Age-Associated B Cell Features of the Murine High-Grade B Cell Lymphoma Bc.DLFL1 and Its Extranodal Expansion in Abdominal Adipose Tissues.

Diffuse large B cell lymphoma comprises a heterogeneous group of B cell-derived tumors, with different degrees of aggressiveness, as defined by their cellular origin and tissue microenvironment. Using the spontaneous Bc.DLFL1 lymphoma originating from a BALB/c mouse as a diffuse large B cell lymphoma model, in this study we demonstrate that the lymphoma cells display surface phenotype, IgH V-region somatic mutations, transcription factor characteristics and in vivo location to splenic extrafollicular regions of age-associated B cells (ABCs), corresponding to T-bet+ and Blimp-1+/CD138- plasmablasts derivation. The expansion of lymphoma cells within lymphoid tissues took place in a close arrangement with CD11c+ dendritic cells, whereas the extranodal infiltration occurred selectively in the mesentery and omentum containing resident gp38/podoplanin+ fibroblastic reticular cells. Antagonizing BAFF-R activity by mBR3-Fc soluble receptor fusion protein led to a significant delay of disease progression. The extranodal expansion of Bc.DLFL1 lymphoma within the omental and mesenteric adipose tissues was coupled with a significant change of the tissue cytokine landscape, including both shared alterations and tissue-specific variations. Our findings indicate that while Bc.DLFL1 cells of ABC origin retain the positioning pattern within lymphoid tissues of their physiological counterpart, they also expand in non-lymphoid tissues in a BAFF-dependent manner, where they may alter the adipose tissue microenvironment to support their extranodal growth.

Clinical Benefits of Decreased Photo-Oxidative Stress on Human Embryo Development.

OBJECTIVE: Early embryonic development is characterized by rapid cell division and gene activation, making the embryo extremely sensitive to environmental influences. Light exposure can affect embryonic development through a direct toxic effect on the embryo via the generation of reactive oxygen species. In a previous study, we demonstrated the positive effect of improved light-protected embryo culture conditions implemented in our laboratory. This study aimed to investigate the changes in human embryo development under light protection during the conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). MATERIALS AND METHODS: We tested the potential beneficial effect of light filters to reduce the risk of toxic effects of light. IVF outcomes were compared between two experimental conditions, light protection with red light filters versus no light protection as a control. RESULTS: Blastocyst development rate in IVF was significantly higher in the light-protected group than in the group treated under conventional conditions (46.6 vs. 26.7%). In the case of ICSI, we obtained a similar result (44.5 vs. 31.6%). The rate of cryopreservation with at least one embryo was higher in the light-protected phase (32.8%) than in the conventionally manipulated phase (26.8%). The abortion rate was also significantly lower during the light-protected period in IVF, resulting in a higher live birth rate. CONCLUSIONS: The implementation of light protection to reduce the embryotoxic wavelengths of light in IVF centers may improve the blastocyst development rate and embryo quality while maintaining embryo safety.

miRNA Profiling of Developing Rat Retina in the First Three Postnatal Weeks.

The morphogenesis of the mammalian retina depends on the precise control of gene expression during development. Small non-coding RNAs, including microRNAs play profound roles in various physiological and pathological processes via gene expression regulation. A systematic analysis of the expression profile of small non-coding RNAs in developing Wistar rat retinas (postnatally day 5 (P5), P7, P10, P15 and P21) was executed using IonTorrent PGM next-generation sequencing technique to reveal the crucial players in the early postnatal retinogenesis. Our analysis reveals extensive regulatory potential of microRNAs during retinal development. We found a group of microRNAs that show constant high abundance (miR-19, miR-101; miR-181, miR-183, miR-124 and let-7) during the development process. Others are present only in the early stages (miR-20a, miR-206, miR-133, miR-466, miR-1247, miR-3582), or at later stages (miR-29, miR-96, miR-125, miR-344 or miR-664). Further miRNAs were detected which are differentially expressed in time. Finally, pathway enrichment analysis has revealed 850 predicted target genes that mainly participate in lipid-, amino acid- and glycan metabolisms in the examined time-period (P5-P21). P5-P7 transition revealed the importance of miRNAs in glutamatergic synapse and gap junction pathways. Significantly downregulated miRNAs rno-miR-30c1 and 2, rno-miR-205 and rno-miR-503 were detected to target Prkx (ENSRNOG00000003696), Adcy6 (ENSRNOG00000011587), Gnai3 (ENSRNOG00000019465) and Gja1 (ENSRNOG00000000805) genes. The dataset described here will be a valuable resource for clarifying new regulatory mechanisms for retinal development and will greatly contribute to our understanding of the divergence and function of microRNAs.

Long-term Effects of the pituitary-adenylate cyclase-activating Polypeptide (PACAP38) in the Adult Mouse Retina: Microglial Activation and Induction of Neural Proliferation.

The degenerative retinal disorders characterized by progressive cell death and exacerbating inflammation lead ultimately to blindness. The ubiquitous neuropeptide, PACAP38 is a promising therapeutic agent as its proliferative potential and suppressive effect on microglia might enable cell replacement and attenuate inflammation, respectively. Our previous finding that PACAP38 caused a marked increase of the amacrine cells in the adult (1-year-old) mouse retina, served as a rationale of the current study. We aimed to determine the proliferating elements and the inflammatory status of the PACAP38-treated retina. Three months old mice were intravitreally injected with 100 pmol PACAP38 at 3 months intervals (3X). Retinas of 1-year-old animals were dissected and effects on cell proliferation, and expression of inflammatory regulators were analyzed. Interestingly, both mitogenic and anti-mitogenic actions were detected after PACAP38-treatment. Further analysis of the mitogenic effect revealed that proliferating cells include microglia, endothelial cells, and neurons of the ganglion cell layer but not amacrine cells. Furthermore, PACAP38 stimulated retinal microglia to polarize dominantly into M2-phenotype but also might cause subsequent angiogenesis. According to our results, PACAP38 might dampen pro-inflammatory responses and help tissue repair by reprogramming microglia into an M2 phenotype, nonetheless, with angiogenesis as a warning side effect.

Total sleep deprivation decreases saliva ghrelin levels in adolescents.

Ghrelin, a regulator of food intake and energy expenditure, has been shown to be associated with insufficient sleep. The goal of the present study was to investigate the effect of a single night of total sleep deprivation on fasting saliva ghrelin and on nocturnal variation of saliva ghrelin concentration. A further aim of the study was to investigate the influence of body mass index on changes in saliva ghrelin levels. Altogether 35 adolescents (18 boys; age: 13.8 ± 1.14 years) were studied on two subsequent days (sleep and total sleep deprivation). Saliva samples were collected during the two experimental nights at 21:00 hours, 01:00 hours and 06:00 hours. Total-ghrelin concentration showed a continuous increase from the evening until 06:00 hours. This increase was blunted significantly (p = 0.003) by total sleep deprivation. Total-ghrelin level was significantly lower (p = 0.02) during total sleep deprivation at 06:00 hours (median 403.6 pg ml-1 ; 95% confidence interval: 343.1-468.9 pg ml-1 ) as compared with values during the sleep condition (median 471.2 pg ml-1 ; 95% confidence interval: 205.4-1578.7 pg ml-1 ). Acyl-ghrelin levels did not present any change at the three time points, and were not affected by total sleep deprivation. Stratifying the study population according to body mass index (normal weight and overweight/obese groups), the blunting effect of total sleep deprivation was more pronounced in the obese/overweight group (sleep: median 428.2 pg ml-1 ; 95% confidence interval: 331.3-606.9 pg ml-1 versus total sleep deprivation: median 333.1 pg ml-1 ; 95% confidence interval: 261.5-412.9 pg ml-1 ; p = 0.0479). Saliva total-ghrelin concentrations gradually increased during the night, and total sleep deprivation significantly blunted this increase. This blunting effect was mainly observed in subjects with overweight/obesity. The physiological and clinical implications of the present observation are to be clarified by further studies.

An Optimized Flow Cytometric Method to Demonstrate the Differentiation Stage-Dependent Ca2+ Flux Responses of Peripheral Human B Cells.

Calcium (Ca2+) flux acts as a central signaling pathway in B cells, and its alterations are associated with autoimmune dysregulation and B-cell malignancies. We standardized a flow-cytometry-based method using various stimuli to investigate the Ca2+ flux characteristics of circulating human B lymphocytes from healthy individuals. We found that different activating agents trigger distinct Ca2+ flux responses and that B-cell subsets show specific developmental-stage dependent Ca2+ flux response patterns. Naive B cells responded with a more substantial Ca2+ flux to B cell receptor (BCR) stimulation than memory B cells. Non-switched memory cells responded to anti-IgD stimulation with a naive-like Ca2+ flux pattern, whereas their anti-IgM response was memory-like. Peripheral antibody-secreting cells retained their IgG responsivity but showed reduced Ca2+ responses upon activation, indicating their loss of dependence on Ca2+ signaling. Ca2+ flux is a relevant functional test for B cells, and its alterations could provide insight into pathological B-cell activation development.

Amino Acid Profiling of Follicular Fluid in Assisted Reproduction Reveals Important Roles of Several Amino Acids in Patients with Insulin Resistance.

The global prevalence of insulin resistance (IR) is increasing continuously, influencing metabolic parameters and fertility. The metabolic changes due to IR can alter the molecular composition of plasma and other body fluids. Follicular fluid (FF) is derived mainly from plasma, and it is a critical microenvironment for the developing oocytes. It contains various metabolites and amino acids, and the quality of the oocytes is linked at least partially to amino acid metabolism. Our goal was to quantitatively determine the amino acid (AA) profile of FF in IVF patients and to compare IR and non-insulin resistance (NIR) groups to investigate the AA changes in their FF. Using UHPLC-based methods, we quantified the main 20 amino acids from human FF samples in the IR and NIR groups. Several amino acids (aspartate, glycine, glutamate, and cysteine) differed significantly (p < 0.05 or less) between the two groups. The most significant alterations between the IR and NIR groups were related to the glutathione metabolic pathway involving glycine, serine, and threonine. Since insulin resistance alters the amino acid composition of the FF, the oocytes may undergo metabolism-induced changes resulting in poor oocyte quality and less fertility in the insulin resistance groups.

PACAP-38 in Acute ST-Segment Elevation Myocardial Infarction in Humans and Pigs: A Translational Study.

Acute myocardial infarction (MI) is one of the most common causes of death worldwide. Pituitary adenylate cyclase activating polypeptide (PACAP) is a cardioprotective neuropeptide expressing its receptors in the cardiovascular system. The aim of our study was to examine tissue PACAP-38 in a translational porcine MI model and plasma PACAP-38 levels in patients with ST-segment elevation myocardial infarction (STEMI). Significantly lower PACAP-38 levels were detected in the non-ischemic region of the left ventricle (LV) in MI heart compared to the ischemic region of MI-LV and also to the Sham-operated LV in porcine MI model. In STEMI patients, plasma PACAP-38 level was significantly higher before percutaneous coronary intervention (PCI) compared to controls, and decreased after PCI. Significant negative correlation was found between plasma PACAP-38 and troponin levels. Furthermore, a significant effect was revealed between plasma PACAP-38, hypertension and HbA1c levels. This was the first study showing significant changes in cardiac tissue PACAP levels in a porcine MI model and plasma PACAP levels in STEMI patients. These results suggest that PACAP, due to its cardioprotective effects, may play a regulatory role in MI and could be a potential biomarker or drug target in MI.

Identification of disease- and headache-specific mediators and pathways in migraine using blood transcriptomic and metabolomic analysis.

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.

Identification, presence, and possible multifunctional regulatory role of invertebrate gonadotropin-releasing hormone/corazonin molecule in the great pond snail (Lymnaea stagnalis).

In the last years, our interpretation of the origin and function of the gonadotropin-releasing hormone (GnRH) neuropeptide superfamily has changed substantially. A main driver for these conceptual changes came from increased investigations into functions and evolutionary lineage of previously identified molluscan GnRH molecules. Emerging evidence suggests not only reproductive, but also diverse biological effects of these molecules and proposes they should most likely be called corazonin (CRZ). Clearly, a more global understanding requires further exploration of species-specific functions and structure of invGnRH/CRZ peptides. Towards this goal, we have identified the full-length cDNA of invGnRH/CRZ peptide in an invertebrate model species, the great pond snail Lymnaea stagnalis, termed ly-GnRH/CRZ, and characterized the transcript and peptide distribution in the central nervous system (CNS) and peripheral organs. Our results are consistent with previous data that molluscan GnRHs are more related to CRZs and serve diverse functions. Hence, our findings support the notion that peptides originally termed molluscan GnRH are multifunctional modulators and that nomenclature change should be taken into consideration.

Morphotype of bacteroids in different legumes correlates with the number and type of symbiotic NCR peptides.

In legume nodules, rhizobia differentiate into nitrogen-fixing forms called bacteroids, which are enclosed by a plant membrane in an organelle-like structure called the symbiosome. In the Inverted Repeat-Lacking Clade (IRLC) of legumes, this differentiation is terminal due to irreversible loss of cell division ability and is associated with genome amplification and different morphologies of the bacteroids that can be swollen, elongated, spherical, and elongated-branched, depending on the host plant. In Medicago truncatula, this process is orchestrated by nodule-specific cysteine-rich peptides (NCRs) delivered into developing bacteroids. Here, we identified the predicted NCR proteins in 10 legumes representing different subclades of the IRLC with distinct bacteroid morphotypes. Analysis of their expression and predicted sequences establishes correlations between the composition of the NCR family and the morphotypes of bacteroids. Although NCRs have a single origin, their evolution has followed different routes in individual lineages, and enrichment and diversification of cationic peptides has resulted in the ability to impose major morphological changes on the endosymbionts. The wide range of effects provoked by NCRs such as cell enlargement, membrane alterations and permeabilization, and biofilm and vesicle formation is dependent on the amino acid composition and charge of the peptides. These effects are strongly influenced by the rhizobial surface polysaccharides that affect NCR-induced differentiation and survival of rhizobia in nodule cells.

Hemokinin-1 Gene Expression Is Upregulated in Trigeminal Ganglia in an Inflammatory Orofacial Pain Model: Potential Role in Peripheral Sensitization.

A large percentage of primary sensory neurons in the trigeminal ganglia (TG) contain neuropeptides such as tachykinins or calcitonin gene-related peptide. Neuropeptides released from the central terminals of primary afferents sensitize the secondary nociceptive neurons in the trigeminal nucleus caudalis (TNC), but also activate glial cells contributing to neuroinflammation and consequent sensitization in chronic orofacial pain and migraine. In the present study, we investigated the newest member of the tachykinin family, hemokinin-1 (HK-1) encoded by the Tac4 gene in the trigeminal system. HK-1 had been shown to participate in inflammation and hyperalgesia in various models, but its role has not been investigated in orofacial pain or headache. In the complete Freund's adjuvant (CFA)-induced inflammatory orofacial pain model, we showed that Tac4 expression increased in the TG in response to inflammation. Duration-dependent Tac4 upregulation was associated with the extent of the facial allodynia. Tac4 was detected in both TG neurons and satellite glial cells (SGC) by the ultrasensitive RNAscope in situ hybridization. We also compared gene expression changes of selected neuronal and glial sensitization and neuroinflammation markers between wild-type and Tac4-deficient (Tac4-/-) mice. Expression of the SGC/astrocyte marker in the TG and TNC was significantly lower in intact and saline/CFA-treated Tac4-/- mice. The procedural stress-related increase of the SGC/astrocyte marker was also strongly attenuated in Tac4-/- mice. Analysis of TG samples with a mouse neuroinflammation panel of 770 genes revealed that regulation of microglia and cytotoxic cell-related genes were significantly different in saline-treated Tac4-/- mice compared to their wild-types. It is concluded that HK-1 may participate in neuron-glia interactions both under physiological and inflammatory conditions and mediate pain in the trigeminal system.

How to Reduce the Potential Harmful Effects of Light on Blastocyst Development during IVF.

PURPOSE: Earlier findings revealed the damaging effect of visible light on zygotes and gametes. The aim of our study is to eliminate or significantly reduce the potentially harmful effects of light exposure during in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and to investigate the effect of light protection on embryo development and implantation. MATERIALS AND METHODS: To protect sperm cells, oocytes, and embryos from the potential harmful effects of light exposure during laboratory procedures, we created a dark environment for the cells and applied red filters on laboratory lamps and UV or infrared filters in the microscopes in order to eliminate white light exposure of the cells throughout all work stages. RESULTS: The fertilization rate was significantly (p = 0.011) higher in light-protected ICSI cycles. Blastocyst development rates (blastocyst/embryo) were significantly (p < 0.001) higher in light-protected embryos than in those manipulated in conventional light conditions both in IVF (20.9% difference) and ICSI (38.6% difference). Numbers of clinical pregnancies/transfers of ICSI fertilized day 5 blastocysts were also significantly (p = 0.040) higher in light-protected conditions. CONCLUSIONS: These data show that light protection has a positive effect on fertilization rate and increases the blastocyst development as well as the number of clinical pregnancies/transfers. Implementation of this light protection method in IVF centers may improve the success rate while maintaining maximal embryo safety.

First molecular detection of Apis mellifera filamentous virus in honey bees (Apis mellifera) in Hungary.

Western honey bees (Apis mellifera) are important pollinators in the ecosystem and also play a crucial economic role in the honey industry. During the last decades, a continuous decay was registered in honey bee populations worldwide, including Hungary. In our study, we used metagenomic approaches and conventional PCR screening on healthy and winter mortality affected colonies from multiple sites in Hungary. The major goal was to discover presumed bee pathogens with viral metagenomic experiments and gain prevalence and distribution data by targeted PCR screening. We examined 664 honey bee samples that had been collected during winter mortality from three seemingly healthy colonies and from one colony infested heavily by the parasitic mite Varroa destructor in 2016 and 2017. The subsequent PCR screening of honey bee samples revealed the abundant presence of Apis mellifera filamentous virus (AmFV) for the first time in Central Europe. Based on phylogeny reconstruction, the newly-detected virus was found to be most closely related to a Chinese AmFV strain. More sequence data from multiple countries would be needed for studying the detailed phylogeographical patterns and worldwide spreading process of AmFV. Here we report the prevalent presence of this virus in Hungarian honey bee colonies.

Emergence and evolution of an international cluster of MDR Bacteroides fragilis isolates.

OBJECTIVES: The aim of this study was to examine the antibiotic resistance profiles, antibiotic resistance mechanisms and possible 'clonal' nature of some MDR Bacteroides fragilis strains that simultaneously harboured cfiA, nimB, IS1186 and IS4351. METHODS: Antibiotic susceptibilities were determined by Etests and antibiotic resistance genes and different genetic elements were detected by applying PCR methods. The environments of the cfiA and nimB genes were also determined by sequencing. The transferability of the cfiA, nimB and tet(Q) genes was tested by conjugation. The genetic relatedness of the test strains was tested by ERIC-PCR or PFGE. The complete genome sequences of two strains (B. fragilis BF8 and O:21) were determined by next-generation sequencing. RESULTS: Most of the seven B. fragilis strains tested displayed multidrug resistance phenotypes; five strains were resistant to at least five types of antibiotics. Besides the common genetic constitution, ERIC-PCR implied high genetic relatedness. Similarities in some of the antibiotic resistance mechanisms [carbapenems (cfiA) and metronidazole (nimB)] also confirmed their common origin, but some other resistance mechanisms {MLSB [erm(F)] and tetracycline [tet(Q)]} and PFGE typing revealed differences. In B. fragilis BF8 and O:21, erm(F) and tet(X) genes were found with IS4351 borders, thus constituting Tn4351. All the strains were tet(Q) positive and transferred this gene in conjugation experiments, but not the cfiA and nimB genes. CONCLUSIONS: An international cluster of MDR B. fragilis strains has been identified and characterized. This 'clone' may have emerged early in the evolution of division II B. fragilis strains, which was suggested by the low-complexity ERIC profiles and differences in the PFGE patterns.

Co-infecting viruses of species Bovine rhinitis B virus (Picornaviridae) and Bovine nidovirus 1 (Tobaniviridae) identified for the first time from a post-mortem respiratory sample of a sheep (Ovis aries) in Hungary.

In this study, a picornavirus and a nidovirus were identified from a single available nasopharyngeal swab (NPS) sample of a freshly deceased sheep, as the only vertebrate viruses found with viral metagenomics and next-generation sequencing methods. The sample was originated from a mixed feedlot farm in Hungary where sheep and cattle were held together but in separate stalls. Most of the sheep had respiratory signs (coughing and increased respiratory effort) at the time of sampling. Other NPS were not, but additional enteric samples were collected from sheep (n = 27) and cattle (n = 11) of the same farm at that time. The complete/nearly complete genomes of the identified viruses were determined using RT-PCR and Nanopore (MinION-Flonge) / Dye-terminator sequencing techniques. The results of detailed genomic and phylogenetic analyses indicate that the identified picornavirus most likely belongs to a type 4 genotype of species Bovine rhinitis B virus (BRBV-4, OR885914) of genus Aphthovirus, family Picornaviridae while the ovine nidovirus (OvNV, OR885915) - as a novel variant - could belong to the recently created Bovine nidovirus 1 (BoNV) species of genus Bostovirus, family Tobaniviridae. None of the identified viruses were detectable in the enteric samples using RT-PCR and generic screening primer pairs. Both viruses are well-known respiratory pathogens of cattle, but their presence was not demonstrated before in other animals, like sheep. Furthermore, neither BRBV-4 nor BoNVs were investigated in European cattle and/or sheep flocks, therefore it cannot be determined whether the presence of these viruses in sheep was a result of a single host species switch/spillover event or these viruses are circulating in not just cattle but sheep populations as well. Further studies required to investigate the spread of these viruses in Hungarian and European sheep and cattle populations and to identify their pathogenic potential in sheep.

COVID-19 mortality prediction in Hungarian ICU settings implementing random forest algorithm.

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.

The genomic and epidemiological investigations of enteric viruses of domestic caprine (Capra hircus) revealed the presence of multiple novel viruses related to known strains of humans and ruminant livestock species.

IMPORTANCE: Compared with other domestic animals, the virome and viral diversity of small ruminants especially in caprine are less studied even of its zoonotic potential. In this study, the enteric virome of caprine was investigated in detail using next-generation sequencing and reverse transcription PCR techniques. The complete or nearly complete genomes of seven novel viruses were determined which show a close phylogenetic relationship to known human and ruminant viruses. The high similarity between the identified caprine tusavirus (family Parvoviridae) and an unassigned CRESS DNA virus with closely related human strains could indicate the (reverse) zoonotic potential of these viruses. Others, like astroviruses (family Astroviridae), enteroviruses, or novel caripiviruses (named after the term caprine picornavirus) of family Picornaviridae found mostly in multiple co-infections in caprine and ovine, could indicate the cross-species transmission capabilities of these viruses between small ruminants.

Characterization of antibiotic resistomes by reprogrammed bacteriophage-enabled functional metagenomics in clinical strains.

Functional metagenomics is a powerful experimental tool to identify antibiotic resistance genes (ARGs) in the environment, but the range of suitable host bacterial species is limited. This limitation affects both the scope of the identified ARGs and the interpretation of their clinical relevance. Here we present a functional metagenomics pipeline called Reprogrammed Bacteriophage Particle Assisted Multi-species Functional Metagenomics (DEEPMINE). This approach combines and improves the use of T7 bacteriophage with exchanged tail fibres and targeted mutagenesis to expand phage host-specificity and efficiency for functional metagenomics. These modified phage particles were used to introduce large metagenomic plasmid libraries into clinically relevant bacterial pathogens. By screening for ARGs in soil and gut microbiomes and clinical genomes against 13 antibiotics, we demonstrate that this approach substantially expands the list of identified ARGs. Many ARGs have species-specific effects on resistance; they provide a high level of resistance in one bacterial species but yield very limited resistance in a related species. Finally, we identified mobile ARGs against antibiotics that are currently under clinical development or have recently been approved. Overall, DEEPMINE expands the functional metagenomics toolbox for studying microbial communities.

Plasmid sequence dataset of multidrug-resistant Enterobacterales isolated from hospital effluents and wastewater treatment plant.

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.