Literature related to antimicrobial resistant (AMR) bacteria in exotic pets is minimal, being essential to report objective data on this topic, which represents a therapeutic challenge for veterinary medicine and public health. Between 2016 and 2020, laboratory records of 3156 exotic pet specimens’ microbiological diagnoses and antibiotic susceptibility testing (AST) results were examined. The samples were classified into three animal classes: birds (n = 412), mammalia (n = 2399), and reptilian (n = 345). The most prevalent bacteria in birds and mammals were Staphylococcus spp. (15% and 16%), while in reptiles they were Pseudomonas spp. (23%). Pseudomonas was the genus with the highest levels of AMR in all animal groups, followed by Enterococcus spp. By contrast, Gram-positive cocci and Pasteurella spp. were the most sensitive bacteria. Moreover, in reptiles, Stenotrophomonas spp., Morganella spp., and Acinetobacter spp. presented high levels of AMR. Multidrug-resistant (MDR) bacteria were isolates from reptiles (21%), birds (17%), and mammals (15%). The Enterobacterales had the highest MDR levels: S. marcescens (94.4%), C. freundii (50%), M. morganii (47.4%), K. pneumoniae (46.6%), E. cloacae (44%), and E. coli (38.3%). The prevalence of MDR P. aeruginosa strains was 8%, detecting one isolate with an XDR profile. Regarding antimicrobial use, many antibiotics described as critically important for human use had significant AMR prevalence in bacteria isolated from exotic pets. Under the One-Health approach, these results are alarming and of public health concern since potential transmission of AMR bacteria and genes can occur from exotic pets to their owners in both senses. For this reason, the collaboration between veterinarians and public health professionals is crucial.

Light-sheet fluorescence microscopy has revolutionized biology by visualizing dynamic cellular processes in three dimensions. However, light scattering in thick tissue and photobleaching of fluorescent reporters limit this method to studying thin or translucent specimens. In this study, we applied nondiffractive ultrasound beams in conjunction with a cross-amplitude modulation sequence and nonlinear acoustic reporters to enable fast and volumetric imaging of targeted biological functions. We reported volumetric imaging of tumor gene expression at the cubic centimeter scale using genetically encoded gas vesicles and localization microscopy of cerebral capillary networks using intravascular microbubble contrast agents. Nonlinear sound-sheet microscopy provides a ~64× acceleration in imaging speed, ~35× increase in imaged volume, and ~4× increase in classical imaging resolution compared with the state of the art in biomolecular ultrasound.

The development of fully noninvasive, transcranial functional ultrasound (fUS) would increase the translatability and clinical potential of this neuroimaging modality. Unfortunately, transcranial fUS is hindered by skull-induced aberrations which degrade power Doppler image quality and lower sensitivity. As a result, a majority of fUS imaging studies rely on craniotomies or acoustically transparent cranial windows. To advance fUS technology further, we present an adaptive aberration correction method based on ray-tracing through 4 tissue layers (transducer lens, gel & skin, bone and brain tissue). Our method segments these layers, and estimates ultrasound wave speeds in each layer iteratively. Once a velocity model of the imaging plane of interest is retrieved, ultrafast power Doppler imaging of the brain is performed using a ray-tracing beamformer that accounts for wave refraction. We tested our method in three adult rats, and estimated wave speeds for the skin/gel layer (1628±7m/s), skull bone (3247±110m/s), and brain tissue (1526 ± 55m/s). After aberration correction, we measured an average adult rat skull thickness of 388±41 μm in agreement with anatomical records. The largest improvements in Doppler imaging quality were observed in cortical brain layers adjacent to the skull, specifically lateral spatial resolution was improved by 32%. Our method consistently outperformed Doppler imaging based on traditional delay-and-sum (DAS) beamforming, which assumes a uniform sound speed.

Genetically encoded pH sensors based on fluorescent proteins enable dynamic optical imaging of cellular processes such as endocytosis and exocytosis. To date, light scattering in thick tissue as well as photobleaching of fluorescent proteins prevent deep cellular imaging over sustained periods of time. To visualize intracellular pH variations across opaque organs, we introduce a genetically encoded acoustic pH sensor dubbed ‘pHonon’. We modified the outer gas vesicle protein (GvpC) of echogenic protein nanostructures via histidine point mutations. At low pH, engineered gas vesicles exhibit an increased shell stiffness which switched their acoustic response from nonlinear to linear. By combining pHonons with nonlinear ultrasound imaging, we captured dynamic deep tissue images of lysosomal acidification by macrophages in murine liver. The combination of pHonon with nonlinear ultrasound creates the possibility for basic studies of endo- and exocytic activity in deep tissue of living opaque organisms.

Gas vesicles (GVs) are air-filled protein nanostructures (∼85 nm diameter, ∼500 nm length) with the physical property to scatter sound. This new class of contrast agent serves as an acoustic analog to green fluorescent proteins and enables ultrasound imaging of cells that have been genetically labelled with GVs. To date, methods to produce GVs rely on expensive CO ₂ shakers, limiting accessibility and scalability. In this study, we present a cost-effective and scalable protocol to produce GVs using an adapted bubble column photobioreactor design. This production method operates at approximately 10% of the cost of the state-of-the-art, while utilizing off-the-shelf components for broader accessibility and dissemination. We characterized GVs produced with both photobioreactor and shaker-incubator production methods using hydrostatic collapse pressure measurements, hydrodynamic diameter measurements, and ultrasound imaging. Our results demonstrate that GVs produced with both methods exhibit identical physicochemical properties, ensuring intercompatibility. In summary, this new protocol to produce GVs lowers the barrier to producing GVs in research labs, thereby creating the possibility of a broader use of GVs as ultrasound contrast agents and biosensors for a wide array of biomedical applications.

Functional ultrasound (fUS) is a sensitive neuroimaging technique that uses high frame rate ultrasound to monitor brain hemo-dynamics as a proxy for neural activity. Recent studies have demonstrated its potential for brain-machine interfaces (BMIs) in both primates and humans. However, current 2D fUS approaches are limited to a single brain slice, restricting the ability to decode widespread neural activity. While 3D fUS could overcome this, it demands high data throughput, increased computation, and higher temperature increases due to the requirement of a higher number of transmitted waves. To address this, we present selective-plane fUS, a method that leverages the wide field of view of row-column addressed (RCA) transducer arrays to capture activity in targeted brain regions without moving the probe. By electronically selecting imaging planes, this approach achieves higher spatio-temporal resolution with lower data and pulse repetition rate compared to 3D fUS, while preserving sensitivity to neurovascular signals. Our pipeline begins with a 3D functional activation scan to guide plane selection, followed by high frame rate focused wave (FW) imaging in coronal or sagittal slices. This method offers robust detection of visually evoked responses in rodents and reduces signal variability compared to 3D fUS. By imaging only functional regions of interest, selective-plane fUS cuts computational load by an order of magnitude, enables continuous 1000 Hz recordings, and reduces functional signal variability fivefold. We envision that this method will allow tailored continuous functional imaging of widespread neuronal activity in the human brain in a BMI context.