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ACS Infectious Diseases Jun 2024Antimicrobial peptides (AMPs) are becoming next-generation alternative antibacterial agents because of the rapid increase in resistance in bacteria against existing...
Antimicrobial peptides (AMPs) are becoming next-generation alternative antibacterial agents because of the rapid increase in resistance in bacteria against existing antibiotics, which can also be attributed to the formation of resilient biofilms. However, their widespread use is limited because of their poor absorption, higher dosage requirements, and delayed onset of the bioactivity to elicit a desired response. Here we developed a short AMP that specifically targeted . We conjugated 23R to a statherin-derived peptide (SDP) through rational design; this conjugate binds to FomA, a major porin protein of . The SDP-tagged 23R exhibited rapid and highly specific bactericidal efficacy against . Further, IC values were in the nanomolar range, and they were 100-fold lower than those obtained with unconjugated 23R. In a human gut microbiota model, 0.1 nM SDP-23R achieved 99% clearance of ATCC 25586 without markedly altering resident microbiota. Here we demonstrated that binding-peptide-coupled AMPs show increased killing efficacy and specificity for the target pathogen without affecting the resident microbiota.
PubMed: 38922179
DOI: 10.1021/acsinfecdis.4c00387 -
Pathogens (Basel, Switzerland) Jun 2024is a concerning pathogen, responsible for hospital-associated outbreaks. Multi drug resistant (MDR) strains are especially hard to treat. We conducted whole-genome...
Complete Genome Sequence of a Strain Carrying Novel Variant , Cross-Resistant to Ceftazidime/Avibactam and Cefiderocol, but Susceptible to Carbapenems, Isolated in Italy, 2023.
BACKGROUND
is a concerning pathogen, responsible for hospital-associated outbreaks. Multi drug resistant (MDR) strains are especially hard to treat. We conducted whole-genome sequencing on a MDR strain in order to identify genomic features potentially linked to its phenotype.
METHODS
DNA sequencing was performed on the Illumina iSeq 100 platform. Genome assembly was carried out with SPAdes. The genome was annotated with RASTtk. Typing was performed with MLST and Kaptive. Antibiotic resistance genes were detected with AMRFinderPlus and Abricate, and further verified with BLAST.
RESULTS
The strain exhibited resistance to ceftazidime/avibactam and cefiderocol, but remained susceptible to carbapenems. The strain belonged to sequence type ST101, serotype O1:K17. The analysis of antibiotic resistance genes indicated that the strain carried a novel KPC variant, designated as KPC-203, featuring a EL deletion at amino acid position 166-167, within the Ω-loop, and a nine-amino-acid insertion (LAVYTRAPM) at position 259. Sequence alterations were found in porin genes and . Unlike molecular testing, which was able to detect the KPC-203 variant, all phenotypic carbapenemase detection methods achieved negative results.
CONCLUSIONS
KPC-203, a novel KPC variant, showed a sequence modification in a cephalosporin resistance-associated hotspot. Interestingly, such alterations typically correlate with the restoration of carbapenem susceptibility. We hypothesize that KPC-203 likely led to resistance to ceftazidime/avibactam and cefiderocol, while maintaining susceptibility to carbapenems.
PubMed: 38921804
DOI: 10.3390/pathogens13060507 -
Cell Death & Disease Jun 2024Multiple sevoflurane exposures may damage the developing brain. The neuroprotective function of dexmedetomidine has been widely confirmed in animal experiments and human...
Multiple sevoflurane exposures may damage the developing brain. The neuroprotective function of dexmedetomidine has been widely confirmed in animal experiments and human studies. However, the effect of dexmedetomidine on the glymphatic system has not been clearly studied. We hypothesized that dexmedetomidine could alleviate sevoflurane-induced circulatory dysfunction of the glymphatic system in young mice. Six-day-old C57BL/6 mice were exposed to 3% sevoflurane for 2 h daily, continuously for 3 days. Intraperitoneal injection of either normal saline or dexmedetomidine was administered before every anaesthesia. Meanwhile the circulatory function of glymphatic system was detected by tracer injection at P8 and P32. On P30-P32, behavior tests including open field test, novel object recognition test, and Y-maze test were conducted. Primary astrocyte cultures were established and treated with the PI3K activator 740Y-P, dexmedetomidine, and small interfering RNA (siRNA) to silence ΔFosB. We propose for the first time that multiple exposure to sevoflurane induces circulatory dysfunction of the glymphatic system in young mice. Dexmedetomidine improves the circulatory capacity of the glymphatic system in young mice following repeated exposure to sevoflurane through the PI3K/AKT/ΔFosB/AQP4 signaling pathway, and enhances their long-term learning and working memory abilities.
Topics: Animals; Dexmedetomidine; Sevoflurane; Glymphatic System; Proto-Oncogene Proteins c-akt; Mice; Mice, Inbred C57BL; Phosphatidylinositol 3-Kinases; Aquaporin 4; Signal Transduction; Astrocytes; Male
PubMed: 38918408
DOI: 10.1038/s41419-024-06845-w -
Nature Communications Jun 2024The incidence of beta-lactam resistance among clinical isolates is a major health concern. A key method to study the emergence of antibiotic resistance is adaptive...
The incidence of beta-lactam resistance among clinical isolates is a major health concern. A key method to study the emergence of antibiotic resistance is adaptive laboratory evolution. However, in the case of the beta-lactam ampicillin, bacteria evolved in laboratory settings do not recapitulate clinical-like resistance levels, hindering efforts to identify major evolutionary paths and their dependency on genetic background. Here, we used the Microbial Evolution and Growth Arena (MEGA) plate to select ampicillin-resistant Escherichia coli mutants with varying degrees of resistance. Whole-genome sequencing of resistant isolates revealed that ampicillin resistance was acquired via a combination of single-point mutations and amplification of the gene encoding beta-lactamase AmpC. However, blocking AmpC-mediated resistance revealed latent adaptive pathways: strains deleted for ampC were able to adapt through combinations of changes in genes involved in multidrug resistance encoding efflux pumps, transcriptional regulators, and porins. Our results reveal that combinations of distinct genetic mutations, accessible at large population sizes, can drive high-level resistance to ampicillin even independently of beta-lactamases.
Topics: beta-Lactamases; Ampicillin Resistance; Escherichia coli; Ampicillin; Bacterial Proteins; Anti-Bacterial Agents; Microbial Sensitivity Tests; Whole Genome Sequencing; Evolution, Molecular; Mutation
PubMed: 38918379
DOI: 10.1038/s41467-024-49621-2 -
Microbiology Spectrum Jun 2024The aim of this study was to evaluate the proportion of resistance to a temocillin, tigecycline, ciprofloxacin, and chloramphenicol phenotype called t2c2 that resulted...
The aim of this study was to evaluate the proportion of resistance to a temocillin, tigecycline, ciprofloxacin, and chloramphenicol phenotype called t2c2 that resulted from mutations within the locus among extended-spectrum β-lactamases (ESBL-E) isolated in three intensive care units for 3 years in a French university hospital. Two parallel approaches were performed on all 443 ESBL-E included: (i) the minimal inhibitory concentrations of temocillin, tigecycline, ciprofloxacin, and chloramphenicol were determined and (ii) the genomes obtained from the Illumina sequencing platform were analyzed to determine multilocus sequence types, resistomes, and diversity of several -associated genes including operon. Among the 443 ESBL-E strains included, isolates of ( = 194), ( = 122), and complex () ( = 127) were found. Thirty-one ESBL-E strains (7%), 16 . (13.1%), and 15 (11.8%) presented the t2c2 phenotype in addition to their ESBL profile, whereas no presented these resistances. The t2c2 phenotype was invariably reversible by the addition of Phe-Arg-β-naphthylamide, indicating a role of resistance-nodulation-division pumps in these observations. Mutations associated with the t2c2 phenotype were restricted to RamR, the intergenic region (IR), and AcrR. Mutations in RamR consisted of C- or N-terminal deletions and amino acid substitutions inside its DNA-binding domain or within key sites of protein-substrate interactions. The IR showed nucleotide substitutions involved in the RamR DNA-binding domain. This diversity of sequences suggested that RamR and the IR represent major genetic events for bacterial antimicrobial resistance.IMPORTANCEMorbimortality caused by infectious diseases is very high among patients hospitalized in intensive care units (ICUs). A part of these outcomes can be explained by antibiotic resistance, which delays the appropriate therapy. The transferable antibiotic resistance gene is a well-known mechanism to explain the high rate of multidrug resistance (MDR) bacteria in ICUs. This study describes the prevalence of chromosomal mutations, which led to additional antibiotic resistance among MDR bacteria. More than 12% of and complex strains presented mutations within the locus associated with a dysregulation of an efflux pump called AcrAB-TolC and a porin: OmpF. These dysregulations led to an increase in antibiotic output notably tigecycline, ciprofloxacin, and chloramphenicol associated with a decrease of input for beta-lactam, especially temocillin. Mutations within transcriptional regulators such as locus played a major role in antibiotic resistance dissemination and need to be further explored.
PubMed: 38916360
DOI: 10.1128/spectrum.03548-23 -
Frontiers in Cellular and Infection... 2024Ceftazidime/avibactam (CZA) is indicated against multidrug-resistant , particularly those that are carbapenem resistant. CZA resistance in producing PER, a class A...
INTRODUCTION
Ceftazidime/avibactam (CZA) is indicated against multidrug-resistant , particularly those that are carbapenem resistant. CZA resistance in producing PER, a class A extended-spectrum β-lactamase, has been well documented . However, data regarding clinical isolates are scarce. Our aim was to analyze the contribution of PER to CZA resistance in non-carbapenemase-producing clinical isolates that were ceftazidime and/or carbapenem non-susceptible.
METHODS
Antimicrobial susceptibility was determined through agar dilution and broth microdilution, while gene was screened through PCR. All PER-positive isolates and five PER-negative isolates were analyzed through Whole Genome Sequencing. The mutational resistome associated to CZA resistance was determined through sequence analysis of genes coding for PBPs 1b, 3 and 4, MexAB-OprM regulators MexZ, MexR, NalC and NalD, AmpC regulators AmpD and AmpR, and OprD porin. Loss of gene was induced in a PER-positive isolate by successive passages at 43°C without antibiotics.
RESULTS
Twenty-six of 287 isolates studied (9.1%) were CZA-resistant. Thirteen of 26 CZA-resistant isolates (50%) carried . One isolate carried but was CZA-susceptible. PER-producing isolates had significantly higher MICs for CZA, amikacin, gentamicin, ceftazidime, meropenem and ciprofloxacin than non-PER-producing isolates. All PER-producing isolates were ST309 and their gene was associated to ISCR1, an insertion sequence known to mobilize adjacent DNA. PER-negative isolates were classified as ST41, ST235 (two isolates), ST395 and ST253. PER-negative isolates carried genes for narrow-spectrum β-lactamases and the mutational resistome showed that all isolates had one major alteration in at least one of the genes analyzed. Loss of gene restored susceptibility to CZA, ceftolozane/tazobactam and other β-lactamsin the evolved isolate.
DISCUSSION
PER-3-producing ST309 is a successful multidrug-resistant clone with gene implicated in resistance to CZA and other β-lactams.
Topics: Ceftazidime; Pseudomonas aeruginosa; Azabicyclo Compounds; Microbial Sensitivity Tests; Humans; Drug Combinations; beta-Lactamases; Anti-Bacterial Agents; Pseudomonas Infections; Bacterial Proteins; Drug Resistance, Multiple, Bacterial; Chile; Whole Genome Sequencing; Mutation
PubMed: 38903939
DOI: 10.3389/fcimb.2024.1410834 -
Chemical Science Jun 2024Bacterial membrane porins facilitate the translocation of small molecules while restricting large molecules, and this mechanism remains elusive at the molecular level....
Bacterial membrane porins facilitate the translocation of small molecules while restricting large molecules, and this mechanism remains elusive at the molecular level. Here, we investigate the selective uptake of large cyclic sugars across an unusual passive membrane transporter, CymA, comprising a charged zone and a constricting N terminus segment. Using a combination of electrical recordings, protein mutagenesis and molecular dynamics simulations, we establish substrate translocation across CymA governed by the electrostatic pore properties and conformational dynamics of the constriction segment. Notably, we show that the variation in pH of the environment resulted in reversible modulation of the substrate binding site in the pore, thereby regulating charge-selective transport of cationic, anionic and neutral cyclic sugars. The quantitative kinetics of cyclic sugar translocation across CymA obtained in electrical recordings at different pHs are comparable with molecular dynamics simulations that revealed the transport pathway, energetics and favorable affinity sites in the pore for substrate binding. We further define the molecular basis of cyclic sugar translocation and establish that the constriction segment is flexible and can reside inside or outside the pore, regulating substrate translocation distinct from the ligand-gated transport mechanism. Our study provides novel insights into energy-independent large molecular membrane transport for targeted drug design strategies.
PubMed: 38903220
DOI: 10.1039/d4sc00345d -
BioRxiv : the Preprint Server For... May 2024Treatment of pulmonary disease requires multiple antibiotics including intravenous β-lactams (e.g., imipenem, meropenem). produces a β-lactamase (Bla ) that...
Treatment of pulmonary disease requires multiple antibiotics including intravenous β-lactams (e.g., imipenem, meropenem). produces a β-lactamase (Bla ) that inactivates β-lactam drugs but less efficiently carbapenems. Due to intrinsic and acquired resistance in and poor clinical outcomes, it is critical to understand the development of antibiotic resistance both within the host and in the setting of outbreaks. We compared serial longitudinally collected subsp. isolates from the index case of a CF center outbreak and four outbreak-related strains. We found strikingly high imipenem resistance in the later patient isolates, including the outbreak strain (MIC >512 µg/ml). The phenomenon was recapitulated upon exposure of intracellular bacteria to imipenem. Addition of the β-lactamase inhibitor avibactam abrogated the resistant phenotype. Imipenem resistance was caused by an increase in β-lactamase activity and increased mRNA level. Concurrent increase in transcription of preceding gene indicated upregulation of the entire operon in the resistant strains. Deletion of the porin coincided with the first increase in MIC (from 8 to 32 µg/ml). A frameshift mutation in responsible for the rough colony morphology, and a SNP in ATP-dependent helicase co-occurred with the second increase in MIC (from 32 to 256 µg/ml). Increased Bla expression and enzymatic activity may have been due to altered regulation of the - operon by the mutated HrpA alone, or in combination with other genes described above. This work supports using carbapenem/β-lactamase inhibitor combinations for treating , particularly imipenem resistant strains.
PubMed: 38903073
DOI: 10.1101/2024.05.08.593223 -
Nature Communications Jun 2024Extracellular ATP (eATP) signaling through the P2X7 receptor pathway is widely believed to trigger NLRP3 inflammasome assembly in microglia, potentially contributing to...
Extracellular ATP (eATP) signaling through the P2X7 receptor pathway is widely believed to trigger NLRP3 inflammasome assembly in microglia, potentially contributing to depression. However, the cellular stress responses of microglia to both eATP and stress itself remain largely unexplored. Mitochondria-associated membranes (MAMs) is a platform facilitating calcium transport between the endoplasmic reticulum (ER) and mitochondria, regulating ER stress responses and mitochondrial homeostasis. This study aims to investigate how MAMs influence microglial reaction and their involvement in the development of depression-like symptoms in response to chronic social defeat stress (CSDS). CSDS induced ER stress, MAMs' modifications, mitochondrial damage, and the formation of the IP3R3-GRP75-VDAC1 complex at the ER-mitochondria interface in hippocampal microglia, all concomitant with depression-like behaviors. Additionally, exposing microglia to eATP to mimic CSDS conditions resulted in analogous outcomes. Furthermore, knocking down GRP75 in BV2 cells impeded ER-mitochondria contact, calcium transfer, ER stress, mitochondrial damage, mitochondrial superoxide production, and NLRP3 inflammasome aggregation induced by eATP. In addition, reduced GRP75 expression in microglia of Cx3cr1Hspa9 mice lead to reduce depressive behaviors, decreased NLRP3 inflammasome aggregation, and fewer ER-mitochondria contacts in hippocampal microglia during CSDS. Here, we show the role of MAMs, particularly the formation of a tripartite complex involving IP3R3, GRP75, and VDAC1 within MAMs, in facilitating communication between the ER and mitochondria in microglia, thereby contributing to the development of depression-like phenotypes in male mice.
Topics: Animals; Mitochondria; Depression; Microglia; Mice; Social Defeat; Male; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Stress, Psychological; Mice, Inbred C57BL; NLR Family, Pyrin Domain-Containing 3 Protein; Voltage-Dependent Anion Channel 1; Hippocampus; Adenosine Triphosphate; Inflammasomes; Inositol 1,4,5-Trisphosphate Receptors; Calcium; Membrane Proteins; Behavior, Animal; Mitochondria Associated Membranes; HSP70 Heat-Shock Proteins
PubMed: 38890305
DOI: 10.1038/s41467-024-49597-z -
Frontiers in Immunology 2024Aquaporin-4 antibodies (AQP4-Abs) are a diagnostic marker for patients with a demyelinating disease called neuromyelitis optica spectrum disorder (NMOSD). Anti-Argonaute...
Aquaporin-4 antibodies (AQP4-Abs) are a diagnostic marker for patients with a demyelinating disease called neuromyelitis optica spectrum disorder (NMOSD). Anti-Argonaute antibodies (AGO-Abs) present as potential biomarkers of the overlap syndrome between NMOSD and other autoimmune diseases. In this paper, we present the case of an adult woman with numbness, tingling, and burning sensations in her arms and subsequent bilateral internuclear ophthalmoplegia. Brain-cervical-thoracic magnetic resonance imaging (MRI) showed T2 hyperintensities in the dorsal brainstem and around the midbrain aqueduct and longitudinally transverse myelitis with homogeneous enhancement on gadolinium-enhanced MRI. The contemporaneous detection of AQP4- and AGO-Abs led to a definite diagnosis of overlap syndrome of NMOSD with AGO-Abs. The patient was treated with immunosuppressive agents, including corticosteroids and immunoglobulins, and achieved remission. This case highlights a novel phenotype of NMOSD with AGO-Abs overlap syndrome, which presents with relapsing brainstem syndrome and longitudinally extensive myelitis with acute severe neurological involvement. The promising prognosis of the disease could serve as a distinct clinical profile. Broad screening for antibodies against central nervous system autoimmune antigens is recommended in suspected patients with limited or atypical clinical manifestations.
Topics: Humans; Neuromyelitis Optica; Female; Autoantibodies; Aquaporin 4; Adult; Biomarkers; Magnetic Resonance Imaging; Middle Aged; Immunosuppressive Agents
PubMed: 38887290
DOI: 10.3389/fimmu.2024.1366531