Characterisation of Early Positive mcr-1 Resistance Gene and Plasmidome in Escherichia coli Pathogenic Strains Associated with Variable Phylogroups under Colistin Selection.

An antibiotic susceptibility monitoring programme was conducted from 2004 to 2010, resulting in a collection of 143 Escherichia coli cultured from bovine faecal samples (diarrhoea) and milk-aliquots (mastitis). The isolates were subjected to whole-genome sequencing and were distributed in phylogroups A, B1, B2, C, D, E, and G with no correlation for particular genotypes with pathotypes. In fact, the population structure showed that the strains belonging to the different phylogroups matched broadly to ST complexes; however, the isolates are randomly associated with the diseases, highlighting the necessity to investigate the virulence factors more accurately in order to identify the mechanisms by which they cause disease. The antimicrobial resistance was assessed phenotypically, confirming the genomic prediction on three isolates that were resistant to colistin, although one isolate was positive for the presence of the gene mcr-1 but susceptible to colistin. To further characterise the genomic context, the four strains were sequenced by using a single-molecule long read approach. Genetic analyses indicated that these four isolates harboured complex and diverse plasmids encoding not only antibiotic resistant genes (including mcr-1 and bla) but also virulence genes (siderophore, ColV, T4SS). A detailed description of the plasmids of these four E. coli strains, which are linked to bovine mastitis and diarrhoea, is presented for the first time along with the characterisation of the predicted antibiotic resistance genes. The study highlighted the diversity of incompatibility types encoding complex antibiotic resistance elements such as Tn6330, ISEcp1, Tn6029, and IS5075. The mcr-1 resistance determinant was identified in IncHI2 plasmids pCFS3273-1 and pCFS3292-1, thus providing some of the earliest examples of mcr-1 reported in Europe, and these sequences may be a representative of the early mcr-1 plasmidome characterisation in the EU/EEA.

Protection of Hamsters from Mortality by Reducing Fecal Moxifloxacin Concentration with DAV131A in a Model of Moxifloxacin-Induced Clostridium difficile Colitis.

Lowering the gut exposure to antibiotics during treatments can prevent microbiota disruption. We evaluated the effects of an activated charcoal-based adsorbent, DAV131A, on the fecal free moxifloxacin concentration and mortality in a hamster model of moxifloxacin-induced Clostridium difficile infection. A total of 215 hamsters receiving moxifloxacin subcutaneously (day 1 [D1] to D5) were orally infected at D3 with C. difficile spores. They received various doses (0 to 1,800 mg/kg of body weight/day) and schedules (twice a day [BID] or three times a day [TID]) of DAV131A (D1 to D8). Moxifloxacin concentrations and C. difficile counts were determined at D3, and mortality was determined at D12 We compared mortality rates, moxifloxacin concentrations, and C. difficile counts according to DAV131A regimen and modeled the links between DAV131A regimen, moxifloxacin concentration, and mortality. All hamsters that received no DAV131A died, but none of those that received 1,800 mg/kg/day died. Significant dose-dependent relationships between DAV131A dose and (i) mortality, (ii) moxifloxacin concentration, and (iii) C. difficile count were evidenced. Mathematical modeling suggested that (i) lowering the moxifloxacin concentration at D3, which was 58 µg/g (95% confidence interval [CI] = 50 to 66 µg/g) without DAV131A, to 17 µg/g (14 to 21 µg/g) would reduce mortality by 90%; and (ii) this would be achieved with a daily DAV131A dose of 703 mg/kg (596 to 809 mg/kg). In this model of C. difficile infection, DAV131A reduced mortality in a dose-dependent manner by decreasing the fecal free moxifloxacin concentration.

Multidrug-Resistant Escherichia coli in Bovine Animals, Europe.

Of 150 Escherichia coli strains we cultured from specimens taken from cattle in Europe, 3 had elevated MICs against colistin. We assessed all 3 strains for the presence of the plasmid-mediated mcr-1 gene and identified 1 isolate as mcr-1-positive and co-resistant to ß-lactam, florfenicol, and fluoroquinolone antimicrobial compounds.

In vitro antibacterial activity of the ceftazidime-avibactam combination against enterobacteriaceae, including strains with well-characterized ß-lactamases.

The novel ß-lactamase inhibitor avibactam is a potent inhibitor of class A, class C, and some class D enzymes. The in vitro antibacterial activity of the ceftazidime-avibactam combination was determined for a collection of Enterobacteriaceae clinical isolates; this collection was enriched for resistant strains, including strains with characterized serine ß-lactamases. The inhibitor was added either at fixed weight ratios to ceftazidime or at fixed concentrations, with the latter type of combination consistently resulting in greater potentiation of antibacterial activity. In the presence of 4 µg/ml of avibactam, the ceftazidime MIC50 and MIC90 (0.25 and 2 µg/ml, respectively) were both below the CLSI breakpoint for ceftazidime. Further comparisons with reference antimicrobial agents were performed using this fixed inhibitor concentration. Against most ceftazidime-susceptible and -nonsusceptible isolates, the addition of avibactam resulted in a significant increase in ceftazidime activity, with MICs generally reduced 256-fold for extended-spectrum ß-lactamase (ESBL) producers, 8- to 32-fold for CTX-M producers, and >128-fold for KPC producers. Overall, MICs of a ceftazidime-avibactam combination were significantly lower than those of the comparators piperacillin-tazobactam, cefotaxime, ceftriaxone, and cefepime and similar or superior to those of imipenem.

Efficacy of a Ceftazidime-Avibactam combination in a murine model of Septicemia caused by Enterobacteriaceae species producing ampc or extended-spectrum ß-lactamases.

Avibactam is a novel non-ß-lactam ß-lactamase inhibitor that has been shown in vitro to inhibit class A, class C, and some class D ß-lactamases. It is currently in phase 3 of clinical development in combination with ceftazidime. In this study, the efficacy of ceftazidime-avibactam was evaluated in a murine septicemia model against five ceftazidime-susceptible (MICs of 0.06 to 0.25 µg/ml) and 15 ceftazidime-resistant (MICs of 64 to >128 µg/ml) species of Enterobacteriaceae, bearing either TEM, SHV, CTX-M extended-spectrum, or AmpC ß-lactamases. In the first part of the study, ceftazidime-avibactam was administered at ratios of 4:1 and 8:1 (wt/wt) to evaluate the optimal ratio for efficacy. Against ceftazidime-susceptible isolates of Klebsiella pneumoniae and Escherichia coli, ceftazidime and ceftazidime-avibactam demonstrated similar efficacies (50% effective doses [ED50] of <1.5 to 9 mg/kg of body weight), whereas against ceftazidime-resistant ß-lactamase-producing strains (ceftazidime ED50 of >90 mg/kg), the addition of avibactam restored efficacy to ceftazidime (ED50 dropped to <5 to 65 mg/kg). In a subsequent study, eight isolates (two AmpC and six CTX-M producers) were studied in the septicemia model. Ceftazidime-avibactam was administered at a 4:1 (wt/wt) ratio, and the efficacy was compared to that of the 4:1 (wt/wt) ratio of either piperacillin-tazobactam or cefotaxime-avibactam. Against the eight isolates, ceftazidime-avibactam was the more effective combination, with ED50 values ranging from 2 to 27 mg/kg compared to >90 mg/kg and 14 to >90 mg/kg for piperacillin-tazobactam and cefotaxime-avibactam, respectively. This study demonstrates that the potent in vitro activity observed with the ceftazidime-avibactam combination against ceftazidime-resistant Enterobacteriaceae species bearing class A and class C ß-lactamases translated into good efficacy in the mouse septicemia model.

Activities of ceftazidime and avibactam against ß-lactamase-producing Enterobacteriaceae in a hollow-fiber pharmacodynamic model.

Avibactam is a novel non-ß-lactam ß-lactamase inhibitor that is currently undergoing phase 3 clinical trials in combination with ceftazidime. Ceftazidime is hydrolyzed by a broad range of ß-lactamases, but avibactam is able to inhibit the majority of these enzymes. The studies described here attempt to provide insight into the amount of avibactam required to suppress bacterial growth in an environment where the concentrations of both agents are varying as they would when administered to humans. Following the simulation of a single intravenous dose of the drug, ceftazidime alone had no effect on any test organism, but a ceftazidime-avibactam combination resulted in rapid killing of all of the strains, with growth suppressed for the 8 h of the study. For seven of eight strains, this was achieved with a 1-g-250-mg profile, but a 2-g-500-mg profile was necessary to completely suppress a high-level-AmpC-producing isolate. When ceftazidime was infused continuously for 24 h with a single bolus dose of avibactam, rapid killing of all of the strains was again observed, with growth suppressed for 10 to >24 h. Regrowth appeared to commence once the avibactam concentration dropped below a critical concentration of approximately 0.3 µg/ml. In a third series of studies, ceftazidime was administered every 8 h for 24 h with avibactam administered at fixed concentrations for short periods during each ceftazidime dose profile. Simulating a 1-g dose of ceftazidime, an avibactam pulse of >0.25 and <0.5 µg/ml was required to suppress growth for 24 h.

The ß-lactamase inhibitor avibactam (NXL104) does not induce ampC ß-lactamase in Enterobacter cloacae.

Induction of ampC ß-lactamase expression can often compromise antibiotic treatment and is triggered by several ß-lactams (such as cefoxitin and imipenem) and by the ß-lactamase inhibitor clavulanic acid. The novel ß-lactamase inhibitor avibactam (NXL104) is a potent inhibitor of both class A and class C enzymes. The potential of avibactam for induction of ampC expression in Enterobacter cloacae was investigated by ampC messenger ribonucleic acid quantitation. Cefoxitin and clavulanic acid were confirmed as ampC inducers, whereas avibactam was found to exert no effect on ampC expression. Thus, avibactam is unlikely to diminish the activity of any partner ß-lactam antibiotic against AmpC-producing organisms.

In vitro antibacterial activity of the ceftazidime-avibactam (NXL104) combination against Pseudomonas aeruginosa clinical isolates.

The ß-lactamase inhibitor avibactam (NXL104) displays potent inhibition of both class A and C enzymes. The in vitro antibacterial activity of the combination ceftazidime-avibactam was evaluated against a clinical panel of Pseudomonas aeruginosa isolates. Avibactam offered efficient protection from hydrolysis since 94% of isolates were susceptible to ceftazidime when combined with 4 µg/ml avibactam, compared with 65% susceptible to ceftazidime alone. Ceftazidime-avibactam also demonstrated better antipseudomonal activity than imipenem (82% susceptibility), a common reference treatment.

Mechanistic studies of the inactivation of TEM-1 and P99 by NXL104, a novel non-beta-lactam beta-lactamase inhibitor.

NXL104 is a potent inhibitor of class A and C serine ß-lactamases, including KPC carbapenemases. Native and NXL104-inhibited TEM-1 and P99 ß-lactamases analyzed by liquid chromatography-electrospray ionization-time of flight mass spectrometry revealed that the inactivated enzymes formed a covalent adduct with NXL104. The principal inhibitory characteristics of NXL104 against TEM-1 and P99 ß-lactamases were determined, including partition ratios, dissociation constants (K), rate constants for deactivation (k(2)), and reactivation rates. NXL104 is a potent inhibitor of TEM-1 and P99, characterized by high carbamylation efficiencies (k(2)/K of 3.7 × 10(5) M(-1) s(-1) for TEM-1 and 1 × 10(4) M(-1) s(-1) for P99) and slow decarbamylation. Complete loss of ß-lactamase activity was obtained at a 1/1 enzyme/NXL104 ratio, with a k(3) value (rate constant for formation of product and free enzyme) close to zero for TEM-1 and P99. Fifty percent inhibitory concentrations (IC(50)s) were evaluated on selected ß-lactamases, and NXL104 was shown to be a very potent inhibitor of class A and C ß-lactamases. IC(50)s obtained with NXL104 (from 3 nM to 170 nM) were globally comparable on the ß-lactamases CTX-M-15 and SHV-4 with those obtained with the comparators (clavulanate, tazobactam, and sulbactam) but were far lower on TEM-1, KPC-2, P99, and AmpC than those of the comparators. In-depth studies on TEM-1 and P99 demonstrated that NXL104 had a comparable or better affinity and inactivation rate than clavulanate and tazobactam and in all cases an improved stability of the covalent enzyme/inhibitor complex.

In vitro activity of the {beta}-lactamase inhibitor NXL104 against KPC-2 carbapenemase and Enterobacteriaceae expressing KPC carbapenemases.

BACKGROUND: NXL104 is a novel-structure beta-lactamase inhibitor with potent activity against both class A and class C enzymes. Among the class A carbapenemases, KPC-type enzymes are now spreading rapidly and KPC-related carbapenemase resistance is an emerging phenomenon of great clinical importance. The activity of NXL104 against KPC beta-lactamases was examined. METHODS: Enzymatic activity of purified recombinant KPC-2 was measured with nitrocefin as reporter substrate and inhibition by NXL104 was measured by determination of IC(50) values. Antimicrobial susceptibility testing of various beta-lactams combined with a fixed concentration of NXL104 at 4 mg/L against strains producing KPC enzymes was performed by the broth microdilution method. RESULTS: NXL104 was a potent inhibitor of KPC-2 with an IC(50) of 38 nM. NXL104 restored the antimicrobial activity of ceftazidime, ceftriaxone, imipenem and piperacillin against Enterobacteriaceae strains producing KPC-2 or KPC-3. MIC values of ceftazidime against KPC producers were reduced by up to 1000-fold by combination with NXL104. CONCLUSIONS: NXL104 inhibitory activity is unique in terms of spectrum, encompassing class A extended-spectrum beta-lactamases, class C enzymes and class A carbapenemases. Given the limited therapeutic options available for infections caused by multiresistant Enterobacteriaceae isolates, NXL104 beta-lactamase inhibitor is a promising agent to be used in combination with a beta-lactam to protect its antibacterial activity.

NXL104 combinations versus Enterobacteriaceae with CTX-M extended-spectrum beta-lactamases and carbapenemases.

BACKGROUND: The beta-lactamase landscape is changing radically, with CTX-M types now the most prevalent extended-spectrum beta-lactamases (ESBLs) worldwide, except maybe in the USA. In addition, there are growing numbers of Enterobacteriaceae with KPC and metallo-carbapenemases. We examined whether combinations of oxyimino-cephalosporins with NXL104, a novel non-beta-lactam beta-lactamase inhibitor, overcame these resistances. METHODS: NXL104 was tested at 4 mg/L in combination with cefotaxime and ceftazidime versus: (i) Escherichia coli transconjugants and wild-type Enterobacteriaceae with CTX-M ESBLs; (ii) Enterobacteriaceae with ertapenem resistance contingent on combinations of impermeability and ESBLs or AmpC; and (iii) Enterobacteriaceae with KPC, SME, metallo- or OXA-48 carbapenemases. RESULTS: MICs of cefotaxime + NXL104 were < or = 1 mg/L for most Enterobacteriaceae with CTX-M, KPC or OXA-48 enzymes and were < or = 2 mg/L for those that also had ertapenem resistance contingent on combinations of beta-lactamase and impermeability. MICs of the ceftazidime + NXL104 combination were < or = 4 mg/L, except for a single Enterobacter aerogenes with KPC and AmpC enzymes together with porin loss, which required an MIC of 32 mg/L. The major gap was that NXL104 could not potentiate cephalosporins against Enterobacteriaceae with IMP or VIM metallo-enzymes. CONCLUSIONS: Oxyimino-cephalosporin + NXL104 combinations have potential against strains with the prevalent ESBLs and non-metallo-carbapenemases.

Mechanism of action of the antibiotic NXL101, a novel nonfluoroquinolone inhibitor of bacterial type II topoisomerases.

NXL101 is one of a new class of quinoline antibacterial DNA gyrase and topoisomerase IV inhibitors showing potent activity against gram-positive bacteria, including methicillin- and fluoroquinolone-resistant strains. NXL101 inhibited topoisomerase IV more effectively than gyrase from Escherichia coli, whereas the converse is true of enzymes from Staphylococcus aureus. This apparent target preference is opposite to that which is associated with most fluoroquinolone antibiotics. In vitro isolation of S. aureus mutants resistant to NXL101 followed by cloning and sequencing of the genes encoding gyrase and topoisomerase IV led to the identification of several different point mutations within, or close to, the quinolone resistance-determining region (QRDR) of GyrA. However, the mutations were not those that are most frequently associated with decreased sensitivity to quinolones. A fluoroquinolone-resistant mutant variant of gyrase generated in vitro was highly resistant to inhibition by the fluoroquinolones ciprofloxacin and moxifloxacin but remained fully susceptible to inhibition by NXL101. Two mutant gyrases constructed in vitro, with mutations in gyrA engineered according to those most frequently found in S. aureus strains resistant to NXL101, were insensitive to inhibition by NXL101 and had a diminished sensitivity to ciprofloxacin and moxifloxacin. Certain combinations of mutations giving rise to NXL101 resistance and those giving rise to fluoroquinolone resistance may be mutually exclusive.