mcr-3 and mcr-4 Variants in Carbapenemase-Producing Clinical Enterobacteriaceae Do Not Confer Phenotypic Polymyxin Resistance

The worldwide distribution of plasmid-mediated colistin resistance determinants (mcr-1, mcr-2, mcr-3, and mcr-4) coupled to the emerging observation that colistin resistance is more prevalent in carbapenem-resistant Enterobacteriaceae (CRE) (1, 2) presents a daunting challenge in combatting antimicrobial resistance. Undoubtedly, next-generation sequencing approaches have expedited the discovery of mobile colistin resistance determinants (3). In this study, we undertook the in silico screening of 500 phenotypically carbapenem-resistant carbapenemase-producing Enterobacteriaceae whole genomes for the presence of the mcr gene, using CLC Genomics Workbench (CLC Bio-Qiagen, Aarhus, Denmark). The isolates comprised clinical and screening pure cultures submitted to the national reference laboratory for mandatory CRE surveillance. Locally, the presence of mcr-1 as well as its cocarriage with KPC-2 had been previously well described (4, 5); hence, we did not look further into the distribution of mcr-1. mcr-2 was not detected among the genomes analyzed. mcr-3 was identified in one Escherichia coli genome (ENT1955) by the use of both read mapping and de novo assembly.MOH (Min. of Health, S’pore)Published versio

Genomic study of blaIMI-positive Enterobacter cloacae complex in Singapore over a five-year study period

The blaIMI gene is rarely detected outside the Enterobacter genus. Genomic characterization of 87 blaIMI-positive Enterobacter cloacae complex members revealed that the largest phylogenomic clade was made up of E. cloacae subsp. cloacae (71.3%), followed by the newly described species E. bugandensis (13.8%), E. sichuanensis (10.3%), and E. roggenkampii (4.6%). IMI-1 was the predominant carbapenemase variant (86/87, 98.9%). All the blaIMI genes were associated with chromosomally integrated Xer-dependent integrative mobile elements (IMEXs), with two new variants detected.Ministry of Education (MOE)National Medical Research Council (NMRC)Published versionGrant support was provided by the NMRC Clinician-Scientist Individual ResearchGrant (NMRC/CIRG/1463/2016), Singapore Ministry of Education Academic ResearchFund Tier 2 grant: New Delhi Metallo-Beta-Lactamase: A global multicenter, whole-genome study (MOE2015-T2-2-096), NMRC Collaborative Grant: Collaborative SolutionsTargeting Antimicrobial Resistance Threats in Health Systems (CoSTAR-HS) (NMRCCGAug16C005), and NMRC Clinician Scientist Award (NMRC/CSA-INV/0002/2016)

Genetic signatures of HIV-1 envelope-mediated bystander apoptosis

The envelope (Env) glycoprotein of HIV is an important determinant of viral pathogenesis. Several lines of evidence support the role of HIV-1 Env in inducing bystander apoptosis that may be a contributing factor in CD4+ T cell loss. However, most of the studies testing this phenomenon have been conducted with laboratory-adapted HIV-1 isolates. This raises the question of whether primary Envs derived from HIV-infected patients are capable of inducing bystander apoptosis and whether specific Env signatures are associated with this phenomenon. We developed a high throughput assay to determine the bystander apoptosis inducing activity of a panel of primary Envs. We tested 38 different Envs for bystander apoptosis, virion infectivity, neutralizing antibody sensitivity, and putative N-linked glycosylation sites along with a comprehensive sequence analysis to determine if specific sequence signatures within the viral Env are associated with bystander apoptosis. Our studies show that primary Envs vary considerably in their bystander apoptosis-inducing potential, a phenomenon that correlates inversely with putative N-linked glycosylation sites and positively with virion infectivity. By use of a novel phylogenetic analysis that avoids subtype bias coupled with structural considerations, we found specific residues like Arg-476 and Asn-425 that were associated with differences in bystander apoptosis induction. A specific role of these residues was also confirmed experimentally. These data demonstrate for the first time the potential of primary R5 Envs to mediate bystander apoptosis in CD4+ T cells. Furthermore, we identify specific genetic signatures within the Env that may be associated with the bystander apoptosis-inducing phenotype.Published versio

Validation of self-collected buccal swab and saliva as a diagnostic tool for COVID-19

10.1016/j.ijid.2020.12.080International Journal of Infectious Diseases104255-26

Comparison of Circulating Tumour Cells and Circulating Cell-Free Epstein-Barr Virus DNA in Patients with Nasopharyngeal Carcinoma Undergoing Radiotherapy

10.1038/s41598-016-0006-3Scientific Reports611

T-cell lymphoma clonality by copy number variation analysis of T-cell receptor genes

T-cell lymphomas arise from a single neoplastic clone and exhibit identical patterns of deletions in T-cell receptor (TCR) genes. Whole genome sequencing (WGS) data represent a treasure trove of information for the development of novel clinical applications. However, the use of WGS to identify clonal T-cell proliferations has not been systematically studied. In this study, based on WGS data, we identified monoclonal rearrangements (MRs) of T-cell receptors (TCR) genes using a novel segmentation algorithm and copy number computation. We evaluated the feasibility of this technique as a marker of T-cell clonality using T-cell lymphomas (TCL, n = 44) and extranodal NK/T-cell lymphomas (ENKTLs, n = 20), and identified 98% of TCLs with one or more TCR gene MRs, against 91% detected using PCR. TCR MRs were absent in all ENKTLs and NK cell lines. Sensitivity-wise, this platform is sufficiently competent, with MRs detected in the majority of samples with tumor content under 25% and it can also distinguish monoallelic from biallelic MRs. Understanding the copy number landscape of TCR using WGS data may engender new diagnostic applications in hematolymphoid pathology, which can be readily adapted to the analysis of B-cell receptor loci for B-cell clonality determination

Impact of smoking and brain metastasis on outcomes of advanced EGFR mutation lung adenocarcinoma patients treated with first line epidermal growth factor receptor tyrosine kinase inhibitors

10.1371/journal.pone.0123587PLoS ONE105e012358

Designer broad-spectrum polyimidazolium antibiotics

© 2020 National Academy of Sciences. All rights reserved. For a myriad of different reasons most antimicrobial peptides (AMPs) have failed to reach clinical application. Different AMPs have different shortcomings including but not limited to toxicity issues, potency, limited spectrum of activity, or reduced activity in situ. We synthesized several cationic peptide mimics, main-chain cationic polyimidazoliums (PIMs), and discovered that, although select PIMs show little acute mammalian cell toxicity, they are potent broad-spectrum antibiotics with activity against even pan-antibiotic-resistant gram-positive and gram-negative bacteria, and mycobacteria. We selected PIM1, a particularly potent PIM, for mechanistic studies. Our experiments indicate PIM1 binds bacterial cell membranes by hydrophobic and electrostatic interactions, enters cells, and ultimately kills bacteria. Unlike cationic AMPs, such as colistin (CST), PIM1 does not permeabilize cell membranes. We show that a membrane electric potential is required for PIM1 activity. In laboratory evolution experiments with the gram-positive Staphylococcus aureus we obtained PIM1-resistant isolates most of which had menaquinone mutations, and we found that a sitedirected menaquinone mutation also conferred PIM1 resistance. In similar experiments with the gram-negative pathogen Pseudomonas aeruginosa, PIM1-resistant mutants did not emerge. Although PIM1 was efficacious as a topical agent, intraperitoneal administration of PIM1 in mice showed some toxicity. We synthesized a PIM1 derivative, PIM1D, which is less hydrophobic than PIM1. PIM1D did not show evidence of toxicity but retained antibacterial activity and showed efficacy in murine sepsis infections. Our evidence indicates the PIMs have potential as candidates for development of new drugs for treatment of pan-resistant bacterial infections