Novel quinazolinone disulfide analogues as pqs quorum sensing inhibitors against Pseudomonas aeruginosa.

It is well established that the quorum sensing (QS) in Pseudomonas aeruginosa is primarily responsible for the synthesis and the release of several virulence factors including pyocyanin and are involved in biofilm formation. In the Pseudomonas quinolone signal (PQS) system, autoinducers such as PQS and HHQ bind and activate the transcription regulator protein receptor PqsR (MvfR). Targeting PqsR with competitive inhibitors could be a promising strategy to inhibit QS in P. aeruginosa to overcome antimicrobial resistance. In this study, we have designed and synthesized a series of novel quinazolinone disulfide-containing competitive inhibitor of PqsR. The most potent analogue 8q efficiently inhibited the pqs system with an IC50 value of 4.5 µM. It also showed complete suppression of pyocyanin production and a significant reduction in biofilm formation by P. aeruginosa (PAO1) with low cytotoxicity. Additionally, 8q produced synergy in combination with known antibiotics such as ciprofloxacin and tobramycin. Finally, molecular docking analysis suggested that compound 8q could bind with the ligand-binding domain of PqsR in a similar fashion to the native ligand.

The role of staphopain a in Staphylococcus aureus keratitis.

Staphylococcus aureus is a common bacterial isolate from cases of microbial keratitis. The virulence factors that contribute to its pathogenicity during this disease have not been fully resolved. The aim of the current study was to examine the effects of the extracellular protease Staphopain A on corneal virulence. Two strains were used, one Staph 38 that gives a high pathology score during keratitis and a less virulent strain ATCC 8325-4. The effect of inhibition of Staphopain by general or specific protease inhibitors on adhesion of strains to fibronectin-coated glass or PMMA was determined. This was followed by an analysis of the effect of Staphopain A on the ability of the bacteria to adhere to and invade corneal epithelial cells. Finally, the effect of inhibiting Staphopain A on pathogenesis in a mouse model of keratitis was studied. Staphopain A increased the adhesion of strains to fibronectin-coated substrata and inhibition of Staphopain A reduced adhesion. The inhibition of Staphopain A by staphostatin A significantly decreased both association with and invasion into human corneal epithelial cells by 15-fold for strain Saur38. Inhibition of Staphopain A significantly reduced the pathology associated with S. aureus keratitis, reducing the infecting numbers of bacteria from 1.8x105 to <1x104 cells/cornea (p ≤ 0.001), significantly reducing the corneal pathology score (p ≤ 0.038) and reducing the numbers of infiltrating PMNs. This study shows that Staphopain increases adhesion and invasion of corneal cells due to increasing fibronectin binding and its inhibition has a significant impact on pathogenicity of S. aureus during keratitis.

A New Era of Antibiotics: The Clinical Potential of Antimicrobial Peptides.

Antimicrobial resistance is a multifaceted crisis, imposing a serious threat to global health. The traditional antibiotic pipeline has been exhausted, prompting research into alternate antimicrobial strategies. Inspired by nature, antimicrobial peptides are rapidly gaining attention for their clinical potential as they present distinct advantages over traditional antibiotics. Antimicrobial peptides are found in all forms of life and demonstrate a pivotal role in the innate immune system. Many antimicrobial peptides are evolutionarily conserved, with limited propensity for resistance. Additionally, chemical modifications to the peptide backbone can be used to improve biological activity and stability and reduce toxicity. This review details the therapeutic potential of peptide-based antimicrobials, as well as the challenges needed to overcome in order for clinical translation. We explore the proposed mechanisms of activity, design of synthetic biomimics, and how this novel class of antimicrobial compound may address the need for effective antibiotics. Finally, we discuss commercially available peptide-based antimicrobials and antimicrobial peptides in clinical trials.

Esculentin-1a derived peptides kill Pseudomonas aeruginosa biofilm on soft contact lenses and retain antibacterial activity upon immobilization to the lens surface.

Contact lens (CL) wear is a risk factor for development of microbial keratitis, a vision threatening infection of the eye. Adverse events associated with colonization of lenses, especially by the multi-drug resistant and biofilm forming bacterium Pseudomonas aeruginosa remain a major safety issue. Therefore, novel strategies and compounds to reduce the onset of CL-associated ocular infections are needed. Recently, the activity of the frog skin-derived antimicrobial peptide Esc(1-21) and its diastereomer Esc(1-21)-1c was evaluated against both planktonic and sessile forms of this pathogen. Furthermore, Esc(1-21) was found to significantly reduce the severity of P. aeruginosa keratitis in a mouse model and preserve antipseudomonal activity in the presence of human basal tears. Here, we have analyzed the activity of the peptides on P. aeruginosa biofilm formed on soft CLs. Microbiological assays and scanning electron microscopy analysis indicated that the peptides were able to disrupt the bacterial biofilm, with the diastereomer having the greater efficacy (up to 85% killing vs no killing at 4 µM for some strains). Furthermore, upon covalent immobilization to the CL, the two peptides were found to cause more than four log reduction in the number of bacterial cells within 20 minutes and to reduce bacterial adhesion to the CL surface (77%-97% reduction) in 24 hours. Importantly, peptide immobilization was not toxic to mammalian cells and did not affect the lens characteristics. Overall, our data suggest that both peptides have great potential to be developed as novel pharmaceuticals for prevention and treatment of CL-associated P. aeruginosa keratitis.

Ciprofloxacin resistance and tolerance of Pseudomonas aeruginosa ocular isolates.

PURPOSE: Tolerance to antibiotics may occur due to changes in bacterial growth patterns and can be a precursor to development of resistance. However, there is a lack of information on the ability of ocular bacteria isolates to develop tolerance. This paper explores the tolerance to 8 different antibiotics of 61 microbial keratitis isolates of Pseudomonas aeruginosa from Australia and India using the MBC/MIC ratio, with tolerance defined by a ratio ≥ 32, and tolerance to ciprofloxacin by an agar diffusion assay. METHODS: Antibiotics used were ciprofloxacin, levofloxacin, gentamicin, tobramycin, piperacillin, imipenem, ceftazidime and polymyxin B. Isolates were sourced from microbial keratitis infections in Australia and India. Minimum bactericidal and minimum inhibitory concentration (MBC and MIC) were obtained using broth microdilution and compared to breakpoints from the Clinical Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) to determine bacterial susceptibility. Tolerance was assessed as MBC/MIC ≥ 32. An alternative method for tolerance detection (TD) was assessed with 13P. aeruginosa sensitive isolates by agar disk diffusion assay of ciprofloxacin followed by application of glucose to the agar and observation of re-growth of colonies. RESULTS: Thirty-three isolates were resistant to imipenem, 20 to ciprofloxacin, 14 to tobramycin and piperacillin, 12 to levofloxacin and ceftazidime, 8 to gentamicin, and 5 to polymyxin B. The percentage of strains resistant to levofloxacin (7 vs 30 %; p = 0.023), gentamicin (0 vs 24 %; p = 0.005) and tobramycin (4 vs 33 %; p = 0.004) was significantly greater in isolates from India.On average, strains from India exhibited notably greater MIC and MBC values compared to strains obtained from Australia. Out of 61 isolates, none displayed an MBC/MIC ratio ≥ 32. However, three sensitive isolates had low tolerance, nine had medium tolerance and one had high tolerance to ciprofloxacin with the TDtest. CONCLUSIONS: This study used two methods to determine whether P. aeruginosa strains could show tolerance to antibiotics. Using the MBC/MIC criteria no strain was considered tolerant to any of the eight antibiotics used. When 13 strains were tested for tolerance against ciprofloxacin, the most commonly used monotherapy for keratitis, one had high tolerance and nine had medium tolerance. This demonstrates the capacity of P. aeruginosa to develop tolerance which may result in therapeutic failures if inappropriate dosing regimens are used to treat keratitis.

Longevity of hand sanitisers on fingers.

CLINICAL RELEVANCE: Hand hygiene is important to reduce the spread of microbes in clinical settings. Hand sanitisers that last longer may be beneficial. BACKGROUND: Longevity of hand sanitisation products on fingers and hands may be important to help reduce microbial transmission. The current study evaluated the persistence of disinfection of three hand sanitisers. METHODS: Initially the minimum inhibitory concentrations of the hand sanitisers were determined using strains of Staphylococcus epidermidis and S. aureus. Then a cross-over study with participants randomly assigned to use three different hand sanitisers for 30 seconds was undertaken. The number of bacteria and fungi on fingers was assessed 10 and 20 minutes and 4 hours after use. The type of microbial inhibition of the capric acid sanitiser was studied by examining the effects of adding Tween 80 and lecithin to microbial agar. RESULTS: The minimum inhibitory concentration of an alcohol-based sanitiser (AS) was 10%, for the capric acid-based (CS) sanitiser was 70%, and for the quaternary ammonium-based (QS) sanitiser was < 10%. AS significantly reduced the number of microbes on fingers 10 minutes after hand washing (18.2 cfu/mL) compared to CS (59.7 cfu/mL; p < 0.0001) or QS (64.5 cfu/mL; p < 0.0001). Twenty minutes after use, microbes on fingers after AS (23 cfu/mL) or CS (16.7 cfu/mL) were significantly reduced compared to QS (72.2  cfu/mL; p < 0.0001) and the numbers on fingers after CS was significantly less than after AS (p = 0.002). Four hours after use of any hand sanitiser, the number of microbes increased to near baseline levels. The reduction in bacterial numbers was not affected by the use of neutralisers in agar (48 ± 28% reduction with, 47 ± 49% reduction without; p = 0.876). CONCLUSIONS: Hand sanitisers containing capric acid or alcohol out-performed one containing quaternary ammonium in the clinical trial and may help reduce the spread of microbes.

Antimicrobial resistance of ocular microbes and the role of antimicrobial peptides.

Isolation of antimicrobial-resistant microbes from ocular infections may be becoming more frequent. Infections caused by these microbes can be difficult to treat and lead to poor outcomes. However, new therapies are being developed which may help improve clinical outcomes. This review examines recent reports on the isolation of antibiotic-resistant microbes from ocular infections. In addition, an overview of the development of some new antibiotic therapies is given. The recent literature regarding antibiotic use and resistance, isolation of antibiotic-resistant microbes from ocular infections and the development of potential new antibiotics that can be used to treat these infections was reviewed. Ocular microbial infections are a global public health issue as they can result in vision loss which compromises quality of life. Approximately 70 per cent of ocular infections are caused by bacteria including Chlamydia trachomatis, Staphylococcus aureus, and Pseudomonas aeruginosa and fungi such as Candida albicans, Aspergillus spp. and Fusarium spp. Resistance to first-line antibiotics such as fluoroquinolones and azoles has increased, with resistance of S. aureus isolates from the USA to fluoroquinolones reaching 32 per cent of isolates and 35 per cent being methicillin-resistant (MRSA). Lower levels of MRSA (seven per cent) were isolated by an Australian study. Antimicrobial peptides, which are broad-spectrum alternatives to antibiotics, have been tested as possible new drugs. Several have shown promise in animal models of keratitis, especially treating P. aeruginosa, S. aureus or C. albicans infections. Reports of increasing resistance of ocular isolates to mainstay antibiotics are a concern, and there is evidence that for ocular surface disease this resistance translates into worse clinical outcomes. New antibiotics are being developed, but not by large pharmaceutical companies and mostly in university research laboratories and smaller biotech companies. Antimicrobial peptides show promise in treating keratitis.

The ocular surface, coronaviruses and COVID-19.

The ocular surface has been suggested as a site of infection with Coronavirus-2 (SARS-CoV-2) responsible for the coronavirus disease-19 (COVID-19). This review examines the evidence for this hypothesis, and its implications for clinical practice. Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), responsible for the COVID-19 pandemic, is transmitted by person-to-person contact, via airborne droplets, or through contact with contaminated surfaces. SARS-CoV-2 binds to angiotensin converting enzyme-2 (ACE2) to facilitate infection in humans. This review sets out to evaluate evidence for the ocular surface as a route of infection. A literature search in this area was conducted on 15 April 2020 using the Scopus database. In total, 287 results were returned and reviewed. There is preliminary evidence for ACE2 expression on corneal and conjunctival cells, but most of the other receptors to which coronaviruses bind appear to be found under epithelia of the ocular surface. Evidence from animal studies is limited, with a single study suggesting viral particles on the eye can travel to the lung, resulting in very mild infection. Coronavirus infection is rarely associated with conjunctivitis, with occasional cases reported in patients with confirmed COVID-19, along with isolated cases of conjunctivitis as a presenting sign. Coronaviruses have been rarely isolated from tears or conjunctival swabs. The evidence suggests coronaviruses are unlikely to bind to ocular surface cells to initiate infection. Additionally, hypotheses that the virus could travel from the nasopharynx or through the conjunctival capillaries to the ocular surface during infection are probably incorrect. Conjunctivitis and isolation of the virus from the ocular surface occur only rarely, and overwhelmingly in patients with confirmed COVID-19. Necessary precautions to prevent person-to-person transmission should be employed in clinical practice throughout the pandemic, and patients should be reminded to maintain good hygiene practices.

Tear film, contact lenses and tear biomarkers.

This article summarises research undertaken since 1993 in the Willcox laboratory at the University of New South Wales, Sydney on the tear film, its interactions with contact lenses, and the use of tears as a source of biomarkers for ocular and non-ocular diseases. The proteome, lipidome and glycome of tears all contribute to important aspects of the tear film, including its structure, its ability to defend the ocular surface against microbes and to help heal ocular surface injuries. The tear film interacts with contact lenses in vivo and interactions between tears and lenses can affect the biocompatibility of lenses, and may be important in mediating discomfort responses during lens wear. Suggestions are made for follow-up research.

A laboratory assessment of factors that affect bacterial adhesion to contact lenses.

Adhesion of pathogenic microbes, particularly bacteria, to contact lenses is implicated in contact lens related microbial adverse events. Various in vitro conditions such as type of bacteria, the size of initial inoculum, contact lens material, nutritional content of media, and incubation period can influence bacterial adhesion to contact lenses and the current study investigated the effect of these conditions on bacterial adhesion to contact lenses. There was no significant difference in numbers of bacteria that adhered to hydrogel etafilcon A or silicone hydrogel senofilcon A contact lenses. Pseudomonas aeruginosa adhered in higher numbers compared to Staphylococcus aureus. Within a genera/species, adhesion of different bacterial strains did not differ appreciably. The size of initial inoculum, nutritional content of media, and incubation period played significant roles in bacterial adhesion to lenses. A set of in vitro assay conditions to help standardize adhesion between studies have been recommended.

Management and treatment of contact lens-related Pseudomonas keratitis.

Pubmed and Medline were searched for articles referring to Pseudomonas keratitis between the years 2007 and 2012 to obtain an overview of the current state of this disease. Keyword searches used the terms "Pseudomonas" + "Keratitis" limit to "2007-2012", and ["Ulcerative" or "Microbial"] + "Keratitis" + "Contact lenses" limit to "2007-2012". These articles were then reviewed for information on the percentage of microbial keratitis cases associated with contact lens wear, the frequency of Pseudomonas sp. as a causative agent of microbial keratitis around the world, the most common therapies to treat Pseudomonas keratitis, and the sensitivity of isolates of Pseudomonas to commonly prescribed antibiotics. The percentage of microbial keratitis associated with contact lens wear ranged from 0% in a study from Nepal to 54.5% from Japan. These differences may be due in part to different frequencies of contact lens wear. The frequency of Pseudomonas sp. as a causative agent of keratitis ranged from 1% in Japan to over 50% in studies from India, Malaysia, and Thailand. The most commonly reported agents used to treat Pseudomonas keratitis were either aminoglycoside (usually gentamicin) fortified with a cephalosporin, or monotherapy with a fluoroquinolone (usually ciprofloxacin). In most geographical areas, most strains of Pseudomonas sp. (≥95%) were sensitive to ciprofloxacin, but reports from India, Nigeria, and Thailand reported sensitivity to this antibiotic and similar fluoroquinolones of between 76% and 90%.

Role of carnitine in disease.

Carnitine is a conditionally essential nutrient that plays a vital role in energy production and fatty acid metabolism. Vegetarians possess a greater bioavailability than meat eaters. Distinct deficiencies arise either from genetic mutation of carnitine transporters or in association with other disorders such as liver or kidney disease. Carnitine deficiency occurs in aberrations of carnitine regulation in disorders such as diabetes, sepsis, cardiomyopathy, malnutrition, cirrhosis, endocrine disorders and with aging. Nutritional supplementation of L-carnitine, the biologically active form of carnitine, is ameliorative for uremic patients, and can improve nerve conduction, neuropathic pain and immune function in diabetes patients while it is life-saving for patients suffering primary carnitine deficiency. Clinical application of carnitine holds much promise in a range of neural disorders such as Alzheimer's disease, hepatic encephalopathy and other painful neuropathies. Topical application in dry eye offers osmoprotection and modulates immune and inflammatory responses. Carnitine has been recognized as a nutritional supplement in cardiovascular disease and there is increasing evidence that carnitine supplementation may be beneficial in treating obesity, improving glucose intolerance and total energy expenditure.

Secretory immunoglobulin A in tears:functions and changes during contact lens wear.

INTRODUCTION: Secretory immunoglobulin A (sIgA) is the predominant immunoglobulin in tears. The role of sIgA in defending the eye against pathogens has not been established clearly. There have been conflicting reports about the effect of contact lens wear on the concentration of sIgA in tears. This study was conducted to elucidate the role of sIgA in ocular defence and to determine the effect of contact lens wear on sIgA concentration. METHODS: Tears were collected from contact lens wearers and non-wearers using micro-capillary tubes. The concentration of sIgA was evaluated using an in-house ELISA. The specificity of sIgA to a strain of P. aeruginosa was examined using a fluorescent assay and the ability of neutrophils to phagocytose sIgA coated bacteria were assessed by plate counts. RESULTS: Tears contained sIgA that reacted to P. aeruginosa. P. aeruginosa coated with sIgA was phagocytosed by the neutrophils. The level of sIgA and the level of sIgA specific to P. aeruginosa in the tears of contact lens wearers were significantly reduced. CONCLUSIONS: These results indicate that contact lens wear significantly alters the level of sIgA in tears which may lead to changes in the ability of the ocular surface to defend itself against potential pathogens.