Molecular mechanisms driving the in vivo development of OXA-10-mediated resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of XDR Pseudomonas aeruginosa infections

2020 ◽  
Vol 76 (1) ◽  
pp. 91-100
Author(s):  
Jorge Arca-Suárez ◽  
Cristina Lasarte-Monterrubio ◽  
Bruno-Kotska Rodiño-Janeiro ◽  
Gabriel Cabot ◽  
Juan Carlos Vázquez-Ucha ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Molecular Mechanisms ◽  
Genomic Analysis ◽  
Resistance Mechanisms ◽  
Comparative Genomic ◽  
Development Of Resistance ◽  
Structural Impact ◽  
Genetic Context

Abstract Background The development of resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of Pseudomonas aeruginosa infections is concerning. Objectives Characterization of the mechanisms leading to the development of OXA-10-mediated resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of XDR P. aeruginosa infections. Methods Four paired ceftolozane/tazobactam- and ceftazidime/avibactam-susceptible/resistant isolates were evaluated. MICs were determined by broth microdilution. STs, resistance mechanisms and genetic context of β-lactamases were determined by genotypic methods, including WGS. The OXA-10 variants were cloned in PAO1 to assess their impact on resistance. Models for the OXA-10 derivatives were constructed to evaluate the structural impact of the amino acid changes. Results The same XDR ST253 P. aeruginosa clone was detected in all four cases evaluated. All initial isolates showed OprD deficiency, produced an OXA-10 enzyme and were susceptible to ceftazidime, ceftolozane/tazobactam, ceftazidime/avibactam and colistin. During treatment, the isolates developed resistance to all cephalosporins. Comparative genomic analysis revealed that the evolved resistant isolates had acquired mutations in the OXA-10 enzyme: OXA-14 (Gly157Asp), OXA-794 (Trp154Cys), OXA-795 (ΔPhe153-Trp154) and OXA-824 (Asn143Lys). PAO1 transformants producing the evolved OXA-10 derivatives showed enhanced ceftolozane/tazobactam and ceftazidime/avibactam resistance but decreased meropenem MICs in a PAO1 background. Imipenem/relebactam retained activity against all strains. Homology models revealed important changes in regions adjacent to the active site of the OXA-10 enzyme. The blaOXA-10 gene was plasmid borne and acquired due to transposition of Tn6746 in the pHUPM plasmid scaffold. Conclusions Modification of OXA-10 is a mechanism involved in the in vivo acquisition of resistance to cephalosporin/β-lactamase inhibitor combinations in P. aeruginosa.

scholarly journals Molecular and biochemical insights into the in vivo evolution of AmpC-mediated resistance to ceftolozane/tazobactam during treatment of an MDR Pseudomonas aeruginosa infection

2020 ◽  
Vol 75 (11) ◽  
pp. 3209-3217 ◽  
Author(s):  
Jorge Arca-Suárez ◽  
Juan Carlos Vázquez-Ucha ◽  
Pablo Arturo Fraile-Ribot ◽  
Emilio Lence ◽  
Gabriel Cabot ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Conformational Changes ◽  
Catalytic Efficiency ◽  
The Novel ◽  
Development Of Resistance ◽  
New Variant ◽  
Ic50 Values ◽  
Dynamics Simulations

Abstract Background Pseudomonas aeruginosa may develop resistance to novel cephalosporin/β-lactamase inhibitor combinations during therapy through the acquisition of structural mutations in AmpC. Objectives To describe the molecular and biochemical mechanisms involved in the development of resistance to ceftolozane/tazobactam in vivo through the selection and overproduction of a novel AmpC variant, designated PDC-315. Methods Paired susceptible/resistant isolates obtained before and during ceftolozane/tazobactam treatment were evaluated. MICs were determined by broth microdilution. Mutational changes were investigated through WGS. Characterization of the novel PDC-315 variant was performed through genotypic and biochemical studies. The effects at the molecular level of the Asp245Asn change were analysed by molecular dynamics simulations using Amber. Results WGS identified mutations leading to modification (Asp245Asn) and overproduction of AmpC. Susceptibility testing revealed that PAOΔC producing PDC-315 displayed increased MICs of ceftolozane/tazobactam, decreased MICs of piperacillin/tazobactam and imipenem and similar susceptibility to ceftazidime/avibactam compared with WT PDCs. The catalytic efficiency of PDC-315 for ceftolozane was 10-fold higher in relation to the WT PDCs, but 3.5- and 5-fold lower for piperacillin and imipenem. IC50 values indicated strong inhibition of PDC-315 by avibactam, but resistance to cloxacillin inhibition. Analysis at the atomic level explained that the particular behaviour of PDC-315 is linked to conformational changes in the H10 helix that favour the approximation of key catalytic residues to the active site. Conclusions We deciphered the precise mechanisms that led to the in vivo emergence of resistance to ceftolozane/tazobactam in P. aeruginosa through the selection of the novel PDC-315 enzyme. The characterization of this new variant expands our knowledge about AmpC-mediated resistance to cephalosporin/β-lactamase inhibitors in P. aeruginosa.

scholarly journals Hemolysis in the spleen drives erythrocyte turnover

Blood ◽  
2020 ◽  
Author(s):  
Thomas robert leon Klei ◽  
Jill Jasmine Dalimot ◽  
Benjamin Nota ◽  
Martijn Veldthuis ◽  
Erik Mul ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Matrix Proteins ◽  
Human Spleen ◽  
Erythrocyte Adhesion ◽  
Subsequent Degradation ◽  
Macrophage Subpopulations ◽  
Senescent Erythrocytes ◽  
Red Pulp

Red pulp macrophages of the spleen mediate turnover of billions of senescent erythrocytes per day. However, the molecular mechanisms involved in sequestration of senescent erythrocytes, their recognition and their subsequent degradation by red pulp macrophages remain unclear. In this study we provide evidence that the splenic environment is of substantial importance in facilitating erythrocyte turnover through induction of hemolysis. Upon isolating human spleen red pulp macrophages we noted a substantial lack of macrophages that were in the process of phagocytosing intact erythrocytes. Detailed characterization of erythrocyte and macrophage subpopulations from human spleen tissue led to the identification of erythrocytes that are devoid of hemoglobin, so-called erythrocyte ghosts. By in vivo imaging and transfusion experiments we further confirmed that senescent erythrocytes that are retained in the spleen are subject to hemolysis. Additionally, we show that erythrocyte adhesion molecules, which are specifically activated on aged erythrocytes, cause senescent erythrocytes to interact with extracellular matrix proteins that are exposed within the splenic architecture. Such adhesion molecule-driven retention of senescent erythrocytes, under low shear conditions, was found to result in steady shrinkage of the cell and ultimately resulted in hemolysis. In contrast to intact senescent erythrocytes, the remnant erythrocyte ghost shells were prone to recognition and breakdown by red pulp macrophages. These data identify hemolysis as a key event in the turnover of senescent erythrocytes, which alters our current understanding of how erythrocyte degradation is regulated.

scholarly journals Development of Resistance in Wild-Type and Hypermutable Pseudomonas aeruginosa Strains Exposed to Clinical Pharmacokinetic Profiles of Meropenem and Ceftazidime Simulated In Vitro

2007 ◽  
Vol 51 (10) ◽  
pp. 3642-3649 ◽  
Author(s):  
Beate Henrichfreise ◽  
Irith Wiegand ◽  
Ingeborg Luhmer-Becker ◽  
Bernd Wiedemann
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Parent Strain ◽  
Resistance Mechanisms ◽  
In Vitro Models ◽  
Wild Type ◽  
Resistance Development ◽  
Development Of Resistance ◽  
Pharmacokinetic Profiles ◽  
Mutant Prevention Concentration

ABSTRACT In this study we investigated the interplay of antibiotic pharmacokinetic profiles and the development of mutation-mediated resistance in wild-type and hypermutable Pseudomonas aeruginosa strains. We used in vitro models simulating profiles of the commonly used therapeutic drugs meropenem and ceftazidime, two agents with high levels of antipseudomonal activity said to have different potentials for stimulating resistance development. During ceftazidime treatment of the wild-type strain (PAO1), fully resistant mutants overproducing AmpC were selected rapidly and they completely replaced wild-type cells in the population. During treatment with meropenem, mutants of PAO1 were not selected as rapidly and showed only intermediate resistance due to the loss of OprD. These mutants also replaced the parent strain in the population. During the treatment of the mutator P. aeruginosa strain with meropenem, the slowly selected mutants did not accumulate several resistance mechanisms but only lost OprD and did not completely replace the parent strain in the population. Our results indicate that the commonly used dosing regimens for meropenem and ceftazidime cannot avoid the selection of mutants of wild-type and hypermutable P. aeruginosa strains. For the treatment outcome, including the prevention of resistance development, it would be beneficial for the antibiotic concentration to remain above the mutant prevention concentration for a longer period of time than it does in present regimens.

scholarly journals Interactions between the Prophage 919TP and Its Vibrio cholerae Host: Implications of gmd Mutation for Phage Resistance, Cell Auto-Aggregation, and Motility

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2342
Author(s):  
Na Li ◽  
Yigang Zeng ◽  
Bijie Hu ◽  
Tongyu Zhu ◽  
Sine Lo Svenningsen ◽  
...  
Keyword(s):  
Bacterial Pathogen ◽  
Genomic Analysis ◽  
Resistant Mutant ◽  
Resistance Mechanisms ◽  
Phage Resistance ◽  
Comparative Genomic ◽  
Lytic Phage ◽  
O Antigen ◽  
Trade Offs ◽  
Base Pair Deletion

Prophage 919TP is widely distributed among Vibrio cholera and is induced to produce free φ919TP phage particles. However, the interactions between prophage φ919TP, the induced phage particle, and its host remain unknown. In particular, phage resistance mechanisms and potential fitness trade-offs, resulting from phage resistance, are unresolved. In this study, we examined a prophage 919TP-deleted variant of V. cholerae and its interaction with a modified lytic variant of the induced prophage (φ919TP cI-). Specifically, the phage-resistant mutant was isolated by challenging a prophage-deleted variant with lytic phage φ919TP cI-. Further, the comparative genomic analysis of wild-type and φ919TP cI--resistant mutant predicted that phage φ919TP cI- selects for phage-resistant mutants harboring a mutation in key steps of lipopolysaccharide (LPS) O-antigen biosynthesis, causing a single-base-pair deletion in gene gmd. Our study showed that the gmd-mediated O-antigen defect can cause pleiotropic phenotypes, e.g., cell autoaggregation and reduced swarming motility, emphasizing the role of phage-driven diversification in V. cholerae. The developed approach assists in the identification of genetic determinants of host specificity and is used to explore the molecular mechanism underlying phage-host interactions. Our findings contribute to the understanding of prophage-facilitated horizontal gene transfer and emphasize the potential for developing new strategies to optimize the use of phages in bacterial pathogen control.

scholarly journals AAV9-mediated delivery of miR-23a reduces disease severity in Smn2B/−SMA model mice

2019 ◽  
Vol 28 (19) ◽  
pp. 3199-3210 ◽  
Author(s):  
Kevin A Kaifer ◽  
Eric Villalón ◽  
Benjamin S O'Brien ◽  
Samantha L Sison ◽  
Caley E Smith ◽  
...  
Keyword(s):  
Motor Neuron ◽  
Motor Neurons ◽  
Molecular Mechanisms ◽  
Viral Vector ◽  
Muscular Atrophy ◽  
Induced Pluripotent Stem Cell ◽  
Survival Motor Neuron ◽  
Virus Serotype

Abstract Spinal muscular atrophy (SMA) is a neuromuscular disease caused by deletions or mutations in survival motor neuron 1 (SMN1). The molecular mechanisms underlying motor neuron degeneration in SMA remain elusive, as global cellular dysfunction obscures the identification and characterization of disease-relevant pathways and potential therapeutic targets. Recent reports have implicated microRNA (miRNA) dysregulation as a potential contributor to the pathological mechanism in SMA. To characterize miRNAs that are differentially regulated in SMA, we profiled miRNA levels in SMA induced pluripotent stem cell (iPSC)-derived motor neurons. From this array, miR-23a downregulation was identified selectively in SMA motor neurons, consistent with previous reports where miR-23a functioned in neuroprotective and muscle atrophy-antagonizing roles. Reintroduction of miR-23a expression in SMA patient iPSC-derived motor neurons protected against degeneration, suggesting a potential miR-23a-specific disease-modifying effect. To assess this activity in vivo, miR-23a was expressed using a self-complementary adeno-associated virus serotype 9 (scAAV9) viral vector in the Smn2B/− SMA mouse model. scAAV9-miR-23a significantly reduced the pathology in SMA mice, including increased motor neuron size, reduced neuromuscular junction pathology, increased muscle fiber area, and extended survival. These experiments demonstrate that miR-23a is a novel protective modifier of SMA, warranting further characterization of miRNA dysfunction in SMA.

scholarly journals Functional and Genomic Characterization of Ligilactobacillus salivarius TUCO-L2 Isolated From Lama glama Milk: A Promising Immunobiotic Strain to Combat Infections

2020 ◽  
Vol 11 ◽  
Author(s):  
Sandra Quilodrán-Vega ◽  
Leonardo Albarracin ◽  
Flavia Mansilla ◽  
Lorena Arce ◽  
Binghui Zhou ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Functional Characterization ◽  
Comparative Genomic ◽  
Cytokines And Chemokines ◽  
Isolation And Characterization ◽  
Intestinal Pathogens ◽  
Lama Glama

Potential probiotic or immunobiotic effects of lactic acid bacteria (LAB) isolated from the milk of the South American camelid llama (Lama glama) have not been reported in published studies. The aim of the present work was to isolate beneficial LAB from llama milk that can be used as potential probiotics active against bacterial pathogens. LAB strains were isolated from llama milk samples. In vitro functional characterization of the strains was performed by evaluating the resistance against gastrointestinal conditions and inhibition of the pathogen growth. Additionally, the adhesive and immunomodulatory properties of the strains were assessed. The functional studies were complemented with a comparative genomic evaluation and in vivo studies in mice. Ligilactobacillus salivarius TUCO-L2 showed enhanced probiotic/immunobiotic potential compared to that of other tested strains. The TUCO-L2 strain was resistant to pH and high bile salt concentrations and demonstrated antimicrobial activity against Gram-negative intestinal pathogens and adhesion to mucins and epithelial cells. L. salivarius TUCO-L2 modulated the innate immune response triggered by Toll-like receptor (TLR)-4 activation in intestinal epithelial cells. This effect involved differential regulation of the expression of inflammatory cytokines and chemokines mediated by the modulation of the negative regulators of the TLR signaling pathway. Moreover, the TUCO-L2 strain enhanced the resistance of mice to Salmonella infection. This is the first report on the isolation and characterization of a potential probiotic/immunobiotic strain from llama milk. The in vitro, in vivo, and in silico investigation performed in this study reveals several research directions that are needed to characterize the TUCO-L2 strain in detail to position this strain as a probiotic or immunobiotic that can be used against infections in humans or animals, including llama.

scholarly journals Advanced Resistance Studies Identify Two Discrete Mechanisms in Staphylococcus aureus to Overcome Antibacterial Compounds that Target Biotin Protein Ligase

Antibiotics ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 165 ◽  
Author(s):  
Andrew J. Hayes ◽  
Jiulia Satiaputra ◽  
Louise M. Sternicki ◽  
Ashleigh S. Paparella ◽  
Zikai Feng ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Transcriptional Repression ◽  
Molecular Mechanisms ◽  
Resistance Mechanism ◽  
Resistance Mechanisms ◽  
Resistance Rate ◽  
Biotin Protein Ligase ◽  
Essential Enzyme

Biotin protein ligase (BPL) inhibitors are a novel class of antibacterial that target clinically important methicillin-resistant Staphylococcus aureus (S. aureus). In S. aureus, BPL is a bifunctional protein responsible for enzymatic biotinylation of two biotin-dependent enzymes, as well as serving as a transcriptional repressor that controls biotin synthesis and import. In this report, we investigate the mechanisms of action and resistance for a potent anti-BPL, an antibacterial compound, biotinyl-acylsulfamide adenosine (BASA). We show that BASA acts by both inhibiting the enzymatic activity of BPL in vitro, as well as functioning as a transcription co-repressor. A low spontaneous resistance rate was measured for the compound (<10−9) and whole-genome sequencing of strains evolved during serial passaging in the presence of BASA identified two discrete resistance mechanisms. In the first, deletion of the biotin-dependent enzyme pyruvate carboxylase is proposed to prioritize the utilization of bioavailable biotin for the essential enzyme acetyl-CoA carboxylase. In the second, a D200E missense mutation in BPL reduced DNA binding in vitro and transcriptional repression in vivo. We propose that this second resistance mechanism promotes bioavailability of biotin by derepressing its synthesis and import, such that free biotin may outcompete the inhibitor for binding BPL. This study provides new insights into the molecular mechanisms governing antibacterial activity and resistance of BPL inhibitors in S. aureus.

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