Enterobacterales represent the largest group of bacterial pathogens in humans and are responsible for severe, deep-seated infections, often resulting in sepsis or death. They are also a prominent cause of multidrug-resistant (MDR) infections, and some species are recognized as biothreat pathogens. Tools for noninvasive, whole-body analysis that can localize a pathogen with specificity are needed, but no such technology currently exists. We previously demonstrated that positron emission tomography (PET) with 2-deoxy-2-[18F]fluoro-d-sorbitol (18F-FDS) can selectively detect Enterobacterales infections in murine models. Here, we demonstrate that uptake of 18F-FDS by bacteria occurs via a metabolically conserved sorbitol-specific pathway with rapid in vitro 18F-FDS uptake noted in clinical strains, including MDR isolates. Whole-body 18F-FDS PET/computerized tomography (CT) in 26 prospectively enrolled patients with either microbiologically confirmed Enterobacterales infection or other pathologies demonstrated that 18F-FDS PET/CT was safe, could rapidly detect and localize Enterobacterales infections due to drug-susceptible or MDR strains, and differentiated them from sterile inflammation or cancerous lesions. Repeat imaging in the same patients monitored antibiotic efficacy with decreases in PET signal correlating with clinical improvement. To facilitate the use of 18F-FDS, we developed a self-contained, solid-phase cartridge to rapidly (<10 min) formulate ready-to-use 18F-FDS from commercially available 2-deoxy-2-[18F]fluoro-d-glucose (18F-FDG) at room temperature. In a hamster model, 18F-FDS PET/CT also differentiated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia from secondary Klebsiella pneumoniae pneumonia—a leading cause of complications in hospitalized patients with COVID-19. These data support 18F-FDS as an innovative and readily available, pathogen-specific PET technology with clinical applications.

Background: Intraocular tuberculosis (IOTB), an extrapulmonary manifestation of tuberculosis of the eye, has unique and varied clinical presentations with poorly understood pathogenesis. As it is a significant cause of inflammation and visual morbidity, particularly in TB endemic countries, it is essential to study the pathogenesis of IOTB. Clinical and histopathologic studies suggest the presence of Mycobacterium tuberculosis in retinal pigment epithelium (RPE) cells.Methods: A human retinal pigment epithelium (ARPE-19) cell line was infected with a virulent strain of M. tuberculosis (H37Rv). Electron microscopy and colony forming units (CFU) assay were performed to monitor the M. tuberculosis adherence, invasion, and intracellular replication, whereas confocal microscopy was done to study its intracellular fate in the RPE cells. To understand the pathogenesis, the transcriptional profile of M. tuberculosis in ARPE-19 cells was studied by whole genome microarray. Three upregulated M. tuberculosis transcripts were also examined in human IOTB vitreous samples.Results: Scanning electron micrographs of the infected ARPE-19 cells indicated adherence of bacilli, which were further observed to be internalized as monitored by transmission electron microscopy. The CFU assay showed that 22.7 and 8.4% of the initial inoculum of bacilli adhered and invaded the ARPE-19 cells, respectively, with an increase in fold CFU from 1 dpi (0.84) to 5dpi (6.58). The intracellular bacilli were co-localized with lysosomal-associated membrane protein-1 (LAMP-1) and LAMP-2 in ARPE-19 cells. The transcriptome study of intracellular bacilli showed that most of the upregulated transcripts correspond to the genes encoding the proteins involved in the processes such as adherence (e.g., Rv1759c and Rv1026), invasion (e.g., Rv1971 and Rv0169), virulence (e.g., Rv2844 and Rv0775), and intracellular survival (e.g., Rv1884c and Rv2450c) as well as regulators of various metabolic pathways. Two of the upregulated transcripts (Rv1971, Rv1230c) were also present in the vitreous samples of the IOTB patients.Conclusions: M. tuberculosis is phagocytosed by RPE cells and utilizes these cells for intracellular multiplication with the involvement of late endosomal/lysosomal compartments and alters its transcriptional profile plausibly for its intracellular adaptation and survival. The findings of the present study could be important to understanding the molecular pathogenesis of IOTB with a potential role in the development of diagnostics and therapeutics for IOTB.

Bedaquiline is a promising drug against tuberculosis (TB), but limited data are available on its intralesional pharmacokinetics. Moreover, current techniques rely on invasive tissue resection, which is difficult in humans and generally limited even in animals. In this study, we developed a novel radiosynthesis for 76 Br-bedaquiline and performed noninvasive, longitudinal whole-body positron emission tomography (PET) in live, Mycobacterium tuberculosis-infected mice over 48 h. After the intravenous injection, 76 Br-bedaquiline distributed to all organs and selectively localized to adipose tissue and liver, with excellent penetration into infected lung lesions (86%) and measurable penetration into the brain parenchyma (15%). Ex vivo high resolution, two-dimensional autoradiography, and same section hematoxylin/eosin and immunofluorescence provided detailed intralesional drug biodistribution. PET bioimaging and high-resolution autoradiography are novel techniques that can provide detailed, multicompartment, and intralesional pharmacokinetics of new and existing TB drugs. These technologies can significantly advance efforts to optimize drug dosing.