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Porin deficiency or plasmid copy number increase mediated carbapenem-resistant resistance evolution.Emerging Microbes & Infections Dec 2024This study investigated resistance evolution mechanisms of conjugated plasmids and bacterial hosts under different concentrations of antibiotic pressure. Ancestral...
This study investigated resistance evolution mechanisms of conjugated plasmids and bacterial hosts under different concentrations of antibiotic pressure. Ancestral strain ECNX52 was constructed by introducing the -carrying IncX3 plasmid into C600, and was subjected to laboratory evolution under different concentrations of meropenem pressure. Minimal inhibitory concentrations and conjugation frequency were determined. Fitness of these strains was assessed. Whole genome sequencing and transcriptional changes were performed. Ancestral host or plasmids were recombined with evolved hosts or plasmids to verify plasmid or host factors in resistance evolution. Role of the mutation on plasmid copy number was determined. Two out of the four clones (EM2N1 and EM2N3) exhibited four-fold increase in MIC when exposed to a continuous pressure of 2 μg/mL MEM (1/32 MIC), by down regulating expression of outer membrane protein . Besides, all four clones displayed four-fold increase in MIC and higher conjugation frequency when subjected to a continuous pressure of 4 μg/mL MEM (1/16 MIC), attributing to increasing plasmid copy number generated by D140Y (GAT→TAT) mutation. Bacterial hosts and conjugative plasmids can undergo resistance evolution under certain concentrations of antimicrobial pressure by reducing the expression of outer membrane proteins or increasing plasmid copy numbers.
Topics: Escherichia coli; Plasmids; Microbial Sensitivity Tests; Anti-Bacterial Agents; Porins; Escherichia coli Proteins; Carbapenems; Meropenem; Mutation; Evolution, Molecular; Conjugation, Genetic; Carbapenem-Resistant Enterobacteriaceae; Whole Genome Sequencing; Gene Dosage; beta-Lactamases
PubMed: 38712634
DOI: 10.1080/22221751.2024.2352432 -
Neurology May 2024Knowledge of the evolution of CNS demyelinating lesions within attacks could assist diagnosis. We evaluated intra-attack lesion dynamics in patients with myelin... (Observational Study)
Observational Study
BACKGROUND AND OBJECTIVES
Knowledge of the evolution of CNS demyelinating lesions within attacks could assist diagnosis. We evaluated intra-attack lesion dynamics in patients with myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) vs multiple sclerosis (MS) and aquaporin-4 antibody seropositive neuromyelitis optica spectrum disorder (AQP4+NMOSD).
METHODS
This retrospective observational multicenter study included consecutive patients from Mayo Clinic (USA) and Great Ormond Street Hospital for Children (UK). Inclusion criteria were as follows: (1) MOGAD, MS, or AQP4+NMOSD diagnosis; (2) availability of ≥2 brain MRIs (within 30 days of attack onset); and (3) brain involvement (i.e., ≥1 T2 lesion) on ≥1 brain MRI. The initial and subsequent brain MRIs within a single attack were evaluated for the following: new T2 lesions(s); resolved T2 lesion(s); both; or no change. This was compared between MOGAD, MS, and AQP4+NMOSD attacks. We used the Mann-Whitney test and χ/Fisher exact test for statistical analysis.
RESULTS
Our cohort included 55 patients with MOGAD (median age, 14 years; interquartile range [IQR] 5-34; female sex, 29 [53%]) for a total of 58 attacks. The comparison groups included 38 patients with MS, and 19 with AQP4+NMOSD. In MOGAD, the initial brain MRI (median of 5 days from onset [IQR 3-9]) was normal in 6/58 (10%) attacks despite cerebral symptoms (i.e., radiologic lag). The commonest reason for repeat MRI was clinical worsening or no improvement (33/56 [59%] attacks with details available). When compared with the first MRI, the second intra-attack MRI (median of 8 days from initial scan [IQR 5-13]) showed the following: new T2 lesion(s) 27/58 (47%); stability 24/58 (41%); resolution of T2 lesion(s) 4/58 (7%); or both new and resolved T2 lesions 3/58 (5%). Findings were similar between children and adults. Steroid treatment was associated with resolution of ≥1 T2 lesion (6/28 [21%] vs 1/30 [3%], = 0.048) and reduced the likelihood of new T2 lesions (9/28 vs 18/30, = 0.03). Intra-attack MRI changes favored MOGAD (34/58 [59%]) over MS (10/38 [26%], = 0.002) and AQP4+NMOSD (4/19 [21%], = 0.007). Resolution of ≥1 T2 lesions was exclusive to MOGAD (7/58 [12%]).
DISCUSSION
Radiologic lag is common within MOGAD attacks. Dynamic imaging with frequent appearance and occasional disappearance of lesions within a single attack suggest MOGAD diagnosis over MS and AQP4+NMOSD. These findings have implications for clinical practice, clinical trial attack adjudication, and understanding of MOGAD pathogenesis.
Topics: Humans; Female; Male; Myelin-Oligodendrocyte Glycoprotein; Magnetic Resonance Imaging; Adolescent; Child; Retrospective Studies; Brain; Multiple Sclerosis; Aquaporin 4; Neuromyelitis Optica; Young Adult; Autoantibodies; Adult; Disease Progression
PubMed: 38710000
DOI: 10.1212/WNL.0000000000209303 -
European Journal of Paediatric... May 2024Patients with myelin oligodendrocyte glycoprotein antibody-associated disorders (MOGAD) clinically present e.g. with acute disseminated encephalomyelitis (ADEM), optic...
BACKGROUND AND OBJECTIVES
Patients with myelin oligodendrocyte glycoprotein antibody-associated disorders (MOGAD) clinically present e.g. with acute disseminated encephalomyelitis (ADEM), optic neuritis (ON), transverse myelitis (TM) or aquaporin-4-IgG (AQP4-IgG) negative neuromyelitis optica spectrum disorders (NMOSD)-like phenotypes. We aimed to analyze and compare blood parameters in children with MOGAD, AQP4-IgG-positive NMOSD (hence NMOSD), multiple sclerosis (MS) and healthy controls (HC).
METHODS
We evaluated differences in complete blood counts (CBC), neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), monocyte-to-lymphocyte ratio (MLR) and C-reactive protein (CRP) between these four groups and within the groups between clinical attack, acute treatment and remission.
RESULTS
Our cohort consisted of 174 children and adolescents with a total of 550 timepoints: 66 patients had MOGAD (202 timepoints), 11 NMOSD (76 timepoints), 58 MS (219 timepoints) and 39 were HC (53 timepoints). At clinical attack, leukocyte counts were elevated in MOGAD compared to remission (p < 0.001) and compared to all other groups (p < 0.001). NLR was high in MOGAD and NMOSD, and PLR was high in NMOSD, however, after correction for multiple testing these findings did not remain significant. While glucocorticoids caused an increase of leukocyte counts and NLR in NMOSD and MS, these values remained stable during acute treatment in MOGAD. In remission, NLR normalized in MOGAD, while it stayed high in NMOSD. PLR increased in NMOSD and was significantly higher compared to all other groups.
DISCUSSION
Some blood parameters, mainly leukocyte and differential counts, might help clinicians to evaluate disease activity, differentiate relapses from pseudo-relapses and even distinguish between different disease entities.
Topics: Humans; Child; Myelin-Oligodendrocyte Glycoprotein; Female; Male; Adolescent; Neuromyelitis Optica; Autoantibodies; Multiple Sclerosis; Child, Preschool; Aquaporin 4; C-Reactive Protein; Encephalomyelitis, Acute Disseminated
PubMed: 38705015
DOI: 10.1016/j.ejpn.2024.04.011 -
BMC Microbiology May 2024Pseudomonas aeruginosa is a common cause of nosocomial infections. However, the emergence of multidrug-resistant strains has complicated the treatment of P. aeruginosa...
BACKGROUND
Pseudomonas aeruginosa is a common cause of nosocomial infections. However, the emergence of multidrug-resistant strains has complicated the treatment of P. aeruginosa infections. While polymyxins have been the mainstay for treatment, there is a global increase in resistance to these antibiotics. Therefore, our study aimed to determine the prevalence and molecular details of colistin resistance in P. aeruginosa clinical isolates collected between June 2019 and May 2023, as well as the genetic linkage of colistin-resistant P. aeruginosa isolates.
RESULTS
The resistance rate to colistin was 9% (n = 18) among P. aeruginosa isolates. All 18 colistin-resistant isolates were biofilm producers and carried genes associated with biofilm formation. Furthermore, the presence of genes encoding efflux pumps, TCSs, and outer membrane porin was observed in all colistin-resistant P. aeruginosa strains, while the mcr-1 gene was not detected. Amino acid substitutions were identified only in the PmrB protein of multidrug- and colistin-resistant strains. The expression levels of mexA, mexC, mexE, mexY, phoP, and pmrA genes in the 18 colistin-resistant P. aeruginosa strains were as follows: 88.8%, 94.4%, 11.1%, 83.3%, 83.3%, and 38.8%, respectively. Additionally, down-regulation of the oprD gene was observed in 44.4% of colistin-resistant P. aeruginosa strains.
CONCLUSION
This study reports the emergence of colistin resistance with various mechanisms among P. aeruginosa strains in Ardabil hospitals. We recommend avoiding unnecessary use of colistin to prevent potential future increases in colistin resistance.
Topics: Colistin; Pseudomonas aeruginosa; Anti-Bacterial Agents; Humans; Microbial Sensitivity Tests; Bacterial Proteins; Pseudomonas Infections; Prevalence; Drug Resistance, Multiple, Bacterial; Biofilms; Hospitals; Drug Resistance, Bacterial; Cross Infection; Membrane Transport Proteins; Porins; Transcription Factors
PubMed: 38702660
DOI: 10.1186/s12866-024-03309-1 -
Virulence Dec 2024This study aimed at revealing the underlying mechanisms of the loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing hypervirulent...
OBJECTIVES
This study aimed at revealing the underlying mechanisms of the loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing hypervirulent (hvKp).
METHODS
Here we longitudinally recovered 3 non-carbapenemase-producing K1-ST23 hvKp strains at a one-month interval (KP29105, KP29499 and KP30086) from an elderly male. Antimicrobial susceptibility testing, whole genome sequencing, transcriptomic sequencing, gene cloning, plasmid conjugation, quantitative real-time PCR (qRT-PCR), and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were conducted.
RESULTS
Among the 3 hvKp strains, KP29105 was resistant to the third- and fourth-generation cephalosporins, KP29499 acquired resistance to both ceftazidime-avibactam and carbapenems, while KP30086 restored its susceptibility to ceftazidime-avibactam, imipenem and meropenem but retained low-level resistance to ertapenem. KP29105 and KP29499 carried plasmid-encoded genes and , respectively, but KP30086 lost both. Cloning of gene and conjugation experiment of -carrying plasmid showed that the transformant and transconjugant were susceptible to ceftazidime-avibactam but had a more than 8-fold increase in MICs. Supplementation with an outer membrane permeabilizer could reduce the MIC of ceftazidime-avibactam by 32 folds, indicating that porins play a key role in ceftazidime-avibactam resistance. The OmpK35 of the 3 isolates was not expressed, and the OmpK36 of KP29499 and KP30086 had a novel amino acid substitution (L359R). SDS-PAGE and qRT-PCR showed that the expression of porin OmpK36 of KP29499 and KP30086 was significantly down-regulated compared with KP29105.
CONCLUSIONS
In summary, we reported the rare ceftazidime-avibactam resistance in a non-carbapenemase-producing hvKp strain. Resistance plasmid carrying and mutated OmpK36 had a synergetic effect on the resistance.
Topics: Ceftazidime; Klebsiella pneumoniae; Azabicyclo Compounds; Drug Combinations; Anti-Bacterial Agents; Male; Microbial Sensitivity Tests; Klebsiella Infections; Humans; Bacterial Proteins; beta-Lactamases; Aged; Drug Resistance, Multiple, Bacterial; Virulence; Plasmids; Whole Genome Sequencing
PubMed: 38697754
DOI: 10.1080/21505594.2024.2348251 -
PLoS Biology May 2024BAK and BAX execute intrinsic apoptosis by permeabilising the mitochondrial outer membrane. Their activity is regulated through interactions with pro-survival BCL-2...
BAK and BAX execute intrinsic apoptosis by permeabilising the mitochondrial outer membrane. Their activity is regulated through interactions with pro-survival BCL-2 family proteins and with non-BCL-2 proteins including the mitochondrial channel protein VDAC2. VDAC2 is important for bringing both BAK and BAX to mitochondria where they execute their apoptotic function. Despite this important function in apoptosis, while interactions with pro-survival family members are well characterised and have culminated in the development of drugs that target these interfaces to induce cancer cell apoptosis, the interaction between BAK and VDAC2 remains largely undefined. Deep scanning mutagenesis coupled with cysteine linkage identified key residues in the interaction between BAK and VDAC2. Obstructive labelling of specific residues in the BH3 domain or hydrophobic groove of BAK disrupted this interaction. Conversely, mutating specific residues in a cytosol-exposed region of VDAC2 stabilised the interaction with BAK and inhibited BAK apoptotic activity. Thus, this VDAC2-BAK interaction site can potentially be targeted to either inhibit BAK-mediated apoptosis in scenarios where excessive apoptosis contributes to disease or to promote BAK-mediated apoptosis for cancer therapy.
Topics: Voltage-Dependent Anion Channel 2; bcl-2 Homologous Antagonist-Killer Protein; Apoptosis; Humans; Protein Binding; Mitochondria; Animals; HEK293 Cells
PubMed: 38696533
DOI: 10.1371/journal.pbio.3002617 -
Acta Medica Okayama Apr 2024Brain edema causes abnormal fluid retention and can be fatal in severe cases. Although it develops in various diseases, most treatments for brain edema are classical. We...
Brain edema causes abnormal fluid retention and can be fatal in severe cases. Although it develops in various diseases, most treatments for brain edema are classical. We analyzed the impacts of age and gender on the characteristics of a water intoxication model that induces pure brain edema in mice and examined the model's usefulness for research regarding new treatments for brain edema. C57BL/6J mice received an intraperitoneal administration of 10% body weight distilled water, and we calculated the brain water content by measuring the brain-tissue weight immediately after dissection and after drying. We analyzed 8-OHdG and caspase-3 values to investigate the brain damage. We also applied this model in aquaporin 4 knockout (AQP4-) mice and compared these mice with wild-type mice. The changes in water content differed by age and gender, and the 8-OHdG and caspase-3 values differed by age. Suppression of brain edema by AQP4- was also confirmed. These results clarified the differences in the onset of brain edema by age and gender, highlighting the importance of considering the age and gender of model animals. Similar studies using genetically modified mice are also possible. Our findings indicate that this water intoxication model is effective for explorations of new brain edema treatments.
Topics: Animals; Brain Edema; Water Intoxication; Male; Mice; Mice, Inbred C57BL; Female; Disease Models, Animal; Aquaporin 4; Age Factors; Sex Factors; Mice, Knockout; Caspase 3; Brain
PubMed: 38688829
DOI: 10.18926/AMO/66914 -
Nanoscale May 2024Aquaporin-4 (AQP4) facilitates water transport across astrocytic membranes in the brain, forming highly structured nanometric arrays. AQP4 has a central role in...
Aquaporin-4 (AQP4) facilitates water transport across astrocytic membranes in the brain, forming highly structured nanometric arrays. AQP4 has a central role in regulating cerebrospinal fluid (CSF) circulation and facilitating the clearance of solutes from the extracellular space of the brain. Adrenergic signaling has been shown to modulate the volume of the extracellular space of the brain AQP4 localized at the end-feet of astrocytes, but the mechanisms by which AQP4 regulates CSF inflow and outflow in the brain remain elusive. Using advanced imaging techniques, including super-resolution microscopy and single-molecule tracking, we investigated the hypothesis that β-adrenergic receptor activation induces cellular changes that regulate AQP4 array size and mobility, thus influencing water transport in the brain. We report that the β-adrenergic agonist, isoproterenol hydrochloride, decreases AQP4 array size and enhances its membrane mobility, while hyperosmotic conditions induce the formation of larger, less mobile arrays. These findings reveal that AQP4 arrays are dynamic structures, responsive to adrenergic signals and osmotic changes, highlighting a novel regulatory mechanism of water transport in the brain. Our results provide insights into the molecular control of CSF circulation and extracellular brain space volume, laying the groundwork for understanding the relationship between astrocyte water transport, sleep physiology, and neurodegeneration.
Topics: Aquaporin 4; Astrocytes; Animals; Isoproterenol; Single Molecule Imaging; Mice; Water; Cells, Cultured; Receptors, Adrenergic, beta; Adrenergic beta-Agonists; Brain
PubMed: 38682293
DOI: 10.1039/d4nr00330f -
Microorganisms Apr 2024To date, three carbapenem resistance mechanisms have been identified: carbapenemase released from the pathogen, changes in the expression of the outer membrane OprD...
To date, three carbapenem resistance mechanisms have been identified: carbapenemase released from the pathogen, changes in the expression of the outer membrane OprD porin, and overexpression of the efflux pump MexAB-OprM. Twelve carbapenemase-negative carbapenem-resistant strains, isolated from patients hospitalized at the University Hospital of Larissa, Central Greece, during 2023, which belonged to various sequence types (STs), were selected and were studied focusing on the characterization of their -lactamases, on changes to OprD and its regulator MexT proteins, and on alterations to the MexAB-OprM regulator proteins encoded by the , , and genes. Whole genome sequencing analysis revealed the presence of -lactamase encoding genes, with present in all isolates. Additionally, seven different genes of the oxacillinase family (, , , , , , ) were identified, with each strain harboring one to three of these. Regarding the OprD, five strains had truncated structures, at Loop 2, Loop 3, Loop 4, and Loop 9, while the remaining strains carried previously reported amino acid changes. Further, an additional strain had a truncated MexR; whereas, two other strains had totally modified NalC sequences. The active form of MexT, responsible for the downregulation of OprD production, as the intact sequence of the NalD protein, was found in all the strains studied. It is concluded that the truncated OprD, MexR, and NalC proteins, detected in eight strains, probably led to inactive proteins, contributing to carbapenem resistance. However, four strains carried known modifications in OprD, MexR, and NalC, as previously reported in both susceptible and resistant strains, a finding that indicates the complexity of carbapenem resistance in .
PubMed: 38674749
DOI: 10.3390/microorganisms12040805 -
International Journal of Molecular... Apr 2024Demyelinating Charcot-Marie-Tooth 4G (CMT4G) results from a recessive mutation in the 5'UTR region of the Hexokinase 1 (HK1) gene. HK participates in mitochondrial...
The 5'-UTR Mutation in Charcot-Marie-Tooth 4G Disease Alters Hexokinase 1 Binding to Voltage-Dependent Anion Channel-1 and Leads to Dysfunctional Mitochondrial Calcium Buffering.
Demyelinating Charcot-Marie-Tooth 4G (CMT4G) results from a recessive mutation in the 5'UTR region of the Hexokinase 1 (HK1) gene. HK participates in mitochondrial calcium homeostasis by binding to the Voltage-Dependent Anion Channel (VDAC), through its N-terminal porin-binding domain. Our hypothesis is that CMT4G mutation results in a broken interaction between mutant HK1 and VDAC, disturbing mitochondrial calcium homeostasis. We studied a cohort of 25 CMT4G patients recruited in the French gypsy population. The disease was characterized by a childhood onset, an intermediate demyelinating pattern, and a significant phenotype leading to becoming wheelchair-bound by the fifth decade of life. Co-IP and PLA studies indicated a strong decreased interaction between VDAC and HK1 in the patients' PBMCs and sural nerve. We observed that either wild-type HK1 expression or a peptide comprising the 15 aa of the N-terminal wild-type HK1 administration decreased mitochondrial calcium release in HEK293 cells. However, mutated CMT4G HK1 or the 15 aa of the mutated HK1 was unable to block mitochondrial calcium release. Taken together, these data show that the CMT4G-induced modification of the HK1 N-terminus disrupts HK1-VDAC interaction. This alters mitochondrial calcium buffering that has been shown to be critical for myelin sheath maintenance.
Topics: Adolescent; Adult; Child; Female; Humans; Male; Middle Aged; Young Adult; 5' Untranslated Regions; Calcium; Charcot-Marie-Tooth Disease; HEK293 Cells; Hexokinase; Mitochondria; Mutation; Protein Binding; Voltage-Dependent Anion Channel 1
PubMed: 38673950
DOI: 10.3390/ijms25084364