These findings support avoidance of fluoroquinolones for first-line UTI therapy in primary care, and suggest potential for interventions that interrupt household circulation of resistant Enterobacteriaceae.

e Nitrofurantoin has been used for decades for the treatment of urinary tract infections (UTIs), but clinically significant resistance in Escherichia coli is uncommon. Nitrofurantoin concentrations in the gastrointestinal tract tend to be low, which might facilitate selection of nitrofurantoin-resistant (NIT-R) strains in the gut flora. We subjected two nitrofurantoin-susceptible intestinal E. coli strains (ST540-p and ST2747-p) to increasing nitrofurantoin concentrations under aerobic and anaerobic conditions. Whole-genome sequencing was performed for both susceptible isolates and selected mutants that exhibited the highest nitrofurantoin resistance levels aerobically (ST540-a and ST2747-a) and anaerobically (ST540-an and ST2747-an). ST540-a/ST540-an and ST2747-a (aerobic MICs of >64 g/ml) harbored mutations in the known nitrofurantoin resistance determinants nfsA and/or nfsB, which encode oxygen-insensitive nitroreductases. ST2747-an showed reduced nitrofurantoin susceptibility (aerobic MIC of 32 g/ml) and exhibited remarkable growth deficits but did not harbor nfsA/nfsB mutations. We identified a 12-nucleotide deletion in ribE, encoding lumazine synthase, an essential enzyme involved in the biosynthesis of flavin mononucleotide (FMN), which is an important cofactor for NfsA and NfsB. Complementing ST2747-an with a functional wild-type lumazine synthase restored nitrofurantoin susceptibility. Six NIT-R E. coli isolates (NRCI-1 to NRCI-6) from stools of UTI patients treated with nitrofurantoin, cefuroxime, or a fluoroquinolone harbored mutations in nfsA and/or nfsB but not ribE. Sequencing of the ribE gene in six intestinal and three urinary E. coli strains showing reduced nitrofurantoin susceptibility (MICs of 16 to 48 g/ml) also did not identify any relevant mutations. NRCI-1, NRCI-2, and NRCI-5 exhibited up to 4-fold higher anaerobic MICs, compared to the mutants generated in vitro, presumably because of additional mutations in oxygen-sensitive nitroreductases.

No significant impact was observed other than a temporary increase in the beneficial Bifidobacterium genus following nitrofurantoin treatment, which supports its reintroduction into clinical use.

Widespread multidrug resistance in Escherichia coli has necessitated the reintroduction of older antibiotics, such as nitrofurantoin. However, mechanisms by which resistance to nitrofurantoin emerges in E. coli are not well elucidated. Toward this aim, we sequenced two nitrofurantoin-sensitive E. coli sequence types (ST540 and ST2747) and their four nitrofurantoin-resistant derivatives generated in vitro under aerobic and anaerobic growth conditions.

The purposes of this study were to investigate the intestinal carriage of extended-spectrum β-lactamase-harbouring Enterobacteriaceae (ESBL-EN) and associated fluoroquinolone resistance (FQ-R) in 120 hospitalised patients with antibiotic-associated diarrhoea, and to investigate a correlation between Clostridium difficile (C. difficile) infection and intestinal colonisation with ESBL-EN in these patients. Stool samples were screened for C. difficile infection by toxin A/B enzyme-linked immunosorbent assay (ELISA) and for the presence of enterobacterial isolates producing β-lactamases by plating on β-lactamase screening (BLSE) agar. Recovered isolates were confirmed pheno- and genotypically for the presence of ESBL genes (bla CTX-M, bla TEM, bla SHV) by the double-disc synergy test and polymerase chain reaction (PCR) sequencing, and tested for the presence of topoisomerase mutations (gyrA, parC) and plasmid-mediated quinolone resistance (PMQR) determinants [qnrA, qnrB, qnrS, qepA, aac(6')-Ib-cr] by PCR sequencing. ESBL-EN were detected in 44/120 (37 %) stool samples. C. difficile-infected patients showed a significantly higher frequency of intestinal colonisation with ESBL-EN compared to C. difficile non-infected patients (62 % vs. 31 %, p = 0.008). Of the 73 ESBL-EN recovered, 46 (63 %) showed high-level FQ-R [ciprofloxacin minimum inhibitory concentration (MIC) ≥32 mg/L]. The largest group consisted of 21 bla CTX-M-15-harbouring Enterobacteriaceae (ciprofloxacin MIC ≥64 mg/L) with multiple topoisomerase mutations in gyrA and parC, in combination with co-carriage of aac(6')-Ib-cr. Most of them were Escherichia coli isolates belonging to sequence types ST131 and ST410. We found remarkably high rates of intestinal colonisation with high-level FQ-R ESBL-EN in hospitalised patients with antibiotic-associated diarrhoea, especially among C. difficile-infected patients. These data underscore the need for stringent infection control to contain this potentially infectious and multidrug-resistant reservoir.

ObjectivesWe characterized two new CTX-M-type extended-spectrum β-lactamase (ESBL) variants in Escherichia coli isolates from stool samples of two elderly patients admitted at the Tel Aviv Sourasky Medical Center, Israel. Both patients underwent treatment with cephalosporins prior to isolation of the E. coli strains.MethodsESBLs were detected by the double-disk synergy test and PCR-sequencing of β-lactamase genes. The bla CTX-M genes were cloned into the pCR-BluntII-TOPO vector in E. coli TOP10. The role of amino-acid substitutions V77A and D240G was analyzed by site-directed mutagenesis of the bla CTX-M-94 and bla CTX-M-100 genes and comparative characterization of the resulting E. coli recombinants. MICs of β-lactams were determined by Etest. Plasmid profiling, mating experiments, replicon typing and sequencing of bla CTX-M flanking regions were performed to identify the genetic background of the new CTX-M variants.ResultsThe novel CTX-M β-lactamases, CTX-M-94 and -100, belonged to the CTX-M-25-group. Both variants differed from CTX-M-25 by the substitution V77A, and from CTX-M-39 by D240G. CTX-M-94 differed from all CTX-M-25-group enzymes by the substitution F119L. Glycine-240 was associated with reduced susceptibility to ceftazidime and leucine-119 with increased resistance to ceftriaxone. bla CTX-M-94 and bla CTX-M-100 were located within ISEcp1 transposition units inserted into ∼93 kb non-conjugative IncFI and ∼130 kb conjugative IncA/C plasmids, respectively. The plasmids carried also different class 1 integrons.ConclusionsThis is the first report on CTX-M-94 and -100 ESBLs, novel members of the CTX-M-25-group.

Carriage dynamics of drug-resistant bacteria, especially within households, are poorly un- derstood. This limits the ability to develop effective interventions for controlling the spread of antimicrobial resistance in the community. Two groups consisting of: (i) patients with urinary tract infection requiring antimicrobial treatment; and (ii) patients who were not pre- scribed antimicrobial treatment were prospectively recruited at three European sites: Antwerp (Belgium), Geneva (Switzerland) and Lodz (Poland). Each index patient and up to three additional household members provided faecal samples at baseline, completion of antimicro- bial therapy (or 7-10 days after the first sample for the non-exposed) and 28 days after the second sample. We analysed household-level and individual-level fluoroquinolone resistant Enterobacteriaceae (FQR-E) acquisition and carriage data using Bayesian multi-state Markov models. At the individual level, we estimated a median baseline FQR-E acquisition rate of 0.006 (95%CrI = [0.004, 0.01]) per day, and a median duration of carriage of 24.4 days (95% CrI=[15.23,41.38]). Nitrofurantoin exposure was associated with a reduced rate of FQR-E acquisition (HR=0.28, 95%CrI=[0.14,0.56]), while fluoroquinolone exposure had no clear as- sociation with rates of FQR-E acquisition (HR=1.43, 95% CrI=[0.81,2.53]) at individual level. There was evidence that rates of FQR-E acquisition varied by site, and coming from Lodz was associated with a higher acquisition rate (HR=3.56, 95% CrI=[1.92, 6.34]). Prolonged duration of carriage was associated with exposure to fluoroquinolone or nitrofurantoin during the study, use of any antimicrobial agent in the prior 12 months and travel to endemic regions. At household level, we found strong evidence of positive association between FQR-E acquisi- tion and fluoroquinolone exposure (HR=3.43, 95% CrI=[1.51,7.74]). There was weak evidence of negative association between FQR-E acquisition and nitrofurantoin exposure (HR=0.42, 95%CrI=[0.12, 1.24]. Similar to the individual level, carriage duration was also associated with antimicrobial exposure at the household level. Our study has identified within household contacts as an important route for FQR-E transmission and highlights the need for prioritising household focused interventions to control FQR-E spread.

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