Occurrence of High Levels of Cefiderocol Resistance in Carbapenem-Resistant Escherichia coli before Its Approval in China: a Report from China CRE-Network.

Cefiderocol has been approved in the United States and Europe but not in China. We aim to evaluate carbapenem-resistant Enterobacterales (CRE) susceptibility to cefiderocol to provide baseline data and investigate the resistance mechanism. From 2018 to 2019, 1,158 CRE isolates were collected from 23 provinces and municipalities across China. The MICs of antimicrobials were determined via the agar dilution and broth microdilution methods. Whole-genome sequencing was performed for 26 cefiderocol-resistant Escherichia coli isolates to investigate the resistance mechanism. Clone transformations were used to explore the function of cirA, pbp3, and blaNDM-5 in resistance. Among the 21 antimicrobials tested, aztreonam-avibactam had the highest antibacterial activity (98.3%), followed by cefiderocol (97.3%) and colistin (95.3%). A total of 26 E. coli isolates harboring New Delhi metallo-beta-lactamase 5 (NDM-5) showed high levels of cefiderocol resistance, of which sequence type 167 (ST167) accounted for 76.9% (20/26). We found 4 amino-acid insertions (YRIN/YRIK) at position 333 of penicillin-binding protein 3 (PBP3) in the 26 E. coli isolates, and 22 isolates had a siderophore receptor cirA premature stop codon. After obtaining the wild-type cirA supplementation, the MIC of the transformants decreased by 8 to 16 times in two cefiderocol-resistant isolates. A cefiderocol-susceptible isolate harboring NDM-5 has an MIC increased from 1 µg/mL to 64 µg/mL after cirA deletion, and the MIC decreased from 64 µg/mL to 0.5 µg/mL after blaNDM-5 deletion. The MIC of the E. coli DH5α, from which the pbp3 mutant was obtained, increased from 0.064 µg/mL to 0.25 µg/mL. Cefiderocol showed activity against most CRE in China. The resistance of ST167 E. coli to cefiderocol is a combination of the premature stop codon of cirA, pbp3 mutation, and blaNDM-5 existence. IMPORTANCE Cefiderocol, a new siderophore cephalosporin, has been approved in the United States and Europe but not in China. At present, there are almost no antimicrobial susceptibility evaluation data on cefiderocol in China. We evaluated the in vitro susceptibility of 1,158 strains of carbapenem-resistant Enterobacterales to cefiderocol and other antibiotics. We found that a high proportion of Escherichia coli showed high-level resistance to cefiderocol. Whole-genome sequencing (WGS) and molecular cloning experiments confirmed that the synergistic effect of the cirA gene premature stop codon, blaNDM-5 existence, and the pbp3 mutation is associated with high levels of cefiderocol resistance.

Down-regulation of c-Myc expression inhibits the invasion of bile duct carcinoma cells.

Cholangiocarcinoma is the second most common primary hepatic tumour originating from biliary tract epithelial cells with poor prognosis. Enhanced c-Myc protein expression contributes to many aspects of tumour cell biology. Although the ability of c-Myc to drive unrestricted cell proliferation and to inhibit cell differentiation had been well recognized, whether down-regulated c-Myc expression can inhibit tumour cell invasion still remains to be explored. The c-Myc ASODN (antisense oligodeoxyribonucleotide) and NSODN (nonsense oligodeoxyribonucleotide) were designed, synthesized and transfected into human QBC939 bile duct carcinoma cells using the Lipofectamine 2000 reagent. The protein expression of c-Myc was detected by Western blot. A transwell experiment was applied to evaluate the invasive capacity of the QBC939 cells. c-Myc ASODN could significantly suppress the c-Myc protein expression (P<0.05) and the invasion (P<0.01) of QBC939 cells transfected with c-Myc ASODN compared with that in the control and c-Myc NSODN-transfected group. Thus in the present study we show that down-regulation of c-Myc expression can inhibit the invasion of QBC939 cells in vitro.

Wilms' tumor suppressor (WT1) is a mediator of neuronal degeneration associated with the pathogenesis of Alzheimer's disease.

Wilms' tumor suppressor (WT1), a 52- to 54-kda transcription factor, is the gene product of Wilms' tumor 1 (wt1), one of at least three genes involved in the development of a pediatric kidney cancer. Expression patterns of WT1 indicate that it is not restricted to the kidney but may play a role in the development and homeostasis of other tissues as well. WT1 has been implicated in various cellular processes including proliferation, differentiation, and apoptosis. High levels of WT1 induce apoptosis independent of p53, whereas low levels of WT1 inhibit apoptosis. Because apoptosis has been suggested to play a role in neurodegeneration in Alzheimer's disease (AD), immunohistochemistry of WT1 and paired helical filament (PHF) in serial sections was carried out. Immunohistochemical localization of WT1 and PHF showed the presence of WT1 in approximately 42% of PHF-positive neurofibrillary tangle containing-neurons. Laser confocal microscopy of hippocampal neuron cultures undergoing apoptosis induced by amyloid beta peptide (Abeta) or staurosporine demonstrated significant time-dependent elevations of WT1 correlating with increased levels of apoptosis. Blockade of WT1 transcription by antisense oligonucleotide reduced WT1 expression and prevented neuronal apoptosis in both Abeta- and staurosporine-treated cultures. Together, these data suggest a role for WT1 in the neurodegeneration observed in AD brain.

A novel type of factor XI deficiency showing compound genetic abnormalities: a nonsense mutation and an impaired transcription.

We studied a 29-year-old Japanese male patient with factor XI deficiency; we also studied his parents and one sibling. Factor XI coagulation activity and antigen levels were extremely low (less than 1% of normal level) in both the patient and his brother, and they were half the normal levels in both parents. Sequence analysis of all 15 exons and the exon-intron boundaries of the factor XI gene amplified by polymerase chain reaction revealed a nonsense mutation in exon 8 (Gln263-->Stop). Although the parents are first cousins, the mutation was unexpectedly heterozygous in all the family members except the father, who showed the homozygous wild type, indicating that this mutation alone was not sufficient to account for the factor XI deficiency. To explore the genetic abnormality in the father, we analyzed allele-specific expression of the platelet factor XI gene using reverse transcription-polymerase chain reaction and subsequent restriction enzyme digestion. As a result, gene expression from only one allele was severely impaired in the father. This result implies an additional mutation in some regulatory element of the factor XI gene from paternal inheritance. We concluded that the factor XI deficiency of the patient was caused by compound heterozygous genetic abnormalities.

Nonsense mutations inhibit RNA splicing in a cell-free system: recognition of mutant codon is independent of protein synthesis.

Mutations resulting in premature termination codons reduce the corresponding mRNA levels. We describe a cell-free system in which depletion of the mutant immunoglobulin kappa mRNA pool correlates with inefficient splicing and not with RNA decay. Splicing deficiency does not depend on the sequence surrounding the in-frame nonsense codon and can be partially corrected by mutating the methionine initiation codon. Despite the apparent link between translation and low mutant mRNA levels, inefficient splicing is not dependent on protein synthesis. Abnormal splicing of mutant immunoglobulin RNA is observed with B-cell but not with HeLa or T-cell extracts. A nonsense mutant beta-globin RNA is normally spliced by B-cell extract. We propose that the phenomenon exhibits tissue and gene specificity.