Highly Efficient Photodynamic Hydrogel with AIE-Active Photosensitizers toward Methicillin-Resistant Staphylococcus aureus Ultrafast Imaging and Killing.

Methicillin-resistant Staphylococcus aureus (MRSA) causes great health hazards to society because most antibiotics are ineffective. Photodynamic treatment (PDT) has been proposed to combat MRSA due to the advantage of imaging-guided no-drug resistance therapy. However, the traditional photosensitizers for PDT are limited by aggregation-caused quenching for imaging and low photodynamic antibacterial efficiency. In this work, we synthesize a new aggregation-induced emission (AIE) photosensitizer (APNO), which can ultrafast distinguish between Gram-positive and Gram-negative bacteria within 3 s by AIE-active photosensitizer imaging. Meanwhile, APNO can generate antibacterial reactive oxygen species under light irradiation, which holds potential for antibacterial PDT. Then, APNO is loaded by PHEAA hydrogel to obtain a highly efficient photodynamic hydrogel (APNO@gel). In vitro results show complete inhibition of MRSA by APNO@gel under lower-power light irradiation. Transcriptome analysis is performed to investigate antibacterial mechanism of APNO@gel. Most importantly, APNO@gel also exhibits significant inhibition and killing ability of MRSA in the MRSA wound infection model, which will further promote rapid wound healing. Therefore, the photodynamic hydrogel provides a promising strategy toward MRSA ultrafast imaging and killing.

Radiosynthesis and Bioevaluation of 99mTc-Labeled Isocyanide Ubiquicidin 29-41 Derivatives as Potential Agents for Bacterial Infection Imaging.

To develop a novel 99mTc-labeled ubiquicidin 29-41 derivative for bacterial infection single-photon emission computed tomography (SPECT) imaging with improved target-to-nontarget ratio and lower nontarget organ uptake, a series of isocyanide ubiquicidin 29-41 derivatives (CNnUBI 29-41, n = 5-9) with different carbon linkers were designed, synthesized and radiolabeled with the [99mTc]Tc(I)+ core, [99mTc][Tc(I)(CO)3(H2O)3]+ core and [99mTc][Tc(V)N]2+ core. All the complexes are hydrophilic, maintain good stability and specifically bind Staphylococcus aureus in vitro. The biodistribution in mice with bacterial infection and sterile inflammation demonstrated that [99mTc]Tc-CN5UBI 29-41 was able to distinguish bacterial infection from sterile inflammation, which had an improved abscess uptake and a greater target-to-nontarget ratio. SPECT imaging study of [99mTc]Tc-CN5UBI 29-41 in bacterial infection mice showed that there was a clear accumulation in the infection site, suggesting that this radiotracer could be a potential radiotracer for bacterial infection imaging.

Findaureus: An open-source application for locating Staphylococcus aureus in fluorescence-labelled infected bone tissue slices.

Staphylococcus aureus (S. aureus) is a facultative pathogenic bacterium that can cause infections in various tissue types in humans. Fluorescence imaging techniques have been employed to visualize the bacteria in ex-vivo samples mostly in research, aiding in the understanding of the etiology of the pathogen. However, the multispectral images generated from fluorescence microscopes are complex, making it difficult to locate bacteria across image files, especially in consecutive planes with different imaging depths. To address this issue, we present Findaureus, an open-source application specifically designed to locate and extract critical information about bacteria, especially S. aureus. Due to the limited availability of datasets, we tested the application on a dataset comprising fluorescence-labelled infected mouse bone tissue images, achieving an accuracy of 95%. We compared Findaureus with other state-of-the-art image analysis tools and found that it performed better, given its specificity toward bacteria localization. The proposed approach has the potential to aid in medical research of bone infections and can be extended to other tissue and bacteria types in the future.

Longitudinal intravital imaging of the bone marrow for analysis of the race for the surface in a murine osteomyelitis model.

Critical knowledge gaps of orthopedic infections pertain to bacterial colonization. The established dogma termed the Race for the Surface posits that contaminating bacteria compete with host cells for the implant post-op, which remains unproven without real-time in vivo evidence. Thus, we modified the murine longitudinal intravital imaging of the bone marrow (LIMB) system to allow real-time quantification of green fluorescent protein (GFP+) host cells and enhanced cyan fluorescent protein (ECFP+) or red fluorescent protein (RFP+) methicillin-resistant Staphylococcus aureus (MRSA) proximal to a transfemoral implant. Following inoculation with ~105 CFU, an L-shaped metal implant was press-fit through the lateral cortex at a 90° angle ~0.150 mm below a gradient refractive index (GRIN) lens. We empirically derived a volume of interest (VOI) = 0.0161 ± 0.000675 mm3 during each imaging session by aggregating the Z-stacks between the first (superior) and last (inferior) in-focus LIMB slice. LIMB postimplantation revealed very limited bacteria detection at 1 h, but by 3 h, 56.8% of the implant surface was covered by ECFP+ bacteria, and the rest were covered by GFP+ host cells. 3D volumetric rendering of the GFP+ and ECFP+ or RFP+ voxels demonstrated exponential MRSA growth between 3 and 6 h in the Z-plane, which was validated with cross-sectional ex vivo bacterial burden analyses demonstrating significant growth by ~2 × 104 CFU/h on the implant from 2 to 12 h post-op (p < 0.05; r2 > 0.98). Collectively, these results show the competition at the surface is completed by 3 h in this model and demonstrate the potential of LIMB to elucidate mechanisms of bacterial colonization, the host immune response, and the efficacy of antimicrobials.

Elevated 68 Ga-FAPI-04 Activity Due to Staphylococcus aureus Intracranial Infection.

ABSTRACT: A 37-year-old man was admitted to our hospital after experiencing syncope. An MRI of his head revealed multiple foci of abnormal signal, which could potentially be metastases. The patient then underwent a 68 Ga-FAPI-04 PET/MRI examination, which revealed multiple FAPI-avid foci in the brain. Upon analyzing the cerebrospinal fluid, it was found that the patient had a Staphylococcus aureus infection. The results suggest that 68 Ga-FAPI-04 PET has the potential to be a valuable tool in the visualization of intracranial infectious lesions.

Evaluating the Performance of Pathogen-Targeted Positron Emission Tomography Radiotracers in a Rat Model of Vertebral Discitis-Osteomyelitis.

BACKGROUND: Vertebral discitis-osteomyelitis (VDO) is a devastating infection of the spine that is challenging to distinguish from noninfectious mimics using computed tomography and magnetic resonance imaging. We and others have developed novel metabolism-targeted positron emission tomography (PET) radiotracers for detecting living Staphylococcus aureus and other bacteria in vivo, but their head-to-head performance in a well-validated VDO animal model has not been reported. METHODS: We compared the performance of several PET radiotracers in a rat model of VDO. [11C]PABA and [18F]FDS were assessed for their ability to distinguish S aureus, the most common non-tuberculous pathogen VDO, from Escherichia coli. RESULTS: In the rat S aureus VDO model, [11C]PABA could detect as few as 103 bacteria and exhibited the highest signal-to-background ratio, with a 20-fold increased signal in VDO compared to uninfected tissues. In a proof-of-concept experiment, detection of bacterial infection and discrimination between S aureus and E coli was possible using a combination of [11C]PABA and [18F]FDS. CONCLUSIONS: Our work reveals that several bacteria-targeted PET radiotracers had sufficient signal to background in a rat model of S aureus VDO to be potentially clinically useful. [11C]PABA was the most promising tracer investigated and warrants further investigation in human VDO.

A metal-organic framework-based fluorescence resonance energy transfer nanoprobe for highly selective detection of Staphylococcus Aureus.

Survival and infection of pathogenic bacteria, such as Staphylococcus aureus (S. aureus), pose a serious threat to human health. Efficient methods for recognizing and quantifying low levels of bacteria are imperiously needed. Herein, we introduce a metal-organic framework (MOF)-based fluorescence resonance energy transfer (FRET) nanoprobe for ratiometric detection of S. aureus. The nanoprobe utilizes blue-emitting 7-hydroxycoumarin-4-acetic acid (HCAA) encapsulated inside zirconium (Zr)-based MOFs as the energy donor and green-emitting fluorescein isothiocyanate (FITC) as the energy acceptor. Especially, vancomycin (VAN) is employed as the recognition moiety to bind to the cell wall of S. aureus, leading to the disassembly of VAN-PEG-FITC from MOF HCAA@UiO-66. As the distance between the donor and acceptor increases, the donor signal correspondingly increases as the FRET signal decreases. By calculating the fluorescence intensity ratio, S. aureus can be quantified with a dynamic range of 1.05 × 103-1.05 × 107 CFU mL-1 and a detection limit of 12 CFU mL-1. Due to the unique high affinity of VAN to S. aureus, the nanoprobe shows high selectivity and sensitivity to S. aureus, even in real samples like lake water, orange juice, and saliva. The FRET-based ratiometric fluorescence bacterial detection method demonstrated in this work has a prospect in portable application and may reduce the potential threat of pathogens to human health.

Imaging of bacterial infection: Harnessing positron emission tomography and Cherenkov luminescence imaging with UBI-derived octapeptide.

Noninvasive imaging techniques for the early detection of infections are in high demand. In this study, we present the development of an infection imaging agent consisting of the antimicrobial peptide fragment UBI (31-38) conjugated to the chelator 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), which allows for labeling with the positron emitter Ga-68. The preclinical evaluation of [68 Ga]Ga-NODAGA-UBI (31-38) was conducted to investigate its potential for imaging bacterial infections caused by Staphylococcus aureus. The octapeptide derived from ubiquicidin, UBI (31-38), was synthesized and conjugated with the chelator NODAGA. The conjugate was then radiolabeled with Ga-68. The radiolabeling process and the stability of the radio formulation were confirmed through chromatography. The study included both in vitro evaluations using S. aureus and in vivo evaluations in an animal model of infection and inflammation. Positron emission tomography (PET) and Cherenkov luminescence imaging (CLI) were performed to visualize the targeted localization of the radio formulation at the site of infection. Ex vivo biodistribution studies were carried out to quantify the uptake of the radio formulation in different organs and tissues. Additionally, the uptake of [18 F]Fluorodeoxyglucose ([18 F] FDG) in the animal model was also studied for comparison. The [68 Ga]Ga-NODAGA-UBI (31-38) complex consistently exhibited high radiochemical purity (>90%) after formulation. The complex demonstrated stability in saline, phosphate-buffered saline, and human serum for up to 3 h. Notably, the complex displayed significantly higher uptake in S. aureus, which was inhibited in the presence of unconjugated UBI (29-41) peptide, confirming the specificity of the formulation for bacterial membranes. Bacterial imaging capability was also observed in PET and CLI images. Biodistribution results indicated a substantial target-to-nontarget ratio of approximately 4 at 1 h postinjection of the radio formulation. Conversely, the uptake of [18 F]FDG in the animal model did not allow for the discrimination of infected and inflamed sites. Our studies have demonstrated that [68 Ga]Ga-NODAGA-UBI (31-38) holds promise as a radiotracer for imaging bacterial infections caused by S. aureus.

Predictors of infectious foci on FDG PET/CT in Staphylococcus aureus bacteremia.

We looked for predicting factors for the detection of infectious foci on 18F-fluorodeoxyglucose-positron emission tomography in combination with computed tomography (FDG PET/CT) among patients with Staphylococcus aureus bacteremia (SAB) who participated in an interventional study that was conducted at Rambam Health Care Campus, between July 1, 2015 and February 1, 2019. The primary outcome was an infectious focus detected by FDG PET/CT. Independent predictors for detection of focal infection were identified using univariate followed by a logistic regression multivariate analysis. We included 149 patients with 151 separate episodes of SAB who underwent FDG-PET/CT. Focal infections were detected in 107 patients (70.8%). Independent predictors for focal infection detection were community acquisition of bacteremia with odds ratio (OR) 3.03 [95% confidence interval (CI) 1.04-8.77], p-0.042 and C reactive protein (CRP) with OR 1.09 [95% CI 1.04-1.14], p < 0.001. Primary bacteremia was inversely associated with focal infection detection with OR 0.27 [0.10-0.69], p = 0.007, as were the pre-scan blood glucose levels OR 0.9 [0.98-0.99], p-0.004. The latter stayed significant in the subgroup of patients with diabetes mellitus. To conclude, patients with community-acquired bacteremia or high CRP levels should be carefully investigated for focal infection. Patients who present with primary bacteremia seem to be at low risk for focal infection.

In vivo behavior of technetium-99m labeled ibuprofen in infection and inflamation animal models.

OBJECTIVE: The objective of this study was to develop radiolabeled ibuprofen (99mTc-ibu) for imaging and discrimination of inflammation and infection and compare its biodistribution in two different animal models. SIGNIFICANCE: The development of radiolabeled ibuprofen as an imaging agent for inflammation and infection may have significant clinical implications for the diagnosis and management of various inflammatory and infectious diseases. This study provides a promising approach to the detection of sterile infections. METHODS: Ibuprofen was radiolabeled with 99mTc using the stannous chloride method with a yield of 99.05 ± 0.83% (n = 5). The in vivo biological behavior of radiolabeled ibuprofen was determined in Wistar albino rat models of sterile inflammation and bacterial infection with Staphylococcus aureus gram-positive bacteria. Biodistribution studies were carried out at different time points, and the results were compared between the two animal models. RESULTS: The uptake of 99mTc-ibu in sterile inflammation sites at all time points was higher than that in the infection sites. This suggests that 99mTc-ibu can be used to discriminate between sterile inflammation and bacterial infection. CONCLUSIONS: The results of this study suggest that the detection of sterile infections with 99mTc-ibu is possible and highly encouraging.

A comparative 18F-FDG and an anti-PD-L1 probe PET/CT imaging of implant-associated Staphylococcus aureus osteomyelitis.

Background: Early and accurate diagnosis of infection-induced osteomyelitis, which often involves increased PD-L1 expression, is crucial for better treatment outcomes. Radiolabeled anti-PD-L1 nuclear imaging allows for sensitive and non-invasive whole-body assessments of PD-L1 expression. This study aimed to compare the efficacy of 18F-FDG and an 18F-labeled PD-L1-binding peptide probe (18F-PD-L1P) in PET imaging of implant-associated Staphylococcus aureus osteomyelitis (IAOM). Methods: In this study, we synthesized an anti-PD-L1 probe and compared its efficacy with 18F-FDG and 18F-PD-L1P in PET imaging of implant-associated Staphylococcus aureus osteomyelitis (IAOM). The %ID/g ratios (i.e., radioactivity ratios between the infected and non-infected sides) of both probes were evaluated for sensitivity and accuracy in post-infected 7-day tibias and post-infected 21 days, and the intensity of 18F-PD-L1P uptake was compared with pathological changes measured by PD-L1 immunohistochemistry (IHC). Results: Compared with 18F-FDG, 18F-PDL1P demonstrated higher %ID/g ratios for both post-infected 7-day tibias (P=0.001) and post-infected 21 days (P=0.028). The intensity of 18F-PD-L1P uptake reflected the pathological changes of osteomyelitic bones. In comparison to 18F-FDG, 18F-PDL1P provides earlier and more sensitive detection of osteomyelitis caused by S. aureus. Conclusion: Our findings suggest that the 18F-PDL1P probe is a promising tool for the early and accurate detection of osteomyelitis caused by S. aureus.

Endocarditis Presenting as Right Atrial Mass, Diagnosed with 18F-Fluorodeoxyglucose-PET: A Case Report.

We report a case of a 55-year-old male with a history of methicillin-resistant staphylococcus aureus bacteremia whose initial transesophageal echocardiography revealed a cardiac mass attached to the right atrium. Because of the uncommon location of the mass, 18F-fluorodeoxyglucose-PET was used to confirm the diagnosis of infective endocarditis.

Positron Emission Tomography-Computed Tomography and Magnetic Resonance Imaging Assessments in a Mouse Model of Implant-Related Bone and Joint Staphylococcus aureus Infection.

Osteomyelitis is an infection of the bone, associated with an inflammatory process. Imaging plays an important role in establishing the diagnosis and the most appropriate patient management. However, data are lacking regarding the use of preclinical molecular imaging techniques to assess osteomyelitis progression in experimental models. This study aimed to compare structural and molecular imaging to assess disease progression in a mouse model of implant-related bone and joint infections caused by Staphylococcus aureus. In SWISS mice, the right femur was implanted with a resorbable filament impregnated with S. aureus (infected group, n = 10) or sterile culture medium (uninfected group, n = 6). Eight animals (5 infected, 3 uninfected) were analyzed with magnetic resonance imaging (MRI) at 1, 2, and 3 weeks postintervention, and 8 mice were analyzed with [18F]fluorodeoxyglucose (FDG)-positron emission tomography (PET)-computed tomography (CT) at 48 h and at 1, 2, and 3 weeks postintervention. In infected animals, CT showed bone lesion progression, mainly in the distal epiphysis, although some uninfected animals presented evident bone sequestra at 3 weeks. MRI showed a lesion in the articular area that persisted for 3 weeks in infected animals. This lesion was smaller and less evident in the uninfected group. At 48 h postintervention, FDG-PET showed higher joint uptake in the infected group than in the uninfected group (P = 0.025). Over time, the difference between groups increased. These results indicate that FDG-PET imaging was much more sensitive than MRI and CT for differentiating between infection and inflammation at early stages. FDG-PET clearly distinguished between infection and postsurgical bone healing (in uninfected animals) from 48 h to 3 weeks after implantation. IMPORTANCE Our results encourage future investigations on the utility of the model for testing different therapeutic procedures for osteomyelitis.

Positive Impact of [18F]FDG-PET/CT on Mortality in Patients With Staphylococcus aureus Bacteremia Explained by Immortal Time Bias.

BACKGROUND: Several studies have suggested that in patients with Staphylococcus aureus bacteremia (SAB) [18F] fluorodeoxyglucose positron emission tomography/computed tomography ([18F]FDG-PET/CT) improves outcome. However, these studies often ignored possible immortal time bias. METHODS: Prospective multicenter cohort study in 2 university and 5 non-university hospitals, including all patients with SAB. [18F]FDG-PET/CT was performed on clinical indication as part of usual care. Primary outcome was 90-day all-cause mortality. Effect of [18F]FDG-PET/CT was modeled with a Cox proportional hazards model using [18F]FDG-PET/CT as a time-varying variable and corrected for confounders for mortality (age, Charlson score, positive follow-up cultures, septic shock, and endocarditis). Secondary outcome was 90-day infection-related mortality (assessed by adjudication committee) using the same analysis. In a subgroup-analysis, we determined the effect of [18F]FDG-PET/CT in patients with high risk of metastatic infection. RESULTS: Of 476 patients, 178 (37%) underwent [18F]FDG-PET/CT. Day-90 all-cause mortality was 31% (147 patients), and infection-related mortality was 17% (83 patients). The confounder adjusted hazard ratio (aHR) for all-cause mortality was 0.50 (95% confidence interval [CI]: .34-.74) in patients that underwent [18F]FDG-PET/CT. Adjustment for immortal time bias changed the aHR to 1.00 (95% CI .68-1.48). Likewise, after correction for immortal time bias, [18F]FDG-PET/CT had no effect on infection-related mortality (cause specific aHR 1.30 [95% CI .77-2.21]), on all-cause mortality in patients with high-risk SAB (aHR 1.07 (95% CI .63-1.83) or on infection-related mortality in high-risk SAB (aHR for 1.24 [95% CI .67-2.28]). CONCLUSIONS: After adjustment for immortal time bias [18F]FDG-PET/CT was not associated with day-90 all-cause or infection-related mortality in patients with SAB.

Imaging Diverse Pathogenic Bacteria In Vivo with 18F-Fluoromannitol PET.

Infectious disease remains the main cause of morbidity and mortality throughout the world. Of growing concern is the rising incidence of multidrug-resistant bacteria, derived from various selection pressures. Many of these bacterial infections are hospital-acquired and have prompted the Centers for Disease Control and Prevention in 2019 to reclassify several pathogens as urgent threats, its most perilous assignment. Consequently, there is an urgent need to improve the clinical management of bacterial infection via new methods to specifically identify bacteria and monitor antibiotic efficacy in vivo. In this work, we developed a novel radiopharmaceutical, 2-18F-fluoro-2-deoxy-mannitol (18F-fluoromannitol), which we found to specifically accumulate in both gram-positive and gram-negative bacteria but not in mammalian cells in vitro or in vivo. Methods: Clinical isolates of bacteria were serially obtained from wounds of combat service members for all in vitro and in vivo studies. Bacterial infection was quantified in vivo using PET/CT, and infected tissue was excised to confirm radioactivity counts ex vivo. We used these same tissues to confirm the presence of bacteria by extracting and correlating radioactive counts with colony-forming units of bacteria. Results: 18F-fluoromannitol was able to differentiate sterile inflammation from Staphylococcus aureus and Escherichia coli infections in vivo in a murine myositis model using PET imaging. Our study was extended to a laceration wound model infected with Acinetobacter baumannii, an important pathogen in the nosocomial and battlefield setting. 18F-fluoromannitol PET rapidly and specifically detected infections caused by A. baumannii and several other important pathogens (Enterococcus faecium, S. aureus, Klebsiella pneumoniae, A. baumannii, Pseudomonas aeruginosa, and Enterobacter spp.). Importantly, 18F-fluoromannitol PET was able to monitor the therapeutic efficacy of vancomycin against S. aureus in vivo. Conclusion: The ease of production of 18F-fluoromannitol is anticipated to facilitate wide radiopharmaceutical dissemination. Furthermore, the broad sensitivity of 18F-fluoromannitol for bacterial infection in vivo suggests that it is an ideal imaging agent for clinical translation to detect and monitor infections and warrants further studies in the clinical setting.

Impact of 18F-FDG-PET/CT on the management of Staphylococcus aureus bacteraemia: a retrospective observational study.

OBJECTIVES: To assess the impact of 18F-FDG-PET/CT on the diagnosis and management of patients with Staphylococcus aureus bacteraemia (SAB). METHODS: Post hoc analysis of a prospective cohort of consecutive adult patients diagnosed with SAB (January 2013-December 2017). Patients who underwent 18F-FDG-PET/CT at the discretion of the attending physician were included. Endpoints were the identification of previously unknown infectious foci and changes in clinical management, defined as changes in the duration or class of antibiotic therapy, a surgical procedure on the source of infection or a change in the decision to remove or retain an implantable device. RESULTS: We included 39 patients (median age: 69 years, IQR:60-79). Fifteen (39%) patients did not have an infectious focus identified before 18F-FDG-PET/CT). Thirty new infectious foci were detected in 22/39 (56%) patients. In 11/15 (73%) patients without an identified focus at least one infectious focus was detected by 18F-FDG-PET/CT. In 22/26 (85%) patients with implantable devices, 18F-FDG-PET/CT confirmed or ruled out infection or detected local complications. Out of 13 device infections, 10 were detected by 18F-FDG-PET/CT (7/10 for the first time). In 19/39 (49%) patients 18F-FDG-PET/CT results led to changes in clinical management (15 changes in antibiotic therapy, 2 device removals, 2 surgical procedures, 1 avoidance of a surgical procedure). CONCLUSIONS: 18F-FDG-PET/CT may be a useful asset in the management of selected SAB cases, allowing the identification of previously undetected infectious foci and optimization of therapy, particularly in patients with endovascular devices. Indication should be made on a case-by-case basis.

Assessment of Keratitis Severity Using Quantitative Image Analysis in an In Vivo Murine Model of Staphylococcus aureus Bacterial Keratitis.

Purpose: Bacterial keratitis (BK) severity in murine models has traditionally been measured by subjective clinical grading or quantification of ocular bacterial burden. This investigation explores an objective and repeatable quantification of slit lamp photography (SLP) images to measure BK severity. Methods: BALB/c strain mice underwent three parallel scratches of the right cornea with subsequent inoculation of 107Staphylococcus aureus cells. SLP imaging and clinical severity grading were performed at 48 hours post-infection. Stromal infiltrate (SI) area on SLP images were quantified. Bacterial burden was determined after enucleation and homogenization. Spearman rank correlations (rs) were used to estimate associations between SI area, clinical severity grades, and bacterial burden. Results: BALB/c strain mice (n = 14) were evaluated with an average SI area of 0.92 mm2 (standard deviation, SD = 0.65) and average bacterial burden of 3.16 × 105 colony forming units per milliliter (CFU/mL) (SD = 8.3 × 105). Clinical severity grade positively correlated with SI area (rs = 0.59, p = 0.0276) and bacterial burden (rs = 0.66, p = 0.0106). There was a trend towards positive association between SI area and bacterial burden (rs = 0.51, p = 0.0543). Conclusions: SLP annotation of SI area is correlated with clinical severity and may provide an objective, quantitative, and repeatable assessment of BK disease severity. Translational Relevance: SLP annotation of SI area is a novel quantitative method to evaluate bacterial keratitis severity longitudinally in mouse models which may be a powerful tool to better understand BK pathogenesis and response to treatments.

Visualizing Staphylococcus aureus pathogenic membrane modification within the host infection environment by multimodal imaging mass spectrometry.

Bacterial pathogens have evolved virulence factors to colonize, replicate, and disseminate within the vertebrate host. Although there is an expanding body of literature describing how bacterial pathogens regulate their virulence repertoire in response to environmental signals, it is challenging to directly visualize virulence response within the host tissue microenvironment. Multimodal imaging approaches enable visualization of host-pathogen molecular interactions. Here we demonstrate multimodal integration of high spatial resolution imaging mass spectrometry and microscopy to visualize Staphylococcus aureus envelope modifications within infected murine and human tissues. Data-driven image fusion of fluorescent bacterial reporters and matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance imaging mass spectrometry uncovered S. aureus lysyl-phosphatidylglycerol lipids, localizing to select bacterial communities within infected tissue. Absence of lysyl-phosphatidylglycerols is associated with decreased pathogenicity during vertebrate colonization as these lipids provide protection against the innate immune system. The presence of distinct staphylococcal lysyl-phosphatidylglycerol distributions within murine and human infections suggests a heterogeneous, spatially oriented microbial response to host defenses.