A lipoglycopeptide antibiotic for Gram-positive biofilm-related infections.

Drug-resistant Gram-positive bacterial infections are still a substantial burden on the public health system, with two bacteria (Staphylococcus aureus and Streptococcus pneumoniae) accounting for over 1.5 million drug-resistant infections in the United States alone in 2017. In 2019, 250,000 deaths were attributed to these pathogens globally. We have developed a preclinical glycopeptide antibiotic, MCC5145, that has excellent potency (MIC90 ≤ 0.06 µg/ml) against hundreds of isolates of methicillin-resistant S. aureus (MRSA) and other Gram-positive bacteria, with a greater than 1000-fold margin over mammalian cell cytotoxicity values. The antibiotic has therapeutic in vivo efficacy when dosed subcutaneously in multiple murine models of established bacterial infections, including thigh infection with MRSA and blood septicemia with S. pneumoniae, as well as when dosed orally in an antibiotic-induced Clostridioides difficile infection model. MCC5145 exhibited reduced nephrotoxicity at microbiologically active doses in mice compared to vancomycin. MCC5145 also showed improved activity against biofilms compared to vancomycin, both in vitro and in vivo, and a low propensity to select for drug resistance. Characterization of drug action using a transposon library bioinformatic platform showed a mechanistic distinction from other glycopeptide antibiotics.

Humanized mice reveal a macrophage-enriched gene signature defining human lung tissue protection during SARS-CoV-2 infection.

The human immunological mechanisms defining the clinical outcome of SARS-CoV-2 infection remain elusive. This knowledge gap is mostly driven by the lack of appropriate experimental platforms recapitulating human immune responses in a controlled human lung environment. Here, we report a mouse model (i.e., HNFL mice) co-engrafted with human fetal lung xenografts (fLX) and a myeloid-enhanced human immune system to identify cellular and molecular correlates of lung protection during SARS-CoV-2 infection. Unlike mice solely engrafted with human fLX, HNFL mice are protected against infection, severe inflammation, and histopathological phenotypes. Lung tissue protection from infection and severe histopathology associates with macrophage infiltration and differentiation and the upregulation of a macrophage-enriched signature composed of 11 specific genes mainly associated with the type I interferon signaling pathway. Our work highlights the HNFL model as a transformative platform to investigate, in controlled experimental settings, human myeloid immune mechanisms governing lung tissue protection during SARS-CoV-2 infection.

In vitro and in vivo methods to study bacterial colonization of hydrogel dermal fillers.

Preclinical in vitro and in vivo methods to study bacterial interactions with dermal fillers and infection pathogenesis are lacking. In this work, first in vitro methods to assess protein biofouling and effective pore size of commercial dermal fillers, including degradable hyaluronic acid (HA)-based fillers and other semi-degradable or permanent fillers (non-HA), were developed. The results were then related to Staphylococcus aureus (S. aureus) adhesion rates in vitro. HA fillers had less protein sorption than non-HA fillers and overall had smaller effective pore sizes. The properties correlated with levels of bacterial adhesion, where the control glass surface had the most rapid increase in bacterial cell adhesion, with a slope of 0.29 cm-2  min-1 , three unique non-HA fillers had intermediate adhesion with slopes of 0.11 and 0.06 cm-2  min-1 , and three unique HA fillers had the least adhesion with slopes of 0.02, 0.02, and 0.01 cm-2  min-1 . S. aureus had greater motility on the HA fillers than on non-HA fillers. Next, a mouse model for dermal filler biofilm and infection was developed. Mice were inoculated with a controlled amount of bioluminescent bacteria (Xen36 S. aureus) and polyacrylamide hydrogels of different stiffness were injected. In vivo bioluminescence was monitored longitudinally for 35 days to ensure that lasting colonization was established. The inoculum was optimized to achieve adequate bioluminescent signal, and bacterial bioburden over time and inter-animal variability in bioburden were determined. These in vitro and in vivo approaches can be used for future studies of antimicrobial interventions for dermal fillers.

Fatal Neurodissemination and SARS-CoV-2 Tropism in K18-hACE2 Mice Is Only Partially Dependent on hACE2 Expression.

Animal models recapitulating COVID-19 are critical to enhance our understanding of SARS-CoV-2 pathogenesis. Intranasally inoculated transgenic mice expressing human angiotensin-converting enzyme 2 under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. We evaluated the clinical and virological dynamics of SARS-CoV-2 using two intranasal doses (104 and 106 PFUs), with a detailed spatiotemporal pathologic analysis of the 106 dose cohort. Despite generally mild-to-moderate pneumonia, clinical decline resulting in euthanasia or death was commonly associated with hypothermia and viral neurodissemination independent of inoculation dose. Neuroinvasion was first observed at 4 days post-infection, initially restricted to the olfactory bulb suggesting axonal transport via the olfactory neuroepithelium as the earliest portal of entry. Absence of viremia suggests neuroinvasion occurs independently of transport across the blood-brain barrier. SARS-CoV-2 tropism was neither restricted to ACE2-expressing cells (e.g., AT1 pneumocytes), nor inclusive of some ACE2-positive cell lineages (e.g., bronchiolar epithelium and brain vasculature). Absence of detectable ACE2 protein expression in neurons but overexpression in neuroepithelium suggest this as the most likely portal of neuroinvasion, with subsequent ACE2 independent lethal neurodissemination. A paucity of epidemiological data and contradicting evidence for neuroinvasion and neurodissemination in humans call into question the translational relevance of this model.

Red blood cell transfusion-induced non-transferrin-bound iron promotes Pseudomonas aeruginosa biofilms in human sera and mortality in catheterized mice.

Transfusion of storage-damaged red blood cells (RBCs) increases non-transferrin-bound iron (NTBI) levels in humans. This can potentially enhance virulence of microorganisms. In this study, Pseudomonas aeruginosa replication and biofilm production in vitro correlated with NTBI levels of transfused subjects (R2 = 0·80; P < 0·0001). Transfusion of stored RBCs into catheterized mice enhanced P. aeruginosa virulence and mortality in vivo, while pre-administration of apotransferrin reduced NTBI levels improving survival (69% vs 27% mortality; P < 0·05). These results suggest that longer RBC storage, by modulating the bioavailability of iron, may increase the risk of P. aeruginosa biofilm-related infections in transfused patients.

Portable bioluminescent platform for in vivo monitoring of biological processes in non-transgenic animals.

Bioluminescent imaging (BLI) is one of the most powerful and widely used preclinical imaging modalities. However, the current technology relies on the use of transgenic luciferase-expressing cells and animals and therefore can only be applied to a limited number of existing animal models of human disease. Here, we report the development of a "portable bioluminescent" (PBL) technology that overcomes most of the major limitations of traditional BLI. We demonstrate that the PBL method is capable of noninvasive measuring the activity of both extracellular (e.g., dipeptidyl peptidase 4) and intracellular (e.g., cytochrome P450) enzymes in vivo in non-luciferase-expressing mice. Moreover, we successfully utilize PBL technology in dogs and human cadaver, paving the way for the translation of functional BLI to the noninvasive quantification of biological processes in large animals. The PBL methodology can be easily adapted for the noninvasive monitoring of a plethora of diseases across multiple species.

Comparison of two fluorescent probes in preclinical non-invasive imaging and image-guided debridement surgery of Staphylococcal biofilm implant infections.

Implant-associated infections are challenging to diagnose and treat. Fluorescent probes have been heralded as a technologic advancement that can improve our ability to non-invasively identify infecting organisms, as well as guide the inexact procedure of surgical debridement. This study's purpose was to compare two fluorescent probes for their ability to localize Staphylococcus aureus biofilm infections on spinal implants utilizing noninvasive optical imaging, then assessing the broader applicability of the more successful probe in other infection animal models. This was followed by real-time, fluorescence image-guided surgery to facilitate debridement of infected tissue. The two probe candidates, a labelled antibiotic that targets peptidoglycan (Vanco-800CW), and the other, a labelled antibody targeting the immunodominant Staphylococcal antigen A (1D9-680), were injected into mice with spine implant infections. Mice were then imaged noninvasively with near infrared fluorescent imaging at wavelengths corresponding to the two probe candidates. Both probes localized to the infection, with the 1D9-680 probe showing greater fidelity over time. The 1D9-680 probe was then tested in mouse models of shoulder implant and allograft infection, demonstrating its broader applicability. Finally, an image-guided surgery system which superimposes fluorescent signals over analog, real-time, tissue images was employed to facilitate debridement of fluorescent-labelled bacteria.

Fatal neuroinvasion and SARS-CoV-2 tropism in K18-hACE2 mice is partially independent on hACE2 expression.

Animal models recapitulating distinctive features of severe COVID-19 are critical to enhance our understanding of SARS-CoV-2 pathogenesis. Transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. The precise mechanisms of lethality in this mouse model remain unclear. Here, we evaluated the spatiotemporal dynamics of SARS-CoV-2 infection for up to 14 days post-infection. Despite infection and moderate pneumonia, rapid clinical decline or death of mice was invariably associated with viral neuroinvasion and direct neuronal injury (including brain and spinal neurons). Neuroinvasion was observed as early as 4 dpi, with virus initially restricted to the olfactory bulb supporting axonal transport via the olfactory neuroepithelium as the earliest portal of entry. No evidence of viremia was detected suggesting neuroinvasion occurs independently of entry across the blood brain barrier. SARS-CoV-2 tropism was not restricted to ACE2-expressing cells (e.g., AT1 pneumocytes), and some ACE2-positive lineages were not associated with the presence of viral antigen (e.g., bronchiolar epithelium and brain capillaries). Detectable ACE2 expression was not observed in neurons, supporting overexpression of ACE2 in the nasal passages and neuroepithelium as more likely determinants of neuroinvasion in the K18-hACE2 model. Although our work incites caution in the utility of the K18-hACE2 model to study global aspects of SARS-CoV-2 pathogenesis, it underscores this model as a unique platform for exploring the mechanisms of SARS-CoV-2 neuropathogenesis that may have clinical relevance acknowledging the growing body of evidence that suggests COVID-19 may result in long-standing neurologic consequences.

Fighting Staphylococcus aureus infections with light and photoimmunoconjugates.

Infections caused by multidrug-resistant Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), are responsible for high mortality and morbidity worldwide. Resistant lineages were previously confined to hospitals but are now also causing infections among healthy individuals in the community. It is therefore imperative to explore therapeutic avenues that are less prone to raise drug resistance compared with today's antibiotics. An opportunity to achieve this ambitious goal could be provided by targeted antimicrobial photodynamic therapy (aPDT), which relies on the combination of a bacteria-specific targeting agent and light-induced generation of ROS by an appropriate photosensitizer. Here, we conjugated the near-infrared photosensitizer IRDye700DX to a fully human mAb, specific for the invariantly expressed staphylococcal antigen immunodominant staphylococcal antigen A (IsaA). The resulting immunoconjugate 1D9-700DX was characterized biochemically and in preclinical infection models. As demonstrated in vitro, in vivo, and in a human postmortem orthopedic implant infection model, targeted aPDT with 1D9-700DX is highly effective. Importantly, combined with the nontoxic aPDT-enhancing agent potassium iodide, 1D9-700DX overcomes the antioxidant properties of human plasma and fully eradicates high titers of MRSA. We show that the developed immunoconjugate 1D9-700DX targets MRSA and kills it upon illumination with red light, without causing collateral damage to human cells.

In vivo Mouse Model of Spinal Implant Infection.

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The purpose of this method is to describe a novel mouse model of spinal implant infection (SII) that was created to provide an inexpensive, rapid, and accurate in vivo tool to test potential therapeutics and treatment strategies for spinal implant infections. In this method, we present a model of posterior-approach spinal surgery in which a stainless-steel k-wire is transfixed into the L4 spinous process of 12-week old C57BL/6J wild-type mice and inoculated with 1 x 103 CFU of a bioluminescent strain of Staphylococcus aureus Xen36 bacteria. Mice are then longitudinally imaged for bioluminescence in vivo on post-operative days 0, 1, 3, 5, 7, 10, 14, 18, 21, 25, 28, and 35. Bioluminescence imaging (BLI) signals from a standardized field of view are quantified to measure in vivo bacterial burden. To quantify bacteria adhering to implants and peri-implant tissue, mice are euthanized and the implant and surrounding soft tissue are harvested. Bacteria are detached from the implant by sonication, cultured overnight and then colony forming units (CFUs) are counted. The results acquired from this method include longitudinal bacterial counts as measured by in vivo S. aureus bioluminescence (mean maximum flux) and CFU counts following euthanasia. While prior animal models of instrumented spine infection have involved invasive, ex vivo tissue analysis, the mouse model of SII presented in this paper leverages noninvasive, real time in vivo optical imaging of bioluminescent bacteria to replace static tissue study. Applications of the model are broad and may include utilizing alternative bioluminescent bacterial strains, incorporating other types of genetically engineered mice to contemporaneously study host immune response, and evaluating current or investigating new diagnostic and therapeutic modalities such as antibiotics or implant coatings.

Novel in vivo mouse model of shoulder implant infection.

BACKGROUND: Animal models are used to guide management of periprosthetic implant infections. No adequate model exists for periprosthetic shoulder infections, and clinicians thus have no preclinical tools to assess potential therapeutics. We hypothesize that it is possible to establish a mouse model of shoulder implant infection (SII) that allows noninvasive, longitudinal tracking of biofilm and host response through in vivo optical imaging. The model may then be employed to validate a targeting probe (1D9-680) with clinical translation potential for diagnosing infection and image-guided débridement. METHODS: A surgical implant was press-fit into the proximal humerus of c57BL/6J mice and inoculated with 2 µL of 1 × 103 (e3), or 1 × 104 (e4), colony-forming units (CFUs) of bioluminescent Staphylococcus aureus Xen-36. The control group received 2 µL sterile saline. Bacterial activity was monitored in vivo over 42 days, directly (bioluminescence) and indirectly (targeting probe). Weekly radiographs assessed implant loosening. CFU harvests, confocal microscopy, and histology were performed. RESULTS: Both inoculated groups established chronic infections. CFUs on postoperative day (POD) 42 were increased in the infected groups compared with the sterile group (P < .001). By POD 14, osteolysis was visualized in both infected groups. The e4 group developed catastrophic bone destruction by POD 42. The e3 group maintained a congruent shoulder joint. Targeting probes helped to visualize low-grade infections via fluorescence. DISCUSSION: Given bone destruction in the e4 group, a longitudinal, noninvasive mouse model of SII and chronic osteolysis was produced using e3 of S aureus Xen-36, mimicking clinical presentations of chronic SII. CONCLUSION: The development of this model provides a foundation to study new therapeutics, interventions, and host modifications.

Preclinical Models and Methodologies for Monitoring Staphylococcus aureus Infections Using Noninvasive Optical Imaging.

In vivo whole-animal optical (bioluminescence and fluorescence) imaging of Staphylococcus aureus infections has provided the opportunity to noninvasively and longitudinally monitor the dynamics of the bacterial burden and ensuing host immune responses in live anesthetized animals. Herein, we describe several different mouse models of S. aureus skin infection, skin inflammation, incisional/excisional wound infections, as well as mouse and rabbit models of orthopedic implant infection, which utilized this imaging technology. These animal models and imaging methodologies provide insights into the pathogenesis of these infections and innate and adaptive immune responses, as well as the preclinical evaluation of diagnostic and treatment modalities. Noninvasive approaches to investigate host-pathogen interactions are extremely important as virulent community-acquired methicillin-resistant S. aureus strains (CA-MRSA) are spreading through the normal human population, becoming more antibiotic resistant and creating a serious threat to public health.

Development of a Staphylococcus aureus reporter strain with click beetle red luciferase for enhanced in vivo imaging of experimental bacteremia and mixed infections.

In vivo bioluminescence imaging has been used to monitor Staphylococcus aureus infections in preclinical models by employing bacterial reporter strains possessing a modified lux operon from Photorhabdus luminescens. However, the relatively short emission wavelength of lux (peak 490 nm) has limited tissue penetration. To overcome this limitation, the gene for the click beetle (Pyrophorus plagiophtalamus) red luciferase (luc) (with a longer >600 emission wavelength), was introduced singly and in combination with the lux operon into a methicillin-resistant S. aureus strain. After administration of the substrate D-luciferin, the luc bioluminescent signal was substantially greater than the lux signal in vitro. The luc signal had enhanced tissue penetration and improved anatomical co-registration with infected internal organs compared with the lux signal in a mouse model of S. aureus bacteremia with a sensitivity of approximately 3 × 104 CFU from the kidneys. Finally, in an in vivo mixed bacterial wound infection mouse model, S. aureus luc signals could be spectrally unmixed from Pseudomonas aeruginosa lux signals to noninvasively monitor the bacterial burden of both strains. Therefore, the S. aureus luc reporter may provide a technological advance for monitoring invasive organ dissemination during S. aureus bacteremia and for studying bacterial dynamics during mixed infections.

Cell-specific image-guided transcriptomics identifies complex injuries caused by ischemic acute kidney injury in mice.

The kidney's inherent complexity has made identifying cell-specific pathways challenging, particularly when temporally associating them with the dynamic pathophysiology of acute kidney injury (AKI). Here, we combine renal cell-specific luciferase reporter mice using a chemoselective luciferin to guide the acquisition of cell-specific transcriptional changes in C57BL/6 background mice. Hydrogen peroxide generation, a common mechanism of tissue damage, was tracked using a peroxy-caged-luciferin to identify optimum time points for immunoprecipitation of labeled ribosomes for RNA-sequencing. Together, these tools revealed a profound impact of AKI on mitochondrial pathways in the collecting duct. In fact, targeting the mitochondria with an antioxidant, ameliorated not only hydrogen peroxide generation, but also significantly reduced oxidative stress and the expression of the AKI biomarker, LCN2. This integrative approach of coupling physiological imaging with transcriptomics and drug testing revealed how the collecting duct responds to AKI and opens new venues for cell-specific predictive monitoring and treatment.

Corrigendum: Elevated Gut Microbiome-Derived Propionate Levels Are Associated With Reduced Sterile Lung Inflammation and Bacterial Immunity in Mice.

[This corrects the article DOI: 10.3389/fmicb.2019.00159.].

Elevated Gut Microbiome-Derived Propionate Levels Are Associated With Reduced Sterile Lung Inflammation and Bacterial Immunity in Mice.

Short-chain fatty acids (SCFA) are important dietary and microbiome metabolites that can have roles in gut immunity as well as further afield. We previously observed that gut microbiome alteration via antibiotics led to attenuated lung inflammatory responses. The rationale for this study was to identify gut microbiome factors that regulate lung immune homeostasis. We first investigated key factors within mouse colonic lumen filtrates (CLF) which could elicit direct inflammatory effects in vitro. We identified lipopolysaccharide (LPS) and SCFAs as key CLF ingredients whose levels and inflammatory capacity changed after antibiotic exposure in mice. Specifically, the SCFA propionate appeared to be a key regulator of LPS responses in vitro. Elevated propionate: acetate ratios, as seen in CLF after antibiotic exposure, strongly blunted inflammatory responses in vitro. In vivo, exposure of lungs to high dose propionate, to mimic how prior antibiotic exposure changed SCFA levels, resulted in diminished immune containment of Staphylococcus aureus pneumonia. Finally, we discovered an enrichment of propionate-producing gut bacteria in mice with reduced lung inflammation following lung ischemia reperfusion injury in vivo. Overall, our data show that propionate levels can distinctly modulate lung immune responses in vitro and in vivo and that gut microbiome increased production of propionate is associated with reduced lung inflammation.

Multimodal imaging guides surgical management in a preclinical spinal implant infection model.

Spine implant infections portend disastrous outcomes, as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. Current imaging modalities can detect anatomical alterations and anomalies but cannot differentiate between infection and aseptic loosening, diagnose specific pathogens, or delineate the extent of an infection. Herein, a fully human monoclonal antibody 1D9, recognizing the immunodominant staphylococcal antigen A on the surface of Staphylococcus aureus, was assessed as a nuclear and fluorescent imaging probe in a preclinical model of S. aureus spinal implant infection, utilizing bioluminescently labeled bacteria to confirm the specificity and sensitivity of this targeting. Postoperative mice were administered 1D9 probe dual labeled with 89-zirconium (89Zr) and a near infrared dye (NIR680) (89Zr-NIR680-1D9), and PET-CT and in vivo fluorescence and bioluminescence imaging were performed. The 89Zr-NIR680-1D9 probe accurately diagnosed both acute and subacute implant infection and permitted fluorescent image-guided surgery for selective debridement of infected tissue. Therefore, a single probe could noninvasively diagnose an infection and facilitate image-guided surgery to improve the clinical management of implant infections.

Mouse model of Gram-negative prosthetic joint infection reveals therapeutic targets.

Bacterial biofilm infections of implantable medical devices decrease the effectiveness of antibiotics, creating difficult-to-treat chronic infections. Prosthetic joint infections (PJI) are particularly problematic because they require prolonged antibiotic courses and reoperations to remove and replace the infected prostheses. Current models to study PJI focus on Gram-positive bacteria, but Gram-negative PJI (GN-PJI) are increasingly common and are often more difficult to treat, with worse clinical outcomes. Herein, we sought to develop a mouse model of GN-PJI to investigate the pathogenesis of these infections and identify potential therapeutic targets. An orthopedic-grade titanium implant was surgically placed in the femurs of mice, followed by infection of the knee joint with Pseudomonas aeruginosa or Escherichia coli. We found that in vitro biofilm-producing activity was associated with the development of an in vivo orthopedic implant infection characterized by bacterial infection of the bone/joint tissue, biofilm formation on the implants, reactive bone changes, and inflammatory immune cell infiltrates. In addition, a bispecific antibody targeting P. aeruginosa virulence factors (PcrV and Psl exopolysaccharide) reduced the bacterial burden in vivo. Taken together, our findings provide a preclinical model of GN-PJI and suggest the therapeutic potential of targeting biofilm-associated antigens.

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer.

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) imaging. Co-mutations in the KRAS proto-oncogene and the LKB1 tumor suppressor gene are frequent events in lung cancer that drive hypermetabolic, glycolytic tumor growth. A critical pathway regulating the growth and metabolism of these tumors is the mechanistic target of the rapamycin (mTOR) pathway, which can be effectively targeted using selective catalytic mTOR kinase inhibitors. The mTOR inhibitor MLN0128 suppresses glycolysis in mice bearing tumors with Kras and Lkb1 co-mutations, referred to as KL mice. The therapy response in KL mice is first measured by 18F-FDG PET and computed tomography (CT) imaging before and after the delivery of MLN0128. By utilizing 18F-FDG PET/CT, researchers are able to measure dynamic changes in the glucose metabolism in genetically engineered mouse models (GEMMs) of lung cancer following a therapeutic intervention with targeted therapies. This is followed by ex vivo autoradiography and a quantitative immunohistochemical (qIHC) analysis using morphometric software. The use of qIHC enables the detection and quantification of distinct changes in the biomarker profiles following treatment as well as the characterization of distinct tumor pathologies. The coupling of PET imaging to quantitative histology is an effective strategy to identify metabolic and therapeutic responses in vivo in mouse models of disease.

Can 3D imaging provide insights into spatial variations in bacterial infection patterns?

Recently developed 3D noninvasive in vivo optical imaging is providing fresh insights into the understanding of the pathogenesis of invasive bacteria in small animal experimental models. Here, we describe the advantages of 3D diffuse light imaging tomography with integrated micro-computed tomography (DLIT-µCT) over more traditional 2D systems, in particular with regard to precise localization of infectious foci within tissues in 3D space. We highlight data from rodent studies that employ experimental infections replicating the course of naturally occurring bacterial disease, such as invasive Escherichia coli infections that arise following colonization of the GI tract in neonatal rats. It is argued that this technology will find increasing utility in the study and diagnosis of infectious disease.