Buttiauxella massiliensis sp. nov., Isolated from a Human Bone Infection.

Strain Marseille-P9829 was isolated from a bone sample collected from an open right fibula fracture from a 46-years old patient. Strain Marseille-P9829 (= CSUR P9829 = DSM 110695) was a Gram-negative, non-spore-forming and non-motile bacterium. This strain had a positive catalase activity but was oxidase-negative. The major fatty acids methyl esters were hexadecanoic acid (45.6%) and 9-hexadecenoic acid (28.4%). Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry analysis suggested that this strain belongs to the species Buttiauxella gaviniae. Since there were few reports of clinical infections with this species in humans, whole genome sequencing was performed and a polyphasic taxono-genomic approach was followed in order to verify the classification of strain Marseille-P9829. The 16S rRNA gene sequence BLAST against the NCBI database yielded the highest similarity of 99.8% with Buttiauxella agrestis, suggesting that strain Marseille-P9829 belongs to this species. However, genomic comparison by digital DNA-DNA hybridization showed that values between strain Marseille-P9829 and other validly published Buttiauxella species were all lower than 70%. Furthermore, all average nucleotide identities were lower than 95-96%. Therefore, these results confirmed that strain Marseille-P9829 belonged to a new Buttiauxella species for which we propose the name Buttiauxella massiliensis sp. nov., with strain Marseille-P9829 as type strain.

Interest of bacterial pangenome analyses in clinical microbiology.

Thanks to the progress and decreasing costs in genome sequencing technologies, more than 250,000 bacterial genomes are currently available in public databases, covering most, if not all, of the major human-associated phylogenetic groups of these microorganisms, pathogenic or not. In addition, for many of them, sequences from several strains of a given species are available, thus enabling to evaluate their genetic diversity and study their evolution. In addition, the significant cost reduction of bacterial whole genome sequencing as well as the rapid increase in the number of available bacterial genomes have prompted the development of pangenomic software tools. The study of bacterial pangenome has many applications in clinical microbiology. It can unveil the pathogenic potential and ability of bacteria to resist antimicrobials as well identify specific sequences and predict antigenic epitopes that allow molecular or serologic assays and vaccines to be designed. Bacterial pangenome constitutes a powerful method for understanding the history of human bacteria and relating these findings to diagnosis in clinical microbiology laboratories in order to optimize patient management.

Confirmatory Virucidal Activity of Ionised Active Water S-100® on the SARS-CoV-2 Virus.

Ionised active water S-100® has been proposed as an original solution for use in dermocosmetics and for the treatment of wounds such as burns and atopic dermatitis. Among the mechanisms of action that are not completely understood, an antimicrobial activity would appear to be important. In the context of the COVID-19 pandemic, we assessed the inactivating efficacy of this solution on SARS-CoV-2 based on the recommendations of the NF-EN-14476+A2 standard. The tests carried out demonstrated that ionised active water S-100® 40% has a virucidal activity on SARS-CoV-2 which is at least 3.1 log after a contact time of 30 seconds and 3.5 log after two minutes at 20°C under clean conditions. Assays were also performed at 4°C and 37°C, and the results obtained are identical to those obtained at 20°C. This demonstration of the virucidal effect of ionised water against SARS-CoV-2 paves the way for the development of usage as an alternative disinfectant in SARS-CoV-2 control.

Description of Virulent Factors and Horizontal Gene Transfers of Keratitis-Associated Amoeba Acanthamoeba Triangularis by Genome Analysis.

Acanthamoeba triangularis strain SH 621 is a free-living amoeba belonging to Acanthamoeba ribo-genotype T4. This ubiquitous protist is among the free-living amoebas responsible for Acanthamoeba keratitis, a severe infection of human cornea. Genome sequencing and genomic comparison were carried out to explore the biological functions and to better understand the virulence mechanism related to the pathogenicity of Acanthamoeba keratitis. The genome assembly harbored a length of 66.43 Mb encompassing 13,849 scaffolds. The analysis of predicted proteins reported the presence of 37,062 ORFs. A complete annotation revealed 33,168 and 16,605 genes that matched with NCBI non-redundant protein sequence (nr) and Cluster of Orthologous Group of proteins (COG) databases, respectively. The Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG) annotation reported a great number of genes related to carbohydrate, amino acid and lipid metabolic pathways. The pangenome performed with 8 available amoeba genomes belonging to genus Acanthamoeba revealed a core genome containing 843 clusters of orthologous genes with a ratio core genome/pangenome of less than 0.02. We detected 48 genes related to virulent factors of Acanthamoeba keratitis. Best hit analyses in nr database identified 99 homologous genes shared with amoeba-resisting microorganisms. This study allows the deciphering the genome of a free-living amoeba with medical interest and provides genomic data to better understand virulence-related Acanthamoeba keratitis.

Vermamoeba vermiformis CDC-19 draft genome sequence reveals considerable gene trafficking including with candidate phyla radiation and giant viruses.

Vermamoeba vermiformis is a predominant free-living amoeba in human environments and amongst the most common amoebae that can cause severe infections in humans. It is a niche for numerous amoeba-resisting microorganisms such as bacteria and giant viruses. Differences in the susceptibility to these giant viruses have been observed. V. vermiformis and amoeba-resisting microorganisms share a sympatric lifestyle that can promote exchanges of genetic material. This work analyzed the first draft genome sequence of a V. vermiformis strain (CDC-19) through comparative genomic, transcriptomic and phylogenetic analyses. The genome of V. vermiformis is 59.5 megabase pairs in size, and 22,483 genes were predicted. A high proportion (10% (n = 2,295)) of putative genes encoded proteins showed the highest sequence homology with a bacterial sequence. The expression of these genes was demonstrated for some bacterial homologous genes. In addition, for 30 genes, we detected best BLAST hits with members of the Candidate Phyla Radiation. Moreover, 185 genes (0.8%) best matched with giant viruses, mostly those related to the subfamily Klosneuvirinae (101 genes), in particular Bodo saltans virus (69 genes). Lateral sequence transfers between V. vermiformis and amoeba-resisting microorganisms were strengthened by Sanger sequencing, transcriptomic and phylogenetic analyses. This work provides important insights and genetic data for further studies about this amoeba and its interactions with microorganisms.

Insight into the Lifestyle of Amoeba Willaertia magna during Bioreactor Growth Using Transcriptomics and Proteomics.

Willaertia magna C2c maky is a thermophilic free-living amoeba strain that showed ability to eliminate Legionella pneumophila, a pathogenic bacterium living in the aquatic environment. The amoeba industry has proposed the use of Willaertia magna as a natural biocide to control L. pneumophila proliferation in cooling towers. Here, transcriptomic and proteomic studies were carried out in order to expand knowledge on W. magna produced in a bioreactor. Illumina RNA-seq generated 217 million raw reads. A total of 8,790 transcripts were identified, of which 6,179 and 5,341 were assigned a function through comparisons with National Center of Biotechnology Information (NCBI) reference sequence and the Clusters of Orthologous Groups of proteins (COG) databases, respectively. To corroborate these transcriptomic data, we analyzed the W. magna proteome using LC-MS/MS. A total of 3,561 proteins were identified. The results of transcriptome and proteome analyses were highly congruent. Metabolism study showed that W. magna preferentially consumed carbohydrates and fatty acids to grow. Finally, an in-depth analysis has shown that W. magna produces several enzymes that are probably involved in the metabolism of secondary metabolites. Overall, our multi-omic study of W. magna opens the way to a better understanding of the genetics and biology of this amoeba.

Intracellular Behaviour of Three Legionella pneumophila Strains within Three Amoeba Strains, Including Willaertia magna C2c Maky.

Legionella pneumophila is a facultative intracellular pathogen found in aquatic environments as planktonic cells within biofilms and as intracellular parasites of free-living amoebae such as Acanthamoeba castellanii. This pathogen bypasses the elimination mechanism to replicate within amoebae; however, not all amoeba species support the growth of L. pneumophila. Willaertia magna C2c Maky, a non-pathogenic amoeba, was previously demonstrated to possess the ability to eliminate the L. pneumophila strain Paris. Here, we study the intracellular behaviour of three L. pneumophila strains (Paris, Philadelphia, and Lens) within W. magna C2c Maky and compare this strain to A. castellanii and W. magna Z503, which are used as controls. We observe the intracellular growth of strain Lens within W. magna Z503 and A. castellanii at 22 °C and 37 °C. Strain Paris grows within A. castellanii at any temperature, while it only grows at 22 °C within W. magna Z503. Strain Philadelphia proliferates only within A. castellanii at 37 °C. Within W. magna C2c Maky, none of the three legionella strains exhibit intracellular growth. Additionally, the ability of W. magna C2c Maky to decrease the number of internalized L. pneumophila is confirmed. These results support the idea that W. magna C2c Maky possesses unique behaviour in regard to L. pneumophila strains.

Proteomics and Lipidomics Investigations to Decipher the Behavior of Willaertia magna C2c Maky According to Different Culture Modes.

Willaertia magna C2c Maky is a free-living amoeba that has demonstrated its ability to inhibit the intracellular multiplication of some Legionella pneumophila strains, which are pathogenic bacteria inhabiting the aquatic environment. The Amoeba, an industry involved in the treatment of microbiological risk in the water and plant protection sectors, has developed a natural biocide based on the property of W. magna to manage the proliferation of the pathogen in cooling towers. In axenic liquid medium, amoebas are usually cultivated in adhesion on culture flask. However, we implemented a liquid culture in suspension using bioreactors in order to produce large quantities of W. magna. In order to investigate the culture condition effects on W. magna, we conducted a study based on microscopic, proteomics and lipidomics analyzes. According to the culture condition, amoeba exhibited two different phenotypes. The differential proteomics study showed that amoebas seemed to promote the lipid metabolism pathway in suspension culture, whereas we observed an upregulation of the carbohydrate pathway in adherent culture. Furthermore, we observed an over-regulation of proteins related to the cytoskeleton for W. magna cells grown in adhesion. Regarding the lipid analysis, suspension and adhesion cell growth showed comparable lipid class compositions. However, the differential lipid analysis revealed differences that confirmed cell phenotype differences observed by microscopy and predicted by proteomics. Overall, this study provides us with a better insight into the biology and molecular processes of W. magna in different culture lifestyles.

Whole-Genome Sequence of French Clinical Peptoniphilus catoniae Strain P8546.

In 2016, Peptoniphilus catoniae was reported as a bacterial species isolated from a healthy Peruvian male. In 2018, a clinical strain from the same species was isolated from the stool of a French patient with kidney cancer. The genome of this strain, P8546, was 1,725,465 bp long, with 33.4% G+C content.

Investigation of potential pathogenicity of Willaertia magna by investigating the transfer of bacteria pathogenicity genes into its genome.

Willaertia magna c2c maky is a thermophilic amoeba closely related to the genus Naegleria. This free-living amoeba has the ability to eliminate Legionella pneumophila, which is an amoeba-resisting bacterium living in an aquatic environment. To prevent the proliferation of L. pneumophila in cooling towers, the use of W. magna as natural biocide has been proposed. To provide a better understanding of the W. magna genome, whole-genome sequencing was performed through the study of virulence factors and lateral gene transfers. This amoeba harbors a genome of 36.5 megabases with 18,519 predicted genes. BLASTp analyses reported protein homology between 136 W. magna sequences and amoeba-resistant microorganisms. Horizontal gene transfers were observed based on the basis of the phylogenetic reconstruction hypothesis. We detected 15 homologs of N. fowleri genes related to virulence, although these latter were also found in the genome of N. gruberi, which is a non-pathogenic amoeba. Furthermore, the cytotoxicity test performed on human cells supports the hypothesis that the strain c2c maky is a non-pathogenic amoeba. This work explores the genomic repertory for the first draft genome of genus Willaertia and provides genomic data for further comparative studies on virulence of related pathogenic amoeba, N. fowleri.

Genomic analysis of a Raoultella ornithinolytica strain causing prosthetic joint infection in an immunocompetent patient.

We sequenced the genome of Raoultella ornithinolytica strain Marseille-P1025 that caused a rare case of prosthetic joint infection in a 67-year-old immunocompetent male. The 6.7-Mb genome exhibited a genomic island (RoGI) that was unique among R. ornithinolytica strains. RoGI was likely acquired by lateral gene transfer from a member of the Pectobacterium genus and coded for a type IVa secretion system found in other pathogenic bacteria and that may have conferred strain Marseille-P1025 an increased virulence. Strain Marseille-P1025 was also able to infect, multiply within, and kill Acanthamoaeba castellanii amoebae.

Orpheovirus IHUMI-LCC2: A New Virus among the Giant Viruses.

Giant viruses continue to invade the world of virology, in gigantic genome sizes and various particles shapes. Strains discoveries and metagenomic studies make it possible to reveal the complexity of these microorganisms, their origins, ecosystems and putative roles. We isolated from a rat stool sample a new giant virus "Orpheovirus IHUMI-LCC2," using Vermamoeba vermiformis as host cell. In this paper, we describe the main genomic features and replicative cycle of Orpheovirus IHUMI-LCC2. It possesses a circular genome exceeding 1.4 Megabases with 25% G+C content and ovoidal-shaped particles ranging from 900 to 1300 nm. Particles are closed by at least one thick membrane in a single ostiole-like shape in their apex. Phylogenetic analysis and the reciprocal best hit for Orpheovirus show a connection to the proposed Pithoviridae family. However, some genomic characteristics bear witness to a completely divergent evolution for Orpheovirus IHUMI-LCC2 when compared to Cedratviruses or Pithoviruses.