Introduction. The fungal pathogen Aspergillus fumigatus can induce prolonged colonization of the lungs of susceptible patients, resulting in conditions such as allergic bronchopulmonary aspergillosis and chronic pulmonary aspergillosis.Hypothesis. Analysis of the A. fumigatus secretome released during sub-lethal infection of G. mellonella larvae may give an insight into products released during prolonged human colonisation.Methodology.Galleria mellonella larvae were infected with A. fumigatus, and the metabolism of host carbohydrate and proteins and production of fungal virulence factors were analysed. Label-free qualitative proteomic analysis was performed to identify fungal proteins in larvae at 96 hours post-infection and also to identify changes in the Galleria proteome as a result of infection.Results. Infected larvae demonstrated increasing concentrations of gliotoxin and siderophore and displayed reduced amounts of haemolymph carbohydrate and protein. Fungal proteins (399) were detected by qualitative proteomic analysis in cell-free haemolymph at 96 hours and could be categorized into seven groups, including virulence (n = 25), stress response (n = 34), DNA repair and replication (n = 39), translation (n = 22), metabolism (n = 42), released intracellular (n = 28) and cellular development and cell cycle (n = 53). Analysis of the Gallerial proteome at 96 hours post-infection revealed changes in the abundance of proteins associated with immune function, metabolism, cellular structure, insect development, transcription/translation and detoxification.Conclusion. Characterizing the impact of the fungal secretome on the host may provide an insight into how A. fumigatus damages tissue and suppresses the immune response during long-term pulmonary colonization.
- MeSH
- Aspergillus fumigatus * metabolismus MeSH
- aspergilóza mikrobiologie metabolismus MeSH
- faktory virulence metabolismus MeSH
- fungální proteiny * metabolismus genetika MeSH
- hemolymfa mikrobiologie metabolismus MeSH
- larva * mikrobiologie MeSH
- můry * mikrobiologie MeSH
- proteom analýza MeSH
- proteomika MeSH
- sekretom metabolismus MeSH
- virulence MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Paenibacillus larvae and Melissococcus plutonius represent the most threatening bacterial diseases of honeybee (Apis mellifera)-American and European foulbrood, respectively. For efficient control of those diseases, rapid and accurate detection of the pathogens is crucial. Therefore, we developed a novel multiplex PCR method simultaneously detecting both pathogens. To design and optimize multiplex PCR reaction, four strains of P. larvae representing four ERIC genotypes I-IV (strain DSM 7030-ERIC I, DSM 25430-ERIC II, LMG 16252-ERIC III, DSM 3615-ERIC IV) were selected. Those strains were fully sequenced using long-read sequencing (Sequel I, Pacific Biosciences). For P. larvae, the multicopy insertion sequence IS256 identified in all genotypes of P. larvae was selected to provide high sensitivity. M. plutonius was detected by plasmid pMP1 sequence and the virulence verified by following detection of ETX/MTX2 toxin responsible for pore formation in the cell membrane. As an internal control, a gene encoding for major royal jelly protein 1 specific for honeybees was selected. The method was validated on 36 clinical specimens collected from the colonies suffering from American and European foulbrood in the Czech Republic. Based on the results, sensitivity of PCR was calculated to 93.75% and specificity to 100% for P. larvae diagnosed from hive debris and 100% sensitivity and specificity for honeybee workers and larval scales as well as for diseased brood infected by M. plutonius.
- MeSH
- Enterococcaceae * MeSH
- larva mikrobiologie MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- Paenibacillus larvae * genetika MeSH
- Paenibacillus * genetika MeSH
- plazmidy genetika MeSH
- transpozibilní elementy DNA MeSH
- včely genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The European tent caterpillar, Malacosoma neustria Linnaeus (Lepidoptera: Lasiocampidae), is a worldwide known pest that causes significant losses by feeding on many woody and shrub-like plants during the larval period. In this study, a Beauveria bassiana strain isolated from M. neustria larvae and characterized according to its morphological and molecular properties. Its insecticidal activity was tested on M. neustria larvae. Sequence results of partial ITS (ITS1-5.8S-ITS2) gene region identified the isolate (named as Mn1) as a B. bassiana strain. Phylogenetic analysis showed that Mn1 strain displays close similarity to B. bassiana ARSEF 1848 and ARSEF 751 isolates. According to the concentration-responce tests performed using 1 × 103 - 1 × 109 conidia per mL concentrations, LC50 value of the new strain was calculated as 1 × 105 conidia per mL within 7 days against the larvae of M. neustria in the laboratory conditions. B. bassiana Mn1 strain, characterized in this study, is recorded as the first entomopathogenic fungus isolated from M. neustria, and the results showed that new strain of B. bassiana has a serious potential to be utilized as a good biocontrol agent against M. neustria.
- MeSH
- Beauveria * genetika MeSH
- biologická kontrola škůdců metody MeSH
- fylogeneze MeSH
- larva mikrobiologie MeSH
- Lepidoptera * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Chironomids are the most abundant aquatic insects in freshwater habitats that can survive in extreme conditions. In this study, as the microbiome provides extended genotype to the host to perform various functions, we explored the microbiota of the Chironomus circumdatus larvae to find out the putative role played by the symbiotic bacteria for the host. The metabarcoding analyses of the larvae revealed that the insect harbors 1771 phylotypes. Out of the various microbial communities found, the majority corresponded to the phyla Proteobacteria (52.59%) and Actinobacteria (20.56%), respectively. The midges also harbored Klebsiella (2.57%), Enterobacter (1.32%), Bacillus (2.29%), and Acinetobacter (2.13%) genera that are involved in detoxification of xenobiotics present in the water. The presence of radiation-resistant genera like Deinococcus, including bacterial species like radiodurans, a highly radiation-resistant bacterium, indicates its potential to support the host's ability to sustain in adverse environments. The functional profiling of the bacteria showed the relative abundance of many enzyme groups, such as transferases (40.62%), oxidoreductases (23.49%), and hydrolases (3.77%). The results indicate that the larvae harbor a considerable variety of bacteria that help the host adapt and survive in the polluted waters. The present study provides thorough insights into the microbiome of the C. circumdatus larvae that can be exploited for the bioremediation of certain pollutants through biomimetic strategies. It also gives us a wake-up call to take a good look at the guts of these disease-carrying insects' inabilities to spread deadly human diseases.
- MeSH
- Bacteria genetika MeSH
- Chironomidae * mikrobiologie MeSH
- larva mikrobiologie MeSH
- lidé MeSH
- mikrobiota * MeSH
- střevní mikroflóra * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Strain CS-1T, a novel facultative anaerobic bacterium, was isolated from the larval gastrointestinal tract of the biting midge, Culicoides sonorensis, a vector of the epizootic haemorrhagic disease virus and the bluetongue virus. Cells were Gram-stain-positive, non-motile, non-spore-forming, pleomorphic rods. Optimal growth occurred at pH 7.5 and 37 °C. The G+C content of the genomic DNA was 38.3 mol%, estimated by using HPLC. The dominant cellular fatty acids were C14 : 0 (45.9 %) and C16 : 0 (26.6 %). The polar lipid profile comprised glycolipids, diphosphatidylglycerol, phospholipids and phosphoglycolipids. Respiratory quinones were not detected. Strain CS-1T had very low 16S rRNA gene similarity to members of the phylum Firmicutes: Macrococcus canis KM45013T (85 % similarity) and Turicibacter sanguinis MOL361T (88 % similarity). Phylogenetic analysis based on 16S rRNA, rpoB, gyrB genes, and conserved protein sequences of the whole genome revealed that strain CS-1T was related to members of the classes Bacilli and Erysipelotrichia within the phylum Firmicutes. Furthermore, average nucleotide identity and digital DNA-DNA hybridization analyses of the whole genome revealed very low sequence similarity to species of Bacilli and Erysipelotrichaceae (Macrococcus canis KM45013T and Turicibacter sp. H121). These results indicate that strain CS-1T belongs to the phylum Firmicutes and represents a new species of a novel genus, family, order and class. Based on the phenotypic, chemotaxonomic, phylogenetic and genomic characteristics, we propose the novel taxon Culicoidibacter larvae gen. nov., sp. nov. with the type strain CS-1T (=CCUG 71726T=DSM 106607T) within the hereby new proposed novel family Culicoidibacteraceae fam. nov., new order Culicoidibacaterales ord. nov. and new class Culicoidibacteria classis nov. in the phylum Firmicutes.
- MeSH
- Ceratopogonidae mikrobiologie MeSH
- DNA bakterií genetika MeSH
- druhová specificita MeSH
- Firmicutes klasifikace genetika MeSH
- fylogeneze * MeSH
- gastrointestinální trakt mikrobiologie MeSH
- larva mikrobiologie MeSH
- mastné kyseliny chemie MeSH
- RNA ribozomální 16S genetika MeSH
- sekvenční analýza DNA MeSH
- techniky typizace bakterií MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Chronic undernutrition leads to growth hormone resistance and poor growth in children, which has been shown to be modulated by microbiota. We studied whether Lactobacillus fermentum CECT5716 (Lf CECT5716), isolated from mother's breast milk, could promote juvenile growth through the modulation of lipid absorption in a model of starvation. METHODS: Germ-free (GF) Drosophila melanogaster larvae were inoculated with Lf CECT5716 in conditions of undernutrition with and without infant formula. The impact of Lf CECT5716 on larval growth was assessed 7 days after egg laying (AED) by measuring the larval size and on maturation by measuring the emergence of pupae during 21 days AED. For lipid absorption test, Caco2/TC7 intestinal cells were incubated with Lf CECT5716 and challenged with mixed lipid micelles. RESULTS: The mono-associated larvae with Lf CECT5716 were significantly longer than GF larvae (3.7 vs 2.5 mm; p < 0.0001). The effect was maintained when Lf CECT5716 was added to the infant formula. The maturation time of larvae was accelerated by Lf CECT5716 (12 vs 13.2 days; p = 0.01). Lf CECT5716 did not have significant impact on lipid absorption in Caco2/TC7 cells. CONCLUSIONS: Lf CECT5716 is a growth-promoting strain upon undernutrition in Drosophila, with a maintained effect when added to an infant formula but without effect on lipid absorption in vitro.
- MeSH
- Caco-2 buňky MeSH
- časové faktory MeSH
- chronická nemoc MeSH
- Drosophila melanogaster MeSH
- enterocyty cytologie MeSH
- kokultivační techniky MeSH
- Lactobacillus plantarum * MeSH
- larva mikrobiologie MeSH
- lidé MeSH
- Limosilactobacillus fermentum * MeSH
- lipidy chemie MeSH
- mateřské mléko mikrobiologie MeSH
- micely MeSH
- mikrobiota MeSH
- modely u zvířat MeSH
- náhražky mateřského mléka MeSH
- novorozenec MeSH
- podvýživa dietoterapie patofyziologie MeSH
- probiotika * MeSH
- techniky in vitro MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- novorozenec MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Insects rely on the innate immune system for defense against pathogens, some aspects of which are under hormonal control. Here we provide direct experimental evidence showing that the juvenile hormone-binding protein (mJHBP) of Aedes aegypti is required for the regulation of innate immune responses and the development of mosquito blood cells (hemocytes). Using an mJHBP-deficient mosquito line generated by means of CRISPR-Cas9 gene editing technology we uncovered a mutant phenotype characterized by immunosuppression at the humoral and cellular levels, which profoundly affected susceptibility to bacterial infection. Bacteria-challenged mosquitoes exhibited significantly higher levels of septicemia and mortality relative to the wild type (WT) strain, delayed expression of antimicrobial peptides (AMPs), severe developmental dysregulation of embryonic and larval hemocytes (reduction in the total number of hemocytes) and increased differentiation of the granulocyte lineage. Interestingly, injection of recombinant wild type mJHBP protein into adult females three-days before infection was sufficient to restore normal immune function. Similarly, injection of mJHBP into fourth-instar larvae fully restored normal larval/pupal hemocyte populations in emerging adults. More importantly, the recovery of normal immuno-activation and hemocyte development requires the capability of mJHBP to bind JH III. These results strongly suggest that JH III functions in mosquito immunity and hemocyte development in a manner that is perhaps independent of canonical JH signaling, given the lack of developmental and reproductive abnormalities. Because of the prominent role of hemocytes as regulators of mosquito immunity, this novel discovery may have broader implications for the understanding of vector endocrinology, hemocyte development, vector competence and disease transmission.
- MeSH
- Aedes genetika růst a vývoj imunologie mikrobiologie MeSH
- hemocyty imunologie mikrobiologie MeSH
- hmyzí proteiny genetika imunologie MeSH
- juvenilní hormony imunologie MeSH
- larva genetika růst a vývoj imunologie mikrobiologie MeSH
- přirozená imunita MeSH
- Serratia marcescens fyziologie MeSH
- transportní proteiny genetika imunologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, N.I.H., Intramural MeSH
Candidatus Neoehrlichia mikurensis, the causative agent of tick-borne "neoehrlichiosis" has recently been reported in humans, mammals and ticks in Europe. The aim of this study was to map the distribution of this bacterium in questing ticks in the Czech Republic. A total of 13,325 Ixodes ricinus including 445 larvae, 5270 nymphs and 7610 adults were collected from vegetation by flagging in 140 Czech towns and villages from every region of the Czech Republic. The ticks were pooled into 2665 groups of 5 individuals respecting life stage or sex and tested for the presence of Ca. Neoehrlichia mikurensis by conventional PCR targeting of the groEL gene. The bacterium was detected in 533/2665 pools and 125/140 areas screened, showing an overall estimated prevalence of 4.4 % in ticks of all life stages. Phylogenetic analysis revealed only small genetic diversity among the strains found. Two pools of questing larvae tested positive, suggesting transovarial transmission. According to this study, Ca. Neoehrlichia mikurensis is another tick-borne pathogen widespread in I. ricinus ticks in the Czech Republic.
- MeSH
- Anaplasmataceae genetika izolace a purifikace MeSH
- genetická variace MeSH
- klíště růst a vývoj mikrobiologie MeSH
- larva růst a vývoj mikrobiologie MeSH
- nymfa růst a vývoj mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
The aim was to evaluate in vitro possible interactions, gene expression, and biofilm formation in species of Candida albicans, Streptococcus mitis, and Streptococcus sanguinis and their in vivo pathogenicity. The in vitro analysis evaluated the effects of S. mitis and S. sanguinis on C. albicans's biofilm formation by CFU count, filamentation capacity, and adhesion (ALS1, ALS3, HWP1) and transcriptional regulatory gene (BCR1, CPH1, EFG1) expression. In vivo studies evaluated the pathogenicity of the interaction of the microorganisms on Galleria mellonella, with analyses of the CFU per milliliter count and filamentation. In vitro results indicated that there was an observed decrease in CFU (79.4-71.5%) in multi-species biofilms. The interaction with S. mitis inhibited filamentation, which seems to increase its virulence factor with over-expression of genes ALS1, ALS3, and HWP1 as well the interaction with S. sanguinis as ALS3 and HWP1. S. mitis upregulated BRC1, CPH1, and EFG1. The histological images of in vivo study indicate an increase in the filamentation of C. albicans when in interaction with the other species. It was concluded that S. mitis interaction suggests increased virulence factors of C. albicans, with periods of lower virulence and proto-cooperation in the interaction with S. sanguinis.
- MeSH
- biofilmy růst a vývoj MeSH
- Candida albicans genetika růst a vývoj patogenita MeSH
- fungální proteiny genetika MeSH
- hyfy růst a vývoj MeSH
- kokultivační techniky MeSH
- larva mikrobiologie MeSH
- mikrobiální interakce fyziologie MeSH
- modely nemocí na zvířatech MeSH
- můry mikrobiologie MeSH
- počet mikrobiálních kolonií MeSH
- regulace genové exprese u hub MeSH
- Streptococcus mitis fyziologie MeSH
- Streptococcus fyziologie MeSH
- virulence genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Knowledge of microbiota composition, persistence, and transmission as well as the overall function of the bacterial community is important and may be linked to honey bee health. This study aimed to investigate the inter-individual variation in the gut microbiota in honey bee larvae and pupae. RESULTS: Individual larvae differed in the composition of major bacterial groups. In the majority of 5th instar bees, Firmicutes showed predominance (70%); however, after larval defecation and during pupation, the abundance decreased to 40%, in favour of Gammaproteobacteria. The 5th instar larvae hosted significantly more (P < 0.001) Firmicutes than black pupae. Power calculations revealed that 11 and 18 replicate-individuals, respectively, were required for the detection of significant differences (P < 0.05) in the Bacteroidetes and Firmicutes abundance between stages, while higher numbers of replicates were required for Actinobacteria (478 replicates) and Gammaproteobacteria (111 replicates). CONCLUSIONS: Although sample processing and extraction protocols may have had a significant influence, sampling is very important for studying the bee microbiome, and the importance of the number of individuals pooled in samples used for microbiome studies should not be underestimated.
- MeSH
- Bacteria klasifikace genetika izolace a purifikace MeSH
- časové faktory MeSH
- fylogeneze MeSH
- kladení vajíček * MeSH
- kukla anatomie a histologie mikrobiologie MeSH
- larva anatomie a histologie mikrobiologie MeSH
- mikrobiota MeSH
- RNA ribozomální 16S genetika MeSH
- sekvenční analýza DNA metody MeSH
- střevní mikroflóra MeSH
- včely anatomie a histologie mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
