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Background: This study provides a comprehensive analysis of Acinetobacter baumannii in aquatic environments and fish microbiota by integrating culture-dependent methods, 16S metagenomics, and antibiotic resistance profiling., Methods: A total of 83 A. baumannii isolates were recovered using culture-dependent methods from intra-hospital infections (IHI) and wastewater (WW) and surface water (SW) samples from two southern Romanian cities in August 2022. The antibiotic susceptibility was screened using disc diffusion, microdilution, PCR, and Whole Genome Sequencing assays., Results: The highest microbial load in the analyzed samples was found in Glina, Bucharest, for both WW and SW samples across all investigated phenotypes. For Bucharest isolates, the resistance levels corresponded to fluoroquinolones > aminoglycosides > β-lactam antibiotics. In contrast, A. baumannii from upstream SW samples in Târgoviște showed the highest resistance to aminoglycosides. The bla OXA-23 gene was frequently detected in IHI, WW, and SW isolates in Bucharest, but was absent in Târgoviște. Molecular phylogeny revealed the presence of ST10 in Târgoviște isolates and ST2 in Bucharest isolates, while other minor STs were not specifically correlated with a sampling point. Using 16S rRNA sequencing, significant differences in microbial populations between the two locations was identified. The low abundance of Alphaproteobacteria and Actinobacteria in both locations suggests environmental pressures or contamination events., Conclusions: These findings indicate significant fecal contamination and potential public health risks, emphasizing the need for improved water quality monitoring and management.

Acinetobacter baumannii , a notorious opportunistic pathogen, presents a formidable challenge in both clinical and environmental fields due to its resilience and ability to acquire resistance. This study undertook a comprehensive analysis of 183 A. baumannii isolates collected between 2019 and 2022 from intra-hospital infections (IHI), hospital sewages (Hs), wastewater treatment plants (WWTP), and adjacent river waters from two Southern cities, focusing on their resistome, virulome, and mobilome through isolation on chromogenic media, identification by MALDI-TOF-MS and antibiotic susceptibility testing by disk diffusion) followed by genotypic characterization [Whole Genome Sequencing (WGS), 3rd generation sequencing through the MinION (ONT) platform, pangenome description, and respectively horizontal gene transfer through conjugation assays]. Our findings reveal significant genomic plasticity and the prevalence of high-risk international clones, underlining the potential of these isolates to act as reservoirs for antibiotic resistance genes (ARGs) that could be dynamically exchanged between clinical and environmental settings through mobile genetic elements (MGEs) such as the pMAL1 plasmids and the critical role of WWTPs in the persistence and spread of A. baumannii . Moreover, our study presents the first report of the co-occurrence of bla OXA-23 and bla OXA-72 in A. baumannii ST2 clone. Thus, our research underscores the necessity for integrated surveillance and targeted interventions across healthcare and environmental sectors to mitigate the risk posed by this adaptable pathogen., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Author(s).)

The emerging opportunistic fungal pathogen Candida auris raises significant concerns for public health due to its outbreak potential, the associated high mortality, increased resistance to antifungals, challenging identification to species level, since commonly used diagnostic methods can confuse this fungus with other Candida spp. The present outbreak report describes probably some of the first Candida auris cases in Romania, providing clinical and epidemiological data, and also whole genome sequencing data. The cases were identified in three hospitals in Bucharest during the first eight months of 2022., (© 2023. BioMed Central Ltd., part of Springer Nature.)

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 bla OXA-23 , bla OXA-24 , bla SHV , bla TEM and bla GES , while Pa strains bla IMP , bla VIM , bla NDM , bla VEB , bla GES and bla TEM , 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 bla OXA-23 or bla OXA-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., (© 2022. The Author(s).)

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.

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., Competing Interests: OS, Anca S-C, and Adrian S-C report being investigators in influenza clinical trials by Shionogi and F. Hoffmann-La Roche, outside the scope of the submitted work. No other authors have competing interests to declare.

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.

Acinetobacter baumannii has emerged worldwide as a dominant pathogen in a broad range of severe infections, raising an acute need for efficient antibacterials. This is the first report on the resistome and virulome of 33 extended drug-resistant and carbapenem-resistant A. baumannii (XDR CRAB) strains isolated from hospitalized and ambulatory patients in Bucharest, Romania. A total of 33 isolates were collected and analyzed using phenotypic antibiotic susceptibility and conjugation assays, PCR, whole-genome sequencing (WGS), pulsed-field gel electrophoresis (PFGE) and MultiLocus Sequence Typing (MLST). All isolates were extensively drug-resistant (XDR), being susceptible only to colistin. The carbapenem resistance was attributed by PCR mainly to bla OXA-24 and bla OXA-23 genes. PFGE followed by MLST analysis demonstrated the presence of nine pulsotypes and six sequence types. WGS of seven XDR CRAB isolates from healthcare-associated infections demonstrated the high diversity of resistance genes repertoire, as well as of mobile genetic elements, carrying ARGs for aminoglycosides, sulphonamides and macrolides. Our data will facilitate the understanding of resistance, virulence and transmission features of XDR AB isolates from Romanian patients and might be able to contribute to the implementation of appropriate infection control measures and to develop new molecules with innovative mechanisms of action, able to fight effectively against these bugs, for limiting the spread and decreasing the infection rate and mortality.

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 [ bla CTX-M- 15 , bla OXA- 48 , bla OXA- 1 , bla SHV- 106 , bla TEM- 150 , aac(3)-IIa , aac(6')-Ib-cr , oqx A10, oqx B17, fos A, cat B3, dfr A14, tet (D)] and chromosomal mutations involved in AR (Δ mgrB , Δ ompK35 , 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Popa, Gheorghe, Barbu, Surleac, Paraschiv, Măruţescu, Popa, Pîrcălăbioru, Talapan, Niţă, Streinu-Cercel, Streinu-Cercel, Oţelea and Chifiriuc.)

Background: The spread of SARS-CoV-2 generated an unprecedented global public health crisis. Soon after Asia, Europe was seriously affected. Many countries, including Romania, adopted lockdown measures to limit the outbreak., Aim: We performed a molecular epidemiology analysis of SARS-CoV-2 viral strains circulating in Romania during the first two months of the epidemic in order to detect mutation profiles and phylogenetic relatedness., Methods: Respiratory samples were directly used for shotgun sequencing., Results: All Romanian sequences belonged to lineage B, with a different subtype distribution between northern and southern regions (subtype B.1.5 and B.1.1). Phylogenetic analysis suggested that the Romanian epidemic started with multiple introduction events from other European countries followed by local transmission. Phylogenetic links between northern Romania and Spain, Austria, Scotland and Russia were observed, as well as between southern Romania and Switzerland, Italy, France and Turkey. One viral strain presented a previously unreported mutation in the Nsp2 gene, namely K489E. Epidemiologically-defined clusters displayed specific mutations, suggesting molecular signatures for strains coming from areas that were isolated during the lockdown., Conclusions: Romanian epidemic was initiated by multiple introductions from European countries followed by local transmissions. Different subtype distribution between northern and southern Romania was observed after two months of the pandemic.

We report on the genomic characterization of 47 multi-drug resistant, carbapenem resistant and ESBL-producing K. pneumoniae isolates from the influent (I) and effluent (E) of three wastewater treatment plants (WWTPs) and from Romanian hospital units which are discharging the wastewater in the sampled WWTPs. The K. pneumoniae whole genome sequences were analyzed for antibiotic resistance genes (ARGs), virulence genes and sequence types (STs) in order to compare their distribution in C, I and E samples. Both clinical and environmental samples harbored prevalent and widely distributed ESBL genes, i.e. blaSHV, blaOXA, blaTEM and blaCTX M. The most prevalent carbapenemase genes were blaNDM-1, blaOXA-48 and blaKPC-2. They were found in all types of isolates, while blaOXA-162, a rare blaOXA-48 variant, was found exclusively in water samples. A higher diversity of carbapenemases genes was seen in wastewater isolates. The aminoglycoside modifying enzymes (AME) genes found in all types of samples were aac(6'), ant(2'')Ia, aph(3'), aaD, aac(3) and aph(6). Quinolone resistance gene qnrS1 and the multi-drug resistance oqxA/B pump gene were found in all samples, while qnrD and qnrB were associated to aquatic isolates. The antiseptics resistance gene qacEdelta1 was found in all samples, while qacE was detected exclusively in the clinical ones. Trimethroprim-sulfamethoxazole (dfrA, sul1 and sul2), tetracyclines (tetA and tetD) and fosfomycin (fosA6, known to be located on a transpozon) resistance genes were found in all samples, while for choramphenicol and macrolides some ARGs were detected in all samples (catA1 and catB3 / mphA), while other (catA2, cmIA5 and aac(6')Ib / mphE and msrE) only in wastewater samples. The rifampin resistance genes arr2 and 3 (both carried by class I integrons) were detected only in water samples. The highly prevalent ARGs preferentially associating with aquatic versus clinical samples could ascribe potential markers for the aquatic (blaSHV-145, qacEdelta1, sul1, aadA1, aadA2) and clinical (blaOXA-1, blaSHV-106,blaTEM-150, aac(3)Iia, dfrA14, oqxA10; oqxB17,catB3, tetD) reservoirs of AR. Moreover, some ARGs (oqxA10; blaSHV-145; blaSHV-100, aac(6')Il, aph(3')VI, armA, arr2, cmlA5, blaCMY-4, mphE, msrE, oqxB13, blaOXA-10) showing decreased prevalence in influent versus effluent wastewater samples could be used as markers for the efficiency of the WWTPs in eliminating AR bacteria and ARGs. The highest number of virulence genes (75) was recorded for the I samples, while for E and C samples it was reduced to half. The most prevalent belong to three functional groups: adherence (fim genes), iron acquisition (ent, fep, fyu, irp and ybt genes) and the secretion system (omp genes). However, none of the genes associated with hypervirulent K. pneumoniae have been found. A total of 14 STs were identified. The most prevalent clones were ST101, ST219 in clinical samples and ST258, ST395 in aquatic isolates. These STs were also the most frequently associated with integrons. ST45 and ST485 were exclusively associated with I samples, ST11, ST35, ST364 with E and ST1564 with C samples. The less frequent ST17 and ST307 aquatic isolates harbored blaOXA-162, which was co-expressed in our strains with blaCTX-M-15 and blaOXA-1., Competing Interests: The authors have declared that no competing interests exist.

Transposases are ubiquitous mobile genetic elements responsible for genome development, driving rearrangements, such as insertions, deletions and translocations. Across species evolution, some transposases are tamed by their host and are made part of complex cellular systems. The proliferation of retroviruses is also dependent on transposase related enzymes termed integrases. Recombination‑activating gene protein (RAG)1 and metnase are just two examples of transposase domestication and together with retroviral integrases (INs), they belong to the DDE polynucleotidyl transferases superfamily. They share mechanistic and structural features linked to the RNase H‑like fold, harboring a DDE(D) metal dependent catalytic motif. Recent antiretroviral compounds target the catalytic domain of integrase, but they also have the potential of inhibiting other related enzymes. In this review, we report the activity of different classes of integrase inhibitors on various DDE transposases. Computational simulations are useful to predict the extent of off‑target activity and have been employed to study the interactions between RAG1 recombinase and compounds from three different pharmacologic classes. We demonstrate that strand‑transfer inhibitors display a higher affinity towards the RAG1 RNase H domain, as suggested by experimental data compared to allosteric inhibitors. While interference with RAG1 and 2 recombination is associated with a negative impact on immune function, the inhibition of metnase or HTLV‑1 integrase opens the way for the development of novel therapies for refractory cancers.

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., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

In all jawed vertebrates RAG (recombination activating gene) recombinase orchestrates V(D)J recombination in B and T lymphocyte precursors, assembling the V, D and J germline gene segments into continuous functional entities which encode the variable regions of their immune receptors. V(D)J recombination is the process by which most of the diversity of our specific immune receptors is acquired and is thought to have originated by domestication of a transposon in the genome of a vertebrate.  RAG acts similarly to the cut and paste transposases, by first binding two recombination signal DNA sequences (RSSs), which flank the two coding genes to be adjoined, in a process called synaptic or paired complex (PC) formation. At these RSS-coding borders, RAG first nicks one DNA strand, then creates hairpins, thus cleaving the duplex DNA at both RSSs. Although RAG reaction mechanism resembles that of insect mobile element transposases and RAG itself can inefficiently perform intramolecular and intermolecular integration into the target DNA, inside the nuclei of the developing lymphocytes transposition is extremely rare and is kept under proper surveillance. Our review may help understand how RAG synaptic complex organization prevents deleterious transposition. The phosphoryl transfer reaction mechanism of RNAseH-like fold DDE motif enzymes, including RAG, is discussed accentuating the peculiarities described for various transposases from the light of their available high resolution structures (Tn5, Mu, Mos1 and Hermes). Contrasting the structural 3D organization of DNA in these transpososomes with that of the RSSs-DNA in RAG PC allows us to propose several clues for how evolutionarily RAG may have become "specialized" in recombination versus transposition., Competing Interests: Conflict of interests: There are no conflicts of interest., (Copyright © 2014, Applied Systems.)