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Abstract Extracellular vesicle (EV) secretion is mediated by purinergic receptor P2X7 (P2RX7), an ATP‐gated cation channel highly expressed in microglia. We have previously shown that administration of GSK1482160, a P2RX7 selective inhibitor, suppresses EV secretion from murine microglia and prevents tauopathy development, leading to the recovery of the hippocampal function in PS19 mice, expressing P301S tau mutant. It is yet unknown, however, whether the effect of GSK1482160 on EV secretion from glial cells is specifically regulated through P2RX7. Here we tested GSK1482160 on primary microglia and astrocytes isolated from C57BL/6 (WT) and P2rx7–/– mice and evaluated their EV secretion and phagocytotic activity of aggregated human tau (hTau) under ATP stimulation. GSK1482160 treatment and deletion of P2rx7 significantly reduced secretion of small and large EVs in microglia and astrocytes in both ATP stimulated or unstimulated condition as determined by nanoparticle tracking analysis, CD9 ELISA and immunoblotting of Tsg101 and Flotilin 1 using isolated EVs. GSK1482160 treatment had no effect on EV secretion from P2rx7–/– microglia while we observed significant reduction in the secretion of small EVs from P2rx7–/– astrocytes, suggesting its specific targeting of P2RX7 in EV secretion except small EV secretion from astrocytes. Finally, deletion of P2rx7 suppressed IL‐1β secretion and phagocytosed misfolded tau from both microglia and astrocytes. Together, these findings show that GSK1482160 suppresses EV secretion from microglia and astrocytes in P2RX7‐dependment manner, and P2RX7 critically regulates secretion of IL‐1β and misfolded hTau, demonstrating as the viable target of suppressing EV‐mediated neuroinflammation and tau propagation.

Prevalence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has compromised antimicrobial efficacy against severe infections worldwide. To monitor global spread, we conducted a comprehensive genomic epidemiologic study comparing sequences from 21 blaOXA-232–carrying CRKP isolates from China with K. pneumoniae sequence type (ST) 15 strains from 68 countries available in GenBank. Phylogenetic and phylogeographic analyses revealed all blaOXA-232–carrying CRKP isolates belonged to ST15 lineage and exhibited multidrug resistance. Analysis grouped 330 global blaOXA-232–carrying ST15 CRKP strains into 5 clades, indicating clonal transmission with small genetic distances among multiple strains. The lineage originated in the United States, then spread to Europe, Asia, Oceania, and Africa. Most recent common ancestor was traced back to 2000; mutations averaged ≈1.7 per year per genome. Our research helps identify key forces driving global spread of blaOXA-232–carrying CRKP ST15 lineage and emphasizes the importance of ongoing surveillance of epidemic CRKP.

Colistin is considered as one of the last-resort antimicrobial agents for treating multidrug-resistant bacterial infections. Multidrug-resistant E. asburiae has been increasingly isolated from clinical patients, which posed a great challenge for antibacterial treatment. This study aimed to report a mcr-10 and blaNDM-1 co-carrying E. asburiae clinical isolate 5549 conferred a high-level resistance against colistin. Antibiotic susceptibility testing was performed using the microdilution broth method. Transferability of mcr-10 and blaNDM-1-carrying plasmids were investigated by conjugation experiments. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to identify modifications in lipid A. Whole genome sequencing and phylogenetic analysis between strain 5549 and a total of 301 E. asburiae genomes retrieved from NCBI database were performed. The genetic characteristics of mcr-10 and blaNDM-1-bearing plasmids were also analyzed. Our study indicated that strain 5549 showed extensively antibiotic-resistant trait, including colistin and carbapenem resistance. The mcr-10 and blaNDM-1 were carried by IncFIB/IncFII type p5549_mcr-10 (159417 bp) and IncN type p5549_NDM-1 (63489 bp), respectively. Conjugation assays identified that only the blaNDM-1-carrying plasmid could be successfully transferred to E. coli J53. Interestingly, mcr-10 did not mediate colistin resistance when it was cloned into E. coli DH5α. Mass spectrometry analysis showed the lipid A palmitoylation of the C-lacyl-oxo-acyl chain to the chemical structure of lipid A at m/z 2063 in strain 5549. In summary, this study is the first to report a mcr-10 and blaNDM-1 co-occurrence E. asburiae recovered from China. Our investigation revealed the distribution of different clonal lineage of E. asburiae with epidemiology perspective and the underlying mechanisms of colistin resistance. Active surveillance is necessary to control the further dissemination of multidrug-resistant E. asburiae.

Abstract Antibiotic resistance is spreading rapidly around the world and seriously impeding efforts to control microbial infections. Although nucleic acid testing is widely deployed for the detection of antibiotic resistant bacteria, the current techniques—mainly based on polymerase chain reaction (PCR)—are time-consuming and laborious. There is an urgent need to develop new strategies to control bacterial infections and the spread of antimicrobial resistance (AMR). The CRISPR-Cas system is an adaptive immune system found in many prokaryotes that presents attractive opportunities to target and edit nucleic acids with high precision and reliability. Engineered CRISPR-Cas systems are reported to effectively kill bacteria or even revert bacterial resistance to antibiotics (resensitizing bacterial cells to antibiotics). Strategies for combating antimicrobial resistance using CRISPR (i.e., Cas9, Cas12, Cas13, and Cas14) can be of great significance in detecting bacteria and their resistance to antibiotics. This review discusses the structures, mechanisms, and detection methods of CRISPR-Cas systems and how these systems can be engineered for the rapid and reliable detection of bacteria using various approaches, with a particular focus on nanoparticles. In addition, we summarize the most recent advances in applying the CRISPR-Cas system for virulence modulation of bacterial infections and combating antimicrobial resistance. Graphical Abstract

ABSTRACT: Objectives: The emergence and dissemination of colistin-resistant Enterobacterales has become a major global public-health threat. Here we investigated the genomic and phylogenetic characteristics of a multidrug-resistant Klebsiella pneumoniae strain (KP4823) carrying the mcr-1 gene recovered from a urinary tract infection in China. Methods: Antimicrobial susceptibility of K. pneumoniae KP4823 was determined by broth microdilution. Whole genomic DNA was extracted and sequenced using Oxford Nanopore MinION and Illumina NovaSeq 6000 platforms. Hybrid assembly with long and short reads was performed using Unicycler, and the genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The sequence type (ST), capsular type, and antimicrobial resistance and virulence genes were identified from the genome sequence. Core genome multilocus sequence typing (cgMLST) analysis was performed by BacWGSTdb 2.0 server. Results: Klebsiella pneumoniae KP4823 was resistant to colistin, ceftazidime, cefepime, cefotaxime, fosfomycin and aztreonam. The complete genome sequence of KP4823 consists of five contigs comprising 5 445 519 bp, including one chromosome and four plasmids. The isolate was assigned to ST101 with capsular serotype KL106. Several antimicrobial resistance genes were identified, including the colistin resistance gene mcr-1, which was located on a 34 685-bp IncX4 plasmid. The closest relative of K. pneumoniae KP4823 was another ST101 isolate (08EU827) recovered from Sweden in 2008, which differed by 191 cgMLST loci. Conclusion: Our study reports the genome sequence of a multidrug-resistant mcr-1-carrying K. pneumoniae in China. These data may help to understand the antimicrobial resistance mechanisms, genomic features and transmission dynamics of colistin resistance in clinical settings.

ABSTRACT The in vitro activity of two new fluoroquinolones, delafloxacin and finafloxacin, were evaluated against M. hominis and Ureaplasma spp. The MICs of delafloxacin, finafloxacin, and two classical fluoroquinolones (moxifloxacin and levofloxacin) were tested against 29 M. hominis and 67 Ureaplasma spp. isolates using the broth microdilution method. The molecular mechanisms underlying fluoroquinolone resistance were also investigated. Delafloxacin exhibited low MICs against M. hominis and Ureaplasma spp., including the levofloxacin-resistant isolates. For M. hominis, delafloxacin showed low MIC90 value of 1 μg/mL (MIC range, 32 μg/mL for levofloxacin. The two mutations GyrA S153L and ParC S91I were commonly identified in fluoroquinolone-resistant M. hominis, and ParC S83L was the most frequent mutation identified in fluoroquinolone-resistant Ureaplasma spp. Delafloxacin displayed lower MICs against fluoroquinolone-resistant isolates of both M. hominis and Ureaplasma spp. that have mutations in the quinolone resistance determining regions (QRDRs) than the two classical fluoroquinolones. Delafloxacin is a promising fluoroquinolone with low MICs against fluoroquinolone-resistant M. hominis and Ureaplasma spp. Our study confirms the potential clinical use of delafloxacin in treating antimicrobial-resistant M. hominis and Ureaplasma spp. infections. IMPORTANCE Fluoroquinolone resistance in Mycoplasma hominis and Ureaplasma spp. is on the rise globally, which has compromised the efficacy of the currently available antimicrobial agents. This study evaluated the antimicrobial activity of two new fluoroquinolones, delafloxacin and finafloxacin, for the first time, against M. hominis and Ureaplasma spp. clinical isolates. Delafloxacin and finafloxacin displayed different antimicrobial susceptibility profiles against M. hominis and Ureaplasma spp. in vitro. Delafloxacin was found to be more effective against M. hominis and Ureaplasma spp. than three classical fluoroquinolones (finafloxacin, moxifloxacin, and levofloxacin). Finafloxacin displayed activity similar to moxifloxacin but superior to levofloxacin against M. hominis and Ureaplasma spp. Our findings demonstrate that delafloxacin is a promising fluoroquinolone with outstanding activity against fluoroquinolone-resistant M. hominis and Ureaplasma spp.

ABSTRACT The wide spread of plasmid-borne mobilized colistin resistance (mcr) genes from animals to humans broadly challenges the clinical use of polymyxins. Here, we evaluated the incidence of a recently reported mcr variant, mcr-10, in animals and humans in the same area. Our results revealed the presence of novel mcr-10-carrying plasmids in two Klebsiella pneumoniae isolates from chickens, one Escherichia coli isolate from slaughterhouse workers, and a chromosome-borne mcr-10 gene in Enterobacter kobei from a healthy resident in the same region. It is worth mentioning that the multidrug-resistant ST11 K. pneumoniae isolates coharboring mcr-10 and mcr-8 genes in two separate plasmids not only were resistant to polymyxins (MIC = 8 mg/L) but also showed reduced susceptibility to tigecycline (MIC ≥ 2 mg/L) due to the tet(A) mutation or the tmexCD1-toprJ1 gene cluster. The structure xerC-mcr10-insCinsD-like was found in genetic environments of both the plasmid and chromosome carrying mcr-10. We compared genomic epidemiological characteristics of mcr-10-harboring bacteria available in 941,449 genomes in the NCBI database (including strains of K. pneumoniae, E. coli, and E. kobei) with isolates in this study. The results indicated a sporadic distribution of mcr-10 all around the world and in a variety of sources, including humans, environments, and animals, which confirms that mcr-10 has spread among various hosts and warrants close monitoring and further future studies. IMPORTANCE We discovered mcr-10-harboring isolates in the “one health” approach and reported for the first time multidrug-resistant clinically threatening ST11 K. pneumoniae isolates coharboring mcr-10 and mcr-8 genes that are resistant to polymyxins and show reduced susceptibility to tigecycline. The exhaustive screening of 941,449 bacterial genomes in the GenBank database discovered a sporadic distribution of mcr-10-harboring isolates all around the world in a variety of sources, especially humans, which warrants close monitoring and a particular concern in clinical settings.

Prevalence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has compromised antimicrobial efficacy against severe infections worldwide. To monitor global spread, we conducted a comprehensive genomic epidemiologic study comparing sequences from 21 blaOXA-232-carrying CRKP isolates from China with K. pneumoniae sequence type (ST) 15 strains from 68 countries available in GenBank. Phylogenetic and phylogeographic analyses revealed all blaOXA-232-carrying CRKP isolates belonged to ST15 lineage and exhibited multidrug resistance. Analysis grouped 330 global blaOXA-232-carrying ST15 CRKP strains into 5 clades, indicating clonal transmission with small genetic distances among multiple strains. The lineage originated in the United States, then spread to Europe, Asia, Oceania, and Africa. Most recent common ancestor was traced back to 2000; mutations averaged -1.7 per year per genome. Our research helps identify key forces driving global spread of blaOXA-232-carrying CRKP ST15 lineage and emphasizes the importance of ongoing surveillance of epidemic CRKP. [ABSTRACT FROM AUTHOR]