Endogenous bacteria inhabiting the Ophiocordyceps highlandensis during fruiting body development

Background The genus Ophiocordyceps, which includes Ophiocordyceps sinensis, has been demonstrated to be one of the most valuable medicinal taxa. The low rate of larval infection and slow development that characterize the cultivation of this genus should be urgently addressed. To identify potential bioinoculants that stimulate the growth of Ophiocordyceps, O. highlandensis was selected as a model system, and a total of 72 samples were collected to systematically compare the microbial communities present during fruiting body development. By applying high-throughput 16S and ITS2 amplicon sequencing technology, the bacterial and fungal communities were identified in O. highlandensis and its surrounding soil, and the functional dynamics of the bacteria were explored. Results The results indicate that the most abundant bacteria across all the samples from O. highlandensis were Proteobacteria, Firmicutes and Bacteroidetes, while members of Ascomycota were detected among the fungi. The pathways enriched in the developmental stages were associated with carbohydrate degradation, nucleotides and pyridoxal biosynthesis, and the TCA cycle. Compared with that in the fungal community, an unexpectedly high taxonomic and functional fluctuation was discovered in the bacterial community during the maturation of O. highlandensis. Furthermore, bipartite network analysis identified four potential supercore OTUs associated with O. highlandensis growth. Conclusions All the findings of this study suggest unexpectedly high taxonomic and functional fluctuations in the bacterial community of O. highlandensis during its maturation. O. highlandensis may recruit different endogenous bacteria across its life cycle to enhance growth and support rapid infection. These results may facilitate Ophiocordyceps cultivation and improve the development of strategies for the identification of potential bioinoculant resources.

Evidence for Vertical Transmission of Babesia odocoilei (Piroplasmida: Babesiidae) in Ixodes scapularis (Acari: Ixodidae)

Limited evidence suggests that the cervid parasite, Babesia odocoilei, is transovarially transmitted from adult female Ixodes scapularis Say to offspring. The prevalence of B. odocoilei in unfed larval I. scapularis and whether vertical transmission is crucial to pathogen maintenance are currently unknown. To investigate these questions, 275 unfed larvae from two Wisconsin counties were tested for B. odocoilei genetic material. Sixteen of 29 pools were positive for the parasite. The maximum likelihood estimation for overall larval infection prevalence was 7.8% (95% confidence interval: 4.7–12). This vertically acquired infection appears to be sustained transstadially in nymphal ticks the following year; however, our relatively small sample and replicate size warrants additional evaluation. Our study revealed further evidence of vertical transmission, a low and consistent infection prevalence in larvae, and the potential importance of vertical transmission in B. odocoilei maintenance.

Galleria mellonella Larvae as an Infection Model to Investigate sRNA-Mediated Pathogenesis in Staphylococcus aureus

Small regulatory RNAs (sRNAs) are key players in bacterial regulatory networks. Monitoring their expression inside living colonized or infected organisms is essential for identifying sRNA functions, but few studies have looked at sRNA expression during host infection with bacterial pathogens. Insufficient in vivo studies monitoring sRNA expression attest to the difficulties in collecting such data, we therefore developed a non-mammalian infection model using larval Galleria mellonella to analyze the roles of Staphylococcus aureus sRNAs during larval infection and to quickly determine possible sRNA involvement in staphylococcal virulence before proceeding to more complicated animal testing. We began by using the model to test infected larvae for immunohistochemical evidence of infection as well as host inflammatory responses over time. To monitor sRNA expression during infection, total RNAs were extracted from the larvae and invading bacteria at different time points. The expression profiles of the tested sRNAs were distinct and they fluctuated over time, with expression of both sprD and sprC increased during infection and associated with mortality, while rnaIII expression remained barely detectable over time. A strong correlation was observed between sprD expression and the mortality. To confirm these results, we used sRNA-knockout mutants to investigate sRNA involvement in Staphylococcus aureus pathogenesis, finding that the decrease in death rates is delayed when either sprD or sprC was lacking. These results demonstrate the relevance of this G. mellonella model for investigating the role of sRNAs as transcriptional regulators involved in staphylococcal virulence. This insect model provides a fast and easy method for monitoring sRNA (and mRNA) participation in S. aureus pathogenesis, and can also be used for other human bacterial pathogens.

Immunohistopathological response against anisakid nematode larvae and a coccidian in Micromesistius poutassou from NE Atlantic waters

A survey on Anisakis simplex (sensu stricto (s.s.)) from blue whiting, Micromesistius poutassou, in the north-eastern Atlantic Ocean revealed the occurrence of high infection levels of third larval stages in visceral organs and flesh. Larvae were genetically identified with a multilocus approach as A. simplex (s.s.). Histochemical, immunohistochemical and ultrastructural observations were conducted on 30 M. poutassou specimens. Gonads, pyloric caeca and flesh harboured encapsulated larvae of A. simplex (s.s.) but no intense host reaction was encountered around the parasite in the above organs. In the liver, the most infected organ, the larvae co-occurred with the coccidian Goussia sp. Within the granuloma around the A. simplex (s.s.) larvae, two concentric layers were recognized, an inner mostly comprising electron-dense epithelioid cells and an outer layer made of less electron-dense epithelioid cells. Macrophages and macrophage aggregates (MAs) were abundant out of the granulomas, scattered in parenchyma, and inside the MAs, the presence of engulfed Goussia sp. was frequent. In liver tissue co-infected with Goussia sp. and A. simplex (s.s.), hepatocytes showed cytoplasmic rarefaction and acute cell swelling. Results suggest that the host-induced encapsulation of A. simplex (s.s.) larvae is a strategic compromise to minimize collateral tissue damage around the larval infection sites, to facilitate the survival of both parasite and host.

In vivo synergism of ampicillin, gentamicin, ceftaroline and ceftriaxone against Enterococcus faecalis assessed in the Galleria mellonella infection model

Background The unfavourable safety profile of aminoglycosides and the synergistic effects observed in vitro have prompted the development of novel dual β-lactam therapies, e.g. ampicillin/ceftriaxone or ampicillin/ceftaroline, for the treatment of Enterococcus faecalis endocarditis. Objectives For comparison with in vitro chequerboard assay results, a partial chequerboard setup of ampicillin/gentamicin, ampicillin/ceftriaxone and ampicillin/ceftaroline against E. faecalis was established in the Galleria mellonella larval infection model. Methods Discrimination of synergistic and additive interactions was based on the evaluation of larval survival, bacterial quantity in the haemolymph and a pathology score index (internal to the workgroup). Single and multiple dosing schemes based on the half-life of ampicillin were applied. Pharmacokinetic data of the antibiotics in the larvae were determined via agar plate diffusion assays. Results Ampicillin and ceftriaxone exhibited strain-specific synergistic interactions in the larvae under both dosing regimens, while the other two combinations showed additive effects. Ampicillin/ceftaroline was inferior to ampicillin/ ceftriaxone. Not all synergistic effects observed in vitro could be replicated in the larvae. Conclusions Our results suggest superior efficacy of ampicillin/ceftriaxone for the treatment of high-inoculum enterococcal infections, for at least some strains, but question the benefit of the current standard of adding the nephrotoxic gentamicin compared with the safer ceftriaxone. This is the first study to develop a scheme for differentiation between additive and synergistic effects in larvae and apply a multiple-antibiotic dosing scheme based on the pharmacokinetics of ampicillin. The model allows the analysis of synergistic effects of antimicrobials in an in vivo setting, but the clinical correlation warrants further study.

In Vitro Effects of Pesticides on European Foulbrood in Honeybee Larvae

Neonicotinoid and fungicide exposure has been linked to immunosuppression and increased susceptibility to disease in honeybees (Apis mellifera). European foulbrood, caused by the bacterium Melissococcus plutonius, is a disease of honeybee larvae which causes economic hardship for commercial beekeepers, in particular those whose colonies pollinate blueberries. We report for the first time in Canada, an atypical variant of M. plutonius isolated from a blueberry-pollinating colony. With this isolate, we used an in vitro larval infection system to study the effects of pesticide exposure on the development of European foulbrood disease. Pesticide doses tested were excessive (thiamethoxam and pyrimethanil) or maximal field-relevant (propiconazole and boscalid). We found that chronic exposure to the combination of thiamethoxam and propiconazole significantly decreased the survival of larvae infected with M. plutonius, while larvae chronically exposed to thiamethoxam and/or boscalid or pyrimethanil did not experience significant increases in mortality from M. plutonius infection in vitro. Based on these results, individual, calculated field-realistic residues of thiamethoxam and/or boscalid or pyrimethanil are unlikely to increase mortality from European foulbrood disease in honeybee worker brood, while the effects of field-relevant exposure to thiamethoxam and propiconazole on larval mortality from European foulbrood warrant further study.