Search

Diabetic nephropathy (DN) is the main cause of morbidity and mortality in diabetes and is characterized by mesangial matrix deposition and podocytopathy, including podocyte loss. The risk factors and mechanisms involved in the pathogenesis of DN are still not completely defined. In the present study, we aimed to understand the cellular mechanisms through which activation of B2 kinin receptors contribute to the initiation and progression of DN. Stimulation of cultured rat podocytes with bradykinin (BK) resulted in a significant increase in ROS generation, and this was associated with a significant increase in NADPH oxidase (NOX)1 and NOX4 protein and mRNA levels. BK stimulation also resulted in a signicant increase in the phosphorylation of ERK1/2 and Akt, and this effect was inhibited in the presence of NOX1 and Nox4 small interfering (si)RNA. Furthermore, podocytes stimulated with BK resulted in a significant increase in protein and mRNA levels of connective tissue growth factor (CTGF) and, at the same time, a significant decrease in protein and mRNA levels of nephrin. siRNA targeted against NOX1 and NOX4 significantly inhibited the BK-induced increase in CTGF. Nephrin expression was increased in response to BK in the presence of NOX1 and NOX4 siRNA, thus implicating a role for NOXs in modulating the BK response in podocytes. Moreover, nephrin expression in response to BK was also significantly increased in the presence of siRNA targeted against CTGF. These findings provide novel aspects of BK signal transduction pathways in pathogenesis of DN and identify novel targets for interventional strategies. [ABSTRACT FROM AUTHOR]

As the global burden of disease caused by multidrug resistant bacteria is a major source of concern, credible clinical alternatives to antibiotic therapy, such as personalized phage therapy, are actively explored. Although phage therapy has been used for more than a century, the issue of an easy to implement diagnostic tool for determining phage susceptibility that meets current routine clinical needs is still open. In this Review, we summarize the existing methods used for determining phage activity on bacteria, including the three reference methods: the spot test, the double agar overlay plaque assay, and the Appelmans method. The first two methods rely on the principle of challenging the overnight growth of a lawn of bacteria in an agar matrix to a known relative phage to bacteria concentration and represent good screening tools to determine if the tested phage can be used for a "passive" and or "active" treatment. Beside these methods, several techniques, based on "real-time" growth kinetics assays (GKA) have been developed or are under development. They all monitor the growth of clinical isolates in the presence of phages, but use various detection methods, from classical optical density to more sophisticated techniques such as computer-assisted imagery, flow-cytometry, quantitative real-time polymerase chain reaction (qPCR) or metabolic indicators. Practical considerations as well as information provided about phage activity are reviewed for each technique. Finally, we also discuss the analytical and interpretative requirements for the implementation of a phage susceptibility testing tool in routine clinical microbiology., Competing Interests: The handling editor GR declared a past co-authorship with the author(s) MM, JP and BB., (Copyright © 2022 Daubie, Chalhoub, Blasdel, Dahma, Merabishvili, Glonti, De Vos, Quintens, Pirnay, Hallin and Vandenberg.)

Achromobacter genus (including Achromobacter xylosoxidans , the most prevalent Achromobacter species in patients with cystic fibrosis) is poorly susceptible to most conventional antibiotics. Contribution of efflux by AxyABM, AxyXY-OprZ, and AxyEF-OprN and of target mutations were studied in clinical isolates of A. xylosoxidans and Achromobacter insuavis. Forty-one isolates longitudinally collected from 21 patients with CF were studied by whole-genome sequencing (WGS)-typing, determination of minimum inhibitory concentrations (MICs) of β-lactams, aminoglycosides, colistin, azithromycin, ciprofloxacin, chloramphenicol, and doxycycline, and expression (quantitative RT-PCR) and function (measure of the uptake of a fluorescent substrate) of efflux pumps. WGS-based typing resulted in 10 clusters comprising 2 or 3 isolates and 20 singletons. The efflux activity was high in strains with elevated MICs for amikacin or azithromycin. This work sheds a new light on the impact of efflux and target mutations in resistance of Achromobacter to several drugs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chalhoub, Kampmeier, Kahl and Van Bambeke.)

Development of novel therapeutics to treat antibiotic-resistant infections, especially those caused by ESKAPE pathogens, is urgent. One of the most critical pathogens is P. aeruginosa , which is able to develop a large number of factors associated with antibiotic resistance, including high level of impermeability. Gram-negative bacteria are protected from the environment by an asymmetric Outer Membrane primarily composed of lipopolysaccharides (LPS) at the outer leaflet and phospholipids in the inner leaflet. Based on a large hemi-synthesis program focusing on amphiphilic aminoglycoside derivatives, we extend the antimicrobial activity of 3',6-dinonyl neamine and its branched isomer, 3',6-di(dimethyloctyl) neamine on clinical P. aeruginosa , ESBL, and carbapenemase strains. We also investigated the capacity of 3',6-homodialkyl neamine derivatives carrying different alkyl chains (C7-C11) to interact with LPS and alter membrane permeability. 3',6-Dinonyl neamine and its branched isomer, 3',6-di(dimethyloctyl) neamine showed low MICs on clinical P. aeruginosa , ESBL, and carbapenemase strains with no MIC increase for long-duration incubation. In contrast from what was observed for membrane permeability, length of alkyl chains was critical for the capacity of 3',6-homodialkyl neamine derivatives to bind to LPS. We demonstrated the high antibacterial potential of the amphiphilic neamine derivatives in the fight against ESKAPE pathogens and pointed out some particular characteristics making the 3',6-dinonyl- and 3',6-di(dimethyloctyl)-neamine derivatives the best candidates for further development.

In Pseudomonas aeruginosa (P. aeruginosa) collected from cystic fibrosis (CF) patients, 24% resistance to ceftazidime-avibactam in isolates negative for carbapenemases and extended-spectrum β-lactamases (ESBLs) has previously been observed. The current study aimed to unravel the underlying mechanism(s). Using the laboratory strain PAO1 and derivatives thereof, with ampC expression induced by a sub-minimum inhibitory concentration (MIC) of imipenem, a higher MIC of ceftazidime-avibactam was found for those overexpressing MexAB-OprM (quantitative polymerase chain reaction (PCR) of mexA) and, to a lesser extent, MexEF-OprN (PCR of mexE), or without OprD expression (SDS-Page and Coomassie blue staining). This was ascribed to (i) an efflux of avibactam (efflux mutants) and (ii) a lack of avibactam penetration (OprD mutants), respectively. We then used 10 CF clinical isolates resistant to ceftazidime (MIC ≥ 128 mg/L) and with (i) variable basal levels of ampC overexpression, (ii) mutations in mexA or mexB inactivating to variable extent the MexAB-OprM transport capacity (assessed by extrusion of N-phenyl-1-naphthylamine [NPN]), and (iii) expression or not of mexE and of OprD porin. The reduction of ceftazidime MIC in the presence of avibactam was partially lost for isolates with large efflux activity of MexAB-OprM and/or increased ampC expression, but not significantly with mexE expression or lack of OprD (non-parametric and parametric tests). This identified MexAB-OprM as a main avibactam efflux transporter in P. aeruginosa that, together with ampC overexpression, reduced avibactam potency. Since about 30% of CF isolates show mutations in MexAB-OprM compromising efflux (Chalhoub, et al. Sci Reports 2017;7:40208), routine susceptibility testing of CF P. aeruginosa with ceftazidime-avibactam is warranted., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The β-lactam antibiotic temocillin (6-α-methoxy-ticarcillin) shows stability to most extended spectrum β-lactamases, but is considered inactive against Pseudomonas aeruginosa. Mutations in the MexAB-OprM efflux system, naturally occurring in cystic fibrosis (CF) isolates, have been previously shown to reverse this intrinsic resistance. In the present study, we measured temocillin activity in a large collection (n = 333) of P. aeruginosa CF isolates. 29% of the isolates had MICs ≤ 16 mg/L (proposed clinical breakpoint for temocillin). Mutations were observed in mexA or mexB in isolates for which temocillin MIC was ≤512 mg/L (nucleotide insertions or deletions, premature termination, tandem repeat, nonstop, and missense mutations). A correlation was observed between temocillin MICs and efflux rate of N-phenyl-1-naphthylamine (MexAB-OprM fluorescent substrate) and extracellular exopolysaccharide abundance (contributing to a mucoid phenotype). OpdK or OpdF anion-specific porins expression decreased temocillin MIC by ~1 two-fold dilution only. Contrarily to the common assumption that temocillin is inactive on P. aeruginosa, we show here clinically-exploitable MICs on a non-negligible proportion of CF isolates, explained by a wide diversity of mutations in mexA and/or mexB. In a broader context, this work contributes to increase our understanding of MexAB-OprM functionality and help delineating how antibiotics interact with MexA and MexB., Competing Interests: P.M.T. is an unpaid advisor to Eumedica. F.V.B. has obtained a private public partnership financial support (TEMOEXPAND program) from the Region Wallonne for the performance of the present work, with Eumedica as industrial partner.

High-level carbapenem resistance is worryingly increasing in clinical isolates and is often attributed to carbapenemase expression. This study aimed to determine the mechanisms leading to high-level meropenem resistance in six carbapenemase-negative Pseudomonas aeruginosa isolated from cystic fibrosis (CF) patients and seven carbapenemase-positive isolates from patients suffering from hospital-acquired pneumonia (HAP). MICs were determined in the absence or presence of l-arginine or glycine-glutamate as competitive substrates for OprD (OccD1) or OpdP (OccD3), respectively, or the efflux pump inhibitor Phe-Arg β-naphthylamide (PAβN). β-Lactamases were screened by phenotypic tests and/or PCR. The oprD gene and its promoter were sequenced; protein expression was evidenced by SDS-PAGE. mexA, mexX, mexC and mexE transcripts were evaluated by real-time and semiquantitative PCR. Meropenem/imipenem MICs were 64-128/16-32 mg/L and 128/128-256 mg/L in CF and HAP isolates, respectively; PAβN reduced meropenem MICs to 4-16 mg/L only and specifically in CF isolates; porin competitors had no effect on MICs. All isolates showed an increase in transcription levels of mexA, mexX and/or mexC and mutations in oprD leading to production of truncated proteins. AmpC-type cephalosporinases were overexpressed in CF isolates and VIM-2 was expressed in HAP isolates. Antibiotic exclusion from bacteria by concomitant efflux and reduced uptake is sufficient to confer high-level resistance to meropenem in isolates overexpressing AmpC-type cephalosporinases. As efflux is preponderant in these isolates, it confers a paradoxical phenotype where meropenem is less active than imipenem. Concomitant susceptibility testing of both carbapenems and rapid elucidation of the most probable resistance mechanisms is thus warranted., (Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

Pseudomonas aeruginosa is a major cause of morbidity and mortality in cystic fibrosis patients. This study compared the antimicrobial susceptibilities of 153 P. aeruginosa isolates from the United Kingdom (UK) (n = 58), Belgium (n = 44), and Germany (n = 51) collected from 118 patients during routine visits over the period from 2006 to 2012. MICs were measured by broth microdilution. Genes encoding extended-spectrum β-lactamases (ESBL), metallo-β-lactamases, and carbapenemases were detected by PCR. Pulsed-field gel electrophoresis and multilocus sequence typing were performed on isolates resistant to ≥3 antibiotic classes among the penicillins/cephalosporins, carbapenems, fluoroquinolones, aminoglycosides, and polymyxins. Based on EUCAST/CLSI breakpoints, susceptibility rates were ≤30%/≤40% (penicillins, ceftazidime, amikacin, and ciprofloxacin), 44 to 48%/48 to 63% (carbapenems), 72%/72% (tobramycin), and 92%/78% (colistin) independent of patient age. Sixty percent of strains were multidrug resistant (MDR; European Centre for Disease Prevention and Control criteria). Genes encoding the most prevalent ESBL (BEL, PER, GES, VEB, CTX-M, TEM, SHV, and OXA), metallo-β-lactamases (VIM, IMP, and NDM), or carbapenemases (OXA-48 and KPC) were not detected. The Liverpool epidemic strain (LES) was prevalent in UK isolates only (75% of MDR isolates). Four MDR sequence type 958 (ST958) isolates were found to be spread over the three countries. The other MDR clones were evidenced in ≤3 isolates and localized in a single country. A new sequence type (ST2254) was discovered in one MDR isolate in Germany. Clonal and nonclonal isolates with different susceptibility profiles were found in 20 patients. Thus, resistance and MDR are highly prevalent in routine isolates from 3 countries, with meropenem, tobramycin, and colistin remaining the most active drugs., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Diabetic nephropathy (DN), the leading cause of end-stage renal failure, is clinically manifested by albuminuria and a progressive decline in glomerular filtration rate. The risk factors and mechanisms that contribute to the development and progression of DN are still incompletely defined. To address the involvement of bradykinin B(2)-receptors (B(2)R) in DN, we used a genome wide approach to study the effects of diabetes on differential renal gene expression profile in wild type and B(2)R knockout (B(2)R(-/-)) mice. Diabetes was induced with streptozotocin and plasma glucose levels and albumin excretion rate (AER) were measured at predetermined times throughout the 23 week study period. Longitudinal analysis of AER indicated that diabetic B(2)R(-/-)D null mice had a significantly decreased AER levels compared to wild type B(2)R(+/+)D mice (P = 0.0005). Results from the global microarray study comparing gene expression profiles among four groups of mice respectively: (B(2)R(+/+)C, B(2)R(+/+)D, B(2)R(-/-)C and B(2)R(-/-)D) highlighted the role of several altered pathological pathways in response to disruption of B(2)R and to the diabetic state that included: endothelial injury, oxidative stress, insulin and lipid metabolism and inflammatory process with a marked alteration in the pro-apoptotic genes. The findings of the present study provide a global genomics view of biomarkers that highlight the mechanisms and putative pathways involved in DN.

Every model species requires its own developmental table. Astyanax mexicanus, a teleost fish comprising both sighted river and blind cave populations, is becoming more and more important in the field of developmental and evolutionary biology. As such, a developmental staging table is increasingly necessary, particularly since comparative analysis of early developmental events is widely employed by researchers. We collected freshly spawned embryos from surface fish and Pachón cavefish populations. Embryos were imaged every 10-12 min during the first day of development, and less frequently in the following days. The results provide an illustrated comparison of selected developmental stages from one cell to hatching of these two populations. The two morphs show an essentially synchronous development regarding major events such as epiboly, neurulation, somitogenesis, heart beating, or hatching. We also present data on particular morphological characters appearing during larval development, such as eye size, yolk regression, swim bladder, and fin development. Some details about the development of F1 Pachón cave×surface hybrids are also given. Comparisons are made with Danio rerio (zebrafish) development.