Endophthalmitis Model Research Articles

Omiganan-Based Synthetic Antimicrobial Peptides for the Healthcare of Infectious Endophthalmitis.

Bacterial endophthalmitis is a severe infection of the aqueous or vitreous humor of the eye that can lead to permanent vision loss. Due to the rapid emergence of antibiotic resistance and dose-limiting toxicities, the standard treatment of bacterial endophthalmitis via the intravitreal injection of broad-spectrum antibiotics remains inadequate. Membrane active cationic antimicrobial peptides (AMPs) have emerged as a promising class of effective and broad-spectrum antimicrobial agents with potential to overcome antibiotic resistance. In this work, we investigate, for the first time, the use of omiganan (IK-12), a 12-amino acid indolicidin derivative for the treatment of bacterial endophthalmitis. Additionally, IK-12 was used as a template to perform amino acid rearrangements, without altering the length or type of amino acids, to yield a series of new derivative AMPs with varying extents of secondary structure formation under membrane mimicking conditions. IK-12 and its derivatives demonstrated strong and broad-spectrum antibacterial activities against a panel of clinically isolated Gram-positive and Gram-negative bacteria, including methicillin-resistant Staphylococcus aureus commonly implicated in bacterial endophthalmitis. Interestingly, two of the new IK-12 derivatives, IP-12 and WP-12, showed lower geometric mean minimum inhibitory concentration and higher 50% hemolysis concentration values, which effectively translated into 2- to 3.4-fold higher bacterial selectivity than the parent IK-12. Furthermore, the intravitreal injection of IK-12, IP-12, and WP-12 in a rabbit model of MRSA-induced endophthalmitis led to considerably improved clinical presentation and reduced recruitment of inflammatory cells. In all, these results demonstrate the potential of IK-12 and its derivatives, IP-12 and WP-12, as promising candidates for the treatment of bacterial endophthalmitis.

Intravitreal chlorhexidine for sterilizing the vitreous cavity in an animal model of bacterial endophthalmitis

Purpose To evaluate the efficacy and safety of intravitreal chlorhexidine (CHX) for sterilising the vitreous cavity in bacterial endophthalmitis. Methods For in-vitro experiments, full-thickness retina explants were harvested from freshly enucleated pig eyes. Six-millimeter circular sensory retina patches were then incubated in varying concentrations of CHX (0.625-800 µg/mL) for 24 hours. Retinal cell viability was determined at the end of the incubation period with a live-dead assay. The bactericidal effects of the tested CHX concentrations were determined using a quantitative suspension test on Staphylococcus epidermidis. The safety of CHX was also tested by injecting varying doses of CHX (50-400 µg/mL) into the vitreous cavity of albino rabbits followed by flash electroretinography (ERG) and light microscopy. The bactericidal effect of the non-toxic CHX doses was determined using the rabbit model of endophthalmitis created by injecting 3000 CFU/0.1 mL of Staphylococcus epidermidis. Results In vitro concentrations of CHX greater than 6.25 µgr/mL exerted a bactericidal effect, while concentrations of CHX less than 200 µg/mL did not impair retinal cell viability. Intravitreal concentrations of CHX between 20-100 µg/mL were adequate to sterilise the infected rabbit vitreous cavity in the animal model. No significant functional or anatomical deleterious effect was observed with ERG or light microscopy. Conclusion CHX can sterilise the vitreous cavity in an animal model of bacterial endophthalmitis without impairing retinal cell viability. Our results encourage further research for clinical use of chlorhexidine in treatment of bacterial endophthalmitis.

In vivo and in vitro efficacy of the ithmid kohl/zinc-oxide nanoparticles, ithmid kohl/Aloe vera, and zinc-oxide nanoparticles/Aloe vera for the treatment of bacterial endophthalmitis

The aim of this study was to investigate the efficacy of the ithmid kohl/zinc-oxide nanoparticles (ZnONPs), ithmid kohl/Aloe vera, and ZnONPs/Aloe vera in the treatment of bacterial endophthalmitis. The endophthalmitis model was prepared by contaminating both eyes of 24 healthy adult male albino rabbits with a clinical isolate of Klebsiella pneumoniae. The animals were randomly divided into eight groups (A-H) according to the treatment. Group A received 1 ml of ithmid kohl/ZnONPs ointment, group B received 1 ml of ithmid kohl/Aloe vera gel ointment, group C received 1 ml of ZnONPs/Aloe vera gel ointment, and groups D, E, and F were treated with 1 ml of ithmid kohl solution (0.5 g/ml in distilled water), 1 ml of ZnONPs (0.5 g/ml) colloidal dispersion, and 1 ml of Aloe vera gel, respectively. Group G received 100 μl of a tetracycline antibiotic solution (final concentration: 16 µg/ml), and group H received sterile distilled water (no treatment). In vitro antibacterial activity was evaluated against K. pneumoniae using the agar well diffusion. The combination of ithmid kohl/ZnONPs was the most effective formulation for treating endophthalmitis model in infected rabbits within 2 days. In vitro antibacterial assay confirmed the potential of the ithmid kohl/ZnONPs formulation, which had the largest zone of inhibition (31 mm) among the compounds tested. The preparation of the ithmid kohl/ZnONPs formulation and its in vivo experiment in albino rabbits for the treatment of bacterial endophthalmitis was an innovative approach that has shown promise and may potentially serve as a viable alternative in clinical practice.

Comparative extracellular vesicles proteomics unravels host-pathogen interactions: New insights in bacterial and fungal endophthalmitis in murine models

PurposeEndophthalmitis, an intraocular infection, often results in devastating outcomes due to frequent misdiagnosis and delayed treatment initiation. Extracellular Vesicles (EVs) have long been lauded for its potential as a biomarker in several infections. Previously, we have profiled EVs in bacterial and fungal infected murine model of endophthalmitis. In this study, we reported a comparative analysis of bacterial and fungal endophthalmitis to understand the disease pathobiology and identify a potential signature molecule for diagnosis of infectious endophthalmitis. MethodsEndophthalmitis was induced by intravitreal injection of bacteria (S. aureus and P. aeruginosa) and fungus (A. flavus and C. albicans) in C57BL/6 mice followed by enucleation at 24 h. EVs were isolated by ultracentrifugation and total proteins were subjected to LC-MS/MS. A comparative analysis of the EV protein cargo during bacterial and fungal endophthalmitis was performed in this study along with the pathway assessment by Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology. ResultsProteome analysis of EVs revealed the presence of over 1500 proteins in each of the infected and control groups. Comparative (bacteria versus fungal) study revealed that the protein cargo of the EVs was very distinct among the infected sets with a very few common proteins. Crucial differentially expressed proteins (DEPs) common to both infections were Complement Cascade 8-alpha (C8α), chymase, sorting nexin-29 and glutathione-S-transferase and common pathways were metabolic pathways, Staphylococcus aureus infection, PPAR signalling pathway, cAMP signalling pathway, TGFβ signalling pathway and HIF-1 signalling pathway. ConclusionThis study comprehensively investigates the global proteome of EVs derived from mice model of bacterial and fungal endophthalmitis. Proteomic analysis reveals the disparate EV protein cargo underlining the differences in the pathogenesis of bacterial and fungal endophthalmitis with C8α, chymase, sorting nexin 29 and glutathione-S-transferase common to both infections.

Virulence-related genotypic differences among Bacillus cereus ocular and gastrointestinal isolates and the relationship to endophthalmitis pathogenesis.

Bacillus cereus (Bc) can cause self-limiting gastrointestinal infections, but when infecting the eye, can cause rapid and irreversible blindness. This study investigated whether clinical ocular and gastrointestinal Bc isolates differed in terms of virulence-related genotypes and endophthalmitis virulence. Twenty-eight Bc ocular, gastrointestinal, and laboratory reference isolates were evaluated. Hemolysis assays were performed to assess potential differences in hemolytic activity. The presence of twenty Bc virulence-related genes was assessed by PCR. A subset of ocular and gastrointestinal isolates differing in PCR positivity for 5 virulence genes was compared to strain ATCC14579 in an experimental murine model of endophthalmitis. At 8 hours post infection, retinal function was evaluated by electroretinography, and intraocular bacterial concentrations were determined by plate counts. Gastrointestinal Bc isolates were more hemolytic than the Bc ocular isolates and ATCC14579 (p < 0.0001). Bc ocular isolates were more frequently PCR-positive for capK, cytK, hblA, hblC, and plcR compared to the gastrointestinal isolates (p ≤ 0.0002). In the endophthalmitis model, mean A-wave retention did not differ significantly between eyes infected with ATCC14579 and eyes infected with the selected ocular or gastrointestinal isolates (p ≥ 0.3528). Similar results were observed for mean B-wave retention (p ≥ 0.0640). Only one diarrheal isolate showed significantly greater B-wave retention when compared to ATCC14579 (p = 0.0303). No significant differences in mean A-wave (p ≥ 0.1535) or B-wave (p ≥ 0.0727) retention between the selected ocular and gastrointestinal isolates were observed. Intraocular concentrations of ATCC14579 were significantly higher than the selected ocular isolate and 3 of the gastrointestinal isolates (p ≤ 0.0303). Intraocular concentrations of the selected ocular isolate were not significantly different from the gastrointestinal isolates (p ≥ 0.1923). Among the subset of virulence-related genes assessed, 5 were significantly enriched among the ocular isolates compared to gastrointestinal isolates. While hemolytic activity was higher among gastrointestinal isolates, retinal function retention and intraocular growth was not significantly different between the selected ocular and gastrointestinal isolates. These results suggest that Bc strains causing gastrointestinal infections, while differing from ocular isolates in hemolytic activity and virulence-related gene profile, are similarly virulent in endophthalmitis.

Oral administration of S-nitroso-L-glutathione (GSNO) provides anti-inflammatory and cytoprotective effects during ocular bacterial infections.

Bacterial endophthalmitis is a severe complication of eye surgeries that can lead to vision loss. Current treatment involves intravitreal antibiotic injections that control bacterial growth but not inflammation. To identify newer therapeutic targets to promote inflammation resolution in endophthalmitis, we recently employed an untargeted metabolomics approach. This led to the discovery that the levels of S-nitroso-L-glutathione (GSNO) were significantly reduced in an experimental murine Staphylococcus aureus (SA) endophthalmitis model. In this study, we tested the hypothesis whether GSNO supplementation via different routes (oral, intravitreal) provides protection during bacterial endophthalmitis. Our results show that prophylactic administration of GSNO via intravitreal injections ameliorated SA endophthalmitis. Therapeutically, oral administration of GSNO was found to be most effective in reducing intraocular inflammation and bacterial burden. Moreover, oral GSNO treatment synergized with intravitreal antibiotic injections in reducing the severity of endophthalmitis. Furthermore, in vitro experiments using cultured human retinal Muller glia and retinal pigment epithelial (RPE) cells showed that GSNO treatment reduced SA-induced inflammatory mediators and cell death. Notably, both in-vivo and ex-vivo data showed that GSNO strengthened the outer blood-retinal barrier during endophthalmitis. Collectively, our study demonstrates GSNO as a potential therapeutic agent for the treatment of intraocular infections due to its dual anti-inflammatory and cytoprotective properties.

A Novel and Low-cost Approach for Intravitreal Injection in an Experimental Model of Endophthalmitis.

Animal models are necessary in understanding the pathogenesis of endophthalmitis and are also necessary to assist the development of new therapeutics for this sight-threatening ocular inflammation. Hamilton syringes are usually preferred to inject pathogens when performing experiments on test subjects, however, this method has technical and financial disadvantages. In this study, we report the findings and assess the related benefits of applying a novel low-cost intravitreal injection technique to initiate endophthalmitis in a mouse model while using the Eppendorf tip and a 26G needle. The 18-hr culture of clinical isolates of bacteria (Staphylococcus aureus and Pseudomonas aeruginosa) and fungus (Aspergillus flavus and Candida albicans) were resuspended to a final concentration of 10,000 colony forming units (CFU)/1 µL which were separately injected intravitreally into C57BL/6 mice (6-8 weeks) using a 0.1-2.5µL pipette attached to the modified Eppendorf tip with a 26G needle. The contralateral eye served as vehicle/uninjected control. Disease progression was determined by assessing the corneal haze, opacity, bacterial burden, and retinal histology of the eyes used in the model. Following euthanization, bacteria-infected mice were enucleated after 24 hr of the initial injection, and fungus-infected mice after 72 hr. Of the 50 mice injected, the modified technique was successful in 48 mice. Two mice were excluded due to cataract formed by accidental injury to the lens. The experimental endophthalmitis mice model successfully mimicked the natural clinical course. Clinical assessment and histopathology confirmed the influx of inflammatory cells into the posterior segment of the eye along with dissolution of retinal architecture. Our novel method of injection using a modified Eppendorf tip and 26G needle yielded a cost-effective mouse model of clinical endophthalmitis, resulting in reproducible infection for understanding various aspects of its pathobiology.

OculusGraphy: Signal Analysis of the Electroretinogram in a Rabbit Model of Endophthalmitis Using Discrete and Continuous Wavelet Transforms.

The electroretinogram is a clinical test used to assess the function of the photoreceptors and retinal circuits of various cells in the eye, with the recorded waveform being the result of the summated response of neural generators across the retina. The present investigation involved an analysis of the electroretinogram waveform in both the time and time-frequency domains through the utilization of the discrete wavelet transform and continuous wavelet transform techniques. The primary aim of this study was to monitor and evaluate the effects of treatment in a New Zealand rabbit model of endophthalmitis via electroretinogram waveform analysis and to compare these with normal human electroretinograms. The wavelet scalograms were analyzed using various mother wavelets, including the Daubechies, Ricker, Wavelet Biorthogonal 3.1 (bior3.1), Morlet, Haar, and Gaussian wavelets. Distinctive variances were identified in the wavelet scalograms between rabbit and human electroretinograms. The wavelet scalograms in the rabbit model of endophthalmitis showed recovery with treatment in parallel with the time-domain features. The study compared adult, child, and rabbit electroretinogram responses using DWT and CWT, finding that adult signals had higher power than child signals, and that rabbit signals showed differences in the a-wave and b-wave depending on the type of response tested, while the Haar wavelet was found to be superior in visualizing frequency components in electrophysiological signals for following the treatment of endophthalmitis and may give additional outcome measures for the management of retinal disease.

Therapeutic potential of Bacillus phage lysin PlyB in ocular infections.

Bacteriophage lytic enzymes (i.e., phage lysins) are a trending alternative for general antibiotics to combat growing antimicrobial resistance. Gram-positive Bacillus cereus causes one of the most severe forms of intraocular infection, often resulting in complete vision loss. It is an inherently β-lactamase-resistant organism that is highly inflammogenic in the eye, and antibiotics are not often beneficial as the sole therapeutic option for these blinding infections. The use of phage lysins as a treatment for B. cereus ocular infection has never been tested or reported. In this study, the phage lysin PlyB was tested in vitro, demonstrating rapid killing of vegetative B. cereus but not its spores. PlyB was also highly group specific and effectively killed the bacteria in various bacterial growth conditions, including ex vivo rabbit vitreous (Vit). Furthermore, PlyB demonstrated no cytotoxic or hemolytic activity toward human retinal cells or erythrocytes and did not trigger innate activation. In in vivo therapeutic experiments, PlyB was effective in killing B. cereus when administered intravitreally in an experimental endophthalmitis model and topically in an experimental keratitis model. In both models of ocular infection, the effective bactericidal property of PlyB prevented pathological damage to ocular tissues. Thus, PlyB was found to be safe and effective in killing B. cereus in the eye, greatly improving an otherwise devastating outcome. Overall, this study demonstrates that PlyB is a promising therapeutic option for B. cereus eye infections.IMPORTANCEEye infections from antibiotic-resistant Bacillus cereus are devastating and can result in blindness with few available treatment options. Bacteriophage lysins are an alternative to conventional antibiotics with the potential to control antibiotic-resistant bacteria. This study demonstrates that a lysin called PlyB can effectively kill B. cereus in two models of B. cereus eye infections, thus treating and preventing the blinding effects of these infections.

Temporal Transcriptome Analysis Suggests Modulation of Key Pathways and Hub Genes in a Mice Model of Multi-Drug Resistant (MDR) Pseudomonas aeruginosa Endophthalmitis

Purpose Increasing incidence of multidrug-resistant Pseudomonas aeruginosa (MDR-PA) causing endophthalmitis challenges our ability to manage this vision-threatening condition. In this study, temporal dynamics of immune response in a mouse model of MDR-PA endophthalmitis was investigated by whole transcriptome analysis. Methods C57BL/6 mice were infected with MDR-PA and antibiotic susceptible (S-PA) clinical strains and disease severity were monitored at 6 and 24-h postinfection (p.i), following which eyeballs were enucleated. Microarray analysis was performed using SuperPrint G3 Mouse Gene Expression v2 chip and the differential gene expression analysis was performed with limma package in R (v4.0.0.)/Bioconductor (v3.11). Results Histopathological analysis revealed a significant difference in retinal architecture and vitreous infiltrates at 6 and 24 h. In comparison to S-PA, MDR-PA revealed altered expression of 923 genes at 6 h and 2220 genes at 24 h. Further, 23 and 76% of these altered genes and its downstream interacting proteins showed time-specific expression (6 and 24 h respectively), indicating their association with disease progression. At 24 hours, MDR-PA induced endophthalmitis showed aberrant immune response with the enrichment inflammasome signalling, dysregulated ubiquitination, complement cascade, MMPs NF-κβ and IL-1 signalling. Conclusion The rapid development of transcriptional differences between the two-time points reveals that distinct genes contribute to disease severity. The results from this study highlighted a link between innate and adaptive immune responses and provided novel insights in the pathogenesis of MDR-PA endophthalmitis by extending the number of molecular determinants and functional pathways that underpin host-associated damage.

Proteomic profiling of exosomes in a mouse model of Candida albicans endophthalmitis

Exosomes play pivotal roles in intercellular communication, and pathophysiological functions. In this study, we aimed to understand the role of exosomal proteome derived from C. albicans infected mice (C57BL/6) eyeball. Exosomes were characterized by Dynamic Light Scattering and Western blot, quantified and subjected to LC-MS/MS and cytokine quantification by ELISA. The average size of exosomes was 170–200 nm with number of exosomes amounted to 1.42 × 1010 in infected set compared to control (1.24 × 109). Western blot was positive for CD9, CD63 and CD81 confirming the presence of exosomes. IL-6, IL1β, TNF-α, and IFN-γ levels were significantly elevated in infected eye at 72 h.p.i. Proteomic analysis identified 42 differentially expressed proteins, of these 37 were upregulated and 5 were downregulated. Gene Ontology (GO) revealed enrichment of cell adhesion, cytoskeleton organisation, and cellular response proteins such as aquaporin-5, gasdermin-A, CD5 antigen-like, Catenin, V-ATPase, and vesicle associated protein. Additionally, KEGG pathway analysis indicated the association of metabolic and carbon signalling pathways with exosomes from C. albicans infected eye. The protein cargo in exosomes released during endophthalmitis with C. albicans seems to play a unique role in the pathogenesis of the disease and further validations with larger cohort of patients is required to confirm them as biomarkers.

Transcriptomic and Histological Analysis of Exacerbated Immune Response in Multidrug-Resistant Pseudomonas aeruginosa in a Murine Model of Endophthalmitis.

Multidrug-resistant (MDR) endophthalmitis is a serious threat to the whole spectrum of therapeutic procedures associated with the risk of managing and preventing vision loss. We have earlier shown the interplay of immune mediators in patients with MDR Pseudomonas aeruginosa (PA) endophthalmitis leading to worse outcome. Expanding on these findings, a murine model of endophthalmitis was developed to explore the effects of drug resistance on the pathogenesis by analyzing the temporal changes in retinal morphology along with its transcriptomic signatures. Clinical isolates of susceptible (S-PA) and multidrug-resistant PA (MDR-PA) were injected intravitreally in C57BL/6 mice followed by enucleation at 6 and 24 h time points postinfection. Disease progression and retinal changes were monitored by clinical and histological assessment and transcriptome analysis in a pair-wise manner. Histological assessment of MDR-PA eyeball revealed higher disease severity (p < 0.05), CD45+ cells (p = 0.007), MPO+ cells (p = 0.01), GFAP+ (p = 0.02), along with higher retinal cell death in mice infected with MDR-PA (p = 0.008). Temporal transcriptome analysis revealed differential expression of nearly 923 genes at 6 h p.i. and 2,220 genes at 24 h p.i. (FC ≥2, adjusted p-value <0.05). Pathway enrichment analysis identified differential regulation of chemokine- and cytokine-mediated, MAPK, and NF-кβ signaling pathways. In conclusion, rapid deterioration of retinal architecture and immune exacerbation was significantly associated with the MDR endophthalmitis, suggesting the need for immunomodulatory agents to strengthen host cell functions and support antibiotics to save the retinal structure from inevitable deterioration and restoration of the vision.

Sequential drug release of co-assembled supramolecular hydrogel as synergistic therapy against Staphylococcus aureus endophthalmitis

Although endophthalmitis is a serious sight-threatening disorder, combining anti-inflammatory agents and antibiotics has provided new methods for effectively controlling it. This paper reports that the co-assembly of a sparingly soluble antibiotic (levofloxacin; Lev) and dexamethasone-peptide amphiphile (Dex-SA-RGD) instantly generates a macroscopically supramolecular hydrogel (Lev/Dex-SA-RGD) without any external stimulus under physiological conditions, which could be used in synergistic therapy to cure endophthalmitis induced by Staphylococcus aureus (i.e., S. aureus). The resulting co-assembled supramolecular hydrogel was thoroughly characterized using rheology, circular dichroism, and transmission electron microscopy. An in vitro drug release study showed that Lev was rapidly released from the supramolecular hydrogel within 6 h, while Dex-SA-RGD escaped from the supramolecular hydrogel as a result of matrix erosion, and then acted as a prodrug to provide the sequentially slow release of active dexamethasone via the hydrolytic route. The Lev/Dex-SA-RGD supramolecular hydrogel showed high antibacterial activities against both gram-negative and gram-positive strains. Moreover, a rabbit endophthalmitis model induced by S. aureus showed that the intravitreal injection of the Lev/Dex-SA-RGD supramolecular hydrogel resulted in excellent therapeutic efficacy with no apparent damage or destruction of the retina, and dramatically suppressed inflammatory responses (e.g., leukocyte and lymphocyte infiltration, microglial activation, cytokine production, and retinal cell death). As a rational approach to investigate and apply the co-assembled hydrogel, this work presents the potential of synergistic intervention to prevent retinal tissue damage and vision impairment, and ultimately shows the clinical benefits of using supramolecular hydrogels for ocular drug delivery.

The Bacillus virulome in endophthalmitis.

Bacillus cereus is recognized as a causative agent of gastrointestinal syndromes, but can also cause a devastating form of intraocular infection known as endophthalmitis. We have previously reported that the PlcR/PapR master virulence factor regulator system regulates intraocular virulence, and that the S-layer protein (SlpA) contributes to the severity of B. cereus endophthalmitis. To better understand the role of other B. cereus virulence genes in endophthalmitis, expression of a subset of factors was measured at the midpoint of disease progression in a murine model of endophthalmitis by RNA-Seq. Several cytolytic toxins were expressed at significantly higher levels in vivo than in BHI. The virulence regulators codY, gntR, and nprR were also expressed in vivo. However, at this timepoint, plcR/papR was not detectable, although we previously reported that a B. cereus mutant deficient in PlcR was attenuated in the eye. The motility-related genes fla, fliF, and motB, and the chemotaxis-related gene cheA were detected during infection. We have shown previously that motility and chemotaxis phenotypes are important in B. cereus endophthalmitis. The sodA2 variant of manganese superoxide dismutase was the most highly expressed gene in vivo. Expression of the surface layer protein gene, slpA, an activator of Toll-like receptors (TLR)-2 and -4, was also detected during infection, albeit at low levels. Genes expressed in a mouse model of Bacillus endophthalmitis might play crucial roles in the unique virulence of B. cereus endophthalmitis, and serve as candidates for novel therapies designed to attenuate the severity of this often blinding infection.

An Integrated Theranostic Nanomaterial for Targeted Photodynamic Therapy of Infectious Endophthalmitis

Infectious endophthalmitis is a growing concern that causes irreversible intraocular tissue and optic nerve damage. Here, we report a theranostic nanoplatform based on UiO-66-NH2 to combine photodynamic therapy (PDT) and lipopolysaccharide (LPS) targeting through polypeptide (YVLWKRKRKFCFI-NH2) modification. Toluidine blue (TB), serving as a photosensitizer (PS), is loaded into UiO-66-NH2 nanoparticles (NPs) for both self-imaging and PDT functions. The accelerated TB release at pH 5.5 compared to pH 7.4 demonstrates the responsive drug release feature. In vitro antibacterial tests, including contact incubation-agar plate counting, bacterial live/dead staining, and scanning electron microscopy observations, confirm the utility of the [email protected]2@PEP&PEG NPs (UTPP NPs) in biofilms removal and sterilization of Pseudomonas aeruginosa and Staphylococcus epidermidis. Furthermore, the UTPP NPs also show good biocompatibility toward human retinal pigment epithelial cells, as well as in situ self-imaging property. Finally, an in vivo infectious endophthalmitis model supports the application of the UTPP NPs in infectious endophthalmitis treatment.

Rapid pathogen identification and antimicrobial susceptibility testing in in vitro endophthalmitis with matrix assisted laser desorption-ionization Time-of-Flight Mass Spectrometry and VITEK 2 without prior culture.

Prompt clinical diagnosis and initiation of treatment are critical in the management of infectious endophthalmitis. Current methods used to identify causative agents of infectious endophthalmitis are mostly inefficient, owing to suboptimal sensitivity, length, and cost. Matrix Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) can be used to rapidly identity pathogens without a need for culture. Similarly, automated antimicrobial susceptibility test systems (AST, VITEK 2) provide accurate antimicrobial susceptibility profiles. In this proof-of-concept study, we apply these technologies for the direct identification and characterization of pathogens in vitreous samples, without culture, as an in vitro model of infectious endophthalmitis. Vitreous humor aspirated from freshly enucleated porcine eyes was inoculated with different inocula of Staphylococcus aureus (S. aureus) and incubated at 37°C. Vitreous endophthalmitis samples were centrifuged and pellets were directly analyzed with MALDI-TOF MS and VITEK 2 without prior culture. S. aureus colonies that were conventionally grown on culture medium were used as control samples. Time-to-identification, minimum concentration of bacteria required for identification, and accuracy of results compared to standard methods were determined. MALDI-TOF MS achieved accurate pathogen identification from direct analysis of intraocular samples with confidence values of up to 99.9%. Time from sample processing to pathogen identification was <30 minutes. The minimum number of bacteria needed for positive identification was 7.889x106 colony forming units (cfu/μl). Direct analysis of intraocular samples with VITEK 2 gave AST profiles that were up to 94.4% identical to the positive control S. aureus analyzed per standard protocol. Our findings demonstrate that the direct analysis of vitreous samples with MALDI-TOF MS and VITEK 2 without prior culture could serve as new, improved methods for rapid, accurate pathogen identification and targeted treatment design in infectious endophthalmitis. In vivo models and standardized comparisons against other microbiological methods are needed to determine the value of direct analysis of intraocular samples from infectious endophthalmitis with MALDI-TOF MS and VITEK 2.

Efficacy of Tamponade of Vitreal Cavity by Perfluororganic Compound Emulsion with Antibiotics Solutions in Treatment of Gram Negative Endopthalmitis (Experimental Study)

The purpose of investigation was assessment of efficacy of the method suggested for treatment of postoperative endophthalmitis on the experimental model of gram negative endophthalmitis. Materials and methods . In order to create experimental model of endophthalmitis phacoemulsification of lens was rendered on 30 rabbits (30 eyes). At the end of surgery 0.1 ml of Escherichia coli microbial suspension containing 100 cells of pathogen was injected intravitreally to all. In 12 hours after the contamination signs of heavy postoperative endophthalmitis appeared in all rabbits. All the rabbits were divided into two groups in dependence of the method of further surgical treatment. 15 rabbits (15 eyes) were in the first group, vitrectomy with intravitreal injection of 1 mg vancomicin + 2.25 mg ceftazidim antibiotics combination was rendered. For 15 rabbits (15 eyes) of the second group vitrectomy with tamponade of vitreal cavity by PFOC and 1 mg vancomicin + 2.25 mg ceftazidim antibiotics combination intravitreal injection was rendered. Results. Treatment of the second group rabbits was more effective in comparison with the first group ( p = 0.0018). According to ERG data visual functions were saved in 6 rabbits of the first group (40 %) and in 10 rabbits of the second group (66.67 %). Besides functional loss anatomic loss of eye was observed in 33.33 % cases in the first group. While eyeball was saved in all 100 % cases of the second group with use of the suggested method. Conclusion. Experimental investigation proved efficacy of the suggested method of endophthalmitis treatment. This method of treatment is universal regardless of disease etiological cause.

Study of the Effectiveness of Precision Doses of Antibiotics in the Treatment of Induced Acute Bacterial Postoperative Endophthalmitis in an Experiment

Objective: to conduct a clinical and histomorphological study of the efficacy and safety of individually calculated doses of antibiotic in an experimental model of acute endophthalmitis in laboratory animals. Materials and methods. The study group included 15 adult laboratory white rabbits of the Chinchilla breed, male, aged from 2 to 3 months (2.6 ± 0.44 months). Determination of the anatomical parameters of the eyeball was performed on an ultrasound biometer — Compact Touch Quantel Medical (France). The obtained data were exported to the original program of an electronic computing machine of its own design, for automatic calculation of the dose of antibacterial drug for intravitreal injection. In the course of the experiment, the native lens was removed from animals with intraoperative administration of a culture of epidermal staphylococcus in a volume of 0.1 ml (10,000 colony forming units). After surgery, the animals were divided into 4 treatment groups: group 1 — without treatment, group 2 — standard dose of vancomycin for intravitreal administration (1 mg / 0.1 ml), group 3 — double dose (2 mg / 0.1 ml), group 4 — individual a dose of 0.25 mg / 0.1 ml (according to the calculation of the computer program). After a period of clinical observation, the enucleation of the eyeball was performed with a histomorphological study. Results . Group 1 — increase of the inflammation signs, deep irreversible structural damage to all intraocular structures according to histomorphology. Group 2 — regression of clinical manifestations by the first week of observation, the processes of alteration and initial regeneration of intraocular structures according to histomorphology. Group 3 — diffuse opacification of the cornea with the impossibility of further analysis, signs of toxic damage to intraocular structures according to histomorphology. Group 4 — complete regression of clinical symptoms, residual signs of aseptic inflammation according to histomorphology. Conclusions . Clinical and histomorphological studies of enucleated eyeballs in experimental animals demonstrate clinical efficacy and safety of vancomycin concentration in the vitreal cavity equal to or close to 128 μg/ml. Individual (personalized) calculation of the antibacterial drug dose eliminates the risk of toxic damage to the retina when the antibiotic concentration is excessive in the vitreal cavity. The toxic effects of excessive doses of antibiotics increase with increasing concentration of the active substance.

Therapeutic Effects of Intravitreously Administered Bacteriophage in a Mouse Model of Endophthalmitis Caused by Vancomycin-Sensitive or -Resistant Enterococcus faecalis

Endophthalmitis due to infection with Enterococcus spp. progresses rapidly and often results in substantial and irreversible vision loss. Given that the frequency of this condition caused by vancomycin-resistant Enterococcus faecalis has been increasing, the development of novel therapeutics is urgently required. We have demonstrated the therapeutic potential of bacteriophage ΦEF24C-P2 in a mouse model of endophthalmitis caused by vancomycin-sensitive (EF24) or vancomycin-resistant (VRE2) strains of E. faecalis Phage ΦEF24C-P2 induced rapid and pronounced bacterial lysis in turbidity reduction assays with EF24, VRE2, and clinical isolates derived from patients with E. faecalis-related postoperative endophthalmitis. Endophthalmitis was induced in mice by injection of EF24 or VRE2 (1 × 104 cells) into the vitreous. The number of viable bacteria in the eye increased to >1 × 107 CFU, and neutrophil infiltration into the eye was detected as an increase in myeloperoxidase activity at 24 h after infection. A clinical score based on loss of visibility of the fundus as well as the number of viable bacteria and the level of myeloperoxidase activity in the eye were all significantly decreased by intravitreous injection of ΦEF24C-P2 6 h after injection of EF24 or VRE2. Whereas histopathologic analysis revealed massive infiltration of inflammatory cells and retinal detachment in vehicle-treated eyes, the number of these cells was greatly reduced and retinal structural integrity was preserved in phage-treated eyes. Our results thus suggest that intravitreous phage therapy is a potential treatment for endophthalmitis caused by vancomycin-sensitive or -resistant strains of E. faecalis.

Vitreous D-Lactate Levels as a Biomarker in the Diagnosis of Presumed Infectious Culture Negative Endophthalmitis

ABSTRACTPurpose: Microbiological investigations of vitreous fluid have often failed to detect the causative agent in infectious endophthalmitis resulting in a clinical dilemma. D-Lactate is a byproduct of bacterial metabolism, and its accumulation in sterile body fluids indicates bacterial infection. The aim of the study was to evaluate the measurement of vitreous fluid D-lactate for the diagnosis of infectious endophthalmitis and to define an optimal D-lactate concentration for the differentiation from non-infectious samples.Methods: Vitreous samples of 41 patients clinically diagnosed as endophthalmitis and 20 patients with non-infectious disorders, as controls, between October 2018 and February 2019 were included in the study. D-lactate levels were determined by a D-lactate colorimetric assay kit (MAK058 Sigma-Aldrich) and the receiver operating characteristic curves (ROC) of D-lactate were calculated. The clinical finding of D-lactate production in bacterial endophthalmitis was also verified in a mouse model of bacterial endophthalmitis.Results: Of the 41 patients included in the infectious group, 25 had culture-positive infections of which 13/25 were gram-positive organisms and 12/25 grew gram-negative bacilli. Based on the ROC curve, the sensitivity of D-lactate was found to be 80% and specificity 100% and a cut-off value of above 47.06 ng/µl for D-lactate was defined as positive or true infectious in vitreous samples for diagnosis of endophthalmitis. In-vivo, a mouse model of bacterial endophthalmitis showed the significant production of D-lactate levels in retina and vitreous. Interestingly the levels were elevated in Gram-negative infections compared to Gram-positive bacterial endophthalmitis.Conclusion: Our clinical and in-vivo mouse model data showed that vitreous fluid D-lactate could be used as a bacterial-specific biomarker in the diagnosis of most infectious endophthalmitis and could be implemented for the evaluation of treatment success.