Pierre Edouard Fournier

Identification of Rare Pathogenic Bacteria in a Clinical Microbiology Laboratory: Impact of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

ABSTRACT During the past 5 years, matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) has become a powerful tool for routine identification in many clinical laboratories. We analyzed our 11-year experience in routine identification of clinical isolates (40 months using MALDI-TOF MS and 91 months using conventional phenotypic identification [CPI]). Among the 286,842 clonal isolates, 284,899 isolates of 459 species were identified. The remaining 1,951 isolates were misidentified and required confirmation using a second phenotypic identification for 670 isolates and using a molecular technique for 1,273 isolates of 339 species. MALDI-TOF MS annually identified 112 species, i.e., 36 species/10,000 isolates, compared to 44 species, i.e., 19 species/10,000 isolates, for CPI. Only 50 isolates required second phenotypic identifications during the MALDI-TOF MS period (i.e., 4.5 reidentifications/10,000 isolates) compared with 620 isolates during the CPI period (i.e., 35.2/10,000 isolates). We identified 128 bacterial species rarely reported as human pathogens, including 48 using phenotypic techniques (22 using CPI and 37 using MALDI-TOF MS). Another 75 rare species were identified using molecular methods. MALDI-TOF MS reduced the time required for identification by 55-fold and 169-fold and the cost by 5-fold and 96-fold compared with CPI and gene sequencing, respectively. MALDI-TOF MS was a powerful tool not only for routine bacterial identification but also for identification of rare bacterial species implicated in human infectious diseases. The ability to rapidly identify bacterial species rarely described as pathogens in specific clinical specimens will help us to study the clinical burden resulting from the emergence of these species as human pathogens, and MALDI-TOF MS may be considered an alternative to molecular methods in clinical laboratories.

Gene Sequence-Based Criteria for Identification of New Rickettsia Isolates and Description of Rickettsia heilongjiangensis sp. nov.

ABSTRACT We propose genetic guidelines for the classification of rickettsial isolates at the genus, group, and species levels by using sequences of the 16S rRNA (rrs) gene and four protein-coding genes, the gltA, ompA, and ompB genes and gene D. To be classified as a member of the genus Rickettsia, an isolate should exhibit degrees of rrs and gltA homology with any of the 20 Rickettsia species studied of ≥98.1 and ≥86.5%, respectively. A member of the typhus group should fulfill at least two of the following four criteria: pairwise nucleotide sequence homologies with rrs, gltA, ompB, and gene D of either Rickettsia typhi or Rickettsia prowazekii of ≥99.4, ≥96.6, ≥92.4, and ≥91.6%, respectively. A member of the spotted fever group should either possess the ompA gene or fulfill at least two of the following four criteria: pairwise nucleotide sequence homologies with rrs, gltA, ompB, and gene D of any member of this group of ≥98.8, ≥92.7, ≥85.8, and ≥82.2%, respectively. The existence of a distinct “ancestral” group should be questioned. To be classified as a new Rickettsia species, an isolate should not exhibit more than one of the following degrees of nucleotide similarity with the most homologous validated species: ≥99.8 and ≥ 99.9% for the rrs and gltA genes, respectively, and, when amplifiable, ≥98.8, ≥99.2, and ≥99.3% for the ompA and ompB genes and gene D, respectively. By use of our classification scheme, “Rickettsia heilongjiangii” belongs to a new species for which we officially propose the name Rickettsia heilongjiangensis sp. nov.

Multispacer Typing To Study the Genotypic Distribution of Bartonella henselae Populations

ABSTRACT Bartonella henselae, a worldwide fastidious bacterium, has a feline reservoir and is pathogenic for humans. However, the relationship between human and cat isolates of B. henselae, as well as its population dynamics and geographic heterogeneity, is not fully understood, in part because of the absence of appropriate typing methods. Multilocus sequence typing (MLST), the most discriminatory genotyping method for B. henselae, identified seven genotypes and suggested that human isolates arose from a limited number of cat isolates. Herein, we estimated the discriminatory power of multispacer typing (MST) by studying 126 B. henselae cat isolates from various areas of Europe, Asia, and the United States. We identified the nine most variable intergenic spacers conserved by both B. henselae and Bartonella quintana genomes. By comparing the sequences obtained from these nine spacers for each studied isolate, we identified 39 MST genotypes. The distribution of isolates into MST genotypes matched their phylogenetic organization into four clusters. MST showed that European and Asian isolates were different, in contrast with American isolates, but failed to identify pandemic strains. Our study demonstrated that MST is a powerful method for genotyping B. henselae at the strain level and may serve in studying the population dynamics of this bacterium and understanding the relationships between cat and human isolates. Finally, we provide a free-access MST-Rick online software program (http://ifr48.timone.univ-mrs.fr/MST_BHenselae/mst ) that investigators may use to compare their own MST sequences to our database.

Non-contiguous finished genome sequence and description of Bacillus massiliogorillae sp. nov.

Strain G2T sp. nov. is the type strain of B. massiliogorillae, a proposed new species within the genus Bacillus. This strain, whose genome is described here, was isolated in France from the fecal sample of a wild western lowland gorilla from Cameroon. B. massiliogorillae is a facultative anaerobic, Gram-variable, rod-shaped bacterium. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 5,431,633 bp long genome (1 chromosome but no plasmid) contains 5,179 protein-coding and 98 RNA genes, including 91 tRNA genes.

Role of second-line injectable antituberculosis drugs in the treatment of MDR/XDR tuberculosis

Second-line injectable antituberculosis drugs (aminoglycosides and capreomycin) are the main drugs used in the management of resistant tuberculosis. Their preserved efficacy in the case of multi-drug-resistant bacillus is counterbalanced by frequent adverse events. Adverse events are linked to the drugs themselves and to the recommended 8-month period of parenteral administration. In numerous countries facing a high incidence of multi-drug-resistant tuberculosis, treatment is administered by intramuscular injection. This procedure is painful and restrictive, and therefore treatment adherence is limited. This study reports the follow-up of 11 patients diagnosed with multi-drug- and extensively-resistant tuberculosis and treated with parenteral amikacin, and discusses the role of amikacin in the treatment of resistant tuberculosis.

Miniphocibacter massiliensis gen. nov., sp. nov., a new species isolated from the human gut and its taxono-genomics description

With the aim of describing the human microbiota by the means of culture methods, culturomics was developed in order to target previously un‐isolated bacterial species and describe it via the taxono‐genomics approach. While performing a descriptive study of the human gut microbiota of the pygmy people, strain Marseille‐P4678T has been isolated from a stool sample of a healthy 39‐year‐old pygmy male. Cells of this strain were Gram‐positive cocci, spore‐forming, non‐motile, catalase‐positive and oxidase‐negative, and grow optimally at 37°C under anaerobic conditions. Its 16S rRNA gene sequence exhibited 89.69% of sequence similarity with Parvimonas micra strain 3119BT (NR 036934.1), its phylogenetically closest species with standing in nomenclature. The genome of strain Marseille‐P4678T is 2,083,161 long with 28.26 mol% of G+C content. Based on its phenotypic, biochemical, genotypic and proteomic profile, this bacterium was classified as a new bacterial genus and species Miniphocibacter massiliensis gen. nov., sp. nov. with the type strain Marseille‐P4678T.

‘Bacillus massiliglaciei’, a new bacterial species isolated from Siberian permafrost

We describe here the main characteristics of a new species isolated from Siberian permafrost dated around 10 million years. This species was named ‘Bacillus massiliglaciei’ strain Marseille-P2600T (= CSUR P2600 = DSM 102861).