Infection of fish with the facultative intracellular bacterium Francisella noatunensis remains an unresolved problem for aquaculture industry worldwide as it is difficult to vaccinate against without using live attenuated vaccines. Outer membrane vesicles (OMVs) are biological structures shed by Gramnegative bacteria in response to various environmental stimuli. OMVs have successfully been used to vaccinate against both intracellular and extracellular pathogens, due to an ability to stimulate innate, cell-mediated and humoral immune responses. We show by using atomic force and electron microscopy that the fish pathogenic bacterium E noatunensis subspecies noatunensis (F.n.n.) shed OMVs both in vitro into culture medium and in vivo in a zebrafish infection model. The main protein constituents of the OMV are IgIC, PdpD and PdpA, all known Francisella virulence factors, in addition to the outer membrane protein FopA and the chaperonin GroEL, as analyzed by mass spectrometry. The vesicles, when used as a vaccine, reduced proliferation of the bacterium and protected zebrafish when subsequently challenged with a high dose of F.n.n. without causing adverse effects for the host. Also granulomatous responses were reduced in F.n.n.-challenged zebrafish after OMV vaccination. Taken together, the data support the possible use of OMVs as vaccines against francisellosis in fish. (C) 2014 The Authors. Published by Elsevier Ltd.

Antibiotics seize an effect on bacterial composition and diversity and have been demonstrated to induce disruptions on gut microbiomes. This may have implications for human health and wellbeing, and an increasing number of studies suggest a link between the gut microbiome and several diseases. Hence, reducing antibiotic treatments may be beneficial for human health status. Further, antimicrobial resistance (AMR) is an increasing global problem that can be counteracted by limiting the usage of antibiotics. Longer antibiotic treatments have been demonstrated to increase the development of AMR. Therefore, shortening of antibiotic treatment durations, provided it is safe for patients, may be one measure to reduce AMR. In this study, the objective was to investigate effects of standard and reduced antibiotic treatment lengths on gut microbiomes using a murine model. Changes in the murine gut microbiome was assessed after using three different treatment durations of amoxicillin (3, 7 or 14 days) as well as a control group not receiving amoxicillin. Fecal samples were collected before and during the whole experiment, until three weeks past end of treatment. These were further subject for 16S rRNA Illumina MiSeq sequencing. Our results demonstrated significant changes in bacterial diversity, richness and evenness during amoxicillin treatment, followed by a reversion in terms of alpha-diversity and abundance of major phyla, after end of treatment. However, a longer restitution time was indicated for mice receiving amoxicillin for 14 days, and phylum Patescibacteria did not fully recover. In addition, an effect on the composition of Firmicutes was indicated to last for at least three weeks in mice treated with amoxicillin for 14 days. Despite an apparently reversion to a close to original state in overall bacterial diversity and richness, the results suggested more durable changes in lower taxonomical levels. We detected several families, genera and ASVs with significantly altered abundance three weeks after exposure to amoxicillin, as well as bacterial taxa that appeared significantly affected by amoxicillin treatment length. This may strengthen the argument for shorter antibiotic treatment regimens to both limit the emergence of antibiotic resistance and risk of gut microbiome disturbance.

Vaccine development against extracellular bacteria has been important for the sustainability of the aquaculture industry. In contrast, infections with intracellular pathogens remain largely an unresolved problem. Francisella noatunensis subsp. orientalis is a Gram-negative, facultative intracellular bacterium that causes the disease francisellosis in fish. Francisellosis is commonly characterized as a chronic granulomatous disease with high morbidity and can result in high mortality depending on the host. In this study, we explored the potential of bacterial membrane vesicles (MVs) as a vaccine agent against F. noatunensis subsp. orientalis . Bacterial MVs are spherical structures naturally released from the membrane of bacteria and are often enriched with selected bacterial components such as toxins and signaling molecules. MVs were isolated from broth-cultured F. noatunensis subsp. orientalis in the present work, and proteomic analysis by mass spectrometry revealed that MVs contained a variety of immunogenic factors, including the intracellular growth proteins IglC and IglB, known to be part of a Francisella pathogenicity island (FPI), as well as outer membrane protein OmpA, chaperonin GroEL, and chaperone ClpB. By using flow cytometry and electron microscopy, we observed that F. noatunensis subsp. orientalis mainly infects myelomonocytic cells, both in vivo and in vitro . Immunization with MVs isolated from F. noatunensis subsp. orientalis protects zebrafish from subsequent challenge with a lethal dose of F. noatunensis subsp. orientalis . To determine if MVs induce a typical acute inflammatory response, mRNA expression levels were assessed by quantitative real-time PCR. Expression of tnfa , il1b , and ifng , as well as mhcii , mpeg1.1 , and ighm , was upregulated, thus confirming the immunogenic properties of F. noatunensis subsp. orientalis -derived MVs.

The mycobiome, representing the fungal component of microbial communities, is increasingly acknowledged as an integral part of the gut microbiome. However, research in this area remains relatively limited. The characterization of mycobiome taxa from metagenomic data is heavily reliant on the quality of the software and databases. In this study, we evaluated the feasibility of mycobiome profiling using existing bioinformatics tools on simulated fungal metagenomic data. We identified seven tools claiming to perform taxonomic assignment of fungal shotgun metagenomic sequences. One of these was outdated and required substantial modifications of the code to be functional and was thus excluded. To evaluate the accuracy of identification and relative abundance of the remaining tools (Kraken2, MetaPhlAn4, EukDetect, FunOMIC, MiCoP, and HumanMycobiomeScan), we constructed 18 mock communities of varying species richness and abundance levels. The mock communities comprised up to 165 fungal species belonging to the phyla Ascomycota and Basidiomycota, commonly found in gut microbiomes. Of the tools, FunOMIC and HumanMycobiomeScan needed source code modifications to run. Notably, only one species, Candida orthopsilosis, was consistently identified by all tools across all communities where it was included. Increasing community richness improved precision of Kraken2 and the relative abundance accuracy of all tools on species, genus, and family levels. MetaPhlAn4 accurately identified all genera present in the communities and FunOMIC identified most species. The top three tools for overall accuracy in both identification and relative abundance estimation were EukDetect, MiCoP, and FunOMIC, respectively. Adding 90% and 99% bacterial background did not significantly impact these tools’ performance. Among the whole genome reference tools (Kraken2, HMS, and MiCoP), MiCoP exhibited the highest accuracy when the same reference database was used. Our survey of mycobiome-specific software revealed a very limited selection of such tools and their poor robustness due to error-prone software, along with a significant lack of comprehensive databases enabling characterization of the mycobiome. None of the implemented tools fully agreed on the mock community profiles. FunOMIC recognized most of the species, but EukDetect and MiCoP provided predictions that were closest to the correct compositions. The bacterial background did not impact these tools’ performance.

Extracellular vesicles secreted by Gram-negative bacteria have proven to be important in bacterial defense, communication and host–pathogen relationships. They resemble smaller versions of the bacterial mother cell, with similar contents of proteins, LPS, DNA, and RNA. Vesicles can elicit a protective immune response in a range of hosts, and as vaccine candidates, it is of interest to properly characterize their cargo. Genetic sequencing data is already available for vesicles from several bacterial strains, but it is not yet clear how the genetic makeup of vesicles differ from that of their parent cells, and which properties may characterize enriched genetic material. The present study provides evidence for DNA inside vesicles from Vibrio cholerae O395, and key characteristics of their genetic and proteomic content are compared to that of whole cells. DNA analysis reveals enrichment of fragments containing ToxR binding sites, as well as a positive correlation between AT-content and enrichment. Some mRNAs were highly enriched in the vesicle fraction, such as membrane protein genes ompV, ompK, and ompU, DNA-binding protein genes hupA, hupB, ihfB, fis, and ssb, and a negative correlation was found between mRNA enrichment and transcript length, suggesting mRNA inclusion in vesicles may be a size-dependent process. Certain non-coding and functional RNAs were found to be enriched, such as VrrA, GcvB, tmRNA, RNase P, CsrB2, and CsrB3. Mass spectrometry revealed enrichment of outer membrane proteins, known virulence factors, phage components, flagella and extracellular proteins in the vesicle fraction, and a low, negative correlation was found between transcript-, and protein enrichment. This result opposes the hypothesis that a significant degree of protein translation occurs in vesicles after budding. The abundance of viral-, and flagellar proteins in the vesicle fraction underlines the importance of purification during vesicle isolation.

Membrane vesicles (MVs) are spherical particles naturally released from the membrane of Gram-negative bacteria. Bacterial MV production is associated with a range of phenotypes including biofilm formation, horizontal gene transfer, toxin delivery, modulation of host immune responses and virulence. This study reports comparative profiling of MVs from bacterial strains isolated from three widely disperse geographical areas. Mass spectrometry identified 119, 159 and 142 proteins in MVs from three different strains of Piscirickettsia salmonis isolated from salmonids in Chile (LF-89), Norway (NVI 5692) and Canada (NVI 5892), respectively. MV comparison revealed several strain-specific differences related to higher virulence capability for LF-89 MVs, both in vivo and in vitro, and stronger similarities between the NVI 5692 and NVI 5892 MV proteome. The MVs were similar in size and appearance as analyzed by electron microscopy and dynamic light scattering. The MVs from all three strains were internalized by both commercial and primary immune cell cultures, which suggest a potential role of the MVs in the bacterium’s utilization of leukocytes. When MVs were injected into an adult zebrafish infection model, an upregulation of several pro-inflammatory genes were observed in spleen and kidney, indicating a modulating effect on the immune system. The present study is the first comparative analysis of P. salmonis derived MVs, highlighting strain-specific vesicle characteristics. The results further illustrate that the MV proteome from one bacterial strain is not representative of all bacterial strains within one species.