Olivier Croce

Clostridium butyricum strains and dysbiosis linked to necrotizing enterocolitis in preterm neonates

BACKGROUND Necrotizing enterocolitis (NEC) is the most common and serious gastrointestinal disorder among preterm neonates. We aimed to assess a specific gut microbiota profile associated with NEC. METHODS Stool samples and clinical data were collected from 4 geographically independent neonatal intensive care units, over a 48-month period. Thirty stool samples from preterm neonates with NEC (n = 15) and controls (n = 15) were analyzed by 16S ribosomal RNA pyrosequencing and culture-based methods. The results led us to develop a specific quantitative polymerase chain reaction (qPCR) assay for Clostridium butyricum, and we tested stool samples from preterm neonates with NEC (n = 93) and controls (n = 270). We sequenced the whole genome of 16 C. butyricum strains, analyzed their phylogenetic relatedness, tested their culture supernatants for cytotoxic activity, and searched for secreted toxins. RESULTS Clostridium butyricum was specifically associated with NEC using molecular and culture-based methods (15/15 vs 2/15; P < .0001) or qPCR (odds ratio, 45.4 [95% confidence interval, 26.2-78.6]; P < .0001). Culture supernatants of C. butyricum strains from preterm neonates with NEC (n = 14) exhibited significant cytotoxic activity (P = .008), and we identified in all a homologue of the β-hemolysin toxin gene shared by Brachyspira hyodysenteriae, the etiologic agent of swine dysentery. The corresponding protein was secreted by a NEC-associated C. butyricum strain. CONCLUSIONS NEC was associated with C. butyricum strains and dysbiosis with an oxidized, acid, and poorly diversified gut microbiota. Our findings highlight the plausible toxigenic mechanism involved in the pathogenesis of NEC.

Epigenetic switch drives the conversion of fibroblasts into proinvasive cancer-associated fibroblasts

Carcinoma-associated fibroblasts (CAF) mediate the onset of a proinvasive tumour microenvironment. The proinflammatory cytokine LIF reprograms fibroblasts into a proinvasive phenotype, which promotes extracellular matrix remodelling and collective invasion of cancer cells. Here we unveil that exposure to LIF initiates an epigenetic switch leading to the constitutive activation of JAK1/STAT3 signalling, which results in sustained proinvasive activity of CAF. Mechanistically, p300-histone acetyltransferase acetylates STAT3, which, in turn, upregulates and activates the DNMT3b DNA methyltransferase. DNMT3b methylates CpG sites of the SHP-1 phosphatase promoter, which abrogates SHP-1 expression, and results in constitutive phosphorylation of JAK1. Sustained JAK1/STAT3 signalling is maintained by DNA methyltransferase DNMT1. Consistently, in human lung and head and neck carcinomas, STAT3 acetylation and phosphorylation are inversely correlated with SHP-1 expression. Combined inhibition of DNMT activities and JAK signalling, in vitro and in vivo, results in long-term reversion of CAF-associated proinvasive activity and restoration of the wild-type fibroblast phenotype.

Zamilon, a Novel Virophage with Mimiviridae Host Specificity

Virophages, which are potentially important ecological regulators, have been discovered in association with members of the order Megavirales. Sputnik virophages target the Mimiviridae, Mavirus was identified with the Cafeteria roenbergensis virus, and virophage genomes reconstructed by metagenomic analyses may be associated with the Phycodnaviridae. Despite the fact that the Sputnik virophages were isolated with viruses belonging to group A of the Mimiviridae, they can grow in amoebae infected by Mimiviridae from groups A, B or C. In this study we describe Zamilon, the first virophage isolated with a member of group C of the Mimiviridae family. By co-culturing amoebae with purified Zamilon, we found that the virophage is able to multiply with members of groups B and C of the Mimiviridae family but not with viruses from group A. Zamilon has a 17,276 bp DNA genome that potentially encodes 20 genes. Most of these genes are closely related to genes from the Sputnik virophage, yet two are more related to Megavirus chiliensis genes, a group B Mimiviridae, and one to Moumouvirus monve transpoviron.

Identification of Novel Zoonotic Activity of Bartonella spp., France

These bacteria may cause paucisymptomatic bacteremia and endocarditis in humans.

Whole-genome assembly of Akkermansia muciniphila sequenced directly from human stool

BackgroundAlterations in gut microbiota composition under antibiotic pressure have been widely studied, revealing a restricted diversity of gut flora, including colonization by organisms such as Enterococci, while their impact on bacterial load is variable. High-level colonization by Akkermansia muciniphila, ranging from 39% to 84% of the total bacterial population, has been recently reported in two patients being treated with broad-spectrum antibiotics, although attempts to cultivate this microorganism have been unsuccessful.ResultsHere, we propose an original approach of genome sequencing for Akkermansia muciniphila directly from the stool sample collected from one of these patients. We performed and assembly using metagenomic data obtained from the stool sample. We used a mapping method consisting of aligning metagenomic sequencing reads against the reference genome of the Akkermansia muciniphila MucT strain, and a De novo assembly to support this mapping method. We obtained draft genome of the Akkermansia muciniphila strain Urmite with only 56 gaps. The absence of particular metabolic requirement as possible explanation of our inability to culture this microorganism, suggests that the bacterium was dead before the inoculation of the stool sample. Additional antibiotic resistance genes were found following comparison with the reference genome, providing some clues pertaining to its survival and colonization in the gut of a patient treated with broad-spectrum antimicrobial agents. However, no gene coding for imipenem resistance was detected, although this antibiotic was a part of the patient’s antibiotic regimen.ConclusionsThis work highlights the potential of metagenomics to facilitate the assembly of genomes directly from human stool.ReviewersThis article was reviewed by Eric Bapteste, William Martin and Vivek Anantharaman.

Developmental Cycle and Genome Analysis of “Rubidus massiliensis,” a New Vermamoeba vermiformis Pathogen

The study of amoeba-associated Chlamydiae is a dynamic field in which new species are increasingly reported. In the present work, we characterized the developmental cycle and analyzed the genome of a new member of this group associated with Vermamoeba vermiformis, we propose to name “Rubidus massiliensis.” This bacterium is well-adapted to its amoeba host and do not reside inside of inclusion vacuoles after phagocytosis. It has a developmental cycle typical of this family of bacteria, with a transition from condensed elementary bodies to hypodense replicative reticulate bodies. Multiplication occurs through binary fission of the reticulate bodies. The genome of “R. massiliensis” consists of a 2.8 Mbp chromosome and two plasmids (pRm1, pRm2) consisting of 39,075 bp and 80,897 bp, respectively, a feature that is unique within this group. The Re-analysis of the Chlamydiales genomes including the one of “R. massiliensis” slightly modified the previous phylogeny of the tlc gene encoding the ADP/ATP translocase. Our analysis suggested that the tlc gene could have been transferred to plant and algal plastids before the transfer to Rickettsiales, and that this gene was probably duplicated several times.

Pan-genomic analysis to redefine species and subspecies based on quantum discontinuous variation: the Klebsiella paradigm

BackgroundVarious methods are currently used to define species and are based on the phylogenetic marker 16S ribosomal RNA gene sequence, DNA-DNA hybridization and DNA GC content. However, these are restricted genetic tools and showed significant limitations.ResultsIn this work, we describe an alternative method to build taxonomy by analyzing the pan-genome composition of different species of the Klebsiella genus. Klebsiella species are Gram-negative bacilli belonging to the large Enterobacteriaceae family. Interestingly, when comparing the core/pan-genome ratio; we found a clear discontinuous variation that can define a new species.ConclusionsUsing this pan-genomic approach, we showed that Klebsiella pneumoniae subsp. ozaenae and Klebsiella pneumoniae subsp. rhinoscleromatis are species of the Klebsiella genus, rather than subspecies of Klebsiella pneumoniae. This pan-genomic analysis, helped to develop a new tool for defining species introducing a quantic perspective for taxonomy.ReviewersThis article was reviewed by William Martin, Pierre Pontarotti and Pere Puigbo (nominated by Dr Yuri Wolf).

Noncontiguous finished genome sequence and description of Virgibacillus massiliensis sp. nov., a moderately halophilic bacterium isolated from human gut

Strain Vm-5T was isolated from the stool specimen of a 10-year-old Amazonian boy. This bacterium is a Gram-positive, strictly aerobic rod, motile by a polar flagellum. Here we describe its phenotypic characteristics and complete genome sequence. The 4 353 177 bp long genome exhibits a G + C content of 36.87% and contains 4394 protein-coding and 125 predicted RNA genes. Phylogenetically and genetically, strain Vm-c is a member of the genus Virgibacillus but is distinct enough to be classified as a new species. We propose the creation of V. massiliensis sp. nov., whose type strain is strain Vm-5T (CSUR P971 = DSM 28587).

Non-contiguous finished genome sequence and description of Fenollaria massiliensis gen. nov., sp. nov., a new genus of anaerobic bacterium

Fenollaria massiliensis strain 9401234T, is the type strain of Fenollaria massiliensis gen. nov., sp. nov., a new species within a new genus Fenollaria. This strain, whose genome is described here, was isolated from an osteoarticular sample. F. massiliensis strain 9401234T is an obligate anaerobic Gram-negative bacillus. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 1.71 Mbp long genome exhibits a G+C content of 34.46% and contains 1,667 protein-coding and 30 RNA genes, including 3 rRNA genes.

Genomic Insights into a New Citrobacter koseri Strain Revealed Gene Exchanges with the Virulence-Associated Yersinia pestis pPCP1 Plasmid

The history of infectious diseases raised the plague as one of the most devastating for human beings. Far too often considered an ancient disease, the frequent resurgence of the plague has led to consider it as a reemerging disease in Madagascar, Algeria, Libya, and Congo. The genetic factors associated with the pathogenicity of Yersinia pestis, the causative agent of the plague, involve the acquisition of the pPCP1 plasmid that promotes host invasion through the expression of the virulence factor Pla. The surveillance of plague foci after the 2003 outbreak in Algeria resulted in a positive detection of the specific pla gene of Y. pestis in rodents. However, the phenotypic characterization of the isolate identified a Citrobacter koseri. The comparative genomics of our sequenced C. koseri URMITE genome revealed a mosaic gene structure resulting from the lifestyle of our isolate and provided evidence for gene exchanges with different enteric bacteria. The most striking was the acquisition of a continuous 2 kb genomic fragment containing the virulence factor Pla of the Y. pestis pPCP1 plasmid; however, the subcutaneous injection of the CKU strain in mice did not produce any pathogenic effect. Our findings demonstrate that fast molecular detection of plague using solely the pla gene is unsuitable and should rather require Y. pestis gene marker combinations. We also suggest that the evolutionary force that might govern the expression of pathogenicity can occur through the acquisition of virulence genes but could also require the loss or the inactivation of resident genes such as antivirulence genes.