Crystal structure of the Pseudomonas aeruginosa BEL-1 extended-spectrum β-lactamase and its complexes with moxalactam and imipenem

BEL-1 is an acquired class A extended-spectrum β-lactamase (ESBL) found in Pseudomonas aeruginosa clinical isolates from Belgium which is divergent from other ESBLs (maximum identity of 54% with GES-type enzymes). This enzyme is efficiently inhibited by clavulanate, imipenem, and moxalactam. Crystals of BEL-1 were obtained at pH 5.6, and the structure of native BEL-1 was determined from orthorhombic and monoclinic crystal forms at 1.60-Å and 1.48-Å resolution, respectively. By soaking native BEL-1 crystals, complexes with imipenem (monoclinic form, 1.79-Å resolution) and moxalactam (orthorhombic form, 1.85-Å resolution) were also obtained. In the acyl-enzyme complexes, imipenem and moxalactam differ by the position of the α- substituent and of the carbonyl oxygen (in or out of the oxyanion hole). More surprisingly, the Ω-loop, which includes the catalytically relevant residue Glu166, was found in different conformations in the various subunits, resulting in the Glu166 side chain being rotated out of the active site or even in displacement of its Cα atom up to approximately 10 Å. A BEL-1 variant showing the single Leu162Phe substitution (BEL- 2) confers a higher level of resistance to CAZ, CTX, and FEP and shows significantly lower Km values than BEL-1, especially with oxyiminocephalosporins. BEL-1 Leu162 is located at the beginning of the Ω-loop and is surrounded by Phe72, Leu139, and Leu148 (contact distances, 3.5 to 3.9 Å). This small hydrophobic cavity could not reasonably accommodate the bulkier Phe162 found in BEL-2 without altering neighboring residues or the Ω-loop itself, thus likely causing an important alteration of the enzyme kinetic properties

Expanding the Repertoire of Carbapenem-Hydrolyzing Metallo-ß-Lactamases by Functional Metagenomic Analysis of Soil Microbiota

Carbapenemases are bacterial enzymes that hydrolyze carbapenems, a group of last-resort β-lactam antibiotics used for treatment of severe bacterial infections. They belong to three β-lactamase classes based amino acid sequence (A, B, and D). The aim of this study was to elucidate occurrence, diversity and functionality of carbapenemase-encoding genes in soil microbiota by functional metagenomics. Ten plasmid libraries were generated by cloning metagenomic DNA from agricultural (n = 6) and grassland (n = 4) soil into Escherichia coli. The libraries were cultured on amoxicillin-containing agar and up to 100 colonies per library were screened for carbapenemase production by CarbaNP test. Presumptive carbapenemases were characterized with regard to DNA sequence, minimum inhibitory concentration (MIC) of β-lactams, and imipenem hydrolysis. Nine distinct class B carbapenemases, also known as metallo-beta-lactamases (MBLs), were identified in six soil samples, including two subclass B1 (GRD23-1 and SPN79-1) and seven subclass B3 (CRD3-1, PEDO-1, GRD33-1, ESP-2, ALG6-1, ALG11-1, and DHT2-1). Except PEDO-1 and ESP-2, these enzymes were distantly related to any previously described MBLs (33 to 59% identity). RAIphy analysis indicated that six enzymes (CRD3-1, GRD23-1, DHT2-1, SPN79-1, ALG6-1, and ALG11-1) originated from Proteobacteria, two (PEDO-1 and ESP-2) from Bacteroidetes and one (GRD33-1) from Gemmatimonadetes. All MBLs detected in soil microbiota were functional when expressed in E. coli, resulting in detectable imipenem-hydrolyzing activity and significantly increased MICs of clinically relevant β-lactams. Interestingly, the MBLs yielded by functional metagenomics generally differed from those detected in the same soil samples by antibiotic selective culture, showing that the two approaches targeted different subpopulations in soil microbiota. © 2016 Gudeta, Bortolaia, Pollini, Docquier, Rossolini, Amos, Wellington and Guardabassi.Grant HEALTH-F3-2011-282004(EvoTAR

Discovering NDM-1 inhibitors using molecular substructure embeddings representations

NDM-1 (New-Delhi-Metallo-beta-lactamase-1) is an enzyme developed by bacteria that is implicated in bacteria resistance to almost all known antibiotics. In this study, we deliver a new, curated NDM-1 bioactivities database, along with a set of unifying rules for managing different activity properties and inconsistencies. We define the activity classification problem in terms of Multiple Instance Learning, employing embeddings corresponding to molecular substructures and present an ensemble ranking and classification framework, relaying on a k-fold Cross Validation method employing a per fold hyper-parameter optimization procedure, showing promising generalization ability. The MIL paradigm displayed an improvement up to 45.7 %, in terms of Balanced Accuracy, in comparison to the classical Machine Learning paradigm. Moreover, we investigate different compact molecular representations, based on atomic or bi-atomic substructures. Finally, we scanned the Drugbank for strongly active compounds and we present the top-15 ranked compounds

Optimization of Pyrazole Compounds as Antibiotic Adjuvants Active against Colistin- and Carbapenem-Resistant Acinetobacter baumannii

The diffusion of antibiotic-resistant, Gram-negative, opportunistic pathogens, an increasingly important global public health issue, causes a significant socioeconomic burden. Acinetobacter baumannii isolates, despite causing a lower number of infections than Enterobacterales, often show multidrug-resistant phenotypes. Carbapenem resistance is also rather common, prompting the WHO to include carbapenem-resistant A. baumannii as a "critical priority" for the discovery and development of new antibacterial agents. In a previous work, we identified several series of compounds showing either direct-acting or synergistic activity against relevant Gram-negative species, including A. baumannii. Among these, two pyrazole compounds, despite being devoid of any direct-acting activity, showed remarkable synergistic activity in the presence of a subinhibitory concentration of colistin on K. pneumoniae and A. baumannii and served as a starting point for the synthesis of new analogues. In this work, a new series of 47 pyrazole compounds was synthesized. Some compounds showed significant direct-acting antibacterial activity on Gram-positive organisms. Furthermore, an evaluation of their activity as potential antibiotic adjuvants allowed for the identification of two highly active compounds on MDR Acinetobacter baumannii, including colistin-resistant isolates. This work confirms the interest in pyrazole amides as a starting point for the optimization of synergistic antibacterial compounds active on antibiotic-resistant, Gram-negative pathogens

Resistencia a carbapenemes en aislamientos de Pseudomonas aeruginosa: un ejemplo de interacción entre distintos mecanismos

Objetivo. Identificar la proteína de membrana externa ausente en los aislamientos resistentes y determinar tanto las causas de su ausencia en la membrana, como la presencia de otros mecanismos de resistencia a carbapenemes en aislamientos clínicos de Pseudomonas aeruginosa. Métodos. Se estudió un brote de 20 aislamientos de P. aeruginosa previamente caracterizados como productores de la metalobetalactamasa IMP-13. Estos aislamientos presentaron igual expresión de la enzima IMP-13, pero solo cinco de ellos fueron resistentes a carbapenemes. En esos cinco aislamientos resistentes se confirmó la ausencia de una proteína de membrana externa. Se secuenciaron oprD y ampC; se identificaron las proteínas de membrana externa por desorción/ionización láser asistida por matriz/espectometría de masa tiempo de vuelo (MALDI-TOF); se determinó el nivel de expresión de OprD, de AmpC y de los sistemas de eflujo tipo Mex, por reacción en cadena de polimerasa en tiempo real, y por último, se determinó la contribución del déficit de OprD a la resistencia a carbapenemes. Resultados. La proteína de la membrana externa ausente en el grupo R (resistentes a ambos carbapenemes) fue identificada como OprD-TS, pero no se observaron variaciones en su expresión. El gen oprD presentó mutaciones en los cinco aislamientos resistentes. Se observó la misma producción de la enzima tipo AmpC PDC-5 y del sistema de eflujo Mex AB-OprM entre los aislamientos sensibles y resistentes a carbapenemes. Se analizó cómo la presencia conjunta de IMP-13 y el déficit de OprD contribuyen al aumento de la resistencia. Conclusiones. Distintos mecanismos contribuyen a la resistencia de aislamientos productores de IMP-13 a carbapenemes. La posibilidad de no detectar estos aislamientos productores de IMP-13 representa un riesgo latente de selección de mutantes con mecanismos de resistencia que se suman para aumentar la resistencia a carbapenemes.Fil: Santella, Gisela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Pollini, Simona. Università degli Studi di Siena; ItaliaFil: Docquier, Jean Denis. Università degli Studi di Siena; ItaliaFil: Almuzara, Marisa. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "Eva Perón"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gutkind, Gabriel Osvaldo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rossolini, Gian Maria. Università degli Studi di Siena; ItaliaFil: Radice, Marcela Alejandra. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Understanding transmission and control of Cystic Echinococcosis and other taeniid infections in the Falkland Islands

Cystic echinococcosis, caused by the larval form of the cestode parasite Echinococcus granulosus, has been identified as an important public health risk in the Falkland Islands since the early 1940s. This prompted the instigation of an intensive control scheme in the mid-1960s, comprised of regular dosing of domestic dogs with the anthelmintic praziquantel and education of local people about safe disposal of potentially infected offal. This scheme has remained in place to the current day and is generally considered to be a successful programme– resulting in a reduction in the prevalence of infection in sheep has reduced from >50% in the 1950s to less than 1 % now and there has not been a case of human hydatid disease for more than 20 years. However, concerns remain that hydatid cysts are still identified in a small number of sheep at slaughter (0.004% in 2017) and occurring every year subsequently suggesting transmission is still occurring. This is also supported by the observation that sheep continue to be infected at higher levels with the (non-zoonotic) cestode Taenia hydatigena, also transmitted by dogs. In 2010, all dogs on the Falkland Islands were tested by Copro-PCR, resulting in eight dogs (1.4%) testing positive. The dog population was tested again in 2012, where there were no cases but when tested in 2014 by Coproantigen testing, six (1.04%) were positive for E. granulosus coproantigens. This project used questionnaires, coproantigen and coproPCR analysis, abattoir data surveillance, DNA sequencing, environmental sample analysis and mathematical modelling to study Echinococcus granulosus and other taeniids endemic in the Falklands and investigate how their continued transmission can occur in the face of the prolonged intensive control programme. A questionnaire survey identified possible methods of disposal of offal that in a previous study, were associated with canine coproantigen positivity. The entire dog population was analysed via coproantigen techniques in 2018, and four (0.68%) dogs were coproantigen positive, though none of these were confirmed by PCR. From 2018 to 2020, five cases of CE were identified in sheep at the Sand Bay abattoir in the Falklands (0.01%), with one of the cases coming from a positive farm in 2018. There were two cases from farms with positive dogs in 2010 and one from a farm with a positive dog in 2014. To investigate environmental contamination on farms and potentially identify historical dog infections, soil samples taken from kennel sites were analysed for the presence of coproantigens, with five farms having positive results, one farm matching with a positive dog in 2018. To identify key processes fuelling the transmission of E. granulosus in the Falklands, a mechanically informed compartmental model was created, estimating the basic reproduction number (R0) for the parasite, and identifying scenarios where this estimate increased above one suggesting continued transmission could occur. Seven scenarios where lapses in control measures could result in the R0 estimate increasing above one and continued transmission of E. granulosus could occur. The results of this project show clear evidence of dogs still being involved in the transmission of taeniid parasites in the Falklands, with key areas of the eradication programme such as the inadequate disposal of offal and dogs gaining access to offal allowing the transmission cycle to be completed and transmission of E. granulosus and other taeniids to occur. Rectifying these lapses in control measures and focussing control and surveillance to a more localised control approach will help strengthen the control programme and move the Falklands closer towards the complete eradication of Cystic Echinococcosis

Chemical Optimization of Selective Pseudomonas aeruginosa LasB Elastase Inhibitors and Their Impact on LasB-Mediated Activation of IL-1β in Cellular and Animal Infection Models

LasB elastase is a broad-spectrum exoprotease and a key virulence factor of Pseudomonas aeruginosa, a major pathogen causing lung damage and inflammation in acute and chronic respiratory infections. Here, we describe the chemical optimization of specific LasB inhibitors with druglike properties and investigate their impact in cellular and animal models of P. aeruginosa infection. Competitive inhibition of LasB was demonstrated through structural and kinetic studies. In vitro LasB inhibition was confirmed with respect to several host target proteins, namely, elastin, IgG, and pro-IL-1 beta. Furthermore, inhibition of LasBmediated IL-1 beta activation was demonstrated in macrophage and mouse lung infection models. In mice, intravenous administration of inhibitors also resulted in reduced bacterial numbers at 24 h. These highly potent, selective, and soluble LasB inhibitors constitute valuable tools to study the proinflammatory impact of LasB in P. aeruginosa infections and, most importantly, show clear potential for the clinical development of a novel therapy for life-threatening respiratory infections caused by this opportunistic pathogen

Biological Characterization and in Vivo Assessment of the Activity of a New Synthetic Macrocyclic Antifungal Compound

We recently identified a novel family of macrocyclic amidinoureas showing potent antifungal activity against Candida spp. In this study, we demonstrate the fungicidal effect of these compounds as well as their killing activity in a dose-dependent manner. Transcriptional analysis data indicate that our molecules induce a significant change in the transcriptome involving ATP binding cassette (ABC) transporter genes. Notably, experiments against Candida albicans mutants lacking those genes showed resistance to the compound, suggesting the involvement of ABC transporters in the uptake or intracellular accumulation of the molecule. To probe the mode of action, we performed fluorescence microscopy experiments on fungal cells treated with an ad-hoc synthesized fluorescent derivative. Fluorescence microscopy images confirm the ability of the compound to cross the membrane and show a consistent accumulation within the cytoplasm. Finally, we provide data supporting the in vivo efficacy in a systemic infection murine model setup with a drug-resistant strain of C. albicans

SAR Studies Leading to the Identification of a Novel Series of Metallo-β-lactamase Inhibitors for the Treatment of Carbapenem-Resistant Enterobacteriaceae Infections That Display Efficacy in an Animal Infection Model

The clinical effectiveness of carbapenem antibiotics such as meropenem is becoming increasingly compromised by the spread of both metallo-β-lactamase (MBL) and serine-β-lactamase (SBL) enzymes on mobile genetic elements, stimulating research to find new β-lactamase inhibitors to be used in conjunction with carbapenems and other β-lactam antibiotics. Herein, we describe our initial exploration of a novel chemical series of metallo-β-lactamase inhibitors, from concept to efficacy, in a survival model using an advanced tool compound (ANT431) in conjunction with meropenem