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Background Romania is one of the European countries reporting very high antimicrobial resistance (AMR) rates and consumption of antimicrobials. We aimed to characterize the AMR profiles and clonality of 304 multi-drug resistant (MDR) Acinetobacter baumannii (Ab) and Pseudomonas aeruginosa (Pa) strains isolated during two consecutive years (2018 and 2019) from hospital settings, hospital collecting sewage tanks and the receiving wastewater treatment plants (WWTPs) located in the main geographical regions of Romania. Methods The strains were isolated on chromogenic media and identified by MALDI-TOF-MS. Antibiotic susceptibility testing and confirmation of ESBL- and CP- producing phenotypes and genotypes were performed. The genetic characterization also included horizontal gene transfer experiments, whole-genome sequencing (WGS), assembling, annotation and characterization. Results Both clinical and aquatic isolates exhibited high MDR rates, especially the Ab strains isolated from nosocomial infections and hospital effluents. The phenotypic resistance profiles and MDR rates have largely varied by sampling point and geographic location. The highest MDR rates in the aquatic isolates were recorded in Galați WWTP, followed by Bucharest. The Ab strains harbored mostly blaOXA-23, blaOXA-24, blaSHV, blaTEM and blaGES, while Pa strains blaIMP, blaVIM, blaNDM, blaVEB, blaGES and blaTEM, with high variations depending on the geographical zone and the sampling point. The WGS analysis revealed the presence of antibiotic resistance genes (ARGs) to other antibiotic classes, such as aminoglycosides, tetracyclines, sulphonamides, fosfomycin, phenicols, trimethoprim-sulfamethoxazole as well as class 1 integrons. The molecular analyses highlighted: (i) The presence of epidemic clones such as ST2 for Ab and ST233 and ST357 for Pa; (ii) The relatedness between clinical and hospital wastewater strains and (iii) The possible dissemination of clinical Ab belonging to ST2 (also proved in the conjugation assays for blaOXA-23 or blaOXA-72 genes), ST79 and ST492 and of Pa strains belonging to ST357, ST640 and ST621 in the wastewaters. Conclusion Our study reveals the presence of CP-producing Ab and Pa in all sampling points and the clonal dissemination of clinical Ab ST2 strains in the wastewaters. The prevalent clones were correlated with the presence of class 1 integrons, suggesting that these isolates could be a significant reservoir of ARGs, being able to persist in the environment.

The occurrence of the omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has importantly impacted surveillance and diagnosis, and has changed the therapeutic landscape of coronavirus disease 2019 (COVID-19). We present the first documented case of locally acquired SARS-CoV-2 omicron variant in Romania in a patient with no recent travel outside the country. We also present the full results of the epidemiological investigation that led to the identification of the index case in a co-worker who had developed mild symptoms shortly after returning from the UK and who had undergone multiple rapid antigen tests with negative results prior to being tested by RT-PCR. We highlight potential lessons learned and describe further directions for actionable research and development in the field of COVID-19.

Currently, used antiretroviral HIV therapy drugs exclusively target critical groups in the enzymes essential for the viral life cycle. Increased mutagenesis of their genes changes these viral enzymes, which once mutated can evade therapeutic targeting, effects which confer drug resistance. To circumvent this, our review addresses a strategy to design and derive HIV-Integrase (HIV-IN) inhibitors which simultaneously target two IN functional domains, rendering it inactive even if the enzyme accumulates many mutations. First we review the enzymatic role of IN to insert the copied viral DNA into a chromosome of the host T lymphocyte, highlighting its main functional and structural features to be subjected to inhibitory action. From a functional and structural perspective we present all classes of HIV-IN inhibitors with their most representative candidates. For each chosen compound we also explain its mechanism of IN inhibition. We use the recently resolved cryo EM IN tetramer intasome DNA complex onto which we dock various reference IN inhibitory chemical scaffolds such as to target adjacent functional IN domains. Pairing compounds with complementary activity, which dock in the vicinity of a IN structural microdomain, we design bifunctional new drugs which may not only be more resilient to IN mutations but also may be more potent inhibitors than their original counterparts. In the end of our review we propose synthesis pathways to link such paired compounds with enhanced synergistic IN inhibitory effects.

New SARS-CoV-2 variants are constantly emerging and putting a strain on public health systems by spreading faster and potentially evading immune protection through vaccination. One of these strains is the B.1.1.7 variant that has initially been described in the United Kingdom and has subsequently spread to several countries. Monitoring the amplification of the S gene-a major hotspot for molecular evolution-by reverse transcription polymerase chain reaction (RT-PCR) allows rapidly screening for such variants. This report describes the detection of sequence variants in Romania by using this strategy followed by next-generation sequencing of the entire genome for confirmation and further characterization. One B.1.1.7 and three B.1.258 sequences were confirmed. Each of these strains presented additional mutations with possible impact on the replicative capacity. Public health strategies should be devised to ensure molecular monitoring of SARS-CoV-2 evolution during the pandemic and allow adequate and rapid reaction.

EDEM3 recognizes and directs misfolded proteins to the ER-associated protein degradation (ERAD) process. EDEM3 was predicted to act as lectin or as a mannosidase because of its homology with the GH47 catalytic domain of the Man1B1, but the contribution of the other regions remained unresolved. Here, we dissect the molecular determinants governing EDEM3 function and its cellular interactions. LC/MS analysis indicates very few stable ER interactors, suggesting EDEM3 availability for transient substrate interactions. Sequence analysis reveals that EDEM3 consists of four consecutive modules defined as GH47, intermediate (IMD), protease-associated (PA), and intrinsically disordered (IDD) domain. Using an EDEM3 knock-out cell line, we expressed EDEM3 and domain deletion mutants to address EDEM3 function. We find that the mannosidase domain provides substrate binding even in the absence of mannose trimming and requires the IMD domain for folding. The PA and IDD domains deletions do not impair the trimming, but specifically modulate the turnover of two misfolded proteins, NHK and the soluble tyrosinase mutant. Hence, we demonstrate that EDEM3 provides a unique ERAD timing to misfolded glycoproteins, not only by its mannose trimming activity, but also by the positive and negative feedback modulated by the protease-associated and intrinsically disordered domain, respectively.

In this paper we describe the transmission of a multi-drug resistant Klebsiella pneumoniae ST101 clone from hospital to wastewater and its persistence after chlorine treatment. Water samples from influents and effluents of the sewage tank of an infectious diseases hospital and clinical strains collected from the intra-hospital infections, during a period of 10 days prior to wastewater sampling were analyzed. Antibiotic resistant K. pneumoniae strains from wastewaters were recovered on selective media. Based on antibiotic susceptibility profiles and PCR analyses of antibiotic resistance (AR) genetic background, as well as whole-genome sequencing (Illumina MiSeq) and subsequent bioinformatic analyses, 11 ST101 K. pneumoniae strains isolated from hospital wastewater influent, wastewater effluent and clinical sector were identified as clonally related. The SNP and core genome analyses pointed out that five strains were found to be closely related (with ≤18 SNPs and identical cgMLST profile). The strains belonging to this clone harbored multiple acquired AR genes [blaCTX–M–15, blaOXA–48, blaOXA–1, blaSHV–106, blaTEM–150, aac(3)-IIa, aac(6′)-Ib-cr, oqxA10, oqxB17, fosA, catB3, dfrA14, tet(D)] and chromosomal mutations involved in AR (ΔmgrB, ΔompK35, amino acid substitutions in GyrA Ser83Tyr, Asp87Asn, ParC Ser80Tyr). Twenty-nine virulence genes involved in iron acquisition, biofilm and pili formation, adherence, and the type six secretion system – T6SS-III were identified. Our study proves the transmission of MDR K. pneumoniae from hospital to the hospital effluent and its persistence after the chlorine treatment, raising the risk of surface water contamination and further dissemination to different components of the trophic chain, including humans.

Neurofibromatosis type 1 (NF1) is caused by mutations in the NF1 gene encoding neurofibromin, which negatively regulates Ras signaling. NF1 patients have an increased risk of developing early onset breast cancer, however, the association between NF1 and high grade serous ovarian cancer (HGSOC) is unclear. Since most NF1-related tumors exhibit early biallelic inactivation of NF1, we evaluated the evolution of genetic alterations in HGSOC in an NF1 patient. Somatic variation analysis of whole exome sequencing of tumor samples from both ovaries and a peritoneal metastasis showed a clonal lineage originating from an ancestral clone within the left adnexa, which exhibited copy number (CN) loss of heterozygosity (LOH) in the region of chromosome 17 containing TP53, NF1, and BRCA1 and mutation of the other TP53 allele. This event led to biallelic inactivation of NF1 and TP53 and LOH for the BRCA1 germline mutation. Subsequent CN alterations were found in the dominant tumor clone in the left ovary and nearly 100% of tumor at other sites. Neurofibromin modeling studies suggested that the germline NF1 mutation could potentially alter protein function. These results demonstrate early, biallelic inactivation of neurofibromin in HGSOC and highlight the potential of targeting RAS signaling in NF1 patients., Highlights • A link between neurofibromatosis type 1 and ovarian cancer has emerged. • A genetic study of high grade serous ovarian cancer in a patient with NF1 is presented. • Biallelic inactivation of the NF1 gene is an early event in HGSOC tumorigenesis. • The NF1 pathway is a potential therapeutic target in women with NF1 that develop HGSOC.

Two main mechanisms contribute to the continuous evolution of influenza viruses: accumulation of mutations in the hemagglutinin and neuraminidase genes (antigenic drift) and genetic re-assortments (antigenic shift). Epidemiological surveillance is important in identifying new genetic variants of influenza viruses with potentially increased pathogenicity and transmissibility. In order to characterize the 2019/20 influenza epidemic in Romania, 1042 respiratory samples were collected from consecutive patients hospitalized with acute respiratory infections in the National Institute for Infectious Diseases “Prof. Dr. Matei Balș”, Bucharest Romania and tested for influenza A virus, influenza B virus and respiratory syncytial virus (RSV) by real-time PCR. Out of them, 516 cases were positive for influenza, with relatively equal distribution of influenza A and B. Two patients had influenza A and B co-infection and 8 patients had influenza-RSV co-infection. The most severe cases, requiring supplemental oxygen administration or intensive care, and the most deaths were reported in patients aged 65 years and over. Subtyping showed the predominance of A(H3N2) compared to A(H1N1)pdm09 pdm09 (60.4% and 39.6% of all subtyped influenza A isolates, respectively), and the circulation of Victoria B lineage only. Influenza B started to circulate first (week 47/2019), with influenza A appearing slightly later (week 50/2019), followed by continued co-circulation of A and B viruses throughout the season. Sixty-eight samples, selected to cover the entire influenza season and all circulating viral types, were analysed by next generation sequencing (NGS). All A(H1N1)pdm09 sequences identified during this season in Romania were clustered in the 6b1.A clade (sub-clades: 6b1.A.183P -5a and 6b1.A.187A). For most A(H1N1)pdm09 sequences, the dominant epitope was Sb (pepitope = 0.25), reducing the vaccine efficacy by approximately 60%. According to phylogenetic analysis, influenza A(H3N2) strains circulating in this season belonged predominantly to clade 3C.3A, with only few sequences in clade 3C.2A1b. These 3C.2A1b sequences, two of which belonged to vaccinated patients, harbored mutations in antigenic sites leading to potential reduction of vaccine efficacy. Phylogenetic analysis of influenza B, lineage Victoria, sequences showed that the circulating strains belonged to clade V1A3. As compared to the other viral types, fewer mutations were observed in B/Victoria strains, with limited impact on vaccine efficiency based on estimations.

Emerging SARS-CoV-2 strains continue to generate difficulties for authorities and health care professionals worldwide due to enhanced transmissibility and/or immune response evasion. The appearance of the Alpha and Delta strains has been associated with substantial increases in the number of COVID-19 cases and associated deaths. Whole Genome Sequencing (WGS) continues to be the gold standard for molecular surveillance of the pandemics but other assays such as mutation genotyping can be used to reduce costs and allocated time. This study investigates the efficiency of mutation screening tests compared to WGS and their predictive value to anticipate future waves. A very high degree of fidelity for this type of assay was found, regardless of the method used. The positive predictive value (PPV) of 4/5 markers was over 95% for the detection of Alpha and Delta variants. By estimating the prevalence of the Alpha and Delta strains using genotyping assays and fitting the data to a mathematical model, a five week period between the point of exponential growth of variant prevalence and a drastic increase in case numbers was found. For that reason, raising awareness about the efficacy of mutation screening could help authorities adopt better measures in the future.

Topoisomerase (topo) IIα and IIβ maintain genome stability and are targets for anti-tumor drugs. In this study, we demonstrate that the decatenation checkpoint is regulated, not only by topo IIα, as previously reported, but also by topo IIβ. The decatenation checkpoint is most efficient when both isoforms are present. Regulation of this checkpoint and sensitivity to topo II-targeted drugs is influenced by the C-terminal domain (CTD) of the topo II isoforms and by a conserved non-catalytic tyrosine, Y640 in topo IIα and Y656 in topo IIβ. Deletion of most of the CTD of topo IIα, while preserving the nuclear localization signal (NLS), enhances the decatenation checkpoint and sensitivity to topo II-targeted drugs. In contrast, deletion of most of the CTD of topo IIβ, while preserving the NLS, and mutation of Y640 in topo IIα and Y656 in topo IIβ inhibits these activities. Structural studies suggest that the differential impact of the CTD on topo IIα and topo IIβ function may be due to differences in CTD charge distribution and differential alignment of the CTD with reference to transport DNA. Together these results suggest that topo IIα and topo IIβ cooperate to maintain genome stability, which may be distinctly modulated by their CTDs.

Romania has faced an HIV outbreak among people who inject drugs (PWID) since 2011. The introduction of so-called ‘legal highs’ (amphetamine-type stimulants) on the drug market a few years prior contributed substantially to this outbreak. Next-generation sequencing (NGS) provides the possibility to detect drug resistance mutations with higher sensitivity than Sanger sequencing. The aim of this study was to search for transmitted drug resistance (TDR) mutations in strains from PWID recently diagnosed with HIV infection by parallel use of Sanger sequencing and NGS. The study was conducted on strains from 34 PWID diagnosed with HIV infection between 2016 and 2017. Sequencing was performed for the pol (PR, RT, and INT) and env (V2-V3 loop) regions. Sanger sequencing was performed with the commercial ViroseqTMHIV-1 Genotyping system (Abbott Laboratories) and with an in-house protocol for the env gene. NGS was performed in the same genomic regions using Nextera DNA Library Preparation Kit (Illumina) and the Miseq instrument (Illumina). NGS data were processed for error correction, read mapping, and detection of drug resistance mutations with HIV-1 Deepchek analysis software. Geno2pheno algorithm was used for viral tropism prediction and the WHO 2009 list for TDRM analysis. By using NGS, we detected seven cases (20.6%) of TDR in PWID and only two cases (5.8%) with standard sequencing. The TDR mutations detected by NGS were K103N, K101EN, Y181C, T215S in RT gene, I54V and M46L in PR, and none in INT. Two NNRTI mutations (K103N and K101EN) were detected in the same sample. Most of the TDR identified were present in the minority population (between 1% and 2% of the total reads) explaining the higher sensitivity of NGS method compared with standard sequencing. No significant differences were observed between these two methods when tropism prediction was analyzed. The majority of the viruses circulating in this group were R5-tropic. All strains showed more resistance mutations when analyzed by deep sequencing than by Sanger sequencing and more than previously observed in other risk groups. NGS proved to be a sensitive tool to detect TDR in newly infected PWID.

V(D)J recombination is initiated by RAG1 and RAG2, which together with HMGB1 bind to a recombination signal sequence (12RSS or 23RSS) to form the signal complex (SC) and then capture a complementary partner RSS, yielding the paired complex (PC). Little is known regarding the structural changes that accompany the SC to PC transition or the structural features that allow RAG to distinguish its two asymmetric substrates. To address these issues, we analyzed the structure of the 12RSS in the SC and PC using fluorescence resonance energy transfer (FRET) and molecular dynamics modeling. The resulting models indicate that the 12RSS adopts a strongly bent V-shaped structure upon RAG/HMGB1 binding and reveal structural differences, particularly near the heptamer, between the 12RSS in the SC and PC. Comparison of models of the 12RSS and 23RSS in the PC reveals broadly similar shapes but a distinct number and location of DNA bends as well as a smaller central cavity for the 12RSS. These findings provide the most detailed view yet of the 12RSS in RAG–DNA complexes and highlight structural features of the RSS that might underlie activation of RAG-mediated cleavage and substrate asymmetry important for the 12/23 rule of V(D)J recombination.

Decapping scavenger (DcpS) assists in precluding inhibition of cap-binding proteins by hydrolyzing cap species remaining after mRNA 3'→5' degradation. Its significance was reported in splicing, translation initiation and microRNA turnover. Here we examine the structure and binding mode of DcpS from Caenorhabditis elegans (CeDcpS) using a large collection of chemically modified methylenebis(phosphonate), imidodiphosphate and phosphorothioate cap analogs. We determine that CeDcpS is a homodimer and propose high accuracy structural models of apo- and m(7) GpppG-bound forms. The analysis of CeDcpS regioselectivity uncovers that the only site of hydrolysis is located between the β and γ phosphates. Structure-affinity relationship studies of cap analogs for CeDcpS reveal molecular determinants for efficient cap binding: a strong dependence on the type of substituents in the phosphate chain, and reduced binding affinity for either methylated hydroxyl groups of m(7) Guo or an extended triphosphate chain. Docking analysis of cap analogs in the CeDcpS active site explains how both phosphate chain mobility and the orientation in the cap-binding pocket depend on the number of phosphate groups, the substituent type and the presence of the second nucleoside. Finally, the comparison of CeDcpS with its well known human homolog provides general insights into DcpS-cap interactions.