Editor’s Choice Articles

Lactococcus garvieae is the etiological agent of lactococcosis, a clinically and economically significant infectious disease affecting farmed rainbow trout. L. garvieae had been considered the only cause of lactococcosis for a long time; however, L. petauri, another species of the genus Lactococcus, has lately been linked to the same disease. The genomes and biochemical profiles of L. petauri and L. garvieae have a high degree of similarity. Traditional diagnostic tests currently available cannot distinguish between these two species. The aim of this study was to use the transcribed spacer (ITS) region between 16S rRNA and 23S rRNA as a potential useful molecular target to differentiate L. garvieae from L. petauri, saving time and money compared to genomics methods currently used as diagnostic tools for accurate discrimination between these two species. The ITS region of 82 strains was amplified and sequenced. The amplified fragments varied in size from 500 to 550 bp. Based on the sequence, seven SNPs were identified that separate L. garvieae from L. petauri. The 16S-23S rRNA ITS region has enough resolution to distinguish between closely related L. garvieae and L. petauri and it can be used as a diagnostic marker to quickly identify the pathogens in a lactococcosis outbreak. Full article

In aquatic environments, the consensus of viral impact on bacterial carbon metabolism with the nutrient environment as an important axis is limited. Henceforth, we explored the viral regulation of carbon-based bacterial growth efficiency (BGE) in a set of freshwater systems from French Massif Central, which were broadly classified based on two trophic statuses: eutrophic and non-eutrophic lakes. Comparative analysis showed that microbial abundances (viruses and bacteria) were 3-fold higher in eutrophic compared with non-eutrophic lakes, and so were bacterial production and viral lytic infection. The observed variability in BGE (10–60%) was explained by the uncoupling between bacterial respiration and production. Viruses through selective lysis of susceptible host communities had an antagonistic impact on BGE in the eutrophic lakes, whereas the release of substrates via viral shunt exerted a synergistic influence on the carbon metabolism of non-targeted host populations in non-eutrophic lakes. The decisive effect of the two individual processes (i.e., lysis and substrate release) on BGE was supported by regressions of bacterial abundance as a function of bacterial production, which is considered as a proxy of top-down processes. The role of viruses through their negative impact via mortality and positive impact via substrate supply can eventually have implications on carbon transfer through bacterioplankton in freshwaters. Full article

Brown trout (Salmo trutta) is an important aquaculture species in Germany, but its production faces challenges due to global warming and a high embryo mortality. Climate factors might influence the fish’s bacterial community (BC) and thus increase embryo mortality. Yet, knowledge of the physiological BC during ontogeny in general is scarce. In this project, the BC of brown trout has been investigated in a period from unfertilized egg to 95 days post fertilization (dpf) using 16S rRNA gene amplicon sequencing. Developmental changes differed between early and late ontogeny and major differences in BC occurred especially during early developmental stages. Thus, analysis was conducted separately for 0 to 67 dpf and from 67 to 95 dpf. All analyzed stages were sampled in toto to avoid bias due to different sampling methods in different developmental stages. The most abundant phylum in the BC of all developmental stages was Pseudomonadota, while only two families (Comamonadaceae and Moraxellaceae) occurred in all developmental stages. The early developmental stages until 67 dpf displayed greater shifts in their BC regarding bacterial richness, microbial diversity, and taxonomic composition. Thereafter, in the fry stages, the BC seemed to stabilize and changes were moderate. In future studies, a reduction in the sampling time frames during early development, an increase in sampling numbers, and an attempt for biological reproduction in order to characterize the causes of these variations is recommended. Full article

The clinical courses of COVID-19 in children are often mild and may remain undiagnosed, but prolonged intestinal virus shedding has been documented, thus potentially enabling fecal–oral transmission. However, the infectious potential of SARS-CoV-2 viruses excreted with feces has remained unclear. Here, we investigated 247 stool specimens from 213 pediatric patients to assess the prevalence of intestinal SARS-CoV-2 shedding in hospitalized children without or with COVID-19 and determined the infectious capacity of stool-borne viruses. Upon RT-qPCR screening, the infectivity of virus-positive samples was tested in cell culture using the Vero-E6 permissive cell line. SARS-CoV-2 RNA was detected by RT-qPCR in 32 (13%) stool specimens, but the analysis of virus-positive samples in cell culture revealed no cytopathic effects attributable to SARS-CoV-2-related cell damage. Our findings do not support the notion of potential fecal–oral SARS-CoV-2 spreading, thus questioning the role of hygienic measures designed to prevent this mode of viral transmission. Full article

β-Mannan is abundant in the human diet and in hemicellulose derived from softwood. Linear or galactose-substituted β-mannan-oligosaccharides (MOS/GMOSs) derived from β-mannan are considered emerging prebiotics that could stimulate health-associated gut microbiota. However, the underlying mechanisms are not yet resolved. Therefore, this study investigated the cross-feeding and metabolic interactions between Bifidobacterium adolescentis ATCC 15703, an acetate producer, and Roseburia hominis A2-183 DSMZ 16839, a butyrate producer, during utilization of MOS/GMOSs. Cocultivation studies suggest that both strains coexist due to differential MOS/GMOS utilization, along with the cross-feeding of acetate from B. adolescentis E194a to R. hominis A2-183. The data suggest that R. hominis A2-183 efficiently utilizes MOS/GMOS in mono- and cocultivation. Notably, we observed the transcriptional upregulation of certain genes within a dedicated MOS/GMOS utilization locus (RhMosUL), and an exo-oligomannosidase (RhMan113A) gene located distally in the R. hominis A2-183 genome. Significantly, biochemical analysis of β-1,4 mannan-oligosaccharide phosphorylase (RhMOP130A), α-galactosidase (RhGal36A), and exo-oligomannosidase (RhMan113A) suggested their potential synergistic role in the initial utilization of MOS/GMOSs. Thus, our results enhance the understanding of MOS/GMOS utilization by potential health-promoting human gut microbiota and highlight the role of cross-feeding and metabolic interactions between two secondary mannan degraders inhabiting the same ecological niche in the gut. Full article

Exopolysaccharide (EPS)-producing Lacticaseibacillus paracasei EPS DA-BACS was isolated from healthy human feces and its probiotic properties, as well as the structure and prebiotic activity of the EPS from this strain were examined. EPS from L. paracasei EPS DA-BACS had a ropy phenotype, which is known to have potential health benefits and is identified as loosely cell-bounded glucomannan-type EPS with a molecular size of 3.7 × 10⁶ Da. EPS promoted the acid tolerance of L. paracasei EPS DA-BACS and provided cells with tolerance to gastrointestinal stress. The purified EPS showed growth inhibitory activity against Clostridium difficile. L. paracasei EPS DA-BACS cells completely inhibited the growth of Bacillus subtilis, Pseudomonas aeruginosa, and Aspergillus brasiliensis, as well as showed high growth inhibitory activity against Staphylococcus aureus and Escherichia coli. Treatment of lipopolysaccharide-stimulated RAW 264.7 cells with heat-killed L. paracasei EPS DA-BACS cells led to a decrease in the production of nitric oxide, indicating the anti-inflammatory activity of L. paracasei EPS DA-BACS. Purified EPS promoted the growth of Lactobacillus gasseri, Bifidobacterium bifidum, B. animalis, and B. faecale which showed high prebiotic activity. L. paracasei EPS DA-BACS harbors no antibiotic resistance genes or virulence factors. Therefore, L. paracasei EPS DA-BACS exhibits anti-inflammatory and antimicrobial activities with high gut adhesion ability and gastrointestinal tolerance and can be used as a potential probiotic. Full article

We previously reported the emergence of amitraz-resistant Rhipicephalus (Boophilus) decoloratus ticks in the western region of Uganda. This study characterized the octopamine/tyramine receptor gene (OCT/Tyr) of amitraz-resistant and -susceptible R. (B.) decoloratus ticks from four regions of Uganda. The OCT/Tyr gene was amplified from genomic DNA of 17 R. (B.) decoloratus larval populations of known susceptibility to amitraz. The amplicons were purified, cloned and sequenced to determine mutations in the partial coding region of the OCT/Tyr gene. The amplified R. (B.) decoloratus OCT/Tyr gene was 91–100% identical to the R. (B.) microplus OCT/Tyr gene. Up to 24 single nucleotide polymorphisms (SNPs) were found in the OCT/Tyr gene from ticks obtained from high acaricide pressure areas, compared to 8 from the low acaricide pressure areas. A total of eight amino acid mutations were recorded in the partial OCT/Tyr gene from ticks from the western region, and four of them were associated with amitraz-resistant tick populations. The amino acid mutations M1G, L16F, D41G and V72A were associated with phenotypic resistance to amitraz with no specific pattern. Phylogenetic analysis revealed that the OCT/Tyr gene sequence from this study clustered into two distinct groups that separated the genotype from high acaricide pressure areas from the susceptible populations. In conclusion, this study is the first to characterize the R. (B.) decoloratus OCT/Tyr receptor gene and reports four novel amino acid mutations associated with phenotypic amitraz resistance in Uganda. However, lack of mutations in the ORF of the OCT/Tyr gene fragment for some of the amitraz-resistant R. (B.) decoloratus ticks could suggest that other mechanisms of resistance may be responsible for amitraz resistance, hence the need for further investigation. Full article

Astronauts remote from Earth, not least those who will inhabit the Moon or Mars, are vulnerable to disease due to their reduced immunity, isolation from clinical support, and the disconnect from any buffering capacity provided by the Earth. Here, we explore potential risks for astronaut health, focusing on key aspects of the biology of Bacillus anthracis and other anthrax-like bacilli. We examine aspects of Bacillus cereus group genetics in relation to their evolutionary biology and pathogenicity; a new clade of the Bacillus cereus group, close related to B. anthracis, has colonized the International Space Station (ISS), is still present, and could in theory at least acquire pathogenic plasmids from the other B. cereus group strains. The main finding is that the genomic sequence alignments of the B. cereus group ISS strains revealed a high sequence identity, indicating they originated from the same strain and that a close look to the genetic variations among the strains suggesting they lived, or they are living, in a vegetative form in the ISS enough time to accumulate genetic variations unique for each single strains. Full article

Aspergillus fumigatus is a disease-causing, opportunistic fungus that can establish infection due to its capacity to respond to a wide range of environmental conditions. Secreted proteins and metabolites, which play a critical role in fungal–host interactions and pathogenesis, are modulated by epigenetic players, such as bromodomain and extraterminal domain (BET) proteins. In this study, we evaluated the in vitro and in vivo capability of the BET inhibitor JQ1 to modulate the extracellular proteins and virulence of A. fumigatus. The abundance of 25 of the 76 extracellular proteins identified through LC-MS/MS proteomic analysis changed following JQ1 treatment. Among them, a ribonuclease, a chitinase, and a superoxide dismutase were dramatically downregulated. Moreover, the proteomic analysis of A. fumigatus intracellular proteins indicated that Abr2, an intracellular laccase involved in the last step of melanin synthesis, was absent in the JQ1-treated group. To investigate at which level this downregulation occurred and considering the ability of JQ1 to modulate gene expression we checked the level of ABR2, Chitinase, and Superoxide dismutase mRNA expression by qRT-PCR. Finally, the capacity of JQ1 to reduce the virulence of A. fumigatus has been proved using Galleria mellonella larvae, which are an in vivo model to evaluate fungal virulence. Overall, the promising activity exhibited by JQ1 suggests that A. fumigatus is sensitive to BET inhibition and BET proteins may be a viable target for developing antifungal agents. Full article

Mastitis due to Mycoplasma bovis is a worldwide problem, which leads to significant economic losses and affects animal welfare. However, the mechanisms by which M. bovis establishes and maintains intra-mammary infections (IMI) in dairy cows are largely unknown. To study in further detail the pathogenesis of M. bovis IMI, time- and cost-effective experimental models are needed. To this end, we established and characterized an in vitro murine mammary alveolar epithelial (EpH4) cell-based model and an in vivo murine mastitis model. Our results showed that live and UV-treated M. bovis field strain 161791 and its lipid-associated membrane proteins (LAMP) activated nuclear factor kappa B (NF-kB) in EpH4 cells in a dose-dependent manner. In the murine mastitis model, temporal and spatial dynamics of inflammation in the mammary tissues were evident. Live M. bovis elicited diffuse inflammation affecting the whole challenged gland peaking at 48 h post infection (pi) in contrast to LAMP challenge, which elicited only focal inflammation peaking at 24 h and resolving at 48 h pi. Inflammation was characterized by massive neutrophil recruitment into the milk spaces and by elevated expression of the inflammatory mediators TNF-α, KC, iNOS and NF-kB dependent genes: A20 and IkBα. Moreover, the presence of intraepithelial bacterial communities in glands challenged with live M. bovis bacteria was shown. The developed models can be used efficiently for future characterization of M. bovis virulence factors and host immune response to IMI. Full article

Tick-borne encephalitis (TBE) is the most important viral tick-borne infection in Europe and Asia. It is emerging in new areas. The mechanisms of emergence are fairly unknown or speculative. In the Ravensburg district in southern Germany, TBE emerged, mainly over the last five years. Here, we analyzed the underlying epidemiology in humans. The resulting identified natural foci of the causal TBE virus (TBEV) were genetically characterized. We sampled 13 potential infection sites at these foci and detected TBEV in ticks (Ixodes ricinus) at eight sites. Phylogenetic analysis spurred the introduction of at least four distinct TBEV lineages of the European subtype into the Ravensburg district over the last few years. In two instances, a continuous spread of these virus strains over up to 10 km was observed. Full article

Bile acids are synthesized from cholesterol and play an important role in regulating intestinal microflora. The different degrees of hydrophobicity and acidity of individual bile acids may affect their antimicrobial properties. We examined the antimicrobial effects of different bile acids on various microorganisms in vitro and confirmed whether these remain consistent in vivo. Using human bile acids, including ursodeoxycholic acid, cholic acid, chenodeoxycholic acid, deoxycholic acid, and lithocholic acid, a disc diffusion test was performed, and a rodent model was created to determine the antimicrobial effects of each bile acid. The fecal bacterial population was analyzed using a real-time polymerase chain reaction. Each bile acid showed different microbial inhibitory properties. The inhibitory activity of bile acids against microbiota which normally resides in the gastrointestinal tract and biliary system, was low; however, normal flora of other organs was significantly inhibited. Changes in microbial counts after bile acid administration in a rodent model differed in the colon and cecum. The in vivo and in vitro results show that the antimicrobial effects of bile acids against intestinal microbiota were similar. In conclusion, bile acids could be a novel treatment strategy to regulate gut microbiota. Full article

Anaerobic fungi from the herbivore digestive tract (Neocallimastigomycetes) are primary lignocellulose modifiers and hold promise for biotechnological applications. Their molecular detection is currently difficult due to the non-specificity of published primer pairs, which impairs evolutionary and ecological research with environmental samples. We developed and validated a Neocallimastigomycetes-specific PCR primer pair targeting the D2 region of the ribosomal large subunit suitable for screening, quantifying, and sequencing. We evaluated this primer pair in silico on sequences from all known genera, in vitro with pure cultures covering 16 of the 20 known genera, and on environmental samples with highly diverse microbiomes. The amplified region allowed phylogenetic differentiation of all known genera and most species. The amplicon is about 350 bp long, suitable for short-read high-throughput sequencing as well as qPCR assays. Sequencing of herbivore fecal samples verified the specificity of the primer pair and recovered highly diverse and so far unknown anaerobic gut fungal taxa. As the chosen barcoding region can be easily aligned and is taxonomically informative, the sequences can be used for classification and phylogenetic inferences. Several new Neocallimastigomycetes clades were obtained, some of which represent putative novel lineages such as a clade from feces of the rodent Dolichotis patagonum (mara). Full article

Background: Since 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic (COVID-19) has caused millions of deaths worldwide and is the second most serious pandemic after the Spanish flu. Despite SARS-CoV-2 infection having a dominant effect on morbidity and life-threatening outcomes, the role of bacterial co-infection in patients with COVID-19 is poorly understood. The present study aimed to verify the existence of bacterial co-infections and their possible role as cofactors worsening COVID-19-related clinical manifestations. Methods: All patients with suspected SARS-CoV-infection, hospitalised in COVID-19 wards at the Sant’Anna University Hospital of Ferrara, were retrospectively included in this single-centre study and their specific bacterial serologies were assessed. Univariate and logistic regression analyses were performed. Results: A total of 1204 individual records were retrieved. Among them, 959 were excluded because of a negative nasopharyngeal swab or missing data; of the eligible 245 patients, 51 were co-infected. Compared to patients with SARS-CoV-2 infection alone, those with Chlamydia pneumoniae or Mycoplasma pneumoniae co-infections had worse respiratory/radiological features and more intensive care unit admissions. However, the co-infection did not result in a higher mortality rate. Conclusions: The present study, comparing clinical, laboratory and radiological findings between patients with COVID-19 vs. those with co-infections (C. pneumoniae or M. pneumoniae) showed that, on admission, these features were worse in co-infected patients, although the mortality rate did not differ between the two groups. Full article

Hospital outbreaks with vancomycin-resistant enterococci (VRE) pose a serious health threat and a challenge to infection prevention and control (IPC). We herein report on a VRE outbreak of unprecedented extent in Southern Germany (October 2015–November 2019). We used descriptive epidemiology and whole-genome sequencing (WGS) for a detailed outbreak investigation. Of the 2905 cases, 2776 (95.3%) were colonized, whereas from 127 (3.7%), VRE could be isolated from otherwise sterile body fluids or sites unlikely for enterococci colonization. Cases had a median age of 78 years (IQR 68–84) and 1339/2905 (46%) were female. The majority of isolates sequenced belonged to the clonal lineage ST80/CT1013 (212/397, 53%). Nosocomial transmission was observed as well as the constant import of VRE into the hospital. Extensive IPC measures were implemented and terminated the outbreak in late 2019, eventually. Our study shows that the combination of epidemiological and genomic analyses is indispensable for comprehensive outbreak investigations. The adaptation of IPC measures to these findings, their timely implementation, and strict execution also allow containment of large VRE outbreaks in hospital settings. Full article

The spread of carbapenemase-producing (CP) Enterobacterales is currently a worldwide concern, especially in the elderly. Twelve CP-E. coli isolated from rectal swabs of colonized inpatients aged ≥65 years from four hospitals in two Italian cities (Milan and Rome) were analyzed by whole genome sequencing (WGS) to obtain multi-locus sequence typing (MLST), identification of carbapenemase-encoding genes, resistome, plasmid content, and virulence genes. MLST analysis showed the presence of 10 unrelated lineages: ST410 (three isolates from three different hospitals in two cities) and ST12, ST38, ST69, ST95, ST131, ST189, ST648, ST1288, and ST1598 (one isolate each). Most isolates (9/12, 75%) contained a serine-β-lactamase gene (5 bla_KPC-3, 2 bla_KPC-2, and 2 bla_OXA-181), while three isolates harbored a metallo-β-lactamase gene (two bla_NDM-5 and one bla_VIM-1). In most CP-E. coli, the presence of more than one plasmid was observed, with the predominance of IncF. Several virulence genes were detected. All isolates contained genes enhancing the bacterial fitness, such as gad and terC, and all isolates but one, fimH, encoding type 1 fimbriae. In conclusion, CP-E. coli clones colonizing elderly patients showed heterogeneous genetic backgrounds. We recommend strict surveillance to monitor and prevent the spread of successful, high-risk clones in healthcare settings. Full article

About half a billion people worldwide are infected with herpes simplex virus-2 (HSV-2). Prolonged treatment with acyclovir (ACV) and its analogs leads to the development of resistant strains. The aim of this study was to investigate the antiviral potential of epigallocatechin gallate (EGCG) from Camellia sinensis and a stable analog EGCG-stearate (EGCG-S) against HSV-2 in cultured Vero cells. Cell viability and cell proliferation assays were used to determine the non-cytotoxic concentrations on cultured Vero cells. HSV-2 with a green fluorescent protein (GFP) fusion protein of VP26 virions were treated with non-cytotoxic concentrations of EGCG and EGCG-S. The effects on infectivity and mechanisms were determined by plaque assay, attachment and penetration assays, confocal microscopy, qPCR, and in silico modeling analysis. Our results demonstrate that treatment of HSV-2 virions with EGCG and EGCG-S at a concentration of 75 µM showed greater than 99.9% inhibition by inhibiting the attachment of HSV-2 virions to host cells. The bioinformatic analysis indicated high binding affinity of EGCG-S for glycoprotein D; thus EGCG-S may block fusion of HSV-2 and the cell membrane, preventing entry of HSV-2 into the cell. Full article

The emergence of drug resistant Mycobacterium tuberculosis (MTB) strains has become a global public health problem, while, at the same time, there has been development of new antimicrobial agents. The main goals of this study were to determine new variants associated with drug resistance in MTB and to observe which polymorphisms emerge in MTB genomes after anti-tuberculosis treatment. We performed whole-genome sequencing of 152 MTB isolates including 70 isolates as 32 series of pre- and post-treatment MTB. Based on genotypes and phenotypic drug susceptibility, we conducted phylogenetic convergence-based genome-wide association study (GWAS) with streptomycin-, isoniazid-, rifampicin-, ethambutol-, fluoroquinolones-, and aminoglycosides-resistant MTB against susceptible ones. GWAS revealed statistically significant associations of SNPs within Rv2820c, cyp123 and indels in Rv1269c, Rv1907c, Rv1883c, Rv2407, Rv3785 genes with resistant MTB phenotypes. Comparisons of serial isolates showed that treatment induced different patterns of intra-host evolution. We found indels within Rv1435c and ppsA that were not lineage-specific. In addition, Beijing-specific polymorphisms within Rv0036c, Rv0678, Rv3433c, and dop genes were detected in post-treatment isolates. The appearance of Rv3785 frameshift insertion in 2 post-treatment strains compared to pre-treatment was also observed. We propose that the insertion within Rv3785, which was a GWAS hit, might affect cell wall biosynthesis and probably mediates a compensatory mechanism in response to treatment. These results may shed light on the mechanisms of MTB adaptation to chemotherapy and drug resistance formation. Full article

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel virus that belongs to the Coronoviridae family, emerged in December 2019, causing the COVID-19 pandemic in March 2020. Unlike previous SARS and Middle East respiratory syndrome (MERS) outbreaks, this virus has a higher transmissibility rate, albeit a lower case fatality rate, which results in accumulation of a significant number of mutations and a faster evolution rate. Genomic studies on the mutation rate of the virus, as well as the identification of mutations that prevail and their impact on disease severity, are of great importance for pandemic surveillance and vaccine and drug development. Here, we aim to identify mutations on the SARS-CoV-2 viral genome and their effect on the proteins they are located in, in Greek patients infected in the first wave of the pandemic. To this end, we perform SARS-CoV-2 amplicon-based NGS sequencing on nasopharyngeal swab samples from Greek patients and bioinformatic analysis of the results. Although SARS-CoV-2 is considered genetically stable, we discover a variety of mutations on the viral genome. In detail, 18 mutations are detected in total on 10 SARS-CoV-2 isolates. The mutations are located on ORF1ab, S protein, M protein, ORF3a and ORF7a. Sixteen are also detected in patients from other regions around the world, and two are identified for the first time in the present study. Most of them result in amino acid substitutions. These substitutions are analyzed using computational tools, and the results indicate minor or major impact on the proteins’ structural stability, which could probably affect viral transmissibility and pathogenesis. The correlation of these variations with the viral load levels is examined, and their implication for disease severity and the biology of the virus are discussed. Full article

Extreme habitats often harbor specific communities that differ substantially from non-extreme habitats. In many cases, these communities are characterized by archaea, bacteria and protists, whereas the number of species of metazoa and higher plants is relatively low. In extremely acidic habitats, mostly prokaryotes and protists thrive, and only very few metazoa thrive, for example, rotifers. Since many studies have investigated the physiology and ecology of individual species, there is still a gap in research on direct, trophic interactions among extremophiles. To fill this gap, we experimentally studied the trophic interactions between a predatory protist (Actinophrys sol, Heliozoa) and its prey, the rotifers Elosa woralli and Cephalodella sp., the ciliate Urosomoida sp. and the mixotrophic protist Chlamydomonas acidophila (a green phytoflagellate, Chlorophyta). We found substantial predation pressure on all animal prey. High densities of Chlamydomonas acidophila reduced the predation impact on the rotifers by interfering with the feeding behaviour of A. sol. These trophic relations represent a natural case of intraguild predation, with Chlamydomonas acidophila being the common prey and the rotifers/ciliate and A. sol being the intraguild prey and predator, respectively. We further studied this intraguild predation along a resource gradient using Cephalodella sp. as the intraguild prey. The interactions among the three species led to an increase in relative rotifer abundance with increasing resource (Chlamydomonas) densities. By applying a series of laboratory experiments, we revealed the complexity of trophic interactions within a natural extremophilic community. Full article

The type III secretion system (T3SS) effector EseN is encoded on the Edwardsiella ictaluri chromosome and is homologous to a family of T3SS effector proteins with phosphothreonine lyase activity. Previously we demonstrated that E. ictaluri invasion activates extracellular signal-regulated kinases 1 and 2 (ERK1/2) early in the infection, which are subsequently inactivated by EseN. Comparative transcriptomic analysis showed a total of 753 significant differentially expressed genes in head-kidney-derived macrophages (HKDM) infected with an EseN mutant (∆EseN) compared to HKDM infected with wild-type (WT) strains. This data strongly indicates classical activation of macrophages (the M1 phenotype) in response to E. ictaluri infection and a significant role for EseN in the manipulation of this process. Our data also indicates that E. ictaluri EseN is involved in the modulation of pathways involved in the immune response to infection and expression of several transcription factors, including NF-κβ (c-rel and relB), creb3L4, socs6 and foxo3a. Regulation of transcription factors leads to regulation of proinflammatory interleukins (IL-8, IL-12a, IL-15, IL-6) and cyclooxygenase-2 (COX-2) expression. Inhibition of COX-2 mRNA by WT E. ictaluri leads to decreased production of prostaglandin E2 (PGE2), which is the product of COX-2 activity. Collectively, our results indicate that E. ictaluri EseN is an important player in the modulation of host immune responses to E.ictaluri infection. Full article

For five decades it has been known that the pentamer of B subunits (choleragenoid) of the cholera toxin (CT) of Vibrio cholerae binds with high preference to the ganglioside GM1 (II³Neu5Ac-Gg4Cer). However, the exact structures of CT-binding GM1 lipoforms of primary human colon epithelial cells (pHCoEpiCs) have not yet been described in detail. The same holds true for generating further GM1 receptor molecules from higher sialylated gangliosides with a GM1 core through the neuraminidase of V. cholerae. To avoid the artificial incorporation of exogenous gangliosides from animal serum harboring GM1 and higher sialylated ganglio-series gangliosides, pHCoEpiCs were cultured in serum-free medium. Thin-layer chromatography overlay binding assays using a choleragenoid combined with electrospray ionization mass spectrometry revealed GM1 lipoforms with sphingosine (d18:1) as the sole sphingoid base linked to C14:0, C16:0, C18:0 or C20:0 fatty acyl chains forming the ceramide (Cer) moieties of the main choleragenoid-binding GM1 species. Desialylation of GD1a (IV³Neu5Ac,II³Neu5Ac-Gg4Cer) and GT1b (IV³Neu5Ac,II³(Neu5Ac)₂-Gg4Cer) of pHCoEpiCs by V. cholerae neuraminidase was observed. GD1a-derived GM1 species with stable sphingosine (d18:1) and saturated fatty acyl chains varying in chain length from C16:0 up to C22:0 could be identified, indicating the ingenious interplay between CT and the neuraminidase of V. cholerae recruiting additional GM1 receptors of pHCoEpiCs. Full article

Contrary to the majority of other Trematoda, Schistosoma species are gonochoric. Consequently, in endemic areas where several schistosome species overlap and can co-infect the same definitive host, there may be frequent opportunities for interspecific pairing. Our experimental study provides novel insight on the pairing behavior between Schistosoma bovis and S. mansoni in mixed infections in mice. We used six mate choice experiments to assess mating interactions between the two schistosome species. We show that mating between the two Schistosoma species is not random and that S. mansoni exhibits greater mate recognition compared to S. bovis. We also performed reciprocal crosses (male S. mansoni × female S. bovis) and (female S. mansoni × male S. bovis) that produce active swimming miracidia. These miracidia were genotyped by ITS2 sequencing and proposed for mollusc infection. Molecular analyses show that all the miracidia are parthenogenetically produced (i.e., their harbor the mother ITS2 genotype) and as a consequence can only infect the mollusc of the maternal species. Offspring produced by male S. mansoni × female S. bovis pairing can only infect Bulinus truncatus whereas offspring produced by female S. mansoni × male S. bovis can only infect Biomphalaria glabrata snails. Evolutionary and epidemiological consequences are discussed. Full article

The inoculation of arbuscular mycorrhizal (AM) fungi and rhizobia in legumes has been proven to increase plant growth and yield. To date, studies of the effects of these interactions on phytophagous insects have shown them to be context-dependent depending on the inoculant strain, the plant, and the insect species. Here, we document how a symbiosis involving an AM fungus, Rhizophagus irregularis; a rhizobium, Bradyrhizobium japonicum; and soybean, Glycine max, influences the soybean aphid, Aphis glycines. Soybean co-inoculated with the AM fungus–rhizobium pair increased the plant’s biomass, nodulation, mycorrhizal colonization, nitrogen, and carbon concentrations, but decreased phosphorus concentration. Similar effects were observed with rhizobium alone, with the exception that root biomass was unaffected. With AM fungus alone, we only observed an increase in mycorrhizal colonization and phosphorus concentration. The aphids experienced an increased reproductive rate with the double inoculation, followed by rhizobium alone, whereas no effect was observed with the AM fungus. The size of individual aphids was not affected. Furthermore, we found positive correlation between nitrogen concentration and aphid population density. Our results confirm that co-inoculation of two symbionts can enhance both plant and phytophagous insect performance beyond what either symbiont can contribute alone. Full article

Microorganisms capable of decomposing hydrophobic substrates in cold climates are of considerable interest both in terms of studying adaptive reactions to low temperatures and in terms of their application in biotechnologies for cleaning up oil spills in a crude-oil polluted soil. The aim of this work was to investigate the genome of Rhodococcus opacus S8 and explore behavior traits of this strain grown in the presence of hexadecane. The genome size of strain S8 is 8.78 Mb, of which the chromosome size is 7.75 Mb. The S8 strain contains 2 circular plasmids of 135 kb and 105 kb and a linear plasmid with a size of 788 kb. The analysis of the genome revealed the presence of genes responsible for the degradation of alkanes and synthesis of biosurfactants. The peculiarities of morphology of microbial cells when interacting with a hydrophobic substrate were revealed. An adaptive mechanism responsible in the absence of oxygen for maintaining the process of degradation of hexadecane is discussed. The data obtained show that the strain S8 has great potential to be used in biotechnologies. Full article

Infancy is a critical life stage for the establishment of the gut microbiome. Human milk contains a unique microbial ecosystem that serves as a continuous source of commensal bacteria for the infant. However, the origin of the human milk microbiota, how it is influenced by breastfeeding exclusivity, and its role in infant gut microbiota assembly are not clear. To interrogate these questions, we examined the relationships among fecal, oral, breast skin, and human milk microbiota of 33 exclusively breastfeeding (EBF) and mixed-feeding (MF; human milk + infant formula) mother–infant pairs at 6 weeks postpartum. Here, we show that MF infants have a significantly more diverse oral microbiome comprised of lower relative abundances of Streptococcus and Gemella and higher abundances of Veillonella. Using both SourceTracker2 and FEAST, we demonstrate breast skin and infant saliva as the principal contributing sources to the human milk microbiota. Of the sampled sites, human milk and maternal stool were predicted to contribute the largest fraction to the infant fecal microbiome, but the majority of the community was estimated to arise from unknown sources. Lastly, we identified twenty-one significant co-occurrence relationships between bacteria in human milk and on other maternal and infant body sites. These results demonstrate several unique microbial interrelationships between breastfeeding dyads, providing insight into potential mechanisms of microbial assembly in early life. Full article

The gastrointestinal microbiota of cattle is important for feedstuff degradation and feed efficiency determination. This study evaluated the fecal microbiome of Angus steers with distinct feed efficiencies during the feedlot-finishing phase. Angus steers (n = 65), fed a feedlot-finishing diet for 82 days, had growth performance metrics evaluated. Steers were ranked based upon residual feed intake (RFI), and the 5 lowest RFI (most efficient) and 5 highest RFI (least efficient) steers were selected for evaluation. Fecal samples were collected on 0-d and 82-d of the finishing period and microbial DNA was extracted and evaluated by 16S rRNA gene sequencing. During the feedlot trial, inefficient steers had decreased (p = 0.02) Ruminococcaceae populations and increased (p = 0.01) Clostridiaceae populations. Conversely, efficient steers had increased Peptostreptococcaceae (p = 0.03) and Turicibacteraceae (p = 0.01), and a trend for decreased Proteobacteria abundance (p = 0.096). Efficient steers had increased microbial richness and diversity during the feedlot period, which likely resulted in increased fiber-degrading enzymes in their hindgut, allowing them to extract more energy from the feed. Results suggest that cattle with better feed efficiency have greater diversity of hindgut microorganisms, resulting in more enzymes available for digestion, and improving energy harvest in the gut of efficient cattle. Full article

The intestinal virus community contributes to health and disease. Runting and stunting syndrome (RSS) is associated with enteric viruses and leads to economic losses in the poultry industry. However, many viruses that potentially cause this syndrome have also been identified in healthy animals. To determine the difference in the virome of healthy and diseased broilers, samples from 11 healthy and 17 affected broiler flocks were collected at two time points and analyzed by Next-Generation Sequencing. Virus genomes of Parvoviridae, Astroviridae, Picornaviridae, Caliciviridae, Reoviridae, Adenoviridae, Coronaviridae, and Smacoviridae were identified at various days of poultry production. De novo sequence analysis revealed 288 full or partial avian virus genomes, of which 97 belonged to the novel genus Chaphamaparvovirus. This study expands the knowledge of the diversity of enteric viruses in healthy and RSS-affected broiler flocks and questions the association of some viruses with the diseases. Full article

In urban wastewater treatment plants, bacteria lead the biological component of the depuration process, but the microbial community is also rich in fungi (mainly molds, yeasts and pseudo-yeasts), whose taxonomical diversity and relative frequency depend on several factors, e.g., quality of wastewater input, climate, seasonality, and depuration stage. By joining morphological and molecular identification, we investigated the fungal diversity in two different plants for the urban wastewater treatment in the suburbs of the two major cities in Lombardia, the core of industrial and commercial activities in Italy. This study presents a comparison of the fungal diversity across the depuration stages by applying the concepts of α-, β- and ζ-diversity. Eurotiales (mainly with Aspergillus and Penicillium), Trichosporonales (Trichosporon sensu lato), Saccharomycetales (mainly with Geotrichum) and Hypocreales (mainly with Fusarium and Trichoderma) are the most represented fungal orders and genera in all the stages and both the plants. The two plants show different trends in α-, β- and ζ-diversity, despite the fact that they all share a crash during the secondary sedimentation and turnover across the depuration stages. This study provides an insight on which taxa potentially contribute to each depuration stage and/or keep viable propagules in sludges after the collection from the external environment. Full article

Dengue fever, caused by any of four dengue viruses (DENV1-4), is a major global burden. Currently, there is no effective vaccine that prevents infection in dengue naïve populations. We tested the ability of two novel adjuvants (Advax-PEI and Advax-2), using aluminum hydroxide (alum) as control, to enhance the immunogenicity of formalin- or psoralen-inactivated (PIV or PsIV) DENV2 vaccines in mice. Mice were vaccinated on days 0 and 30, and serum samples were collected on days 30, 60, 90, and 101. Neutralizing antibodies were determined by microneutralization (MN) assays, and the geometric mean 50% MN (MN₅₀) titers were calculated. For the PIV groups, after one dose MN₅₀ titers were higher in the novel adjuvant groups compared to the alum control, while MN₅₀ titers were comparable between the adjuvant groups after the second dose. For the PsIV groups, both novel adjuvants induced higher MN₅₀ titers than the alum control after the second dose. Spleen cells were collected on days 45 and 101 for enzyme-linked immunospot (ELISPOT) for IFNγ and IL4. Both PIV and PsIV groups elicited different degrees of IFNγ and IL4 responses. Overall, Advax-2 gave the best responses just ahead of Advax-PEI. Given Advax-2’s extensive human experience in other vaccine applications, it will be pursued for further development. Full article

The contamination of coastal marine sediments with heavy metals (HMs) is a widespread phenomenon that requires effective remediation actions. Bioremediation based on the use of bacteria is an economically and environmentally sustainable effective strategy for reducing HM contamination and/or toxicity in marine sediments. However, information on the efficiency of marine-derived fungi for HM decontamination of marine sediments is still largely lacking, despite evidence of the performance of terrestrial fungal strains on other contaminated matrixes (e.g., soils, freshwater sediments, industrial wastes). Here, we carried out for the first time an array of parallel laboratory experiments by using different combinations of chemical and microbial amendments (including acidophilic autotrophic and heterotrophic bacteria, as well as filamentous marine fungi) for the bioremediation of highly HM-contaminated sediments of the Portman Bay (NW Mediterranean Sea), an area largely affected by long-term historical discharges of mine tailings. Our results indicate that the bioleaching performance of metals from the sediment is based on the addition of fungi (Aspergillus niger and Trichoderma sp.), either alone or in combination with autotrophic bacteria, was higher when compared to other treatments. In particular, fungal addition allowed obtaining bioleaching yields for As eight times higher than those by chemical treatments and double compared with the addition of bacteria alone. Moreover, in our study, the fungal addition was the only treatment allowing effective bioleaching of otherwise not mobile fractions of Zn and Cd, thus overtaking bacterial treatments. We found that the lower the sediment pH reached by the experimental conditions, as in the case of fungal addition, the higher the solubilization yield of metals, suggesting that the specific metabolic features of A. niger and Trichoderma sp. enable lowering sediment pH and enhance HM bioleaching. Overall, our findings indicate that fungi can be more effective than acidophilic autotrophic and heterotrophic bacteria in HM bioleaching, and as such, their use can represent a promising and efficient strategy for the bioremediation of marine sediments highly contaminated with heavy metals. Full article

BackgroundSince the appearance of COVID-19 in January 2020, invasive bacterial infections have decreased significantly worldwide. However, alterations in age and sex distributions, clinical forms, phenotypes, and genotypes of isolates have not been analyzed. Our goal is to present and discuss these data considering the current COVID-19 pandemic situation. Methods: The data of the national reference center for meningococci and Haemophilus influenzae in France were mined to examine the above aspects of invasive bacterial infection before (2018–2019) and after (2020–2021) the COVID-19 pandemic. Detailed epidemiological, clinical, and microbiological data were collected, and whole genome sequencing was carried out on meningococcal isolates (n = 1466). Results: In addition to the overall decline in the number of cases, various changes in age, sex, and phenotypes of isolates were also noted. As for N. meningitidis, more cases were observed in adults, as well as more invasive pneumopathies. Furthermore, fewer hyperinvasive meningococcal genotypes have circulated since COVID-19 emerged. The situation has been different for H. influenzae, as the number of invasive cases among adults decreased due to a reduction in non-typeable isolates. In contrast, cases due to serotypeable isolates, particularly serotypes a and b, increased in children <5 years-old. Conclusions: It is possible that measures implemented to stop COVID-19 may have reduced the circulation of N. meningitidis and H. influenzae isolates, but to a variable extent. This may be due to differences in circulation between these two species according to age groups. Vaccination schedules against these two species may have also influenced the evolution of these invasive bacterial infections since the emergence of the COVID-19 pandemic. Full article

Shiga toxin-producing Escherichia coli (STEC) O145:H28 can cause severe disease in humans and is a predominant serotype in STEC O145 environmental isolates. Here, comparative genomics was applied to a set of clinical and environmental strains to systematically evaluate the pathogenicity potential in environmental strains. While the core genes-based tree separated all O145:H28 strains from the non O145:H28 reference strains, it failed to segregate environmental strains from the clinical. In contrast, the accessory genes-based tree placed all clinical strains in the same clade regardless of their genotypes or serotypes, apart from the environmental strains. Loss-of-function mutations were common in the virulence genes examined, with a high frequency in genes related to adherence, autotransporters, and the type three secretion system. Distinct differences in pathogenicity islands LEE, OI-122, and OI-57, the acid fitness island, and the tellurite resistance island were detected between the O145:H28 and reference strains. A great amount of genetic variation was detected in O145:H28, which was mainly attributed to deletions, insertions, and gene acquisition at several chromosomal “hot spots”. Our study demonstrated a distinct virulence gene repertoire among the STEC O145:H28 strains originating from the same geographical region and revealed unforeseen contributions of loss-of-function mutations to virulence evolution and genetic diversification in STEC. Full article

Freshwater salinization is an ongoing concern for north temperate lakes; however, little is known about its impacts on microbial communities, particularly for bacteria. We tested the hypotheses that road de-icing salt induces changes in the microbial community structure of lake plankton, and that changes due to chloride would differ from those due to urban snowmelt because of additional chemicals in the snowmelt. In a laboratory incubator experiment, an overwintering plankton community in lake water was exposed for two weeks to either NaCl or municipal road snow with the same level of chloride. Microbial community structure as determined by 16S (prokaryotes) and 18S (eukaryotes) rRNA transcript analysis showed changes in response to the chloride-only enrichment, with some rare taxa becoming more prominent. Consistent with our hypothesis, the salt and the snow treatments induced different community changes. These results indicate that ecotoxicology assays based on a single salt addition may not reflect the in situ effects of salt-contaminated urban snow, and that the combined chemical effects of urban snowmelt require direct testing. Full article

Since March 2020, the COVID-19 pandemic forced hospitals worldwide to intensify their infection control measures to prevent health care-associated transmission of SARS-CoV-2. The correct use of personal protective equipment, especially the application of masks, was quickly identified as priority to reduce transmission with this pathogen. Here, we report a nosocomial cluster of methicillin-resistant Staphylococcus aureus (MRSA) that occurred during the COVID-19 pandemic in a gynecology/obstetrics department, despite these intensified contact precautions. Five MRSA originating from clinical samples after surgical intervention led to an outbreak investigation. Firstly, this included environmental sampling of the operation theatre (OT) and, secondly, a point prevalence screening of patients and health care workers (HCW). All detected MRSA were subjected to whole genome sequencing (WGS) and isolate relatedness was determined using core genome multilocus sequence typing (cgMLST). WGS revealed one MRSA cluster with genetically closely related five patient and two HCW isolates differing in a single cgMLST allele at maximum. The outbreak was terminated after implementation of infection control bundle strategies. Although contact precaution measures, which are also part of MRSA prevention bundle strategies, were intensified during the COVID-19 pandemic, this MRSA outbreak could take place. This illustrates the importance of adherence to classical infection prevention strategies. Full article

There are uncertainties with respect to the transmission of methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MSSA and MRSA) and Staphylococcus pseudintermedius between dogs and humans. In this study, we investigated concomitant nasal colonization of dogs and humans in three cohorts. Cohort I, households owning dogs: In 42 of 84 households, 66 humans (36.9%) and 10 dogs (8.9%) carried S. aureus. MRSA, attributed to sequence type (ST) 22 and ST130, were detected in two (1.1%) of the humans but in none of the dogs. Typing by means of spa-typing and whole-genome sequencing (WGS) indicated eight transmissions of S. aureus between humans and dogs in 8 of 42 (19.0%) households with human S. aureus carriers, whereas in 11 of 38 (29.0%) households with ≥two persons and S. aureus colonization of humans, 15 human-to-human transmissions were observed (p = 0.43). S. pseudintermedius was isolated from 42 dogs (37.5%), but from only one human (0.6%). In this case, WGS-based typing indicated strong relatedness of this isolate with a canine isolate from the same household. Cohort II, dogs and their owners visiting a veterinary practice: Among 17 humans and 17 dogs attending a veterinary practice, MSSA was detected in three humans and two dogs, and S. pseudintermedius in only six dogs. Cohort III, dogs used for animal-assisted interventions in human healthcare facilities and their owners: MSSA was obtained in 1 of 59 dogs (1.7%) and in 17 of 60 (28.3%) of the dog owners, while S. pseudintermedius was isolated from seven (12%) dogs and one (1.7%) human owner. We conclude that the risk of exchanging S. aureus/MRSA between humans and dogs is higher than that for S. pseudintermedius. Full article

Gut microbiota are involved in both host health and disease and can be stratified based on bacteriological composition. However, gut microbiota clustering data are limited for Asians. In this study, fecal microbiota of 1803 Japanese subjects, including 283 healthy individuals, were analyzed by 16S rRNA sequencing and clustered using two models. The association of various diseases with each community type was also assessed. Five and fifteen communities were identified using partitioning around medoids (PAM) and the Dirichlet multinominal mixtures model, respectively. Bacteria exhibiting characteristically high abundance among the PAM-identified types were of the family Ruminococcaceae (Type A) and genera Bacteroides, Blautia, and Faecalibacterium (Type B); Bacteroides, Fusobacterium, and Proteus (Type C); and Bifidobacterium (Type D), and Prevotella (Type E). The most noteworthy community found in the Japanese subjects was the Bifidobacterium-rich community. The odds ratio based on type E, which had the largest population of healthy subjects, revealed that other types (especially types A, C, and D) were highly associated with various diseases, including inflammatory bowel disease, functional gastrointestinal disorder, and lifestyle-related diseases. Gut microbiota community typing reproducibly identified organisms that may represent enterotypes peculiar to Japanese individuals and that are partly different from those of indivuals from Western countries. Full article

Several species of fungi from the orders Chaetothyriales and Pleosporales have been reported to produce swainsonine and be associated as symbionts with plants of the Convolvulaceae and Fabaceae, respectively. An endosymbiont belonging to the Chaetothyriales produces swainsonine and grows as an epibiont on the adaxial leaf surfaces of Ipomoea carnea, but how the symbiont passes through plant growth and development is unknown. Herein, different types of microscopy were used to localize the symbiont in seeds and in cross sections of plant parts. The symbiont was found in several tissues including the hilum, the sclereids, and the hypocotyl of seeds. In five-day old seedlings and mature plants, the symbiont was found in the shoot apical meristem (SAM) and the adaxial surface of immature folded leaves. The mycelia generally formed a close association with peltate glandular trichomes. This report provides further data explaining the relationship between the seed transmitted Chaetothyriales symbiont and Ipomoea carnea. These results provide a possible explanation for how this symbiont, and others like Periglandula may persist and are transmitted over time. Full article

Hematopoietic stem/progenitor cells (HSPC) are responsible for the generation of most immune cells throughout the lifespan of the organism. Inflammation can activate bone marrow HSPCs, leading to enhanced myelopoiesis to replace cells, such as neutrophils, which are attracted to inflamed tissues. We have previously shown that HSPC activation promotes parasite persistence and expansion in experimental visceral leishmaniasis through the increased production of permissive monocytes. However, it is not clear if the presence of the parasite in the bone marrow was required for infection-adapted myelopoiesis. We therefore hypothesized that persistent forms of Leishmania major (cutaneous leishmaniasis) could also activate HSPCs and myeloid precursors in the C57Bl/6 mouse model of intradermal infection in the ear. The accrued influx of myeloid cells to the lesion site corresponded to an increase in myeloid-biased HSPCs in the bone marrow and spleen in mice infected with a persistent strain of L. major, together with an increase in monocytes and monocyte-derived myeloid cells in the spleen. Analysis of the bone marrow cytokine and chemokine environment revealed an attenuated type I and type II interferon response in the mice infected with the persistent strain compared to the self-healing strain, while both strains induced a rapid upregulation of myelopoietic cytokines, such as IL-1β and GM-CSF. These results demonstrate that an active infection in the bone marrow is not necessary for the induction of infection-adapted myelopoiesis, and underline the importance of considering alterations to the bone marrow output when analyzing in vivo host-pathogen interactions. Full article

With the current advancements in DNA sequencing technology, the limiting factor in long-read metagenomic assemblies is now the quantity and quality of input DNA. Although these requirements can be met through the use of axenic bacterial cultures or large amounts of biological material, insect systems that contain unculturable bacteria or that contain a low amount of available DNA cannot fully utilize the benefits of third-generation sequencing. The citrus greening disease insect vector Diaphorina citri is an example that exhibits both of these limitations. Although endosymbiont genomes have mostly been closed after the short-read sequencing of amplified template DNA, creating de novo long-read genomes from the unamplified DNA of an insect population may benefit communities using bioinformatics to study insect pathosystems. Here all four genomes of the infected D. citri microbiome were sequenced to closure using unamplified template DNA and two long-read sequencing technologies. Avoiding amplification bias and using long reads to assemble the bacterial genomes allowed for the circularization of the Wolbachia endosymbiont of Diaphorina citri for the first time and paralleled the annotation context of all four reference genomes without utilizing a traditional hybrid assembly. The strategies detailed here are suitable for the sequencing of other insect systems for which the input DNA, time, and cost are an issue. Full article

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), which is involved in the calcium signaling pathway, is an important regulator of cancer cell proliferation, motility, growth, and metastasis. The effects of CaMKII on hepatitis B virus (HBV) replication have never been evaluated. Here, we found that phosphorylated, active CaMKII is reduced during HBV replication. Similar to other members of the AMPK/AKT/mTOR signaling pathway associated with HBV replication, CaMKII, which is associated with this pathway, was found to be a novel regulator of HBV replication. Overexpression of CaMKII reduced the expression of covalently closed circular DNA (cccDNA), HBV RNAs, and replicative intermediate (RI) DNAs while activating AMPK and inhibiting the AKT/mTOR signaling pathway. Findings in HBx-deficient mutant-transfected HepG2 cells showed that the CaMKII-mediated AMPK/AKT/mTOR signaling pathway was independent of HBx. Moreover, AMPK overexpression reduced HBV cccDNA, RNAs, and RI DNAs through CaMKII activation. Although AMPK acts downstream of CaMKII, AMPK overexpression altered CaMKII phosphorylation, suggesting that CaMKII and AMPK form a positive feedback loop. These results demonstrate that HBV replication suppresses CaMKII activity, and that CaMKII upregulation suppresses HBV replication from cccDNA via AMPK and the AKT/mTOR signaling pathway. Thus, activation or overexpression of CaMKII may be a new therapeutic target against HBV infection. Full article

The Indian Ocean Lineage (IOL) of the chikungunya virus (CHIKV) East/Central/South African (ECSA) genotype, which originated in Kenya, spread to the Indian ocean and the Indian subcontinent, and then expanded through Southeast Asia in the previous decade. It carried an adaptive mutation E1-A226V, which enhances CHIKV replication in Aedes albopictus. However, the IOL CHIKV of the most recent outbreaks during 2016–2020 in India, Pakistan, Bangladesh, the Maldives, Myanmar, Thailand, and Kenya lacked E1-A226V but carried E1-K211E and E2-V264A. Recent CHIKV genome sequences of the Maldives and Thailand were determined, and their phylogenetic relationships were further investigated together with IOL sequences reported in 2004–2020 in the database. The results showed that the ancestral IOLs diverged to a sub-lineage E1-K211E/E2-V264A, probably in India around 2008, and caused sporadic outbreaks in India during 2010–2015 and in Kenya in 2016. The massive expansion of this new sub-lineage occurred after the acquisition of E1-I317V in other neighboring and remote regions in 2014–2020. Additionally, the phylogenetic tree indicated that independent clades formed according to the geographical regions and introduction timing. The present results using all available partial or full sequences of the recent CHIKVs emphasized the dynamics of the IOL sub-lineages in the Indian subcontinent, Southeast Asia, and Eastern Africa. Full article

The pandemic caused by SARS-CoV-2 is not over yet, despite all the efforts from the scientific community. Vaccination is a crucial weapon to fight this virus; however, we still urge the development of antivirals to reduce the severity and progression of the COVID-19 disease. For that, a deep understanding of the mechanisms involved in viral replication is necessary. nsp15 is an endoribonuclease critical for the degradation of viral polyuridine sequences that activate host immune sensors. This enzyme is known as one of the major interferon antagonists from SARS-CoV-2. In this work, a biochemical characterization of SARS-CoV-2 nsp15 was performed. We saw that nsp15 is active as a hexamer, and zinc can block its activity. The role of conserved residues from SARS-CoV-2 nsp15 was investigated, and N164 was found to be important for protein hexamerization and to contribute to the specificity to degrade uridines. Several chemical groups that impact the activity of this ribonuclease were also identified. Additionally, FDA-approved drugs with the capacity to inhibit the in vitro activity of nsp15 are reported in this work. This study is of utmost importance by adding highly valuable information that can be used for the development and rational design of therapeutic strategies. Full article

The anthropogenic release of oil hydrocarbons into the cold marine environment is an increasing concern due to the elevated usage of sea routes and the exploration of new oil drilling sites in Arctic areas. The aim of this study was to evaluate prokaryotic community structures and the genetic potential of hydrocarbon degradation in the metagenomes of seawater, sea ice, and crude oil encapsulating the sea ice of the Norwegian fjord, Ofotfjorden. Although the results indicated substantial differences between the structure of prokaryotic communities in seawater and sea ice, the crude oil encapsulating sea ice (SIO) showed increased abundances of many genera-containing hydrocarbon-degrading organisms, including Bermanella, Colwellia, and Glaciecola. Although the metagenome of seawater was rich in a variety of hydrocarbon degradation-related functional genes (HDGs) associated with the metabolism of n-alkanes, and mono- and polyaromatic hydrocarbons, most of the normalized gene counts were highest in the clean sea ice metagenome, whereas in SIO, these counts were the lowest. The long-chain alkane degradation gene almA was detected from all the studied metagenomes and its counts exceeded ladA and alkB counts in both sea ice metagenomes. In addition, almA was related to the most diverse group of prokaryotic genera. Almost all 18 good- and high-quality metagenome-assembled genomes (MAGs) had diverse HDGs profiles. The MAGs recovered from the SIO metagenome belonged to the abundant taxa, such as Glaciecola, Bermanella, and Rhodobacteracea, in this environment. The genera associated with HDGs were often previously known as hydrocarbon-degrading genera. However, a substantial number of new associations, either between already known hydrocarbon-degrading genera and new HDGs or between genera not known to contain hydrocarbon degraders and multiple HDGs, were found. The superimposition of the results of comparing HDG associations with taxonomy, the HDG profiles of MAGs, and the full genomes of organisms in the KEGG database suggest that the found relationships need further investigation and verification. Full article

Metagenome profiling research using next-generation sequencing (NGS), a technique widely used to analyze the diversity and composition of microorganisms living in the human body, especially the gastrointestinal tract, has been actively conducted, and there is a growing interest in the quantitative and diagnostic technology for specific microorganisms. According to recent trends, quantitative real-time PCR (qRT-PCR) is still a considerable technique in detecting and quantifying bacteria associated with the human oral and nasal cavities, due to the analytical cost and time burden of NGS technology. Here, based on NGS metagenome profiling data produced by utilizing 100 gut microbiota samples, we conducted a comparative analysis for the identification and quantification of five bacterial genera (Akkermansia, Bacteroides, Bifidobacterium, Phascolarctobacterium, and Roseburia) within same metagenomic DNA samples through qRT-PCR assay in parallel. Genus-specific primers, targeting the particular gene of each genus for qRT-PCR assay, allowed a statistically consistent quantification pattern with the metagenome profiling data. Furthermore, results of bacterial identification through Sanger validation demonstrated the high genus-specificity of each primer set. Therefore, our study suggests that an approach to quantifying specific microorganisms by applying the qRT-PCR method can compensate for the concerns (potential issues) of NGS while also providing efficient benefits to various microbial industries. Full article

Pneumococcal infections have increasingly high mortality rates despite the availability of vaccines and antibiotics. Therefore, the identification of new virulence determinants and the understanding of the molecular mechanisms behind pathogenesis have become of paramount importance in the search of new targets for drug development. The exoribonuclease RNase R has been involved in virulence in a growing number of pathogens. In this work, we used Galleria mellonella as an infection model to demonstrate that the presence of RNase R increases the pneumococcus virulence. Larvae infected with the RNase R mutant show an increased expression level of antimicrobial peptides. Furthermore, they have a lower bacterial load in the hemolymph in the later stages of infection, leading to a higher survival rate of the larvae. Interestingly, pneumococci expressing RNase R show a sudden drop in bacterial numbers immediately after infection, resembling the eclipse phase observed after intravenous inoculation in mice. Concomitantly, we observed a lower number of mutant bacteria inside larval hemocytes and a higher susceptibility to oxidative stress when compared to the wild type. Together, our results indicate that RNase R is involved in the ability of pneumococci to evade the host immune response, probably by interfering with internalization and/or replication inside the larval hemocytes. Full article

Toxocara canis and Toxocara cati are globally occurring zoonotic roundworms of dogs and cats. Migration and persistence of Toxocara larvae in the central nervous system of paratenic hosts including humans may cause clinical signs of neurotoxocarosis (NT). As pathomechanisms of NT and host responses against Toxocara larvae are mostly unknown, whole-genome microarray transcription analysis was performed in cerebra and cerebella of experimentally infected C57Bl/6J mice as paratenic host model at days 14, 28, 70, 98, and 120 post-infection. Neuroinvasion of T. cati evoked 220 cerebral and 215 cerebellar differentially transcribed genes (DTGs), but no particular PANTHER (Protein ANalysis THrough Evolutionary Relationships) pathway was affected. In T. canis-infected mice, 1039 cerebral and 2073 cerebellar DTGs were identified. Statistically significant dysregulations occurred in various pathways, including cholesterol biosynthesis, apoptosis signaling, and the Slit/Robo mediated axon guidance as well as different pathways associated with the immune and defense response. Observed dysregulations of the cholesterol biosynthesis, as well as the Alzheimer disease-amyloid secretase pathway in conjunction with previous histopathological neurodegenerative findings, may promote the discussion of T. canis as a causative agent for dementia and/or Alzheimer’s disease. Furthermore, results contribute to a deeper understanding of the largely unknown pathogenesis and host-parasite interactions during NT, and may provide the basis for prospective investigations evaluating pathogenic mechanisms or designing novel diagnostic and therapeutic approaches. Full article

Background: Toxoplasma gondii (T. gondii) is a parasite that infects more than 40 million Americans and causes toxoplasmosis. Most cases of toxoplasmosis are asymptomatic; however, T. gondii is capable of invading organs like the kidney, causing chronic infections and cell destruction. Methods: This study focused on evaluating the association between T. gondii exposure and chronic kidney disease (CKD) using data from the 2009–2010 National Health and Nutrition Examination Survey (NHANES). T. gondii exposure was assessed using Toxoplasma gondii IgG antibody status, and the status of CKD was assessed using the CKD biomarkers. The evaluation of risk rate and population prevalence was performed. In addition, multivariable regression models were used to further investigate this association after adjusting for sociodemographic, anthropometric, behavioral, and clinical covariates commonly associated with kidney dysfunction. Results: The positive T. gondii IgG antibody participants had significantly higher levels of CKD biomarkers, including second albumin-to-creatinine ratio (p = 0.0376), second albuminuria (p = 0.0005), and persistent albuminuria (p < 0.0001) compared to the negative participants. Furthermore, there were statistical associations between T. gondii exposure and the status of CKD (negative vs. positive) (p = 0.0001), and between T. gondii exposure and the CKD stage (negative, stage 1, …, stage 5) (p = 0.0004). Without adjusting for age, the positive T. gondii participants had a significantly higher risk (27% higher) of having CKD than the negative participants (RRcrude = 1.27, 95% CI: 1.09–1.49). The age-adjusted prevalence of CKD was higher among Toxoplasma-positive participants compared to the Toxoplasma-negative participants (10.45 vs. 8.99). T. gondii infection was significantly associated with CKD (OR = 1.40, 95% CI = 1.06–1.84, p = 0.00447) after adjusting for age, gender, race/ethnicity, and BMI. Age was positively associated with CKD (OR = 8.89, 95% CI = 6.31–12.51, p < 0.0001) with the participants 45+ years old being 8.89 times more likely to have CKD than those who are <45 years old, after adjusting for T. gondii infection, gender, race/ethnicity, and BMI. Moreover, positive T. gondii increased the odds of CKD progression (OR = 1.41, 95% CI = 1.07–1.86, p = 0.0424). Conclusions: Positive T. gondii IgG antibody is associated with CKD and the progression of CKD stages. This association is more apparent among older people. Further investigations are needed to examine these findings in different geographical locations and among differentially exposed populations. Full article

Trees are able to colonize, establish and survive in a wide range of soils through associations with ectomycorrhizal (EcM) fungi. Proper functioning of EcM fungi implies the differentiation of structures within the fungal colony. A symbiotic structure is dedicated to nutrient exchange and the extramatricular mycelium explores soil for nutrients. Eventually, basidiocarps develop to assure last stages of sexual reproduction. The aim of this study is to understand how an EcM fungus uses its gene set to support functional differentiation and development of specialized morphological structures. We examined the transcriptomes of Laccaria bicolor under a series of experimental setups, including the growth with Populus tremula x alba at different developmental stages, basidiocarps and free-living mycelium, under various conditions of N, P and C supply. In particular, N supply induced global transcriptional changes, whereas responses to P supply seemed to be independent from it. Symbiosis development with poplar is characterized by transcriptional waves. Basidiocarp development shares transcriptional signatures with other basidiomycetes. Overlaps in transcriptional responses of L. bicolor hyphae to a host plant and N/C supply next to co-regulation of genes in basidiocarps and mature mycorrhiza were detected. Few genes are induced in a single condition only, but functional and morphological differentiation rather involves fine tuning of larger gene sets. Overall, this transcriptomic atlas builds a reference to study the function and stability of EcM symbiosis in distinct conditions using L. bicolor as a model and indicates both similarities and differences with other ectomycorrhizal fungi, allowing researchers to distinguish conserved processes such as basidiocarp development from nutrient homeostasis. Full article

Antimicrobial resistance is an emerging condition that increases the risk of spreading and prolonging infectious diseases globally. Therefore, a new alternative strategy for antibiotics is required urgently to control pathogens spreading. Probiotics are considered as an alternative for antibiotics that inhibit pathogens. In the present study, potent lactic acid bacteria (LAB) were isolated and screened for their probiotic characteristics and antagonistic activity against intestinal pathogens by agar well diffusion, Time and Dose-dependent killing assay, minimum inhibitor, and minimum bactericidal concentration (MIC/MBC), and co-culture methods. The Lactococcus lactis RWP-3 and RWP-7 fermented the different carbohydrate substrates and produced different extracellular enzymes. Both isolates showed significant tolerant capability in the gastric, duodenal, and intestinal juices. In addition, RWP-3 and RWP-7 had hydrophobicity and aggregation properties in a time-dependent manner. Furthermore, the cell-free secondary metabolites (CFS) of RWP-3 and RWP-7 showed strong antibacterial activity against Escherichia coli,Staphylococcus aureus, Pseudomonas aeruginosa and Enterococcus faecalis. A co-culture study revealed that the RWP-3 and RWP-7 strongly compete with pathogen growths. RWP-3 and RWP-7 showed strong antagonistic activities against tested pathogens with significant probiotic characteristics, suggesting that these strains obtained could be used as an alternative strategy for the antibiotic to control infectious pathogens. Full article