Lori A. S. Snyder

Bacterial ocular infections are a major cause of vision loss, with antimicrobial resistance limiting treatment efficacy. This study investigated oregano oil and its phenolic components, carvacrol and thymol, as alternative antibacterial agents for ophthalmic use. Gas chromatography–mass spectrometry analysis identified 27 volatile compounds, dominated by carvacrol (~ 75%), O- cymene (~ 7%), and thymol (~ 3%). Antibacterial activity against Neisseria gonorrhoeae , Staphylococcus aureus , and Pseudomonas aeruginosa was assessed using disc diffusion and broth microdilution assays. Minimum inhibitory and bactericidal concentrations (MICs/MBCs) ranged from 0.003 to 0.1% (v/v) for oregano oil and carvacrol, and from 0.003 to 0.1% (w/v) for thymol. Fractional inhibitory concentration (FIC) analysis revealed additive antibacterial interactions (FICI > 0.5–1) between carvacrol and thymol, suggesting these phenolic constituents could serve as adjunct antibacterial agents. Ocular safety was evaluated using the bovine corneal opacity and permeability (BCOP), hen’s egg chorioallantoic membrane (HET-CAM), and red blood cell haemolysis assays. At concentrations exceeding MICs (0.5% v/v oregano oil, 0.1% v/v carvacrol, 0.1% w/v thymol), no corneal irritation occurred in the BCOP model, although mild to moderate irritation appeared in conjunctival and membrane assays. Safety index analysis showed oregano oil had the widest safety margin, supporting its potential as an antibacterial for ocular and wider use.

The aim of this study was to assess whether a Type VI Secretion System (T6SS) present in the commensal, non-pathogenic Neisseria subflava strain KU1003-01 plays a role in competition between these bacteria and other commensal and pathogenic Gram-negative microorganisms and the yeast Candida albicans . Using an overlap extension PCR strategy, the core T6SS gene encoding membrane protein TssM was knocked out for N. subflava strain KU1003-01. Co-culture competition assays were conducted with either the N. subflava or N. subflava tssM knock-out strains as the attackers and N. gonorrhoeae strain NCCP11945, N. subflava strain KU1003-02, or C. albicans strain 3153 as the targets. There was a significant difference in recoverable target cell CFUs observed following co-culture with the T6SS knock-out strain in comparison to the parent strain. TssM has been identified as critical to T6SS function for a number of species, and our findings indicate that the T6SS in N. subflava contributes to competition with these other species. Key points: • Neisseria subflava Type 6 Secretion System is functional • Neisseria subflava attacks other microbes of the same niche • Neisseria subflava is competent for transformation

Bacterial culture on solid media is the crucial step in diagnosing Neisseria gonorrhoeae infections and is the gold standard for determining their antimicrobial resistance profile. However, culture of Neisseria spp. can be challenging in resource poor areas, relying on specialist incubators or other methods of supplying 5% CO ₂ for growth of the bacteria. Even when such incubators are available, the CO ₂ to run them may be scarce; there were CO ₂ shortages during the COVID-19 pandemic, for example. Although culture jars with gas packs or candles can be used, these are inefficient in terms of use of incubator space and researcher time. To achieve simplicity in culturing of N. gonorrhoeae, the standard Oxoid GC agar base medium, made with the Kellogg’s glucose and iron supplements was improved with the addition of 0.75 g/l sodium bicarbonate (NaHCO ₃ ), which is inexpensive and readily available. This improved media in a standard incubator performed as well as standard Oxoid GC agar media with supplements in a 5% CO ₂ incubator. Chocolate agar and Thayer-Martin agar with sodium bicarbonate were also developed, with all showing good growth of N. gonorrhoeae without the need for atmospheric CO ₂ . Key points: • Neisseria spp. (N. gonorrhoeae, N. meningitidis) require atmospheric CO ₂ to grow. • Sources of CO ₂ may be scarce depending on geography and lab supply availability. • We have developed GC, Chocolate, and Thayer-Martin media that does not need CO ₂ .

Bacterial culture is the crucial step in diagnosing Neisseria gonorrhoeae infections and is the gold standard for determining their antimicrobial resistance profile. However, culture of Neisseria spp. can be challenging in resource poor areas, relying on specialist incubators supplying 5% CO ₂ for growth of the bacteria. Even when such incubators are available, the CO ₂ to run them may be scarce; there were CO ₂ shortages during the COVID-19 pandemic, for example. Although culture jars with gas packs or candles can be used, these are inefficient in terms of use of incubator space and researcher time. To achieve simplicity in culturing of N. gonorrhoeae , the standard Oxoid GC base medium, made with the Kellogg’s glucose and iron supplements was improved with the addition of 0.75 g/L sodium bicarbonate (NaHCO ₃ ), which is inexpensive and really available. This improved media was able to sustain gonococcal growth as well as standard GC media in 5% CO ₂ . Chocolate agar and Thayer-Martin agar with sodium bicarbonate was also developed, with all showing good growth of N. gonorrhoeae without the need for atmospheric CO ₂ .

Ocular infections caused by bacterial pathogens may damage the cornea and rapidly progress to permanent blindness. Topical application of an ophthalmic formulation is often used to treat corneal infections. The animal models used in many preclinical studies frequently involve expensive in vivo experiments that compromises the corneal epithelium to mimic real life conditions such as during contact lens wear, but these do not consider other instances where infection occurs in intact corneas such as in ophthalmia neonatorum. To develop an ex vivo model of infection, bovine eyes from human food chain waste were processed and the corneas inoculated with Neisseria gonorrhoeae strain NCCP 11945 , Staphylococcus aureus strain 6571, and Pseudomonas aeruginosa strain ATCC 15442 for 1 hour, 4 hours, and 6 hours, respectively. Inoculation included intact bovine corneas and those compromised with scalpel, needle, and blot methods. Recovery of N. gonorrhoeae , S. aureus, and P. aeruginosa colonies demonstrated that infection of bovine corneas was achieved with intact and compromised corneas using this model. In addition, corneas inoculated with the bacteria were treated with a suitable antibiotic, demonstrating clearance of the bacterial infections with at least 5 log ₁₀ reduction. This model is appropriate for both establishing infection and testing the ability of antimicrobial agents to clear bacterial eye infections. The bovine ex vivo model is reliable, cost-effective, suitable for different bacteria species, and reduces the need for further animal exploitation in laboratory research. Author Summary: Bacterial pathogens such as Neisseria gonorrhoeae , Staphylococcus aureus, and Pseudomonas aeruginosa infect the eyes, damage the clear transparent cornea and may eventually cause blindness. Several in vivo animal models that have been used to investigate corneal infections in preclinical studies involve compromising the integrity of the corneal epithelium, which predisposes the eye to infection and simulates conditions of corneal abrasion suggested to be seen during contact lens wear. However, corneal infection in infants during ophthalmia neonatorum occurs with intact corneal epithelium and as such may not be explained by abrasion simulating models. Also, in vivo experiments are expensive, involve invasive corneal procedures despite efforts at ethical compliance, and may be time consuming. Reliable models that are quicker, cost effective, cause less (or no) discomfort to animals, and simulate a wide range of corneal infection scenarios need to be explored. Here, we demonstrate the use of a novel ex vivo bovine eye model to establish bacterial infection of the cornea, with and without compromising the corneal epithelium, and clearance of the infection with selected antimicrobial agents. The optimisation of the ex vivo bovine corneal infection model may serve as a bridge between in vitro and in vivo models of corneal infection.

Complete Type VI Secretion Systems were identified in the genome sequence data of Neisseria subflava isolates sourced from throat swabs of human volunteers. The previous report was the first to describe two complete Type VI Secretion Systems in these isolates, both of which were distinct in terms of their gene organization and sequence homology. Since publication of the first report, Type VI Secretion System subtypes have been identified in Neisseria spp. The characteristics of each type in N. subflava are further investigated here and in the context of the other Neisseria spp., including identification of the lineages containing the different types and subtypes. Type VI Secretion Systems use VgrG for delivery of toxin effector proteins; several copies of vgrG and associated effector / immunity pairs are present in Neisseria spp. Based on sequence similarity between strains and species, these core Type VI Secretion System genes, vgrG, and effector / immunity genes may diversify via horizontal gene transfer, an instrument for gene acquisition and repair in Neisseria spp.

Notwithstanding their 3 to 5% mortality, the 2.7 million envenomation-related injuries occurring annually—predominantly across Africa, Asia, and Latin America—are also major causes of morbidity. Venom toxin-damaged tissue will develop infections in some 75% of envenomation victims, with E. faecalis being a common culprit of disease; however, such infections are generally considered to be independent of envenomation.

Ophthalmia neonatorum is a severe, sight-threatening condition that occurs in neonates worldwide. Etiological factors include chemical agents, viruses, and bacteria, such as Neisseria gonorrhoeae a...

Commensal non-pathogenic Neisseria spp. live within the human host alongside the pathogenic Neisseria meningitidis and Neisseria gonorrhoeae and due to natural competence, horizontal gene transfer within the genus is possible and has been observed. Four distinct Neisseria spp. isolates taken from the throats of two human volunteers have been assessed here using a combination of microbiological and bioinformatics techniques. Three of the isolates have been identified as Neisseria subflava biovar perflava and one as Neisseria cinerea . Specific gene clusters have been identified within these commensal isolate genome sequences that are believed to encode a Type VI Secretion System, a newly identified CRISPR system, a Type IV Secretion System unlike that in other Neisseria spp., a hemin transporter, and a haem acquisition and utilization system. This investigation is the first to investigate these systems in either the non-pathogenic or pathogenic Neisseria spp. In addition, the N. subflava biovar perflava possess previously unreported capsule loci and sequences have been identified in all four isolates that are similar to genes seen within the pathogens that are associated with virulence. These data from the four commensal isolates provide further evidence for a Neisseria spp. gene pool and highlight the presence of systems within the commensals with functions still to be explored.

Neisseria gonorrhoeae bacteria are acknowledged as an urgent threat to human health because this species has developed resistances to all of the antibiotics used clinically to treat its infections. N. gonorrhoeae causes the sexually transmitted disease gonorrhoea, but also causes blindness when the bacteria infect the eyes. Infants are particularly susceptible, acquiring the infection from their mothers at birth. We have shown that the monoglyceride monocaprin rapidly kills N. gonorrhoeae and other bacterial species and is non-irritating in ocular assays. Here we show that the physical and chemical properties of monocaprin make it ideal for use in a thickened eye drop formulation to combat eye infections. Monocaprin-containing formulations were assessed using analytical techniques and for antimicrobial activity in vitro and in ex vivo infections. Monocaprin-containing formulations retained activity after three years and are non-irritating, unlike preparations of povidone iodine in our assays. A recommended formulation for further development and investigation is 0.25% monocaprin in 1% HPMC with 1% polysorbate 20.