The role of naturally acquired intracellular Pseudomonas aeruginosa in the development of Acanthamoeba keratitis in an animal model.

BACKGROUND: Acanthamoeba is an environmental host for various microorganisms. Acanthamoeba is also becoming an increasingly important pathogen as a cause of keratitis. In Acanthamoeba keratitis (AK), coinfections involving pathogenic bacteria have been reported, potentially attributed to the carriage of microbes by Acanthamoeba. This study assessed the presence of intracellular bacteria in Acanthamoeba species recovered from domestic tap water and corneas of two different AK patients and examined the impact of naturally occurring intracellular bacteria within Acanthamoeba on the severity of corneal infections in rats. METHODOLOGY/PRINCIPAL FINDINGS: Household water and corneal swabs were collected from AK patients. Acanthamoeba strains and genotypes were confirmed by sequencing. Acanthamoeba isolates were assessed for the presence of intracellular bacteria using sequencing, fluorescence in situ hybridization (FISH), and electron microscopy. The viability of the bacteria in Acanthamoeba was assessed by labelling with alkyne-functionalized D-alanine (alkDala). Primary human macrophages were used to compare the intracellular survival and replication of the endosymbiotic Pseudomonas aeruginosa and a wild type strain. Eyes of rats were challenged intrastromally with Acanthamoeba containing or devoid of P. aeruginosa and evaluated for the clinical response. Domestic water and corneal swabs were positive for Acanthamoeba. Both strains belonged to genotype T4F. One of the Acanthamoeba isolates harboured P. aeruginosa which was seen throughout the Acanthamoeba's cytoplasm. It was metabolically active and could be seen undergoing binary fission. This motile strain was able to replicate in macrophage to a greater degree than strain PAO1 (p<0.05). Inoculation of Acanthamoeba containing the intracellular P. aeruginosa in rats eyes resulted in a severe keratitis with increased neutrophil response. Acanthamoeba alone induced milder keratitis. CONCLUSIONS/SIGNIFICANCE: Our findings indicate the presence of live intracellular bacteria in Acanthamoeba can increase the severity of acute keratitis in vivo. As P. aeruginosa is a common cause of keratitis, this may indicate the potential for these intracellular bacteria in Acanthamoeba to lead to severe polymicrobial keratitis.

Antiviral Activity of Anthranilamide Peptidomimetics against Herpes Simplex Virus 1 and a Coronavirus.

The development of potent antiviral agents is of utmost importance to combat the global burden of viral infections. Traditional antiviral drug development involves targeting specific viral proteins, which may lead to the emergence of resistant strains. To explore alternative strategies, we investigated the antiviral potential of antimicrobial peptidomimetic compounds. In this study, we evaluated the antiviral potential of 17 short anthranilamide-based peptidomimetic compounds against two viruses: Murine hepatitis virus 1 (MHV-1) which is a surrogate of human coronaviruses and herpes simplex virus 1 (HSV-1). The half-maximal inhibitory concentration (IC50) values of these compounds were determined in vitro to assess their potency as antiviral agents. Compounds 11 and 14 displayed the most potent inhibitory effects with IC50 values of 2.38 µM, and 6.3 µM against MHV-1 while compounds 9 and 14 showed IC50 values of 14.8 µM and 13 µM against HSV-1. Multiple antiviral assessments and microscopic images obtained through transmission electron microscopy (TEM) collectively demonstrated that these compounds exert a direct influence on the viral envelope. Based on this outcome, it can be concluded that peptidomimetic compounds could offer a new approach for the development of potent antiviral agents.

A review of the antiviral activity of cationic antimicrobial peptides.

Viral epidemics are occurring frequently, and the COVID-19 viral pandemic has resulted in at least 6.5 million deaths worldwide. Although antiviral therapeutics are available, these may not have sufficient effect. The emergence of resistant or novel viruses requires new therapies. Cationic antimicrobial peptides are agents of the innate immune system that may offer a promising solution to viral infections. These peptides are gaining attention as possible therapies for viral infections or for use as prophylactic agents to prevent viral spread. This narrative review examines antiviral peptides, their structural features, and mechanism of activity. A total of 156 cationic antiviral peptides were examined for information of their mechanism of action against both enveloped and non-enveloped viruses. Antiviral peptides can be isolated from various natural sources or can be generated synthetically. The latter tend to be more specific and effective and can be made to have a broad spectrum of activity with minimal side effects. Their unique properties of being positively charged and amphipathic enable their main mode of action which is to target and disrupt viral lipid envelopes, thereby inhibiting viral entry and replication. This review offers a comprehensive summary of the current understanding of antiviral peptides, which could potentially aid in the design and creation of novel antiviral medications.

Human tear metabolites associated with nucleoside-signalling pathways in bacterial keratitis.

OBJECTIVE: The study aimed to profile and quantify tear metabolites associated with bacterial keratitis using both untargeted and targeted metabolomic platforms. METHODS: Untargeted metabolomic analysis using liquid-chromatography-Q Exactive-HF mass-spectrometry explored tear metabolites significantly associated with bacterial keratitis (n = 6) compared to healthy participants (n = 6). Differential statistics and principal component analysis determined meaningful metabolite differences between cases and controls. Purines and nucleosides were further quantified and compared between 15 cases and 15 controls in the targeted metabolomic platform using TSQ quantum access triple quadrupole mass spectrometry. Compound quantification was done by plotting the calibration curves and the difference in the compound levels was evaluated using the Wilcoxon rank-sum test. RESULTS: In the untargeted analysis, 49 tear metabolites (27 upregulated and 22 downregulated) were differentially expressed between cases and controls. The untargeted analysis indicated that the purine metabolism pathway was the most affected by bacterial keratitis. Metabolite quantification in the targeted analysis further confirmed the upregulation of xanthine (P = 0.02) and downregulation of adenine (P < 0.0001), adenosine (P < 0.0001) and cytidine (P < 0.0001) in the tears of participants with bacterial keratitis compared to that of healthy participants. CONCLUSIONS: Bacterial keratitis significantly changes the tear metabolite profile, including five major compound classes such as indoles, amino acids, nucleosides, carbohydrates, and steroids. This study also indicates that tear fluids can be used to map the metabolic pathways and uncover metabolic markers associated with bacterial keratitis. Conceivably, the inhibition of nucleoside synthesis may contribute to the pathophysiology of bacterial keratitis because nucleosides are required for maintaining cellular energy homeostasis and immune adaptability.

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.

The Relationship between Ciprofloxacin Resistance and Genotypic Changes in S. aureus Ocular Isolates.

Staphylococcus aureus (S. aureus) is a frequent cause of eye infections with some isolates exhibiting increased antimicrobial resistance to commonly prescribed antibiotics. The increasing resistance of ocular S. aureus to ciprofloxacin is a serious concern as it is a commonly used as a first line antibiotic to treat S. aureus keratitis. This study aimed to analyse genetic mutations in the genomes of 25 S. aureus isolates from infections or non-infectious ocular conditions from the USA and Australia and their relationship to ciprofloxacin resistance. Overall, 14/25 isolates were phenotypically resistant to ciprofloxacin. All isolates were analyzed for mutations in their quinolone resistance-determining regions (QRDRs) and efflux pump genes. Of the fourteen resistant isolates, 9/14 had ciprofloxacin resistance mutations within their QRDRs, at codons 80 or 84 within the parC subunit and codon 84 within the gyrA subunit of DNA gyrase. The highest resistance (MIC = 2560 µg/mL) was associated with two SNPs in both gyrA and parC. Other resistant isolates (3/14) had mutations within norB. Mutations in genes of other efflux pumps and their regulator (norA, norC, mepA, mdeA, sepA, sdrM, mepR, arlR, and arlS) or the DNA mismatch repair (MMR) system (mutL and mutS) were not associated with increased resistance to ciprofloxacin. The functional mutations associated with ciprofloxacin resistance in QRDRs (gyrA and parC) and norB suggests that these are the most common reasons for ciprofloxacin resistance in ocular isolates. Novel SNPs of gyrA Glu-88-Leu, Asn-860-Thr and Thr-845-Ala and IIe-855-Met, identified in this study, need further gene knock out/in studies to better understand their effect on ciprofloxacin resistance.

Genomics of Staphylococcus aureus Strains Isolated from Infectious and Non-Infectious Ocular Conditions.

Staphylococcus aureus is a major cause of ocular infectious (corneal infection or microbial keratitis (MK) and conjunctivitis) and non-infectious corneal infiltrative events (niCIE). Despite the significant morbidity associated with these conditions, there is very little data about specific virulence factors associated with the pathogenicity of ocular isolates. A set of 25 S. aureus infectious and niCIEs strains isolated from USA and Australia were selected for whole genome sequencing. Sequence types and clonal complexes of S. aureus strains were identified by using multi-locus sequence type (MLST). The presence or absence of 128 virulence genes was determined by using the virulence finder database (VFDB). Differences between infectious (MK + conjunctivitis) and niCIE isolates from USA and Australia for possession of virulence genes were assessed using the chi-square test. The most common sequence types found among ocular isolates were ST5, ST8 while the clonal complexes were CC30 and CC1. Virulence genes involved in adhesion (ebh, clfA, clfB, cna, sdrD, sdrE), immune evasion (chp, esaD, esaE, esxB, esxC, esxD), and serine protease enzymes (splA, splD, splE, splF) were more commonly observed in infectious strains (MK + conjunctivitis) than niCIE strains (p = 0.004). Toxin genes were present in half of infectious (49%, 25/51) and niCIE (51%, 26/51) strains. USA infectious isolates were significantly more likely to possess splC, yent1, set9, set11, set36, set38, set40, lukF-PV, and lukS-PV (p < 0.05) than Australian infectious isolates. MK USA strains were more likely to possesses yent1, set9, set11 than USA conjunctivitis strains (p = 0.04). Conversely USA conjunctivitis strains were more likely to possess set36 set38, set40, lukF-PV, lukS-PV (p = 0.03) than MK USA strains. The ocular strain set was then compared to 10 fully sequenced non-ocular S. aureus strains to identify differences between ocular and non-ocular isolates. Ocular isolates were significantly more likely to possess cna (p = 0.03), icaR (p = 0.01), sea (p = 0.001), set16 (p = 0.01), and set19 (p = 0.03). In contrast non-ocular isolates were more likely to possess icaD (p = 0.007), lukF-PV, lukS-PV (p = 0.01), selq (p = 0.01), set30 (p = 0.01), set32 (p = 0.02), and set36 (p = 0.02). The clones ST5, ST8, CC30, and CC1 among ocular isolates generally reflect circulating non-ocular pathogenic S. aureus strains. The higher rates of genes in infectious and ocular isolates suggest a potential role of these virulence factors in ocular diseases.

Virulence Genes of Staphylococcus aureus Associated With Keratitis, Conjunctivitis, and Contact Lens-Associated Inflammation.

Purpose: Staphylococcus aureus, cause a range of ocular diseases in humans, including noninfectious corneal infiltrative events (niCIE), infectious conjunctivitis and sight threatening microbial keratitis (MK). This study aimed to determine the possession of known virulence genes of S. aureus associated with MK and conjunctivitis, in strains isolated from these conditions and niCIE. Methods: Sixty-three S. aureus strains-23 from MK, 26 from conjunctivitis, and 14 from niCIE-were evaluated for possession of genes. Polymerase chain reaction was used for the detection of mecA and 10 known virulence genes involved in MK (clfA, fnbpA, eap, coa, scpA, sspB, sspA, hla, hld, and hlg), 2 associated with conjunctivitis (pvl and seb). Results: mecA was present in 35% of infections and 7% of niCIE strains (P = 0.05). It was not seen in infection strains from Australia. Adhesion genes were found in all strains except clfA, which was found in 75% of infection and 93% of niCIE strains. Invasion genes were found in higher frequency in infections strains-hlg (100% vs. 85%; P = 0.04) and hld (94% vs. 50%; P = 0.005)-compared with niCIE strains. Evasion genes were common in infection strains except scpA, which was found at a significantly higher frequency in niCIE strains (86%) compared with infection strains (45%; P = 0.001). Conclusions: The higher rates of hlg and hld in strains isolated from infections than niCIE may have a role in pathogenesis, whereas scpA may be an important virulence factor during niCIEs. Translational Relevance: This study has identified virulence factors involved in the ocular pathogenesis of S. aureus infections and niCIE.

Ability of Essential Oil Vapours to Reduce Numbers of Culturable Aerosolised Coronavirus, Bacteria and Fungi.

Transmission of pathogens present in the indoor air can occur through aerosols. This study evaluated the efficacy of an evaporated mix of essential oils to reduce the numbers of culturable aerosolized coronavirus, bacterium and fungus. The essential oil-containing gel was allowed to vaporize inside a glass chamber for 10 or 20 min. Aerosols of a surrogate of SARS-CoV-2, murine hepatitis coronavirus MHV-1, Escherichia coli or Aspergillus flavus spores were produced using a collision nebuliser and passed through the essential oil vapours, then collected on a six-stage Andersen sampler. The six-stages of the impact sampler capture aerosols in sizes ranging from 7 to 0.65 µm. The number of culturable microbes present in the aerosols collected in the different stages were enumerated and compared to the number of culturable microbes in control microbial aerosols that were not exposed to the evaporated essential oils. After 10 and 20 min evaporation, the essential oils reduced the numbers of culturable aerosolized coronavirus by 48% (log10 reduction = 0.3; p = 0.002 vs. control) and 53% (log10 reduction = 0.3; p = 0.001 vs. control), respectively. The essential oils vaporised for 10 min, reduced the number of viable E. coli by 51% (log10 reduction = 0.3; p = 0.032 vs. control). The Aspergillus flavus spores were mostly observed in the larger aerosols (7.00 µm to 2.10 µm) and the essential oils vaporised for 10 min reduced the number of viable spores by 72% (log10 reduction = 0.6; p = 0.008 vs. control). The vapours produced by a gel containing naturally occurring essential oils were able to significantly reduce the viable numbers of aerosolized coronavirus, bacteria and fungal spores. The antimicrobial gel containing the essential oils may be able to reduce aerosol transmission of microbes when used in domestic and workplace settings.

Susceptibility of Ocular Staphylococcus aureus to Antibiotics and Multipurpose Disinfecting Solutions.

Staphylococcus aureus is a frequent cause of ocular surface infections worldwide. Of these surface infections, those involving the cornea (microbial keratitis) are most sight-threatening. S. aureus can also cause conjunctivitis and contact lens-related non-infectious corneal infiltrative events (niCIE). The aim of this study was to determine the rates of resistance of S. aureus isolates to antibiotics and disinfecting solutions from these different ocular surface conditions. In total, 63 S. aureus strains from the USA and Australia were evaluated; 14 were from niCIE, 26 from conjunctivitis, and 23 from microbial keratitis (MK). The minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC) of all the strains to ciprofloxacin, ceftazidime, oxacillin, gentamicin, vancomycin, chloramphenicol, azithromycin, and polymyxin B were determined. The MIC and MBC of the niCIE strains to contact lens multipurpose disinfectant solutions (MPDSs) was determined. All isolates were susceptible to vancomycin (100%). The susceptibility to other antibiotics decreased in the following order: gentamicin (98%), chloramphenicol (76%), oxacillin (74%), ciprofloxacin (46%), ceftazidime (11%), azithromycin (8%), and polymyxin B (8%). In total, 87% of all the isolates were multidrug resistant and 17% of the isolates from microbial keratitis were extensively drug resistant. The microbial keratitis strains from Australia were usually susceptible to ciprofloxacin (57% vs. 11%; p = 0.04) and oxacillin (93% vs. 11%; p = 0.02) compared to microbial keratitis isolates from the USA. Microbial keratitis isolates from the USA were less susceptible (55%) to chloramphenicol compared to conjunctivitis strains (95%; p = 0.01). Similarly, 75% of conjunctivitis strains from Australia were susceptible to chloramphenicol compared to 14% of microbial keratitis strains (p = 0.04). Most (93%) strains isolated from contact lens wearers were killed in 100% MPDS, except S. aureus 27. OPTI-FREE PureMoist was the most active MPDS against all strains with 35% of strains having an MIC ≤ 11.36%. There was a significant difference in susceptibility between OPTI-FREE PureMoist and Biotrue (p = 0.02). S. aureus non-infectious CIE strains were more susceptible to antibiotics than conjunctivitis strains and conjunctivitis strains were more susceptible than microbial keratitis strains. Microbial keratitis strains from Australia (isolated between 2006 and 2018) were more susceptible to antibiotics in comparison with microbial keratitis strains from the USA (isolated in 2004). Most of the strains were multidrug-resistant. There was variability in the susceptibility of contact lens isolates to MPDSs with one S. aureus strain, S. aureus 27, isolated from niCIE, in Australia in 1997 being highly resistant to all four MPDSs and three different types of antibiotics. Knowledge of the rates of resistance to antibiotics in different conditions and regions could help guide treatment of these diseases.

Effect of Hygiene Procedures on Lens Case Contamination with Povidone-Iodine or Multipurpose Disinfecting Solutions.

SIGNIFICANCE: A multipurpose disinfecting solution (MPDS), which contains povidone-iodine (PI) as a disinfectant, has high disinfecting efficacy not only on planktonic bacterium but also on the case biofilms. The addition of case hygiene practice removed more bacteria from cases than MPDS alone. PURPOSE: This study compared the ability of two MPDSs, one containing PI and another containing polyaminopropyl biguanide and polyquaternium, to reduce bacterial numbers in solution or adhered to the cases following case hygiene procedures. METHODS: Bacterial strains (Delftia acidovorans, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Serratia marcescens, Staphylococcus aureus, and Staphylococcus epidermidis) were exposed to the MPDSs for the recommended disinfection times, and the viable number evaluated according to ISO 14729. Cases were inoculated with bacterial strains and incubated for 24 hours to allow for biofilm formation. Cases were disinfected with both disinfecting solutions for 4 hours and rinsed, followed by recapping or air-drying, or tissue-wiping and air-drying for 18 hours. The number of survivors was counted using standard culture techniques. RESULTS: Both products exceeded the recommended 3-log reduction against planktonic bacteria. Regarding biofilm, after rinsing and recapping wet, the numbers of D. acidovorans (mean difference [95% confidence interval] log10 colony-forming units per case, -2.9 [0.8 to -4.6], P < .01), P. aeruginosa (-2.0 [0.5 to -3.1], P < .01), S. marcescens (-1.7 [0.8 to -3.5], P < .05), and S. epidermidis (-2.1 [0.6 to -3.5], P < .05) in PI cases were significantly lower than in the dual-disinfectant MPDS storage cases. After air-drying, the PI storage cases had significantly lower numbers of S. maltophilia (-2.6 [0.6 to -4.0], P < .01), D. acidovorans (-1.6 [0.7 to -3.3], P < .05), and S. aureus (-1.6 [0.7 to -3.1], P < .05). The addition of tissue-wiping reduced the bacterial numbers in the MPDS storage cases to levels in the PI storage cases. CONCLUSIONS: Contact lens users should be recommended to tissue-wipe and air-dry their lens storage cases after disinfection with regular MPDS.

Factors Affecting Microbial Contamination on the Back Surface of Worn Soft Contact Lenses.

SIGNIFICANCE: The results of this study demonstrate that Smart Touch Technology packaging, which is designed to reduce and simplify contact lens handling before insertion, is effective in reducing the frequency of bacterial contamination of the back surface of contact lenses after short-term wear. PURPOSE: The purpose of this study was to investigate the effect of lens packaging type, chelating agent, and finger contamination on microbial contamination on the back surface of worn soft contact lenses. METHODS: Twenty-five subjects completed each contralateral lens wear comparison in this randomized study: Smart Touch Technology versus conventional blister packaging for (1) silicone hydrogel lenses with ethylenediaminetetraacetic acid (EDTA) and (2) hydrogel lenses without EDTA in the packaging, and (3) silicone hydrogel lenses without EDTA versus hydrogel lenses with EDTA both in Smart Touch Technology packaging. Participants washed hands, underwent finger swabs, and inserted the lenses. After 45 minutes, lenses were removed aseptically and the posterior lens surfaces cultured. RESULTS: Thirty-eight subjects (average age, 30.9 ± 12.5 years) participated in this study. Overall, the level of back surface contamination was low for both lens materials, ranging from 0 to 43 colony-forming unit (CFU)/lens for the silicone hydrogel and 0 to 17 CFU/lens for the hydrogel lenses. The proportion of lenses with zero back surface contamination ranged from 16 to 64% for silicone hydrogel lenses and 28 to 64% for hydrogel lenses. Contact lenses from conventional packaging containing EDTA had 3.38 times increased risk (95% confidence interval [CI], 1.02 to 11.11; P = .05) of contamination being present compared with lenses from Smart Touch packaging with EDTA. Contact lenses from conventional packaging without EDTA had 3.4 times increased risk (95% CI, 1.02 to 11.36; P = .05) of contamination being present compared with Smart Touch packaging without EDTA, and silicone hydrogel lenses had a 6.28 times increased risk (95% CI, 1.65 to 23.81; P = .007) of contamination being present compared with hydrogels. The median (interquartile range) number of bacteria isolated from fingers used to perform lens insertion after handwashing but before lens insertion was not significantly different between the silicone hydrogel and hydrogel lenses (63.7 [204.2] vs. 59 [84.5], P = .09). Finger contamination was not significantly associated with lens contamination in the presence or absence of EDTA. CONCLUSIONS: Smart Touch Technology packaging was effective in reducing the proportion of contaminated lenses. Although silicone hydrogel lenses were more likely to be contaminated, the presence of EDTA ameliorated this effect. Finger contamination was not associated with lens contamination.

CLEAR - Contact lens complications.

Contact lens-related complications are common, affecting around one third of wearers, although most are mild and easily managed. Contact lenses have well-defined anatomical and physiological effects on the ocular surface and can result in other consequences due to the presence of a biologically active material. A contact lens interacts with the tear film, ocular surface, skin, endogenous and environmental microorganisms, components of care solutions and other antigens which may result in disease specific to contact lens wear, such as metabolic or hypersensitivity disorders. Contact lens wear may also modify the epidemiology or pathophysiology of recognised conditions, such as papillary conjunctivitis or microbial keratitis. Wearers may also present with intercurrent disease, meaning concomitant or pre-existing conditions unrelated to contact lens wear, such as allergic eye disease or blepharitis, which may complicate the diagnosis and management of contact lens-related disease. Complications can be grouped into corneal infection (microbial keratitis), corneal inflammation (sterile keratitis), metabolic conditions (epithelial: microcysts, vacuoles, bullae, tight lens syndrome, epithelial oedema; stromal: superficial and deep neovascularisation, stromal oedema [striae/folds], endothelial: blebs, polymegethism/ pleomorphism), mechanical (corneal abrasion, corneal erosion, lens binding, warpage/refractive error changes; superior epithelial arcuate lesion, mucin balls, conjunctival epithelial flaps, ptosis, discomfort), toxic and allergic disorders (papillary conjunctivitis, solution-induced corneal staining, incomplete neutralisation of peroxide, Limbal Stem Cell Deficiency), tear resurfacing disorders/dry eye (contact lens-induced dry eye, Meibomian gland dysfunction, lid wiper epitheliopathy, lid parallel conjunctival folds, inferior closure stain, 3 and 9 o'clock stain, dellen, dimple veil) or contact lens discomfort. This report summarises the best available evidence for the classification, epidemiology, pathophysiology, management and prevention of contact lens-related complications in addition to presenting strategies for optimising contact lens wear.

Understanding clinical and immunological features associated with Pseudomonas and Staphylococcus keratitis.

Pseudomonas aeruginosa and Staphylococcus aureus are the two dominant Gram-negative and -positive species, respectively, isolated from patients with contact lens-related bacterial keratitis. The clinical features of bacterial keratitis vary, such that timely differential diagnosis can be challenging, which may cause a delay in diagnosis resulting in poorer outcome. This review aims to explore the current understanding of clinical and immunological features associated with contact lens-related P. aeruginosa and S. aureus keratitis based on currently available evidence. Firstly, the review characterises contact lens-related P. aeruginosa and S. aureus keratitis, based on clinical features and prognostic factors. Secondly, the review describes the primary immune response associated with a bacterial infection in in-vivo non-scratch contact lens-wearing animal models, colonised by bacteria on contact lens and topical administration of bacteria on the cornea. Finally, the review discusses the role of macrophage inflammatory protein-2 (MIP-2) and intercellular adhesion molecule (ICAM-1) in neutrophil recruitment based on both in-vivo scratch models of bacterial keratitis and bacterial challenged in cell culture models.

Bacterial biofilm in silver-impregnated contact lens cases.

PURPOSE: This study investigated the efficacy of pre-conditioning lens cases on bacterial biofilm formation and removal. METHODS: Silver impregnated (MicroBlock / ProGuard™ & i-Clean) and control storage cases were pre-conditioned for 24 h with their respective multipurpose solutions (MPDSs). Cases were then inoculated with 2 ml of 106 CFU/mL of ocular isolates of either P. aeruginosa or S. aureus and incubated for 48 h. Cases were subsequently disinfected (4-6 hours) as per the manufacturer's recommended disinfecting time (MRDT) followed by the recommended case hygiene procedures - recapping wet (MicroBlock / ProGuard™ cases only) or rinse and air-dry or rinse, tissue-wipe and air dry (mechanical disruption). Surviving bacteria were enumerated using standard techniques. RESULTS: Pre-conditioning the MicroBlock / ProGuard™ cases with MPDS significantly reduced biofilm formation (-1.1 log10 CFU, p < 0.01 for P. aeruginosa & -1.3 log10, p < 0.001, CFU for S. aureus) compared to the i-Clean lens cases. Maintaining the MicroBlock / ProGuard™ lens cases wet after the MRDT resulted in partial removal of bacterial biofilms (-2.9 log10 CFU, p < 0.001 for P. aeruginosa and -2.6 log10 CFU, p < 0.001 for S. aureus). Air-drying of all three types of lens storage cases after MRDT significantly reduced the bacterial biofilm (-5.4 log10 CFU, p < 0.001 for P. aeruginosa and -3.5 log10 CFU, p < 0.001 for S. aureus). Mechanical disruption produced the greatest reduction in the levels of bacterial biofilm in all 3 types of lens cases tested (-6.8 log10 CFU, p < 0.001 for P. aeruginosa and -4.5 log10 CFU, p < 0.001 for S. aureus). Synergi MPDS was significantly better than AQuify MPDS in removing bacterial biofilm from all 3 lens case types for case hygiene treatments with an air-drying step. CONCLUSION: Pre-conditioning of silver-impregnated ProGuard™ lens cases inhibited initial bacterial biofilm formation. Synergi MPDS was more effective than AQuify MPDS in removing bacterial biofilm in silver impregnated cases and tissue-wiping significantly improved biofilm removal.

Accessory genome of the multi-drug resistant ocular isolate of Pseudomonas aeruginosa PA34.

Bacteria can acquire an accessory genome through the horizontal transfer of genetic elements from non-parental lineages. This leads to rapid genetic evolution allowing traits such as antibiotic resistance and virulence to spread through bacterial communities. The study of complete genomes of bacterial strains helps to understand the genomic traits associated with virulence and antibiotic resistance. We aimed to investigate the complete accessory genome of an ocular isolate of Pseudomonas aeruginosa strain PA34. We obtained the complete genome of PA34 utilising genome sequence reads from Illumina and Oxford Nanopore Technology followed by PCR to close any identified gaps. In-depth genomic analysis was performed using various bioinformatics tools. The susceptibility to heavy metals and cytotoxicity was determined to confirm expression of certain traits. The complete genome of PA34 includes a chromosome of 6.8 Mbp and two plasmids of 95.4 Kbp (pMKPA34-1) and 26.8 Kbp (pMKPA34-2). PA34 had a large accessory genome of 1,213 genes and had 543 unique genes not present in other strains. These exclusive genes encoded features related to metal and antibiotic resistance, phage integrase and transposons. At least 24 genomic islands (GIs) were predicated in the complete chromosome, of which two were integrated into novel sites. Eleven GIs carried virulence factors or replaced pathogenic genes. A bacteriophage carried the aminoglycoside resistance gene (AAC(3)-IId). The two plasmids carried other six antibiotic resistance genes. The large accessory genome of this ocular isolate plays a large role in shaping its virulence and antibiotic resistance.

The Effect of Microblepharon Exfoliation on Clinical Correlates of Contact Lens Discomfort.

SIGNIFICANCE: Microblepharon exfoliation improved eyelid signs and tear film characteristics after a single in-office treatment in symptomatic contact lens wearers. PURPOSE: The purpose of this study was to assess the effect of two eyelid hygiene treatments-microblepharon exfoliation and a hypoallergenic foam cleanser (LidHygenix)-on clinical signs of the eyelids, meibomian glands, and tear film in contact lens discomfort. METHODS: A randomized, interventional, unmasked, crossover trial was conducted on 30 experienced daily-wear soft contact lens wearers. Assessment of clinical signs of the eyelid margin, meibomian gland morphology and secretion, and tear film biophysical properties was performed (baseline 1), and participants were randomly assigned to receive one of the two treatments (microblepharon exfoliation or foam cleansing using LidHygenix) as a single in-office procedure. Symptoms were evaluated using the Contact Lens Dry Eye Questionnaire-8 immediately after treatment, and assessment of all the study variables was repeated at the follow-up visit 7 to 10 days after treatment. After 28 to 30 days of washout, participants returned for reassessment of the study variables (baseline 2) and were crossed over to receive the alternate treatment. Follow-up was repeated 7 to 10 days after the second treatment. RESULTS: Seven to 10 days after treatment with microblepharon exfoliation, symptomatic wearers showed significant improvement in anterior blepharitis (mean difference, 0.60; P = .04), lid wiper staining (0.50; P = .06), and lid-parallel conjunctival folds (0.68, P = .02) along with orifice capping (median difference, 0.65; P < .001), foam (0.90; P < .001), secretion volume (0.69; P < .001), quality (0.74; P < .001), and expressibility (0.49; P = .002), which were also clinically significant changes. However, in tear properties, significant improvements were observed in tear volume (LidHygenix, -1.25 mm; microblepharon exfoliation, -1.62 mm), break-up time (-0.14 seconds; -0.14 seconds), tear evaporation rate without contact lenses (21.52 g m h; 45.43 g m h), and lipid layer thickness (-20.61 nm; -25.13 nm) after both treatments but in symptomatic lens wearers only (P < .05). CONCLUSIONS: Microblepharon exfoliation improved eyelid signs and tear film characteristics in symptomatic contact lens wearers after a single in-office treatment.

Predictive Potential of Eyelids and Tear Film in Determining Symptoms in Contact Lens Wearers.

SIGNIFICANCE: Early diagnosis of clinical markers of contact lens discomfort can help clinicians set realistic expectations and monitor and provide prophylactic management for contact lens wearers. PURPOSE: The purpose of this study was to evaluate the potential of eyelid- and tear film-related clinical markers to be used as predictive factors for diagnosing discomfort in contact lens wearers. METHODS: A cross-sectional study was performed on 30 contact lens wearers (6 male, 24 female) with median age of 23 years (range, 18 to 41 years). Eyelid signs and tear film characteristics were evaluated during a single visit, and subjects completed the Contact Lens Dry Eye Questionnaire to evaluate ocular discomfort. Area under the curve (AUC) statistics and sensitivity and specificity values from receiver operating characteristic curves were analyzed to evaluate the predictive potential of clinical signs in discriminating symptoms of contact lens discomfort. RESULTS: The presence of foam at meibomian gland orifices (AUC, 0.944; P < .05; sensitivity >0.7), meibomian gland secretion volume (AUC, 0.935; P < .05; sensitivity >0.7), quality (AUC, 0.969; P < .05; sensitivity >0.7), and expressibility (AUC, 0.933; P < .05; sensitivity >0.7) were significant and strong predictors of discomfort in lens wear. Tear evaporation rates with (AUC, 0.779; P < .05; sensitivity >0.7) or without contact lenses (AUC, 0.788; P < .05; sensitivity >0.7), palpebral conjunctival roughness (AUC, 0.859; P < .05; sensitivity >0.7), palpebral conjunctival staining (AUC, 0.817; P < .05; sensitivity >0.7), palpebral conjunctival hyperemia (AUC, 0.746; P < .05; sensitivity >0.7), meibomian gland orifice capping (AUC, 0.873; P < .05; sensitivity >0.7), pouting (AUC, 0.891; P < .05; sensitivity >0.7), and lid-parallel conjunctival folds (AUC, 0.770; P < .05; sensitivity >0.7) were other acceptable discriminators of symptoms of discomfort during contact lens wear. An equation was developed to identify symptomatic from asymptomatic lens wearers based on the significant predictors: Symptom discriminant function score = 3.378 (meibomian gland secretion grade) + 0.224 (meibomian gland morphology grade) + 0.61 (tear evaporation rate without contact lenses) + 0.439 (lid-parallel conjunctival folds grade) - 0.346 (palpebral conjunctival health grade) - 4.625. CONCLUSIONS: This study demonstrated that clinical signs related to meibomian gland secretions and morphology, tear evaporation, lid-parallel conjunctival folds, and palpebral conjunctival health may successfully predict symptoms of discomfort in contact lens wearers.

Study of Disinfectant Resistance Genes in Ocular Isolates of Pseudomonas aeruginosa.

BACKGROUND: The prevalence of disinfectant resistance in Pseudomonas aeruginosa is on the rise. P. aeruginosa is the most common bacteria isolated from cases of microbial keratitis. Many multi-purpose contact lens disinfectant solutions are available to decontaminate contact lenses before use and to help reduce the incidence of infections. However, with increasing disinfectant resistance, the effect of multi-purpose disinfectant solutions may diminish. The goal of this study was to examine genes associated with disinfectant resistance in ocular isolates of P. aeruginosa and understand the strain's susceptibility to different multipurpose disinfectant solutions. METHODS: Seven potential disinfectant resistance genes were used in BLASTn searches against the whole genomes of 13 eye isolates of P. aeruginosa. A microdilution broth method was used to examine susceptibility to four different multipurpose disinfectant solutions. RESULTS: All strains possessed the sugE2, sugE3 and emrE (qacE) genes. The sugE1 and qacEdelta1 genes were present in 6/13 isolates. No strains contained the qacF or qacG genes. All tested disinfectant solutions had the ability to kill all test strains at 100% concentration, with some strains being susceptible at 1:8 dilutions of the disinfecting solutions. However, the presence of disinfectant resistance genes was not associated with susceptibility to multi-purpose disinfectants. CONCLUSION: All four tested contact lens disinfectant preparations are effective against P. aeruginosa isolates regardless of the presence of disinfectant resistance genes.