Rita Zgheib

Analyse génomique comparative en taxonomie : classification et reclassification des espèces bactériennes: Le monde regorge de bactéries. Le nombre estimé de bactéries non identifiées dépasse de loin les taxons actuellement validement publiés. La taxonomie est la première étape vers l'organisation de cette grande diversité de bactéries. Au début, les bactéries ont été classées en fonction de leurs caractéristiques morphologiques, physiologiques et biochimiques, et par hybridation de l'ADN. Dans les années 1980, les taxonomistes ont connu une révolution avec la proposition du gène de l'ARNr 16S comme marqueur phylogénétique. Ensuite, le séquençage du génome a été introduit et a montré une plus grande précision dans la discrimination entre les espèces bactériennes que le gène de l'ARNr 16S. En 2019, l'utilisation de séquences génomiques pour la description de nouveaux taxons bactériens est devenue obligatoire.Dans cette thèse, nous avons souligné l'importance des méthodes basées sur le génome dans la taxonomie bactérienne. Nous l'avons démontré en revisitant la taxonomie complexe du genre Pseudomonas à l'aide de critères génomiques. Nous avons analysé 558 génomes et avons constaté qu'au sein de ce genre plusieurs espèces nécessitaient une reclassification.Un deuxième aspect de cette thèse est l'utilisation de l'approche taxono-génomique polyphasique qui combine les caractéristiques phénotypiques bactériennes et l'analyse génomique pour décrire et caractériser de nouveaux taxons bactériens.Sur la base des résultats globaux présentés dans cette thèse, nous avons pu confirmer que l'utilisation de séquences de génomes en taxonomie représente un avantage même si certaines limitations sont encore rencontrées.

In 2018, Corynebacterium sanguinis strain Marseille-P8776 was isolated from the blood of a 64-year-old woman suffering from breast cancer who had undergone chemotherapy and radiotherapy. Following whole-genome sequencing, the chromosome of strain Marseille-P8776 was 2,613,836 bp long with a G+C content of 64.92%, 2,568 protein-coding genes, and 64 RNA genes.

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.

Se.ne.ga.li.mas.si'li.a N.L. fem. n. Senegalimassilia , combination of Senegal, where the stool sample was collected and Massilia, the Latin name of Marseille, where strain JC110 ^T was cultivated. The genus Senegalimassilia englobes nonmotile, Gram‐positive, and oxidase‐negative coccobacilli. It has two species with validly published names, Senegalimassilia anaerobia and Senegalimassilia faecalis , that were isolated from healthy human gut samples. Senegalimassilia spp. grow best on blood‐enriched Columbia agar, at 37°C in strictly anaerobic atmosphere. The major fatty acid methyl esters are C _16:0 and C _16:0 dimethyl acetal, and the polar lipids include diphosphatidylglycerol, phosphatidylglycerol, unidentified phospholipids, and unidentified glycolipids. In addition to the fatty acid methyl esters and polar lipid composition, the species belonging to this genus can also be differentiated from closely related genera by their asaccharolytic trait. They can also be identified by genomic criteria, using the published limits that delineate new species. They can be identified by 16S rRNA gene sequence phylogeny and similarity (95–98.65% species limit), average nucleotide identity (ANI) (95–96%), and digital DNA–DNA hybridization (dDDH) (70%). They have an average genome size of 2,573,031 bp. DNA G + C content (mol%) : 60.9–61.2 (genome analysis). Type species : Senegalimassilia anaerobia Lagier et al. 2013, VL155 emend. Han et al. 2020. Taxonomic and Nomenclature Notes: According to the List of Prokaryotic names with Standing in Nomenclature (LPSN), the taxonomic status of the genus Senegalimassilia is: correct name (last update, February 2025) ^* . LPSN classification: Bacteria / Bacillati / Actinomycetota / Coriobacteriia / Eggerthellales / Eggerthellaceae / Senegalimassilia The genus Senegalimassilia can also be recovered in the Genome Taxonomy Database (GTDB) as g__Senegalimassilia (version v220) ^** . GTDB classification: d__Bacteria / p__Actinomycetota / c__Coriobacteriia / o__Coriobacteriales / f__Eggerthellaceae / g__Senegalimassilia ^* Meier‐Kolthoff et al. ( 2022 ). Nucleic Acids Res , 50 , D801 – D807 ; DOI: 10.1093/nar/gkab902 ^** Parks et al. ( 2022 ). Nucleic Acids Res , 50 , D785 – D794 ; DOI: 10.1093/nar/gkab776

Introduction: We studied the trend of antimicrobial resistance and consumption at Saint George Hospital University Medical Center (SGHUMC), a tertiary care center in Beirut, Lebanon, with a focus on the SARS-CoV-2 pandemic. Materials and Methods: We calculated the isolation density/1000 patient-days (PD) of the most isolated organisms from 1 January 2015–31 December 2020 that included: E. coli (Eco), K. pneumoniae (Kp), P. aeruginosa (Pae), A. baumannii (Ab), S. aureus (Sau), and E. faecium (Efm). We considered March–December 2020 a surrogate of COVID-19. We considered one culture/patient for each antimicrobial susceptibility and excluded Staphylococcus epidermidis, Staphylococcus coagulase-negative, and Corynebacterium species. We analyzed the trends of the overall isolates, the antimicrobial susceptibilities of blood isolates (BSI), difficult-to-treat (DTR) BSI, carbapenem-resistant Enterobacteriaceae (CRE) BSI, and restricted antimicrobial consumption as daily-defined-dose/1000 PD. DTR implies resistance to carbapenems, beta-lactams, fluoroquinolones, and additional antimicrobials where applicable. Results and Discussion: After applying exclusion criteria, we analyzed 1614 blood cultures out of 8314 cultures. We isolated 85 species, most commonly Eco, at 52%. The isolation density of total BSI in 2020 decreased by 16%: 82 patients were spared from bacteremia, with 13 being DTR. The isolation density of CRE BSI/1000 PD decreased by 64% from 2019 to 2020, while VREfm BSI decreased by 34%. There was a significant decrease of 80% in Ab isolates (p-value < 0.0001). During COVID-19, restricted antimicrobial consumption decreased to 175 DDD/1000 PD (p-value < 0.0001). Total carbapenem consumption persistently decreased by 71.2% from 108DDD/1000 PD in 2015–2019 to 31 DDD/1000 PD in 2020. At SGHUMC, existing epidemics were not worsened by the pandemic. We attribute this to our unique and dynamic collaboration of antimicrobial stewardship, infection prevention and control, and infectious disease consultation.

Living organisms interact with each other during their lifetime, leading to genomes rearrangement and sequences transfer. These well-known phenomena give these organisms mosaic genomes, which challenge their classification. Moreover, many findings occurred between the IXXth and XXIst century, especially the discovery of giant viruses and candidate phyla radiation (CPR). Here, we tried to provide an updated classification, which integrates 216 representative genomes of the current described organisms. The reclassification was expressed through a genetic network based on the total genomic content, not on a single gene to represent the tree of life. This rhizomal exploration represents, more accurately, the evolutionary relationships among the studied species. Our analyses show a separated branch named fifth TRUC (Things Resisting Uncompleted Classifications). This taxon groups CPRs together, independently from Bacteria, Archaea (which regrouped also Nanoarchaeota and Asgard members), Eukarya, and the giant viruses (recognized recently as fourth TRUC). Finally, the broadening of analysis methods will lead to the discovery of new organisms, which justify the importance of updating the classification at every opportunity. In this perspective, our pragmatic representation could be adjusted along with the progress of evolutionary studies.

The reason why severe cases of Buruli ulcers caused by Mycobacterium ulcerans are emerging in some South Australia counties has not been determined. In this study, we measured the diversity of M. ulcerans complex whole genome sequences (WGS) and reported a marker of this diversity. Using this marker as a probe, we compared WGS diversity in Buruli ulcer-epidemic South Australia counties versus non-epidemic Australian counties and further refined comparisons at the level of counties where severe Buruli ulcer cases have been reported. Analyzing 218 WGS (35 complete and 183 reconstructed WGS, including 174 Australian WGS) yielded 15 M. ulcerans complex genotypes, including three genotypes specific to Australia and one genotype specific to South Australia. A 1,068-bp PPE family protein gene exhibiting genotype-specific sequence variations was employed to further probe 13 minority clones hidden in sequence reads. The repartition of these clones significantly differed between South Australia and the rest of Australia. In addition, a significantly higher prevalence of 3/13 clones was observed in South Australia counties of the Mornington Peninsula, Melbourne and Bellarine Peninsula than in other South Australia counties. The data presented in this report suggest that the microevolution of three M. ulcerans complex clones drove the emergence of severe Buruli ulcer cases in some South Australia counties. Sequencing one specific PPE gene served to efficiently probe M. ulcerans complex clones. Further functional studies may balance the environmental adaptation and virulence of these clones.