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We present plasmid sequences of 21 multidrug resistant isolates of Enterobacterales belonging to Escherichia coli (n=10), Klebsiella pneumoniae (n=9), Klebsiella oxytoca (n=1), and Citrobacter freundii (n=1). The isolates originated from effluent collected from hospital sewer pipes and from a wastewater treatment plant (WWTP) in a southwestern Hungarian city. Isolation was carried out using eosin methylene blue agar supplemented with ceftriaxone and the isolates were identified with MALDI-TOF MS. Screening for multidrug resistance was conducted by determining susceptibility to four chemical classes namely, beta-lactams, aminoglycoside, fluoroquinolone, and sulfonamide. Plasmid DNA was isolated by alkaline lysis method using the Monarch plasmid DNA miniprep kit from freshly grown pure colonies. Molecular typing and Illumina sequencing of plasmid DNA of multiresistant strains were performed. After the assembly of contigs, genes localized on plasmid sequences were determined and functionally annotated. These reconstructed plasmid sequences supplemented with gene functional annotations were deposited in the Mendeley data. Using these datasets different plasmid incompatibility groups were identified. These conjugative plasmids appear to play a key role in the transmission of multiple resistance genes in enteric bacteria via wastewater. The presented data may provide useful insight on the correlations between environmental antibiotic contamination and the development of bacterial resistance, which poses a serious public health threat.

Exercise initiates systemic adaptation to promote health and prevent various lifestyle-related chronic diseases. Emerging evidence suggests that circulating exosomes mediate some of the beneficial effects of exercise via the transfer of microRNAs between tissues. Yet to date, a comprehensive profile of the exosomal miRNA (exomiR) content released following short-term (0.5 year in this study) and long-term (25 + years in this study) regular bouts of exercise is still lacking. However, a better understanding of these miRNA species would assist in clarifying the role of regular exercise at the molecular level in the prevention of chronic diseases. In the present pilot studies we analyzed serum exomiR expression in healthy young, sedentary participants (n = 14; age: 23 ± 2 years) at baseline and following a half year-long moderate-intensity regular exercise training. We also analyzed serum exomiR expression in older, healthy trained participants (seniors, n = 11; age: 62 ± 6 years) who engaged in endurance activities for at least 25 years. Following the isolation and enrichment of serum exosomes using Total Exosome Isolation Reagent (TEI) their exomiR levels were determined using the amplification-free Nanostring platform. Hierarchical cluster analysis revealed that the majority of exomiRs overlap for short-term (0.5 year in this study) and long-term (25 + years in this study) regular bouts of exercise. The top 12 significantly altered exomiRs (let-7a-5p; let-7g-5p; miR-130a-3p; miR-142-3p; miR-150-5p; miR-15a-5p; miR-15b-5p; miR-199a-3p; miR-199b-3p; miR-223-3p; miR-23a-3p, and miR-451a-3p) were used for further evaluation. According to KEGG pathway analysis a large portion of the exomiRs target chronic diseases including cancer, neurodegenerative and metabolic diseases, and viral infections. Our results provide evidence that exosomal miRNA modulation is the molecular mechanism through which regular exercise prevents various chronic diseases. The possibility of using such exomiRs to target diseases is of great interest. While further validation is needed, our comprehensive exomiR study presents, for the first time, the disease-preventive molecular pattern of both short and long-term regular exercise.

Antimicrobials in wastewater promote the emergence of antibiotic resistance, facilitated by selective pressure and transfer of resistant genes. Enteric bacteria belonging to Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Citrobacter species (n = 126) from hospital effluents and proximate wastewater treatment plant were assayed for susceptibility to four antimicrobial classes. The β-lactamase encoding genes harbored in plasmids were genotyped and the plasmids were sequenced. A multidrug resistance phenotype was found in 72% (n = 58) of E. coli isolates, 70% (n = 43) of Klebsiella species isolates, and 40% (n = 25) of Enterobacter and Citrobacter species. Moreover, 86% (n = 50) of E. coli, 77% (n = 33) of Klebsiella species, and 25% (n = 4) of Citrobacter species isolates phenotypically expressed extended spectrum β-lactamase. Regarding ESBL genes, blaCTX-M-27 and blaTEM-1 were found in E. coli, while Klebsiella species harbored blaCTX-M-15, blaCTX-M-30, or blaSHV-12. Genes coding for aminoglycoside modifying enzymes, adenylyltransferases (aadA1, aadA5), phosphotransferases (aph(6)-1d, aph(3″)-Ib), acetyltransferases (aac(3)-IIa), (aac(6)-Ib), sulfonamide/trimethoprim resistant dihydropteroate synthase (sul), dihydrofolate reductase (dfrA), and quinolone resistance protein (qnrB1) were also identified. Monitoring wastewater from human sources for acquired resistance in clinically important bacteria may provide a cheaper alternative in regions facing challenges that limit clinical surveillance.

Studies support that regular physical activity (PA) decelerates senescence-related decline of physiological and molecular parameters in the elderly. We have addressed the other end of this spectrum: healthy and young, inactive individuals participated in a 6-month long personal trainer-guided lifestyle program. We have measured physiological and molecular parameters (differentiating high- and low responders) and their correlation with PA (sedentary status). Cluster analysis helped to distinguish individuals with high- or low PA and differentiate high- and low-responders of each parameter. The assessed cardiovascular parameters (heart rate, blood pressure, 6-min walking distance, relative VO2max), body composition parameters (body fat and muscle mass percentage) metabolic parameters (glucose, insulin, HDL, LDL), immune parameters (cortisol, CRP, lymphocyte counts, hTREC) all showed improvement. Artificial neural network analysis (ANN) showed correlation efficiencies of physiological and molecular parameters using a concept-free approach. ANN analysis appointed PA as the mastermind of molecular level changes. Besides sedentary status, insulin and hTREC showed significant segregation. Biostatistics evaluation also supported the schism of participants for their sedentary status, insulin concentration and hTREC copy number. In the future ANN and biostatistics, may predict individual responses to regular exercise. Our program reveals that high responder individuals of certain parameters may be low responders of others. Our data show that moderate regular PA is essential to counteract senescence in young and healthy individuals, despite individual differences in responsiveness. Such PA may not seem important in the everyday life of young and healthy adults, but shall become the base for healthy aging.

Abstract The emergence of newer SARS-CoV-2 variants of concern (VOCs) profoundly changed the ICU demography; this shift in the virus’s genotype and its correlation to lethality in the ICUs is still not fully investigated. We aimed to survey ICU patients’ clinical and laboratory parameters in correlation with SARS-CoV-2 variant genotypes to lethality. 503 COVID-19 ICU patients were included in our study beginning in January 2021 through November 2022 in Hungary. Furthermore, we implemented random forest (RF) as a potential predictor regarding SARS-CoV-2 lethality among 649 ICU patients in two ICU centers. Survival analysis and comparison of hypertension (HT), diabetes mellitus (DM), and vaccination effects were conducted. Logistic regression identified DM as a significant mortality risk factor (OR: 1.55, 95% CI 1.06–2.29, p = 0.025), while HT showed marginal significance. Additionally, vaccination demonstrated protection against mortality (p = 0.028). RF detected lethality with 81.42% accuracy (95% CI 73.01–88.11%, [AUC]: 91.6%), key predictors being PaO2/FiO2 ratio, lymphocyte count, and chest Computed Tomography Severity Score (CTSS). Although a smaller number of patients require ICU treatment among Omicron cases, the likelihood of survival has not proportionately increased for those who are admitted to the ICU. In conclusion, our RF model supports more effective clinical decision-making among ICU COVID-19 patients.

Caliciviruses including noro- and sapoviruses of family Caliciviridae are important enteric human and swine pathogens, while others, like valoviruses, are less known. In this study, we developed a detection and typing pipeline for the most prevalent swine enteric caliciviruses—sapovirus GIII (Sw-SaV), norovirus GII (Sw-NoV), and valovirus GI (Sw-VaV). The pipeline integrates triplex RT-qPCR, 3′RACE semi-nested PCR, and next-generation sequencing (NovaSeq, Illumina) techniques. A small-scale epidemiological investigation was conducted on archived enteric and, for the first time, on oral fluid/saliva samples of diarrheic and asymptomatic swine of varying ages from Hungary and Slovakia. In enteric samples, Sw-SaV was the most prevalent, detected in 26.26% of samples, primarily in diarrheic pigs with low Cq values, followed by Sw-NoV (2.53%) in nursery pigs. In oral fluid samples, Sw-NoV predominated (7.46%), followed by Sw-SaV (4.39%). Sw-VaVs were sporadically found in both sample types. A natural, asymptomatic Sw-SaV outbreak was retrospectively detected where the transient shedding of the virus was n = 59; 43 Sw-SaV, 13 Sw-NoV, and 3 Sw-VaV) including multiple (up to five) co-infecting variants were identified. Sw-SaV sequences belong to seven genotypes, while Sw-NoV and Sw-VaV strains clustered into distinct sub-clades, highlighting the complex diversity of these enteric caliciviruses in swine.

ABSTRACT In this study, the enteric virome of diarrheic caprine was investigated using next-generation sequencing, reverse transcription PCR methods, and bioinformatics analyses. The complete or nearly complete genomes of seven novel viruses including (i) a caprine astrovirus (OQ758025) of family Astroviridae, four picornaviruses in genera (ii) Boosepivirus (OQ758026), (iii) Enterovirus (OQ758028), (iv) Kobuvirus (OQ758029), and (v) an unassigned picornavirus provisionally called as capripivirus (from the term caprine picornavirus) of the family Picornaviridae (OQ758027), as well as (vi) a tusavirus of family Parvoviridae (OL692339.2) and an (vii) unassigned CRESS DNA virus (OQ758030) have been identified. Structural analyses of the internal ribosomal entry sites revealed the presence of unique motifs and suggest the modular exchange of certain elements between co-infecting viruses. Epidemiological investigations and genotyping PCR reactions of identified RNA virus groups were also conducted using multiple “in-house” developed SYBRgreen-based screening quantitative PCR (qPCR) assays and generic capsid primers on additional fecal samples from ruminant livestock species (n = 62 caprine, n = 32 ovine, and n = 94 cattle) of three different age groups from n = 12 geographically distant sampling sites in Hungary. The results of qPCR screening assays as well as K-means cluster and phylogenetic analyses of the determined sequences suggest that diverse members of the above RNA virus groups were found in all age groups, but mostly in

Abstract Background Recent data suggest that gene expression profiles of peripheral white blood cells can reflect changes in the brain. We aimed to analyze the transcriptome of peripheral blood mononuclear cells (PBMC) and changes of plasma metabolite levels of migraineurs in a self-controlled manner during and between attacks. Methods Twenty-four patients with migraine were recruited and blood samples were collected in a headache-free (interictal) period and during headache (ictal) to investigate disease- and headache-specific alterations. Control samples were collected from 13 age- and sex-matched healthy volunteers. RNA was isolated from PBMCs and single-end 75 bp RNA sequencing was performed using Illumina NextSeq 550 instrument followed by gene-level differential expression analysis. Functional analysis was carried out on information related to the role of genes, such as signaling pathways and biological processes. Plasma metabolomic measurement was performed with the Biocrates MxP Quant 500 Kit. Results We identified 144 differentially-expressed genes in PBMCs between headache and headache-free samples and 163 between symptom-free patients and controls. Network analysis revealed that enriched pathways included inflammation, cytokine activity and mitochondrial dysfunction in both headache and headache-free samples compared to controls. Plasma lactate, succinate and methionine sulfoxide levels were higher in migraineurs while spermine, spermidine and aconitate were decreased during attacks. Conclusions It is concluded that enhanced inflammatory and immune cell activity, and oxidative stress can play a role in migraine susceptibility and headache generation.

Background The mosquito Aedes koreicus (Edwards, 1917) is a recent invader on the European continent that was introduced to several new places since its first detection in 2008. Compared to other exotic Aedes mosquitoes with public health significance that invaded Europe during the last decades, this species’ biology, behavior, and dispersal patterns were poorly investigated to date. Methodology/Principal findings To understand the species’ population relationships and dispersal patterns within Europe, a fragment of the cytochrome oxidase I (COI or COX1) gene was sequenced from 130 mosquitoes, collected from five countries where the species has been introduced and/or established. Oxford Nanopore and Illumina sequencing techniques were combined to generate the first complete nuclear and mitochondrial genomic sequences of Ae. koreicus from the European region. The complete genome of Ae. koreicus is 879 Mb. COI haplotype analyses identified five major groups (altogether 31 different haplotypes) and revealed a large-scale dispersal pattern between European Ae. koreicus populations. Continuous admixture of populations from Belgium, Italy, and Hungary was highlighted, additionally, haplotype diversity and clustering indicate a separation of German sequences from other populations, pointing to an independent introduction of Ae. koreicus to Europe. Finally, a genetic expansion signal was identified, suggesting the species might be present in more locations than currently detected. Conclusions/Significance Our results highlight the importance of genetic research of invasive mosquitoes to understand general dispersal patterns, reveal main dispersal routes and form the baseline of future mitigation actions. The first complete genomic sequence also provides a significant leap in the general understanding of this species, opening the possibility for future genome-related studies, such as the detection of ‘Single Nucleotide Polymorphism’ markers. Considering its public health importance, it is crucial to further investigate the species’ population genetic dynamic, including a larger sampling and additional genomic markers.

Background: Effective testing is an essential tool for controlling COVID-19. We aimed to analyse the data from first-wave PCR test results in Hungary's Southern Transdanubian region to improve testing strategies.Methods: We performed a retrospective analysis of all suspected COVID-19 cases between 17 March and 8 May 2020, collecting epidemiological, demographic, clinical and outcome data (ICU admission and mortality) with RT-qPCR test results. Descriptive and comparative statistical analyses were conducted.Results: Eighty-six infections were confirmed among 3,657 tested patients. There was no difference between the positive and negative cases in age and sex distribution; however, ICU admission (8.1 vs. 3.1%, p = 0.006) and in-hospital mortality (4.7 vs. 1.6%, p = 0.062) were more frequent among positive cases. Importantly, none of the initially asymptomatic patients (n = 20) required ICU admission, and all survived. In almost all cases, if the first test was negative, second and third tests were performed with a 48-h delay for careful monitoring of disease development. However, the positive hit rate decreased dramatically with the second and third tests compared to the first (0.3 vs. 2.1%, OR = 0.155 [0.053–0.350]). Higher E-gene copy numbers were associated with a longer period of PCR positivity.Conclusion: In our immunologically naïve suspected COVID-19 population, coronavirus infection increased the need for intensive care and mortality by 3–4 times. In the event of the exponential phase of the pandemic involving a bottleneck in testing capacity, a second or third test should be reconsidered to diagnose more coronavirus infections.

BackgroundEarly childhood adversity is a strong predictor of the development of major depressive disorder (MDD), but not all depressed patients experience early life stress (ELS). Cardio-metabolic diseases and cognitive deficits often coincide in MDD and worsen its course and outcome. Adverse childhood experiences have been associated with elevated risk for cardiovascular disease (CVD), but little is known on the impact of ELS on cardiovascular risk factors in MDD. Here, we examined MDD patients with and without ELS to explore the effects of ELS on serum lipid and lipoprotein levels and on cognitive performances of the patients.MethodsParticipants with a mean age of 35 years (18–55 years) were recruited from the university mental health clinic and general community. Three groups, matched in age, gender and lifestyle were examined: MDD patients with ELS (n = 21), MDD patients without ELS (n = 21), and healthy controls (n = 20). The following CVD risk factors were assessed: serum lipids (total cholesterol, triglycerides, high- and low-density lipoproteins), body mass index and exercise in a typical week. MDD severity was measured by the Beck Depression Inventory. Childhood Trauma Questionnaire was used to assess early life adversities. Executive functions and attentional processes were assessed by the Wisconsin Card Sorting and Conners’ Continuous Performance tests.ResultsMajor depressive disorder patients with ELS had higher serum triglyceride and lower HDL-cholesterol concentrations compared to MDD patients without ELS. Linear regression analysis revealed that the severity of ELS had a significant negative association with HDL-cholesterol levels and significant positive associations with the serum levels of TG and TC/HDL-cholesterol index. We also found significant associations between some specific trauma types and lipid profiles. Finally, we could detect significant associations between depression severity and specific domains of the cognitive tests as well as between lipid profiles and certain domains of the Wisconsin Card Sorting Test. However, we could not detect any association between the severity of ELS and cognitive performance.ConclusionAfter controlling for depressive symptom severity and lifestyle variables, ELS was found to be a strong predictor of serum lipid alterations. Several, inter-correlated pathways may mediate the undesirable effects of ELS on the course and outcome of MDD.

To explore the SARS-CoV-2 pandemic in Algeria, a dataset comprising ninety-five genomes originating from SARS-CoV-2 sampled from Algeria and other countries worldwide, from 24 December 2019, through 4 March 2021, was thoroughly examined. While performing a multi-component analysis regarding the Algerian outbreak, the toolkit of phylogenetic, phylogeographic, haplotype, and genomic analysis were effectively implemented. We estimated the Time to the Most Recent Common Ancestor (TMRCA) in reference to the Algerian pandemic and highlighted the multiple introductions of the disease and the missing data depicted in the transmission loop. In addition, we emphasized the significant role played by local and international travels in disease dissemination. Most importantly, we unveiled mutational patterns, the effect of unique mutations on corresponding proteins, and the relatedness regarding the Algerian sequences to other sequences worldwide. Our results revealed individual amino-acid replacements such as the deleterious replacement A23T in the orf3a gene in Algeria_EPI_ISL_418241. Additionally, a connection between Algeria_EPI_ISL_420037 and sequences originating from the USA was observed through a USA characteristic amino-acid replacement T1004I in the nsp3 gene, found in the aforementioned Algerian sequence. Similarly, successful tracing could be established, such as Algeria/G37318-8849/2020|EPI_ISL_766863, which was imported from Saudi Arabia during the pilgrimage. Lastly, we assessed the Algerian mitigation measures regarding disease containment using statistical analyses.

The natural hosts of Orthohantaviruses are rodents, soricomorphs and bats, and it is well known that they may cause serious or even fatal diseases among humans worldwide. The virus is persistent among animals and it is shed via urine, saliva and feces throughout the entirety of their lives. We aim to identify the effectiveness of hantavirus detection in rodent tissue samples and urine originating from naturally infected rodents. Initially, animals were trapped at five distinct locations throughout the Transdanubian region in Hungary. Lung, liver, kidney and urine samples were obtained from 163 deceased animals. All organs and urine were tested using nested reverse transcription polymerase chain reaction (nRT-PCR). Furthermore, sera were examined for IgG antibodies against Dobrava–Belgrade virus (DOBV) and Puumala virus (PUUV) by Western blot assay. IgG antibodies against hantaviruses and/or nucleic acid were detected in 25 (15.3%) cases. Among Apodemus, Myodes, and Microtus rodent species, DOBV, PUUV and Tula virus (TULV) were clearly identified. Amid the PCR-positive samples, the nucleic acid of the viruses was detected most effectively in the kidney (100%), while only 55% of screened lung tissues were positive. Interestingly, only three out of 20 rodent urine samples were positive when tested using nRT-PCR. Moreover, five rodents were seropositive without detectable virus nucleic acid in any of the tested organs.

A fundamental question in evolutionary biology is how complex innovations requiring multiple genetic changes arise. Here the authors provide lines of evidence that changing environments facilitate the adaptive evolution of complex metabolic innovations via stepwise acquisition of single reactions.

Severe Acute Respiratory Syndrome Coronavirus 2 is the third highly pathogenic human coronavirus in history. Since the emergence in Hubei province, China, during late 2019, the situation evolved to pandemic level. Following China, Europe was the second epicenter of the pandemic. To better comprehend the detailed founder mechanisms of the epidemic evolution in Central-Eastern Europe, particularly in Hungary, we determined the full-length SARS-CoV-2 genomes from 32 clinical samples collected from laboratory confirmed COVID-19 patients over the first month of disease in Hungary. We applied a haplotype network analysis on all available complete genomic sequences of SARS-CoV-2 from GISAID database as of 21 April 2020. We performed additional phylogenetic and phylogeographic analyses to achieve the recognition of multiple and parallel introductory events into our region. Here, we present a publicly available network imaging of the worldwide haplotype relations of SARS-CoV-2 sequences and conclude the founder mechanisms of the outbreak in Central-Eastern Europe.

Medicinal plants are widely used in folk medicine but quite often their composition and biological effects are hardly known. Our study aimed to analyze the composition, cytotoxicity, antimicrobial, antioxidant activity and cellular migration effects of Anthyllis vulneraria, Fuchsia magellanica, Fuchsia triphylla and Lysimachia nummularia used in the Romanian ethnomedicine for wounds. Liquid chromatography with mass spectrometry (LC-MS/MS) was used to analyze 50% (v/v) ethanolic and aqueous extracts of the plants’ leaves. Antimicrobial activities were estimated with a standard microdilution method. The antioxidant properties were evaluated by validated chemical cell-free and biological cell-based assays. Cytotoxic effects were performed on mouse fibroblasts and human keratinocytes with a plate reader-based method assessing intracellular adenosine triphosphate (ATP), nucleic acid and protein contents and also by a flow cytometer-based assay detecting apoptotic−necrotic cell populations. Cell migration to cover cell-free areas was visualized by time-lapse phase-contrast microscopy using standard culture inserts. Fuchsia species showed the strongest cytotoxicity and the highest antioxidant and antimicrobial activity. However, their ethanolic extracts facilitated cell migration, most probably due to their various phenolic acid, flavonoid and anthocyanin derivatives. Our data might serve as a basis for further animal experiments to explore the complex action of Fuchsia species in wound healing assays.

In the past ten years, several novel hantaviruses were discovered in shrews, moles, and bats, suggesting the dispersal of hantaviruses in many animal taxa other than rodents during their evolution. Interestingly, the coevolutionary analyses of most recent studies have raised the possibility that nonrodents may have served as the primordial mammalian host and harboured the ancestors of rodent-borne hantaviruses as well. The aim of our study was to investigate the presence of hantaviruses in bat lung tissue homogenates originally collected for taxonomic purposes in Malaysia in 2015. Hantavirus-specific nested RT-PCR screening of 116 samples targeting the L segment of the virus has revealed the positivity of two lung tissue homogenates originating from two individuals, a female and a male of the Murina aenea bat species collected at the same site and sampling occasion. Nanopore sequencing of hantavirus positive samples resulted in partial genomic data from S, M, and L genome segments. The obtained results indicate molecular evidence for hantaviruses in the M. aenea bat species. Sequence analysis of the PCR amplicon and partial genome segments suggests that the identified virus may represent a novel species in the Mobatvirus genus within the Hantaviridae family. Our results provide additional genomic data to help extend our knowledge about the evolution of these viruses.