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Background: Temocillin is a narrow spectrum β-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., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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). The urine pH was 4.5 before infection and 5.5 to 6.0 during infection. Animals were treated for 24 h with fosfomycin (100 mg/kg of body weight subcutaneously every 4 h), and the CFU were enumerated in kidneys 24 h after the last fosfomycin injection. Peak (20.5 μg/ml at 1 h) and trough (3.5 μg/ml at 4 h) levels in plasma were comparable to those obtained in humans after an oral dose of 3 g. Fosfomycin treatment significantly reduced the bacterial loads in kidneys (3.65 log 10 CFU/g [range, 1.83 to 7.03 log 10 CFU/g] and 1.88 log 10 CFU/g [range, 1.78 to 5.74 log 10 CFU/g] 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) or 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, the fosfomycin kidney/plasma concentration ratio increased from 1 to 7.8 (95% confidence interval, 5.2, 10.4) within 24 h in vitro when the pH decreased to 5, (ii) the fosfomycin MICs for the 3 resistant strains (64 to 256 μg/ml) decreased into the susceptible range (16 to 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 favor fosfomycin activity in pyelonephritis, even against resistant strains., (Copyright © 2019 American Society for Microbiology.)

The adherent-invasive Escherichia coli (AIEC), which colonize the ileal mucosa of Crohn's disease patients, adhere to intestinal epithelial cells, invade them and exacerbate intestinal inflammation. The high nutrient competition between the commensal microbiota and AIEC pathobiont requires the latter to occupy their own metabolic niches to survive and proliferate within the gut. In this study, a global RNA sequencing of AIEC strain LF82 has been used to observe the impact of bile salts on the expression of metabolic genes. The results showed a global up-regulation of genes involved in degradation and a down-regulation of those implicated in biosynthesis. The main up-regulated degradation pathways were ethanolamine, 1,2-propanediol and citrate utilization, as well as the methyl-citrate pathway. Our study reveals that ethanolamine utilization bestows a competitive advantage of AIEC strains that are metabolically capable of its degradation in the presence of bile salts. We observed that bile salts activated secondary metabolism pathways that communicate to provide an energy benefit to AIEC. Bile salts may be used by AIEC as an environmental signal to promote their colonization.

Background: Alternative therapeutic regimens are urgently needed against carbapenemase-producing Enterobacteriaceae. Fosfomycin often remains active against KPC and OXA-48 producers, but emergence of resistance is a major limitation. Our aim was to determine whether the association of temocillin with fosfomycin might be useful to treat KPC- or OXA-48-producing Escherichia coli infections., Methods: Isogenic derivatives of E. coli CFT073 with blaKPC-3- or blaOXA-48-harbouring plasmids (named CFT073-KPC-3 and CFT073-OXA-48, respectively) were used. The addition of temocillin to fosfomycin was tested using the chequerboard method and time-kill curves as well as in a fatal peritonitis murine model. Mice were treated for 24 h with fosfomycin alone or in combination with temocillin. Bacterial loads, before and after treatment, were determined in the peritoneal fluid and fosfomycin-resistant mutants were detected., Results: Temocillin MICs were 8, 32 and 256 mg/L for CFT073 (WT), CFT073-KPC-3 and CFT073-OXA-48, respectively. Fosfomycin MIC was 0.5 mg/L for all strains. The chequerboard experiments demonstrated synergy for all three strains. In time-kill curves, combining temocillin with fosfomycin was synergistic, bactericidal and prevented emergence of resistance for CFT073-pTOPO and CFT073-KPC-3, but not CFT073-OXA-48. In vivo, for the three strains, bacterial counts were lower in peritoneal fluid with the combination compared with fosfomycin alone (P < 0.001) and inhibited growth of resistant mutants in all cases., Conclusions: The combination of fosfomycin and temocillin demonstrated a benefit in vitro and in vivo against E. coli strains producing KPC-3 or OXA-48-type carbapenemases. This combination prevented the emergence of fosfomycin resistance and proved to be more bactericidal than fosfomycin alone.

Prevalence of fosfomycin resistance in E. coli clinical isolates from UTIs remains very low. Our hypothesis was that fosfomycin resistance may be associated with a biological cost. Three groups of strains of E. coli belonging to the B2 phylogenetic group were used: clinical wild-type (WT) isolates, clinical multidrug-resistant isolates and in vitro fosfomycin-resistant derivatives from the uropathogen clinical strain E. coli CFT073. In each group fosfomycin-susceptible and -resistant isolates were compared. In vitro, we found a significantly decreased growth rate for fosfomycin-resistant strains as compared with susceptible strains in the WT group. In a murine model of ascending UTI, there was a significant reduction in infection rates with fosfomycin-resistant isolates as compared with susceptible ones, in all 3 study groups, ranging from 28 to 39% (P<0.03). All fosfomycin-susceptible clinical strains were virulent in vivo (13/13), while fosfomycin-resistant clinical strains were either virulent (2/7) or non-virulent (5/7) (P<0.002). This difference was not explained by the number of virulence factors or pathogenicity-associated islands. In conclusion, fosfomycin resistance appears to carry some biological cost in E. coli, which may explain in part the apparent paradox of the low prevalence of fosfomycin resistance despite a high rate of spontaneous mutants., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Objectives: Temocillin is a 6-α-methoxy derivative of ticarcillin that shows in vitro activity against Enterobacteriaceae producing Klebsiella pneumoniae carbapenemase (KPC). Our objective was to assess in vivo temocillin activity against KPC-producing Escherichia coli., Methods: Isogenic derivatives of the WT E. coli CFT073 producing KPC-2, KPC-3 or OXA-48 were constructed. An experimental murine model of intra-abdominal infection with sepsis was used. Mice were treated subcutaneously with temocillin 200 mg/kg every 2 h for 24 h, reproducing the duration of time that the free serum concentration of temocillin exceeded the MIC in humans with a regimen of 2 g every 12 h or 2 g every 8 h. Blood, peritoneal fluid (PF) and spleen were collected; 24 h survival and sterility rates were assessed., Results: Temocillin MICs were 8, 16, 32, and 256 mg/L for the susceptible strain and KPC-2-, KPC-3-, and OXA-48-producing strains, respectively. In mice treated with temocillin, significant bacterial reduction was obtained in PF, blood, and spleen for the susceptible strain and KPC-2- and KPC-3-producing strains (P < 0.001) but not for the OXA-48-producing strain. Sterility rates in PF were 53%, 10%, 0% and 0% (P < 0.001) and sterility rates in blood were 77%, 40%, 3% and 0% (P < 0.001), while survival rates were 97%, 97%, 57%, 0% (P < 0.001) for mice infected with the susceptible strain and KPC-2-, KPC-3- and OXA-48-producing strains, respectively., Conclusions: In a lethal-infection model with bacteraemia from intra-abdominal origin, temocillin retained significant activity in PF, blood and spleen and prevented death in mice by effectively working against KPC-producing E. coli with temocillin MICs ≤16 mg/L., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objectives: To study the impact of qnrA1, qnrB19 and qnrS1 on the ciprofloxacin treatment of urinary tract infection (UTI)., Methods: From a wild-type (wt) Escherichia coli UTI isolate, three isogenic strains were constructed carrying low-level ciprofloxacin resistance genes qnrA1, qnrB19 or qnrS1 (ciprofloxacin MIC range: 0.19-0.38 mg/L). Time-kill studies were performed for all four isogenic strains at the following concentrations: 1×, 2×, 4×, 8× and 16× MIC. Ciprofloxacin serum and urine pharmacokinetics was determined to calculate a murine dose equivalent (AUC(24)) to the standard human dose of 500 mg twice daily, which corresponded to 0.2 mg/mouse four times daily. In the murine UTI model, mice infected with each of the isogenic qnr strains or the wt strain were treated with ciprofloxacin (0.2 mg/mouse) or saline (only the E. coli wt) subcutaneously four times daily for 3 days starting 24 h after bacterial inoculation., Results: In vitro, the strains responded to ciprofloxacin concentrations of 4-16× MIC by several log(10) reductions. In vivo, despite ciprofloxacin reaching urine concentrations far exceeding the MICs for the strains (500 mg/L), ciprofloxacin was significantly less efficient at reducing the urine and bladder bacterial counts of qnrA1-, qnrB19- and qnrS1-positive strains compared with the ciprofloxacin-treated wt strain (P < 0.05). None of the four strains infected the kidneys well, with median cfu counts of <1 log(10)., Conclusions: Although qnr genes only confer low levels of resistance to ciprofloxacin, a reduced bactericidal activity of ciprofloxacin was observed in both urine and bladder in the murine model of UTI.

Objectives: To analyse the in vitro transfer of the qnrA1 gene by a 50 kb (pSZ50) self-transferable plasmid that derives from a 300 kb plasmid (pSZ300) and to determine the complete nucleotide sequence of plasmid pSZ50., Methods: Extended-spectrum β-lactamase (ESBL) and plasmid-mediated quinolone resistance (PMQR) genes of an Escherichia coli clinical isolate were analysed. Plasmid analysis included conjugation and selection on seven antibiotics examined by antimicrobial susceptibility testing, RFLP comparison, Southern hybridization, incompatibility group identification and shotgun sequencing., Results: The E. coli 5509 isolate carries the genes encoding the ESBL CTX-M-15 and the quinolone resistance determinants qnrA1, qnrB2 and aac(6')-Ib-cr on a 300 kb plasmid. Seven transfer resistances were analysed by conjugation under two conditions (30 and 37°C), leading to two distinct transconjugant phenotypes with different resistances. Transconjugants of phenotype A harboured a 300 kb plasmid named pSZ300 that conferred resistance to eight antibiotics and harboured the qnrA1, aac(6')-Ib-cr and bla(CTX-M-15) genes. Transconjugants of phenotype B were resistant to three antibiotics and they harboured the qnrA1 gene on an ≈ 50 kb plasmid named pSZ50. Both plasmids were self-transferable at a frequency of 1 × 10(-3). Plasmid pSZ300 was typed to be both an IncF and IncN plasmid, whereas pSZ50 corresponded only to type IncN. Fingerprinting and Southern hybridization showed that plasmid pSZ50 derived from pSZ300. The complete nucleotide sequence of plasmid pSZ50 was determined (51556 bp) and 55 open reading frames were predicted. The qnrA1 gene was identified in a tandem duplicate inside a sul1-type integron structure., Conclusions: The plasmid pSZ300 represented a fusion of two replicons (IncF and IncN), and our observations suggest that the plasmid pSZ50 (IncN) may split and transfer antibiotic resistance determinants. This mechanism could be advantageous in the dissemination of antibiotic resistance genes.

One hundred twenty-one extended-spectrum beta-lactamse-producing enterobacterial clinical isolates were screened for the qepA gene. A single CTX-M-15-positive Escherichia coli isolate (0.8%) that produced the putative pump QepA2 was identified. This qepA2 gene was located onto a 90-kb mobilizable plasmid that conferred reduced susceptibility to hydrophilic fluoroquinolones.

A novel QnrB-like plasmid-mediated resistance determinant, QnrB19, was identified from an Escherichia coli clinical isolate from Colombia. Its gene was associated with an ISEcp1-like insertion element that did not act as a promoter for its expression. Using an in vitro model of transposition, we showed that the ISEcp1-like element was able to mobilize the qnrB19 gene.

Plasmid-mediated quinolone resistance is being reported increasingly worldwide among isolates of enterobacteria. This resistance is mostly associated with Qnr-like determinants that confer resistance to quinolones, e.g., nalidixic acid, and reduced susceptibility to fluoroquinolones. This study investigated whether amino-acid substitutions in QnrA1 and QnrS1 determinants might be responsible for an enhancement of resistance to quinolones and, particularly, to fluoroquinolones. However, random and site-directed mutagenesis failed to identify any single amino-acid substitution that could be responsible for higher levels of resistance to quinolones or fluoroquinolones, indicating that the selection of such mutants in vivo is probably a rare event.

Objectives: To investigate quinolone resistance mechanisms in an Escherichia coli clinical isolate (Ar2) resistant to ofloxacin but susceptible to nalidixic acid selected after 10 days of ofloxacin therapy in a patient with prostatitis., Methods: Molecular typing (ERIC-PCR and RAPD), antibiotic susceptibility and gyrA, gyrB, parC and parE QRDR sequences were compared for E. coli Ar2 and a wild-type E. coli (Ar1) isolated 2 months earlier in the same patient. Ofloxacin-resistant mutants were selected in vitro in order to reproduce the mutations observed and the original phenotype., Results: The two strains were similar with regard to antibiotic susceptibility except quinolones and for ERIC-PCR and RAPD patterns, suggesting a clonal relationship and acquisition of quinolone resistance by chromosomal mutation. Quinolone MICs were 3, 0.12, 0.05 and 0.02 mg/L of nalidixic acid, ofloxacin, levofloxacin and ciprofloxacin, respectively, for E. coli Ar1 and 6, 32, 8 and 1 mg/L, respectively, for E. coli Ar2. The strain Ar2 harboured two substitutions, Gly-81-->Asp in GyrA and Ser-80-->Arg in ParC. Introduction into E. coli Ar2 of the wild-type gyrA fully complemented fluoroquinolone resistance. Although the strain was not a hypermutator, ofloxacin first-step resistant mutants with gyrA mutations were easily obtained from E. coli Ar1 and 25% of them were at codon 81. In vitro stepwise combination of Gly-81-->Asp in GyrA and Ser-80-->Arg in ParC reproduced the original phenotype in E. coli KL16., Conclusions: A double topoisomerase mutant was selected in vivo by 10 days ofloxacin. The mutations were originally combined for a result of ofloxacin resistance but nalidixic acid susceptibility.