Intrapulmonary Pharmacokinetics of Cefepime and Enmetazobactam in Healthy Volunteers: Towards New Treatments for Nosocomial Pneumonia.

Cefepime-enmetazobactam is a novel β-lactam-β-lactamase inhibitor combination with broad-spectrum antimicrobial activity against a range of multidrug-resistant This agent is being developed for a range of serious hospital infections. An understanding of the extent of partitioning of β-lactam-β-lactamase inhibitor combinations into the human lung is required to better understand the potential role of cefepime-enmetazobactam for the treatment of nosocomial pneumonia. A total of 20 healthy volunteers were used to study the intrapulmonary pharmacokinetics of a regimen of 2 g cefepime-1 g enmetazobactam every 8 h intravenously (2 g/1 g q8h i.v.). Each volunteer contributed multiple plasma samples and a single epithelial lining fluid (ELF) sample, obtained by bronchoalveolar lavage. Concentrations of cefepime and enmetazobactam were quantified using liquid chromatography-tandem mass spectrometry. The pharmacokinetic data were modeled using a population methodology, and Monte Carlo simulations were performed to assess the attainment of pharmacodynamic targets defined in preclinical models. The concentration-time profiles of both agents in plasma and ELF were similar. The mean ± standard deviation percentage of partitioning of total drug concentrations of cefepime and enmetazobactam between plasma and ELF was 60.59% ± 28.62% and 53.03% ± 21.05%, respectively. Using pharmacodynamic targets for cefepime of greater than the MIC and free enmetazobactam concentrations of >2 mg/liter in ELF of 20% of the dosing interval, a regimen of cefepime-enmetazobactam of 2 g/0.5 g q8h i.v. infused over 2 h resulted in a probability of target attainment of ≥90% for with cefepime-enmetazobactam MICs of ≤8 mg/liter. This result provides a rationale to further consider cefepime-enmetazobactam for the treatment of nosocomial pneumonia caused by multidrug-resistant

Liver transplantation as a new standard of care in patients with perihilar cholangiocarcinoma?:Results from an international benchmark study

Objective: To define benchmark values for liver transplantation (LT) in patients with perihilar cholangiocarcinoma (PHC) enabling unbiased comparisons.Background: Transplantation for PHC is used with reluctance in many centers and even contraindicated in several countries. Although benchmark values for LT are available, there is a lack of specific data on LT performed for PHC.Methods: PHC patients considered for LT after Mayo-like protocol were analyzed in 17 reference centers in 2 continents over the recent 5-year period (2014–2018). The minimum follow-up was 1 year. Benchmark patients were defined as operated at high-volume centers (≥ 50 overall LT/year) after neoadjuvant chemoradiotherapy, with a tumor diameter &lt;3 cm, negative lymph nodes, and with the absence of relevant comorbidities. Benchmark cutoff values were derived from the 75th to 25th percentiles of the median values of all benchmark centers.Results: One hundred thirty-four consecutive patients underwent LT after completion of the neoadjuvant treatment. Of those, 89.6% qualified as benchmark cases. Benchmark cutoffs were 90-day mortality ≤ 5.2%; comprehensive complication index at 1 year of ≤ 33.7; grade ≥ 3 complication rates ≤ 66.7%. These values were better than benchmark values for other indications of LT. Five-year disease-free survival was largely superior compared with a matched group of nodal negative patients undergoing curative liver resection (n = 106) (62% vs 32%, P &lt; 0.001).Conclusion: This multicenter benchmark study demonstrates that LT offers excellent outcomes with superior oncological results in early stage PHC patients, even in candidates for surgery. This provocative observation should lead to a change in available therapeutic algorithms for PHC.</p

AgSPA2 and AgBOI control landmarks of filamentous growth in the filamentous ascomycete Ashbya gossypii

The morphological requirements for the development of a fungal mycelium include the emergence of germ tubes, a permanent hyphal tip extension, lateral and apical branching and septation. The basis for each of these events, also termed landmarks of fi lamentous growth, is polarised growth. Thus polarised growth is essential for hyphal and mycelial morphogenesis. We wanted to investigate genes in the fi lamentous ascomycete Ashbya gossypii that are involved in polarised growth and control landmarks of fi lamentous growth. In "Chapter 1" and "Chapter 2" I described the identifi cation of two genes that are involved in polarised growth and I highlighted their roles in landmarks of fi lamentous growth. At the end of the fi rst two chapters I draw a refi ned model of the developmental pattern in A.gossypii with the insight gained from these fi rst two chapters. In "Chapter 3" and "Chapter 4" I described two novel tools that were established for the functional analysis of the genes investigated in the fi rst two chapters. These two methods were the construction of a versatile module for C-terminal GFP fusion and an integration module based on the AgADE2 marker. Chapter 1 We wanted to identify genes in A.gossypii that are important for hyphal morphogenesis. As polarised growth is crucial for morphogenesis in A.gossypii and S.cerevisiae we hypothesised that a similar set of genes is required for polarised growth in A.gossypii and S.cerevisiae; however, differences in orthologous proteins might have an impact on the regulation of polarised growth and thus guide the process of fi lamentation in A.gossypii and budding in S.cerevisiae. We screened for A.gossypii proteins that have an orthologue in S.cerevisiae implicated in polarised growth and that display a signifi cant difference in the primary structure compared to the S.cerevisiae orthologue. This revealed AgSpa2p, a homologue of ScSpa2p. AgSpa2p is more than twice as long as ScSpa2p due to an extended internal domain without signifi cant homology to ScSpa2p. AgSpa2p localises permanently to hyphal tips and transiently to sites of septation. We constructed two AgSPA2 mutants, a partial deletion of the internal domain alone and a complete deletion. We could show that these mutant strains display alterations in the branching frequency and in the hyphal tip growth speed potential. The branching frequency and the hyphal tip growth speed potential are two factors that have an impact on the hyphal tip growth speed. The two mutants behave opposing in respect of each of these factors and the two factors have a competing effect on the hyphal tip growth speed. We suggest that AgSpa2p is required to balance these two factors to achieve an effi cient hyphal tip growth speed and the extended internal domain in AgSpa2p plays an important role in this process. Chapter 2 We screened an existing A.gossypii knock out library for strains that show defects in landmarks of fi lamentous growth. We identifi ed a strain that frequently displayed spherically enlarged hyphal tips. The deleted gene in that strain was identifi ed as AgBOI. AgBoip represents an orthologue of the redundant proteins in S.cerevisiae ScBoi1p and ScBoi2p, which are implicated in polarised growth. We could show that AgBoip is required for the emergence of germ tubes and for the initiation of lateral branches. Moreover AgBoip is required for permanent hyphal tip extension, as polarisation of hyphal tips is not permanently maintained in AgboiΔ strains. We could show that prior to a spherical enlargement of hyphal tips in AgboiΔ the polarisation marker AgSpa2p delocalises from the tip. Tips can be repolarised which goes in parallel with a relocalisation of AgSpa2p. In spherically enlarged tips the actin cytoskeleton is also depolarised. AgBoip itself localised to sites of polarisation similar to cortical actin patches. We suggest that AgBoip is required for establishment of cell polarity to initiate germ tubes and lateral branches and for maintenance of cell polarity to allow a permanent hyphal tip extension. Chapter 3 The green fl uorescent protein (GFP) is of extraordinary value in molecular biology. It allows the visualisation of proteins in organisms and together with advanced microscopy techniques it enables dynamic studies in living cells. We constructed a versatile module for PCR based C-terminal GFP-fusion and established microscopy techniques for dynamic GFP studies in A.gossypii. The studies described in "Chapter 1" and "Chapter 2" would not have been possible without the development of this module. Chapter 4 The functional analysis of genes often requires the expression of truncated alleles, e.g. reporter modules as the GFP, tagged proteins for biochemical analyses, mutant alleles or overexpression constructs. As it was of importance in this work to express certain GFP fusion proteins as second copies we constructed an integration module on the basis of the AgADE2 marker. The mode of action of the integration module is the reconstitution of a truncated AgADE2 ORF thereby co-integrating the fragment of interest

The SH3/PH Domain Protein AgBoi1/2 Collaborates with the Rho-Type GTPase AgRho3 To Prevent Nonpolar Growth at Hyphal Tips of Ashbya gossypii

Unlike most other cells, hyphae of filamentous fungi permanently elongate and lack nonpolar growth phases. We identified AgBoi1/2p in the filamentous ascomycete Ashbya gossypii as a component required to prevent nonpolar growth at hyphal tips. Strains lacking AgBoi1/2p frequently show spherical enlargement at hyphal tips with concomitant depolarization of actin patches and loss of tip-located actin cables. These enlarged tips can repolarize and resume hyphal tip extension in the previous polarity axis. AgBoi1/2p permanently localizes to hyphal tips and transiently to sites of septation. Only the tip localization is important for sustained elongation of hyphae. In a yeast two-hybrid experiment, we identified the Rho-type GTPase AgRho3p as an interactor of AgBoi1/2p. AgRho3p is also required to prevent nonpolar growth at hyphal tips, and strains deleted for both AgBOI1/2 and AgRHO3 phenocopied the respective single-deletion strains, demonstrating that AgBoi1/2p and AgRho3p function in a common pathway. Monitoring the polarisome of growing hyphae using AgSpa2p fused to the green fluorescent protein as a marker, we found that polarisome disassembly precedes the onset of nonpolar growth in strains lacking AgBoi1/2p or AgRho3p. AgRho3p locked in its GTP-bound form interacts with the Rho-binding domain of the polarisome-associated formin AgBni1p, implying that AgRho3p has the capacity to directly activate formin-driven actin cable nucleation. We conclude that AgBoi1/2p and AgRho3p support polarisome-mediated actin cable formation at hyphal tips, thereby ensuring permanent polar tip growth

Maximal Polar Growth Potential Depends on the Polarisome Component AgSpa2 in the Filamentous Fungus Ashbya gossypii

We used actin staining and videomicroscopy to analyze the development from a spore to a young mycelium in the filamentous ascomycete Ashbya gossypii. The development starts with an initial isotropic growth phase followed by the emergence of germ tubes. The initial tip growth speed of 6–10 μm/h increases during early stages of development. This increase is transiently interrupted in response to the establishment of lateral branches or septa. The hyphal tip growth speed finally reaches a maximum of up to 200 μm/h, and the tips of these mature hyphae have the ability to split into two equally fast-growing hyphae. A search for A. gossypii homologs of polarisome components of the yeast Saccharomyces cerevisiae revealed a remarkable size difference between Spa2p of both organisms, with AgSpa2p being double as long as ScSpa2p due to an extended internal domain. AgSpa2 colocalizes with sites of polarized actin. Using time-lapse videomicroscopy, we show that AgSpa2p-GFP polarization is established at sites of branch initiation and then permanently maintained at hyphal tips. Polarization at sites of septation is transient. During apical branching the existing AgSpa2p-GFP polarization is symmetrically divided. To investigate the function of AgSpa2p, we generated two AgSPA2 mutants, a partial deletion of the internal domain alone, and a complete deletion. The mutations had an impact on the maximal hyphal tip growth speed, on the hyphal diameter, and on the branching pattern. We suggest that AgSpa2p is required for the determination of the area of growth at the hyphal tip and that the extended internal domain plays an important role in this process

A Ras-like GTPase Is Involved in Hyphal Growth Guidance in the Filamentous Fungus Ashbya gossypii

Characteristic features of morphogenesis in filamentous fungi are sustained polar growth at tips of hyphae and frequent initiation of novel growth sites (branches) along the extending hyphae. We have begun to study regulation of this process on the molecular level by using the model fungus Ashbya gossypii. We found that the A. gossypii Ras-like GTPase Rsr1p/Bud1p localizes to the tip region and that it is involved in apical polarization of the actin cytoskeleton, a determinant of growth direction. In the absence of RSR1/BUD1, hyphal growth was severely slowed down due to frequent phases of pausing of growth at the hyphal tip. During pausing events a hyphal tip marker, encoded by the polarisome component AgSPA2, disappeared from the tip as was shown by in vivo time-lapse fluorescence microscopy of green fluorescent protein-labeled AgSpa2p. Reoccurrence of AgSpa2p was required for the resumption of hyphal growth. In the Agrsr1/bud1Δ deletion mutant, resumption of growth occurred at the hyphal tip in a frequently uncoordinated manner to the previous axis of polarity. Additionally, hyphal filaments in the mutant developed aberrant branching sites by mislocalizing AgSpa2p thus distorting hyphal morphology. These results define AgRsr1p/Bud1p as a key regulator of hyphal growth guidance

Third-generation cephalosporin resistance in clinical isolates of Enterobacterales collected between 2016–2018 from USA and Europe: genotypic analysis of β-lactamases and comparative in vitro activity of cefepime/enmetazobactam

ABSTRACT: Objectives: This study aimed to investigate third-generation cephalosporin (3GC) resistance determinants [extended-spectrum β-lactamases (ESBLs), AmpC β-lactamases and OXA-type β-lactamases] in contemporary clinical Enterobacterales isolates and to determine the in vitro activity of β-lactams and β-lactam/β-lactamase inhibitor combinations, including the investigational combination of cefepime and the novel β-lactamase inhibitor enmetazobactam. Methods: Antibacterial susceptibility of 7168 clinical Enterobacterales isolates obtained between 2016–2018 from North America and Europe was determined according to CLSI guidelines. Phenotypic resistance to the 3GC ceftazidime (MIC ≥ 16 µg/mL) and/or ceftriaxone (MIC ≥ 4 µg/mL) but retaining susceptibility to meropenem (MIC ≤ 1 µg/mL) was determined. β-Lactamase genotyping was performed on clinical isolates with ceftazidime, ceftriaxone, cefepime or meropenem MIC ≥ 1 µg/mL. Results: Phenotypic resistance to 3GCs occurred in 17.5% of tested isolates, whereas 2.1% of isolates were resistant to the carbapenem meropenem. Within the 3GC-resistant subgroup, 60.1% (n = 752) of isolates encoded an ESBL, 25.6% (n = 321) encoded an AmpC-type β-lactamase and 0.9% (n = 11) encoded an OXA-type β-lactamase. Susceptibility of the subgroup to piperacillin/tazobactam (57.5%) and ceftolozane/tazobactam (71.3%) was <90% based on breakpoints established by the CLSI. Projected susceptibility to cefepime/enmetazobactam was 99.6% when applying the cefepime susceptible, dose-dependent breakpoint of 8 µg/mL. Against ESBL-producing isolates (n = 801) confirmed by genotyping, only susceptibility to meropenem (96.0%) and cefepime/enmetazobactam (99.9%) exceeded 90%. Conclusion: This study describes the antibacterial activity of important therapies against contemporary 3GC-resistant clinical Enterobacterales isolates and supports the development of cefepime/enmetazobactam as a carbapenem-sparing option for ESBL-producing pathogens

Phosphatidylinositol-dependent phospholipases C Plc2 and Plc3 of Candida albicans are dispensable for morphogenesis and host-pathogen interaction

International audiencePhospholipases play an important role as virulence factors in human pathogens. Candida albicans, the major fungal pathogen of humans, encodes phospholipases of type A, B, C and D. Type B Plb2 and type D Pld1 phospholipases have been shown to contribute to virulence in this organism. We analyzed, in C. albicans, PLC2 and PLC3, two highly conserved genes coding for phosphatidylinositol-dependent phospholipases C with homology to the known virulence factor PlcA in the human pathogen Listeria monocytogenes. We show that expression of PLC2 and PLC3 is upregulated under different filament-inducing conditions and in the constitutive filamentous mutant tup1Δ. In order to analyze PLC2 and PLC3 function in C. albicans, we constructed strains that carry PLC2 or PLC3 under a constitutive promoter and strains that lack all four PLC2/3 alleles. These strains were not affected in their ability to produce filaments under non-inducing conditions, nor was filamentation modified under inducing conditions, suggesting that PLC2/3 are not critical determinants of the yeast-to-hypha switch. In a cell culture model for macrophage interaction, phagocytosis of C. albicans and subsequent killing were not influenced by PLC2/3. These results demonstrate that C. albicans PLC2 and PLC3 are dispensable for virulence; moreover, they underline the sharp contrast with the function of plcA in L. monocytogenes

The natural diyne-furan fatty acid EV-086 is an inhibitor of fungal delta-9 fatty acid desaturation with efficacy in a model of skin dermatophytosis

Human fungal infections represent a therapeutic challenge. Although effective strategies for treatment are available, resistance is spreading, and many therapies have unacceptable side effects. A clear need for novel antifungal targets and molecules is thus emerging. Here, we present the identification and characterization of the plant-derived diyne-furan fatty acid EV-086 as a novel antifungal compound. EV-086 has potent and broad-spectrum activity in vitro against Candida, Aspergillus, and Trichophyton spp., whereas activities against bacteria and human cell lines are very low. Chemical-genetic profiling of Saccharomyces cerevisiae deletion mutants identified lipid metabolic processes and organelle organization and biogenesis as targets of EV-086. Pathway modeling suggested that EV-086 inhibits delta-9 fatty acid desaturation, an essential process in S. cerevisiae, depending on the delta-9 fatty acid desaturase OLE1. Delta-9 unsaturated fatty acids—but not saturated fatty acids—antagonized the EV-086-mediated growth inhibition, and transcription of the OLE1 gene was strongly upregulated in the presence of EV-086. EV-086 increased the ratio of saturated to unsaturated free fatty acids and phosphatidylethanolamine fatty acyl chains, respectively. Furthermore, EV-086 was rapidly taken up into the lipid fraction of the cell and incorporated into phospholipids. Together, these findings demonstrate that EV-086 is an inhibitor of delta-9 fatty acid desaturation and that the mechanism of inhibition might involve an EV-086–phospholipid. Finally, EV-086 showed efficacy in a guinea pig skin dermatophytosis model of topical Trichophyton infection, which demonstrates that delta-9 fatty acid desaturation is a valid antifungal target, at least for dermatophytoses

Nouveaux composés antifongiques

patent submitter:Selmod GmbHThe present invention refers to a compound having the formula (I): R1–Z–C≡C–C≡C–R3 (I) and pharmaceutical compositions comprising the same that can be used in prevention, treatment and alleviation of a fungal infection. The instant compounds and pharmaceutical compositions are particularly useful in treating, preventing and alleviating fungal infections caused by Candida fungi and Aspergillus fungi