José Manuel Rodríguez-Martínez

Routine susceptibility testing often misses resistance in bla _OXA-1 -producing E. coli , risking therapeutic failure. Among 165 clinical isolates from the Spanish multicenter cohort, MicroScan and disk diffusion versus reference broth microdilution yielded suboptimal categorical agreement (73.9%–86.7%) for piperacillin-tazobactam (PTZ) and cefepime (FEP). MicroScan PTZ very major errors (30%) indicate resistance underestimation; disk diffusion PTZ major errors (23%) and both methods FEP major errors (8.3%–13.8%) indicate resistance overestimation. These errors justify cautious interpretation of routine susceptibility results.

Temocillin, a β-lactam antibiotic, has been proposed as an alternative to carbapenems for the treatment of ESBL-producing Enterobacteriaceae. The present EUCAST breakpoints for temocillin are established based on exposure levels derived from a dosing regimen of 2 g every 8 h via intermittent infusion (II). However, the evidence supporting the efficacy of continuous infusion (CI) against ESBL-producing Klebsiella pneumoniae remains limited. The objective of the present study was to evaluate whether CI improves temocillin activity compared with II in a hollow-fiber infection model (HFIM) against CTX-M-15-producing K. pneumoniae . Four clinical isolates were characterized by whole-genome sequencing and tested for temocillin susceptibility. Mutant frequencies were estimated, and temocillin activity was assessed via time–kill assays and HFIM simulating human pharmacokinetics of temocillin 6 g/day via II and CI. Bacterial counts were performed to detect total and resistant subpopulations. Isolates showing regrowth were sequenced to identify resistance-associated mutations. The results showed that both regimens reduced bacterial burden within 8 h. However, three isolates regrew under II, while only one did under CI. CI achieved more sustained bacterial suppression and delayed or prevented the emergence of resistant subpopulations. Mutations in the cpxA gene were associated with increased temocillin MICs in regrown isolates. While both regimens demonstrated initial bactericidal activity, CI was more effective in sustaining bacterial suppression and limiting resistance emergence. These findings support the potential clinical benefit of CI for treating infections caused by CTX-M-15-producing K. pneumoniae and warrant further clinical validation.

The aim of this study is to evaluate the pH-dependent antimicrobial activities of delafloxacin against isogenic Escherichia coli strains carrying diverse fluoroquinolone resistance mechanisms. Eighty-one isogenic E. coli strains derived from ATCC 25922 were tested, carrying various combinations of chromosomal mutations ( gyrA, parC ), efflux pump overexpression ( marR ), and plasmid-mediated quinolone resistance (PMQR) determinants ( qnrA1, qnrB1, qnrC, qnrD1, qnrS1, qepA2, aac(6')-Ib-cr ). Minimum inhibitory concentrations (MICs) were determined using broth microdilution at pH 7.3, 6.0, and 5.0. Time-killing assays were performed on three representative strains. Semi-mechanism-based mathematical modeling using Monolix quantified bactericidal activity through EC ₅₀ parameters. Ciprofloxacin MIC ₉₀ increased from 8 mg/L at pH 7.3 to 128 mg/L at pH 5.0, while delafloxacin MIC ₉₀ decreased from 64 mg/L to 16 mg/L. At physiological pH, ciprofloxacin demonstrated superior activity (median MIC: 1 mg/L vs 8 mg/L for delafloxacin, P < 0.001), while at pH 5.0, delafloxacin median MIC was 2 mg/L versus 64 mg/L for ciprofloxacin ( P < 0.001). According to EUCAST breakpoints, susceptibility frequencies at pH 5.0 were 25.9% (21/81) for delafloxacin versus 2.5% (2/81) for ciprofloxacin ( P < 0.001). Among PMQR, qnr genes led to 0% delafloxacin susceptibility across all conditions (0/150 strain-pH combinations), while qepA2 (8/30 strain-pH combinations, 26.7%) and aac(6')-Ib-cr (16/30 strain-pH combinations, 53.3%) showed comparable susceptibility frequencies to isogenic strains not carrying PMQR (15/33 strain-pH combinations, 45.5%). Simulated clinical exposures against chromosomal mutant EC02 (GyrA-S83L) demonstrated that ciprofloxacin would fail at pH 5.0, while delafloxacin would maintain antimicrobial activity. Environmental pH deeply modulates fluoroquinolone activity and resistance mechanism impact. Delafloxacin demonstrates clinically relevant activity in acidic infection environments. IMPORTANCE: Fluoroquinolones face increasing resistance challenges, particularly in acidic infection sites, such as the urinary tract, abscesses, and biofilms, where pH can drop below 6.0. This study reveals a critical pH-dependent reversal in antimicrobial effectiveness between ciprofloxacin and delafloxacin against resistant Escherichia coli . While ciprofloxacin loses substantial activity in acidic conditions (90% of strains showing 16-fold minimum inhibitory concentration increases at pH 5.0), delafloxacin maintains and even enhances its antimicrobial potency, with susceptibility rates increasing from 25.9% to over 50% as pH decreases. Using comprehensive resistance profiling across 81 isogenic strains and pharmacokinetic modeling, we demonstrate that delafloxacin could overcome treatment failures in acidic infection sites where conventional fluoroquinolones fail. These findings could have immediate clinical implications for treating urinary tract infections, abscesses, and biofilm-associated infections, potentially expanding therapeutic options against multidrug-resistant bacteria in previously challenging anatomical sites.

Introduction: Targeting bacterial DNA damage responses such as the SOS response represents a promising strategy for enhancing the efficacy of existing antimicrobials. This study focuses on a recently discovered DNA damage response mechanism involving tricarboxylic acid cycle (TCA) fumarases and the adaptive response, crucial for Escherichia coli survival in the presence of genotoxic methyl methanesulfonate (MMS). We investigated whether this pathway contributes to protection against antibiotics, either separately or in combination with the SOS response.Methods: An isogenic collection of E. coli BW25113 mutants was used, including strains deficient in fumarases (ΔfumA, ΔfumB, ΔfumC) and the adaptive response (ΔalkA, ΔalkB, ΔaidB). Additional SOS response inactivation (ΔrecA) was conducted by P1 phage transduction. All mutants were subjected to antimicrobial susceptibility testing, growth curve analysis, survival and evolution assays. To validate the relevance of these findings, experiments were also performed in a quinolone-resistant E. coli ST131 clinical isolate.Results and discussion: Overall, no significant differences or only moderate increases in susceptibility were observed in the single mutants, with ΔfumC and ΔaidB mutants showing the highest susceptibility. To enhance this effect, these genes were then inactivated in combination with the SOS response by constructing ΔfumC/ΔrecA and ΔaidB/ΔrecA mutants. These combinations exhibited significant differences in susceptibility to various antimicrobials, particularly cephalosporins and quinolones, and especially in the ΔfumC/ΔrecA strain. To further assess these results, we constructed an E. coli ST131 ΔfumC/ΔrecA mutant, in which a similar trend was observed. Together, these findings suggest that co-targeting the SOS response together with fumarases or the adaptive response could enhance the effectiveness of antibiotics against E. coli, potentially leading to new therapeutic strategies.

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. To evaluate the efficacy of this dose using a hollow-fibre infection model (HFIM) against six KPC-2-producing clinical isolates of K. pneumoniae. 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. 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. 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.

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. To study the role of baeS/baeR two-component system in temocillin resistance in E. coli. 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). 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. 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.

The suppression of the SOS response has been shown to enhance the in vitro activity of quinolones. Furthermore, Dam-dependent base methylation has an impact on susceptibility to other antimicrobials affecting DNA synthesis.