Bacterial niche colonization relies on multiple factors, among which the metabolic capacity to utilize specific substrates is pivotal. As a gut commensal of humans and other vertebrates acting as an intestinal and extraintestinal opportunistic pathogen, Escherichia coli faces such environmental pressures. We therefore aimed to identify (i) metabolic patterns associated with E. coli lifestyle (commensal versus extraintestinal pathogenic E. coli [ExPEC]) and (ii) ExPEC-specific metabolic features that could help explain the emergence and success of major pandemic clones. Using a pangenomic framework coupled with metabolic pathway prediction, we analyzed 1,498 well-characterized E. coli strains collected over 17 years in France, including adult commensal strains (n = 370) and ExPEC strains involved in severe infections (bloodstream infections from various portals of entry and pneumonia) (n = 1,128). Although metabolism was more conserved than gene content, substantial metabolic diversity was observed, with over 50% pathways being variable, mainly involving biosynthetic and degradation processes. No pathway was specifically associated with lifestyle; metabolic profiles primarily reflected phylogeny. However, several clone-specific metabolic capacities were identified. Some may support extraintestinal survival during infection, such as the 5'-deoxynucleoside recycling pathway enriched in the major ExPEC clone STc69 from phylogroup D. Conversely, in phylogroup B2, clone-specific pathways enabled the degradation of plant-derived compounds, potentially facilitating gut colonization through niche-defining nutrients. Notably, D-apiose degradation pathway analysis revealed a functional pathway strongly associated with the pandemic clones STc131 and STc14. Overall, these lineage-specific metabolic capacities may contribute to the ecological success and dissemination of dominant ExPEC clones.IMPORTANCEAccording to the nutrient-niche hypothesis, bacteria must exploit distinct substrates to grow and persist in their various habitats. Such niche differentiation is at play among the commensal and pathogenic E. coli populations. With this in mind, we search for specific associations between metabolic pathways and strain origin (commensal versus severe extraintestinal infections). Metabolic profiles were predominantly shaped by phylogeny, reflecting the species' clonal structure and the close link between phylogenetic background and lifestyle. Among the lineage-specific determinants, we identified several pathways associated with worldwide spread clones responsible for bloodstream infections, supporting the existence of clone-specific strategies for niche adaptation.

The combination of aztreonam (ATM) and avibactam (AVI) is an attractive option to treat infections caused by extended spectrum beta-lactamase plus NDM-1-producing Enterobacteriaceae. Since ATM activity was shown to be severely impacted by an increase in the inoculum size in vitro, we wondered whether ATM-AVI activity could be impaired in high-inoculum infections. We analyzed the impact of the inoculum size on ATM-AVI activity in vitro and in a murine model of peritonitis due to susceptible Escherichia coli CFT073-pTOPO and its isogenic derivatives producing NDM-1 (E. coli CFT073-NDM1) and CTX-M-15 plus NDM-1 (E. coli CFT073-CTXM15-NDM1). The impact of the inoculum size on bacterial morphology was studied by microscopic examination. In vitro, at standard (10(5)) inoculum, E. coli CFT073-CTXM15-NDM1 was resistant to ATM but susceptible to the ATM-AVI combination. At high (10(7)) inoculum, MICs of ATM alone and of the ATM-AVI combination reached >512 and 64 mg/L, respectively, against all tested strains. ATM led to bacterial filamentation when active against the bacteria, i.e., in monotherapy or in combination with AVI against susceptible E. coli CFT073-pTOPO and only in combination with AVI against E. coli CFT073-CTXM15-NDM1. In vivo, increase in the inoculum led to a drastic decrease in the activity of ATM alone against E. coli CFT073-pTOPO and ATM-AVI against E. coli CFT073-CTXM15-NDM1. Our results suggest a high in vivo impact of the inoculum increase on the activity of ATM alone against ATM-susceptible E. coli and of ATM-AVI against CTX-M-15 plus NDM-1 producing E. coli. Clinicians must be aware of the risk of failures when using ATM-AVI in high-inoculum infections.

AbstractBackgroundThe combination of aztreonam (ATM) and avibactam (AVI) is an attractive option to treat infections caused by extended spectrumβ-lactamase plus NDM-1-producingEnterobacteriaceae. Since ATM activity was shown to be severely impacted by an increase in the inoculum sizein vitro, we wondered whether ATM-AVI activity could be impaired in high-inoculum infections.MethodsWe analyzed the impact of the inoculum size on ATM-AVI activityin vitroand in a murine model of peritonitis due to susceptibleE. coliCFT073-pTOPO and its isogenic derivatives producing NDM-1 (E. coliCFT073-NDM1) and CTX-M-15 plus NDM-1 (E. coliCFT073-CTXM15-NDM1). The impact of the inoculum size on bacterial morphology was studied by microscopic examination.ResultsIn vitro, at standard (105) inoculum,E. coliCFT073-CTXM15-NDM1 was resistant to ATM but susceptible to the ATM-AVI combination. At high (107) inoculum, MICs of ATM alone and of the ATM-AVI combination reached > 512 and 64 mg/L respectively, against all tested strains. ATM led to bacterial filamentation when active against the bacteria, i.e., in monotherapy or in combination with AVI against susceptibleE. coliCFT073-pTOPO, and only in combination with AVI againstE. coliCFT073-CTXM15-NDM1.In vivo, increase in the inoculum led to a drastic decrease in the activity of ATM alone againstE. coliCFT073-pTOPO, and of ATM-AVI againstE. coliCFT073-CTXM15-NDM1.ConclusionOur results suggest a highin vivoimpact of the inoculum increase on the activity of ATM alone against ATM-susceptibleE. coli, and of ATM-AVI against CTX-M-15 plus NDM-1 producingE. coli. Clinicians must be aware of the risk of failures when using AZT-AVI in high inoculum infections.

Background Temocillin is a narrow spectrum beta-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-Delta nmpC (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.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-Delta nmpC (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-Delta nmpC, 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-Delta nmpC, 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-Delta nmpC and CFTbaeS-TP, but inefficient against CFTbaeS-AP (100% mortality).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-Delta nmpC, 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-Delta nmpC, 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-Delta nmpC 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.

Urinary tract infection (UTI), mainly caused by Escherichia coli , are frequent and have a recurrent nature even after antibiotic treatment. Potential bacterial escape mechanisms include growth defects, but probing bacterial division in vivo and establishing its relation to the antibiotic response remain challenging. Using a synthetic reporter of cell division, we follow the temporal dynamics of cell division for different E. coli clinical strains in a UTI mouse model with and without antibiotics. We show that more bacteria are actively dividing in the kidneys and urine compared with the bladder. Bacteria that survive antibiotic treatment are consistently non -dividing in three sites of infection. Additionally, we demonstrate how both the strain in vitro persistence profile and the microenvironment impact infection and treatment dynamics. Understanding the relative contribution of the host environment, growth heterogeneity, non -dividing bacteria, and antibiotic persistence is crucial to improve therapies for recurrent infections.

Objective: Whole genome sequencing (WGS) of extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-E. coli) in developing countries is lacking. Here we describe the population structure and molecular characteristics of ESBL-E. coli faecal isolates in rural Southern Niger. Methods: Stools of 383 healthy participants were collected among which 92.4% were ESBL-Enterobacterales carriers. A subset of 90 ESBL-E. coli containing stools (109 ESBL-E. coli isolates) were further analysed by WGS, using short- and long-reads. Results: Most isolates belonged to the commensalism-adapted phylogroup A (83.5%), with high clonal diversity. The bla(CTX-M-15) gene was the major ESBL determinant (98.1%), chromosome-integrated in approximately 50% of cases, in multiple integration sites. When plasmid-borne, bla(CTX-M-15) was found in IncF (57.4%) and IncY plasmids (26.2%). Closely related plasmids were found in different genetic backgrounds. Genomic environment analysis of bla(CTX-M-15) in closely related strains argued for mobilisation between plasmids or from plasmid to chromosome. Conclusions: Massive prevalence of community faecal carriage of CTX-M-15-producing E. coli was observed in a rural region of Niger due to the spread of highly diverse A phylogroup commensalism-adapted clones, with frequent chromosomal integration of bla(CTX-M-15). Plasmid spread was also observed. These data suggest a risk of sustainable implementation of ESBL in community faecal carriage. (c) 2023 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Objective Whole genome sequencing (WGS) of extended-spectrum β-lactamase-producing Escherichia coli (ESBL- E. coli ) in developing countries is lacking. Here we describe the population structure and molecular characteristics of ESBL- E. coli faecal isolates in rural Southern Niger.Methods Stools of 383 healthy participants were collected among which 92.4% were ESBL- E. coli carriers; 90 of these ESBL- E. coli containing stools (109 ESBL- E. coli isolates) were further analysed by WGS, using short- and long-reads.Results Most isolates belonged to the commensalism-adapted phylogroup A (83.5%), with high clonal diversity. The bla CTX-M-15 gene was the major ESBL determinant (98.1%), chromosome-integrated in approximately 50% of cases, in multiple integration sites. When plasmid-borne, bla CTX-M-15 was found in IncF (57.4%) and IncY plasmids (26.2%). Closely related plasmids were found in different genetic backgrounds. Genomic environment analysis of bla CTX-M-15 in closely related strains argued for mobilisation between plasmids or from plasmid to chromosome.Conclusions Massive prevalence of community faecal carriage of CTX-M-15-producing E. coli was observed in a rural region of Niger due to the spread of highly diverse A phylogroup commensalism-adapted clones, with frequent chromosomal integration of bla CTX-M-15. Plasmid spread was also observed. These data suggest a risk of sustainable implementation of ESBL in community faecal carriage.### Competing Interest StatementThe authors have declared no competing interest.

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.

Chlorhexidine is a widely used antiseptic in hospital and community health care. Decreased susceptibility to this compound has been recently described in Klebsiella pneumoniae and Pseudomonas aeruginosa, together with cross-resistance to colistin. Surprisingly, few data are available for Escherichia coli, the main species responsible for community and health care-associated infections. In order to decipher chlorhexidine resistance mechanisms in E. coli, we studied both in vitro derived and clinical isolates through whole-genome sequence analysis. Comparison of strains grown in vitro under chlorhexidine pressure identified mutations in the gene mlaA coding for a phospholipid transport system. Phenotypic analyses of single-gene mutants from the Keio collection confirmed the role of this mutation in the decreased susceptibility to chlorhexidine. However, mutations in mlaA were not found in isolates from large clinical collections. In contrast, genome wide association studies (GWAS) showed that, in clinical strains, chlorhexidine reduced susceptibility was associated with the presence of tetA genes of class B coding for efflux pumps and located in a Tn10 transposon. Construction of recombinant strains in E. coli K-12 confirmed the role of tetA determinant in acquired resistance to both chlorhexidine and tetracycline. Our results reveal that two different evolutionary paths lead to chlorhexidine decreased susceptibility: one restricted to in vitro evolution conditions and involving a retrograde phospholipid transport system; the other observed in clinical isolates associated with efflux pump TetA. None of these mechanisms provide cross-resistance to colistin. This work demonstrates the GWAS power to identify new resistance mechanisms in bacterial species.

Background: Data on extended-spectrum β--lactamase-producing Escherichia coli (ESBL- E. coli) carriage in community settings, especially in developing countries, are scarce. Here, we describe the population structure and molecular characteristics of resistance of ESBL- E. coli faecal isolates in a rural African population.Methods: Between April and May 2017, stools of 383 healthy participants were collected from 20 villages in rural Southern Niger during a clinical trial on ciprofloxacin prophylaxis carried out during a meningococcal meningitis outbreak. Of 383 individuals, 354 (92.4%) were carriers of ESBL- E. coli before any ciprofloxacin intake. A subset of 90 of these ESBL- E. coli containing stools were selected for further analysis, from which 109 different ESBL- E. coli were recovered and whole genome sequenced by short-(Illumina) and long-(Nanopore) reads.FindingsMost belonged to the commensal-adapted phylogroup A (91, 83.5%), with high clonal diversity (57 distinct clones). One-quarter harboured the high pathogenicity island previously associated with a longer duration of faecal carriage.The bla CTX-M-15 gene was the major ESBL determinant (107, 98.1%). It was chromosome-integrated in approximately half of the cases (48, 44.9%), at multiple integration sites in diverse chromosomal genetic backgrounds. When plasmid-borne, blaCTX-M-15 was found in a large diversity of incompatibility groups. A single genetic background was found for 20 distinct plasmids, whereas very closely related plasmids were found in different genetic backgrounds in six cases, suggesting plasmid spread among strains. No geographical or social links to resistance patterns were observed.Interpretation: Massive prevalence of community faecal carriage of CTX-M-15-producing E. coli was observed in a rural region of Niger without apparent antibiotic selective pressure. E. coli were highly diverse, well adapted commensal strains, with chromosomal integration of CTX-M-15 encoding gene in almost half of the cases. Evidence of clonal and plasmid spread suggest a risk of sustainable implementation in community faecal carriage.Trial Registration Details: The trial is registered at ClinicalTrials.gov (NCT02724046).Funding Information: This work was partially supported by a grant from the “Fondation pour la Recherche Médicale” (Equipe FRM 2016, grant number DEQ20161136698). The parent study was funded by Médecins Sans Frontières. Declaration of Interests: None.Ethics Approval Statement: The parent study protocol, which included this sub-study, was reviewed and approved by the National Consultative Ethics Committee of Niger (Ref: 003/2016/CCNE) and the Ethics Review Board of Médecins Sans Frontières (Ref: 1603). Written informed consent was obtained from individual participants.

SummaryObjectiveWhole genome sequencing (WGS) of extended-spectrum β-lactamase-producingEscherichia coli(ESBL-E. coli) in developing countries is lacking. Here we describe the population structure and molecular characteristics of ESBL-E. colifaecal isolates in rural Southern Niger.MethodsStools of 383 healthy participants were collected among which 92.4% were ESBL-E. colicarriers; 90 of these ESBL-E. colicontaining stools (109 ESBL-E. coliisolates) were further analysed by WGS, using short- and long-reads.ResultsMost isolates belonged to the commensalism-adapted phylogroup A (83.5%), with high clonal diversity. TheblaCTX-M-15gene was the major ESBL determinant (98.1%), chromosome-integrated in approximately 50% of cases, in multiple integration sites. When plasmid-borne,blaCTX-M-15was found in IncF (57.4%) and IncY plasmids (26.2%). Closely related plasmids were found in different genetic backgrounds. Genomic environment analysis ofblaCTX-M-15in closely related strains argued for mobilisation between plasmids or from plasmid to chromosome.ConclusionsMassive prevalence of community faecal carriage of CTX-M-15-producingE. coliwas observed in a rural region of Niger due to the spread of highly diverse A phylogroup commensalism-adapted clones, with frequent chromosomal integration ofblaCTX-M-15. Plasmid spread was also observed. These data suggest a risk of sustainable implementation of ESBL in community faecal carriage.

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 bla(NDM-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 CFT073NDM1, 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.

Lack of association between colistin resistance and chlorhexidine reduced susceptibility in clinical isolates of Escherichia coli Guilhem Royer, Guilhem Royer Université de Paris, IAME, UMR 1137, INSERM, 75018 Paris, FranceLABGeM, Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Université Paris-Saclay, 91000 Evry, FranceDépartement de Prévention, Diagnostic et Traitement des Infections, Hôpital Henri Mondor, APHP, 94000 Créteil, France Search for other works by this author on: Oxford Academic PubMed Google Scholar Laurent Poirel, Laurent Poirel Laboratoire Européen Associé INSERM, Emerging Antibiotic Resistance in Gram-Negative Bacteria, Emerging Antibiotic Resistance Unit, Medical and Molecular Microbiology, Faculty of Science and Medicine, University of Fribourg, Fribourg, SwitzerlandNational Reference Centre for Emerging Antibiotic Resistance (NARA), Fribourg, Switzerland Search for other works by this author on: Oxford Academic PubMed Google Scholar Béatrice La Combe, Béatrice La Combe Université de Paris, IAME, UMR 1137, INSERM, 75018 Paris, FranceAPHP, Hôpital Louis Mourier, DMU ESPRIT, Service de Médecine Intensive Réanimation, 92700 Colombes, FranceService de Réanimation polyvalente, Hôpital du Scorff - Groupe Hospitalier Bretagne Sud Lorient, 5 Avenue Choiseul, 56322 Lorient, France Search for other works by this author on: Oxford Academic PubMed Google Scholar Olivier Clermont, Olivier Clermont Université de Paris, IAME, UMR 1137, INSERM, 75018 Paris, France Search for other works by this author on: Oxford Academic PubMed Google Scholar Françoise Chau, Françoise Chau Université de Paris, IAME, UMR 1137, INSERM, 75018 Paris, France Search for other works by this author on: Oxford Academic PubMed Google Scholar Mélanie Mercier-Darty, Mélanie Mercier-Darty Département de Prévention, Diagnostic et Traitement des Infections, Hôpital Henri Mondor, APHP, 94000 Créteil, France Search for other works by this author on: Oxford Academic PubMed Google Scholar Erick Denamur, Erick Denamur Université de Paris, IAME, UMR 1137, INSERM, 75018 Paris, FranceLaboratoire de Génétique Moléculaire, Hôpital Bichat, APHP, 75018 Paris, France Corresponding author. E-mail: erick.denamur@inserm.fr Search for other works by this author on: Oxford Academic PubMed Google Scholar Patrice Nordmann, Patrice Nordmann Laboratoire Européen Associé INSERM, Emerging Antibiotic Resistance in Gram-Negative Bacteria, Emerging Antibiotic Resistance Unit, Medical and Molecular Microbiology, Faculty of Science and Medicine, University of Fribourg, Fribourg, SwitzerlandNational Reference Centre for Emerging Antibiotic Resistance (NARA), Fribourg, Switzerland Search for other works by this author on: Oxford Academic PubMed Google Scholar Jean-Damien Ricard, Jean-Damien Ricard Université de Paris, IAME, UMR 1137, INSERM, 75018 Paris, FranceAPHP, Hôpital Louis Mourier, DMU ESPRIT, Service de Médecine Intensive Réanimation, 92700 Colombes, France Search for other works by this author on: Oxford Academic PubMed Google Scholar Jean-Winoc Decousser Jean-Winoc Decousser Département de Prévention, Diagnostic et Traitement des Infections, Hôpital Henri Mondor, APHP, 94000 Créteil, FranceEA 7380 Dynamyc, Université Paris-Est Créteil, 94000 Créteil, France Search for other works by this author on: Oxford Academic PubMed Google Scholar Journal of Antimicrobial Chemotherapy, Volume 76, Issue 10, October 2021, Pages 2736–2737, https://doi.org/10.1093/jac/dkab235 Published: 10 July 2021 Article history Received: 09 May 2021 Accepted: 14 June 2021 Published: 10 July 2021

Fosfomycin resistance in Escherichia coli results from chromosomal mutations or acquisition of plasmid-mediated genes. Because these mechanisms may be absent in some resistant isolates, we aimed at decipher the genetic basis of fosfomycin resistance in E. coli. Different groups of isolates were studied: fosfomycin-resistant mutants selected in vitro from E. coli CFT073 (MIC = 1 mg/L) and two groups (wildtype and non-wildtype) of E. coli clinical isolates. Single-nucleotide allelic replacement was performed to confirm the implication of novel mutations into resistance. Induction of uhpT expression by glucose-6-phosphate (G6P) was assessed by RT-qPCR. The genome of all clinical isolates was sequenced by MiSeq (Illumina). Two first-step mutants were obtained in vitro from CFT073 (MICs, 128 mg/L) with single mutations: G469R in uhpB (M3); F384L in uhpC (M4). Second-step mutants (MICs, 256 mg/L) presented additional mutations: R282V in galU (M7 from M3); Q558∗ in lon (M8 from M4). Introduction of uhpB or uhpC mutations by site-directed mutagenesis conferred a 128-fold increase in fosfomycin MICs, whereas single mutations in galU or lon were only responsible for a 2-fold increase. Also, these mutations abolished the induction of uhpT expression by G6P. All 14 fosfomycin-susceptible clinical isolates (MICs, 0.5–8 mg/L) were devoid of any mutation. At least one genetic change was detected in all but one fosfomycin-resistant clinical isolates (MICs, 32 – >256 mg/L) including 8, 17, 18, 5, and 8 in uhpA, uhpB, uhpC, uhpT, and glpT genes, respectively. In conclusion, novel mutations in uhpB and uhpC are associated with fosfomycin resistance in E. coli clinical isolates.

Objectives: Embolic events from vegetations are commonly accepted as the main mechanism involved in neurologic complications of infective endocarditis. The pathophysiology may imply other phenomena, including vasculitis. We aimed to define the cerebral lesion spectrum in an infective endocarditis rat model. Design: Experimental model of Staphylococcus aureus or Enterococcus faecalis infective endocarditis. Neurologic lesions observed in the infective endocarditis model were compared with three other conditions, namely bacteremia, nonbacterial thrombotic endocarditis, and healthy controls. Setting: Research laboratory of a university hospital. Subjects: Male Wistar rats. Interventions: Brain MRI, neuropathology, immunohistochemistry for astrocyte and microglia, and bacterial studies on brain tissue were used to characterize neurologic lesions. Measurements and Main Results: In the infective endocarditis group, MRI revealed at least one cerebral lesion in 12 of 23 rats (52%), including brain infarctions (n = 9/23, 39%) and cerebral microbleeds (n = 8/23, 35%). In the infective endocarditis group, neuropathology revealed brain infarctions (n = 12/23, 52%), microhemorrhages (n = 10/23, 44%), and inflammatory processes (i.e., cell infiltrates including abscesses, vasculitis, meningoencephalitis, and/or ependymitis; n = 11/23, 48%). In the bacteremia group, MRI studies were normal and neuropathology revealed only hemorrhages (n = 2/11, 18%). Neuropathologic patterns observed in the nonbacterial thrombotic endocarditis group were similar to those observed in the infective endocarditis group. Immunochemistry revealed higher microglial activation in the infective endocarditis group (n = 11/23, 48%), when compared with the bacteremia (n = 1/11, 9%; p = 0.03) and nonbacterial thrombotic endocarditis groups (n = 0/7, 0%; p = 0.02). Conclusions: This original model of infective endocarditis recapitulates the neurologic lesion spectrum observed in humans and suggests synergistic mechanisms involved, including thromboembolism and cerebral vasculitis, promoted by a systemic bacteremia-mediated inflammation.

The clinical benefit of carbapenems against carbapenemase-producing Enterobacteriaceae (CPE) remains in question. MICs of imipenem (IMP) and ertapenem (ERT) against isogenic derivatives of the wild-type strain Escherichia coli CFT073 producing KPC-3, OXA-48, or NDM-1 were 0.25, 2, 16, and 64 mg/liter for IMP and 0.008, 0.5, 8, and 64 mg/liter for ERT, respectively. Swiss ICR-strain mice with peritonitis were treated for 24 h with IMP or ERT. Despite a limited duration of time during which free antibiotic concentrations were above the MIC (down to 0% for the NDM-1-producing strain), IMP and ERT significantly reduced bacterial counts in spleen and peritoneal fluid at 24 h ( P < 0.

Fosfomycin-tromethamine activity is well established for oral treatment of uncomplicated lower urinary tract infections but little is known about its potential efficacy in pyelonephritis. Ascending pyelonephritis was induced in mice infected with 6 strains of Escherichia coli (fosfomycin MICs: 1 μg/ml to 256 μg/ml). Urine pH was 4.5 before infection and 5.5-6.0 during infection. Animals were treated for 24h with fosfomycin (100 mg/kg subcutaneously every 4 hours) and CFU were enumerated in kidneys 24h after the last fosfomycin injection. Peak (20.5 μg/ml at 1h) and trough (3.5 μg/ml at 4h) levels in plasma were comparable to those obtained in human after an oral dose of 3 grams. Fosfomycin treatment significantly reduced bacterial loads in kidneys (3.65 log10CFU/g [min-max=1.83-7.03] and 1.88 log10CFU/g [1.78-5.74] in start-of-treatment control mice and treated mice, respectively, P < 10-6). However, this effect was not found to differ across the 6 study strains (P = 0.71) and between the 3 susceptible and the 3 resistant strains (P=0.09). Three phenomena may contribute to explain this unexpected in vivo activity: i) in mice, fosfomycin kidney/plasma concentrations ratio increased from 1 to 7.8 (95% CI, 5.2; 10.4) within 24 hours; in vitro , when pH decreased to 5: (ii) fosfomycin MICs for the 3 resistant strains (64-256 μg/ml) decreased into the susceptible range (16-32 μg/ml) and: iii) maximal growth rates significantly decreased for all strains and were the lowest in urine. These results suggest that local fosfomycin concentrations and physiological conditions may favour fosfomycin activity in pyelonephritis, even against resistant strains.

BackgroundDue to a spectrum restricted to Enterobacteriaceae and stability against ESBL and AmpC enzymes, temocillin is of major interest for the treatment of pyelonephritis. But there are still uncertainties about the optimal regimen and clinical breakpoints.ObjectivesTo study in a murine model of pyelonephritis the activity of temocillin against Escherichia coli isolates with different MICs in order to evaluate clinical breakpoints.MethodsFour clinical uropathogenic E. coli isolates with temocillin MICs of 8 mg/L (Ec8), 16 mg/L (Ec16), 32 mg/L (Ec32) and 64 mg/L (Ec64) were evaluated. Antibiotic 24 h T>MIC achieved in humans was reproduced in mice with either intravenous temocillin (2 g q12h or 2 g q8h) or intravenous imipenem (1 g q8h). Efficacy was assessed by bacterial count in kidneys.ResultsCompared with controls, temocillin at 2 g q12h was highly efficient against Ec8 (-3.32 log10 cfu/g and negative cultures in 93% of mice; P < 0.001); imipenem gave similar results. Temocillin at 2 g q12h also induced high reduction of bacterial count against Ec16 (-2.92 log10 cfu/g; P < 0.001), albeit cultures were negative in only 48% of mice. In contrast, no significant effect was observed in mice infected by Ec32 (-0.01 log10 cfu/g; P = 0.981) or Ec64 (-0.55 log10 cfu/g; P = 0.523). Even temocillin at 2 g q8h failed to control Ec32 infection (-1.55 log10 cfu/g; P = 0.197).ConclusionsThis model suggests a clinical breakpoint up to 16 mg/L for non-severe pyelonephritis treated with temocillin at 2 g q12h, a value consistent with the few previous available data.

Abstract Background A strategy used by bacterial strains to resist β-lactam antibiotics is the expression of metallo-β-lactamases (MBL) requiring zinc for activity. The use of a zinc chelator may restore carbapenem activity against MBL-producing Enterobacteriaceae. DMSA is a heavy metal chelator approved in humans with a satisfactory safety record. Our objective was to evaluate the activity of DMSA in combination with carbapenems, in vitro and in a fatal murine peritonitis model, against MBL-producing Escherichia coli. Methods Isogenic derivatives of wild-type E. coli CFT073 producing the MBL NDM-1, VIM-2, IMP-1, and the serine carbapenemases OXA-48 and KPC-3 were constructed. Minimum inhibitory concentrations (MICs) of imipenem, meropenem, and ertapenem were determined against each strain alone or in combination with DMSA. Mice were infected with E. coli CFT073 or NDM-1 and treated intraperitoneally for 24 hours with imipenem 100 mg/kg every 4 hours, DMSA 200 mg/kg every 4 hours, or both. Mice survival rates and bacterial counts in peritoneal fluid (PF) and spleen were assessed at 24 hours. Results In vitro, DMSA in combination with each carbapenem permitted a significant decrease of the MICs against all MBL-producing strains, in a concentration-dependent manner. The maximum effect was found for the NDM-1 strain with a 6- to 8-fold MIC reduction, depending on the carbapenem used. NDM-1 strain became susceptible to carbapenems with concentrations of DMSA ≥6 mM. Increasing zinc concentrations above 1 mg/L (average human plasma concentration) did not alter this effect. No benefit of DMSA was observed against non-MBL strains. In vivo, when used alone, the DMSA regimen was not toxic in uninfected mice and ineffective against NDM-1-infected mice (100% mortality). Combination of imipenem and DMSA significantly reduced bacterial counts in PF and spleen as compared with imipenem alone (P < 0.001), and reduced mortality, although not significantly (11% vs. 37%, respectively, P = 0.12). No benefit of the combination was observed against CFT073. Conclusion DMSA is highly effective in vitro in reducing carbapenems MICs against MBL-producing E. coli and appears as a promising strategy in combination with carbapenems for the treatment of NDM-1-related infections. Disclosures All authors: No reported disclosures.