Predatory bacteria prevent the proliferation of intraocular Serratia marcescens and fluoroquinolone-resistant Pseudomonas aeruginosa.

Endogenous endophthalmitis caused by Gram-negative bacteria is an intra-ocular infection that can rapidly progress to irreversible loss of vision. While most endophthalmitis isolates are susceptible to antibiotic therapy, the emergence of resistant bacteria necessitates alternative approaches to combat intraocular bacterial proliferation. In this study the ability of predatory bacteria to limit intraocular growth of Pseudomonas aeruginosa, Serratia marcescens, and Staphylococcus aureus was evaluated in a New Zealand white rabbit endophthalmitis prevention model. Predatory bacteria Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus were able to reduce proliferation of keratitis isolates of P. aeruginosa and to a lesser extent S. marcescens. However, it was not able to significantly reduce the number of intraocular S. aureus, which is not a productive prey for these predatory bacteria, suggesting that the inhibitory effect on P. aeruginosa and S. marcescens requires active predation rather than an antimicrobial immune response. Similarly, UV-inactivated B. bacteriovorus were unable to prevent proliferation of P. aeruginosa. Together, these data indicate in vivo inhibition of Gram-negative bacteria proliferation within the intra-ocular environment by predatory bacteria.

How Ophthalmologists Can Decarbonize Eye Care: A Review of Existing Sustainability Strategies and Steps Ophthalmologists Can Take.

TOPIC: Understanding approaches to sustainability in cataract surgery and their risks and benefits. CLINICAL RELEVANCE: In the United States, health care is responsible for approximately 8.5% of greenhouse gas (GHG), and cataract surgery is one of the most commonly performed surgical procedures. Ophthalmologists can contribute to reducing GHG emissions, which lead to a steadily increasing list of health concerns ranging from trauma to food instability. METHODS: We conducted a literature review to identify the benefits and risks of sustainability interventions. We then organized these interventions into a decision tree for use by individual surgeons. RESULTS: Identified sustainability interventions fall into the domains of advocacy and education, pharmaceuticals, process, and supplies and waste. Existing literature shows certain interventions may be safe, cost-effective, and environmentally friendly. These include dispensing medications at home to patients after surgery, multi-dosing appropriate medications, training staff to properly sort medical waste, reducing the number of supplies used during surgery, and implementing immediate sequential bilateral cataract surgery where clinically appropriate. The literature was lacking on the benefits or risks for some interventions, such as switching specific single-use supplies to reusables or implementing a hub-and-spoke-style operating room setup. Many of the advocacy and education interventions have inadequate literature specific to ophthalmology but are likely to have minimal risks. CONCLUSIONS: Ophthalmologists can engage in a variety of safe and effective approaches to reduce or eliminate dangerous GHG emissions associated with cataract surgery. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Cyclopentenylcytosine (CPE-C): In Vitro and In Vivo Evaluation as an Antiviral against Adenoviral Ocular Infections.

Adenoviruses are the major cause of ocular viral infections worldwide. Currently, there is no approved antiviral treatment for these eye infections. Cyclopentenylcytosine (CPE-C) is an antiviral that has demonstrated activity against more than 20 viruses. The goals of the current study were to determine the in vitro and in vivo antiviral activity of CPE-C as well as its ocular toxicity. Antiviral activity was evaluated in vitro using standard plaque reduction assays to determine the 50% effective concentrations (EC50s) and in vivo in the Ad5/NZW rabbit ocular replication model. Ocular toxicity was determined in uninfected rabbit eyes following topical ocular application. The in vitro EC50s for CPE-C ranged from 0.03 to 0.059 µg/mL for nine adenovirus types that commonly infect the eye. Ocular toxicity testing determined CPE-C to be non-irritating or practically non-irritating by Draize scoring. In vivo, 3% CPE-C topically administered 4X or 2X daily for 7 days to adenovirus-infected eyes demonstrated effective antiviral activity compared with the negative control and comparable antiviral activity to the positive control, 0.5% cidofovir, topically administered twice daily for 7 days. We conclude CPE-C was relatively non-toxic to rabbit eyes and demonstrated potent anti-adenoviral activity in vitro and in vivo.

IgaA Protein, GumB, Has a Global Impact on the Transcriptome and Surface Proteome of Serratia marcescens.

Bacterial stress response signaling systems, like the Rcs system are triggered by membrane and cell wall damaging compounds, including antibiotics and immune system factors. These regulatory systems help bacteria survive envelope stress by altering the transcriptome resulting in protective phenotypic changes that may also influence the virulence of the bacterium. This study investigated the role of the Rcs stress response system using a clinical keratitis isolate of Serratia marcescens with a mutation in the gumB gene. GumB, an IgaA ortholog, inhibits activation of the Rcs system, such that mutants have overactive Rcs signaling. Transcriptomic analysis indicated that approximately 15% of all S. marcescens genes were significantly altered with 2-fold or greater changes in expression in the ΔgumB mutant compared to the wild type, indicating a global transcriptional regulatory role for GumB. We further investigated the phenotypic consequences of two classes of genes with altered expression in the ΔgumB mutant expected to contribute to infections: serralysin metalloproteases PrtS, SlpB, and SlpE, and type I pili coded by fimABCD. Secreted fractions from the ΔgumB mutant had reduced cytotoxicity to a corneal cell line, and could be complemented by induced expression of prtS, but not cytolysin shlBA, phospholipase phlAB, or flagellar master regulator flhDC operons. Proteomic analysis, qRT-PCR, and type I pili-dependent yeast agglutination indicated an inhibitory role for the Rcs system in adhesin production. Together these data demonstrate GumB has a global impact on S. marcescens gene expression that had measurable effects on bacterial cytotoxicity and surface adhesin production.

The Rcs Stress Response System Regulator GumB Modulates Serratia marcescens-Induced Inflammation and Bacterial Proliferation in a Rabbit Keratitis Model and Cytotoxicity In Vitro.

In this study, we tested the hypothesis that the conserved bacterial IgaA-family protein, GumB, mediates microbial pathogenesis associated with Serratia marcescens ocular infections through regulation of the Rcs stress response system. The role of the Rcs system and bacterial stress response systems for microbial keratitis is not known, and the role of IgaA proteins in mammalian pathogenesis models has only been tested with partial-function allele variants of Salmonella. Here, we observed that an Rcs-activated gumB mutant had a >50-fold reduction in proliferation compared to the wild type within rabbit corneas at 48 h and demonstrated a notable reduction in inflammation based on inflammatory signs, including the absence of hypopyons, and proinflammatory markers measured at the RNA and protein levels. The gumB mutant phenotypes could be complemented by wild-type gumB on a plasmid. We observed that bacteria with an inactivated Rcs stress response system induced high levels of ocular inflammation and restored corneal virulence to the gumB mutant. The high virulence of the ΔrcsB mutant was dependent upon the ShlA cytolysin transporter ShlB. Similar results were found for testing the cytotoxic effects of wild-type and mutant bacteria on a human corneal epithelial cell line in vitro. Together, these data indicate that GumB regulates virulence factor production through the Rcs system, and this overall stress response system is a key mediator of a bacterium's ability to induce vision-threatening keratitis.

Topical Astodrimer Sodium, a Non-Toxic Polyanionic Dendrimer, Demonstrates Antiviral Activity in an Experimental Ocular Adenovirus Infection Model.

There is no approved antiviral therapy for adenovirus (HAdV) ocular infections. Astodrimer sodium (SPL7013) is a polyanionic dendrimer with antiviral activity. The current study evaluated the ocular tolerability and anti-adenoviral efficacy of topical SPL7013 in rabbit ocular models. In a tolerability study, rabbits were treated with 3% SPL7013, vehicle, or 0.5% cidofovir. Their eyes were graded using the Draize scale. In antiviral efficacy studies, HAdV5 inoculated eyes were treated with 3% SPL7013, vehicle, or 0.5% cidofovir. Eyes were cultured for the virus on days 0, 1, 3, 4, 5, 7, 9, 11, and 14. Viral titers were determined. There were no differences in Draize scores between 3% SPL7013 and vehicle on any day. Cidofovir produced significantly higher Draize scores on day 12 than SPL7013 and vehicle. The 3% SPL7013 and 0.5% cidofovir significantly reduced daily viral titers and positive cultures per total compared with vehicle on several different days. The 3% SPL7013 and 0.5% cidofovir significantly reduced the duration of HAdV5 shedding compared to vehicle. The 3% SPL7013 demonstrated significantly more antiviral activity compared with vehicle in the Ad5/NZW rabbit ocular model. The 3% SPL7013 induced "minimal" to "practically non-irritating" Draize scores in the ocular tolerability study. Further development of astodrimer sodium as a topical antiviral therapy for adenoviral ocular infections is indicated.

The Prevalence of Bacteria, Fungi, Viruses, and Acanthamoeba From 3,004 Cases of Keratitis, Endophthalmitis, and Conjunctivitis.

PURPOSE: The definitive identification of ocular pathogens optimizes effective treatment. Although the types of ocular pathogens are known; there is less definitive information on the prevalence of causative infections including viruses, fungi, and protozoa, which is the focus of this retrospective laboratory review. METHODS: Data used for laboratory certification were reviewed for the detection of bacteria, viruses, fungi, and protozoa, from patients with infectious keratitis, endophthalmitis, and conjunctivitis. The main outcome parameter was laboratory-positive ocular infection. RESULTS: The distribution of infectious agents for keratitis (n=1,387) (2004-2018) was bacteria 72.1% (Staphylococcus aureus 20.3%, Pseudomonas aeruginosa 18%, Streptococcus spp. 8.5%, other gram-positives 12.4%, and other gram-negatives 12.9%), Herpes simplex virus 16%, fungi 6.7%, and Acanthamoeba 5.2%. For endophthalmitis, (n=770) (1993-2018), the bacterial distribution was coagulase-negative Staphylococcus 54%, Streptococcus spp. 21%, S. aureus 10%, other gram-positives 8%, and gram-negatives 7%. The distribution for conjunctivitis (n=847) (2004-2018) was Adenovirus 34%, S. aureus 25.5%, Streptococcus pneumoniae 9%, Haemophilus 9%, other gram-negatives 8.8%, other gram-positives 6%, coagulase-negative Staphylococcus 4.5% and Chlamydia 3.2%. CONCLUSION: An updated monitoring of ocular pathogens creates an awareness of the different infectious etiologies and the importance of laboratory studies. This information can determine treatment needs for infectious ocular diseases.

Postsurgical Cataract Prophylaxis With Intravitreal "Triamcinolone-Moxifloxacin" May Not Be Optimal For Preventing Endophthalmitis.

PURPOSE: Intravitreal injections of antibiotics and anti-inflammatories are used by some cataract surgeons for surgical prophylaxis. To support this prophylaxis, intravitreal triamcinolone-moxifloxacin (TM) and triamcinolone-moxifloxacin-vancomycin (TMV) were tested for preventing Staphylococcus aureus (SA) endophthalmitis in rabbits. METHODS: Trademark formulations of TM (15/1 mg/mL) and TMV (15/1/10 mg/mL) were intravitreally injected into seven groups of eight rabbits each (A-G). Before intravitreal injection, the vitreous was first challenged with clinical SA endophthalmitis isolates (5,000 colony-forming unit) with varying minimum inhibitory concentrations (MICs in µg/mL) to moxifloxacin (denoted by the MIC at the end of each group listed): A) TMV-10, B) TM-10, C) Saline-10, D) TM-2, E) Saline-2, F) TM-0.032, and G) Saline-0.032. After 24 hr, the rabbit eyes were graded for clinical endophthalmitis and cultured for viable SA. RESULTS: Rabbits treated with TMV and challenged by SA with a moxifloxacin MIC of 10 µg/mL did not present with endophthalmitis (0/8, no eyes with endophthalmitis). For SA with moxifloxacin MICs of 10.0 and 2.0 µg/mL, TM did not prevent endophthalmitis (16/16, 100% of eyes with endophthalmitis). For SA with a moxifloxacin MIC of 0.032 µg/mL, endophthalmitis was prevented with TM (0/8, no eyes with endophthalmitis). All saline-treated eyes developed endophthalmitis (23/23, 100% of eyes with endophthalmitis). CONCLUSIONS: Intravitreal monotherapy with TM did not provide consistent prevention of SA endophthalmitis, whereas intravitreal TMV successfully prevented endophthalmitis because of SA with elevated MIC values to moxifloxacin. Cataract surgeons need to be aware that vancomycin seems to be essential for intravitreal prophylaxis to cover moxifloxacin resistance.

In Vitro Evaluation of a Hypochlorous Acid Hygiene Solution on Established Biofilms.

OBJECTIVES: The purpose of this study was to determine whether a commercial formulation of hypochlorous acid hygiene solution (0.01%), Avenova, can destroy existing biofilms formed by ocular clinical bacterial isolates, including blepharitis isolates of Staphylococcus aureus and coagulase-negative staphylococci, and a keratitis isolate of Pseudomonas aeruginosa. METHODS: Biofilms grown in bacterial growth media on disposable contact lens cases were challenged with hypochlorous acid hygiene solution. At various time points, surviving bacteria were quantified by serial dilution and colony counts. Staphylococcus aureus biofilms formed on glass were challenged using a hypochlorous acid hygiene solution and imaged using vital staining and confocal laser scanning microscopy. RESULTS: Bactericidal activity (≥3 Log10; 99.9%) was observed for all tested bacterial species after a 30-min exposure. Staphylococcus aureus biofilms had a bactericidal level of killing by 10 min (P<0.01), Staphylococcus capitis by 5 min (P<0.001), Staphylococcus epidermidis by 30 min (P<0.001), and P. aeruginosa by 10 min (P<0.01). Confocal microscopy and crystal violet staining analysis of bacterial biofilms treated with hypochlorous acid solution both demonstrated that biofilm bacteria were readily killed, but biofilm structure was largely maintained. CONCLUSIONS: Hypochlorous acid (0.01%) hygiene solution was able to achieve bactericidal levels of killing of bacteria in biofilms but did not disrupt biofilm structures. Susceptibility of tested staphylococcal blepharitis isolates varied by species, with S. capitis being the most susceptible and S. epidermidis being the least susceptible.

EepR Mediates Secreted-Protein Production, Desiccation Survival, and Proliferation in a Corneal Infection Model.

Serratia marcescens is a soil- and water-derived bacterium that secretes several host-directed factors and causes hospital infections and community-acquired ocular infections. The putative two-component regulatory system composed of EepR and EepS regulates hemolysis and swarming motility through transcriptional control of the swrW gene and pigment production through control of the pigA-pigN operon. Here, we identify and characterize a role for EepR in regulation of exoenzyme production, stress survival, cytotoxicity to human epithelial cells, and virulence. Genetic analysis supports the model that EepR is in a common pathway with the widely conserved cyclic-AMP receptor protein that regulates protease production. Together, these data introduce a novel regulator of host-pathogen interactions and secreted-protein production.

Diffusion of Antimicrobials Across Silicone Hydrogel Contact Lenses.

OBJECTIVES: To measure the diffusion of topical preparations of moxifloxacin, amphotericin B (AmB), and polyhexamethylene biguanide (PHMB) through silicone hydrogel (SH) contact lenses (CLs) in vitro. METHODS: Using an in vitro model, the diffusion of three antimicrobials through SH CLs was measured. Diffused compounds were measured using a spectrophotometer at set time points over a period of 4 hr. The amount of each diffused antimicrobial was determined by comparing the experimental value with a standard curve. A biological assay was performed to validate the CL diffusion assay by testing antimicrobial activity of diffused material against lawns of susceptible bacteria (Staphylococcus epidermidis) and yeast (Saccharomyces cerevisiae). Experiments were repeated at least two times with a total of at least four independent replicates. RESULTS: Our data show detectable moxifloxacin and PHMB diffusion through SH CLs at 30 min, whereas AmB diffusion remained below the limit of detection within the 4-hr experimental period. In the biological assay, diffused moxifloxacin demonstrated microbial killing starting at 20 min on bacterial lawns, whereas PHMB and AmB failed to demonstrate killing on microbial lawns over the course of the 60-min experiment. CONCLUSIONS: In vitro diffusion assays demonstrate limited penetration of certain anti-infective agents through SH CLs. Further studies regarding the clinical benefit of using these agents along with bandage CL for corneal pathologic condition are warranted.

The Efficacy of Topical Cefiderocol Treatment of Experimental Extensively Drug-Resistant Pseudomonas aeruginosa Keratitis Is Dependent upon the State of the Corneal Epithelium.

Background: An overlooked factor in the efficacy of topical antibiotics to treat bacterial keratitis is the state of the corneal epithelium. Recently, we evaluated topical cefiderocol for the treatment of extensively drug-resistant (XDR) Pseudomonas aeruginosa (PA) keratitis in eyes with the corneal epithelium abraded. The goal of this study was to use the same model with the corneal epithelium left intact to evaluate the efficacy of cefiderocol and other antibiotics and compare the results to those of the previous study. Methods: NZW rabbit corneas with intact epithelium were inoculated with XDRPA. After 16 h, the rabbits were topically treated with cefiderocol 50 mg/mL, ciprofloxacin 0.3%, tobramycin 14 mg/mL, or saline. Following 8 h of treatment, the corneas were harvested for CFU determinations and cefiderocol concentrations. Results: Only cefiderocol significantly decreased CFU of the XDRPA strain compared with saline. The CFU in the cefiderocol and tobramycin-treated corneas for the XDRPA strain with initially intact epithelium were 1.83-1.4 Log10 greater than those produced in corneas with the abraded epithelium (p < 0.05). Cefiderocol concentrations were 5.02× less in corneas with initially intact epithelium. Conclusions: The efficacy of cefiderocol and tobramycin to treat experimental XDRPA keratitis is dependent on the state of the corneal epithelium.

Cefiderocol Is an Effective Topical Monotherapy for Experimental Extensively Drug-Resistant Pseudomonas aeruginosa Keratitis.

Purpose: To test cefiderocol, a siderophore-cephalosporin antibiotic for topical monotherapy treatment of experimental extensively drug-resistant (XDR) Pseudomonas aeruginosa keratitis. Design: Preclinical study. Subjects and Controls: Deidentified P. aeruginosa keratitis isolates, XDR P. aeruginosa from eye drop outbreak, rabbits, saline, cefiderocol 50 mg/ml, ciprofloxacin 0.3%, and tobramycin 14 mg/ml. Methods Intervention or Testing: Cefiderocol antibacterial activity against P. aeruginosa keratitis isolates (n = 135) was evaluated by minimum inhibitory concentration (MIC) testing. Ocular toxicity/tolerability and antibacterial efficacy were tested in vivo with experimental rabbit models. Corneal concentrations and stability were assessed using a bioassay. Main Outcome Measures: Minimum inhibitory concentration analysis for susceptibility, graded tests for ocular toxicity/tolerability, colony-forming unit (CFU) analysis for bacterial burden, corneal cefiderocol concentrations. Results: One hundred percent of P. aeruginosa keratitis isolates were susceptible to cefiderocol (n = 135), the MIC90 was 0.125 µg/ml including the XDR isolate (MIC = 0.125 µg/ml). Topical cefiderocol 50 mg/ml was minimally toxic to the ocular surface and was well tolerated. For the XDR P. aeruginosa isolate, topical cefiderocol 50 mg/ml, significantly decreased corneal CFU compared with ciprofloxacin 0.3%, tobramycin 14 mg/ml, and saline. In addition, tobramycin 14 mg/ml was more effective than the saline control. Mean cefiderocol corneal concentrations were 191× greater than the MIC90 of the P. aeruginosa keratitis isolates. Refrigerated cefiderocol maintained antimicrobial activity over a 1-month period. Conclusions: These results demonstrate that cefiderocol is well tolerated on rabbit corneas and is effective against P. aeruginosa keratitis isolates in vitro and was effective in vivo against an XDR isolate in a rabbit keratitis model. Given the recent outbreak of keratitis caused by this XDR P. aeruginosa, cefiderocol is a promising additional antibiotic that should be further evaluated for topical treatment of keratitis caused by antibiotic resistant P. aeruginosa. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Cefiderocol is an effective topical monotherapy for experimental extensively-drug resistant Pseudomonas aeruginosa keratitis.

Purpose: To test cefiderocol, a siderophore-cephalosporin antibiotic for topical monotherapy treatment of experimental extensively drug resistant (XDR) Pseudomonas aeruginosa keratitis. Design: Preclinical study. Subjects and Controls: Deidentified P. aeruginosa keratitis isolates, XDR P. aeruginosa from eye drop outbreak, rabbits, saline, cefiderocol 50 mg/ml, ciprofloxacin 0.3%, and tobramycin 14 mg/ml. Methods Intervention or Testing: Cefiderocol antibacterial activity against P. aeruginosa keratitis isolates (n=135) was evaluated by minimum inhibitory concentration (MIC) testing. Ocular toxicity/tolerability and antibacterial efficacy were tested in vivo with experimental rabbit models. Corneal concentrations and stability were assessed using a bioassay. Main Outcome Measures: MIC analysis for susceptibility, graded tests for ocular toxicity/tolerability, CFU analysis for bacterial burden, corneal cefiderocol concentrations. Results: 100% of P. aeruginosa keratitis isolates were susceptible to cefiderocol (n=135), the MIC90 was 0.125 µg/ml including the XDR isolate (MIC = 0.125 µg/ml). Topical cefiderocol 50 mg/ml was minimally toxic to the ocular surface and was well tolerated. For the XDR P. aeruginosa isolate, topical cefiderocol 50 mg/ml, significantly decreased corneal CFU compared to ciprofloxacin 0.3%, tobramycin 14 mg/ml, and saline. In addition, tobramycin 14 mg/ml was more effective than the saline control. Mean cefiderocol corneal concentrations were 191x greater than the MIC90 of the P. aeruginosa keratitis isolates. Refrigerated cefiderocol maintained antimicrobial activity over a one-month period. Conclusions: These results demonstrate that cefiderocol is well tolerated on rabbit corneas and is effective against P. aeruginosa keratitis isolates in vitro and was effective in vivo against an XDR isolate in a rabbit keratitis model. Given the recent outbreak of keratitis caused by this XDR P. aeruginosa, cefiderocol is a promising additional antibiotic that should be further evaluated for topical treatment of keratitis caused by antibiotic resistant P. aeruginosa.

Intra-ocular Predation of Fluoroquinolone-Resistant Pseudomonas aeruginosa and Serratia marcescens by Predatory Bacteria.

Endogenous endophthalmitis caused by Gram-negative bacteria is an intra-ocular infection that can rapidly progress to irreversible loss of vision. While most endophthalmitis isolates are susceptible to antibiotic therapy, the emergence of resistant bacteria necessitates alternative approaches to combat intraocular bacterial proliferation. In this study the ability of predatory bacteria to limit intraocular growth of Pseudomonas aeruginosa, Serratia marcescens, and Staphylococcus aureus was evaluated in a New Zealand White rabbit endophthalmitis prevention model. Predatory bacteria Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus were able to reduce proliferation of keratitis isolates of P. aeruginosa and S. marcescens. However, it was not able to significantly reduce S. aureus, which is not a productive prey for these predatory bacteria, suggesting that the inhibitory effect on P. aeruginosa requires active predation rather than an antimicrobial immune response. Similarly, UV-inactivated B. bacteriovorus were unable to prevent proliferation of P. aeruginosa. Together, these data suggest in vivo predation of Gram-negative bacteria within the intra-ocular environment.

Extending the use of biologics to mucous membranes by attachment of a binding domain.

Biologics are almost exclusively administered systemically, but localized delivery is preferable as it minimizes off-target exposure and allows more aggressive treatments. Topical application of biologics to epithelia is generally ineffective because most are covered with fluids and biologics are washed out too quickly to have significant therapeutic effects. Here we explore the idea that attaching a binding domain can serve as an "anchor" to extend the residency time of biologics on wet epithelia, allowing their effective use even with infrequent applications. We use topical application to the ocular surface as a challenging test since foreign substances are washed out especially efficiently by tear flow and blinking. Our results demonstrate that conjugation of antibodies to wheat germ agglutinin, which binds GlcNAc and sialic acid that are ubiquitously present in tissues, increases their half-life 350-fold upon application to the ocular surface in a mouse model of dry eye, a common and onerous disease in humans. Importantly, antibodies to IL-17A, IL-23, and IL-1ß conjugated to the agglutinin reduces manifestations of dry eye, even when applied just once daily. In contrast, unconjugated antibodies are ineffective. Attaching an anchor to biologics is a simple means to overcome washout and to extend their therapeutic use.

Predatory Bacteria can Reduce Pseudomonas aeruginosa Induced Corneal Perforation and Proliferation in a Rabbit Keratitis Model.

Purpose: Pseudomonas aeruginosa keratitis is a severe ocular infection that can lead to perforation of the cornea. In this study we evaluated the role of bacterial quorum sensing in generating corneal perforation and bacterial proliferation and tested whether co-injection of the predatory bacteria Bdellovibrio bacteriovorus could alter the clinical outcome. P. aeruginosa with lasR mutations were observed among keratitis isolates from a study collecting samples from India, so an isogenic lasR mutant strain of P. aeruginosa was included. Methods: Rabbit corneas were intracorneally infected with P. aeruginosa strain PA14 or an isogenic Δ lasR mutant and co-injected with PBS or B. bacteriovorus . After 24 h, eyes were evaluated for clinical signs of infection. Samples were analyzed by scanning electron microscopy, optical coherence tomography, sectioned for histology, and corneas were homogenized for CFU enumeration and for inflammatory cytokines. Results: We observed that 54% of corneas infected by wild-type PA14 presented with a corneal perforation (n=24), whereas only 4% of PA14 infected corneas that were co-infected with B. bacteriovorus perforate (n=25). Wild-type P. aeruginosa proliferation was reduced 7-fold in the predatory bacteria treated eyes. The Δ lasR mutant was less able to proliferate compared to the wild-type, but was largely unaffected by B. bacteriovorus . Conclusion: These studies indicate a role for bacterial quorum sensing in the ability of P. aeruginosa to proliferate and cause perforation of the rabbit cornea. Additionally, this study suggests that predatory bacteria can reduce the virulence of P. aeruginosa in an ocular prophylaxis model.

Predatory bacteria can reduce Pseudomonas aeruginosa induced corneal perforation and proliferation in a rabbit keratitis model.

PURPOSE: Pseudomonas aeruginosa keratitis is a severe ocular infection that can lead to perforation of the cornea. In this study we evaluated the role of bacterial quorum sensing in generating corneal perforation and bacterial proliferation and tested whether co-injection of the predatory bacteria Bdellovibrio bacteriovorus could alter the clinical outcome. P. aeruginosa with lasR mutations were observed among keratitis isolates from a study collecting samples from India, so an isogenic lasR mutant strain of P. aeruginosa was included. METHODS: Rabbit corneas were intracorneally infected with P. aeruginosa strain PA14 or an isogenic ΔlasR mutant and co-injected with PBS or B. bacteriovorus. After 24 h, eyes were evaluated for clinical signs of infection. Samples were analyzed by scanning electron microscopy, optical coherence tomography, sectioned for histology, and corneas were homogenized for CFU enumeration and for inflammatory cytokines. RESULTS: We observed that 54% of corneas infected by wild-type PA14 presented with a corneal perforation (n = 24), whereas only 4% of PA14 infected corneas that were co-infected with B. bacteriovorus perforate (n = 25). Wild-type P. aeruginosa proliferation was reduced 7-fold in the predatory bacteria treated eyes. The ΔlasR mutant was less able to proliferate compared to the wild-type, but was largely unaffected by B. bacteriovorus. CONCLUSION: These studies indicate a role for bacterial quorum sensing in the ability of P. aeruginosa to proliferate and cause perforation of the rabbit cornea. Additionally, this study suggests that predatory bacteria can reduce the virulence of P. aeruginosa in an ocular prophylaxis model.

Rise in frequency of lasR mutant Pseudomonas aeruginosa among keratitis isolates between 1993 and 2021.

Pseudomonas aeruginosa causes severe vision threatening keratitis. LasR is a transcription factor that regulates virulence associated genes in response to the quorum sensing molecule N-3-oxo-dodecanoyl-L-homoserine lactone. P. aeruginosa isolates with lasR mutations are characterized by an iridescent high sheen phenotype caused by a build-up of 2-heptyl-4-quinolone. A previous study indicated a high proportion (22 out of 101) of P. aeruginosa keratitis isolates from India between 2010 and 2016 were sheen positive and had mutations in the lasR gene, and the sheen phenotype correlated with worse clinical outcomes for patients. In this study, a longitudinal collection of P. aeruginosa keratitis isolates from Eastern North America were screened for lasR mutations by the sheen phenotype and sequencing of the lasR gene. A significant increase in the frequency of isolates with the sheen positive phenotype was observed in isolates between 1993 and 2021. Extracellular protease activity was lower among the sheen positive isolates and a defined lasR mutant. Cloned lasR alleles from the sheen positive isolates were loss of function or dominant negative and differed in sequence from previously reported ocular lasR mutant alleles. Insertion elements were present in a subset of independent isolates and may represent an endemic source from some of the isolates. Retrospective analysis of patient information suggested significantly better visual outcomes for patients with infected by sheen positive isolates. Together, these results indicate an increasing trend towards lasR mutations among keratitis isolates at a tertiary eye care hospital in the United States.

A rise in the frequency of lasR mutant Pseudomonas aeruginosa among keratitis isolates between 1993 and 2021.

Introduction: Pseudomonas aeruginosa causes vision threatening keratitis. The LasR transcription factor regulates virulence factors in response to the quorum sensing molecule N-3-oxo-dodecanoyl-L-homoserine lactone. P. aeruginosa isolates with lasR mutations are characterized by an iridescent high sheen phenotype caused by a build-up of 2-heptyl-4-quinolone. A previous study demonstrated 22% (n=101) of P. aeruginosa keratitis isolates from India between 2010 and 2016 were sheen positive lasR mutants, and the sheen phenotype correlated with worse clinical outcomes for patients. In this study, a longitudinal collection of P. aeruginosa keratitis isolates from Eastern North America were screened for lasR mutations by the sheen phenotype and sequencing of the lasR gene. Methods: Keratitis isolates (n=399) were classified by sheen phenotype. The lasR gene was cloned from a subset of isolates, sequenced, and tested for loss of function or dominant-negative status based on an azocasein protease assay. A retrospective chart review compared outcomes of keratitis patients infected by sheen positive and negative isolates. Results: A significant increase in sheen positive isolates was observed between 1993 and 2021. Extracellular protease activity was reduced among the sheen positive isolates and a defined lasR mutant. Cloned lasR alleles from the sheen positive isolates were loss of function or dominant negative and differed in sequence from previously reported ocular lasR mutant alleles. Retrospective analysis of patient information suggested significantly better visual outcomes for patients infected by sheen positive isolates. Discussion: These results indicate an increase in lasR mutations among keratitis isolates in the United States and suggest that endemic lasR mutants can cause keratitis.