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. Full article

The purpose of the study was to assess the antimicrobial activity of an ultraviolet-C (UVC) device against microorganisms implicated in contact lens related adverse events. An UVC device with an emitting 4.5 mm diameter Light Emitting Diode (LED; 265 nm; 1.93 mJ/cm²) was used. Pseudomonas aeruginosa, Staphylococcus aureus, Fusarium solani, and Candida albicans agar plate lawns were exposed to the device beams for 15 and 30 s at 8 mm distance. Following the exposure, the diameter of the growth inhibition zone was recorded. Contact lenses made of Delfilicon-A, Senofilicon-A, Comfilicon-A, Balafilicon-A, Samfilicon-A and Omafilicon-A and a commercially available contact storage case was used. They were exposed to bacterial and fungal strains for 18 h at 37 °C and 25 °C respectively. After this, the samples were exposed to UVC for 30 s at 8 mm distance to determine the antimicrobial efficacy. Samples were then gently washed and plated on appropriate agar for enumeration of colonies. The UVC exposure reduced microbial growth by 100% in agar lawns, and significantly (p < 0.05) reduced microbial contamination to contact lenses and cases, ranging between 0.90 to 4.6 log. Very short UVC exposure has high antimicrobial efficacy against most of the predominant causative microorganisms implicated in contact lens related keratitis. UVC could be readily used as a broad-spectrum antimicrobial treatment for lens disinfection. Full article

It has been previously reported that the improper prescribing of antibiotic eye drops is common among orthokeratology (ortho-k) practitioners. Guidelines have since been developed and disseminated to improve their understanding and implementation of antibiotic prescriptions. This study aimed to investigate the influence of these guidelines on the knowledge, attitude, and prescribing habits of ortho-k practitioners by means of a questionnaire, which was administered nationwide via an official online account to eye care practitioners (ECPs) involved in ortho-k lens fitting, 548 of whom completed the survey. Differences in characteristics before and after the dissemination of the guidelines and between the groups were explored using χ² tests. The relationship between prescribing habits and demographics was analyzed using stepwise logistic regression models. The implementation of the guidelines significantly improved the overall prescribing habits of ECPs (p < 0.001), especially for prophylactic antibiotic use before and after ortho-k lens wear (p < 0.001). Most ECPs who prescribed antibiotics properly displayed significantly better knowledge of correct antibiotic use, which in turn affected the compliance in their ortho-k patients (p < 0.001). The ECPs’ occupations (professionals other than ophthalmologists and optometrists, including nurses and opticians), clinical setting (distributor fitting centers), and age (younger than 25 years) were risk factors for the misuse of antibiotics. Although the implementation of the antibiotic guidelines significantly improved overall prescribing habits, some practitioners’ prescribing behavior still needs improvement. A limitation of this study was that all questions were mandatory, requiring ECPs to recall information, and therefore was subjected to selection and recall bias. Full article

(1) Purpose: This study aimed to investigate the effects of Mel4 antimicrobial contact lenses (MACL) on the ocular surface and comfort during extended wear. (2) Methods: A prospective, randomised, double-masked, contralateral clinical trial was conducted with 176 subjects to evaluate the biocompatibility of contralateral wear of MACL. The wearing modality was 14-day extended lens wear for three months. The participants were assessed at lens dispensing, after one night, two weeks, one month and three months of extended wear and one month after study completion. (3) Results: There were no significant differences (p > 0.05) in ocular redness or palpebral roughness between Mel4 and control eyes at any of the study visits. There was no significant difference (p > 0.05) in corneal staining between Mel4 and control eyes. There were no significant differences in front surface wettability or deposits or back surface debris (p > 0.05). No statistically significant differences (p > 0.05) were found in comfort, dryness, CLDEQ-8 scores lens or edge awareness. There was no evidence for delayed reactions on the ocular surface after cessation of lens wear. (4) Conclusion: The novel MACLs showed similar comfort to control lenses and were biocompatible during extended wear. Thus, these lenses were compatible with the ocular surface. Full article

This study analyzed the clinical features and molecular characteristics of methicillin-susceptible Staphylococcus aureus (MSSA) ocular infections in Taiwan and compared them between community-associated (CA) and health-care-associated (HA) infections. We collected S. aureus ocular isolates from patients at Chang Gung Memorial Hospital between 2010 and 2017. The infections were classified as CA or HA using epidemiological criteria, and the isolates were molecularly characterized using pulsed-field gel electrophoresis, multilocus sequence typing, and Panton-Valentine leukocidin (PVL) gene detection. Antibiotic susceptibility was evaluated using disk diffusion and an E test. A total of 104 MSSA ocular isolates were identified; 46 (44.2%) were CA-MSSA and 58 (55.8%) were HA-MSSA. Compared with HA-MSSA strains, CA-MSSA strains caused a significantly higher rate of keratitis, but a lower rate of conjunctivitis. We identified 14 pulsotypes. ST 7/pulsotype BA was frequently identified in both CA-MSSA (28.3%) and HA-MSSA (37.9%) cases. PVL genes were identified in seven isolates (6.7%). Both CA-MSSA and HA-MSSA isolates were highly susceptible to vancomycin, teicoplanin, tigecycline, sulfamethoxazole–trimethoprim, and fluoroquinolones. The most common ocular manifestations were keratitis and conjunctivitis for CA-MSSA and HA-MSSA, respectively. The MSSA ocular isolates had diverse molecular characteristics; no specific genotype differentiated CA-MSSA from HA-MSSA. Both strains exhibited similar antibiotic susceptibility. Full article

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. Full article

Staphylococcus aureus can develop resistance by mutation, transfection or biofilm formation. Resistance was induced in S. aureus by growth in sub-inhibitory concentrations of ciprofloxacin for 30 days. The ability of the antimicrobials to disrupt biofilms was determined using crystal violet and live/dead staining. Effects on the cell membranes of biofilm cells were evaluated by measuring release of dyes and ATP, and nucleic acids. None of the strains developed resistance to AMPs while only S. aureus ATCC 25923 developed resistance (128 times) to ciprofloxacin after 30 passages. Only peptides reduced biofilms of ciprofloxacin-resistant cells. The antibiofilm effect of melimine with ciprofloxacin was more (27%) than with melimine alone at 1X MIC (p < 0.001). Similarly, at 1X MIC the combination of Mel4 and ciprofloxacin produced more (48%) biofilm disruption than Mel4 alone (p < 0.001). Combinations of either of the peptides with ciprofloxacin at 2X MIC released ≥ 66 nM ATP, more than either peptide alone (p ≤ 0.005). At 2X MIC, only melimine in combination with ciprofloxacin released DNA/RNA which was three times more than that released by melimine alone (p = 0.043). These results suggest the potential use of melimine and Mel4 with conventional antibiotics for the treatment of S. aureus biofilms. Full article

The Rcs phosphorelay is a bacterial stress response system that responds to envelope stresses and in turn controls several virulence-associated pathways, including capsule, flagella, and toxin biosynthesis, of numerous bacterial species. The Rcs system also affects antibiotic tolerance, biofilm formation, and horizontal gene transfer. The Rcs system of the ocular bacterial pathogen Serratia marcescens was recently demonstrated to influence ocular pathogenesis in a rabbit model of keratitis, with Rcs-defective mutants causing greater pathology and Rcs-activated strains demonstrating reduced inflammation. The Rcs system is activated by a variety of insults, including β-lactam antibiotics and polymyxin B. In this study, we developed three luminescence-based transcriptional reporters for Rcs system activity and used them to test whether antibiotics used for empiric treatment of ocular infections influence Rcs system activity in a keratitis isolate of S. marcescens. These included antibiotics to which the bacteria were susceptible and resistant. Results indicate that cefazolin, ceftazidime, polymyxin B, and vancomycin activate the Rcs system to varying degrees in an RcsB-dependent manner, whereas ciprofloxacin and tobramycin activated the promoter fusions, but in an Rcs-independent manner. Although minimum inhibitory concentration (MIC) analysis demonstrated resistance of the test bacteria to polymyxin B and vancomycin, the Rcs system was activated by sub-inhibitory concentrations of these antibiotics. Together, these data indicate that a bacterial stress system that influences numerous pathogenic phenotypes and drug-tolerance is influenced by different classes of antibiotics despite the susceptibility status of the bacterium. Full article

It was previously demonstrated that predatory bacteria are able to efficiently eliminate Gram-negative pathogens including antibiotic-resistant and biofilm-associated bacteria. In this proof-of-concept study we evaluated whether two species of predatory bacteria, Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus, were able to alter the survival of Gram-negative pathogens on the ocular surface. Clinical keratitis isolates of Pseudomonas aeruginosa (strain PAC) and Serratia marcescens (strain K904) were applied to the ocular surface of NZW rabbits followed by application of predatory bacteria. At time intervals, surviving pathogenic bacteria were enumerated. In addition, B. bacteriovorus and S. marcescens were applied to porcine organ culture corneas under contact lenses, and the ocular surface was examined by scanning electron microscopy. The ocular surface epithelial layer of porcine corneas exposed to S. marcescens, but not B. bacteriovorus was damaged. Using this model, neither pathogen could survive on the rabbit ocular surface for longer than 24 h. M. aeruginosavorus correlated with a more rapid clearance of P. aeruginosa but not S. marcescens from rabbit eyes. This study supports previous evidence that predatory bacteria are well tolerated by the cornea, but suggest that predatory bacteria do not considerably change the ability of the ocular surface to clear the tested Gram-negative bacterial pathogens from the ocular surface. Full article

Relatively little is known about how the corneal epithelium responds to vision-threatening bacteria from the Enterobacterales order. This study investigates the impact of Serratia marcescens on corneal epithelial cell host responses. We also investigate the role of a bacterial transcription factor EepR, which is a positive regulator of S. marcescens secretion of cytotoxic proteases and a hemolytic surfactant. We treated transcriptomic and metabolomic analysis of human corneal limbal epithelial cells with wild-type bacterial secretomes. Our results show increased expression of proinflammatory and lipid signaling molecules, while this is greatly altered in eepR mutant-treated corneal cells. Together, these data support the model that the S. marcescens transcription factor EepR is a key regulator of host-pathogen interactions, and is necessary to induce proinflammatory chemokines, cytokines, and lipids. Full article

Coagulase-negative staphylococci (CoNS) are frequently occurring ocular opportunistic pathogens that are not easily identifiable to the species level. The goal of this study was to speciate CoNS and document antibiotic susceptibilities from cases of endophthalmitis (n = 50), keratitis (n = 50), and conjunctivitis/blepharitis (n = 50) for empiric therapy. All 150 isolates of CoNS were speciated using (1) API Staph (biochemical system), (2) Biolog GEN III Microplates (phenotypic substrate system), and (3) DNA sequencing of the sodA gene. Disk diffusion antibiotic susceptibilities for topical and intravitreal treatment were determined based on serum standards. CoNS identification to the species level by all three methods indicated that S. epidermidis was the predominant species of CoNS isolated from cases of endophthalmitis (84–90%), keratitis (80–86%), and conjunctivitis/blepharitis (62–68%). Identifications indicated different distributions of CoNS species among endophthalmitis (6), keratitis (10), and conjunctivitis/blepharitis (13). Antibiotic susceptibility profiles support empiric treatment of endophthalmitis with vancomycin, and keratitis treatment with cefazolin or vancomycin. There was no clear antibiotic choice for conjunctivitis/blepharitis. S. epidermidis was the most frequently found CoNS ocular pathogen, and infection by other CoNS appears to be less specific and random. Antibiotic resistance does not appear to be a serious problem associated with CoNS. Full article