Engineered Mycobacteriophage TM4::GeNL Rapidly Determines Bedaquiline, Pretomanid, Linezolid, Rifampicin, and Clofazimine Sensitivity in Mycobacterium tuberculosis Clinical Isolates.

BACKGROUND: Drug-resistant tuberculosis is a growing public health threat, and early characterization of the resistance phenotype is essential for guiding treatment and mitigating the high mortality associated with the disease. However, the slow growth rate of Mycobacterium tuberculosis, the causative agent of tuberculosis, necessitates several weeks for conventional culture-dependent drug susceptibility testing (DST). In addition, there are no widely available molecular diagnostic assays for evaluating resistance to newer tuberculosis drugs or drugs with complex resistance mechanisms. METHODS: We have developed a luciferase-based reporter mycobacteriophage assay that can determine drug resistance within 48 hours. We engineered the TM4 mycobacteriophage to express green enhanced nanoluciferase (GeNL) cassette and optimized DST for bedaquiline, pretomanid, linezolid, clofazimine, and rifampicin using clinical M. tuberculosis isolates. RESULTS: To assess the feasibility of this assay, we conducted a proof-of-principle study using 53 clinical M. tuberculosis isolates. TM4::GeNL phage DST effectively distinguished between sensitive and resistant isolates for bedaquiline and rifampicin at a concentration of 0.125 µg/mL. Optimal differentiation between sensitive and resistant isolates for pretomanid, clofazimine, and linezolid was achieved at concentrations of 0.5 µg/mL, 0.25 µg/mL, and 1 µg/mL, respectively. Additionally, TM4::GeNL DST identified low-level rifampicin resistance in clinical isolates even though they were classified as sensitive by Mycobacteria Growth Indicator Tube DST. CONCLUSIONS: TM4::GeNL reporter phage DST offers a rapid method to identify M. tuberculosis drug resistance, including resistance to newer tuberculosis drugs.

Neisseria oralis sp. nov., isolated from healthy gingival plaque and clinical samples.

A polyphasic analysis was undertaken of seven independent isolates of gram-negative cocci collected from pathological clinical samples from New York, Louisiana, Florida and Illinois and healthy subgingival plaque from a patient in Virginia, USA. The 16S rRNA gene sequence similarity among these isolates was 99.7-100 %, and the closest species with a validly published name was Neisseria lactamica (96.9 % similarity to the type strain). DNA-DNA hybridization confirmed that these isolates are of the same species and are distinct from their nearest phylogenetic neighbour, N. lactamica. Phylogenetic analysis of 16S and 23S rRNA gene sequences indicated that the novel species belongs in the genus Neisseria. The predominant cellular fatty acids were C16 : 0, summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH) and C18 : 1ω7c. The cellular fatty acid profile, together with other phenotypic characters, further supports the inclusion of the novel species in the genus Neisseria. The name Neisseria oralis sp. nov. (type strain 6332(T)  = DSM 25276(T)  = LMG 26725(T)) is proposed.

Psychrobacter sanguinis sp. nov., recovered from four clinical specimens over a 4-year period.

An analysis of 16S rRNA gene sequences from archived clinical reference specimens identified a novel species of the genus Psychrobacter, of which four strains have been independently isolated from human blood. On the basis of 16S rRNA gene sequence similarity, the closest relatives with validly published names were Psychrobacter arenosus R7(T) (98.7%), P. pulmonis CECT 5989(T) (97.7%), P. faecalis Iso-46(T) (97.6%) and P. lutiphocae IMMIB L-1110(T) (97.2%). Maximum-likelihood phylogenetic analysis of 16S rRNA gene sequences showed that the isolates belonged to the genus Psychrobacter and were members of a cluster associated with Psychrobacter sp. PRwf-1, isolated from a silk snapper fish. DNA-DNA relatedness and partial 23S rRNA gene sequences also supported the finding that the isolates belonged to a species distinct from its closest phylogenetic neighbours. The predominant cellular fatty acids were C(18:1)ω9c, C(16:0), summed feature 3 (C(16:1)ω7c and/or iso-C(15:0) 2-OH), summed feature 5 (C(18:2)ω6,9c and/or anteiso-C(18:0)) and C(18:0). Biochemical and morphological analysis further supported the assignment of the four isolates to a novel species. The name Psychrobacter sanguinis sp. nov. is proposed. The type strain is 13983(T) (=DSM 23635(T)=CCUG 59771(T)).

Neisseria wadsworthii sp. nov. and Neisseria shayeganii sp. nov., isolated from clinical specimens.

An analysis of 16S rRNA gene sequences from archived clinical reference specimens has identified two novel Neisseria species. For each species, two strains from independent sources were identified. Amongst species with validly published names, the closest species to the newly identified organisms were Neisseria canis, N. dentiae, N. zoodegmatis, N. animaloris and N. weaveri. DNA-DNA hybridization studies demonstrated that the newly identified isolates represent species that are distinct from these nearest neighbours. Analysis of partial 23S rRNA gene sequences for the newly identified strains and their nearest neighbours provided additional support for the species designation. Bayesian analysis of 16S rRNA gene sequences suggested that the newly identified isolates belong to distinct but related species of the genus Neisseria, and are members of a clade that includes N. dentiae, N. bacilliformis and N. canis. The predominant cellular fatty acids [16 : 0, summed feature 3 (16 : 1ω7c and/or iso-15 : 0 2-OH) and 18 : 1ω7c], as well as biochemical and morphological analyses further support the designation of Neisseria wadsworthii sp. nov. (type strain 9715(T) =DSM 22247(T) =CIP 109934(T)) and Neisseria shayeganii sp. nov. (type strain 871(T) =DSM 22246(T) =CIP 109933(T)).