Giuseppe D'Auria

The active human gut microbiota differs from the total microbiota.

The human gut microbiota is considered one of the most fascinating reservoirs of microbial diversity hosting between 400 to 1000 bacterial species distributed among nine phyla with Firmicutes, Bacteroidetes and Actinobacteria representing around of the diversity. One of the most intriguing issues relates to understanding which microbial groups are active players in the maintenance of the microbiota homeostasis. Here, we describe the diversity of active microbial fractions compared with the whole community from raw human fecal samples. We studied four healthy volunteers by 16S rDNA gene pyrosequencing. The fractions were obtained by cell sorting based on bacterial RNA concentration. Bacterial families were observed to appear or disappear on applying a cell sorting method in which flow cytometry was used to evaluate the active cells by pyronin-Y staining of RNA. This method was able to detect active bacteria, indicating that the active players differed from that observed in raw fecal material. Generally, observations showed that in the active fractions, the number of reads related to Bacteroidetes decreased whereas several families from Clostridiales (Firmicutes) were more highly represented. Moreover, a huge number of families appeared as part of the active fraction when cell sorting was applied, indicating reads that are simply statistically hidden by the total reads.

Phylogenomic evidence for the presence of a flagellum and cbb3 oxidase in the free-living mitochondrial ancestor

The initiation of the intracellular symbiosis that would give rise to mitochondria and eukaryotes was a major event in the history of life on earth. Hypotheses to explain eukaryogenesis fall into two broad and competing categories: those proposing that the host was a phagocytotic proto-eukaryote that preyed upon the free-living mitochondrial ancestor (hereafter FMA), and those proposing that the host was an archaebacterium that engaged in syntrophy with the FMA. Of key importance to these hypotheses are whether the FMA was motile or nonmotile, and the atmospheric conditions under which the FMA thrived. Reconstructions of the FMA based on genome content of Rickettsiales representatives-generally considered to be the closest living relatives of mitochondria-indicate that it was nonmotile and aerobic. We have sequenced the genome of Candidatus Midichloria mitochondrii, a novel and phylogenetically divergent member of the Rickettsiales. We found that it possesses unique gene sets found in no other Rickettsiales, including 26 genes associated with flagellar assembly, and a cbb(3)-type cytochrome oxidase. Phylogenomic analyses show that these genes were inherited in a vertical fashion from an ancestral α-proteobacterium, and indicate that the FMA possessed a flagellum, and could undergo oxidative phosphorylation under both aerobic and microoxic conditions. These results indicate that the FMA played a more active and potentially parasitic role in eukaryogenesis than currently appreciated and provide an explanation for how the symbiosis could have evolved under low levels of oxygen.

Virulence factor rtx in Legionella pneumophila, evidence suggesting it is a modular multifunctional protein

BackgroundThe repeats in toxin (Rtx) are an important pathogenicity factor involved in host cells invasion of Legionella pneumophila and other pathogenic bacteria. Its role in escaping the host immune system and cytotoxic activity is well known. Its repeated motives and modularity make Rtx a multifunctional factor in pathogenicity.ResultsThe comparative analysis of rtx gene among 6 strains of L. pneumophila showed modularity in their structures. Among compared genomes, the N-terminal region of the protein presents highly dissimilar repeats with functionally similar domains. On the contrary, the C-terminal region is maintained with a fashionable modular configuration, which gives support to its proposed role in adhesion and pore formation. Despite the variability of rtx among the considered strains, the flanking genes are maintained in synteny and similarity.ConclusionIn contrast to the extracellular bacteria Vibrio cholerae, in which the rtx gene is highly conserved and flanking genes have lost synteny and similarity, the gene region coding for the Rtx toxin in the intracellular pathogen L. pneumophila shows a rapid evolution. Changes in the rtx could play a role in pathogenicity. The interplay of the Rtx toxin with host membranes might lead to the evolution of new variants that are able to escape host cell defences.

Genome sequence of Lactococcus garvieae UNIUD074, isolated in Italy from a lactococcosis outbreak.

Lactococcus garvieae is the etiological agent of lactococcosis disease, affecting many cultured fish species worldwide. In addition, this bacterium is currently considered a potential zoonotic microorganism since it is known to cause several opportunistic human infections. Here we present the draft genome sequence of the L. garvieae strain UNIUD074.

Active and secreted IgA-coated bacterial fractions from the human gut reveal an under-represented microbiota core.

Host-associated microbiota varies in distribution depending on the body area inhabited. Gut microbes are known to interact with the human immune system, maintaining gut homoeostasis. Thus, we studied whether secreted-IgA (S-IgA) coat specific microbial taxa without inducing strong immune responses. To do so, we fractionated gut microbiota by flow cytometry. We found that active and S-IgA-coated bacterial fractions were characterized by a higher diversity than those observed in raw faecal suspensions. A long-tail effect was observed in family distribution, revealing that rare bacteria represent up to 20% of total diversity. While Firmicutes was the most abundant phylum, the majority of its sequences were not assigned at the genus level. Finally, the single-cell-based approach enabled us to focus on active and S-IgA-coated bacteria. Thus, we revealed a microbiota core common to the healthy volunteers participating in the study. Interestingly, this core was composed mainly of low frequency taxa (e.g. Sphingomonadaceae).

The Monoclonal Antitoxin Antibodies (Actoxumab-Bezlotoxumab) Treatment Facilitates Normalization of the Gut Microbiota of Mice with Clostridium difficile Infection.

Antibiotics have significant and long-lasting impacts on the intestinal microbiota and consequently reduce colonization resistance against Clostridium difficile infection (CDI). Standard therapy using antibiotics is associated with a high rate of disease recurrence, highlighting the need for novel treatment strategies that target toxins, the major virulence factors, rather than the organism itself. Human monoclonal antibodies MK-3415A (actoxumab–bezlotoxumab) to C. difficile toxin A and toxin B, as an emerging non-antibiotic approach, significantly reduced the recurrence of CDI in animal models and human clinical trials. Although the main mechanism of protection is through direct neutralization of the toxins, the impact of MK-3415A on gut microbiota and its restoration has not been examined. Using a CDI murine model, we compared the bacterial diversity of the gut microbiome of mice under different treatments including MK-3415A, vancomycin, or vancomycin combined with MK-3415A, sampled longitudinally. Here, we showed that C. difficile infection resulted in the prevalence of Enterobacter species. Sixty percent of mice in the vehicle group died after 2 days and their microbiome was almost exclusively formed by Enterobacter. MK-3415A treatment resulted in lower Enterobacter levels and restoration of Blautia, Akkermansia, and Lactobacillus which were the core components of the original microbiota. Vancomycin treatment led to significantly lower survival rate than the combo treatment of MK-3415A and vancomycin. Vancomycin treatment decreased bacterial diversity with predominant Enterobacter and Akkermansia, while Staphylococcus expanded after vancomycin treatment was terminated. In contrast, mice treated by vancomycin combined with MK-3415A also experienced decreased bacterial diversity during vancomycin treatment. However, these animals were able to recover their initial Blautia and Lactobacillus proportions, even though episodes of Staphylococcus overgrowth were detected by the end of the experiments. In conclusion, MK-3415A (actoxumab–bezlotoxumab) treatment facilitates normalization of the gut microbiota in CDI mice. It remains to be examined whether or not the prevention of recurrent CDI by the antitoxin antibodies observed in clinical trials occurs through modulation of microbiota.

Direct sequencing of human gut virome fractions obtained by flow cytometry

The sequence assembly of the human gut virome encounters several difficulties. A high proportion of human and bacterial matches is detected in purified viral samples. Viral DNA extraction results in a low DNA concentration, which does not reach the minimal limit required for sequencing library preparation. Therefore, the viromes are usually enriched by whole genome amplification (WGA), which is, however, prone to the development of chimeras and amplification bias. In addition, as there is a very wide diversity of gut viral species, very extensive sequencing efforts must be made for the assembling of whole viral genomes. We present an approach to improve human gut virome assembly by employing a more precise preparation of a viral sample before sequencing. Particles present in a virome previously filtered through 0.2 μm pores were further divided into groups in accordance with their size and DNA content by fluorescence activated cell sorting (FACS). One selected viral fraction was sequenced excluding the WGA step, so that unbiased sequences with high reliability were obtained. The DNA extracted from the 314 viral particles of the selected fraction was assembled into 34 contigs longer than 1,000 bp. This represents an increase to the number of assembled long contigs per sequenced Gb in comparison with other studies where non-fractioned viromes are sequenced. Seven of these contigs contained open reading frames (ORFs) with explicit matches to proteins related to bacteriophages. The remaining contigs also possessed uncharacterized ORFs with bacteriophage-related domains. When the particles that are present in the filtered viromes are sorted into smaller groups by FACS, large pieces of viral genomes can be recovered easily. This approach has several advantages over the conventional sequencing of non-fractioned viromes: non-viral contamination is reduced and the sequencing efforts required for viral assembly are minimized.

Complete Genome Sequence of Acidaminococcus intestini RYC-MR95, a Gram-Negative Bacterium from the Phylum Firmicutes

Acidaminococcus intestini belongs to the family Acidaminococcaceae, order Selenomonadales, class Negativicutes, phylum Firmicutes. Negativicutes show the double-membrane system of Gram-negative bacteria, although their chromosomal backbone is closely related to that of Gram-positive bacteria of the phylum Firmicutes. The complete genome of a clinical A. intestini strain is here presented.

Live genomics for pathogen monitoring in public health.

Whole genome analysis based on next generation sequencing (NGS) now represents an affordable framework in public health systems. Robust analytical pipelines of genomic data provides in a short lapse of time (hours) information about taxonomy, comparative genomics (pan-genome) and single polymorphisms profiles. Pathogenic organisms of interest can be tracked at the genomic level, allowing monitoring at one-time several variables including: epidemiology, pathogenicity, resistance to antibiotics, virulence, persistence factors, mobile elements and adaptation features. Such information can be obtained not only at large spectra, but also at the “local” level, such as in the event of a recurrent or emergency outbreak. This paper reviews the state of the art in infection diagnostics in the context of modern NGS methodologies. We describe how actuation protocols in a public health environment will benefit from a “streaming approach” (pipeline). Such pipeline would include NGS data quality assessment, data mining for comparative analysis, searching differential genetic features, such as virulence, resistance persistence factors and mutation profiles (SNPs and InDels) and formatted “comprehensible” results. Such analytical protocols will enable a quick response to the needs of locally circumscribed outbreaks, providing information on the causes of resistance and genetic tracking elements for rapid detection, and monitoring actuations for present and future occurrences.

Genome Sequence of Lactobacillus plantarum 19L3, a Strain Proposed as a Starter Culture for Slovenská Bryndza Ovine Cheese

ABSTRACT The genome sequence of Lactobacillus plantarum isolated from ovine cheese is presented here. This bacterium is proposed as a starter strain, named 19L3, for Slovenská bryndza cheese, a traditional Slovak cheese fulfilling European Food Safety Authority (EFSA) requirements.