Distinct miRNA Signatures and Networks Discern Fetal from Adult Erythroid Differentiation and Primary from Immortalized Erythroid Cells

MicroRNAs (miRNAs) are small non-coding RNAs crucial for post-transcriptional and translational regulation of cellular and developmental pathways. The study of miRNAs in erythropoiesis elucidates underlying regulatory mechanisms and facilitates related diagnostic and therapy development. Here, we used DNA Nanoball (DNB) small RNA sequencing to comprehensively characterize miRNAs in human erythroid cell cultures. Based on primary human peripheral-blood-derived CD34+ (hCD34+) cells and two influential erythroid cell lines with adult and fetal hemoglobin expression patterns, HUDEP-2 and HUDEP-1, respectively, our study links differential miRNA expression to erythroid differentiation, cell type, and hemoglobin expression profile. Sequencing results validated by reverse-transcription quantitative PCR (RT-qPCR) of selected miRNAs indicate shared differentiation signatures in primary and immortalized cells, characterized by reduced overall miRNA expression and reciprocal expression increases for individual lineage-specific miRNAs in late-stage erythropoiesis. Despite the high similarity of same-stage hCD34+ and HUDEP-2 cells, differential expression of several miRNAs highlighted informative discrepancies between both cell types. Moreover, a comparison between HUDEP-2 and HUDEP-1 cells displayed changes in miRNAs, transcription factors (TFs), target genes, and pathways associated with globin switching. In resulting TF-miRNA co-regulatory networks, major therapeutically relevant regulators of globin expression were targeted by many co-expressed miRNAs, outlining intricate combinatorial miRNA regulation of globin expression in erythroid cells

Inactivation of mgrB gene regulator and resistance to colistin is becoming endemic in carbapenem-resistant Klebsiella pneumoniae in Greece: A nationwide study from 2014 to 2017

Introduction: In Greece, the spread of carbapenem-resistant Enterobacteriaceae in humans has led to the reintroduction of colistin as a therapeutic agent. Unfortunately, colistin resistance with different mechanisms has emerged. The present work aims to determine the prevalence of carbapenem and colistin resistance and the corresponding mechanisms in Klebsiella pneumoniae clinical isolates from Greece. Methods: From 2014 to 2017, 288 carbapenem-resistant K. pneumoniae clinical strains were gathered from a collection of 973 isolates from eight different hospitals in Greece. Antibiotic susceptibility testing was performed using three different methods. Screening of carbapenem and colistin resistance genes was conducted using polymerase chain reaction (PCR) amplification and sequencing. Results: Among the 288 (29.6 %) carbapenem-resistant isolates, 213 (73.9%) were colistin-resistant (minimum inhibitory concentration [MIC] >2 mg/L). The KPC type was the most common carbapenemase gene (116; 40.3%), followed by VIM (41; 14.2%), NDM (33; 11.5%) and OXA-48 (22; 7.6%). Moreover, 44 (15.3%) strains co-produced two types of carbapenemases. No mcr genes were detected for colistin resistance but mutations in chromosomal genes were found. These included inactivation of the mgrB gene for 148 (69.5%) strains, including insertion sequences for 94 (44.1%), nonsense mutations for 4 (1.9%) and missense mutations for 24 (11.3%). Moreover, PCR amplification of mgrB gene was negative for 26 (12.2%) strains. Finally, 65 (30.5%) colistin-resistant strains exhibited a wild-type mgrB, the mechanisms of which remain to be elucidated. Conclusion: This study shows that K. pneumoniae clinical strains in Greece are resistant to both carbapenems and colistin and this is endemic and is likely chromosomally encoded. (C) 2020 Published by Elsevier B.V

Epidemiology and resistance phenotypes of carbapenemase-producing Klebsiella pneumoniae in Greece, 2014 to 2016

Background and aim: A multicentre nationwide surveillance study was conducted in Greek hospitals to evaluate epidemiology of carbapenemase-producing Klebsiella pneumoniae clinical isolates, and their susceptibilities to last-line antibiotics. Methods: Minimum inhibitory concentrations (MICs) were evaluated by Etest, colistin MICs were also evaluated by broth microdilution SensiTest (now known as ComASP) Colistin. Carbapenemase genes were detected by PCR. Clonal relatedness was assessed by PFGE. Isolates were prospectively collected between November 2014 and April 2016, from 15 hospitals. Results: Among 394 isolates, K. pneumoniae carbepenemase (KPC) remained the most prevalent carbapenemase (66.5%). NDM was the second most prevalent (13.7%), identified in 12 hospitals, followed by VIM (8.6%). OXA-48- and double carbapenemase-producers remained rare (3.6%, 6.3%, respectively). Carbapenemase-producing K. pneumoniae isolates showed high resistance to last-line antibiotics. Gentamicin and colistin were the most active in vitro with 61.9% and 59.6% of the isolates to be inhibited at <= 2mg/L, followed by fosfomycin (susceptibility (S): 58.4%) and tigecycline (S:51.5%). Ceftazidime/avibactam inhibited 99.6% of KPC and 100% of OXA-48-like-producing isolates, while temocillin was active against 58% of KPC isolates at urinary breakpoint of <= 2mg/L and only 2.7% at systemic breakpoint of <= 8mg/L. NDM-producing isolates belonged mainly to one clone, whereas KPC, VIM, OXA-48 and double carbapenemase-producers were mainly polyclonal. Conclusions: KPC remains the predominant carbapenemase among K. pneumoniae in Greece, followed by NDM, whereas changing trends of resistance rates to last-line antimicrobials against carbapenemase-producing K. pneumoniae with the exception of ceftazidime/avibactam mandates continuing surveillance to support clinical practice