipolar analysis reveals in particular all the aspects of the coupling between the dipolar single emitter and the Mie resonances hosted by these simple nanoantennas. This proof of principle opens a path to a genuine and large-scale spatial control of the coupling of punctual quantum nanoemitters to arrays of optimized optically resonant nanoantennas. It paves the way for future fundamental studies in quantum nano-optics and toward integrated photonics applications for quantum technologies.

Web-based prediction of antimicrobial resistance in enterococcal clinical isolates by whole-genome sequencing.

Besides phenotypic antimicrobial susceptibility testing (AST), whole genome sequencing (WGS) is a promising alternative approach for detection of resistance phenotypes. The aim of this study was to investigate the concordance between WGS-based resistance prediction and phenotypic AST results for enterococcal clinical isolates using a user-friendly online tools and databases. A total of 172 clinical isolates (34 E. faecalis, 138 E. faecium) received at the French National Reference Center for enterococci from 2017 to 2020 were included. AST was performed by disc diffusion or MIC determination for 14 antibiotics according to CA-SFM/EUCAST guidelines. The genome of all strains was sequenced using the Illumina technology (MiSeq) with bioinformatic analysis from raw reads using online tools ResFinder 4.1 and LRE-finder 1.0. For both E. faecalis and E. faecium, performances of WGS-based genotype to predict resistant phenotypes were excellent (concordance > 90%), particularly for antibiotics commonly used for treatment of enterococcal infections such as ampicillin, gentamicin, vancomycin, teicoplanin, and linezolid. Note that 100% very major errors were found for quinupristin-dalfopristin, tigecycline, and rifampicin for which resistance mutations are not included in databases. Also, it was not possible to predict phenotype from genotype for daptomycin for the same reason. WGS combined with online tools could be easily used by non-expert clinical microbiologists as a rapid and reliable tool for prediction of phenotypic resistance to first-line antibiotics among enterococci. Nonetheless, some improvements should be made such as the implementation of resistance mutations in the database for some antibiotics.

On the in situ 3D electrostatic directed assembly of CdSe/CdZnS colloidal quantum nanoplatelets towards display applications.

HYPOTHESIS: Due to their unique quantum yield and photostability performances, quantum nanoplatelets are very promising building blocks for future generations of displays. The directed assembly of such colloidal nano-objects in the shape of micro-pixels is thus the next mandatory step to reach this goal. Selectively trapping them on electrostatically charged patterns by nanoxerography could be a versatile and appealing strategy but requires a full understanding of the assembly mechanisms in order to make the most of their integration. EXPERIMENTS: We propose an experimental platform based on a smart resealable microfluidic chip coupled to an inverted optical fluorescence microscope and a high-speed camera for in situ access of such assembly mechanisms, using CdSe/CdZnS quantum nanoplatelets as model nano-objects. The photoluminescence signal of the nanoplatelet patterns is thus recorded in real time during their assembly and data extracted after image processing. FINDINGS: The coupling of experimental results and numerical simulations evidences the main role of advection at the origin of this directed nanoparticle trapping. Deep understanding of the involved mechanisms and tuning of experimental parameters allow to make high resolution quantum nanoplatelet based micro-pixels with a fine control of their lateral and vertical dimensions.

Diffusion of amoxicillin into heart valves from infective endocarditis patients.

OBJECTIVES: Amoxicillin is the drug of choice in the management of streptococcal and enterococcal infective endocarditis (IE) but little is known regarding amoxicillin diffusion into infected heart valves. Herein, we assessed amoxicillin valvular distribution and related pharmacokinetic/pharmacodynamic (PK/PD) target attainment in IE patients undergoing heart valve surgery. PATIENTS AND METHODS: In this 2-year prospective study, patients with IE treated by continuous infusion of amoxicillin and undergoing a surgical valve replacement were included. Both amoxicillin plasma and tissue concentrations were measured the day of surgery. Amoxicillin concentration in plasma and crushed heart valves were measured by a validated liquid chromatography method coupled with ultra-violet and tandem mass spectrometry, respectively. MIC and MBC of amoxicillin were determined for all available isolates. The rate of achievement of PK/PD efficacy parameters were assessed. RESULTS: Twenty-two heart valves were removed from 20 patients. Bacterial aetiology was streptococcal (n = 17) and enterococcal (n = 3). Amoxicillin mean daily dose was 12 ±â€Š3 g/24 h, mean plasma concentration was 29 ±â€Š21 mg/L (n = 15), mean tissue concentration was 23 ±â€Š15 mg/L (n = 22). Median diffusion rate was 62%. Patients reached a plasma concentration target >4XCMI (n = 13). Tissue concentrations were bactericidal for all streptococcal IE but not for enterococcal IE. CONCLUSIONS: Amoxicillin intravalvular measurements in IE treated patients showed significant penetration into the infectious site. These data are reassuring that in situ bactericidal concentrations can be largely achieved in the management of streptococcal IE and support the need for combination antibiotic therapy for enterococcal IE.

Exploring Cluster-Dependent Antibacterial Activities and Resistance Pathways of NOSO-502 and Colistin against Enterobacter cloacae Complex Species.

The Enterobacter cloacae complex (ECC) is a group of diverse environmental and clinically relevant bacterial species associated with a variety of infections in humans. ECC have emerged as one of the leading causes of nosocomial infections worldwide. The purpose of this paper is to evaluate the activity of NOSO-502 and colistin (CST) against a panel of ECC clinical isolates, including different Hoffmann's clusters strains, and to investigate the associated resistance mechanisms. NOSO-502 is the first preclinical candidate of a novel antibiotic class, the odilorhabdins (ODLs). MIC50 and MIC90 of NOSO-502 against ECC are 1 µg/mL and 2 µg/mL, respectively, with a MIC range from 0.5 µg/mL to 32 µg/mL. Only strains belonging to clusters XI and XII showed decreased susceptibility to both NOSO-502 and CST while isolates from clusters I, II, IV, and IX were only resistant to CST. To understand this phenomenon, E. cloacae ATCC 13047 from cluster XI was chosen for further study. Results revealed that the two-component system ECL_01761-ECL_01762 (ortholog of CrrAB from Klebsiella pneumoniae) induces NOSO-502 hetero-resistance by expression regulation of the ECL_01758 efflux pump component (ortholog of KexD from K. pneumoniae) which could compete with AcrB to work with the multidrug efflux pump proteins AcrA and TolC. In E. cloacae ATCC 13047, CST-hetero-resistance is conferred via modification of the lipid A by addition of 4-amino-4-deoxy-l-arabinose controlled by PhoPQ. We identified that the response regulator ECL_01761 is also involved in this resistance pathway by regulating the expression of the ECL_01760 membrane transporter.

Small RNA-mediated regulation of the tet(M) resistance gene expression in Enterococcus faecium.

We investigated the role of a novel small RNA expressed in Enterococcus faecium (named Ern0030). We revealed that ern0030 was encoded within the 5'untranslated region of tet(M), a gene conferring tetracycline resistance through ribosomal protection. By RACE mapping, we accurately determined the boundaries of ern0030, which corresponded to Ptet. This upstream sequence of tet(M), Ptet, was previously described within transcriptional attenuation mechanism. Here, Northern blot analyses revealed three transcripts of different lengths (ca. 230, 150 and 100 nucleotides) expressed from Ptet. Phenotypically, the total deletion of ern0030 conferred a decrease in tetracycline MICs that was consistent with gene expression data showing no significant tet(M) induction under tetracycline SIC in ern0030-deleted mutant as opposed to a 10-fold increase of tet(M) expression in the wild-type strain. We investigated the transcriptional attenuation mechanism by toeprint assay. Whereas the expected tet(M) ribosome-binding site (RBS) was detected, the RBS of the putative leader peptide was not highlighted by toeprint assay, suggesting the transcriptional attenuation was unlikely. Here, we demonstrate that Ern0030 has a role in regulation of tet(M) expression and propose a novel model of tet(M) regulation alternative or complementary to transcriptional attenuation.

Activity of the combination of colistin and fosfomycin against NDM-1-producing Escherichia coli with variable levels of susceptibility to colistin and fosfomycin in a murine model of peritonitis.

BACKGROUND: Alternative treatments are needed against NDM-1-producing Escherichia coli. Colistin (COL) and fosfomycin (FOS) often remain active in vitro but selection of resistant mutants is frequent if used separately. We determined whether the combination of colistin and fosfomycin may be useful to treat infections with NDM-1-producing E. coli with varying levels of resistance. METHODS: Isogenic derivatives of E. coli CFT073 with blaNDM-1 and variable levels of resistance to colistin and fosfomycin (CFT073-NDM1, CFT073-NDM1-COL and CFT073-NDM1-FOS, respectively) were used. The combination (colistin + fosfomycin) was tested in vitro and in a fatal peritonitis murine model. Mortality and bacterial loads were determined and resistant mutants detected. RESULTS: Colistin MICs were 0.5, 16 and 0.5 mg/L and fosfomycin MICs were 1, 1 and 32 mg/L against CFT073-NDM1, CFT073-NDM1-COL and CFT073-NDM1-FOS, respectively. In time-kill curves, combining colistin with fosfomycin was synergistic and bactericidal against CFT073-NDM1 and CFT073-NDM1-FOS, with concentrations of 4× MIC (for both drugs), but not against CFT073-NDM1-COL (concentrations of colistin = 0.5× MIC), due to regrowth with fosfomycin-resistant mutants. Mice died less and bacterial counts were lower in spleen with the combination compared with monotherapy against all strains; the combination prevented selection of resistant mutants except for CFT073-NDM1-COL where fosfomycin-resistant mutants were found in all mice. CONCLUSIONS: Combining colistin and fosfomycin was beneficial in vitro and in vivo against NDM-1-producing E. coli, even with strains less susceptible to colistin and fosfomycin. However, the combination failed to prevent the emergence of fosfomycin-resistant mutants against colistin-resistant strains. Combining colistin and fosfomycin constitutes an alternative for treatment of NDM-1 E. coli, except against colistin-resistant strains.