Streptococcus agalactiae or group B Streptococcus (GBS) asymptomatically colonizes the genitourinary tracts of up to 30% of pregnant women. Globally, GBS is an important cause of neonatal morbidity and mortality. GBS has recently been linked to adverse pregnancy outcomes. The potential interactions between GBS and the vaginal microbiome composition remain poorly understood. In addition, little is known about the vaginal microbiota of pregnant Egyptian women.Firmicutes and the family Lactobacillaceae were significantly more abundant in GBS-negative colonizers. In contrast, the phyla Actinobacteria, Tenericutes, and Proteobacteria and the families Bifidobacteriaceae, Mycoplasmataceae, Streptococcaceae, Corynebacteriaceae, Staphylococcaceae, and Peptostreptococcaceae were significantly more abundant in GBS culture-positive colonizers. On the genus and species levels, Lactobacillus was the only genus detected with significantly higher relative abundance in GBS culture-negative status (88%), and L. iners was the significantly most abundant species. Conversely, GBS-positive carriers exhibited a significant decrease in Lactobacillus abundance (56%). In GBS-positive colonizers, the relative abundance of the genera Ureaplasma, Gardnerella, Streptococcus, Corynebacterium, Staphylococcus, and Peptostreptococcus and the species Peptostreptococcus anaerobius was significantly higher. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to the metabolism of cofactors and vitamins, phosphatidylinositol signaling system, peroxisome, host immune system pathways, and host endocrine system were exclusively enriched among GBS culture-positive microbial communities. However, lipid metabolism KEGG pathways, nucleotide metabolism, xenobiotics biodegradation and metabolism, genetic information processing pathways associated with translation, replication, and repair, and human diseases (Staphylococcus aureus infection) were exclusively enriched in GBS culture-negative communities.Using V3-V4 16S rRNA next-generation sequencing, we examined the vaginal microbiome in GBS culture-positive pregnant women (22) and GBS culture-negative pregnant women (22) during the third trimester in Ismailia, Egypt. According to the alpha-diversity indices, the vaginal microbiome of pregnant GBS culture-positive women was significantly more diverse and less homogenous. The composition of the vaginal microbiome differed significantly based on beta-diversity between GBS culture-positive and culture-negative women. The phylum Understanding how perturbations of the vaginal microbiome contribute to pregnancy complications may result in the development of alternative, targeted prevention strategies to prevent maternal GBS colonization. We hypothesized associations between inferred microbial function and GBS status that would need to be confirmed in larger cohorts.The online version contains supplementary material available at 10.1186/s12866-022-02730-8. The human microbiome at various body sites defines the host’s metabolic features and plays an essential role in maintaining the homeostasis of the host immune system and resisting invading pathogens –3. Thus,Streptococcus agalactiae, or group B Streptococcus (GBS), is an encapsulated Gram-positive pathobiont that colonizes the vaginal mucosa and/or rectum of up to 30% of healthy women asymptomatically [The vaginal mucosal surface is one of the primary entry points for invading pathogens and, consequently, a principal immunological barrier . Streptoatically . GBS is atically –8. Throuatically . In addiatically . Recentlatically , 13. To atically .Lactobacillus dominance is a key feature of a healthy vaginal microbiome. They are essential for restoring the vaginal ecosystem and maintaining a low pH, which functions as a barrier against invading and colonizing pathogens [Lactobacillus population is diminished or eradicated [Lactobacillus species has been widely reported [lactobacillus species; L. crispatus dominated CST-I, L. gasseri dominated CST-II, L. iners dominated CST-III, and L. jensenii dominated CST-V. However, the scarcity of lactobacilli with numerous strict and facultative anaerobes was a distinctive feature of the Lactobacillus-poor CST-IV [athogens . The vagadicated , 14. Durreported . This isreported . Five direported . Four ofr CST-IV .Previous reports have investigated the impact of GBS colonization on the vaginal microbiota using classical culture-based methods –21. ThesGBS is an opportunistic vaginal pathobiont whose life cycle involves a transformation from a harmless commensal state to an invading pathogen for both mother and infant –29. ThusUsing 16S rRNA next-generation sequencing technology and advanced microbiome profiling, we compared the vaginal microbial communities of GBS culture-positive pregnant women with those of GBS culture-negative pregnant women during the third trimester in Ismailia, Egypt. We were able to gain a greater understanding of taxonomy and microbial diversity and predict the functional pathways associated with GBS carriage.Vaginal swabs collected from 44 pregnant subjects were sequenced by Illumina MiSeq platform and used for 16S rRNA microbiome analysis. These included 22 GBS culture-positive women and 22 GBS culture-negative women. The ages of the participants ranged from 19 to 44 years, with an average age of 31.11 ± 7.41 years, 31.18 ± 6.62 for GBS culture-positive pregnant women, and 31.05 ± 8.30 for GBS culture-negative pregnant women. Furthermore, the mean parity was 2.90 ± 1.41; 3.18 ± 1.26 for GBS culture-positive pregnant women and 2.64 ± 1.53 for GBS culture-negative pregnant women. There was no significant difference in age and parity of participants between the two groups Table p values = 0.0033461) and Simpson  alpha-diversity indices but not by chao1 . As expected, Lactobacillus was nearly the only genus among GBS culture-negative pregnant women (88%), whereas GBS culture-positive pregnant women had a less homogeneous microbial structure with a significant decrease in Lactobacillus abundance (56%) . Beta diversity is shown in Supplementary Figs. We inferred differences in microbiota profiles using Bray–Curtis and Jaccard dissimilarity indices. We observed a significant difference in the composition of vaginal microbiome between GBS culture-positive and GBS culture-negative pregnant women as assessed by PERMANOVA using the Bray–Curtis and Jaccard dissimilarity indices , this was followed by L. crispatus dominant CST-I , L. johnsonii dominant CST-Other , CST-VII , L. jensenii dominant CST-V , and finally L. gasseri dominant CST-II  and Lactobacillus-poor CST-IV . CST-III and CST-I represented more the 85% (19/22) of GBS-negative communities. The remaining 15% (3/22) was represented as CSTs II, V and VII (One sample for each CST). In contrast, CST-III and CST-I represented less than half of the GBS-positive communities . L. johnsonii dominant CST-Other was exclusive for GBS-positive communities representing more the 20% . The remaining samples were classified as CST-VII , CST-I , CST-V , and CST-IV . None of the samples was assigned to CST-VI. Distribution of samples by GBS status across CSTs is shown in Table Lactobacillus species in vagina of pregnant Egyptian women during the third trimester according to GBS status as revealed by Illumina 16S rRNA microbiome individual reports is shown in Supplementary Fig. Samples could be classified into 7 CST groups. Two samples were not designated (No types). These belonged to the GBS-positive vaginal communities. 52.2% 23/4, this wUreaplasma, Gardnerella, Streptococcus, Corynebacterium, Staphylococcus, Peptostreptococcus, and Campylobacter  Orthology groups) from the 16S rRNA gene data. Predictive functional profiling of microbial communities revealed that 52 KEGG pathways were significantly enriched in GBS culture-positive microbial communities compared to 46 KEGG pathways in GBS culture-negative microbial communities Fig. .Fig. 5LiLactobacillus populations which consequently results in creating a dysbiotic environment with increased vaginal pH [Maternal colonization is a significant risk factor for neonatal GBS transmission and potentially adverse pregnancy outcomes , 6. Althginal pH , 34. Sucginal pH . Beta-heginal pH .Lactobacilli as the sole member of the vaginal community among pregnant women during the third trimester. Pregnant women have a significantly higher prevalence of Lactobacillus vagitypes and a proportionally lower prevalence of vagitypes dominated by other taxa, according to [Lactobacillus at the expense of other taxa, resulting in a vaginal microbiome ecosystem that is less complex [L. iners was the most predominant Lactobacillus species in the vaginal microbiome of our pregnant subjects regardless of GBS status. According to [L. iners was more prevalent in the third trimester. Regarding race and ethnicity, this is consistent with other African studies indicating that L. iners-dominant microbiome CST-III is most prevalent among pregnant women of African descent [L. iners-dominant microbiome in pregnancy in all groups, with a higher prevalence of this vagitype in pregnant women of African ancestry.As previously reported , 36, 37,rding to . This is complex . L. inerrding to , this warding to . Through descent , 36, 37. descent  found anLactobacillus abundance level. Similar results were described by previous culture-based studies [Gardnerella, Streptococcus (which includes GBS), Ureaplasma, Corynebacterium, Peptostreptococcus, and Staphylococcus, as well as Peptostreptococcus anaerobius, were identified as significant biomarkers among GBS culture-positive pregnant women. Conversely, Lactobacillus was the only genus detected whose relative abundance was significantly higher in GBS culture-negative status and L. iners and L. coleohominis were discovered as significant species biomarkers. In line with our results, [L. iners in normal vaginal microbiota than in dysbiota. Interestingly, unlike other lactobacilli, L. iners is only present in the vagina [L. coleohominis is a vaginal lactobacillus species that has been detected frequently in women with normal vaginal flora [L. coleohominis was previously recognized as a minor member of vaginal microbiota in women without bacterial vaginosis, consistent with our findings [GBS culture-positive pregnant carriers had a less homogenous microbial structure with a significant decrease in the  studies , 20, 21  studies , 25, 26. studies –24, 26, results,  found a e vagina , 41. Likal flora , 42–45. findings .L. iners and GBS are antagonistic, persistent species that adapt well to fluctuating vaginal environmental conditions [L. iners and GBS produce pore-forming cytolysins that destabilize the membrane integrity of the host cell. L. iners produces interolysin [L. iners and GBS to directly extract the nutrients they require from host cells. Interestingly, interolysin is active under acidic conditions and inactive at neutral pH [cyl gene cluster necessary for hemolysin release is demonstrably greater under neutral pH conditions than under acidic pH conditions [L. iners than GBS-negative vaginal communities. Moreover, both L. iners and GBS are auxotrophic for many amino acids [Lactobacillus dominance or dysbiosis and GBS proliferation in the vaginal econiche. Interestingly, G. vaginalis is also a cytolysin producer which is named vaginolysin [G. vaginalis. In line with our observation, previous reports demonstrated G. vaginalis as a promoter for GBS vaginal colonization [Notably, nditions , 47. Boterolysin , 48, whierolysin , 50. Thenditions . This suno acids , 51, indinolysin  and our nization . This emthe phosphatidylinositol signaling system. Pathogen entry into host cells requires the activation of lipid kinases, such as phosphoinositide 3-kinases (PI3K3), which are compartmentalized in lipid rafts and activated in host cells upon bacterial growth invasion. In order to invade human endometrial cells, GBS uses lipid rafts and the phosphatidylinositol signaling pathway. This is preferred by bacteria to avoid immune clearance and the intracellular degradation pathways [Gardnerella [E. coli [Gardnerella abundance significantly increased in our GBS-positive communities. Obviously, GBS vaginal colonization is positively correlated with other microbes in this microenvironment. GBS communication and cooperation with other members of the microbiota have been suggested before [Next generation sequencing of 16S RNA marker-gene is among the popular approaches used for profiling various bacterial communities. However, this method does not infer direct functional predictions –57. The pathways . PI3K arpathways . They hadnerella  and E. c[E. coli . This sud before . Taken td before . This isd before , 65, ourd before . Accordid before , 68. In d before . Accordid before . Researcd before , 71. ThiThe current study has several limitations which should be considered in the future. First, the small sample size which may not provide an accurate depiction of diversity and composition of vaginal bacterial community or CST distribution. Second, more detailed demographics for cases and controls would have been useful when comparing vaginal bacterial communities based on GBS status. Third, we used 16S rRNA next-generation sequencing approach to investigate the vaginal microbiome. Such technique has limited taxonomic resolution and detects only part of the vaginal community. It can be less precise at the species level. Besides, it does not provide direct information on functional genes in the sample. This information is not retrievable with 16S rRNA gene sequencing but are rather predicted , 56, 57.L. iners is the predominant Lactobacillus species of the vaginal microbiome in Egypt and was previously reported as the dominant vagitype in Africa and women of African ancestry. L. iners-dominant microbiomes have been previously reported as transition microbiomes that predispose pregnant women to adverse pregnancy outcomes, despite being recognized as a healthy vaginal ecosystem [Lactobacillus probiotic candidates appear promising for preventing GBS colonization during pregnancy [In this study, we demonstrated that cosystem . In addicosystem . Howevercosystem . Understregnancy .All study procedures involving human subjects were reviewed and approved by the Research Ethics Committee at the Faculty of Pharmacy, Suez Canal University, Egypt (Reference number 201811RH2). The study was conducted in accordance with all applicable ethical regulations. All participants provided their consent with knowledge.A cross-sectional study was conducted for a period of 3 months (December 2018 – February 2019) at the Gynecological Clinic of the Suez Canal University Hospital. During this period, 164 consecutive healthy pregnant women in their third trimester provided vaginal swab samples. All subjects underwent GBS screening via culture. The inclusion and exclusion criteria were as stated in , 73. At Vaginal samples were collected from the vaginal introitus . GBS dethttps://support.illumina.com/documentation.html) by amplification of the V3–V4 regions of 16S rRNA [TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG – 3′, Reverse primer 5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC – 3′. Furthermore, negative controls were analyzed to detect the presence of contaminants in swabs, extraction reagents, and during PCR mixture set up to reduce the likelihood of contamination. IGA Technology Services performed library preparation and sequencing on Illumina MiSeq Platform  in 300 bp paired-end mode .DNA was extracted using DNeasy PowerSoil Kit cat no. 12888-100  according to the manufacturer’s instructions. Microbiota profiling was performed according to the Illumina MiSeq standard protocol of 16S rRNA gene amplicons (16S rRNA  with thehttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Quantitative Insights into Microbial Ecology  pipeline [https://www.microbiomeanalyst.ca), downstream processing  was performed [P values less than < 0.05 and LDA scores greater than > 2 were considered statistically significant. MicrobiomeAnalyst is a web-based platform for the comprehensive analysis of common microbiome study data outputs [http://huttenhower.sph.harvard.edu/galaxy) were used to predict enriched microbial functional attributes most likely to explain the differences between GBS culture-positive and GBS culture-negative microbial vaginal communities based on Kyoto Encyclopedia of Genes and Genomes (KEGG). This is a database resource that integrates genomic, chemical and systemic functional information where genes from completely sequenced genomes are linked to higher-level systemic functions of the cell, the organism and the ecosystem [http://huttenhower.sph.harvard.edu/galaxy). The discriminative feature threshold for the logarithmic LDA score was set at > 2.Sequences quality was checked using FastQC  were used for CST assignment. Vaginal bacterial communities were categorized into 5 CSTs: ribed by . A samplribed by , 83. Vag species .https://www.ncbi.nlm.nih.gov/bioproject/PRJNA833599.The raw 16S rRNA sequences were deposited in the NCBI database with SRA under Bio project Accession number PRJNA833599. This information is accessible at Additional file 1:Supplementary Fig. 1. Rarefaction curves. Samples were rarefaied to even sequencing depth. Goods coverage was above 99.9% at 97% similarity cutoff indicating that sequences richness was sufficient for vaginal communities under test in all libraries.Additional file 2: Supplementary Fig. 2. Alpha-diversity indices of the vaginal microbiota across GBS culture-negative and GBS culture-positive pregnant Egyptian women using SILVA database. A Box plot of Shannon alpha-diversity index . B Box plot of Simpson alpha-diversity index . C Box plot of Chao1 alpha-diversity index . Mann-Whitney test and Kruskal-Wallis Test were used to define statistically significant differences between GBS culture-negative and GBS culture-positive pregnant women. A p value < 0.05 was considered statistically significant. Astreks indicate p values < 0.05.Additional file 3: Supplementary Fig. 3. Beta-diversity measurement of the vaginal microbiota across GBS culture-negative and GBS culture-positive pregnant Egyptian carriers using SILVA database. 2D PCoA clustering plot. Ellipses denote significant clustering Beta-diversity was assessed by PERMANOVA using Bray-Curtis dissimilarity matrix . Each dot represents one sample.Additional file 4: Supplementary Fig. 4. Species taxa level relative abundance in vagina of pregnant Egyptian women during the third trimester according to GBS status as revealed by Illumina 16S rRNA microbiome individual reports. Stacked bar charts represent relative proportions of the most predominant Lactobacillus species, Gardnerella, and non-Lactobacillus species in the vaginal microbiome of (A) GBS culture-negative pregnant women and (B) GBS culture-positive pregnant women. Each bar represents one sample.


Obba rivulosa and Gelatoporia subvermispora, during eight-week cultivation on solid spruce wood. Plant cell wall degrading carbohydrate-active enzymes (CAZymes) represented approximately 5% of the total proteins in both species. A core set of orthologous plant cell wall degrading CAZymes was shared between these species on spruce suggesting a conserved plant biomass degradation approach in this clade of basidiomycete fungi. However, differences in time-dependent production of plant cell wall degrading enzymes may be due to differences among initial growth rates of these species on solid spruce wood. The obtained results provide insight into specific enzymes and enzyme sets that are produced during the degradation of solid spruce wood in these fungi. These findings expand the knowledge on enzyme production in nature-mimicking conditions and may contribute to the exploitation of white rot fungi and their enzymes for biotechnological applications. The unique ability of basidiomycete white rot fungi to degrade all components of plant cell walls makes them indispensable organisms in the global carbon cycle. In this study, we analyzed the proteomes of two closely related white rot fungi,  Th. ThO. riradation . The mostivation . As signod decay .−1/1060 cm−1 [G. subvermispora and O. rivulosa cultivation on spruce. G. subvermispora is more efficient than O. rivulosa in lignin degradation after two weeks of cultivation, yet the ratio is similar for both species after eight weeks. Similarly, degradation of lignin and hemicelluloses by G. subvermispora has been detected on oak wood during the first five weeks of cultivation [This is supported by the spruce wood composition analyses after fungal growth by FTIR . Relativ060 cm−1  indicatetivation .O. rivulosa and G. subvermispora encode one and two lignin peroxidase-type enzymes, respectively [O. rivulosa and G. subvermispora cultivations emphasized their importance in degradation of solid spruce wood. This is consistent with the previous studies on the production of MnPs as the main lignin modifying enzymes of O. rivulosa when grown on spruce wood chips [G. subvermispora (strain SS-3) has been shown to produce MnP as its main lignin modifying enzyme on loblolly pine and eucalyptus wood chips [In addition to MnPs, ectively ,18. Howeod chips ,37. Likeod chips ,39.O. rivulosa [G. subvermispora has seven predicted laccase encoding genes [O. rivulosa (Protein ID: 452017) after 2 and 4 weeks, and the corresponding laccase ortholog of G. subvermispora (Protein ID: 118801) was produced after 2 weeks. In addition, this laccase isoenzyme was the only lignin modifying enzyme produced by G. subvermispora after 2-week cultivation , whereas in G. subvermispora they constituted a notably higher fraction making 10% of detected PCWDEs. H2O2-generating family AA3_3 AOX enzymes were the most highly produced PCWDEs in O. rivulosa and G. subvermispora spruce cultures, thus indicating a very potent ligninolytic system in both species studied (G. subvermispora AOX (Protein ID: 80773) also has been abundantly produced in solid-state fermentation (SSF) of Jerusalem artichoke, together with an AA3_2 aryl alcohol oxidase (AAO) (Protein ID: 117387) [2O2-supplying enzyme in the secretome of G. subvermispora liquid ball-milled aspen cultures [In the lignin degradation process by white rot fungi, an array of redox enzymes, such as family AA3 glucose–methanol–choline (GMC) oxidoreductases and AA5 copper radical oxidases (CROs), supplies Hoxidases  as well oxidases . H2O2-su studied . The hig 117387) . Interescultures , but it O. rivulosa (23%)  . The numtivation .O. rivulosa and G. subvermispora produced the highest levels of CBHIs and other cellulases after 2 weeks, similar to lignin modifying enzymes, disputing the hypothesized selective lignin degradation approach in these conditions. Previously, minor depolymerization of cellulose had been detected in G. subvermispora (strain SS-3) eucalyptus wood chip cultures already 15 days of cultivation [O. rivulosa and G. subvermispora with eight and nine putative AA9 LPMO encoding genes, respectively, indicate oxidative capacity for the degradation of plant polysaccharides. Two O. rivulosa LPMOs were among the most abundantly produced proteins in the spruce cultures, which is in line with the high transcript levels of their corresponding genes under similar cultivations [G. subvermispora LPMOs detected from spruce cultivations in this study also have been upregulated at the transcript level on liquid ball-milled aspen medium; but surprisingly, none of those have been detected as proteins from liquid ball-milled aspen, Avicel or glucose cultivations [A full enzymatic repertoire necessary for cellulose hydrolysis was produced by both species studied. The most highly produced cellulolytic enzymes in both species were GH7 cellobiohydrolase I (CBHI) targeted toward degradation of crystalline cellulose . Interestivation , which stivation ,48 . Family GH5_7 mannan hydrolyzing β-1,4-endomannanase (MAN) (Protein ID: 641261) was the most abundant hemicellulase in the O. rivulosa spruce cultivations, indicating that this fungus has a good ability to convert spruce that has mannan as the main hemicellulose polymer [O. rivulosa spruce cultures after two weeks [G. subvermispora with all its six XLN isoenzymes detected. The two most highly produced XLNs on spruce wood  also were detected from the ball-milled aspen liquid cultivations of G. subvermispora [G. subvermispora from Jerusalem artichoke SSF cultivations [Xylanases accounted for the majority (28%) of the detected PCWDEs in ivations b, while  polymer . We prevwo weeks . Althougwo weeks , GH10 XLrmispora , whereasivations , implyinw/w) in the primary cell walls) [O. rivulosa cultivations, and two GH10 XLNs and one CE15 glucuronoyl esterase (GE) in G. subvermispora cultivations, all of which were most abundant after 8 weeks  . Most of 8 weeks .O. rivulosa and 18% in G. subvermispora , GH35 β-galactosidases (LACs), and AA9 LPMOs, putatively acting on several substrates, constituted 22% of all detected CAZymes in rmispora . The numn period . InteresG. subvermispora ABF (Protein ID: 162360) has been detected from SSF cultivation on Jerusalem artichoke that has higher arabinose content (5%) [GH51 ABFs act on side-chains of different arabinose-containing polysaccharides, such as xyloglucans, arabinoxylans, and pectin . Althougent (5%)  as well ent (5%) . ABF hasent (5%)  althoughO. rivulosa and G. subvermispora produced all the necessary lignocellulolytic enzymes for the white rot type of decay of solid spruce wood, which involved an array of secreted cellulases, hemicellulases, lignin modifying enzymes, and auxiliary oxidoreductases. A core set of orthologous PCWDEs was shared suggesting similar approach for plant biomass degradation. Dynamics of the detected protein levels during the 8-week cultivation revealed that several lignin modifying class II peroxidases together with other oxidoreductases were abundantly produced after 2-week cultivation, suggesting an oxidative attack on lignin during the early growth stage on wood. Similar trends in abundances of class II peroxidases have previously been noticed for O. rivulosa [G. subvermispora [In conclusion, both rivulosa , G. subvrmispora , and othrmispora .O. rivulosa and G. subvermispora, in the conditions used in this study the obtained results did not corroborate these monokaryotic strains as selective lignin degraders. While enzymatic mechanisms behind selective lignin degradation remain still largely unsolved, it is known that this strategy can be affected by multiple parameters. One of the most prominent factors is temporal dependency, which limits lignin removal to an early decay stage, whereas hemicellulose and cellulose degradation appear later [O. rivulosa and G. subvermispora showed that even though the majority of CAZymes that they produced during growth on spruce were orthologs, their diversity with respect to time-dependent production of plant cell wall degrading enzymes suggests a different strategy for spruce degradation for these taxonomically related species.Although selective lignin degradation has been postulated for ar later ,54. In aar later ,56,57. H


Escherichia coli, Saccharomyces cerevisiae, Chlamydomonas reinhardtii, etc. For the first time, MIC values were evaluated for phlorotannin-enriched extracts of brown algae of the orders Ectocarpales and Desmarestiales. Phlorotannin extracts of Desmarestia aculeata, Fucus vesiculosus, and Ectocarpus siliculosus showed the lowest MIC values against most of the treated organisms (4–25 μg/mL for bacteria and yeast). Analysis of the survival curves of E. coli showed that massive loss of cells started after 3–4 h of exposure. Microalgae were less susceptible to activity of phlorotannin extracts, with the highest MIC values (≥200 µg/mL) measured for Chlorella vulgaris cells. D. aculeata, E. siliculosus, and three fucalean algae accumulate considerable amounts (4–16% of dry weight) of phlorotannins with MIC values similar to those widely used antibiotics. As these species grow abundantly in polar and temperate seas and have considerable biomass, they may be regarded as promising sources of phlorotannins.Marine seaweeds synthesize a plethora of bioactive metabolites, of which phlorotannins of brown algae currently attract special attention due to their high antibiotic and cytotoxic capacities. Here we measured the minimum inhibitory concentrations (MICs) of several semi-purified phlorotannin preparations of different origins and molecular composition using a set of model unicellular organisms, such as  Marine seaweeds are known as rich sources of specific bioactive substances, such as carrageenans, alginates, fucoidans, and phenols ,2. PhlorFucus, Ascophyllum (Fucaceae), and Carpophyllum (Sargassaceae) [The content of phlorotannins in the cells of brown algae is currently studied in detail only for several species of the orders Laminariales, Fucales, and Dictyotales . All thessaceae) ,10,11. Issaceae) ,16,17,18ssaceae) . Mostly,ssaceae) ,21 was eAscophyllum nodosum, Eisenia bicyclis, Ecklonia cava) [Phlorotannins have both primary and secondary metabolic roles in algal cells ,23,24. Tia cava) ,41,45. Uia cava) . Anotheria cava) .E. bicyclis is especially toxic for microalgae and methicillin-resistant strains of Staphylococcus aureus [Candida species, streptomycin-resistant Listeria monocytogenes, and acne-related bacteria [Escherichia coli for prokaryotes, Saccharomyces cerevisiae for fungi, Chlamydomonas reinhardtii for microalgae, etc.). The benefits of this approach include: (1) the assessment of specific responses of organisms with different cell structures and surface composition to a variety of phlorotannin extracts from different algal species and (2) the possibility of using well-established, standardized methods on model organisms for further deeper molecular studies and revealing the mechanisms of phlorotannin action.Despite numerous studies of phlorotannin cytotoxicity, our understanding of the underlying mechanisms is still very limited. Traditionally, antibiotic effects of phlorotannins were regarded as the consequence of their ability to precipitate proteins, a general feature of phenolic compounds ,49. Thiss aureus ,41. At tbacteria ,44,50. Tbacteria . In addibacteria ,39. Altobacteria ). As a rAs the first and crucial step in every toxicological research is usually a precise determination of MIC values, the objective of our study is measuring MICs of several phlorotannin preparations of different origins using a set of model unicellular organisms. In addition, we analyzed the total phenolic content of the easily extractable intracellular fraction of phlorotannins in several brown algae representing different taxonomic groups.Ch. plumosa) to 16.2% DW (F. vesiculosus) .The total content of phlorotannins in the studied brown algae varied from 2.4% DW (culosus) . RepreseF. vesiculosus, F. serratus, and P. canaliculata) carried out in our previous study [A comprehensive HPLC-MS analysis of three of the most biologically active extracts  to 300 (for Chlorella) µg/mL. Phlorotannins extracted from fucoid algae also had relatively low MIC values (5–20 µg/mL) for bacteria and yeast but were considerably less toxic for microalgae. Polyphenols of the Ectocarpales demonstrated high variation in their toxicity: extract of E. siliculosus had the lowest MIC values, comparable to those of fucoid algae, extracts D. foeniculaceus and Ch. flagelliformis showed moderate antibiotic activity, and extracts of P. littoralis were the least toxic against all the tested microorganisms.The toxicity of phlorotannin extracts varied considerably depending on both the source of polyphenols and the organism used as the test object . ExtractChlorella demonstrated the highest tolerance . The lowest MIC values of most phlorotannin preparations were registered in the experiments with yeast.Generally, microalgae were less susceptible to phlorotannins compared to bacteria and yeast . ChlorelPhloroglucinol, the monomer of phlorotannins, at concentrations up to 1 mg/mL, was not toxic to any tested organism .Desmarestia and fucoid algae showed the highest antibiotic activity, these preparations were additionally tested in experiments with non-growing and growing cultures of E. coli. The results of a time-kill assay with non-dividing bacterial cells (incubated in 0.9% NaCl solution) are shown in D. aculeata) or 4 (for F. serratus) hours of exposure, and more than 98% of E. coli cells died after 5 h-long exposure to any of the phlorotannin preparations , cultures of E. coli stopped growing completely for the first 12 h  also contained fewer phlorotannins (0.16% DW) compared to our data on Ch. plumosa  fucols and fucophlorethols, as was reported in our previous work [eight DP –10 fucolous work ,44,45,68ous work ,39,50) cE. bicyclis and E. cava) gained particular interest due to the high toxicity of their phlorotannins. The dominating phlorotannin molecules of these algae are low-molecular-weight eckols  were reported for extracts of A. nodosum [Fucus vesiculosus, F. serratus, and Pelvetia canaliculata, with extracts of F. vesiculosus being the most toxic  against marine fouling diatoms [Dictyosiphon foeniculaceus and Chordaria flagelliformis (Chordariaceae) contain polyphenols with ether-linked phloroglucinol units with a high degree of hydroxylation , whereas Pylaiella littoralis (Acinetosporaceae) synthesizes almost exclusively phlorotannins with aryl linkages (fucols). Though the mentioned study was focused on high-molecular-weight polyphenols, the authors noted that all available data indicated a structural similarity of low- and high-molecular-weight phlorotannins of the same origin [Ectocarpus is still not studied, though given their high antibiotic activity  showed moderate toxicity comparable to that of some ectocarpalean species  . We may suggest two possible reasons for this discrepancy. First, sphacelarialean algae may differ considerably in both structure and biological activity of their phlorotannins, as shown for the Ectocarpales. Then, data inconsistency may be a result of different methods of MIC calculation, as discussed above.The phlorotannin-enriched extract of the only representative of the order Sphacelariales used in this study (iformis) . This reF. vesiculosus, F. serratus, P. canaliculata, D. aculeata, and E. siliculosus) demonstrating MIC values similar to those of widely used antibiotics, fungicides and algicides. For comparison, MIC of tetracycline, ampicillin, amoxicillin, and ceftiofur is 0.5–128 μg/mL for different isolates and strains of E. coli [S. cerevisiae are 0.25–8 μg/mL [Chlamydomonas and Chlorella are 2–11 μg/mL [D. aculeata and E. siliculosus were determined here for the first time.According to our data, we may select five brown algal species . In particular, a set of mannoproteins linked to the cell wall polysaccharides forms the outer layer of the wall, the first structure facing the phlorotannin impact [S. cerevisiae are also known to interact readily with tannins of vascular plants, and this precipitation reaction is widely exploited in wineries . The major constituents of the walls of Ch. vulgaris are neutral sugars, gluconic acids, and glucosamine [Most probably, the first step in the progress of the reported damage to cell surface structures is the precipitation of surface-exposed proteins by phlorotannin oligomers. The ability of phlorotannins to precipitate proteins is higher compared to that of the tannins of terrestrial plants. Due to their susceptibility to spontaneous oxidation, phlorotannins, under natural conditions, cannot only form hydrogen bonds and non-polar interactions with proteins but also precipitate them via covalent bonds . It was nd yeast . The outsistance ,90. Moren impact ,92. Manns . Apparereatment  is also esistant . Notablycosamine .According to the comprehensive study of Stern et al. , phlorotFucus vesiculosus L., F. serratus L., Pelvetia canaliculata (L.) Dcne and Thur., Pylaiella littoralis (L.) Kjellm., Chordaria flagelliformis (O. F. Müll.) C. Ag., Dictyosiphon foeniculaceus (Huds.) Grev., Ectocarpus siliculosus (Dillw.) Lyngb., Desmarestia aculeata (L.) J. V. Lamour., Chorda filum (L.) Stackh., and Chaetopteris plumosa (Lyngb.) Kütz.) were collected in the Keret Archipelago  in July–August 2020–2022. All algal species are named according to AlgaeBase [Brown algae (lgaeBase . Mature v/v) mixture, ground with mortar and pestle and left soaking in 1 mL aqueous acetone for one hour to extract intracellular phenolics. Then, the extract was centrifuged , the supernatant was transferred to another tube, and the pellet was re-extracted with another 1 mL of aqueous acetone. The supernatants of five extraction rounds were combined.Phlorotannin extraction was carried out based on the standard protocol of Koivikko et al. ,21. For v/v) and left soaking for one hour. Then, each extract was centrifuged , the supernatant was transferred to another tube, and the pellet was re-extracted with another 10 mL of aqueous acetone. The supernatants of five extraction rounds were combined, and acetone was evaporated in a speed vac . Then the extracts were defatted three times, partitioning against dichloromethane , and phlorotannins were extracted by five successive portions of ethyl acetate . Ethyl acetate extracts were dried and resuspended in 1 mL water.For toxicity tests, concentrated and semi-purified extracts were used. Samples of 1–2 g frozen algal material were homogenized using a cryogenic laboratory mill Freezer/Mill 6870 , transferred to the falcon tubes, poured with 10 mL of acetone:water  was carried out in our previous study [A comprehensive HPLC-MS analysis of three exemplary extracts  aqueous Na2CO3 was incubated for 20 min at 45 °C, and then the absorbance was measured at 750 nm using a SPEKOL 1300 spectrophotometer .A modification of the Folin–Ciocalteu micro-method was used to measure the total phlorotannin content in the crude and purified extracts . PhlorogEscherichia coli strain KA796 (ara thi D(pro-lac)) [Saccharomyces cerevisiae haploid strain LAN201-ura3Δ  [Euglena gracilis Klebs strain Z, and two green microalgae, Chlamydomonas reinhardtii P. A. Dang. strain CC-124 and Chlorella vulgaris Beijer. strain Pringsheim. E. coli, S. cerevisiae, and Ch. reinhardtii were obtained from the collection of the Department of Genetics and Biotechnology, St. Petersburg State University. E. gracilis and Ch. vulgaris were obtained from the Resource Centre “Culture Collection of Microorganisms” of St. Petersburg State University.The following unicellular organisms were used for the bioassays: Gram-negative bacteria ro-lac)) , ascomyc2-3,112) , euglenoE. coli was cultured in a complete LB medium [S. cerevisiae was grown in complete YPD medium or minimal SD media  containing adenine (20 mg/L), uracil (20 mg/L), lysine (30 mg/L), tryptophan (20 mg/L), histidine (20 mg/L), and leucine (60 mg/L) [Euglena, Chlamydomonas, and Chlorella were grown phototrophycally under continuous light (50 μM photons/m2c) in the mineral Cramer-Myers, TAP, and BBM media, respectively [B medium  or minimB medium . S. cere60 mg/L) . All treectively ,103,104.4 cells/mL of bacteria and yeast, and 105 cells/mL of microalgae, respectively, were inoculated into each well. The final concentration of phlorotannin extracts/phloroglucinol ranged from 1 to 1000 μg/mL; pure growth medium was used as a negative control. For E. coli and S. cerevisiae, the MIC values were visually determined after 24 or 48 h of incubation, respectively. For microalgae, the MIC values were estimated after 4 days of incubation by counting the cells in a hemocytometer.MIC values were determined as the lowest concentrations of tested phlorotannin extracts, completely inhibiting the growth of the microorganism cultures. In addition to phlorotannins isolated from brown algae, an aqueous solution of phloroglucinol was also tested. The broth dilution method was used for the MIC assays . First, F. serratus and D. aculeata against E. coli.Two variants of bioassays were carried out for a more detailed study of the antibiotic effect of phlorotannins extracted from E. coli cells. An overnight culture of E. coli was diluted 4 × 105 times with 0.9% NaCl solution supplemented with phlorotannin-enriched extracts at the minimum inhibitory concentrations (20 μg/mL for F. serratus extract and 5 μg/mL for D. aculeata extract). Pure NaCl solution was used as a negative control. Then, the cell suspensions were incubated at 37 °C for 6 h, and 50 μL aliquots were plated on LB media every hour. The plates were incubated overnight at 37 °C, and the viable colony-forming units (CFU) were counted.Bactericidal activity was tested using a time-kill assay for non-dividing E. coli were analyzed. Overnight culture of E. coli was diluted 4 × 105 times with fresh media supplemented with phlorotannins at half-MIC and MIC (10–20 μg/mL for F. serratus extract and 2.5–5 μg/mL for D. aculeata extract). A pure growth medium was used as a negative control. Then, cell suspensions were incubated at 37 °C for 12 h, and 100 μL aliquots (diluted to contain ~400 CFU) were plated on LB media every hour. The plates were incubated overnight at 37 °C, and the number of viable CFU was counted.To test both the bacteriostatic and bactericidal activity of phlorotannins for the growing microbial culture, the growth curves of Measurements were performed with four  to twelve  biological replicates. An individual seaweed organism was used to obtain each analyzed phlorotannin sample. Excel 2016 , Photoshop CS2 , and ChemDraw Ultra  software was used for data processing and creating of figures. Where appropriate, data were tested for normality using the Kolmogorov–Smirnov test and for homogeneity of variance using the Levene test. Student’s t-test was used to confirm the significant differences between the means. All values are expressed as means and standard deviations.E. coli, S. cerevisiae, Ch. reinhardtii, E. gracilis, and Ch. vulgaris. For the first time, MIC values were evaluated for phlorotannin-enriched extracts of the brown algae of the orders Ectocarpales and Desmarestiales. Five of the tested algal extracts  demonstrated high toxicity with MIC values similar to those of widely used antibiotics. As all these species grow abundantly in polar and temperate seas and have considerable biomass and relatively high phlorotannin content, they may be regarded as promising sources of these valuable metabolites. Using common model organisms of molecular biology and genetics for the toxicological assays established the basis required for further mechanistic investigations of phlorotannin bioactivity. Certainly, one of the future imperatives of phlorotannin research should also be the production and availability of single phlorotannin standards for more systematic investigations of structure-activity relationships (SAR).In our study, we investigated MICs of several partially purified phlorotannin preparations of different origins using a set of unicellular model organisms:


Shigella species  have been reported, S. sp. PAMC 28760, an Antarctica isolate, is the only one with a complete genome deposited in NCBI database as an uncharacterized isolate. Because it is the world’s driest, windiest, and coldest continent, Antarctica provides an unfavourable environment for microorganisms. Computational analysis of genomic sequences of four Shigella species and our uncategorized Antarctica isolates Shigella sp. PAMC28760 was performed using MP3 (offline version) program to predict trehalase encoding genes as a pathogenic or non-pathogenic form. Additionally, we employed RAST and Prokka (offline version) annotation programs to determine locations of periplasmic (treA) and cytoplasmic (treF) trehalase genes in studied genomes. Our results showed that only 56 out of 134 Shigella strains had two different trehalase genes (treF and treA). It was revealed that the treF gene tends to be prevalent in Shigella species. In addition, both treA and treF genes were present in our strain S. sp. PAMC28760. The main objective of this study was to predict the prevalence of two different trehalase genes (treF and treA) in the complete genome of Shigella sp. PAMC28760 and other complete genomes of Shigella species. Till date, it is the first study to show that two types of trehalase genes are involved in Shigella species, which could offer insight on how the bacteria use accessible carbohydrate like glucose produced from the trehalose degradation pathway, and importance of periplasmic trehalase involvement in bacterial virulence.Although four  Shigella is a Gram-negative bacterium that is genetically related to Escherichia coli  ,21. It hmilarity . Neutralotection . Experimus study . Similarirulence . Despitethe host . HoweverShigella species were obtained from the NCBI database (https://www.ncbi.nlm.nih.gov/) [Shigella strains deposited in NCBI by September 2021 were analysed, including our Antarctica isolate S. sp. PAMC28760, whose genome size was 4,558,287 bp [The complete genome and amino acid sequences of ih.gov/) . A totalS. sp. PAMC28760 with those in other complete genomes of Shigella strains (133 strains), phylogenetic analysis was performed using the ClustalW alignment tool and the Molecular Evolutionary Genetic Analysis (MEGA X) (https://www.megasoftware.net/) tools [https://itol.embl.de/) was used to analyse phylogenetic trees [www.ezibiocloud.net) [Shigella or Escherichia, EziBio cloud 16S rRNA sequencing was used. Secondary data was used to identify the cytoplasmic trehalase or periplasmic trehalase from the characterized strains E. coli strain K-12 substrain MG1655 (NC 000913.3) as a reference for the construction of a phylogenetic tree for trehalase genes (treA and treF) in those studied strains who possess both trehalase genes. NCBI, RAST, and Prokka were used to find the cytoplasmic and periplasmic genes. MUSCLE [To compare 16S rRNA sequences of /) tools . MEGAX w/) tools  and 1,00/) tools . The onlic trees . Ortholostrains 1 strains,oud.net) . To dete. MUSCLE ,36 was uhttp://metagenomics.iiserb.ac.in/mp3/index.php) [https://rast.nmpdr.org/rast.cgi) [www.cgview.ca) was used to better visualization of location predicted trehalase genes [The prevalence of trehalase genes in the studied genome, as well as to predict pathogenic and non-pathogenic factors, were carried out using the MP3 (offline version) program (dex.php) . This prast.cgi)  and Prokast.cgi)  were usese genes .S. sp. PAMC28760 and S. dysenteriae ATCC12037 belonged to the same branch (S. sp. PAMC28760 had a close relationship with strains S. flexneri ATCC29903(T) (99.80%), S. sonnei CECT4887(T) (99.70%), E. coli ATCC11775(T) and S. boydii GTC779(T) (99.19%), E. fergusonii ATCC35469(T) (99.70%), S. dysenteriae ATCC13313 (T) (98.99%), and E. albertii TW07627 (T) (98.89%)  . These rShigella strains including our strain PAMC28760 were studied, only 56 strains were found to have two types of trehalase (treF and treA) genes. Furthermore, we employed RAST annotation database and, Prokka annotation to differentiate cytoplasmic (treF) and periplasmic (treA) trehalase. In addition, the CGview online server  of E. coli K-12 substrain MG655, S. sp. PAMC28760 was found to also encode the same genes involved in trehalose degradation  and treA .When complete genomes of 134 e server  visualizradation . While 4ectively . ResultstreF and treA) with a characterized E. coli K-12 substrain MG 1655 revealed that treA of S. sp. PAMC28760 and E. coli K-12 substrain MG1655 shared the same clade with 100% sequence identity, whereas S. sp. PAMC28760 did not share the same clade as E. coli K-12 substrain MG1655, although both shared 99.82% sequence identity (treA and treF) of S. sp. PAMC28760 could be distinctly divided into two major clades. It was found that treA and treF genes from studied genome clustered together more closely with both genes of S. flexneri. The treA gene is clustered with S. flexneri FDAARGOS-74 and S. flexneri WW1 whereas treF is clustered with S. flexneri 2016AM–0877 and S. flexneri 74–1170.Phylogenetic tree analysis of trehalase genes  [Mycobacterium smegmatis and Mycobacterium tuberculosis possessed trehalase that belonged to the GH15 family [Six routes of trehalose degradation pathways  have been found in organisms depending on their subcellular locations. These pathways have been reported in the MetaCyc pathway database . They arzy.org/) . The GH35 family .Figure S. sp. PAMC28760 possesses GH37 trehalase with two signature motifs (motifs 1 and 2) as well as highly conserved regions (CR3-CR5), which have also been found in E. coli. Further study confirms that S. sp. PAMC28760 possesses trehalase enzyme, a member of the GH37 CAZyme family (Bacillus subtilis (non-pathogenic) and Clostridioidess difficile (pathogenic) share a pathway in which exogenous trehalose can be imported by a PTS to produce glucose and glucose-6-phosphate via the phosphotreahalose TerA . Due to the acquisition of an additional cluster of trehalose metabolism genes, namely a second PTS that mediates high-efficiency trehalose uptake from the environment, epidemic C. difficile strains can also grow on low trehalose. By increasing toxin levels, both modified trehalose utilization systems contributed to the growth and toxicity of these epidemic C. difficile strains [Variovorax sp. PAMC28711 [Trehalase belonging to the GH37 family can hydrolyse a molecule of ∝,∝-trehalose into two molecules of glucose by inverting the anomeric orientation. Trehalase belonging to the GH37 family have been found in different species, including bacteria, fungi, yeasts, plants, insects, and vertebrates . GH famie family . The Gra strains . There hAMC28711 , the preShigella species (strains) from the NCBI database along with our S. sp. PAMC28760 isolates from Antarctica. Our strain S. sp. PAMC28760 showed pathogenic proteins of 1,136 (based on SVM model) out of 4329 total proteins  can predict the presence of pathogenic and non-pathogenic proteins in a complete genome of a microbe based on two models, SVM and HMM, and their hybrids (integrated SVM and HMM models). To predict pathogenic and non-pathogenic trehalase, we retrieved complete genomes of 134 proteins , with peE. coli (ExPEC) that are defective in binding to non-phagocytic cells is an unexpected finding on the probable role of periplasmic trehalase (treA) in virulence [treA) mutant in Burkholderia pseudomallei shows increased sensitivity to oxidative stress despite elevated trehalose levels in the mutant, which is expected to protect against this stress [Clostridioides difficile TreA, whereas trehalose derivatives such as epimers containing hydroxyl groups (2- and 4-positions), and thiotrehalose derivatives showed promise as TreA inhibitors with a larger spectrum. The efficacy of these drugs in treating specific bacterial infections is currently being studied [Shigella strains were analysed for pathogenic and non-pathogenic trehalase genes in this study for the first time. It is assumed that studying trehalase in one pathogenic bacterium like Shigella species could be important for further studies. Trehalase (TreA) from the pathogenic strain of extraintestinal E. coli known as MT78 has also been identified as a member of glycoside hydrolase 37 (GH37). Similarly, deletion of these genes in the meningoencephalitis-causing yeast Crytococcus neoformans resulted in severe defects in spore production, a decrease in spore germination, and an increase in the production of alternative development structures, which spores forms are plausible infectious particles [In addition, the detection of transposon mutants in extraintestinal pathogenic irulence . Furtherirulence ,53. Likes stress . Another studied . It has  studied . Genome articles . Trehaloarticles . Glycinearticles . Many spShigella species based on two different trehalase genes (treF and treA) and pathogenicity. Most Shigella species , as well as our strain S. sp. PAMC28760, have cytoplasmic trehalase, and all periplasmic trehalase predicted in the studied strains showed up as pathogenic proteins using MP3, RAST, and Prokka tools. Notably, treF was detected in all strains of S. sonnei, but treA was identified in only two strains. This sort of research on pathogenic and non-pathogenic trehalase could help researchers to elucidate how and why Shigella species have certain traits. Furthermore, before performing any kinds of wet lab work, these bioinformatics tools are important in determining the nature of proteins present in a complete genome of bacteria.Although there are many studies on trehalase, it was not studied in


Bartonella spp. and found a low frequency (7.9%) of positive animals. This study showed that European bison may be infected by at least two Bartonella species, namely B. bovis and B. schoenbuchensis. Our results demonstrated that further research is needed to determine the distribution of Bartonella species in wild and domestic ruminants, and it is important to identify the transmission route.The European bison is the largest ruminant in Europe. The population of European bison in Lithuania is increasing and several free-ranging herds exist. Owing to their protected status, studies of vector-borne pathogens in European bison are still lacking. By analyzing European bison and ticks collected from them in Lithuania, we checked for the presence of Bartonella bacteria infect the erythrocytes and endothelial cells of mammalians. The spread of the Bartonella infection occurs mainly via bloodsucking arthropod vectors. Studies on Bartonella infection in European bison, the largest wild ruminant in Europe, are lacking. They are needed to clarify their role in the maintenance and transmission of Bartonella spp. The aim of this study was to investigate the presence of the Bartonella pathogen in European bison and their ticks in Lithuania. A total of 38 spleen samples from bison and 258 ticks belonging to the Ixodes ricinus and Dermacentor reticulatus species were examined. The bison and tick samples were subjected to ssrA, 16S–23S rRNA ITS, gltA, and rpoB partial gene fragment amplification using various variants of PCR. Bartonella DNA was detected in 7.9% of the tissue samples of European bison. All tick samples were negative for Bartonella spp. The phylogenetic analysis of 16S–23S rRNA ITS, gltA, and rpoB partial gene fragment revealed that European bison were infected by B. bovis (2.6%) and B. schoenbuchensis (5.3%). This is the first report addressing the occurrence of B. bovis and B. schoenbuchensis in European bison in Europe. Bartonella species are Gram-negative bacteria that infect mammalian erythrocytes and endothelial cells. They are transmitted via direct contact  and by bloodsucking arthropod vectors such as fleas, lice, ticks, sand flies, and deer keds [Bartonella bovis, Bartonella capreoli, Bartonella chomelii, Bartonella schoenbuchensis, and Bartonella melophagi [Bartonella henselae and ‘Candidatus Bartonella davousti’ have been detected in domestic cattle [Bartonella species detected in ruminants are rarely implicated in animal diseases. To date, only B. bovis has been associated with bovine endocarditis [eer keds ,2. Domeselophagi ,4,5,6. Ic cattle ,8. Bartocarditis .Bartonella have been detected in domestic and wild animals and their ectoparasites. Bartonella grahamii, B. tailorii, B. rochalimae, B. tribocorum, B. coopersplainsensis, B. doshiae, and B. washoensis have been identified in several species of small rodents and in the fleas, ticks or mites collected from them [B. henselae, B. clarridgeiae and the Bartonella sp., closely related to B. schoenbuchensis have been detected in domestic cats and their fleas [Bartonella pathogens in domestic or wild ruminants in Lithuania.In Lithuania, various species of rom them ,11,12,13ir fleas . HoweverBison bonasus), the largest ruminant in Europe, is known for its status as an animal under species protection. Recent studies have demonstrated that bison may play a role as a natural reservoir of Anaplasma phagocytophilum, Babesia divergens, and Babesia venatorum [Bartonella infection in European bison in Europe. In the present study, we aimed to investigate the presence of the Bartonella pathogen in European bison and their ticks (Ixodes ricinus and Dermacentor reticulatus) in Lithuania using real-time PCR targeting the ssrA gene, and to characterize Bartonella strains by the PCR and sequence analysis of the 16S–23S rRNA ITS (the 16S–23S rRNA intergenic species region), gltA (citrate synthase gene) and rpoB (RNA polymerase β-subunit) genes.The European bison . Due to the status of animals under species protection, the tissue samples of European bison were taken from animals found dead in the field (n = 6), accidentally killed on roads (n = 10), and eliminated from nature under a protection plan due to diseases or genetic disorders (n = 22). The bison sampling was conducted with permission from the Environmental Protection Department under the Ministry of Environment . A total of 258 ticks were collected from 6 animals that had died due to unknown causes. Ticks were placed in 1.5 mL tubes with 70% ethanol and kept at 4 °C until investigation. Tick species were identified based on morphological criteria [Ixodes ricinus  and Dermacentor reticulatus . A total of 116 ticks were engorged (85 I. ricinus and 31 D. reticulatus) and 143 non-engorged (45 I. ricinus and 98 D. reticulatus).A total of 38 spleen samples of European bison  were collected in the period 2019 to 2022 in Central and Northern Lithuania (Kėdainiai and Panevėžys regions). Bison samples were collected in all seasons  according to the manufacturer’s recommendations. DNA from non-engorged ticks was extracted using 2.5% ammonium hydroxide .Bartonella DNA (124 bp product of ssrA gene) was conducted by using TaqMan real-time PCR with ssrA-F1 and ssrA-R1 primers and a ssrA-P1 probe, as previously described [Bartonella-positive samples in real-time PCR were further analyzed using nested PCR assays that amplify partial sequences of the 16S–23S rRNA ITS region  and conventional PCR assays of gltA (primers BhCS.781p and BhCS.1137n) and rpoB (primers 1400 F and 2300 R) genes [Bartonella-infected rodents, confirmed by sequencing) and negative  controls were included in each PCR run. PCR products were identified by electrophoresis on 1.5 % agarose gel.Screening for the presence of escribed . BartoneR) genes ,20,21,22Bartonella-positive samples were extracted from agarose gel and purified using the GeneJET™ Gel Extraction Kit . The obtained partial 16S–23S rRNA ITS region, gltA, and rpoB gene sequences were analyzed using the MegaX software package [PCR products of all gltA, and rpoB gene sequences for representative samples obtained in this study were submitted to the GenBank database under accession numbers OP888096 for the ITS region, OP894362–OP894364 for the gltA gene, and OP894359–OP894361 for the rpoB gene.Partial 16S–23S rRNA ITS region, Bartonella DNA was detected in three out of the thirty-eight (7.9%) tissue samples of European bison. Positive results were obtained in each animal when applying all three PCR techniques . The Bartonella infection was detected in samples collected during winter. All ticks (n = 258) collected from the six European bison specimens not infected with Bartonella were negative for Bartonella spp. All positive samples were subjected to sequence analysis. B. bovis sequences isolated from domestic cattle from France, Guatemala, and Israel   was 100% identical to M371094) .gltA partial gene fragments were obtained from three European bison samples. The sequence analysis revealed that the Bartonella strain (GenBank: OP894362) derived from one individual European bison clustered with B. bovis isolated from cattle from various countries  with an identity score 100%. The other two sequences  demonstrated a 100% similarity with B. schoenbuchensis isolated from roe deer , deer ked from Germany (GenBank: AJ564632), and a human sample from France (GenBank: HG977196)  .rpoB gene sequence (GenBank: OP894361) clustered with other B. bovis sequences. The highest similarity (99.9%) was shared with B. bovis, previously isolated from cattle from France (GenBank: KF218217). Meanwhile, two sequences derived from two bison specimens  were 99.1% similar to the B. schoenbuchensis sequences isolated from roe deer from Germany (GenBank: CP019789) and human (GenBank: HG977196), differing at six nucleotide positions (B. capreoli isolated from red deer from USA (GenBank: HM167505), differing at fifteen nucleotide positions.The ositions . AdditioB. bovis and B. schoenbuchensis in European bison in Europe. Furthermore, we tested I. ricinus and D. reticulatus ticks collected from bison and did not detect the presence of Bartonella species. The role of ticks in the transmission of Bartonella is not fully understood [Bartonella spp. in ruminants [This study reports the first molecular detection of derstood ,6. Otheruminants .Bartonella bovis is mainly detected in cattle. Infection with B. bovis is usually asymptomatic, but it is known that it can be associated with bovine endocarditis [B. bovis in domestic cattle ranges from 7% to 36% in Europe and from 5% to 90% in other continents [B. bovis infections also have been reported in wild ruminants such as moose, red deer, and roe deer [B. bovis DNA has been identified in 3.2% of ticks collected from cattle [Bartonella infection in ticks has not been detected. This study presents a low frequency of B. bovis (2.6%) in European bison in Lithuania. A previous study conducted in Poland has shown that 6.8% of cattle (Bos taurus), a closely related species to European bison, had asymptomatic B. bovis infection [Bartonella in Lithuanian ruminants, an epidemiological study with a larger sample size is needed.carditis ,9. The pntinents ,26,27. Froe deer ,29. A reB. schoenbuchensis. This result is not unexpected because previous studies reported cases of B. schoenbuchensis in domestic and wild ruminants from Georgia, Poland, France, and Norway [Bartonella spp. highly similar to B. capreoli, B. chomelii, and B. schoenbuchensis had been identified in deer ked distribution areas [Bartonella species. Furthermore, outside the deer ked distribution area, other vectors such as ticks or culicoides biting midges could also be involved in Bartonella transmission [The present study revealed that two individuals of European bison (5.3%) were infected with d Norway ,28,30,31d Norway . Accordion areas ,31,33. Tsmission ,34. Eurosmission .Bartonella species, namely B. bovis and B. schoenbuchensis. Further research is needed to determine the vectors of transmission of Bartonella species among bison and other ruminant species.The results of this study suggest that European bison may be infected by at least two


Melanotus communis larvae attack grain, root/ tuber, and vegetable crops. Our objectives were to characterize the pathogenicity and virulence of fungal and nematode entomopathogens on M. communis wireworms, and determine if wireworm size affected virulence. Pathogens tested included five entomopathogenic nematodes, Steinernema carpocapsae (All strain), S. feltiae (SN strain), S. riobrave (355 strain), Heterorhabditis bacteriophora (VS strain), and H. indica (HiHom1 strain); and two entomopathogenic fungi, Beauveria bassiana (GHA strain) and Cordyceps javanica (WF-GA17 strain). None of the pathogens tested caused >15% mortality at 7 or 14 days post-inoculation. Mortality was highest in S. carpocapsae (All strain); the other entomopathogens did not cause higher mortality than the untreated control. Overall, smaller wireworms were more susceptible than larger wireworms. Our results suggested that M. communis wireworms have defenses that limit the ability of the entomopathogens we tested to infect the wireworms. Conceivably, other entomopathogen strains or species may be more effective. Natural populations of entomopathogens may contribute to wireworm population reduction, but further studies are warranted before entomopathogens can be used for M. communis management.Wireworms, the soil-borne larvae of click beetles (Coleoptera: Elateridae), are important crop pests throughout the world. In the eastern U.S.,  Melanotus communis  (Coleoptera: Elateridae) is an important pest of maize, small grains, sugarcane, vegetables, and root and tuber crops in much of the United States east of the Rocky Mountains  before emergence as adult click beetles in the spring and summer , thus research on development of alternative and/or complementary control strategies is warranted. Entomopathogens that live in the soil offer an attractive solution for the management of M. communis larvae.ountains . In pota harvest . Moreoved summer . Mating d summer . Adult bd summer . CurrentSteinernema Travassos and Heterorhabditis Poinar are obligate parasites of insects . Under field conditions, EPNs can be applied with most standard agricultural equipment, thus facilitating their usefulness in pest management  in the genera  insects . The EPN insects . Recentl insects , use of nagement .Beauveria bassiana  Vuill. and Cordyceps javanica are EPFs that live in the soil and have been effective in controlling numerous arthropods  than did the other species tested . Recently developed strategies for use of EPFs include “attract-and-kill” (Entomopathogenic fungi (EPF) have also shown promise for insect pest management . Beauvernd-kill” .M. communis larvae and to determine the effect of host size  on susceptibility to these pathogens. To our knowledge, EPF and EPN biocontrol agents have not been tested for pathogenicity or virulence to M. communis.The objectives of this study were to screen EPNs and EPFs on M. communis occur in the soil; thus, it is expected to find a range of size classes. After collection, wireworms were identified (Wireworms (larvae) were collected from November 2020 to April 2021 by digging under sugarcane stools (plants) at the Everglades Research and Education Center, University of Florida, in Palm Beach County, Florida. These collections included wireworms of all sizes because multiple generations of entified  and storM. communis: two EPFs, viz., B. bassiana  Vuill.  (GHA strain) and C. javanica  Kepler, B. Shrestha & Spatafora  (WF-GA17 strain); and five EPNs, viz., S. carpocapsae (Weiser) (strain All), S. feltiae (Filipjev) (SN strain), S. riobrave Cabanillas, Poinar, and Raulston (strain 355), H. bacteriophora Poinar (VS strain), and H. indica Poinar, Karunakar, and David (Hom1 strain). We chose these EPNs because they are all commercially available and hence could conceivably be used in biocontrol applications if the results supported such action.All pathogens were obtained from the USDA-ARS collection in Byron, Georgia. The following pathogens were tested for virulence to 8 conidia ml-1) was determined using a hemocytometer under the microscope. Conidial viability of each isolate was assessed by plating 0.1 ml of solution of each of the two isolates on small Sabouraud dextrose agar yeast (SDAY) petri plates (60 mm diameter)  petri plates (100 mm diameter) and incubated at 25°C at light:dark cycle of 14:10 hr. Fungi were scraped from the inoculated plates 7 d to 10 d postincubation, and the resulting conidia were placed inside conical tubes (50 ml) that contained 30 ml of sterile 0.05% Silwet L-77  solution. Eight glass beads were added to each tube and vortexed for approximately 5 min, and the desired concentration   at 25°C, and the IJs were collected on White traps  plus a control group. Experimental arenas consisted of plastic cups (30 ml) that were filled with 20 g of soil giving a surface area of approximately 28 cm2. The soil was obtained from the USDA-ARS pecan orchard ; it was loamy sand  with 2.8% organic matter and a pH of 6.1. Prior to experimentation, the soil was autoclaved (275°C for 30 min) and held at room temperature for at least 2 wk.Effect of entomopathogens on M. communis was tested in both trials . In each trial, three replicates of each treatment were tested, and each replicate consisted of five larvae in individual cups. These larvae ware added to cups with moistened soil and a 2.5-cm piece of carrot. Wireworms that did not burrow into the soil within 12 hr were replaced. Treatments were subsequently added in 1 ml suspensions; the final soil moisture level was at field capacity (16%). The treatments were pipetted onto the soil surface, and the control cups received only 1 ml of tap water. The concentration of EPFs was 108 viable conidia per milliliter and, for EPNs, the concentration was 2,800 IJs (100 IJs/cm2). These rates were chosen because they are comparable to field rates used for the pathogens tested , data were pooled among trials. Multiple treatment differences were elucidated with Tukey’s studentized range (honestly significant difference [HSD]) test  before analysis. Using a factorial design with treatment and wireworm size as the main factors, data were analyzed using PROC GLM . Within D]) test .F = 1.05; df = 7, 72; P = 0.407), wireworm size and treatment effects were analyzed independently. In trials with wireworms of both size classes, significant differences  were detected among the treatments. Treatment mortality ranged from 1.7% to 15% and was the highest in S. carpocapsae (strain All)  (Given that there was no interaction between main factors (ain All) . In tria 0.0021) . Figure S. carpocapsae (strain All) was the only treatment found to be pathogenic to M. communis larvae in small cup laboratory bioassays; mortality observed in the other pathogen treatments was not significantly different from that in the control. While we measured only mortality for assessment of pathogenicity, the pathogens in our treatments may have indirectly affected wireworm feeding, growth, or other aspects of fitness. Overall mortality levels in M. communis larvae were low compared to similar studies with the same pathogens on other insect taxa, with reported mortality ranging from 25% to 80% , was moderately susceptible  to the EPN H. bacteriophora, and was not significantly affected by B. bassiana. M. brunneum has shown promise in the management of Agriotes spp. using an “attract-and-kill” strategy  are more susceptible to entomopathogens than the larger wireworms. Contrary to our study, A. obscurus wireworms were more susceptible to M. anisopliae than smaller wireworms. Studies with other insect taxa indicate that susceptibility of insects to pathogens can be affected by host size . Conversely, Phyllopertha horticola L. (Coleoptera: Scarabaeidae) to Heterorhabditis spp. and Steinernema spp. increased with larval development. Interestingly, in our study, the very-small-sized M. communis (0.02 g and 0.03 g) were also not susceptible to infection. Little is known about the effects of wireworm size on pathogenicity to EPNs and EPFs. The current study found that M. communis. Research areas with potential include exploration for entomopathogens more closely linked to M. communis larvae (M. communis wireworm management practices. Genetic improvement of pathogen strains through hybridization is promising (Our findings indicate that further work is necessary before a management strategy relying on entomopathogens can be developed for s larvae . The facromising . Additio


Toxoplasma gondii (T. gondii) is an obligate intracellular parasite. During the parasitic invasion, T. gondii creates a parasitophorous vacuole, which enables the modulation of cell functions, allowing its replication and host infection. It has effective strategies to escape the immune response and reach privileged immune sites and remain inactive in a controlled environment in tissue cysts. This current review presents the factors that affect host cells and the parasite, as well as changes in the immune system during host cell infection. The secretory organelles of T. gondii  are responsible for these processes. They are involved with proteins secreted by micronemes and rhoptries  that mediate the recognition and entry into host cells. Effector proteins (ROP and GRA) that modify the STAT signal or GTPases in immune cells determine their toxicity. Interference byhost autonomous cells during parasitic infection, gene expression, and production of microbicidal molecules such as reactive oxygen species (ROS) and nitric oxide (NO), result in the regulation of cell death. The high level of complexity in host cell mechanisms prevents cell death in its various pathways. Many of these abilities play an important role in escaping host immune responses, particularly by manipulating the expression of genes involved in apoptosis, necrosis, autophagy, and inflammation. Here we present recent works that define the mechanisms by which T. gondii interacts with these processes in infected host cells. Toxoplasma gondii (T. gondii) is an obligate intracellular pathogenic protozoanwith sexual and asexual life cycles that can infect any nucleated cell of most animals. The reason for the parasite’s success is partly due to its ability to manipulate host cells and escape immune mechanisms, resulting in chronic (lifelong) infections . Th. Th85]. . gondii . Specifi. gondii . They marasitism . T. gondsurvival .T. gondii tachyzoites typically lyse infected cells after 72 h, depending on the type of organism [T. gondii can supply the required nutrients for growth, protection against host defenses, and replication and transmission [Growth and replication of organism . Importaorganism ,94. An ismission ,96,97. Tsmission .T. gondii infection simultaneously blocks the activation and activity of caspase-3. Despite the presence of caspase-3, other factors related to apoptosis were observed in Toxoplasma-infected cells, such as mitogen-activated protein (MAP) kinase , JAK/STAT (ROP16), NF-κB (GRA15), and P53 (GRA16). One of the underlying mechanisms by which T. gondii block caspase activity is through the secretion of the inhibitory apoptotic protein (IAP) in host cells, which effectively inhibits apoptosis [T. gondii may inhibit caspase activity, triggering probiotic and anti-apoptotic responses in host cells [T. gondii on apoptosis can be controlled by regulating various signaling pathways, including the NF-κB and c-Jun N-terminal kinase (JNK) pathways [Studies have shown that intracellular parasitic infection leads to the inhibition of caspases, notably caspase 3, the bulge where signals converge along both pathways . Considepoptosis . There ist cells . The inhpathways ,100.T. gondii infection in mice [T. gondii (type I-RH strain) modifies gene expression in mouse spleen cells. Gene expression was positively correlated with immune responses in the mice models [Reactive oxygen species (ROS) can trigger trophoblast apoptosis by activating the JNK homologous protein, Anti-C/EBP (CHOP), and the caspase 12 pathway in  in mice . It has e models .T.gondii excretory/secretory antigens (TgESAs), the proliferation and phagocytosis capacity of Ana-1 cells was decreased, and apoptosis was induced in a dose-dependent manner [T. gondii inhibits caspase-3 and regulates the activation of different caspases upstream in the intrinsic or extrinsic pathway. Cells infected with T. gondii reduce levels of caspase-3, caspase-8, caspase-9, and caspase-12 [T. gondii is correlated with increased expression of anti-apoptotic genes such as the IAPs and Bcl-2 families [Wang et al. reported that after treatment of Ana-1 cells with different concentrations of t manner . T. gondspase-12 ,103. A rspase-12 . A variefamilies .T. gondii modifies the expression of apoptosis-related genes to maintain survival in host cells [T. gondii. They found that intracellular tachyzoites of T. gondii were resistant to multiple inducers of apoptosis, including Fas-induced cytotoxicity, IL-2 deprivation, irradiation, UV, calcium ionophores, and beauvericin [+ T cells) acts directly on caspase and accelerates the induction of apoptosis. T. gondii reduces apoptosis in Granzyme B-dependent apoptosis in host lymphocytes [T. gondii blocks host cell apoptosis by inhibiting caspases-3 and Granzyme B activity. The parasite-mediated inhibition of Granzyme B demonstrates that this inhibition is associated with the protection of infected host cells from cytotoxic lymphocyte-mediated apoptosis [st cells . Nash etuvericin . Granzymphocytes ,108. T. poptosis .T. gondii infection induces NF-κB activation. Therefore, the induced activation of NF-κB also enhances the activation of anti-apoptotic genes [T. gondii ME49 strain. This strain upregulates A20, resulting in the inhibition of NF-κB activation and induction of apoptosis in human T cell lines [T. gondii infection inhibits actinomycin D in mouse spleen cells by inactivating caspases and activating NF-κB-mediated apoptosis [It has already been shown that ic genes . For exall lines ,111. Trepoptosis .T. gondii depends on IL-12, p40, and FasL, which may play a role in the pathogenesis of fatal infections [T. gondii-infected cells [Gavrilescu et al. showed that the activation of several apoptotic pathways induced by fections . Nishikaed cells .+ enriched splenocytes from immunized mice with concanamycin A, but not monoclonal anti-Fas ligand, significantly decreased their anti-proliferative and lethal abilities. Thisimplies that CD8+ T cells induced by immunization with RH antigen and live Beverley strain bradyzoites may exert protection against T. gondii infection, at least in part by granule-dependent cytotoxic activities [It has been shown that NF-κB, which induces the anti-apoptotic pathway, induces the upregulation of Fas and FasL . Treatmetivities .T. gondii-infected cells. HSP65 synthesis is inhibited via activation of the HSP65 antisense oligonucleotide by IFN-γ plus Tumor necrosis factor (TNF)-α. Hence, HSP65 appears to contributeto immune function by preventing apoptosis of infected macrophages and decreasing parasite survival and virulence. Under in vivo conditions, the Beverly strain, unlike the RH strain of T. gondii, induces the expression of HSP65 in the host gd-T cell [It has also been suggested that Heat shock proteins (HSPs) are involved in the regulation of apoptosis in d-T cell .T. gondii infection. One of the potential targets is an apoptosome because HSPs have been shown to interfere with the development of apoptosomes [Under in vitro conditions, the forced inhibition of HSP65 expression with HSP65 antisense oligonucleotides caused apoptosis during infection with the less contagious Beverly strain. Inhibition of apoptosis is only observed when HSP65 is expressed before cell infection, indicating that the timing of HSP expression may be crucial . It is cptosomes .T. gondii [T. gondii infection in vitro and in vivo induced the phosphorylation of PKB/Akt and Bcl-2-associated death promoter (Bad). Anti-apoptosis by T. gondii occurs partly through phosphorylative inactivation of Bad [T. gondii can cause apoptosis and lead to the spread of infection [Inhibition of apoptosis is also transcriptionally regulated in favor of the parasite’s survival. Infection of mouse splenocytes activates host NF-κB transcription and anti-apoptotic genes . Mitocho. gondii . After c. gondii . In addin of Bad . In the nfection .T. gondii causes host immunization and neuron apoptosis via endoplasmic reticulum (ER) stress [According to a previous study, the ROP18 protein of ) stress . ROP18 i) stress . ROP18 h) stress ,124, but) stress ,126, and) stress .In the murine model, ROP18 modifies innate and adaptive immune responses. ROP18 phosphorylates immunity-related GTPase (IRGs) and prevents the breakdown of the parasitophorous vacuole membrane (PVM) . ROP18 hT. gondii inhibits apoptosis by generating signals and activating transcriptional molecules. Serine protease, such as SERPIN B3/B4, is extensively expressed in T. gondii-infected macrophages upon STAT6 activation. In T. gondii-infected macrophages, upregulation of the squamous cell carcinoma antigen 1/2 (SCCA1/2) gene by the host transcription factor STAT6 may be an important mechanism for T. gondii-mediated apoptosis in host cells [T. gondii infection. In other words, STAT3 mediates a prosurvival pathway by upregulating the miR-17–92 miRNAs that in turn targets Bim, leading to the survival of host cells with Toxoplasma infection [Studies have also shown that st cells . The STAnfection .T. gondii, particularly in murine models. In this regard, it is important to produce IFN-γ in the early phases of infection [T. gondii PVM [T. gondii , it has been shown that the parasite-secreted antigens, such as ROP5, ROP17, and ROP18, are involved in maintaining the integrity of the PVM. The ROP18 kinase function begins after initiating the IFN-γ cascade and protects the degradation of PVM by blocking IRG-dependent killing through the inactivation of IRG proteins by phosphorylation of the nucleotide site [Experimental evidence suggests that autophagy can kill many pathogens such as nfection . Influennfection . In a mondii PVM . In addiide site ,135. Thiide site .T. gondii can also be killed independently of IFN-γ by autophagy in mouse macrophages by the involvement of the TNF receptor superfamily, AMP-activated kinase, calcium/calmodulin-dependent kinase β (CaMKKβ), activation of JNK, and Unc-51-like autophagy activating kinase 1 (ULK1) [1 (ULK1) .In murine cells, STAT1 signaling initiates the expression of inducible nitric oxide synthase (iNOS), generating ROS and nitric oxide (NO), as well as the upregulation of GBP and IRG. The recruitment of GBP and IRG in PV depends on the main set of autophagy proteins, containing the Atg5-12-16 complex. Suwanti et al. demonstrated that murine congenital toxoplasmosis increases skull apoptotic index and skull apoptosis associated with increased IFN-γ expression, but it decreases TNF-α expression . BlockadT. gondii provides an excellent model for determining whether the immune system can target pathogens to lysosomal degradation. CD40 is a major regulator of cell-mediated immunity, which kills T. gondii by macrophages and requires the recruitment of autophagosomes around the PVs, leading to lysosomal degradation of the parasite [parasite .T. gondii-specific T cells [T. gondii can inhibit the function of IFN-γ by blocking IRG and IFN-γ activation and stimulating the formation of PVM and destroying PVP [T. gondii type II and III PVS to surround and destroy them through the process of autophagy. This pathway uses the human-specific nuclear domain protein (NDP) 52 and p62-binding proteins and is dependent on Atg16L1 and Atg7 [T. gondii and has only a marginal effect on the recruitment of GBP. In human foreskin fibroblasts (HFFs), the destruction of Atg5 did not affect the IFN-γ-mediated growth of T. gondii type I. Moreover, in human umbilical vein endothelial cells (HUVECs), autophagy and deletion of T. gondii type II were observed. Atg9 is required for the survival of T. gondii in immune cells, and the general parasite virulence may be promoted by interference with a canonical pathway of autophagy [According to Choi and colleagues (2014), IFN-γ can increase the degradation of PV and parasite antigens; hence, it can be identified by  T cells . Recent ying PVP ,142,143.ying PVP ,144. In and Atg7 . In contutophagy .T. gondii IFN-γ-mediated restriction is mediated by IDO upregulation, which degrades L-tryptophan and inhibits the progression of auxotrophic tryptophan. In human cells, upregulation of IDO and ROS may lead to the control of parasite proliferation. The activity of inorganic nanoparticles (NPs) may be due to alterations in the redox condition and potential of the parasite’s mitochondrial membrane [In many types of human cells, membrane .-parasitic effect of NPs [Toxoplasma is restricted in a human cell might depend on the phagocytic ability of the cell versus the induced GBP and IDO levels. Surprisingly, GBP versus IDO-mediated restriction of Toxoplasma has not been studied. It is thus possible that these pathways counter-regulate each other and co-exist [Adeyemiet al. showed the interaction between several host cellular processes, including Hypoxia-inducible factor 1-alpha (HIF-1α) activity, IDO activity, and, more broadly, the tryptophan pathway, contribute to theantit of NPs . Therefoco-exist .T. gondii PVM are known as linear M1, K48, and K63 polyubiquitin chains [T. gondii PVM after IFN-γ treatment. However, the effect of TRAF6 in stimulating IFN-γ to suppress T. gondii is controversial [T. gondii PVM can be achieved by other E3 ligases, such as TRAF6 [The molecular mechanism of PVM detection by GTPases is still an open question. Studies show that the C-terminal isoprenylation of GBP2 regulates GBP2 use in PVM. However, isoprenylation alone cannot differentiate PVs from host organelles. The detection of ubiquitin in intracellular organisms creates an important host mechanism . More tyn chains ,149. TNFoversial ,148. PVMas TRAF6 .T. gondii could be a result of cell activation by CD40. Stimulation of CD40 may lead to vacuolar ATPase activation followed by fusion of pjosphoinositide-3-class 3 (PIK3C3) and Rab7 on PVM with late lysosomes or endosomes [T. gondii PVM is controversial in response to IFN-γ activities [T. gondii. However, other ATG proteins that regulate the pathway contain Atg9, ULK1, and PI3K. Therefore, anti-IFN-γ-mediated T. gondii responses can be regulated by these Atg proteins independently of autophagy [T. gondii are increased in host cells without mitochondrial fusion, required for efficient mitochondrial oxidation of FA, or when mitochondrial oxidation of FA is pharmacologically inhibited [Atg8 is aubiquitin-like protein, essential for the formation of autophagosome membranes. In mammals, Atg8 includes the subclass of LC3, such as A, B, and C types, and GABARAP subunits, including Gabarap, Gabarapl1, and Gabarapl2/Gate-16 . Studiesndosomes . Howevertivities . It is ptivities . The exputophagy ,156,157.utophagy . GBP is utophagy . Conversnhibited . The assnhibited . LC3 is nhibited ,163,164.T. gondii may pave the way for disease control. GTPases such as GRI and GBP are two families of proteins that have evolved as effective mechanisms for controlling anti-T. gondii autophagy pathways. The homeostasis of these two families is controlled by a set of autophagy proteins involved in the prolonged phase of autophagosome formation. Removing any of these proteins leads to the natural activation and combination of IRGs and the disruption of T. gondii infection control [Understanding the mechanisms of autophagy in  control ,165.T. gondii is a tryptophan auxotroph, tryptophan degradation and indoleamine oxidase induction are important for parasite control in specific cell types [T. gondii could be inhibited by the expression of the dominant negative protein kinase R(PKR). Thus, T. gondii activates a Focal adhesion kinase (FAK)→ Src → Y845-EGFR → STAT3 signaling axis within mammalian cells, which allows the parasite to survive by avoiding autophagy through a mechanism that may block the activation of PKR and the Eukaryotic Initiation Factor 2 alpha (eIF2α) [T. gondii micronemal proteins containing EGF domains appeared to promote EGFR activation [In addition, because ll types . Muniz-Fll types . Portill (eIF2α) . In muritivation .T. gondii infection due to the presence of the rhoptry protein [T. gondii is designed to secrete Cre recombinase into host cells. Cre interrupts a stop codon, and then GFP can be identified in infected and uninfected cells, respectively [T. gondii microenvironment offers two important advantages to fighting off this parasite [In addition to plasma membrane receptors, intracellular proteins may also be targeted during  protein . Previou protein . This paectively . Compareectively . In addiectively . Importaparasite .Toxoplasma infection [The first activation of the host cellular process during the parasitic invasion gives the parasite opportunity to carry out the propagation process. This involves altering host-cell metabolism to help parasites access essential nutrients and activating mechanisms to prevent inherent immune defense such as apoptosis, pyroptosis, and autophagy. Second, it would allow the parasite to disarm IFN-γ and other mechanisms of destruction of immune effectors before entering the host cell. The ability to access and regulate immune responses may provide another mechanism for the parasite to escape the immune response. For example, activation of HIF-1 may promote the development of effector and regulatory T cells . Downregnfection .CD40 is expressed on the surface of antigen-presenting cells (APCs), such as macrophages and many non-hemopoietic cells. In this case, these agents activate pro-inflammatory mediators, such as IL-12, and ultimately lead to IFN-γ secretion. In human cells, CD40 is associated with its ligands expressed on the surface of T cells and leads to the production of IFN-γ from T cells, dependingon the production of IL-12 from macrophages ,178.T. gondii containing PVs by combining LC3 and lysosome proteins. Ubiquitination in human cells and recruitment of automated adapters are not required in the presence of GBP. The recruitment of IRGs and GBP for PVM depends on the Atgs. HeLa epithelial cells target ubiquitinated PVs to improve retardation in cells through non-canonical autophagy. This host defense pathway and the one described in endothelial cells are avoided by type I parasites, suggesting the presence of a human T. gondii virulence factor [The interactions of CD40-CD40L result in the production of TNF-α required for CD40-CD40L signaling to eliminate e factor .T. gondii type I. In other words, Toxoplasma resistance is not significantly altered in cells deficient in ATG5 [T. gondii type II clearance has been observed [T. gondii in human macrophages has not been established. Autophagy is a pathway closely linked to cellular metabolism. In many cell types, the IFN-γ-mediated limitation of T. gondii is implemented by the overregulation of IDO, which, by mortifying L-tryptophan, prevents the evolution of auxotrophic tryptophan in T. gondii [In HFFs, the destruction of ATG5 does not affect the IFN-γ-mediated growth restriction of  in ATG5 . In humaobserved . The rol. gondii .T. gondii infection. Similarly, the gene Arachidonate 12-lipoxygenase, type 12S (ALOX12), which encodes the arachidonate 12-lipoxygenase enzyme, has alleles related to toxoplasmosis, and ALOX12 knockdown prevents the proliferation of T. gondii tachyzoites by aggregating RCD [T. gondii infection in people with the loss of P2X7R function [NACHT leucine-rich-repeat protein 1 (NALP1) alleles produce pyroptosis in human cells during ting RCD . An impofunction .T. gondii proliferation, which requires replication and survival of the host cell. Therefore, it is not astounding that T. gondii inhibits host cells’ apoptosis; this inhibition can occur via various pathways , and these effects depend on GRA15, which increases IL-1 expression by activating NF-κB [T. gondii types I, II, or III do not cause rapid cell death [T. gondii, in which the activation of NLRP1 inflammation leads to rapid cell death. Strains of mice that are susceptible to T. gondii confer a difference from a 1.7-cM genetic source, the toxol locus [T. gondii do not undergo pyroptosis and do not secrete IL-1 [T. gondii, as shown by the induction of IL-1 and its secretion by macrophages. NLRP1 and NLRP3 are innate immune sensors for Toxoplasma infection, activated via a novel mechanism that corresponds to a host-protective innate immune response to the parasite [T. gondii on activating pyroptosis/inflammation highlights the importance of the host cell life for parasite survival.The contribution of interactions between human toxoplasmosis and the inflammatory process indicates that the expression of IL-1 in human monocytes depends on ASC and caspase-1, which is an adapter protein that binds caspase-1 to NLRP3/CARDB or NLRP1/caspase-5 in the flammomasome . IL-1 exng NF-κB . ROP16 kng NF-κB ,57. Studll death ,198. Theol locus . In suscete IL-1 , whereasete IL-1 . This diete IL-1 . In miceparasite ,189. Conparasite . Severalparasite ,189. TheT. gondii. Various evidence has shown that T. gondii infection plays a role in thyroid autoimmunity and rheumatoid arthritis using severalmechanisms [S) by using HSPGS and SAG1 as receptors, infecting various cell types [Autoimmune diseases can be caused by different factors, with genetics and environment being the most important factors, and the effective mechanisms are molecular mimicry and superantigens . Some pachanisms . As mentll types . We can ll types ,203. Thell types . As mentll types ,206.T. gondii immuneregulates host cell death will boost our comprehension of the host–parasite interplay in toxoplasmosis. The various secretory organelles of T. gondii are involved in the invasion and modulation of host cell functions. In general, a set of RCD processes is used by the parasite for this purpose. The recognition, contact, and adhesion to the host cell seem to be mediated mainly by SAG and MIC proteins. Different cell lines of the same species can vary significantly in the expression of RIPK3, NLR, and IRG, which probably explains the cell type and species differences in response to T. gondii. Critical to clarifying the in vitro and in vivo role of RCD blockade may be the identification of defective parasite mutants and the modulation of cell signaling pathways. Thus, the parasite achieves an adequate balance between the RCD response and its evasion to achieve maximum dissemination in the body. Pharmacological approaches to boost autophagy for therapeutic aims may be intricate by the potential role of autophagy in several cellular functions, the intricacy of autophagy pathways, and the specificity of pharmacological agents. Strategies to prevent T. gondii from inhibiting autophagic targeting may represent a novel strategy to improve the treatment for toxoplasmosis in immunocompetent individuals and reactivated toxoplasmosis in immunosuppressed individuals. The description of the molecular processes by which different strains of T. gondii manipulate autophagy and cell death pathways may also serve as an advantage to investigate new pathways in normal cells and in diseases in which autophagy and other RCD processes play a key role.A better understanding of the cellular processes by which


Streptococcus mutans, Streptococcus sanguinis, Streptococcus oralis, Streptococcus mitis, Staphylococcus aureus, Enterococcus faecalis, Lactobacillus rhamnosus and Candida albicans on the surface of hydroxyapatite – major mineral component of a tooth. Therefore, such antimicrobials as chlorhexidine, cetylpyridine chloride, polyhexanide, silver nanoparticles, sulphonated phenolics, and natural antiplaque essential oils and coconut oil were analyzed. Applied experimental settings in in vitro models were designed to reflect accurately the recommended use of the tested substances, therefore four types of eradication procedure were conducted. Sialorrhea simulation was also performed to evaluate antibiofilm potential of diluted mouth rinses. Biofilm was investigated with quantitative method where absorbance values were measured. Statistical differences were assessed using the Kruskal–Wallis test with a post-hoc Dunnett’s analysis. Results have shown that biofilms displayed a diversified sensitivity to the tested antimicrobials. The highest antibiofilm activity was detected for cetylpyridine chloride while the lowest for chlorhexidine. However the differences in E. faecalis biofilm reduction observed after the use of these two compounds were not statistically significant (p > 0.05), whereas all observed differences in S. aureus survival after exposure to the examined antimicrobial agents were statistically significant (p < 0.5). The PHMB, both in standard and in sialorrhea simulated conditions had the highest potential against streptococci. The coconut oil reduced C. albicans fungus biofilm by 65.48% but low eradication level was observed in case of bacterial biofilms. The dehydrating mechanism of action of sulfonated phenolics turned out to be ineffective against streptococcal biofilm which in turn was effectively eradicated by silver nanoparticles. The implementation of Antibiofilm Dressing's Activity Measurement method allowed to observe strain-related differences in terms of antimicrobial sensitivity. The obtained results may be introduced in everyday out-patient dental plaque prophylaxis as well as clinical environment.Oral health is a window to a patient’s general well-being. Balance in oral microbiome functions is crucial for health maintenance. A state of oral dysbiosis may lead to a variety of local and systemic pathological conditions. The presence of dental plaque is related to the majority of oral infections. Proper oral hygiene is crucial and the most economic practice contributing to oral health prophylaxis. Aside from prophylactic treatments provided by dental practitioners, mouth rinses, containing antimicrobial agents, are one of the possible tools used for oral care. Our study was to determine whether available mouth rinses and selected products dedicated for professional use are efficient to eradicate biofilm formed by reference and clinical strains of The online version contains supplementary material available at 10.1186/s12903-022-02532-4. The pivotal role of oral microbiome in human health and disease is widely recognized. The disruption of the oral microbiome leads to dysbiosis resulting in the development of numerous local pathological conditions including dental caries, periodontal diseases or oral cancers , 2. AccoIt is well-established that the presence of dental plaque  correlates with incidences of oral diseases .The oral health prophylaxis struggles with biofilm localized on teeth surface. The dental practitioners provide professional prophylactic treatments to combat dental plaque. Unfortunately, these procedures are not available for the majority of patients due to the high economic cost –12. TherStreptococcus mutans, Streptococcus sanguinis, Streptococcus oralis, Streptococcus mitis, Staphylococcus aureus, Enterococcus faecalis, Lactobacillus rhamnosus and Candida albicans using a spectrum of adequate techniques for biofilm measurement . O. OC. albS. aureus species [C. albicans biofilm (65.48%)  as lauric acid and caprylic acid are responsible for the antimicrobial activity to the greatest extent. The killing effect may be caused by putative mechanisms, including disruption of glycolysis on the Embden-Meyerhof-Parnas pathway and direct impairment of cell wall as demonstrated on  species . In coco biofilm 5.48% , the average level of biofilm eradication, being result of CPC and PHMB application, was comparable  discs. Figure 3. Procedure diagram of the A.D.A.M. method A-cutting of agar tunnels, B - placing HA disks in agar tunnels, C – flooding HA discs with artificial saliva, D -covering tunnels with biocellulose saturated with tested solutions. Figure 4. Biofilm stained withTTC. A - S. mitis; B - E.faecalis.Figure 5.Two-species biofilm formed by L.rhamnosus and S.mutans (magnification 9 999x).